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UNIVERSIDADE FEDERAL DE MATO GROSSO
CAcircMPUS UNIVERSITAacuteRIO DE SINOP
INSTITUTO DE CIEcircNCIAS NATURAIS HUMANAS E SOCIAIS
PROGRAMA DE POacuteS-GRADUACcedilAtildeO EM CIEcircNCIAS AMBIENTAIS - PPGCAM
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
CAMILA CRISTINA PEREIRA DE SOUZA
Sinop ndash Mato Grosso
fevereiro de 2017
2
CAMILA CRISTINA PEREIRA DE SOUZA
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Orientador Prof Dr Adilson Paulo Sinhorin
Co-orientadora Profa Dr
a Valeacuteria Dornelles Gindri Sinhorin
Dissertaccedilatildeo apresentada ao PPGCAM como
parte dos requisitos para obtenccedilatildeo do tiacutetulo
de Mestre em Ciecircncias Ambientais
Aacuterea de concentraccedilatildeo Bioprospecccedilatildeo
Sinop ndash Mato Grosso
fevereiro de 2017
ii
iii
iv
Sinopse
Estudou-se o perfil fitoquiacutemico de extratos de folhas de Caesalpinia
ferrea avaliou-se a atividade radicalar in vitro e atividade
antioxidante em camundongos pelo modelo de estresse oxidativo
induzido via intoxicaccedilatildeo aguda por paracetamol
Palavras-chave Plantas medicinais flavonoides hepatotoxicidade
radicais livres
v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
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Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
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of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
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Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
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Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
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21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
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Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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24
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Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
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2
CAMILA CRISTINA PEREIRA DE SOUZA
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Orientador Prof Dr Adilson Paulo Sinhorin
Co-orientadora Profa Dr
a Valeacuteria Dornelles Gindri Sinhorin
Dissertaccedilatildeo apresentada ao PPGCAM como
parte dos requisitos para obtenccedilatildeo do tiacutetulo
de Mestre em Ciecircncias Ambientais
Aacuterea de concentraccedilatildeo Bioprospecccedilatildeo
Sinop ndash Mato Grosso
fevereiro de 2017
ii
iii
iv
Sinopse
Estudou-se o perfil fitoquiacutemico de extratos de folhas de Caesalpinia
ferrea avaliou-se a atividade radicalar in vitro e atividade
antioxidante em camundongos pelo modelo de estresse oxidativo
induzido via intoxicaccedilatildeo aguda por paracetamol
Palavras-chave Plantas medicinais flavonoides hepatotoxicidade
radicais livres
v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
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Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
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international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
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author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
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Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
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ii
iii
iv
Sinopse
Estudou-se o perfil fitoquiacutemico de extratos de folhas de Caesalpinia
ferrea avaliou-se a atividade radicalar in vitro e atividade
antioxidante em camundongos pelo modelo de estresse oxidativo
induzido via intoxicaccedilatildeo aguda por paracetamol
Palavras-chave Plantas medicinais flavonoides hepatotoxicidade
radicais livres
v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
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Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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These keywords will be used for indexing purposes
Chemical compounds
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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30
EPS (or PDF) Vector drawings embed all used fonts
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iii
iv
Sinopse
Estudou-se o perfil fitoquiacutemico de extratos de folhas de Caesalpinia
ferrea avaliou-se a atividade radicalar in vitro e atividade
antioxidante em camundongos pelo modelo de estresse oxidativo
induzido via intoxicaccedilatildeo aguda por paracetamol
Palavras-chave Plantas medicinais flavonoides hepatotoxicidade
radicais livres
v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
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from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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24
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7 Conference announcements and news
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25
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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30
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31
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iv
Sinopse
Estudou-se o perfil fitoquiacutemico de extratos de folhas de Caesalpinia
ferrea avaliou-se a atividade radicalar in vitro e atividade
antioxidante em camundongos pelo modelo de estresse oxidativo
induzido via intoxicaccedilatildeo aguda por paracetamol
Palavras-chave Plantas medicinais flavonoides hepatotoxicidade
radicais livres
v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
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The rules of 5
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discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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v
Dedicatoacuteria
A minha matildee irmatildeos familiares professores e amigos
pelo incentivo e companheirismo Ao meu padrinho
Geraldo de Almeida pelo constante estiacutemulo pela busca
do saber
vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
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Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
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applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
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Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
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international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
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Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
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modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
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appropriate Avoid extensive citations and discussion of published literature
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Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
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bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Acknowledgements
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writing assistance or proof reading the article etc)
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vi
AGRADECIMENTOS
A Deus pelo existir
Ao professor Dr Adilson Paulo Sinhorin pela confianccedila paciecircncia e orientaccedilatildeo
exercida na transmissatildeo dos conhecimentos proporcionando o desenvolvimento desse trabalho
e o aprendizado inerente
A professora Dra Valeacuteria Dornelles Gindri Sinhorin pela confianccedila oportunidade e
ensinamentos para desenvolver as pesquisas no Laboratoacuterio de Bioquiacutemica
A Universidade Federal de Mato Grosso Cacircmpus de Sinop em especial ao Programa
de Poacutes Graduaccedilatildeo em Ciecircncias Ambientais coordenador professores colaboradores e
secretaacuteria Aos colegas e amigos de turma em especial Liacutelian Marigo Magalhatildees pelo
companheirismo paciecircncia e estiacutemulo durante todo o desenvolvimento da pesquisa Aos
colegas e amigos Fernando Gomes Barbosa Naieacutele Sartori Patias e Fernando Rafael De
Moura por toda ajuda companheirismo durante o desenvolvimento das aulas e dos trabalhos
laboratoriais
Aos amigos dos Laboratoacuterios Integrados de Pesquisas Quiacutemicas (LIPEQ)
Laboratoacuterios de Fitoquiacutemica e Bioquiacutemica Thaniara Costa Barbosa Tatiane Cordeiro Luz
Ritane Rose Ana Paula Simotildees da Cunha Kamila Brentegani e Aline Gemelli pelas
contribuiccedilotildees prestadas para que a pesquisa fosse desenvolvida pela ajuda nos experimentos
pelas longas horas de trabalho pelos feriados domingos e longos periacuteodos em que se
dispuseram para estar comigo no laboratoacuterio realizando as anaacutelises
A Ms Deacutebora Linsbinski Pereira e Ms Jacqueline Kerkhoffk por toda ajuda no
desenvolvimento laboratorial da pesquisa
Agradecimento especial a todas contribuiccedilotildees dos integrantes das bancas de
qualificaccedilatildeo e defesa Dra Marina Mariko Sugui Dr
a Stela Regina Ferrarini Dr
a Carla
Regina Andrighetti Dra Patriacutecia da Costa Marisco Dr
a Cibele Bonacorsi e Dr
Rogeacuterio de
Campos Bicudo
A Embrapa Agrossilvipastoril Sinop-MT e ao Rogeacuterio de Campos Bicudo por toda
ajuda teacutecnica no desenvolvimento desse trabalho
Ao meus amigos do Mato Grosso Rosana Ceconello Bento Gislaine de Oliveira
Flaacutevio Menezes Filipe Ceconello por todo companheirismo durante o desenvolvimento dessa
jornada
Agradecimento a agecircncia de financiamento FAPEMAT pelo apoio agrave pesquisa
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
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Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
For subscription articles
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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26
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Language (usage and editing services)
Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
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visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
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Symbol or use fonts that look similar
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You are urged to visit this site some excerpts from the detailed information are given here
Formats
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
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below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
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bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
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Color artwork
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Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
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Figure captions
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Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
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are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
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MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
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that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
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athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
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All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
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bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
vii
A diferenccedila entre o sonho e a realidade eacute a
quantidade certa de tempo e trabalho
William Douglas
viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
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Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
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Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
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6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
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Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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31
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viii
RESUMO
Caesalpinia ferrea Mart eacute uma planta usada na medicina popular para o tratamento de
doenccedilas como bronquite diabetes coacutelica intestinal diarreias gengivite e siacutefilis Estudos
demonstram que C ferrea possui atividade antimicrobiana cicatrizante antiinflamatoacuteria e
antitumoral Esse trabalho trata do estudo da atividade radicalar pelo radical DPPH e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C ferrea
coletadas em SinopMT frente estresse oxidativo induzido com paracetamol (PCM) em
camundongos Swiss machos As folhas de C ferrea foram coletadas em SinopMT para o
preparo dos extratos etanoacutelico (EE) e acetato de etila (EA) EE e EA tiveram a atividade
radicalar in vitro determinada por espectofotometria UV-VIS e flavonoides identificados por
cromatografia liacutequida-espectrometria de massa (LC-MSMS) No estudo bioloacutegico avaliou-se
o efeito de EE e EA (50 mgKg) comparado aos controles com administraccedilatildeo de aacutegua e PCM
(250 mgKg) na determinaccedilatildeo da dosagem de biomarcadores (superoacutexido dismutase (SOD)
catalase (CAT) glutationa-S-transferase (GST) glutationa reduzida (GSH) proteiacutenas
carboniladas (PC) aacutecido ascoacuterbico (ASA) espeacutecies reativas do aacutecido tiobarbituacuterico
(TBARS)) do estresse oxidativo nos tecidos hepaacutetico eou renal Avaliou-se os paracircmetros
plasmaacuteticos alanina aminotransferase (ALT) aspartato aminotransferase (AST) glicemia
trigliceriacutedeos e colesterol Foram identificados em ambos os extratos os flavonoides rutina
amentoflavona quercetina-3-β-D-glicosiacutedeo taxifolina apigenina e luteolina Aleacutem desses
quercitrina foi encontrada em EE A atividade radicalar de EE e EA demonstrou EC50 460
microgmL e 343 microgmL respectivamente A atividade de enzimas hepaacuteticas sofreu reduccedilatildeo CAT
(376 ) e GST (206 ) no grupo intoxicado com PCM e EA foi hepatoprotetor sobre essas
enzimas As taxas de PC elevaram-se no grupo PCM em 768 (fiacutegado) e 452 (rim) EE e
EA reduziram esse efeito toacutexico do PCM sobre as PC em ambos tecidos avaliados GSH
sofreu depleccedilatildeo de 271 (hepaacutetico) e 424 (renal) no grupo PCM e EA (fiacutegado) e EE e
EA (rim) foram efetivos em restaurar os niacuteveis de GSH ASA sofreu reduccedilatildeo de 200 no
grupo exposto ao PCM e EA foi capaz de restaurar esses niacuteveis Foi observada elevaccedilatildeo (444
) nos niacuteveis de TBARS no grupo PCM natildeo havendo alteraccedilotildees significativas nos demais
grupos Houve aumento dos niacuteveis seacutericos de ALT (1197 ) e AST (687 ) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EE e EA foram hipolipidemiantes e EA foi hipoglicemiante Logo o maior teor de
fenoacuteis totais de EA somado ao seu melhor potencial antioxidante in vitro corroboram tambeacutem
com seu melhor efeito antioxidante bioloacutegico monitorados pelos biomarcadores avaliados
nesse estudo
Palavras-chave Plantas medicinais Flavonoide Antioxidante Paracetamol Estresse
oxidativo
ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Food Chem 166 179ndash191 doi101016jfoodchem201406011
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
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of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
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Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
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Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
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doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
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Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
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Click here to see an example
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
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with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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30
EPS (or PDF) Vector drawings embed all used fonts
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References
Citation in text
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Reference management software
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(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
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ix
ABSTRACT
Caesalpinia ferrea is used in folk medicine for the treatment of conditions bronchitis
diabetes intestinal colic diarrhea gingivitis and syphilis Scientific studies show that C
ferrea has antimicrobial healing anti-inflammatory and anti-tumor activity
This work deals with the radical radical DPPH activity and antioxidant activity of ethanolic
(EE) and ethyl acetate (EA) extracts of C ferrea leaves collected in Sinop MT against
oxidative stress induced with paracetamol (PCM) in Swiss mice Males Leaves of C ferrea
were collected in Sinop MT to prepare extracts of ethyl acetate (EA) and ethanolic (EE) EA
and EE had determined in vitro antioxidant potential by spectrophotometry UV-VIS and
flavonoids identified by liquid chromatography-tandem mass spectrometry (LC-MS MS) In
the biological study the effect of EE and EA (50 mg kg) compared to controls with water
and PCM administration (250 mg kg) was evaluated in the determination of biomarkers
dosage superoxide dismutase (SOD) catalase (GST) reduced glutathione (GSH)
carbonylated proteins (PC) ascorbic acid (ASA) thiobarbituric acid derivatives (TBARS)
from oxidative stress in hepatic and or renal tissues Plasma alanine aminotransferase (ALT)
aspartate aminotransferase (AST) glycemia triglycerides and cholesterol parameters were
evaluated The flavonoids rutin amentoflavone quercetin-3-β-D-glucoside quercetin
taxifoline apigenin and luteolin were identified in both extracts Besides these quercitrin was
found in EE The radical activity of EE and EA demonstrated EC 50 460 μg mL and 343 μg
mL respectively The activity of liver enzymes was reduced by CAT (376) and GST
(206) in the group intoxicated with PCM and EA was hepatoprotective on these enzymes
Rates of CP increased in the PCM group by 768 (liver) and 452 (kidney) EE and EA
reduced this toxic effect of PCM on CP in both tissues evaluated
GSH was depleted 271 (hepatic) and 424 (renal) in the PCM and EA (liver) groups and
