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UNIVERSIDADE ESTADUAL DO CEARÁ
PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
FACULDADE DE VETERINÁRIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS
VETERINÁRIAS
NADJA SOARES VILA NOVA
ALTERNATIVAS FITOTERÁPICAS PARA O TRATAMENTO DA
LEISHMANIOSE
FORTALEZA-CE
2012
2
NADJA SOARES VILA NOVA
ALTERNATIVAS FITOTERÁPICAS PARA O TRATAMENTO DA
LEISHMANIOSE
Tese apresentada ao programa de Pós-Graduação em
Ciências Veterinárias da Faculdade de Veterinária
da Universidade Estadual do Ceará, como requisito
parcial para obtenção do título de Doutor em
Ciências Veterinárias.
Área de Concentração: Reprodução e Sanidade
Animal
Linha de Pesquisa: Reprodução e Sanidade de
carnívoros, onívoros e aves
Orientadora: Selene Maia de Morais
FORTALEZA
2012
3
V695a Vila Nova, Nadja Soares
Alternativas fitoterápicas para o tratamento da leishmaniose / Nadja Soares Vila Nova. — 2012.
CD-ROM. 147f. il. (algumas color) ; 4 ¾ pol.
“CD-ROM contendo o arquivo no formato PDF do trabalho acadêmico, acondicionado em caixa de DVD Slin (19 x 14 cm x 7 mm)”. Tese (doutorado) – Universidade Estadual do Ceará, Faculdade de Veterinária, Programa de Pós-graduação em Ciências Veterinárias, Fortaleza, 2012.
Área de concentração: Reprodução e Sanidade de Carnívoros, Onívoros e Aves.
Orientação: Profa. Dra. Selene Maia de Morais.
1. Leishmania. 2. Alcalóides. 3. Acetogeninas. 4. Cumarinas. 5. Flavonóides. I. Título.
CDD: 636.089
4
5
Ao meu pai que sempre
acreditou mais em mim do que
eu mesma.
Saudades eternas suas!
6
AGRADECIMENTOS
Ao nosso Senhor Jesus por ter me iluminado e me dado força nesses quatro anos.
Minha sábia mãe Nadir Soares Vila Nova, por ser uma mulher incrível e um exemplo de
profissional, esposa, mãe e mulher. Meu amado pai Jarbas Maciel Vila Nova, que tanto
amo, sinto saudades suas todos os dias, o que muitas vezes me moveu foi saber que
sempre se orgulhou de mim. Obrigada aos meus pais por ter enchido a minha vida de
livros, música, estudos e oportunidades. Meus irmãos Alexandre Vila Nova e Helena
Maria Vila Nova por estarem presentes e me apoiarem nessa jornada.
Ao meu melhor amigo e amado marido Matheus Wagner Paulino de Sousa,
obrigada por ser tão paciente, carinhoso, presente e compreensível. O amor e admiração
que sinto por você são indescritíveis, casei com um homem sensível, inteligente,
esforçado, honesto e de um caráter enorme. Não vejo minha vida mais sem você.
Obrigada a minha orientadora Selene Maia de Morais pela orientação e pelos
ensinamentos não somente na área acadêmica mais também na vida. Hoje sou uma
pessoa mais calma, centrada e focada, pois tive uma orientação que foi além do preparo
de um profissional. Fui doutrinada não apenas a realizar pesquisas mais também a
compreender os problemas diários dos alunos, procurar sempre o melhor para o
próximo e entender que a vida dá voltas e que devemos enfrentar as diversidades e nos
adaptar a realidade.
A família do Laboratório de Química de Produtos Naturais – LQPN foi muito
bom ter todos vocês nesses anos, obrigada meus queridos amigos Pablito, Tayse,
Noemia, Daysiane, Igor, Cristiane, Micheline, Adailson, Danyelle, Clécio e todos que
participam desta enorme família. Um agradecimento especial a Maria José Cajazeiras
Falcão por ter se dedicado tanto no desenvolvimento deste trabalho assim como pela
amizade.
Ao professor Heitor Frando de Andrade Jr. e a professora Mary E. Wilson pela
paciência, ensinamentos e principalmente por ter aberto as portas dos seus laboratórios
para que eu pudesse realizar os experimentos, muito obrigada.
Aos meus amigos que mesmo distantes são muito importantes Marianna,
Romena, Bruna, Rosângela, Ana Tereza, Darlete, Iara, Priscila, Rodrigo, Davi, Talícia,
Juliana, sinto a falta das nossas risadas e tempo que passamos juntos.
A todos os funcionários e professores do Programa de Pós-Graduação em
Ciências Veterinárias – PPGCV agradeço por proporcionarem anos tão agradáveis. Um
7
agradecimento especial ao Carlos Lobo e Débora Sales pela ajuda na biologia molecular
e pela inesperada, porém sincera, amizade.
Obrigada CNPq, PPSUS e FULBRIGHT pelo apoio financeiro.
8
RESUMO
A Leishmaniose Visceral (LV) é uma zoonose característica das regiões
tropicais e subtropicais do mundo causada pelo protozoário Leishmania infantum
chagasi. Acomete, além do homem, os canídeos, felídeos, roedores e marsupiais, sendo
transmitida pelo flebotomíneo Lutzomia longipalpis. A LV no estado do Ceará teve seus
primeiros casos registrados no ano de 1934, provenientes de Sobral e atualmente se
encontra até hoje em processo de extensão, tanto em magnitude, como geograficamente.
A observação das propriedades terapêuticas de plantas medicinais tem levado à pesquisa
de princípios ativos de várias espécies vegetais. Metabólitos secundários tais como
alcalóides, terpenóides, flavonóides, considerados no passado como inativos, são hoje
ferramentas importantes no tratamento e investigação clínica da LV. Na procura de
novos compostos com atividade leishmanicida destacam-se os alcalóides como a classe
que tem maior número de compostos, dentre outros como os acetogeninas, flavonóides
e componentes de óleos essenciais. Neste estudo foram utilizados alcalóides e
acetogeninas extraídos da semente da Annona muricata (graviola); rutina e quercetina
isolados das sementes Dimorphandra gardneriana (faveira), o eugenol, timol e seus
derivados sintéticos, além da cumarina isolada do caule e cerne Platymiscium
floribundum (sacambu). Foi realizado um screening in vitro com as formas
promastigotas e amastigotas de L. i. chagasi, L. major, L. donovani e L. mexicana.
