IDENTIFICAÇÃO DE FLAVONOIDES POR LC-MS/MS DE EXTRATOS DE ... · dosage superoxide dismutase (SOD), catalase (GST), reduced glutathione (GSH), ... Identificação de flavonoides

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





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


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


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


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




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


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


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


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


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


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)


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

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

copyright see httpwwwelseviercomcopyright) An e-mail will be sent to the corresponding author confirming receipt of the manuscript

together with a Journal Publishing Agreement form or a link to the online version of this agreement

Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institutions

Permission of the Publisher is required for resale or distribution outside the institution and for all other derivative works including

compilations and translations (please consulthttpwwwelseviercompermissions) If excerpts from other copyrighted works are included

the author(s) must obtain written permission from the copyright owners and credit the source(s) in the article Elsevier has preprinted forms

for use by authors in these cases please consult httpwwwelseviercompermissions

For open access articles Upon acceptance of an article authors will be asked to complete an Exclusive License Agreement (for more

information see httpwwwelseviercomOAauthoragreement) Permitted reuse of open access articles is determined by the authors choice

of user license (see httpwwwelseviercomopenaccesslicenses)

Retained author rights

As an author you (or your employer or institution) retain certain rights For more information on author rights for Subscription articles

please see httpwwwelseviercomjournal-authorsauthor-rights-and-responsibilities Open access articles please

see httpwwwelseviercomOAauthoragreement

Role of the funding source

You are requested to identify who provided financial support for the conduct of the research andor preparation of the article and to briefly

describe the role of the sponsor(s) if any in study design in the collection analysis and interpretation of data in the writing of the report

and in the decision to submit the article for publication If the funding source(s) had no such involvement then this should be stated

Funding body agreements and policies

Elsevier has established agreements and developed policies to allow authors whose articles appear in journals published by Elsevier to

comply with potential manuscript archiving requirements as specified as conditions of their grant awards To learn more about existing

agreements and policies please visithttpwwwelseviercomfundingbodies

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

bull Articles are made available to subscribers as well as developing countries and patient groups through our access programs

(httpwwwelseviercomaccess)

bull No open access publication fee

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

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

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

reputation and license their new adaptations or creations under identical terms (CC BY-NC-SA)

Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) for non-commercial purposes lets others distribute and

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

To provide open access this journal has a publication fee which needs to be met by the authors or their research funders for each article

published open access

Your publication choice will have no effect on the peer review process or acceptance of submitted articles

The open access publication fee for this journal is $3000 excluding taxes Learn more about Elseviers pricing

policy httpwwwelseviercomopenaccesspricing

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

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


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

2





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


fevereiro de 2017

ii

iii

iv

Sinopse






radicais livres

v

Dedicatoacuteria




do saber

vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












ii

iii

iv

Sinopse






radicais livres

v

Dedicatoacuteria




do saber

vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












iii

iv

Sinopse






radicais livres

v

Dedicatoacuteria




do saber

vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












iv

Sinopse






radicais livres

v

Dedicatoacuteria




do saber

vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












v

Dedicatoacuteria




do saber

vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












vi

AGRADECIMENTOS

A Deus pelo existir












laboratoriais












a Carla



Rogeacuterio de

Campos Bicudo





jornada


vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












vii



William Douglas

viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












viii

RESUMO

































nesse estudo


oxidativo

ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












ix

ABSTRACT


































x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












x

SUMAacuteRIO

CAPIacuteTULO I xii

Resumo 1








26 Animais 5







3 Resultados 7





xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












xi


4 Discussatildeo 13



5 Conclusatildeo 17

Agradecimentos 17

Referecircncia 17


xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












xii

CAPIacuteTULO I





1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












1





b



a




Resumo






em camundongos









determinadas





















Medicinais

2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












2















Gonsebatt 2009)



















3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












3































4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












4































5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












5









26 Animais
























dia durante 7 dias

6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












6


















mg proteiacutena-1





mg proteiacutena-1

O coeficiente de











7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












7


ASA g-1

tecido




proteiacutena-1














Brasil)





3 Resultados





8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












8



Extrato ou


Fenois totais

(mg EAGg extrato)

Flavonoides totais


(microgmL)

EE 445 90 460

EA 595 110 343


Rutina - - 435





Fig A-2)


ferrea


2

iacuteons


molecular

1 10 61052 60952 30020 Rutina C27H30O16



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


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


9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












9



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










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












10










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




grupo PCM



Composto 7

Composto 6

SOD CAT GST

mz 13300

mz 11680

6

7

11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












11






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








ao grupo PCM








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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












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-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







GSH CAT


ALT AST

PC

13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












13




(Fig A-6)

4 Discussatildeo






2013)






e Cu+2

Dessa forma

















14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












14



2 similares aos






























15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












15
























seriam detectadas











16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












16






e Cu+2



















(Nijveldt 2001)












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












17

5 Conclusatildeo














Agradecimentos



Referecircncia






Paulo pp 38-58



218 doi101016jcbi201508011



httpdoiorg101590S0100-40422006000100021



248-254 doi1010160003-2697(76)90527-3

18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












18




httpdoiorg101016S0891-5849(96)00351-6







49 2222ndash2228 doi101016jfct201106019








doi101016jjep201407031








135 n 2 p 201ndash208













19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












19




10308 doi103390molecules190710291

































httpdoiorg101016S0003-9861(03)00284-4




20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












20



478 doiorg1010160003-2697(72)90451-4













doi101016jjri201605003























21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












21






doi101016jjep201608047






















22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












22


23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












23
















The rules of 5






Types of paper























24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












24




Before You Begin




Policy and ethics



































25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












25








Copyright


























Open Access


Open Access



Subscription






26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












26























Submission






















27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












27

Preparation

















Article structure





Introduction





Theorycalculation



Results


Discussion



Conclusions



Glossary


Appendices


28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












28


etc













Abstract






and Conclusions


Graphical abstract








Keywords






Chemical compounds









Plant names

29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












29







presented

Acknowledgements




Database linking





Math formulae




Footnotes




Artwork

Electronic artwork

General points













Formats


document format



below)

30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












30





Please do not







Color artwork









Figure captions




Tables




References

Citation in text






Reference links










31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












31







Reference style










Examples




Reference to a book








Video data












Audio Slides






32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












32

Supplementary data













bull E-mail address


bull Phone numbers


bull Keywords












Documents

IDENTIFICAÇÃO DE FLAVONOIDES POR LC-MS/MS DE EXTRATOS DE ... · dosage superoxide dismutase (SOD), catalase (GST), reduced glutathione (GSH), ... Identificação de flavonoides