EE and AI (kidney) were effective in restoring GSH levels ASA suffered a reduction of
200 in the group exposed to PCM and EA was able to restore these levels Elevation
(444) was observed in the TBARS levels in the PCM group and there were no significant
alterations in the other groups There was an increase in the serum levels of ALT (1197)
and AST (687) in the group exposed to PCM however the treatment with EE and EA were
effective in reducing these activities EE and EA were hypolipidemic and EA was
hypoglycemic Therefore the highest total phenol content of EA added to its best antioxidant
potential in vitro also corroborates with its better biological antioxidant effect monitored by
the biomarkers evaluated in this study
Keywords Medicinal plants Flavonoid Antioxidant Acetaminophen Oxidative stress
x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
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Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
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You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
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The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
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The Journal of Ethnopharmacology will accept the following contributions
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papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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x
SUMAacuteRIO
CAPIacuteTULO I xii
Resumo 1
1 Introduccedilatildeo 2
2 Material e meacutetodos 3
21 Compostos quiacutemicos 3
22 Coleta e identificaccedilatildeo botacircnica 3
23 Obtenccedilatildeo dos extratos 4
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar 4
25 Anaacutelise por LC-MSMS 4
26 Animais 5
27 Induccedilatildeo do estresse oxidativo e grupos experimentais 5
28 Anaacutelises bioquiacutemicas 6
281 Antioxidantes enzimaacuteticos 6
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo 6
283 Anaacutelises de paracircmetros plasmaacuteticos 7
29 Anaacutelise estatiacutestica 7
3 Resultados 7
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar 7
32 Anaacutelise por LC-MSMS 8
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico 10
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal 11
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
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from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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Language (usage and editing services)
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visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
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article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
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preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
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Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
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Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
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Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
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In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
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presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
Database linking
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Math formulae
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Footnotes
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Artwork
Electronic artwork
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below)
30
EPS (or PDF) Vector drawings embed all used fonts
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References
Citation in text
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Reference links
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Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
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and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
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Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
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Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
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Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
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32
Supplementary data
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Submission checklist
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For any further information please visit our customer support site at httpsupportelseviercom
xi
35 Paracircmetros metaboacutelicos do plasma 12
4 Discussatildeo 13
41 Anaacutelise por LC-MSMS 13
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo 14
5 Conclusatildeo 17
Agradecimentos 17
Referecircncia 17
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology 22
xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
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Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
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properties Molecules 21 doi103390molecules21030245
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
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discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
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influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
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Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
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Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
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appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
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28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
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Abstract
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
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Click here to see an example
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viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
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chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
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with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
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xii
CAPIacuteTULO I
IDENTIFICACcedilAtildeO DE FLAVONOIDES POR LC-MSMS DE
EXTRATOS DE FOLHAS DE Caesalpinia ferrea Mart E AVALIACcedilAtildeO
DA REVERSAtildeO DO ESTRESSE OXIDATIVO EM CAMUNDONGOS
Artigo a ser submetido ao Journal of Ethnopharmacology
1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
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Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
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Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
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Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
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You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
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The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
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Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
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Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
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below for description and format
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Outlines for potential reviews need to include
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6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
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Before You Begin
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In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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1
Identificaccedilatildeo de flavonoides por LC-MSMS de extratos de folhas de
Caesalpinia ferrea Mart e avaliaccedilatildeo da reversatildeo do estresse oxidativo em camundongos
Camila Cristina Pereira de Souzaa Liacutelian Marigo Magalhatildees
a Thaniara Costa Barbosa
b
Rogeacuterio de Campos Bicudod Valeacuteria Dornelles Gindri Sinhorin
c Adilson Paulo Sinhorin
a
aPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Fitoquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil bCurso de Farmaacutecia Instituto de Ciecircncias da Sauacutede Universidade Federal de Mato Grossso Cacircmpus de Sinop Mato Grosso Brasil cPrograma de Poacutes-Graduaccedilatildeo em Ciecircncias Ambientais Laboratoacuterios Integrados de Pesquisas em Quiacutemica (LIPEQ) Bioquiacutemica Instituto de
Ciecircncias Naturais Humanas e Sociais Universidade Federal de Mato Grossso Campus de Sinop Mato Grosso Brasil dEmbrapa Agrossilvipastoril Rodovia MT-222 Km 25 sn - Zona Rural Sinop - MT Brasil
Resumo
Relevacircncia farmacoloacutegica Caesalpinia ferrea Mart eacute uma planta usada tradicionalmente no
tratamento de bronquite diabetes coacutelica intestinal diarreias e siacutefilis
Objetivo do estudo Identificar flavonoides avaliar o potencial antioxidante in vitro e
atividade antioxidante de extratos etanoacutelico (EE) e acetato de etila (EA) de folhas de C
ferrea coletadas em SinopMT frente ao estresse oxidativo induzido com paracetamol (PCM)
em camundongos
Material e meacutetodos Os teores de fenoacuteis e flavonoides totais foram determinados por
espectofotometria UV-VIS Flavonoides foram identificados por LC-MSMS no modo de
aquisiccedilatildeo de monitoramento de reaccedilotildees muacuteltiplas (MRM) atraveacutes de ionizaccedilatildeo por eletrospray
negativa A atividade radicalar seguiu reaccedilatildeo com radical DPPH No estudo bioloacutegico
camundongos Swiss machos foram subdividos em grupos nos quais foi administrada dose
aguda de PCM (250mgKg) eou tratamento com EE e EA (50 mgKg) Biomarcadores do
estresse oxidativo (SOD CAT GST GSH PC ASA e TBARS) dos tecidos hepaacutetico e renal
e os paracircmetros plasmaacuteticos (ALT e AST glicose trigliceriacutedeos e colesterol) foram
determinadas
Resultados Os flavonoides identificados em EE e EA foram mz-1rarriacuteon produto
60952rarr30020 para rutina 53746rarr37500 para amentoflavona 46338rarr 30000 para
quercetina-3-β-d-glicosiacutedeo 30325rarr 12500 para taxifolina 28524rarr13300 para luteolina
26924rarr11680 para apigenina Aleacutem desses a identificaccedilatildeo de quercitrina em EE seguiu
mz-1 44738rarr 30100 EE e EA demonstraram EC50 460 microgmL e 343 microgmL
respectivamente para o potencial antioxidante com radical DPPH A atividade de enzimas
hepaacuteticas sofreu reduccedilatildeo CAT (376) e GST (206 ) no grupo PCM e EA foi
hepatoprotetor sobre essas enzimas PC elevou-se no grupo PCM em 768 (fiacutegado) e 452
(rim) e EE e EA reduziram esse efeito toacutexico em ambos tecidos estudados GSH sofreu
depleccedilatildeo 271 (fiacutegado) e 424 (rim) no grupo PCM e EA (fiacutegado) e EE e EA (rim) foram
efetivos em restaurar os niacuteveis desse biomarcador ASA sofreu reduccedilatildeo de 200 no grupo
exposto ao PCM e EA foi capaz restaurar esses niacuteveis Foi observada elevaccedilatildeo nos niacuteveis de
TBARS em 444 no grupo intoxicado com PCM natildeo havendo alteraccedilotildees significativas nos
demais grupos Houve aumento dos niacuteveis seacutericos de ALT (1197) e AST (687) no grupo
exposto ao PCM todavia o tratamento com EE e EA foram eficazes na reduccedilatildeo dessas
atividades EA foi hipoglicemiante e EE e EA foram hipolipemiantes
Conclusatildeo O teor de fenoacuteis totais os flavonoides identificados em C ferrea e melhor
potencial antioxidante obtido para esse vegetal corroboram com o melhor efeito antioxidante
bioloacutegico demonstrado pelos biomarcadores avaliados nesse estudo
Palavras-chave Paracetamol Antioxidante Estresse oxidativo Flavonoide Plantas
Medicinais
2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
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Before You Begin
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For information on Ethics in publishing and Ethical guidelines for journal publication
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In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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29
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30
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31
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Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
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March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
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32
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2
1 Introduccedilatildeo
Os organismos aeroacutebios desenvolveram um complexo aparato bioloacutegico em funccedilatildeo da
exposiccedilatildeo a intermediaacuterios reativos oriundos dos processos bioquiacutemicos A condiccedilatildeo de
estresse oxidativo instala-se quando haacute o desequiliacutebrio da homeostase entre formaccedilatildeo de
espeacutecies reativas do oxigecircnio (ERO) e espeacutecies reativas do nitrogecircnio (ERN) com ativaccedilatildeo
das vias bioquiacutemicas envolvidas nos sistemas de detoxificaccedilatildeo celular (Jomova and Valko
2011) O local de atuaccedilatildeo das ERO e ERN na ceacutelula determina seu potencial lesivo A maior
gravidade lesiva estaacute relacionada a proximidade ao DNA devido a possibilidade de mutaccedilotildees
celulares (Saacutenchez-Rodriacuteguez et al 2016)
A formaccedilatildeo de EROs e ERNs estaacute relacionada aos fatores exoacutegenos como dieta e
estilo de vida aleacutem de exposiccedilatildeo a poluentes ambientais radiaccedilatildeo ionizante metais
pesticidas quiacutemicos partiacuteculas do ar e uso recorrente de medicamentos (Rahiminejad et al
2016) Acredita-se que os radicais livres estejam envolvidos nos processos de envelhecimento
celular formaccedilatildeo tumoral diabetes e doenccedilas neurodegenerativas (Limon-Pacheco amp
Gonsebatt 2009)
A associaccedilatildeo do estresse oxidativo a enfermidades da atualidade tem fomentado vaacuterios
estudos bioloacutegicos nesse campo O modelo de intoxicaccedilatildeo aguda por xenobioacuteticos como o
paracetamol (PCM) tem sido amplamente empregado devido a sua farmacocineacutetica
conhecida Dessa forma a overdose de PCM conduz ao acuacutemulo de N-acetil-benzoquinona
imina (NAPQI) composto altamente reativo que configura-se como precursor de efeitos
toacutexicos secundaacuterios observados nos organismos Efeitos esses cujo monitoramento via
biomarcadores enzimaacuteticos e natildeo enzimaacuteticos do estresse oxidativo permite a avaliaccedilatildeo do
grau do dano hepaacutetico e renal associado (Mishra et al 2014)
Estudos em vegetais superiores com integrantes da famiacutelia Fabaceae tem permitido o
isolamento de metaboacutelitos secundaacuterios com accedilatildeo farmacoloacutegica Essas moleacuteculas apresentam
funccedilotildees antioxidante protetora contra raios ultravioleta antifuacutengica e antimicrobiana (Mans
et al 2016) Dentre o grupo dos compostos fenoacutelicos os flavonoides se destacam por sua
atividade antioxidante decorrente do arranjo molecular determinante no sequestro e
estabilizaccedilatildeo de eleacutetrons e radicais livres com interaccedilotildees do tipo mecanismo-especiacutefica em
sistemas bioloacutegicos (Williams et al 2004)
As propriedades farmacoloacutegicas da espeacutecie Caesalpinia ferrea satildeo relacionadas a parte
do vegetal analisada Para os frutos foram descritas as propriedades antioxidante (Da Silva et
al 2011) hipoglicemiante (Ueda et al 2001) antitumoral (Nakamura et al 2002) e
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
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Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
For subscription articles
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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26
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Your publication choice will have no effect on the peer review process or acceptance of submitted articles
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Language (usage and editing services)
Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English
may wish to use the English Language Editing service available from Elseviers WebShop (httpwebshopelseviercomlanguageediting) or
visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
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Symbol or use fonts that look similar
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A detailed guide on electronic artwork is available on our website
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You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
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information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
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placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
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make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
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that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
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one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
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bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
3
antimicrobiana (Sampaio et al 2009) Para o caule observou-se efeito hipoglicemiante
(Vasconcelos et al 2011) antimutagecircnico (Wyrepkowski et al 2014) capacidade de inibir a
topoisomerase II humana (Nozaki et al 2007) e analgeacutesica (De Arauacutejo et al 2014) Para as
sementes observou-se o efeito antiviral (Lopes et al 2013) e para a vagem efeito cicatrizante
(Carvalho et al 2016)
Com vistas nos compostos secundaacuterios com propriedades antioxidantes presentes nos
vegetais e na escassez de estudos na literatura do potencial antioxidante de folhas de C
ferrea o presente estudo objetivou identificar flavonoides determinar o teor de fenoacuteis e
flavonoides totais aleacutem de avaliar a atividade radicalar pelo DPPH e atividade antioxidante
bioloacutegica de extratos de folhas C ferrea em camundongos