Utilizou-se o método colorimétrico MTT ou cepas dependentes de Luciferase para a
avaliação da efetividade das substâncias em promastigotas. E para a avaliação em
amastigotas foi utilizado o método de leishmania in situ. Para os testes in vivo foram
utilizados camundongos BALB/c infectados com promastigotas de L. i. chagasi na
concentração de 107, via intraperitoneal. Os animais foram divididos em grupos de
cinco, e tratados com 100 µg/Kg dos derivados do eugenol e timol assim com as
acetogeninas isoladas das sementes da graviola. Após 30 dias da infecção deu-se inicío
ao tratamento em todos os grupos. Para o controle positivo foi utilizando um veículo de
solubilização por via oral e para o grupo negativo foi utilizado o antimonial N-metil-
glucamina administrado 60mg/kg/dia por via intramuscular durante 30 dias. Os grupos
de animais tratados e não tratados foram eutanasiados após 30 dias de tratamento, e
retirados, de maneira asséptica, o baço e o fígado, para a determinação da carga
parasitária e para os estudos posteriores da quantificação da parasitemia por qPCR.
Palavas chaves: Leishmania. Alcalóides. Acetogeninas. Cumarinas. Flavonóides
9
ABSTRACT
Visceral leishmaniasis (VL) is a characteristic zoonosis of tropical and subtropical
regions of the world caused by a protozoan, Leishmania chagasi. It affects, besides
humans, canines, felines, rodents and marsupials, being transmitted by Lutzomia
longipalpis sandfly. The VL in Ceará State had their first cases recorded in 1934,
reported in Sobral and are found until today in expansion process, in both, magnitude
and geographically. The notice of therapeutic properties of medicinal plants is leading
to the research the active principles of several plant species. Secondary metabolites such
as alkaloids, terpenoids, flavonoids, considered inactive in the past, are now important
tools in clinical investigation and treatment of VL. Alkaloids stand out in search of new
compounds with leishmanicidal activity, as a class with the greatest number of
compounds, among others like acetogenins, flavonoids and components of Essential
Oils. In this study, alkaloids and acetogenins extracted from the seed of Annona
muricata (soursop), rutin and quercetin isolated from the seeds of Dimorphandra
gardneriana (faveira), eugenol, thymol and their synthetic derivates, in addition of
coumarin, isolated from the trunk heartwood Platymiscium floribundum (sacambu) were
used. In vitro Screening was conducted with promastigotes and amastigotes forms of L.
i. chagasi, L. major, L. donovani and L. mexicana, using either the MTT colorimetric
method or Luciferase dependent strains to the evaluation of these studied substances
effectiveness against promastigotes, and the in situ leishmania to the assessment against
amastigotes. To the in vivo tests BALB/c infected with promastigotes of L. i. chagasi
were used at the concentration of 107 intrapetitoneally. The animal were divided into
groups of five, and treated with 100 µg/Kg of thymol and eugenol derivatives, and the
acetogenins isolated from graviola. Thrity days post infections began the treatment in all
groups. As positive control an oral solubilization vehicle was used and to the negative
group N-methyl-glucamine antimony 60mg/kg/day administered intramuscularly for 30
days. The treated and not treated groups were euthanized after 30 days of treatment, and
had spleen and liver aseptically removed, to the determination of parasite load and
further studies of the measurement of parasitemia by qPCR.
Key-words: Leishmania. Alkaloids. Acetogenins. Cumarins. Flavonoids
10
LISTA DE FIGURAS
Pág.
Revisão de Literatura
Figura 1. A planta Annona muricata......................................................................... 27
Figura 2. A planta Annona squamosa....................................................................... 28
Figura 3. Acetogenina triidroxilada com dois anéis tetraidrofurânicos e anel
lactônico ,-insaturada de 37 átomos de carbono isolado das sementes de A.
squamosa..................................................................................................................
30
Figura 4. Estrutura química da (I) Anonaina, (II) Xilopina e (III) O-
metilarmeparvina......................................................................................................
32
Figura 5. A planta e sementes de Dimorphandra gardneriana................................ 33
Figura 6. Representação da estrutura molecular da quercetina................................. 34
Figura 7. Representação da estrutura molecular da Rutina....................................... 35
Figura 8. As flores e planta Platymisciym floribundum............................................ 36
Figura 9. Estrutura química auraptene..................................................................... 37
Figura 10. Estrutura química escoparona.................................................................. 37
Figura 11. Representação da estrutura molecular do Eugenol.................................. 38
Figura 12. Representação da estrutura molecular do Timol..................................... 39
Figura 13. Processos de (a) Acetilação Eugenol e (b) Acetilação Timol................. 40
Figura 14. Processos de (a) Benzoilação Eugenol e (b) Benzolição timol............... 40
Capítulo 1
Figura 1. Chemical structures of leishmanicidal compounds O-methylarmepavine
(I) and C37 trihydroxy adjacent bistetrahydrofuran acetogenin (II) from A.
squamosa and Corossolone (III) and Annonacinone (IV) from A. muricata………
52
Capítulo 2
Figura 1. Toxicity of compounds at 120 µg/mL on murine RAW 264.7
macrophage cells comparing with Anphotericin B and Pentamidine (40 µg/mL
and 100 µg/mL, respectively). P < 0,05…………………………………………
73
11
Capítulo 3
Figura 1. Leishmanicidal activity of corossolone (I), annonacinone (II) and
scoporone (III), against promastigotes of L. donovani, L. major and L. mexicana.
Promastigotes in a logarithimic phase were seeded at 1x106/well and incubated
for 24 h with the isolated compounds, the number of living promastigotes was
determined indirectly by optical density (OD-620nm) and correlated to the
perncentage of survival. Control wells contained DMSO or no additives, and
Pentamidine was used as positive control. Each concentration was tested in
triplicate in replicate experiments.………………………………………………
89
Capítulo 4
Figura 1. Representation of chemical structures of thymol and eugenol
derivatives………………………………………………………………………….
106
Figura 2. Immunohistochemistry from spleen samples of mouse BALB/c infected
with L. i. chagasi during 30 days and treated for 20 days, (a) Infected not trated,
(b) Glucantime, (c) acetyl-eugenol, (d) Benzoyl-eugenol, (e) Acetyl-thymol, (f)
Benzoyl-thymol. Arrows pointing to Leishmania amastigotes stained in brown….
111
Capítulo 5
Figura 1. Relative kDNA mRNA expression detected by qPCR in the liver and
spleen of BALB/c groups infected with L. i. chagasi and treated with
annonacinone, corossolone and glucantime………………………………………..
123
Figura 2. Parasite burden by anti-Leishmania immunohistochemistry (a) Infected
not treated, (b) Glucantime, (c) annonacinone, and (d)
corossolone…………………………………………………………………………
124
12
LISTA DE TABELAS
Capítulo 1
Tabela 1. 1H (CDCl3, 500 MHz) and
13C-NMR (CDCl3, 125 MHz) chemical shifts
of compounds 3 and 4 isolated from Annona muricata…………………………
54
Tabela 2. Effect of A. squamosa and A. muricata compounds and standards on
extra-extracellular promastigote, intra-intracellular amastigote forms of
Leishmania chagasi and their cytotoxicity in mammalian cells……………………
55
Capítulo 2
Tabela 1. NMR 1H and
13C data from 6,7-dimethoxycoumarin…..………………. 71
Tabela 2. Leishmanicidal activity against L. i. chagasi and acetylcholinesterase
inhibition activities of phenolic compounds scoparone, rutin and quercetin and
standard drugs amphotericin B and Pentamidine…………………………………...