2 Material e meacutetodos
21 Compostos quiacutemicos
Albumina de soro bovino Triton X-100 peroacutexido de hidrogecircnio (H2O2) glutationa
reduzida malondialdeiacutedo (MDA) aacutecido 2-tiobarbituacuterico (TBA) dodecil sulfato de soacutedio
(SDS) aacutecido 55-ditio-bis(2-nitrobenzoacuteico) (DTNB) 24 dinitrofenil-hidrazina (DNPH)
reagente de Bradford aacutecido tricloroaceacutetico (TCA) radical 11-difenil-2-picril hidrazila
(DPPH) fosfato de potaacutessio monobaacutesico fosfato de potaacutessio dibaacutesico fosfato de soacutedio
monobaacutesico fosfato de soacutedio dibaacutesico aacutecido etilenodiamino tetra-aceacutetico (EDTA) sal
dissoacutedico dihidratado trisaminometano (Tris) polissorbato 80 reagente de Folin-Ciocalteau
aacutecido gaacutelico quercetina e aacutecido ascoacuterbico (ASA) e os padrotildees rutina amentoflavona
quercetina-3-β-D-glicosiacutedeo taxifolina apigenina quercitrina e luteolina foram adquiridos da
Sigma-Aldrich (St Louis USA) Os solventes aacutelcool etiacutelico acetato de etila aacutecido
trifluoroaceacutetico (TFA) acetonitrila (ACN) utilizados para o preparo e anaacutelises quiacutemicas dos
extratos foram grau PA e grau HPLC (Merck) Os kits para dosagem de colesterol total
glicose trigliceriacutedeos Alanina Transaminase (ALT) e Aspartato Aminotransferase (AST)
foram adquiridos da Labtestreg Diagnoacutestico S A Minas Gerais Brasil
22 Coleta e identificaccedilatildeo botacircnica
As folhas de C ferrea foram coletadas em Sinop ndash Mato Grosso Brasil sob as
coordenadas geograacuteficas 11deg1854S 55deg5754W A exsicata de nuacutemero de registro 2212 foi
depositada no Herbaacuterio Centro Norte Mato-Grossense (CNMT) Acervo Bioloacutegico da
4
Amazocircnia Meridional (Abam) da UFMT Cacircmpus Universitaacuterio de Sinop-MT Brasil onde a
identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
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from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
For subscription articles
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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26
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Language (usage and editing services)
Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
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visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
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A detailed guide on electronic artwork is available on our website
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You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
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bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
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Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
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information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
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Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
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placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
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that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
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one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
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bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
4
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identificaccedilatildeo botacircnica foi realizada
23 Obtenccedilatildeo dos extratos
Folhas de C ferrea foram secas (40degC) trituradas (1133 kg) e maceradas
exaustivamente com adiccedilatildeo de acetato de etila (3x) por 7 dias e o extrato recolhido filtrado e
reservado O material vegetal foi sequencialmente submetido ao processo de maceraccedilatildeo com
adiccedilatildeo de etanol (3x) e o extrato obtido filtrado e reservado Apoacutes a filtraccedilatildeo e rotaevaporccedilatildeo
os extratos brutos acetato de etila e etanoacutelico foram ressuspendidos em metanol-aacutegua (11
VV) posteriormente filtrados para retirada da clorofila resultando nos extratos brutos sem
clorofila etanoacutelico (EE) e acetato de etila (EA)
24 Determinaccedilatildeo de fenoacuteis e flavonoides totais e da atividade radicalar
Os conteuacutedos de fenois e flavonoides totais de EE e EA foram determinados conforme
Woisky e Salatino (1998) Para fenois totais utilizou-se o meacutetodo de Folin-Ciocalteau e o
resultado foi expresso em miligrama de equivalente grama de aacutecido gaacutelico por grama de
extrato (mg EAGg) Os flavonoides totais foram determinados pelo meacutetodo colorimeacutetrico
com cloreto de alumiacutenio (AlCl3) cujo resultado foi expresso em miligrama de equivalente
grama de quercetina por grama de extrato (mg EQg) A atividade radicalar baseou-se em
Sousa et al (2007) cujo reagente radical DPPH e padrotildees analiacuteticos de quercetina e aacutecido
ascoacuterbico foram empregados
25 Anaacutelise por LC-MSMS
Os extratos EE e EA foram submetidos a anaacutelise por cromatografia liacutequida acoplada a
espectrometria de massas (LC-MSMS) usando um sistema UHPLC 1290 Infinnity (Agilent
Technologies) acoplado a um 6460 Triple Quad LCMS (Agilent Technologies) no qual foi
utilizado um sistema binaacuterio de bombas com 20 microL de amostra injetados via sistema de
autoinjeccedilao A separaccedilatildeo dos compostos ocorreu em uma coluna C-18 (Zorbax Eclipse AAA
de diacircmetro 46x150 nm tamanho da partiacutecula 35 microm) Para eluiccedilatildeo das amostras foi
utilizado um fluxo de 05 mLmin e um gradiente de eluiccedilatildeo composto por Solvente A
(aacuteguaaacutecido foacutermico 99901 (vv)) e Solvente B (acetonitrila aacutecido foacutermico 999 01
(vv)) com as seguintes caracteriacutesticas 0-30 min 95-5 B 30-32 min 0-100 B 32-33
min 95-5 B As amostras foram detectadas por espectrometria de massas utilizando
5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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18
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
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discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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For information on Ethics in publishing and Ethical guidelines for journal publication
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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31
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5
ionizaccedilatildeo por eletrospray com os seguintes paracircmetros A identificaccedilatildeo de flavonoides
ocorreu no modo de aquisiccedilatildeo por ionizaccedilatildeo negativa segundo Duan K (2011) com
modificaccedilotildees pelo Monitoramento de Reaccedilotildees Muacuteltiplas (MRM) dos iacuteons de precursor-
produto (mz-1) 60952rarr 30020 para rutina 53746rarr 37500 para amentoflavona
46338rarr 30000 para quercetina-3-β-d-glicosiacutedeo 44738rarr 30100 para quercitrina
30325rarr 12500 para taxifolina 28524rarr 13300 para luteolina 26924rarr 11680 para
apigenina A voltagem capilar empregada foi 35 kV temperatura da fonte 300 degC e
temperatura de dessolvataccedilatildeo 250 degC
26 Animais
Camundongos adultos Swiss machos peso meacutedio 35 plusmn 3 g obtidos do Bioteacuterio Central
da Universidade Federal de Mato Grosso Cuiabaacute foram aclimatados (raccedilatildeo e aacutegua em livre
demanda) em temperatura ambiente (25 plusmn1 degC ) e umidade relativa (55 plusmn2 ) por 7 dias com
controle de foto periacuteodo de 12 h claroescuro Esta pesquisa foi aprovada e seguiu as
orientaccedilotildees do Comitecirc de Eacutetica no Uso de Animais da Universidade Federal do Mato Grosso
protocolo 2310870148414-2 da CEUA-UFMT
27 Induccedilatildeo do estresse oxidativo e grupos experimentais
A dose do PCM selecionada baseou-se em estudo realizado por Olaleye e Rocha
(2008) e a de ambos extratos mediante teste de Malone realizado previamente EE e EA
empregados nesse estudo bioloacutegico foram preparados em soluccedilotildees aquosas contendo Tween
80 a 01 (veiacuteculo) Apoacutes aclimataccedilatildeo os animais foram divididos em 6 grupos (n=7)
conforme tratamento recebido via gavagem
Grupo 1- (Control) Aacutegua filtrada+ veiacuteculo7 dias
Grupo 2- (PCM) PCM (250 mgKg ndash dose uacutenica) + veiacuteculo7 dias
Grupo 3- (PCM +EE) (250 mgKg ndash dose uacutenica) + EE (50 mgKgdia)7 dias
Grupo 4- (PCM +EA) (250 mgKg ndash dose uacutenica) + EA (50 mgKgdia)7 dias
Grupo 5- (EE) Aacutegua filtrada + EE (50 mgKgdia)7 dias
Grupo 6- (EA) Aacutegua filtrada + EA(50 mgKgdia)7 dias
Numa primeira fase experimental aos grupos G1 G5 e G6 foi administrado
tratamento com aacutegua filtrada O estresse oxidativo foi induzido por intoxicaccedilatildeo aguda pela
administraccedilatildeo de soluccedilatildeo aquosa de PCM (250 mgKg) nos grupos G2 G3 e G4
Decorridas 3h da administraccedilatildeo da primeira fase experimental foram administrados
nos grupos G2 (veiacuteculo) G3 e G5 (EE 50 mgKg) e G4 e G6 (EA 50 mgKg) uma dose ao
dia durante 7 dias
6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
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You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
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The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
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Types of paper
The Journal of Ethnopharmacology will accept the following contributions
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Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
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papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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6
Apoacutes 24 h da uacuteltima dose e em jejum preacutevio de 6 h os animais foram anestesiados
(ketamina 50 mgKg xilazina 2 mgKg e acepromazina 2 mgkg) o sangue coletado (com
auxiacutelio de seringas heparinizadas) via punccedilatildeo cardiacuteaca e centrifugado a 1000 g por 10 minutos
para obtenccedilatildeo do plasma Apoacutes os animais sofreram eutanaacutesia por luxaccedilatildeo cervical os oacutergatildeos
fiacutegado e rim foram retirados e lavados com soluccedilatildeo salina (NaCl 015 M) e posteriormente
congelados agrave -85degC
28 Anaacutelises bioquiacutemicas
281 Antioxidantes enzimaacuteticos
A atividade da superoacutexido dismutase (SOD) em amostras de fiacutegado foi determinada agrave
26 ordmC com base em Misra e Fridovich (1972) contendo tampatildeo glicina 50 mM e bitartarato de
adrenalina A velocidade de formaccedilatildeo de adrenocromo foi monitorada
espectofotometricamente a 480 nm sendo o resultado expresso UI SOD mg de proteiacutena-1
As atividades da catalase (CAT) hepaacutetica e renal foram realizadas conforme Nelson e
Kiesow (1972) usando um meio reacional contendo 10 mL de tampatildeo fosfato de potaacutessio
(TFK) 50 mM (pH 70) 0025 mL de homogeneizado e 0025 mL de H2O2 03 M A
mudanccedila da absorbacircncia do H2O2 no tempo de 60 s foi monitorada por espectrofotometria a
240 nm e expressa em micromol H2O2 consumidomin-1
mg proteiacutena-1
A glutationa-S-transferase (GST) foi determinada de acordo com Habig et al (1974)
com amostras hepaacuteticas A formaccedilatildeo de S-24-dinitrofenil glutationa cuja absorbacircncia
espectrofotomeacutetrica eacute lida em 340 nm foi monitorada em intervalos de 10 segundos durante 1
minuto A atividade foi expressa em micromol GS-DNBmin-1
mg proteiacutena-1
O coeficiente de
extinccedilatildeo molar do CDNB foi de 96 mM cm-1
282 Antioxidantes natildeo enzimaacuteticos e marcadores de dano oxidativo
A glutationa reduzida (GSH) foi determinada espectrofotometricamente segundo
Sedlack e Lindsay (1968) com homogeneizados de tecidos hepaacutetico e renal A formaccedilatildeo do
acircnion tiolato foi avaliada em 412 nm e comparada a uma curva padratildeo de GSH O resultado
foi expresso em micromol GSHmg proteiacutena-1
Os niacuteveis hepaacuteticos de aacutecido ascoacuterbico (ASA) foram determinados conforme Roe
(1954) O meio reacional continha a amostra TCA e DNPH em meio aacutecido que foram
incubados em banho maria a 37 ordmC por 3 horas Apoacutes houve interrupccedilatildeo da reaccedilatildeo pela
adiccedilatildeo de H2SO4 65 e a leitura espectrofotomeacutetrica a 520 nm foi realizada e comparada
7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
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Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
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Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
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Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
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bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
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Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
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30
EPS (or PDF) Vector drawings embed all used fonts
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7
com uma curva de calibraccedilatildeo do padratildeo analiacutetico de ASA O resultado foi expresso em micromol
ASA g-1
tecido
A determinaccedilatildeo das proteiacutenas carboniladas (PC) em fiacutegado e rins foi realizada de
acordo com Yan et al (1995) Os homogeneizados foram preparados em TrisHCl 10 mM
(pH 74) A quantidade de proteiacutenas carboniladas foi expressa em nmol de carbonilmg
proteiacutena-1
A peroxidaccedilatildeo lipiacutedica foi determinada pela anaacutelise dos niacuteveis de TBARS (espeacutecies
reativas ao aacutecido tiobarbituacuterico) conforme Buege e Aust (1978) Amostras de fiacutegado foram
incubadas (30 min 100 ordmC) num meio reacional contendo TCA 10 e TBA 067 A leitura
foi realizada a 535nm apoacutes resfriamento e centrifugaccedilatildeo (10 min a 4000 rpm) Os resultados
foram comparados a uma curva de calibraccedilatildeo de MDA 003 mM e a quantidade de
peroxidaccedilatildeo lipiacutedica foi expressa em nmol MDAmg proteiacutena-1
O conteuacutedo proteico das anaacutelises foi determinado pelo meacutetodo de Bradford (1976)
com leituras no UV-VIS em 595 nm sendo o padratildeo analiacutetico para construccedilatildeo da curva de
calibraccedilatildeo a albumina bovina
283 Anaacutelises dos paracircmetros plaacutesmaacuteticos
As dosagens das atividades das enzimas alanina aminotransferase (ALT) e aspartato
aminotransferase (AST) e a quantificaccedilatildeo de glicose colesterol total e trigliceriacutedeos do
plasma foram realizadas via kits comerciais (Labtestreg Diagnoacutestico S A Minas Gerais
Brasil)
29 Anaacutelise estatiacutestica
Os dados foram apresentados como meacutedia plusmn DP (desvio padratildeo) analisados por Anova
de uma via seguida pelo post hoc teste de Tukey O niacutevel de significacircncia estabelecido para
rejeiccedilatildeo da hipoacutetese de nulidade foi de 5 (p lt 005)
3 Resultados
31 Teores de fenoacuteis e flavonoides totais e atividade radicalar
A Tabela A-1 apresenta os resultados do teor de fenois e flavonoides totais de EA que
foi superior ao obtido em EE No ensaio da atividade radicalar os valores de EC50 obtidos de
EA e EE foram similares aos padrotildees analiacuteticos empregados
8
Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
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Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
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of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
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enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
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Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
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Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
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21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
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Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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24
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Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
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Tab A-1 Anaacutelise do teor de fenois totais flavonoides totais e atividade radicalar in vitro de
EE e EA de C ferrea comparado a dois padrotildees analiacuteticos utilizados
Extrato ou
Padratildeo Analiacutetico
Fenois totais
(mg EAGg extrato)
Flavonoides totais
(mg EQg extrato) EC50
(microgmL)
EE 445 90 460
EA 595 110 343
Acido ascoacuterbico - - 383
Rutina - - 435
32 Anaacutelise por LC-MSMS
Foram identificados os flavonoides rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina em ambos extratos Aleacutem dos flavonoides
descritos anteriormente foi identificada a presenccedila de quercitrina em EE (Tab A-2 Fig A-1
Fig A-2)
Tab A-2 Caracterizaccedilatildeo por LC-MSMS dos compostos identificados em EE eou EA de C
ferrea
Composto RT (min) MW [M-H]- MS
2
iacuteons
Composto identificado Foacutermula
molecular
1 10 61052 60952 30020 Rutina C27H30O16
2 169 53846 53746 37500 Amentoflavona C30H18O10
3 103 46438 46338 30000 Quercetina-3-β-D-glicosiacutedeo C6H11O5
4 118 44838 44738 30100 Quercitrina C21H20O11
5 12 30425 30325 12500 Taxifolina C15H12O7
6 15 28624 28524 13300 Luteolina C15H10O6
7 162 27024 26924 11680 Apigenina C15H10O5
O
OOH
HOR2
OH
R3
R1
Compound R1 R2 R3
1 H OH O-(rhamnosyl-glucoside)
2 apigenin H H
3 H OH O-glucoside
4 H OH O-rhamnoside
6 H OH H
7 H H H
O
OOH
HOOH
OH
OH
Compound 5
Fig A-1 Estrutura de sete flavonoides identificados em EE eou EA de folhas de C ferrea
9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
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of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
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Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
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Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
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must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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30
EPS (or PDF) Vector drawings embed all used fonts
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Citation in text
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9
Fig A- 2 LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos compostos
identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos flavonoides
identificados
Composto 3
Composto 1
Composto 2
Composto 4
Composto 5
mz 30020
mz 37500
mz 30000
mz 30100
mz 12500
1
2
3
4
5
10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
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cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
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The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
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10
Fig A- 2 (continuaccedilatildeo) LC-MSMS de amostra de EE de C ferrea Cromatogramas com tempo de retenccedilatildeo dos
compostos identificados massa do iacuteon molecular massa do iacuteon principal e das estruturas quiacutemicas dos
flavonoides identificados
33 Anaacutelise de biomarcadores do estresse oxidativo no tecido hepaacutetico
Natildeo foi observada alteraccedilatildeo significativa na atividade da SOD entre os grupos
avaliados nesse estudo Houve decreacutescimo estatisticamente significativo (p lt 0001) de 376
e 205 nas atividade das enzimas hepaacuteticas CAT e GST respectivamente nos grupos
tratados com PCM O tratamento com EA foi capaz de restaurar a atividade dessas em 539
(CAT) e 310 (GST) respectivamente (Fig A-3)
Con
trol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
UI S
OD
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
00
02
04
06
08
10
microm
ol G
S-D
NB
min
-1m
g p
rote
iacuten-1
Fig A- 3 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores enzimaacuteticos do tecido hepaacutetico (SOD CAT
e GST) n = 7 Anaacutelise Anova seguida por Tukey p lt 0001 comparado ao controle p lt 0001 comparado ao
grupo PCM
A GSH e ASA apresentaram reduccedilotildees estatisticamente significativas (p lt 005) de
271 e 200 respectivamente no grupo exposto ao PCM quando comparado com o
Composto 7
Composto 6
SOD CAT GST
mz 13300
mz 11680
6
7
11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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18
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
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Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
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Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
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Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
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Biochemical and Biophysical Research Communications 479(1) 17ndash21
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
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httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
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from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
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Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
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Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
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ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
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discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
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jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
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Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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11
grupo controle O tratamento com EA foi capaz de restaurar os niacuteveis desses biomarcadores
em 371 (GSH) e 231 (ASA) respectivamente (Fig A-4)
Foi observada elevaccedilatildeo estatisticamente significativa na formaccedilatildeo de TBARS em 444
no grupo exposto ao PCM Natildeo foi observada alteraccedilotildees significativas nos grupos tratados
com EE e EA (Fig A-4)
Contr
ol
PCM
PCM+EE
PCM+EA EE
EA00
06
12
18
m
ol A
SA
g-1
Wei
gh
t
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
00
02
04
06
nm
ol M
DA
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
5
10
15
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 4 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores natildeo enzimaacuteticos e do dano oxidativo no
tecido hepaacutetico (GSH ASA TBARS e PC) n = 7 Anaacutelise Anova seguida por Tukey p lt 005 comparado ao
controle p lt 005 comparado ao grupo PCM
Conforme Fig A-4 a taxa de carbonilaccedilatildeo de proteiacutenas no grupo intoxicado com
PCM elevou-se em 768 comparado ao controle Ambos extratos avaliados EE e EA
foram capazes de reduzir os niacuteveis de PC em 258 e 379 respectivamente comparado
ao grupo PCM
34 Anaacutelise de biomarcadores do estresse oxidativo no tecido renal
Natildeo foram observadas alteraccedilotildees estatisticamente significativas na atividade da CAT
renal entre os grupos testados Os niacuteveis de GSH apresentaram reduccedilatildeo no grupo intoxicado
com PCM (424 ) Ambos extratos avaliados promoveram reversatildeo desse efeito nefrotoacutexico
sendo que EE (706 ) apresentou efeito superior a EA (436 ) A taxa de carbonilaccedilatildeo
proteica renal sofreu elevaccedilatildeo (452 ) no grupo tratado com PCM Esse efeito foi diminuiacutedo
pela administraccedilatildeo de EE (254 ) e EA (269 ) respectivamente (Fig A-5)
ASA
PC TBARS
GSH
12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
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HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
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Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
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Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
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Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
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Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
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of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
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He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
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10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
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Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
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Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
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Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
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Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
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Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
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Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
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Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
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Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
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Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
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Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
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Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
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Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
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Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
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Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
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strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
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layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
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preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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These keywords will be used for indexing purposes
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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Footnotes
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Artwork
Electronic artwork
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Symbol or use fonts that look similar
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You are urged to visit this site some excerpts from the detailed information are given here
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below)
30
EPS (or PDF) Vector drawings embed all used fonts
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Color artwork
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Figure captions
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Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
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References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
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reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
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references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
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12
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
25
m
ol m
in-1
mg
pro
tein
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA E
EEA
0
5
10
15
20
nm
ol carb
on
yl m
g p
rote
iacuten-1
Fig A- 5 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores do tecido renal (CAT GSH e PC) n = 7
Anaacutelise Anova seguida por Tukey p lt 005 comparado ao controle p lt 005 comparado ao grupo PCM
35 Paracircmetros metaboacutelicos do plasma
A intoxicaccedilatildeo aguda com PCM promoveu aumento significativo na atividade das
enzimas plasmaacuteticas sendo que ALT (1197) foi mais afetada que AST (687) Embora os
extratos EE (311) e EA (359) tenham reduzido estatisticamente o dano causado por
PCM na ALT ainda assim esses extratos foram toacutexicos quando comparados ao controle
AST sofreu reduccedilatildeo com o tratamento com EE (391 ) e EA (282 ) respectivamente
(Fig A-6)
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
30
60
90
120
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
40
80
120
160
UL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
70
140
210
280
350
mg
dL
-1
Contr
ol
PCM
PCM
+EE
PCM
+EA EEEA
0
20
40
60
mg
dL
-1
Fig A- 6 Efeito do tratamento de camundongos com EE e EA (50 mgKg) de folhas C ferrea sob estresse
oxidativo induzido com PCM (250 mgKg) sobre os biomarcadores plasma sanguiacuteneo (ALT AST glicose
trigliceriacutedeos e colesterol) n = 7 Anaacutelise Anova seguida por Tukey p lt 00001 comparado ao controle
p lt 00001 comparado ao grupo PCM
Quando comparado ao controle EA apresentou efeito hipoglicecircmico no grupo
intoxicado com PCM e no grupo que recebeu tratamento somente com esse extrato EE e EA
GSH CAT
Glicose Trigliceriacutedeos Colesterol
ALT AST
PC
13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
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doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
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Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
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bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
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Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
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30
EPS (or PDF) Vector drawings embed all used fonts
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13
foram hipolipemiantes uma vez que foram eficazes na reduccedilatildeo dos trigliceriacutedeos em 297 e
458 respectivamente quando comparado ao controle O colesterol total natildeo exibiu
interferecircncia significativa conforme o tratamento executado em nenhum dos grupos testados
(Fig A-6)
4 Discussatildeo
A diversidade dos metaboacutelitos secundaacuterios presentes nos vegetais os torna de grande
interesse como fonte de biomoleacuteculas para fins terapecircuticos Para os compostos fenoacutelicos tem
sido atribuiacutedas propriedades antioxidantes devido a sua estrutura quiacutemica A atividade
antioxidante de flavonoides por sua vez tem sido correlacionada a estabilizaccedilatildeo do radical
flavanoil e ao sequestro de eleacutetrons e estabilizaccedilatildeo por ressonacircncia (Da Silva Portrsquos et al
2013)
Estima-se que os flavonoides exerccedilam atividade antioxidante por mecanismos diversos
como sequestro direto de radicais regulaccedilatildeo da produccedilatildeo de radicais eliminaccedilatildeo de radicais
precursores quelaccedilatildeo de metais inibiccedilatildeo da xantina oxidase e elevaccedilatildeo de antioxidantes
endoacutegenos (Nijveldt 2001) Flavonoides possuem atividade antioxidante superior agraves
vitaminas E e C e possuem potencial de oxidaccedilatildeo inferior aos iacuteons Fe+3
e Cu+2
Dessa forma
os flavonoides satildeo capazes de reduzir caacutetions e impedir a formaccedilatildeo e disseminaccedilatildeo de radicais
livres pelas reaccedilotildees de Fenton e Harber-Weiss (Jomova and Valko 2011)
Caracteriacutesticas intriacutensecas dos flavonoides como a presenccedila de grupos orto-
difenoacutelicos quantidade de hidroxilas e capacidade quelante satildeo relacionadas a estabilizaccedilatildeo
do radical flavanoil (Ravishankar et al 2015) Isso converge com os flavonoides
identificados em C ferrea e com os resultados positivos exibidos nos ensaios realizados do
presente trabalho Apesar do mecanismo especiacutefico de atuaccedilatildeo bioloacutegica desse grupo
fitoquimico natildeo estar bem evidenciado estudos especulam que os flavonoides atuam nos
sistemas bioloacutegicos por mecanismos distintos e inter-relacionados (Williams et al 2004)
41 Anaacutelise por LC-MSMS
No presente trabalho a identificaccedilatildeo dos flavonoides por LC-MSMS foi enfatizada
mediante avaliaccedilatildeo das transiccedilotildees principais de iacuteons precursor-produto Pela anaacutelise quiacutemica
das massas dos fragmentos principais gerados eacute possiacutevel observar que o Composto 1
apresentou iacuteon molecular 60952 (mz -1) e fragmento 30020 (mz ndash30932) o qual
corresponde agrave perda das duas gliconas sendo essa fragmentaccedilatildeo similar a obtida por
Simirgiotis et al (2016) O composto 2 por sua vez apresentou iacuteon molecular 53746 (mz -
14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
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Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
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Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
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Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
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In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
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Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
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discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
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can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
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Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
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Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
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Results
Results should be clear and concise
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appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
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Abstract
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
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Click here to see an example
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viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
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chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
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with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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14
1) e fragmento 37500 (mz ndash 16246) que corresponde a perda do fragmento localizado no
anel C que apresenta as funccedilotildees fenol eacuteter e cetona sendo os iacuteons [M-H]- e MS
2 similares aos
reportados por Abu-Reidah et al (2015)
O Composto 3 apresentou iacuteon molecular 46338 (mz -1) e fragmento 30000 (mz ndash
16338) indicando a quebra do grupamento glicosiacutedeo dessa glicona O Composto 7 mostrou
o iacuteon molecular foi 26924 (mz-1) e fragmento 11680 (mz ndash15244) o qual corresponde a
quebra de ligaccedilatildeo entre os carbonos 2 e 3 do anel C Esses perfis de fragmentaccedilatildeo dos
compostos 3 e 7 foram semelhantes aos descritos por Dai et al (2015) Para o Composto 4
foi identificado iacuteon molecular 44738 (mz -1) e fragmento 30100 (mz ndash14638) que
corresponde agrave ruptura da porccedilatildeo glicona (ramnosiacutedeo) sendo descrita padratildeo de fragmentaccedilatildeo
similar por He et al (2014) O Composto 5 por sua vez apresentou iacuteon molecular de 30325
(mz -1) e o fragmento 12500 (mz ndash17825) que representa a ruptura do anel C entre o
carbono 2 e a carbonila Fragmentaccedilatildeo similar para taxifolina foi descrita por Yang et al
(2016) Em relaccedilatildeo ao Composto 6 que apresentou iacuteon molecular 28524 (mz -1) e
fragmento 13300 (mz ndash15224) o qual representa a quebra do anel C benzeno-diol e que foi
descrita fragmentaccedilatildeo similar em anaacutelise no modo ionizaccedilatildeo negativa por Fabre et al (2001)
42 Avaliaccedilatildeo bioloacutegica do estresse oxidativo
O ensaio da atividade radicalar foi do tipo preditivo cujo menor valor de EC50
reportado para EA em relaccedilatildeo ao padratildeo analiacutetico aacutecido ascoacuterbico e ao EE revelou-se
coerente com a resposta tatildeo somente desse primeiro extrato na reversatildeo da hepatoxicidade em
quatro biomarcadores hepaacuteticos (CAT GST GSH e ASA) analisados nesse estudo
A reversatildeo do efeito toacutexico do PCM por EA no fiacutegado de camundongos nas atividades
de CAT e GST mostram o efeito antioxidante apresentado por C ferrea que pode estar
relacionado ao flavonoide quercetina Em estudo do paracircmetro biodisponibilidade realizado
por (Cao et al 1997) foi observado que a quercetina apresentou concentraccedilatildeo elevada no
plasma de ratos submetidos a uma dieta com esse flavonoide Para a quercetina foram
reportadas as propriedades de antimutagenicidade induccedilatildeo da apoptose inibiccedilatildeo da proteiacutena