72
Capítulo 3
Tabela 1. Leishmania spp. response to secondary metabolites isolated from
Brazilian Northeastern plants and A. salina toxicity……………………………
87
Capítulo 4
Tabela 1. 1H and
13C NMR Spectroscopic data of O-acetyl-thymol (AT), O-
benzoyl-thymol (BT), O-acetyl-eugenol (AE) and O-benzoyl-eugenol (BE)
(CDCl3, 400Mhz)…………………………………………………………………...
105
Tabela 2. Leishmanicidal activity of thymol and eugenol derivatives and toxicity
against L. i. chagasi and RAW 264.7 cells…………………………………………
107
13
LISTA DE ABREVIATURAS E SIGLAS
LV – Leishmaniose Visceral
LVC – Leishmaniose Visceral Canina
LVH – Leishmaniose Visceral Humana
OMS – Organização Mundial da Saúde
MAPA – Ministério da Agricultura, Pecuária e Abastecimento.
MG – Minas Gerais
SP – São Paulo
EUA – Estados Unidos da América
RJ – Rio de Janeiro
SER - Secretarias Executivas Regionais
CCZ – Centro de Controle de Zoonoses
SESA – Secretaria de Saúde
BA – Bahia
Sb-III – Antimonial trivalente
DNA – Ácido desoxirribonucleico
K+ - Potássio
FDA – Food and Drug Administration
iNOS2 - óxido nítrico sintetase 2
NO – Óxido Nítrico
UV – Ultravioleta
ATP – Adenosina Trifosfato
NADH - nicotinamida adenina dinucleótidio hidreto
MTT – diphenyltetrazolium
CE50 – Concenttração Efetiva capaz de matar 50% da população
EC50 – 50% effective concentration
IC50 – 50% Inhibitory Concentration
µg/mL – Microgramas por mililitro
Kg – Quilogramas
CE – Crude Extract
CE – Ceará
ACE - acetogenin-rich extract
TLC - Thin Layer Chromatographic
14
AE - alkaloid extract
CDCL3 – Clorofórmio Delterado
°C – Graus Celsius
% - Por cento
CO2 – Gás Carbônico
µl – Microlitros
mg/mL – Miligramas por mililitros
OD - Optical density
µm – Micrômetro
SDS – Sulfato Dodecil de Sódio
nm – Nanômetros
FCS - Fetal calf serum
M – Molar
HCl – Ácido Clorídrico
CI - Confidence interval
NMR – Nuclear Magnetic Resonance
µM – Micromolar
mg/Kg – Miligrama por quilograma
SbV – Antimoniato pentavalente
CENAUREM – Laboratório de Ressonância Magnética Nuclear
AChE - enzima acetilcolinesterase
GPI - glycosyl phosphatidylinositol
WHO -World Health Organization
g – Gramas
qPCR – Quantitative Polimerase Chain Reaction
15
SUMÁRIO
PG
1 INTRODUÇÃO....................................................................................................... 16
2 REVISÃO DE LITERATURA.............................................................................. 18
2.1 HISTÓRICO DA LEISHMANIOSE VISCERAL.......................................... 18
2.2 RELAÇÕES ENTRE LEISHMANIOSE VISCERAL HUMANA E
LEISHMANIOSE VISCERAL CANINA........................................................
19
2.3 MEDICAMENTOS UTILIZADOS NO TRATAMENTO DA
LEISHMANIOSE VISCERAL............................................................................
21
2.4 USO DE PRODUTOS NATURAIS COMO ALTERNATIVAS PARA O
TRATAMENTO DA LEISHMANIOSE.................................................................
24
2.4.1 PLANTAS MEDICINAIS ............................................................................. 24
2.4.2 ANNONACEAS............................................................................................. 26
2.4.2.1 ACETOGENINAS....................................................................................... 28
2.4.2.2 ALCALÓIDES ............................................................................................ 30
2.4.3 Dimorphandra Gardneriana............................................................................ 32
2.4.3.1 QUERCETINA........................................................................................... 33
2.4.3.2 – RUTINA................................................................................................... 34
2.4.4 Platymiscium Florundum................................................................................. 35
2.4.4.1 CUMARINAS............................................................................................... 36
2.4.5 - COMPONENTES DE ÓLEOS ESSENCIAIS - MONOTERPENOIDES E
FENILPROPANOIDES............................................................................................
37
2.4.5.1 EUGENOL.................................................................................................... 37
2.4.5.2 TIMOL.......................................................................................................... 38
2.4.5.3 DERIVADOS SINTÉTICOS DO EUGENOL E TIMOL........................ 39
3. JUSTIFICATIVA................................................................................................. 41
4. HIPÓTESE.............................................................................................................. 42
5. OBJETIVOS............................................................................................................. 43
5.1 Objetivos Gerais............................................................................................. 43
5.2 Objetivos Específicos................................................................................ 43
6 CAPÍTULO 1........................................................................................................... 44
7 CAPÍTULO 2......................................................................................................... 62
8 CAPÍTULO 3............................................................................................................ 79
9 CAPÍTULO 4............................................................................................................ 96
16
10 CAPÍTULO 5.......................................................................................................... 115
11 CONCLUSÃO......................................................................................................... 126
12 PERSPECTIVAS.................................................................................................... 127
13 REFERÊNCIA BIBLIOGRÁFICAS............................................................... 128
17
1. Introdução:
A Leishmaniose visceral (LV) é uma zoonose emergente e reemergente nas
regiões tropicais e subtropicais do mundo causado por um protozoário Leishmania
infantum chagasi. Acomete além do homem, canídeos, felídeos, roedores e marsupiais,
sendo transmitida pelo flebotomíneo Lutzomia longipalpis (ALENCAR et al., l99l).
No Novo Mundo, a principal forma de transmissão do parasito para o homem e
outros hospedeiros mamíferos é através da picada de fêmeas de dípteros da família
Psychodidae, sub-família Phlebotominae, conhecidos genericamente por flebotomíneos.
O gênero Lutzomyia é o responsável pela transmissão das leishmanioses nas Américas,
existindo 350 espécies catalogadas, distribuídas desde o sul do Canadá até o norte da
Argentina. Destas, pelo menos 200 ocorrem na bacia amazônica (GILL et al., 2003).
Lutzomyia longipalpis é o principal vetor da Leishmania infantum chagasi no Brasil
(GONTIJO; MELO, 2004).