quinase e regulaccedilatildeo do ciclo de modulaccedilatildeo celular (Murota and Terao 2003) Aleacutem disso
acredita-se que a forma conjugada da quercetina com um accediluacutecar influencia na atividade
bioloacutegica devido a glicosilaccedilatildeo possibilitar uma melhor absorccedilatildeo Esse flavonoide tambeacutem eacute
capaz de elevar os niacuteveis de GSH e promover decreacutescimo da peroxidaccedilatildeo lipiacutedica (Huang et
15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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These keywords will be used for indexing purposes
Chemical compounds
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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30
EPS (or PDF) Vector drawings embed all used fonts
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31
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15
al 2011) fato que tambeacutem pode ser correlacionado ao efeito de EA avaliado nesse estudo
frente aos biomarcadores do estresse oxidativo
O efeito antioxidante envolve sistemas complexos cujo modelo praticado nesse estudo
(overdose por PCM) foi escolhido devido a farmacocineacutetica de eliminaccedilatildeo de fase I e II desse
faacutermaco ser bem conhecida Portanto a saturaccedilatildeo das vias de glicuronidaccedilatildeo e sulfataccedilatildeo
conduz a formaccedilatildeo do composto citotoacutexico N-acetil-benzoquinona imina (NAPQI) cujo
excesso promove depleccedilatildeo tecidual de GSH A detoxificaccedilatildeo do metaboacutelito NAPQI envolve
conjugaccedilatildeo com glutationa via rota especiacutefica e formaccedilatildeo de aacutecido mercaptuacuterico destinado a
excreccedilatildeo Todavia a elevada reatividade desse metaboacutelito com proteiacutenas celulares ocasiona
danos severos essencialmente no fiacutegado podendo causar necrose (Hodgman e Garrard 2012)
Logo a reduccedilatildeo experimental da GSH (hepaacutetica e renal) nos animais intoxicados com
PCM estaacute associada ao seu consumo via rota de metabolizaccedilatildeo do NAPQI Uma vez reduzida
a GSH e consequentemente alterado seu sistema de recuperaccedilatildeo a atividade de enzimas de
sua biotransformaccedilatildeo como a GST torna-se afetada Experimentalmente a reduccedilatildeo da
atividade da GST hepaacutetica (grupo PCM) eacute coerente com o exposto
Paralelamente a reduccedilatildeo da atividade da CAT hepaacutetica na condiccedilatildeo de estresse
oxidativo induzido e a indiferenccedila na CAT renal podem estar correlacionadas ao fato das
alteraccedilotildees renais serem detectaacuteveis quando a condiccedilatildeo de dano hepaacutetico estiver bem instalado
(Hodgman e Garrard 2012) Assim quando o consumo da GSH hepaacutetica for de
aproximadamente 70 o NAPQI passa a reagir covalentemente com as proteiacutenas
desencadeando quadro de necrose no fiacutegado (Amaral et al 2008) No presente estudo a taxa
de depleccedilatildeo da GSH hepaacutetica alcanccedilou 271 sendo sugestivo portanto que numa susposta
condiccedilatildeo de maior depleccedilatildeo desse tripeptiacutedeo interferecircncias na atividade da CAT renal
seriam detectadas
O diagnoacutestico cliacutenico de disfunccedilatildeo hepatocelular eacute realizado por avaliaccedilatildeo dos niacuteveis
plasmaacuteticos de ALT e AST No presente estudo a elevaccedilatildeo da concentraccedilatildeo dessas enzimas
foi coerente com a intoxicaccedilatildeo promovida por PCM (250 mgKg) e pode ser relacionada ao
mecanismo dependente da citocromo P450 (Kazemi et al 2016) A reduccedilatildeo experimental
dessas enzimas plasmaacuteticas por EE e EA estaacute relacionada ao efeito antioxidante de C ferrea
mostrado tambeacutem para os demais biomarcadores analisados nesse estudo
As modificaccedilotildees promovidas no sistema GSH acarretam consequentemente reduccedilatildeo
na atividade da GST sendo o flavonoide luteolina associado a depleccedilatildeo na atividade da GST
na presenccedila de tirosina (Balyan et al 2015) Esses resultados bioquiacutemicos e a identificaccedilatildeo de
luteolina em C ferrea pode estar correlacionada com a capacidade desse flavonoide restaurar
16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
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Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
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Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
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Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
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Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
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18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
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Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
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Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
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49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
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De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
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Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
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Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
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Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
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Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
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multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
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Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
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article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
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numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
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In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
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Symbol or use fonts that look similar
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A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
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EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
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placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
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16
os niacuteveis de GSH aleacutem de promover decreacutescimo da atividade das enzimas ALT e AST frente
ao tratamento de ratos com PCM (300 mgkg) (Tai et al 2015)
O estresse oxidativo induzido por xenobioacutetico promove maior consumo e portanto a
reduccedilatildeo hepaacutetica de ASA Estudos mostram que o ascorbato age como agente redutor dos
metais de transiccedilatildeo Fe+3
e Cu+2
presentes no siacutetio ativo de enzimas ou nas formas livres no
organismo transformando-se em semidesidroascorbato pouco reativo Esse uacuteltimo composto
retorna a ascorbato pelo sistema de regeneraccedilatildeo via GSH (Barreiros et al 2006) Logo a
depleccedilatildeo da GSH dificulta a regeneraccedilatildeo do ascorbato e consequentemente reduz os niacuteveis de
ASA Por outro lado a reversatildeo desse efeito por EA eacute indicativo de que C ferrea atua a niacutevel
do sistema semidesidroascorbato ou pelo fato de elevar os niacuteveis de GSH haja vista que para
a quercetina presente em EE e EA jaacute foi descrita essa propriedade (Murota and Terao 2003)
A presenccedila de ERNs e EROs proacuteximos das membranas promove peroxidaccedilatildeo lipiacutedica
ou danos nas proteiacutenas de membranas Isso acarreta interferecircncias a niacutevel de trocas iocircnicas
(Thenmozhi et al 2016) TBARS indica o potencial lesivo ocasionado pela condiccedilatildeo de
estresse oxidativo o que justifica o aumento da peroxidaccedilatildeo lipiacutedica observado nos
camundongos intoxicados com PCM (Fouad et al 2009) O fato dos niacuteveis de TBARS natildeo se
alterarem por EA e EE torna sugestivo inferir que compostos biodisponiacuteveis nesses extratos
possuem menor caraacuteter lipofiacutelico ou maior afinidade com proteiacutenas plasmaacuteticas A polaridade
do acetato de etila e etanol determina o perfil molecular extraiacutedo e consequentemente na
interaccedilatildeo com compartimentos bioloacutegicos Estudos demonstram que cerca de 50 dos
flavonoides ingeridos da dieta satildeo conjugados com glicuronato nas paredes intestinais e
posteriormente ligados a albumina plasmaacutetica para finalmente interagir com os tecidos
(Nijveldt 2001)
EE e EA demonstraram-se hepato e nefroprotetores propriedades essas relacionadas
aos fitoconstituintes que interferem no sistema antioxidante celular O aumento dos niacuteveis de
proteiacutenas carboniladas pela condiccedilatildeo de estresse oxidativo induzido (PCM) eacute decorrente da
depleccedilatildeo da GSH que conduz a saturaccedilatildeo dessa via e acuacutemulo do NAPQI
A reduccedilatildeo da glicemia por EA demonstra o potencial de C ferrea para o tratamento da
diabetes O efeito hipoglicemiante de extrato aquoso das cascas do caule de C ferrea foi
demonstrado e relacionado a presenccedila de taninos e catequinas (Vasconcelos et al 2011) fato
convergente com o uso dessa planta pela medicina popular (Teixeira e De Melo 2006) O
efeito hipolipidecircmico de EE e EA de C ferrea demonstrado no presente estudo tambeacutem
corrobora com estudo realizado no tratamento de ratos com extrato aquoso etanoacutelico de folhas
desse vegetal em concentraccedilotildees de 250 mgKg e 500 mgKg (Hassan et al 2015)
17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
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properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
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24
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
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28
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17
5 Conclusatildeo
O teor de fenois totais nas folhas de C ferrea correlaciona-se ao potencial antioxidante
in vitro e atividade antioxidante bioloacutegica demonstrada Esses resultados satildeo coerentes com os
flavonoides identificados em ambos extratos (rutina amentoflavona quercetina-3-β-D-
glicosideo taxifolina apigenina e luteolina) aleacutem de quercitrina em EE A overdose por
PCM em camundongos promoveu alteraccedilotildees em biomarcadores enzimaacuteticos eou natildeo
enzimaacuteticos nos tecidos hepaacutetico e renal Observou-se que o tratamento com EA foi efetivo
em recuperar do efeito nas dosagens hepaacuteticas de CAT GST GSH e ASA EE e EA
restauraram a GSH (renal) PC (renal e hepaacutetica) e transaminases plasmaacuteticas (ALT e AST)
alteradas por PCM Aleacutem do efeito antioxidante nos tecidos EE e EA apresentaram atividade
antilipecircmica e EA apresentou efeito hipoglicemiante em conformidade com o uso popular de
C ferrea para o tratamento da diabetes Logo o elevado teor de fenois e a identificaccedilatildeo de
flavonoides nos extratos avaliados satildeo indicativos de suas propriedades hepato e
nefroprotetora experimentalmente demonstradas nesse estudo
Agradecimentos
A FAPEMAT pelo financiamento do presente projeto A EMBRAPA
AGROSSILVIPASTORIL pelas anaacutelises por LC-MSMS
Referecircncia
Abu-Reidah IM Ali-Shtayeh MS Jamous RM Arraacuteez-Romaacuten D Segura-Carretero A 2015
HPLC-DAD-ESI-MSMS screening of bioactive components from Rhus coriaria L (Sumac) fruits
Food Chem 166 179ndash191 doi101016jfoodchem201406011
Amaral DA Hernandez EM Barcia SAD 2008 Intoxicaccedilotildees por medicamentos in Oga S
Camargo M M A Batistuzzo J A O (Org) Fundamentos de toxicologia 3ordf ed Atheneu Satildeo
Paulo pp 38-58
Balyan R Kudugunti SK Hamad HA Yousef MS Moridani MY 2015 Bioactivation of
luteolin by tyrosinase selectively inhibits glutathione S-transferase Chem Biol Interact 240 208ndash
218 doi101016jcbi201508011
Barreiros A L B S David J M amp David J P (2006) Estresse oxidativo Relaccedilatildeo entre geraccedilatildeo
de espeacutecies reativas e defesa do organismo Quimica Nova 29(1) 113ndash123
httpdoiorg101590S0100-40422006000100021
Bradford MM 1976 A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72
248-254 doi1010160003-2697(76)90527-3
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
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mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
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Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
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modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
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bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
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Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
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Click here to see an example
Graphical abstract
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
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with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
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Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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30
EPS (or PDF) Vector drawings embed all used fonts
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References
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This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
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31
Reference formatting
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Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
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Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
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presentation after acceptance of their paper
32
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Ensure that the following items are present
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bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
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For any further information please visit our customer support site at httpsupportelseviercom
18
Buege JA Aust SD 1978 Microsomal lipid peroxidation Methods in Enzymology 52 302-309
Cao G Sofic E amp Prior R L (1997) Antioxidant and prooxidant behavior of flavonoids
Structure-activity relationships Free Radical Biology and Medicine 22(5) 749ndash760
httpdoiorg101016S0891-5849(96)00351-6
Carvalho FG da Silva Sampaio JP dos Santos Araujo MM Soares Pinto LS Rocha AJ
2016 Assessment of the healing activity of juca pods [Libidibia ferrea (Mart ex Tul) L P
Queiroz] in cutaneous lesions of rats Acta Sci 38 137ndash143 doi104025actascitechnolv28i228570
Da Silva LCN Da Silva CA De Souza RM Joseacute Macedo A Da Silva MV Dos Santos
Correia MT 2011 Comparative analysis of the antioxidant and DNA protection capacities of
Anadenanthera colubrina Libidibia ferrea and Pityrocarpa moniliformis fruits Food Chem Toxicol
49 2222ndash2228 doi101016jfct201106019
Da Silva Portrsquos P Chisteacute RC Godoy HT Prado MA 2013 The phenolic compounds and the
antioxidant potential of infusion of herbs from the Brazilian Amazonian region Food Res Int 53
875ndash881 doi101016jfoodres201302010
De Arauacutejo AA Soares LAL Assunccedilatildeo Ferreira MR De Souza Neto MA Da Silva GR De
Arauacutejo RF Guerra GCB De Melo MCN 2014 Quantification of polyphenols and evaluation
of antimicrobial analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of
Libidibia ferrea Parapiptadenia rigida and Psidium guajava J Ethnopharmacol 156 88ndash96
doi101016jjep201407031
Dai B Hu Z Li H Yan C Zhang L 2015 Simultaneous determination of six flavonoids from
Paulownia tomentosa flower extract in rat plasma by LC-MSMS and its application to a
pharmacokinetic study J Chromatogr B Anal Technol Biomed Life Sci 978ndash979 54ndash61
doi101016jjchromb201411021
Duan K Yuan Z Guo WMeng Y CuiY kong D Zhang L 2011 LC-MSMS determination
and pharmacokinetic study of five flavone components after solvent extractionacid hydrolysis in rat
plasma after oral administration of Verbena officinalis L extract Journal of Ethnopharmacology v
135 n 2 p 201ndash208
Fabre N Rustan I De Hoffmann E Quetin-Leclercq J 2001 Determination of flavone flavonol
and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass
spectrometry J Am Soc Mass Spectrom 12 707ndash715 doi101016S1044-0305(01)00226-4
Fouad A A Yacoubi M T amp El-Bidawy M H (2009) Therapeutic potential of hemin in
acetaminophen nephrotoxicity in rats Environmental Toxicology and Pharmacology 27(2) 277ndash282
httpdoiorg101016jetap200811002
Habig WH Pabst MJ Jacoby WB 1974 Glutathione S-transferase the first enzymatic step in
mercapturic acid formation Journal of Biological Chemistry 2497130-7139
Hassan S K El-Sammad N M Mousa1 A M Mohammed M H Farrag A el R H Hashim A
N E Werner V Lindequist U Nawwar M A E 2015 Hypoglycemic and antioxidant activities of
Caesalpinia ferrea Martius leaf extract in streptozotocin-induced diabetic rats Asian Paci fi c Journal
of Tropical Medicine Asian Pac J Trop Med 5 1ndash8 doi101016japjtm201509008
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
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can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
minimum of 531 times 1328 pixels (h times w) or proportionally more The image should be readable at a size of 5 times 13 cm using a regular screen
resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
19
He CY Fu J Ma JY Feng R Tan XS Huang M Shou JW Zhao ZX Li XY Zhang
XF Chen Y Wang Y 2014 Biotransformation and in vitro metabolic profile of bioactive extracts
from a traditional miao-nationality herbal medicine polygonum capitatum Molecules 19 10291ndash
10308 doi103390molecules190710291
Hodgman M J amp Garrard A R (2012) A Review of Acetaminophen Poisoning Critical Care
Clinics 28(4) 499ndash516 httpdoiorg101016jccc201207006
Huang J Wang S Zhu M Chen J Zhu X 2011 Effects of Genistein Apigenin Quercetin
Rutin and Astilbin on serum uric acid levels and xanthine oxidase activities in normal and
hyperuricemic mice Food Chem Toxicol 49 1943ndash1947 doi101016jfct201104029
Jomova K Valko M 2011 Advances in metal-induced oxidative stress and human disease
Toxicology 283 65ndash87 doi101016jtox201103001
Kazemi S Mousavi Kani S N Ghasemi-Kasman M Aghapour F Khorasani H amp
Moghadamnia A A (2016) Nonylphenol induces liver toxicity and oxidative stress in rat
Biochemical and Biophysical Research Communications 479(1) 17ndash21
httpdoiorg101016jbbrc201608164
Lima SMA Arauacutejo LCC Sitocircnio MM Freitas ACC Moura SL Correia MTS Malta
DJN Gonccedilalves-Silva T 2011 Anti-inflammatory and analgesic potential of Caesalpinia ferrea
Brazilian J Pharmacogn 22 169ndash175 doi101590S0102
Limon-Pacheco J amp Gonsebatt M E (2009) The role of antioxidants and antioxidant-related
enzymes in protective responses to environmentally induced oxidative stress Mutation Research -
Genetic Toxicology and Environmental Mutagenesis 674(1ndash2) 137ndash147
httpdoiorg101016jmrgentox200809015
Lopes N Faccin-Galhardi LC Espada SF Pacheco AC Ricardo NMPS Linhares REC
Nozawa C 2013 Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and
poliovirus Int J Biol Macromol 60 93ndash99 doi101016jijbiomac201305015
Mans D R A Beerens T Magalli I et al In vitro evaluation of traditionally used Surinamese
medicinal plants for their potential anti-leishmanial efficacy Journal of Ethnopharmacology v 180 p
70ndash77 2016 Elsevier
Mishra G Ratan Lal Khosa Pradeep Singh KKJ 2014 potential of ethanolic extract of C
aesalpenia crista leaves against paracetamol induced hepatotoxicity in rats H epatoprotective J Coast
Life Med 2 575ndash579 doi1012980JCLM22014JCLM-2014-0036
Misra HP Fridovich I 1972 The role of superoxide anion in the auto-oxidation o epinephrine and
a simple assay for superoxide dismutase Journal Biological Chemistry 247 3170ndash3175
httpwwwjbcorgcontent247103170
Murota K amp Terao J (2003) Antioxidative flavonoid quercetin Implication of its intestinal
absorption and metabolism Archives of Biochemistry and Biophysics 417(1) 12ndash17
httpdoiorg101016S0003-9861(03)00284-4
Nakamura ES Kurosaki F Arisawa M Mukainaka T Okuda M Tokuda H Nishino H
Pastore F 2002 Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related
compounds Cancer Lett 177 119ndash124 doi101016S0304-3835(01)00708-X
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
For subscription articles
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As an author you (or your employer or institution) retain certain rights For more information on author rights for Subscription articles
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and in the decision to submit the article for publication If the funding source(s) had no such involvement then this should be stated
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Open Access
This journal offers authors a choice in publishing their research
Open Access
bull Articles are freely available to both subscribers and the wider public with permitted reuse An open access publication fee is payable by
authors or their research funder
Subscription
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(httpwwwelseviercomaccess)
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All articles published open access will be immediately and permanently free for everyone to read and download Permitted reuse is defined
by your choice of one of the following Creative Commons user licenses
26
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Your publication choice will have no effect on the peer review process or acceptance of submitted articles
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Language (usage and editing services)
Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English
may wish to use the English Language Editing service available from Elseviers WebShop (httpwebshopelseviercomlanguageediting) or
visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
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Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
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Symbol or use fonts that look similar
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You are urged to visit this site some excerpts from the detailed information are given here
Formats
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
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below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
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bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
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Figure captions
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Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
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References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
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a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
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are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
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placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
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MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
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Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
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athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
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bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
20
Nelson DP Kiesow LA 1972 Enthalphy of decomposition of hydrogen peroxide by catalase at 25
degC (with molar extinction coefficients of H2O2 solution in the UV) Analytical Biochemistry 49 474ndash
478 doiorg1010160003-2697(72)90451-4
Nijveldt R (2001) Flavonoids a review of probable mechanism of action and potential
applications Am J Clin Nutr 74 418ndash425
Nozaki H Hayashi K ichiro Kido M Kakumoto K Ikeda S Matsuura N Tani H Takaoka
D Iinuma M Akao Y 2007 Pauferrol A a novel chalcone trimer with a cyclobutane ring from
Caesalpinia ferrea mart exhibiting DNA topoisomerase II inhibition and apoptosis-inducing activity
Tetrahedron Lett 48 8290ndash8292 doi101016jtetlet200709130
Olaleye M T amp Rocha B T J (2008) Acetaminophen-induced liver damage in mice Effects of
some medicinal plants on the oxidative defense system Experimental and Toxicologic Pathology
59(5) 319ndash327 httpdoiorg101016jetp200710003
Rahiminejad ME Moaddab A Ganji M Eskandari N Yepez M Rabiee S Wise M Ruano
R Ranjbar A 2016 Oxidative stress biomarkers in endometrial secretions A comparison between
successful and unsuccessful in vitro fertilization cycles J Reprod Immunol 116 70ndash75
doi101016jjri201605003
Ravishankar D Watson KA Boateng SY Green RJ Greco F Osborn HMI 2015
European Journal of Medicinal Chemistry Exploring quercetin and luteolin derivatives as
antiangiogenic agents Eur J Med Chem 97 259ndash274 doi101016jejmech201504056
Roe JH 1954 Chemical determination of ascorbic dehydroascorbic and diketogulonic acids In
Methods of Biochemical Analysis Glick D Ed vol 1 Interscience New York pp115
Sampaio FC Pereira M do S V Dias CS Costa VCO Conde NCO Buzalaf MAR 2009
In vitro antimicrobial activity of Caesalpinia ferrea Martius fruits against oral pathogens J
Ethnopharmacol 124 289ndash294 doi101016jjep200904034
Saacutenchez-Rodriacuteguez C Martiacuten-Sanz E Cuadrado E Granizo J J amp Sanz-Fernaacutendez R (2016)
Protective effect of polyphenols on presbycusis via oxidativenitrosative stress suppression in rats
Experimental Gerontology 83 31ndash36 httpdoiorg101016jexger201607005
Sedlack J Lindsay RH 1968 Estimation of total protein-bound and nonprotein sulfhydryl groups
in tissue with ellmanrsquos reagent Analytical Biochemistry 25 192-205
Simirgiotis MJ Quispe C Areche C Sepuacutelveda B 2016 Phenolic compounds in chilean
mistletoe (quintral Tristerix tetrandus) analyzed by UHPLC-QOrbitrapMSMS and its antioxidant
properties Molecules 21 doi103390molecules21030245
Sousa CMM Silva HR Vieira Jr GM Ayres MCC Costa CLS Arauacutejo DS Cavalcenti
LCD Barros EDS Arauacutejo PBM Brandatildeo MS Chaves MH 2007 Fenoacuteis totais e atividade
antioxidante de cinco plantas medicinais Quiacutemica Nova 30 351-355
Tai M Zhang J Song S Miao R Liu S Pang Q 2015 International Immunopharmacology
Protective effects of luteolin against acetaminophen-induced acute liver failure in mouse Int
Immunopharmacol 27 164ndash170 doi101016jintimp201505009
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
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Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
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Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
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Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
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For each author the contribution to the publication should be mentioned
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All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
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procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
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This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
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proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
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the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
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Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
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immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
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bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
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essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
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In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
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These keywords will be used for indexing purposes
Chemical compounds
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
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writing assistance or proof reading the article etc)
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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30
EPS (or PDF) Vector drawings embed all used fonts
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References
Citation in text
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Reference management software
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(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
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Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
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The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
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presentation after acceptance of their paper
32
Supplementary data
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Submission checklist
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Ensure that the following items are present
One author has been designated as the corresponding author with contact details
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All necessary files have been uploaded and contain
bull Keywords
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Further considerations
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bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
21
Teixeira S A amp De Melo J I M (2006) Plantas medicinais utilizadas no municiacutepio de Jupi
Pernambuco Brasil Iheringia - Serie Botanica 61(1ndash2) 5ndash11
Thenmozhi AJ Dhivyabharathi M Manivasagam T Essa MM 2016 Tannoid principles of
Emblica officinalis attenuated aluminium chloride induced apoptosis by suppressing oxidative stress
and tau pathology via AktGSK-3betasignaling pathway J Ethnopharmacol 194 20ndash29
doi101016jjep201608047
Ueda H Tachibana Y Moriyasu M Kawanishi K Alves SM 2001 Aldose reductase inhibitors
from the fruits of Caesalpinia ferrea Mart Phytomedicine 8 377ndash381 doi1010780944-7113-00043
Vasconcelos C F B Maranhatildeo H M L Batista T M Carneiro E M Ferreira F Costa J
Wanderley a G (2011) Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea
Martius bark extract on streptozotocin-induced diabetes in Wistar rats Journal of Ethnopharmacology
137(3) 1533ndash1541 httpdoiorg101016jjep201108059
Yan LJ Traber MG Packer L 1995 Spectrophotometric method for determination of carbonyls
in oxidatively modified apolipoprotein B of human low-density lipoproteins Analytical Biochemistry
228 349ndash351 doiorg101006abio19951362
Yang P Xu F Li H-F Wang Y Li F-C Shang M-Y Liu G-X Wang X Cai S-Q 2016
Detection of 191 taxifolin metabolites and their distribution in rats using HPLC-ESI-IT-TOF-MSn
Molecules 21 doi103390molecules21091209
Woisky RG Salatino A Analysis of propolis some parameters and procedures for chemical quality
control Journal of Apicultural Research v 37 n2 p 99-105 1998
Williams R J Spencer J P E amp Rice-Evans C (2004) Flavonoids Antioxidants or signalling
molecules Free Radical Biology and Medicine 36(7) 838ndash849
httpdoiorg101016jfreeradbiomed200401001
Wyrepkowski C C Da Costa D L M G Sinhorin A P Vilegas W De Grandis R A Resende
F A hellip Dos Santos L C (2014) Characterization and quantification of the compounds of the
ethanolic extract from caesalpinia ferrea stem bark and evaluation of their mutagenic activity
Molecules 19(10) 16039ndash16057 httpdoiorg103390molecules191016039
22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
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This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
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information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
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This journal offers authors a choice in publishing their research
Open Access
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26
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Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
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strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
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Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
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Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
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You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
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Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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Footnotes
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Artwork
Electronic artwork
General points
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Symbol or use fonts that look similar
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You are urged to visit this site some excerpts from the detailed information are given here
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images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
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References
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31
Reference formatting
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Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
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March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
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32
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22
ANEXO A Normas para publicaccedilatildeo no Journal of Ethnopharmacology
23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
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This journal offers authors a choice in publishing their research
Open Access
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Submission
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Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
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Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
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Footnotes
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Artwork
Electronic artwork
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You are urged to visit this site some excerpts from the detailed information are given here
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30
EPS (or PDF) Vector drawings embed all used fonts
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References
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and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
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Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
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32
Supplementary data
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23
Normas para publicaccedilatildeo no Journal of Ethnopharmacology
Introduccedilatildeo
The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about peoples use of plants fungi
animals microorganisms and minerals and their biological and pharmacological effects based on the principles established through
international conventions Early people confronted with illness and disease discovered a wealth of useful therapeutic agents in the plant and
animal kingdoms The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and
sometimes recorded in herbals and other texts on materia medica Many valuable drugs of today (eg atropine ephedrine tubocurarine
digoxin reserpine) came into use through the study of indigenous remedies Chemists continue to use plant-derived drugs (eg morphine
taxol physostigmine quinidine emetine) as prototypes in their attempts to develop more effective and less toxic medicinals
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also
mandatory to upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Classification of your paper
Please note that upon submitting your article you will have to select at least one classification and at least three of the given keywords
You can preview the list of classifications and keywords (here) This information is needed by the Editors to more quickly process your
article In addition to this you can submit free keywords as described below under Keywords
The rules of 5
The Editors and Editorial Board have developed the Rules of 5 for publishing in JEP We have produced five clear criteria that each author
needs to think about before submitting a manuscript and setting the whole process of editing and reviewing at work Click here