A leishmaniose canina é um problema grave na medicina veterinária e na saúde
pública, pois o cão é considerado o principal reservatório. A Organização Mundial da
Saúde (OMS) indica a eutanásia de cães soroposivos como forma de controle da
leishmaniose, no entanto este tipo de abordagem gera desconforto com os proprietários
e discussões junto com as organizações protetoras de animais, e o seu impacto na
diminuição dos casos em humanos não está totalmente esclarecido (NUNES et al.,
2010). Na Europa é utilizado quatro abordagens para impedir a disseminação da
leishmaniose canina para outros cães e humanos. A primeira é a vacinação, a segunda é
a utilização de repelentes, a terceira é a eutanásia dos animais soropositivos seguida pela
quarta que é o tratamento destes animais (AIT-OUTHIA et al., 2012).
Estudos realizados em cães utilizando diversos medicamentos de uso humano
demonstram a remissão dos sintomas e a cura clínica, no entanto não garante a cura
parasitológica e as recaídas são frequentes (SOLANO-GALEGO et al., 2009). Esta
baixa eficácia pode estar relacionada com o estado imunológico do animal, com as
propriedades farmacocinéticas das drogas e com a sensibilidade das diferentes cepas de
Leishmania além da resistência aos fármacos (AIT-OUTHIA et al., 2012). No entanto, o
tratamento dos animais é capaz de reduzir os sintomas a parasitemia, e diminuir a
possibilidade de transmissão (SOLANO-GALEGO et al., 2009).
Porém no Brasil o Ministério da Saúde, em Nota Técnica - “USO DO
ANTIMONIATO DE MEGLUMINA EM CÃES”, de 20 de janeiro de 2004, baseado
18
no parecer nº 0299/2004 da Advocacia Geral da União, determinou a proibição do uso
de medicamento para o tratamento da leishmaniose em cães, quando o mesmo for de
distribuição do Ministério da Saúde. Uma portaria interministerial MAPA/MS
1.426/2008, proibe em todo o território nacional, o tratamento da leishmaniose visceral
em cães infectados ou doentes, com produtos de uso humano ou produtos não
registrados no Ministério da Agricultura, Pecuária e Abastecimento (MAPA).
19
2 REVISÃO DE LITERATURA
2.1 Histórico da Leishmaniose Visceral
A Leishmaniose visceral (LV) foi descrita na Grécia em 1835 quando então era
denominada "ponos" ou "hapoplinakon". Foi na Índia em 1869 que recebeu o nome
"kala-jwar" que quer dizer febre negra ou "kala-azar" que significa pele negra em
virtude do discreto aumento da pigmentação da pele ocorrido durante a doença
(MARZOCHI et al., 1981).
Em 1900 foi identificada pelo Major W.B. Leishman, que descreveu um caso de
um jovem militar inglês que havia regressado de Dum-Dum na Índia, o paciente
demonstrava sintomas semelhantes a outros que Leishman observou. Na autopsia
constatou-se um aumento excessivo do baço e no exame histológico observaram-se
estruturas redondas ou ovais, com um núcleo redondo, características das formas
amastigotas de Leishmania (GILLESPIE; PEARSON, 2001).
O primeiro caso no Brasil foi descrito por Migone em 1913. O paciente era um
imigrante italiano que vivera muitos anos em Santos, SP, e após viajar para Mato
Grosso, adoeceu, tendo sido diagnosticada a doença no Paraguai (ALENCAR, 1977).
Foi Penna (1934) quem iniciou os estudos sobre a distribuição geográfica da
Leishmaniose Visceral nas Américas, quando comprovou parasitologicamente, 41 casos
dentre as 40.000 viscerotomias examinadas para febre amarela provenientes de vários
estados do Brasil.
A LV no estado do Ceará teve seus primeiros casos registrados por Deane e
Deane (1954a) em 1934, provenientes de Sobral e se encontrava em processo de
expansão, tanto em magnitude, como geograficamente. Na década de 50 também na
cidade de Sobral, houve uma concentração de casos de LV alóctones, onde o número de
diagnóstico na cidade ultrapassou o número de diagnóstico dos anos anteriores no país
(DEANE; DEANE, 1954b).
Entre maio de 1953 e agosto de 1954 foi feito um estudo que abrangeu os
municípios de Sobral, Massapé, Tianguá e Viçosa do Ceará. Na época, a população
destes municípios era de 1550 pessoas e durante o período estudado ocorreram 52 casos
de leishmaniose humana. Também foi realizado um estudo de leishmaniose canina nesta
região, onde foram examinados 174 cães dos quais 7 se mostraram positivos. Além dos
cães estudou-se a incidência da enfermidade em raposas da região identificadas como
20
Cerdocyon thous. Examinaram-se 33 animais dos quais 10 foram provenientes do foco
estudado e 23 apanhadas em zona de sertão ou tope de serra. Entre as 10 raposas
capturadas no foco de leishmaniose em estudo, 3 estavam parasitadas, das outras 23
apenas 1 apresentou parasitismo. Indicando assim que na área em estudo havia três
mamíferos hospedeiros naturais: o homem, o cão doméstico e a raposa (DEANE;
DEANE, 1955).
Os cães freqüentemente acompanhavam as famílias nordestinas em suas
migrações, o que foi comprovado em inquérito sobre a leishmaniose visceral canina
(LVC) em Sobral, onde dentre os animais acometidos, alguns tinham sido trazidos por
seus donos de localidades rurais, algumas das quais eram focos de LV. O que já
indicava que o homem e o cão doentes eram potencialmente responsáveis pelo
aparecimento de LV em localidades para onde migravam ou por onde passavam desde
que nestas áreas existissem os flebotomíneos transmissores (DEANE; DEANE, 1955).
Tal como na cidade de Sobral, no Vale do Jaguaribe, ainda no Estado do Ceará, também
foram detectados alguns casos urbanos da infecção, não somente em humanos como
também em cães (ALENCAR et al., 1956).
2.2 Relações entre Leishmaniose Visceral Humana e Leishmaniose Visceral Canina
A análise deste assunto polêmico leva ao questionamento se a LV não existiria
na ausência de cães, se a erradicação da doença seria possível através do controle da
transmissão em cães. Este aspecto merece ser analisado, pois até o momento, no Brasil,
apesar das medidas implantadas pelos órgãos governamentais, os indicadores
epidemiológicos revelam que ainda não foi observado o impacto positivo esperado no
controle desta doença (FUNASA, 2002).