For more details on how to write a world class paper please visit our Pharmacology Author Resources page
Authors are encouraged to submit video material or animation sequences to support and enhance your scientific research For more
information please see the paragraph on video data below
Types of paper
The Journal of Ethnopharmacology will accept the following contributions
1 Original research articles - whose length is not limited and should include Title Abstract Methods and Materials Results Discussion
Conclusions Acknowledgements and References As a guideline a full length paper normally occupies no more than 10 printed pages of the
journal including tables and illustrations
2 Ethnopharmacological communications (formerly Short Communications) - whose average length is not more than 4 pages in print
(approx 2000-2300 words including abstract and references) A maximum of 2 illustrations (figures or tables) is allowed See paragraph
below for description and format
3 Letters to the Editors
4 Reviews - Authors intending to write review articles should consult and send an outline to the Reviews Editor (see inside front cover for
contact information) before preparing their manuscripts The organization and subdivision of review articles can be arranged at the authors
discretion Authors should keep in mind that a good review sets the trend and direction of future research on the subject matter being
reviewed Tables figures and references are to be arranged in the same way as research articles in the journal Reviews on topics that address
cutting-edge problems are particularly welcome
Outlines for potential reviews need to include
A detailed abstract using the structure provided in the guidelines
An annotated table of contents
A short CV of the lead author
5 Book reviews - Books for review should be sent to the Reviews Editor
6 Commentaries - invited peer-reviewed critical discussion about crucial aspects of the field but most importantly methodological and
conceptual-theoretical developments in the field and should also provide a standard for example for pharmacological methods to be used in
papers in the Journal of Ethnopharmacology The scientific dialogue differs greatly in the social cultural and natural sciences the
discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and
24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
For each author the contribution to the publication should be mentioned
Conflict of interest
All authors are requested to disclose any actual or potential conflict of interest including any financial personal or other relationships with
other people or organizations within three years of beginning the submitted work that could inappropriately influence or be perceived to
influence their work See also httpwwwelseviercomconflictsofinterest Further information and an example of a Conflict of Interest form
can be found at httphelpelseviercomappanswersdetaila_id286p7923
Submission declaration and verification
Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a
published lecture or academic thesis or as an electronic preprint seehttpwwwelseviercompostingpolicy) that it is not under consideration
for publication elsewhere that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the
work was carried out and that if accepted it will not be published elsewhere in the same form in English or in any other language
including electronically without the written consent of the copyright-holder To verify originality your article may be checked by the
originality detection service CrossCheck httpwwwelseviercomeditorsplagdetect
Changes to authorship
This policy concerns the addition deletion or rearrangement of author names in the authorship of accepted manuscripts Before the accepted
manuscript is published in an online issue Requests to add or remove an author or to rearrange the author names must be sent to the Journal
Manager from the corresponding author of the accepted manuscript and must include (a) the reason the name should be added or removed
or the author names rearranged and (b) written confirmation (e-mail fax letter) from all authors that they agree with the addition removal or
rearrangement In the case of addition or removal of authors this includes confirmation from the author being added or removed Requests
that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author who must follow the
procedure as described above Note that (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the
accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
This journal offers authors a choice in publishing their research Open access and Subscription
For subscription articles
Upon acceptance of an article authors will be asked to complete a Journal Publishing Agreement (for more information on this and
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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Language (usage and editing services)
Please write your text in good English (American or British usage is accepted but not a mixture of these) Authors who feel their English
language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English
may wish to use the English Language Editing service available from Elseviers WebShop (httpwebshopelseviercomlanguageediting) or
visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
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Artwork
Electronic artwork
General points
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You are urged to visit this site some excerpts from the detailed information are given here
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Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
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below)
30
EPS (or PDF) Vector drawings embed all used fonts
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References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
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references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
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presentation after acceptance of their paper
32
Supplementary data
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Submission checklist
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Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
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All necessary files have been uploaded and contain
bull Keywords
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Further considerations
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24
multidisciplinary discussion The length should be a maximum of 2-3 printed pages or 2500 words Please contact the Reviews Editor
jethnopharmacolpharmacyacuk with an outline
7 Conference announcements and news
Before You Begin
Ethics in publishing
For information on Ethics in publishing and Ethical guidelines for journal publication
seehttpwwwelseviercompublishingethics and httpwwwelseviercomjournal-authorsethics
Policy and ethics
In the covering letter the author must also declare that the study was performed according to the international national and institutional rules
considering animal experiments clinical studies and biodiversity rights See below for further information The ethnopharmacological
importance of the study must also be explained in the cover letter
Animal and clinical studies - Investigations using experimental animals must state in the Methods section that the research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European
Community guidelines (EEC Directive of 1986 86609EEC) or the US guidelines (NIH publication 85-23 revised in 1985) Investigations
with human subjects must state in the Methods section that the research followed guidelines of the Declaration of Helsinki and Tokyo for
humans and was approved by the institutional human experimentation committee or equivalent and that informed consent was obtained
The Editors will reject papers if there is any doubt about the suitability of the animal or human procedures used
Biodiversity rights - Each country has its own rights on its biodiversity Consequently for studying plants one needs to follow the
international national and institutional rules concerning the biodiversity rights
Author contributions
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Submission declaration and verification
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accepted manuscript in an online issue is suspended until authorship has been agreed
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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Submission
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Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
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strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
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resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
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information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
25
After the accepted manuscript is published in an online issue Any requests to add delete or rearrange author names in an article published
in an online issue will follow the same policies as noted above and result in a corrigendum
Article transfer service
This journal is part of our Article Transfer Service This means that if the Editor feels your article is more suitable in one of our other
participating journals then you may be asked to consider transferring the article to one of those If you agree your article will be transferred
automatically on your behalf with no need to reformat Please note that your article will be reviewed again by the new journal More
information about this can be found herehttpwwwelseviercomauthorsarticle-transfer-service
Copyright
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For subscription articles
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Open Access
This journal offers authors a choice in publishing their research
Open Access
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All articles published open access will be immediately and permanently free for everyone to read and download Permitted reuse is defined
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26
Creative Commons Attribution (CC BY) lets others distribute and copy the article to create extracts abstracts and other revised versions
adaptations or derivative works of or from an article (such as a translation) to include in a collective work (such as an anthology) to text or
data mine the article even for commercial purposes as long as they credit the author(s) do not represent the author as endorsing their
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Creative Commons Attribution-NonCommercial-ShareAlike (CC BY-NC-SA) for non-commercial purposes lets others distribute and
copy the article to create extracts abstracts and other revised versions adaptations or derivative works of or from an article (such as a
translation) to include in a collective work (such as an anthology) to text and data mine the article as long as they credit the author(s) do
not represent the author as endorsing their adaptation of the article do not modify the article in such a way as to damage the authors honor or
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visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
minimum of 531 times 1328 pixels (h times w) or proportionally more The image should be readable at a size of 5 times 13 cm using a regular screen
resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
26
Creative Commons Attribution (CC BY) lets others distribute and copy the article to create extracts abstracts and other revised versions
adaptations or derivative works of or from an article (such as a translation) to include in a collective work (such as an anthology) to text or
data mine the article even for commercial purposes as long as they credit the author(s) do not represent the author as endorsing their
adaptation of the article and do not modify the article in such a way as to damage the authors honor or reputation
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copy the article and to include in a collective work (such as an anthology) as long as they credit the author(s) and provided they do not alter
or modify the article
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visit our customer support site (httpsupportelseviercom) for more information
Submission
Our online submission system guides you stepwise through the process of entering your article details and uploading your files The system
converts your article files to a single PDF file used in the peer-review process Editable files (eg Word LaTeX) are required to typeset your
article for final publication All correspondence including notification of the Editors decision and requests for revision is sent by e-mail
Additional information
Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal for format and style Authors must
include the following contact details on the title page of their submitted manuscript full postal address fax e-mail All manuscripts
submitted are subject to peer review The minimum requirements for a manuscript to qualify for peer review are that it has been prepared by
strictly following the format and style of the journal as mentioned that it is written in good English and that it is complete Manuscripts that
have not fulfilled these requirements will be returned to the author(s)
In addition you are recommended to adhere to the research standards described in the following articles
Cos P Vlietinck AJ Berghe DV et al (2006) Anti-infective potential of natural products how to develop a stronger in vitro proof-of-
concept Journal of Ethnopharmacology 106 290-302
Matteucci E Giampietro O (2008) Proposal open for discussion defining agreed diagnostic procedures in experimental diabetes research
Journal of Ethnopharmacology115 163-172
Froede TSA and YS Medeiros YS (2008) Animal models to test drugs with potential antidiabetic activity Journal of
Ethnopharmacology 115 173-183 Gertsch J (2009) How scientific is the science in ethnopharmacology Historical perspectives and
epistemological problems Journal of Ethnopharmacology 122 177-183
Chan K et al (2012) Good practice in reviewing and publishing studies on herbal medicine with special emphasis on traditional Chinese
medicine and Chinese Materia Medica Journal of Ethnopharmacology 140 469-475
Heinrich M Edwards S Moerman DE and Leonti M (2009) Ethnopharmacological field studies a critical assessment of their
conceptual basis and methods J Ethnopharmacol 124 1-17
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
minimum of 531 times 1328 pixels (h times w) or proportionally more The image should be readable at a size of 5 times 13 cm using a regular screen
resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
27
Preparation
Reference formatting
References in the manuscript should be given with the name of the first author (in case of 2 authors both names and in case of more first
author et al) and the year of publication in the reference list any style or format can be used as long as the style is consistent In the list all
author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook chapter and the pagination must
be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the accepted article by Elsevier at the
proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do wish to format the references
yourself they should be arranged according to the following examples
Use of word processing software
It is important that the file be saved in the native format of the word processor used The text should be in single-column format Keep the
layout of the text as simple as possible Most formatting codes will be removed and replaced on processing the article In particular do not
use the word processors options to justify text or to hyphenate words However do use bold face italics subscripts superscripts etc When
preparing tables if you are using a table grid use only one grid for each individual table and not a grid for each row If no grid is used use
tabs not spaces to align columns The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also
the Guide to Publishing with Elsevier httpwwwelseviercomguidepublication) Note that source files of figures tables and text graphics
will be required whether or not you embed your figures in the text See also the section on Electronic artwork
To avoid unnecessary errors you are strongly advised to use the spell-check and grammar-check functions of your word processor
Article structure
Subdivision - numbered sections
Divide your article into clearly defined and numbered sections Subsections should be numbered 11 (then 111 112 ) 12 etc (the
abstract is not included in section numbering) Use this numbering also for internal cross-referencing do not just refer to the text Any
subsection may be given a brief heading Each heading should appear on its own separate line
Introduction
State the objectives of the work and provide an adequate background avoiding a detailed literature survey or a summary of the results
Material and methods
Provide sufficient detail to allow the work to be reproduced Methods already published should be indicated by a reference only relevant
modifications should be described
Theorycalculation
A Theory section should extend not repeat the background to the article already dealt with in the Introduction and lay the foundation for
further work In contrast a Calculation section represents a practical development from a theoretical basis
Results
Results should be clear and concise
Discussion
This should explore the significance of the results of the work not repeat them A combined Results and Discussion section is often
appropriate Avoid extensive citations and discussion of published literature
Conclusions
The main conclusions of the study may be presented in a short Conclusions section which may stand alone or form a subsection of a
Discussion or Results and Discussion section
Glossary
Please supply as a separate list the definitions of field-specific terms used in your article
Appendices
If there is more than one appendix they should be identified as A B etc Formulae and equations in appendices should be given separate
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
minimum of 531 times 1328 pixels (h times w) or proportionally more The image should be readable at a size of 5 times 13 cm using a regular screen
resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
28
numbering Eq (A1) Eq (A2) etc in a subsequent appendix Eq (B1) and so on Similarly for tables and figures Table A1 Fig A1
etc
Essential title page information
bull Title Concise and informative Titles are often used in information-retrieval systems Avoid abbreviations and formulae where possible
bull Author names and affiliations Where the family name may be ambiguous (eg a double name) please indicate this clearly Present the
authors affiliation addresses (where the actual work was done) below the names Indicate all affiliations with a lower-case superscript letter
immediately after the authors name and in front of the appropriate address Provide the full postal address of each affiliation including the
country name and if available the e-mail address of each author
bull Corresponding author Clearly indicate who will handle correspondence at all stages of refereeing and publication also post-
publication Ensure that phone numbers (with country and area code) are provided in addition to the e-mail address and the
complete postal address Contact details must be kept up to date by the corresponding author
bull Presentpermanent address If an author has moved since the work described in the article was done or was visiting at the time a Present
address (or Permanent address) may be indicated as a footnote to that authors name The address at which the author actually did the work
must be retained as the main affiliation address Superscript Arabic numerals are used for such footnotes
Abstract
A concise and factual abstract is required The abstract should state briefly the purpose of the research the principal results and major
conclusions An abstract is often presented separately from the article so it must be able to stand alone For this reason References should be
avoided but if essential then cite the author(s) and year(s) Also non-standard or uncommon abbreviations should be avoided but if
essential they must be defined at their first mention in the abstract itself
The author should divide the abstract with the headings Ethnopharmacological relevance Materials and Methods Results
and Conclusions
Click here to see an example
Graphical abstract
A Graphical abstract is mandatory for this journal It should summarize the contents of the article in a concise pictorial form designed to
capture the attention of a wide readership online Authors must provide images that clearly represent the work described in the article
Graphical abstracts should be submitted as a separate file in the online submission system Image size please provide an image with a
minimum of 531 times 1328 pixels (h times w) or proportionally more The image should be readable at a size of 5 times 13 cm using a regular screen
resolution of 96 dpi Preferred file types TIFF EPS PDF or MS Office files See httpwwwelseviercomgraphicalabstracts for examples
Authors can make use of Elseviers Illustration and Enhancement service to ensure the best presentation of their images also in accordance
with all technical requirements Illustration Service
Keywords
After having selected a classification in the submission system authors must in the same step select 5 keywords These keywords will help
the Editors to categorize your article accurately and process it more quickly A list of the classifications and set keywords can be found here
In addition you can provide a maximum of 6 specific keywords using American spelling and avoiding general and plural terms and multiple
concepts (avoid for example and of) Be sparing with abbreviations only abbreviations firmly established in the field may be eligible
These keywords will be used for indexing purposes
Chemical compounds
You can enrich your article by providing a list of chemical compounds studied in the article The list of compounds will be used to extract
relevant information from the NCBI PubChem Compound database and display it next to the online version of the article on ScienceDirect
You can include up to 10 names of chemical compounds in the article For each compound please provide the PubChem CID of the most
relevant record as in the following example Glutamic acid (PubChem CID611) The PubChem CIDs can be found
viahttpwwwncbinlmnihgovpccompound Please position the list of compounds immediately below the Keywords section It is strongly
recommended to follow the exact text formatting as in the example below
Chemical compounds studied in this article Ethylene glycol (PubChem CID 174) Plitidepsin (PubChem CID 44152164) Benzalkonium
chloride (PubChem CID 15865)More information is available at httpwwwelseviercomPubChem
Plant names
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
29
In the Materials and Methods section there must be a separate heading for describing the material used That includes official name local
name English name (if known) GPS position in case of collection in the wild or cultivation a voucher specimen must be deposited in an
official herbarium for possible future comparison In the text it should be stated that the plant name has been checked
with wwwtheplantlistorg mentioning the data of accessing that website
In case of commercially procured material should mention the source batch number quality control data Data on chemical characterization
(metabolomics chromatographic methods) should also be presented in case of known active compounds their quantitative analysis should be
presented
Acknowledgements
Collate acknowledgements in a separate section at the end of the article before the references and do not therefore include them on the title
page as a footnote to the title or otherwise List here those individuals who provided help during the research (eg providing language help
writing assistance or proof reading the article etc)
Database linking
Elsevier encourages authors to connect articles with external databases giving their readers one-click access to relevant databases that help
to build a better understanding of the described research Please refer to relevant database identifiers using the following format in your
article Database xxxx (eg TAIR AT1G01020 CCDC 734053 PDB 1XFN) See httpwwwelseviercomdatabaselinking for more
information and a full list of supported databases
Math formulae
Present simple formulae in the line of normal text where possible and use the solidus () instead of a horizontal line for small fractional terms
eg XY In principle variables are to be presented in italics Powers of e are often more conveniently denoted by exp Number
consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text)
Footnotes
Footnotes should be used sparingly Number them consecutively throughout the article using superscript Arabic numbers Many
wordprocessors build footnotes into the text and this feature may be used Should this not be the case indicate the position of footnotes in
the text and present the footnotes themselves separately at the end of the article Do not include footnotes in the Reference list
Artwork
Electronic artwork
General points
bull Make sure you use uniform lettering and sizing of your original artwork
bull Embed the used fonts if the application provides that option
bull Aim to use the following fonts in your illustrations Arial Courier Times New Roman
Symbol or use fonts that look similar
bull Number the illustrations according to their sequence in the text
bull Use a logical naming convention for your artwork files
bull Provide captions to illustrations separately
bull Size the illustrations close to the desired dimensions of the printed version
bull Submit each illustration as a separate file
A detailed guide on electronic artwork is available on our website
httpwwwelseviercomartworkinstructions
You are urged to visit this site some excerpts from the detailed information are given here
Formats
If your electronic artwork is created in a Microsoft Office application (Word PowerPoint Excel) then please supply as is in the native
document format
Regardless of the application used other than Microsoft Office when your electronic artwork is finalized please Save as or convert the
images to one of the following formats (note the resolution requirements for line drawings halftones and linehalftone combinations given
below)
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
30
EPS (or PDF) Vector drawings embed all used fonts
TIFF (or JPEG) Color or grayscale photographs (halftones) keep to a minimum of 300 dpi
TIFF (or JPEG) Bitmapped (pure black amp white pixels) line drawings keep to a minimum of 1000 dpi
TIFF (or JPEG) Combinations bitmapped linehalf-tone (color or grayscale) keep to a minimum of 500 dpi
Please do not
bull Supply files that are optimized for screen use (eg GIF BMP PICT WPG) these typically
have a low number of pixels and limited set of colors
bull Supply files that are too low in resolution
bull Submit graphics that are disproportionately large for the content
Please note that figures and tables should be embedded in the text as close as possible to where they are initially cited It is also mandatory to
upload separate graphic and table files as these will be required if your manuscript is accepted for publication
Color artwork
Please make sure that artwork files are in an acceptable format (TIFF (or JPEG) EPS (or PDF) or MS Office files) and with the correct
resolution If together with your accepted article you submit usable color figures then Elsevier will ensure at no additional charge that
these figures will appear in color on the Web (eg ScienceDirect and other sites) regardless of whether or not these illustrations are
reproduced in color in the printed version For color reproduction in print you will receive information regarding the costs from
Elsevier after receipt of your accepted article Please indicate your preference for color in print or on the Web only For further
information on the preparation of electronic artwork please see httpwwwelseviercomartworkinstructions
Please note Because of technical complications that can arise by converting color figures to gray scale (for the printed version should you
not opt for color in print) please submit in addition usable black and white versions of all the color illustrations
Figure captions
Ensure that each illustration has a caption Supply captions separately not attached to the figure A caption should comprise a brief title
(not on the figure itself) and a description of the illustration Keep text in the illustrations themselves to a minimum but explain all symbols
and abbreviations used
Tables
Number tables consecutively in accordance with their appearance in the text Place footnotes to tables below the table body and indicate
them with superscript lowercase letters Avoid vertical rules Be sparing in the use of tables and ensure that the data presented in tables do
not duplicate results described elsewhere in the article
References
Citation in text
Please ensure that every reference cited in the text is also present in the reference list (and vice versa) Any references cited in the abstract
must be given in full Unpublished results and personal communications are not recommended in the reference list but may be mentioned in
the text If these references are included in the reference list they should follow the standard reference style of the journal and should include
a substitution of the publication date with Unpublished results Personal communication will not be accepted as a reference Citation of a
reference as in press implies that the item has been accepted for publication
Reference links
Increased discoverability of research and high quality peer review are ensured by online links to the sources cited In order to allow us to
create links to abstracting and indexing services such as Scopus CrossRef and PubMed please ensure that data provided in the references
are correct Please note that incorrect surnames journalbook titles publication year and pagination may prevent link creation When copying
references please be careful as they may already contain errors Use of the DOI is encouraged
Reference management software
This journal has standard templates available in key reference management packages EndNote
(httpwwwendnotecomsupportenstylesasp) and Reference Manager (httprefmancomsupportrmstylesasp) Using plug-ins to
wordprocessing packages authors only need to select the appropriate journal template when preparing their article and the list of references
and citations to these will be formatted according to the journal style which is described below
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
31
Reference formatting
There are no strict requirements on reference formatting at submission References can be in any style or format as long as the style is
consistent Where applicable author(s) name(s) journal titlebook title chapter titlearticle title year of publication volume numberbook
chapter and the pagination must be present Use of DOI is highly encouraged The reference style used by the journal will be applied to the
accepted article by Elsevier at the proof stage Note that missing data will be highlighted at proof stage for the author to correct If you do
wish to format the references yourself they should be arranged according to the following examples
Reference style
Text All citations in the text should refer to
1 Single author the authors name (without initials unless there is ambiguity) and the year of publication
2 Two authors both authors names and the year of publication
3 Three or more authors first authors name followed by et al and the year of publication
Citations may be made directly (or parenthetically) Groups of references should be listed first alphabetically then chronologically
Examples as demonstrated (Allan 1996a 1996b 1999 Allan and Jones 1995) Kramer et al (2000) have recently shown
List References should be arranged first alphabetically and then further sorted chronologically if necessary More than one reference from
the same author(s) in the same year must be identified by the letters a b c etc placed after the year of publication
Please use full journal names
Examples
Reference to a journal publication
Van der Geer J Hanraads JAJ Lupton RA 2000 The art of writing a scientific article
Journal of Scientific Communication 163 51-59
Reference to a book
Strunk Jr W White EB 1979 The Elements of Style third ed Macmillan New York
Reference to a chapter in an edited book
Mettam GR Adams LB 1999 How to prepare an electronic version of your article in Jones BS Smith RZ (Eds) Introduction to
the Electronic Age E-Publishing Inc New York pp 281-304
When a web citation with a link is being used as a reference the link should be checked and that date reported with the link as accessed on
March 29 2012 American Chemical Society 2012 Ethical guidelines to publication of chemical
researchhttppubsacsorguserimagesContentEditor1218054468605ethicspdf Accessed on March 31 2012
Video data
Elsevier accepts video material and animation sequences to support and enhance your scientific research Authors who have video or
animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article This
can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be
placed All submitted files should be properly labeled so that they directly relate to the video files content In order to ensure that your video
or animation material is directly usable please provide the files in one of our recommended file formats with a preferred maximum size of 50
MB Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom Please supply stills with your files you can choose any frame from the video or animation or
make a separate image These will be used instead of standard icons and will personalize the link to your video data For more detailed
instructions please visit our video instruction pages at httpwwwelseviercomartworkinstructions Note since video and animation cannot
be embedded in the print version of the journal please provide text for both the electronic and the print version for the portions of the article
that refer to this content
Audio Slides
The journal encourages authors to create an AudioSlides presentation with their published article AudioSlides are brief webinar-style
presentations that are shown next to the online article on ScienceDirect This gives authors the opportunity to summarize their research in
their own words and to help readers understand what the paper is about More information and examples are available
at httpwwwelseviercomaudioslides Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides
presentation after acceptance of their paper
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
32
Supplementary data
Elsevier accepts electronic supplementary material to support and enhance your scientific research Supplementary files offer the author
additional possibilities to publish supporting applications high-resolution images background datasets sound clips and more
Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products including
ScienceDirect httpwwwsciencedirectcom In order to ensure that your submitted material is directly usable please provide the data in
one of our recommended file formats Authors should submit the material in electronic format together with the article and supply a concise
and descriptive caption for each file For more detailed instructions please visit our artwork instruction pages
athttpwwwelseviercomartworkinstructions
Submission checklist
The following list will be useful during the final checking of an article prior to sending it to the journal for review Please consult this Guide
for Authors for further details of any item
Ensure that the following items are present
One author has been designated as the corresponding author with contact details
bull E-mail address
bull Full postal address
bull Phone numbers
All necessary files have been uploaded and contain
bull Keywords
bull All figure captions
bull All tables (including title description footnotes)
Further considerations
bull Manuscript has been spell-checked and grammar-checked
bull References are in the correct format for this journal
bull All references mentioned in the Reference list are cited in the text and vice versa
bull Permission has been obtained for use of copyrighted material from other sources (including the Web)
bull Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print or to be reproduced in
color on the Web (free of charge) and in black-and-white in print
bull If only color on the Web is required black-and-white versions of the figures are also supplied for printing purposes
For any further information please visit our customer support site at httpsupportelseviercom
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