Nunes et al. (2001) afirmam que apesar de a leishmaniose visceral humana
(LVH) nem sempre obedecer a uma distribuição espacial paralela à da LVC, observa-se
que as infecções caninas são mais freqüentes que as humanas e que, normalmente, as
precedem. Na maioria dos estudos sobre epidemias de LVH tem-se encontrado cães
positivos, e ainda mais, não existe nenhum relato na literatura brasileira de epidemias de
LVH sem a presença de cão positivo (período 1953-1997) (FUNASA, 2002). Oliveira et
al. (2001) encontraram evidências que em Belo Horizonte, MG, os casos de LVH
ocorria em locais onde a LVC apresentava altos índices. Em Belo Horizonte e
Araçatuba-SP, locais onde não existia a LVH foram possíveis observar primeiro a
21
introdução da doença canina e depois a doença humana (FUNASA, 2002). Em
Araçatuba, Assis et al. (2008) não obtiveram uma associação significante entre
eutanásia canina e incidência de LVH entre os anos 2000 a 2002, no entanto a
incidência de casos humanos tiveram uma propensão a aumentar, associada com a taxa
de eutanásia em cães. Contudo, a LVC não parece ser a causa suficiente, mas sim a
causa necessária para o aparecimento da LVH em uma região.
Rab et al. (1995) afirmam que no Paquistão, mesmo com altos índices de cães
infectados a LVH não está relacionada com as infecções em cães, que famílias que
possuem cães soropositivo possuem o mesmo risco de infecção daquelas famílias com
cães saudáveis. Dietze et al. (1997) estudaram o impacto da retirada dos cães no Espírito
Santo sobre a transmissão da LV. Neste estudo, foram escolhidas três cidades separadas
através de barreira geográfica (montanhas), todas endêmicas para LV. Em duas delas,
após inquérito sorológico, promoveram a retirada de todos os cães soro-reagentes e na
cidade controle, foram matidos todos os cães. Durante 12 meses as taxas de
soropositividade humanas foram medidas pelo teste Dot–Elisa e aumentaram de 14%
para 54% na cidade controle e de 15% para 54% nas cidades em intervenção. Paranhos-
Silva et al. (1998), estudando, numa coorte de cães, o efeito da migração sobre a
incidência da LV no estado da Bahia, demonstraram que apesar de terem sido
eliminados todos os cães soropositivos da área de estudo, o número de notificações da
doença em humanos nos anos seguintes aumentaram. Em Barra de Guabiraba, RJ, uma
cidade endêmica para LV, foi evidenciada que medidas de controle da doença, como
sacrifício de cães diagnosticados com LV não influencia o número de casos da doença
(CABREIRA et al., 2003). Estes estudos vêm demonstrando, que o critério de
eliminação do cão necessita ser reavaliado.
A LV no município de Fortaleza historicamente apresentou baixas prevalências
tanto de infecção canina como de leishmaniose humana, no entanto a partir de 2001
ocorre um registro crescente no número de casos de LV, com registro de transmissão na
maioria dos bairros da cidade (SMS, 2007).
Rondon et al. (2008) analisaram a situação da LVC, entre os aos de 2005 a 2007,
nas diferentes Secretarias Executivas Regionais (SER) nas quais a cidade de Fortaleza é
dividida. Neste estudo os autores examinaram 1.381 animais, sendo 750 animais
domiciliados e 631 cães de rua, obtendo soroprevalência de 21,4% (135/631) em cães
de rua e 26,2% (197/750) em cães domiciliados. No entanto, de acordo com dados
cedidos pelo Centro de Controle de Zoonoses (CCZ) de Fortaleza, entre os anos de 2005
22
a 2007 foram diagnosticados pelo CCZ, 9002 animais e eutanaziados 6096 animais
soropositivos para leishmaniose.
De acordo com a Secretaria Estadual de Saúde do Estado do Ceará (SESA), de
1986 até agosto de 2012, no estado, o ano de 2006 demonstrou o maior número de casos
de leishmaniose humana (796 casos), seguido pelo ano de 2007 (720 casos) (SESA,
2012). A cidade de Fortaleza se encontra em uma situação epidemiológica de
transmissão intensa, entre os anos de 2009 até 2011 foram diagnosticados 783 casos de
leishmaniose humana com 73 óbitos, e até agosto de 2012 já havia sido diagnosticado
161 casos (SESA, 2012).
Estudos em locais endêmicos em diferentes regiões do país encontram-se índices
distintos de LV. Um estudo realizado em Araçatuba, SP no ano de 2005, mostrou que
16,7% dos 5.861 cães eutanasiados naquele ano eram soropositivos (ASSIS et al.,
2008). Na Bahia, nos municípios de Lauro de Freitas e Camaçaria, entre os anos de
2003 e 2004 constatou-se uma taxa de incidência de LVC de 17,4% e 18,5%
respectivamente (BARBOZA et al., 2006). Moreira et al. (2003), através de um estudo
de coorte na população canina de um bairro do município de Jequié, BA, registraram a
incidência de 11,8%. França- Silva et al. (2003), trabalhando no município de Montes
Claros, MG registraram 6,4% de incidência na população canina e Fisa et al. (1999)
relataram a incidência de 6,7% para LV canina na Catalunha, Espanha.
Apesar da LV ser de notificação compulsória (isto é obrigatória), praticamente
não ocorre registro da doença pelas clínicas e hospitais veterinários particulares, sendo
os casos registrados provenientes de animais examinados pelos CCZs do estado. Isto
dificulta o programa de combate à leishmaniose e o conhecimento real da LVC no
Ceará.
2.3 Medicamentos utilizados no tratamento da Leishmaniose Visceral
A utilização de medicação humana no tratamento de cães não permite a cura
parasitológica do animal, no entanto, diminui os sintomas e a quantidade de parasitas
circulantes e na pele, ocorrendo infecçãoo dos flemobotomíneos em menor grau
(SOLANO-GALLEGO et al., 2009). Sendo assim, o tratamento de animais positivos
para leishmaniose poderia ser considerado uma forma de controle da leishmaniose
visceral.
23
O tártaro emético foi durante muitos anos a única alternativa para o tratamento
da leishmaniose, porém devido aos seus efeitos tóxicos, difícil administração e recidivas
recorrentes, este medicamente foi substituído pelos antimoniais pentavalentes
(MAYRINK et al., 2006). Os antimoniais são utilizados a mais de 50 anos, na América
do Sul é utilizado o antimoniato de meglumina (Glucantime®), que no Brasil, é utilizado
como medicamento de primeira escolha, mas causam alterações hepáticas e distúrbios
cardiológicos como os principais efeitos tóxicos. O antimônio pode ser detectado no
cabelo de pacientes tratados até um ano após o término do tratamento. A utilização
desta droga requer mais de 28 dias de administração por via parenteral e o surgimento
de resistência vem se tornando um grave problema de saúde pública (CROFT;
COOMBS, 2003; AWASTHI et al., 2004; RATH et al., 2003; ANDERSEN et al.,
2005). Mesmo com a alta toxicidade, este medicamento continua sendo usado como
tratamento de primeira escolha. Tempone e Andrade (2008) desenvolveram uma
formulação lipossomal do antimônio com alta eficácia e capaz de diminuir a dose total
administrada em 133 vezes. As rotas de entrada dos antimoniais no macrófago ainda são
incertas, porém acredita-se que a aquagliceroporina parasitária (proteína transportadora
aquaporina 1) é responsável pelo transpote dos antimoniais para dentro das amastigotas
(SINGH et al., 2012). Ao entrar na célula hospedeira, o medicamento sofre uma redução
para sua forma trivalente (antimonial trivalente – SbIII), induzindo então um efluxo de
tripanotiona e glutationa a partir das células e também inibe a tripanotiona redutase,
causando uma grande perda do potencial de redução de tióis nas células (BRUNTON et
al., 2006). O antimônio também possui mecanismos de ação indiretos, elevando os
níveis de citocina, e também os de DNA induzindo ao dano do DNA in vivo (FREITAS-
JUNIOR et al., 2012).
A pentamidina é um quimioterápico que faz parte do grupo das diamidas e é
utilizada como alternativa em casos onde o indivíduo não responde ao tratamento com o
antimoniato, no entato sua nefrotoxicidade e cadiotoxocidade, também leva a uma
limitação na utilização deste medicamento, podento o paciente chegar à morte. Há dois
sais de pentamidina, o isotionato de pentamidina (Pentamidina®), disponível nas
Américas e Europa e o mesilato de pentamidina (Lomidine®). O seu mecanismo de ação
não está totalemente elucidado (RATH et al., 2003; PAULA et al., 2003; BLUM et al.,
2004). Andersen et al. (2005) compararam os tratamentos com o antimoniato e a
pentamidina e observaram que em condições idênticas a pentamidina se mostrou menos
eficaz comparada ao glucantime.
24
A anfoterina B é um antibiótico antifúngico derivado do Streptomyces nodosus e
utilizada no tratamento de infecções fúngicas sistêmicas. A utilização desta droga no
tratamento da leishmaniose é realizada em casos de resistência aos medicamentos de
primeira escolha. Este medicamento é capaz de se ligar ao ergosterol presente na
membrana da Leishmania, implicanto no aumento da permeabilidade celular do parasita
e consequente perda de pequenos cátios como o K+ (ORDONEZ-GUTIÉRREZ et al.,
2007; RATH et al., 2003). No entanto, a anfotericina B possui baixa capacidade de
absorção gastrointestinal e característica hidrofóbica, podendo também interagir em vias
celulares de mamíferos, causando disfunções (ROY et al., 2012), sendo seu principal
efeito colateral a nefrotoxicidade (MOTTA; SAMPAIO, 2012).
Uma forma de tentar reduzir a toxicidade e resistência aos medicamentos
leishmanicidas, é desenvolver novas formulações com o objetivo de direcionar o
medicamento ao macrófago. A utilização de lipossomos vem sendo utilizada juntamente
com a anfotericina B. Os lipossomos são sistemas carreadores de fármacos, que são
capazes de levar altas doses do medicamento até a célula alvo (TEMPONE;
ANDRADE, 2008). A anfotericina B lipossomal reduz os efeitos colaterais e exelentes
índices de eficácia terapêutica inclusive em indivíduos sem resposta ao tratamento com
antimoniais (PAULA et al., 2003; MOTTA; SAMPAIO, 2012). No entanto o alto custo
deste medicamento limita o seu uso em países subdesenvolvidos e em desenvolvimento
(ORDONES-GUTIÉRREZ et al., 2007). Em países desenvolvidos, a anfotericina B
lipossomal é utilizada como medicação de primeira escolha, sendo também o único
medicamento aprovado pela American Food and Drug Administration (FDA) (VAN
GRIENSVEN; DIRO, 2012).
Um grande avanço no tratamento da leishmaniose foi o desenvolvimento da
miltefosine, que foi aprovada em 2002 como o primeiro medicamento leishmanicida
utilizado por via oral (DORLO et al., 2012; PALADIN LABS INC., 2010). A
milefosine, um alquifosfolipídeo, foi originalmente desenvolvida como uma droga para
o tratamento do câncer (CROFT; COOMBS, 2003) e sua ação contra leishmaniose foi
rapidamente reconhecida. Sua toxicidade não é tão elevada, no entantanto, é
extremamente teratogênica e em mulheres o tratamento é realizado em não grávidas que
aceitem tomar anticonceptivos durante, e três meses após o término do tratamento
(SOTO et al., 2004; SUNDAR et al., 2012). Outros efeitos colaterais são disfunções
gastrointestinais, dores de cabeça e aumento das enzimas hepáticas (BLUM et al.,
2004). Seu mecanismo de ação age sobre o metabolismo lipídico, causando apoptose do
25
parasito, e também age diretamente na célula hospedeira, estimulando a produção de
óxido nítrico sintetase 2 (iNOS2), no qual catalisa a produção de óxido nítrico (NO) e
mata o parasita dentro do macrófago (FREITAS-JUNIOR et al., 2012).
Paromomicina é um antibiótico aminoglicosídeo que foi redescoberto como
agente leishmanicida na década de 80 e vem sendo utilizado com sucesso por via
parenteral e tópica (RATH et al., 2003; DORLO et al., 2012). Seu mecanismo de ação
ainda não está totalmente elucidado, no entanto acredita-se que a paromomicina ligue-se
ao glicálice da Leishmania, sugerindo que a mitocôndria seja um alvo primário (SINGH
et al., 2012).
Aloupurinol é um análogo da hipoxantina e normalmente é utilizado em
associação com antimônio (BLUM et al., 2004). Este medicamente possui baixa
toxicidade, no entanto, é ineficaz no controle da infecção (RATH et al., 2003). É um
medicamento utilizado como substrato por várias enzimas na via de purinas, em
tripanossomídeos, sendo incorporada nos nucleotídeos intermediários e ácidos nucleicos
do parasito (CROFT; COOMBS, 2003).
2.4 Uso de produtos naturais como alternativas para o tratamento da
Leishmaniose
O desenvolvimento de drogas segue três linhas, a primeira explora caminhos
metabólicos do parasito para encontrar alvos e desenvolver compostos sintéticos, o
segundo é o estudo de outros medicamentos que já se encontram no mercado até então
com atividade leishmanicida desconhecida (ex. medicamentos contra câncer) e o
terceiro é focado na utilização de plantas medicinais como fonte de moléculas anti-
protozoário (LINDOSO et al., 2012).
2.4.1 Plantas Medicinais
Plantas são importantes fontes de descoberta de drogas, principalmente no que
diz respeito a drogas antiparasitárias, devido à associação entre a coexistência dos
parasitos, seres vivos e plantas mediciais (ANTHONY et al., 2005). Os produtos
naturais oferecem moléculas com impacto profundo na saúde humana, a natureza
produz infinitos metabólitos secundários com propriedades biológicas distintas.
Diversos estudos já validaram o efeito de produtos naturais como potenciais fontes de
26
novos e seletivos agentes para o tratamento de doenças tropicais causados por
protozoários e outros parasitos (MISHRA et al., 2009).
A observação das propriedades terapêuticas de vegetais tem levado à pesquisa
dos princípios ativos de várias espécies vegetais. A exploração dos recursos vegetais
pode levar a identificação de metabólitos secundários valiosos que podem servir como
drogas ou conduzir ao desenvolvimento de novas substâncias terapêuticas (GOBBO-
NETO; LOPES, 2007). O metabolismo das plantas é composto por um conjunto de
reações químicas que estão ocorrendo continuamente nas células. A síntese de
compostos como aminoácidos, açúcares, ácidos graxos e nucleotídeos, essenciais para a
sobrevivência dos vegetais, faz parte do metabolismo primário. Já os compostos
sintetizados por outras vias, que aparentam não ter relação direta com a sobrevivência
do vegetal, fazem parte do metabolismo secundário (MORAIS; BRAZ-FILHO, 2007).
Atualmente, sabe-se que muitas das substâncias produzidas pelo metabolismo
secundário possuem propriedades biológicas importantes e estão diretamente envolvidas
nos mecanismos que permitem a adequação da planta ao seu meio. Muitos metabólitos
secundários possuem diversas funções biológicas, tais como defesa contra herbívoros e
microrganismos, proteção contra raios UV, atração de polinizadores ou animais
dispersores de sementes (FUMAGALI et al., 2008).
Devido à viabilidade limitada de quimioterápicos leishmanicidas eficazes em
áreas endêmicas, uma grande parte da população que vive nestes locais depende de
plantas medicinais que são utilizadas em tratamentos populares para tratar e aliviar os
sintomas da leishmaniose (CHAN-BACAB; PENA-RODRIGUEZ, 2001). Estas plantas
possuem metabólitos secundários que podem agir e destruir agentes invasores, no
entanto muitas vezes essas substâncias são desconhecidas e podem oferecer alternativas
de tratamento para a leishmaniose.
Metabólitos secundários tais como alcalóides, terpenóides e flavonóides,
considerados no passado como inativos são hoje ferramentas importantes no tratamento
de protozoários. Compostos que estimulam o sistema imune são úteis quando usados
como adjuvantes no tratamento de certas doenças causadas por fungos, bactérias e
protozoários, como na leishmaniose. Neste último, estudos químicos e
imunofarmacológicos têm sido realizados com o intuito de encontrar novos compostos
menos tóxicos, economicamente mais viáveis de efeito específico e que reverta à
resistência do parasito às drogas (BERGMANN et al., 1997).
27
O Brasil é o país com maior diversidade genética vegetal do mundo, contando
com mais de 55.000 espécies catalogadas (AZEVEDO; SILVA, 2006). Segundo Simões
et al. (2004), apenas 8% desse percentual biológico foi estudado em busca de compostos
bioativos e 1.100 espécies vegetais foram avaliadas em suas propriedades medicinais.
Dessas, 590 plantas foram registradas no Ministério da Saúde para comercialização.
No entanto, apenas nos últimos 30 anos vem se estudando seriamente a eficácia
e o mecanismo de ação dos medicamentos provenientes de vegetais (ANTHONY et al.,
2005). Alguns autores vem pesquisando novas alternativas para o tratamento da
leishmaniose na natureza, que é uma grande fonte de drogas utilizadas no tratamento de
diversas doenças. Baseado no uso popular, estes autores procuram por medicamentos
com menor toxicidade e custo extraídos de vegetais e microorganismos.
Porém, exitem vários aspectos que limitam a pesquisa em produtos naturais: (1)
viabilidade baixa dos compostos, de uma forma geral as substâncias extraídas dos
vegetais são em baixa quantidade e de difícil extração; (2) alta complexidade estrutural,
vários estereoisômeros; (3) falta de continuidade nas pesquisas, as maiorias das
pesquisas não fazem parte de programas de desenvolvimento de novos medicametos,
ocorre uma falta de continuidade na pesquisa; (4) os compostos isolados muitas vezes
não mostram atividade e requerem um acompanhameno para melhorar estas atividades
(MISHRA et al., 2009).
Como parte da pesquisa por novos e melhores medicamentos com alta
viabilidade e baixa toxicidade, o Programa de Doenças Tropicais da Organização
Mundial de Saúde (OMS) vem considerando a investigação sobre o uso plantas no
tratamento de leishmaniose como essencial e de alta prioridade (OMS, 2012).
2.4.2 Annonaceas
Annona muricata, popularmente conhecida como graviola (Figura 1) é um
vegetal comumente encontrado no Brasil e tradicionalmente utilizado no tratamento de
câncer, pertence à família Annonaceae (GEORGE et al., 2012; HAMIZAH et al., 2012).
É utilizada no combate a verminoses, febre, diarreia e desinteria além de propriedades
antioxidantes (BASKAR et al., 2007). As folhas possuem ação antiespasmótica,
hipotensiva, antiparasitária, antidiarréica e reumatológicas (ARTHUR et al., 2012;
SOUSA et al., 2010). A infusão das folhas também possui ação antiplasmódica,
28
adstringente, e propriedades gástricas, renais e hepatoprotetora além de sua ação contra
icterícia (ARTHUR et al., 2012).
As folhas da A. muricata possuem diversos estudos indicando vários metabólitos
secundários pertencentes à classe das acetogeninas (YUAN et al., 2003). O alto
potencial, seletividade, variedade química, diversidade biológica e ação desta classe de
compostos contra agentes resistentes a antibióticos, podem tornar estas substâncias
importantes no combate a agentes parasitários.
Figura 1. A planta Annona muricata. Fonte: Vila-Nova, 2012
A Annona squamosa (Figura 2) é popularmente chamada de ata, pinha, fruta do
conde e suas preparações farmacológicas são conhecidas mundialmente. É uma
excelente fonte de cálcio e possui propriedades inseticidas. As folhas são usadas como
purgativo e a decocção destas tem uso no tratamento da diabetes e no alívio de dores
reumáticas (PATEL et al., 2012).
29
Figura 2. A planta Annona squamosa. Fonte: Vila-Nova, 2012
Este vegetal é conhecido pelos potentes metabólitos secundários encontrados em
todas as suas partes. Uma de suas propriedades mais conhecida é a anti-câncer
(SRIVASTAVA et al., 2011). Suas sementes que são muitas vezes jogadas fora
possuem ação anti-ovulatória e abortiva (PANDLEY; BARVE, 2011), anti-helmíntica
(SOUZA et al., 2007); o extrato etanólico possui atividade inseticida (KUMAR et al.,
2010), hipoglicêmico (MUJEEB et al., 2009), moluscicida (MRITA et al., 2001),
antibacteriano (PATEL et al., 2012); o extrato aquoso possui ação anti-oxidante
(KALEEM et al., 2006), anti-inflamatória (CHAVAN et al., 2010), estes são apenas
alguns exemplos dentro as diversas aplicações da A. squamosa.
Esta planta é pertencente à família Annonaceae, é produtora de diversos
metabólitos secundários, entre eles os alcaloides e acetogeninas, que são responsáveis
pelas diversas atividades que este vegetal possui.
2.4.2.1 Acetogeninas
As acetogeninas, amplamente encontradas nas Annonaceas, são responsáveis por
grande parte das atividades farmacológicas atribuídas a estes vegetais (UPADHYAY;
AHMAD, 2012). Uma das atividades mais estudadas das acetogeninas é a atividade
anti-câncer. Alvarez-Gonzalez et al. (2008) induziram a formação de criptas no cólon de
camungongos e observou que acetogeninas foram capazes de reduzir estas criptas; um
outro estudo, concluiu que a acetogeninas causam depleção de produção de ATP por
células carcinogênicas pancreáticas (TORRES et al., 2012); em um terceiro estudo
Atawodi et al. (2011) comprovaram a ação de acetogeninas em células cancerígenas da
próstata.
30
Outras ações também podem ser associadas à presença destes metabólitos
secundários presentes em extratos feitos de diferentes partes da Annonaceas. Entre as
mais diversas atividades pode-se citar a ação contra herpes simplex (PADMA et al.,
1998), hipotensiva (NWOKOCHA et al., 2012), larvicida contra larvas de Aedes aegypti
e moluscicida contra Biomphalaria glabrata hospedeiro intermediário de Schistosoma
mansoni (GRZYBOWSKI et al., 2012; LUNA et al., 2005), antiplamosdio contra
Plasmodium falciparum (BOYOM et al., 2011) e anti-microbiano (LIMA et al., 2006).
As acetogeninas das Anonaceas vem demonstrando um grande efeito leishmanicida
contra diversas espécies de Leishmania, e se destacam em quantidade e em variedade.
Das raízes da A. muricata foi isolada a cis-solamina A, acetogenina possuidora
de um anel mono-tetra-hidrofurano e que demonstrou atividade inibitória do complexo
mitocondrial I (KONNO et al., 2008). Também isoladas das raízes descam-se as
cohibinas A e B, e sabadelina todas com ação antipasitária e inibidores do complexo
mitocondrial I (GLEYE et al., 1997; GLEYE et al., 1998 ). Yu et al. (1998) isolaram
das sementes de A. muricata, annonacina A e annonacina. Jaramillo et al. (2000)
isolaram estas mesmas acetogeninas do extrato do pericarpo desta mesma planta, e
comprovou a atividade leishmanicida contra L. panamensis e L. braziliensis. Em outro
estudo, a annonacina foi isolada das sementes deste vegetal e sua atividade
leishmanicida contra L. panamensis foi identificada (ARANGO et al., 2000). Das folhas
desta espécie, Kim et al. (1998) isolaram e identificaram, duas acetogeninas com anel
mono-tetra-hidrofurano, muricoreacina e murihexocina. Calderon et al. (2010),
utilizando extrato bruto desta planta, encontraram atividade leishmanicida contra formas
amastigotas de L. mexicana.
Das sementes de A. squamosa foi isolada uma acetogenina triidroxilada com
dois anéis tetraidrofurânicos e anel lactônico ,-insaturada de 37 átomos de carbono
(Figura 3), dotada de propriedades antihelmínticas contra Haemonchus contortus,
principal nematódeo de ovinos e caprinos do Nordeste brasileiro (SOUZA et al., 2008).
Esta substância mostrou ação leishmanicida frente formas promastigotas e amastigotas
de L. chagasi (VILA-NOVA et al., 2011).
31
Figura 3. Acetogenina triidroxilada com dois anéis tetraidrofurânicos e anel
lactônico ,-insaturada de 37 átomos de carbono isolado das sementes de A.
squamosa.
O mecanismo de ação das acetogeninas não está totalmente elucidado. As
acetogeninas que são potentes inibidores do complexo mitocondrial I são inibidoras da
NADH ubiquinona oxidorredutase, enzima essencial no complexo I, esta inibição induz
uma fosforilação oxidativa na mitocôndria da célula (BERMEJO et al., 2005;
GRANDIC et al., 2004). Estudo na literatura demonstrou que estas substâncias agem
diretamente no sítio catalítico da ubiquinona, dentro do complexo I e na glicose
desidrogenase microbiana. Elas inibem também a NADH oxidase ligada a ubiquinona,
peculiar às membranas plasmáticas das células cancerígenas (BERMEJO et al., 2005).
Mesmo que o complexo mitocondrial não tenha sido caracterizado na Leishmania,
estudos sugerem que os complexos mitocondriais I, II, III e IV estão presentes estão
presente na via de transferência de elétrons e que a fosforilação oxidativa seja
processada com alta eficiência bioenergética (GRANDIC et al., 2004).
2.4.2.2 Alcalóides
Alcalóides são compostos nitrogenados orgânicos de natureza complexa
derivados de aminoácidos que sofrem descarboxilação e normalmente possuem ações
biológicas variadas. Os principais aminoácidos precursores dos alcalóides são a L-
lisina, L-ornitina, L-tirosina, L-triptofano, L-histidina, L-fenilalanina bem como ácido
nicotínico e ácido antranílico (ANISZEWSKI, 2007). Blocos construtores das vias
acetato e chiquimato são também incorporados à estrutura dos alcalóides. Há ainda um
grande número de alcalóides que adquirem o seu nitrogênio via reação de
transaminação, incorporando somente o átomo de nitrogênio do aminoácido. O termo
pseudo-alcalóide é muitas vezes usado para distinguir este grupo (DEWICK, 2002). Os
alcalóides comuns em Annonaceae são do tipo isoquinolinico e benzilisoquinolínico
que são biossinteticamente derivados da tirosina.
32
Os alcalóides constituem a classe de metabólitos que possuem o maior número
de compostos com atividade leishmanicida, e também compreendem a maior classe de
metabólitos secundários provenientes de plantas. Estes metabólitos desempenham um
importante papel na defesa contra vários microorganismos, possuindo uma notável
variedade de atividades famacológicas (Calderon et al, 2009). Entre as atividades dos
alcalóides se encontram anti-tuberculose (KISHORE et al., 2009), anti-câncer (MIN et
al., 2010), contra estágios larvares de nematódeos (SATOU et al., 2002), moluscicida
(BAGALWA et al., 2010) e anti-bacteriana (MANEERAT et al., 2012). Estudos
recentes apontam estes produtos como potenciais fontes de drogas contra leishmaniose.
Diversos alcalóides são relatados com excelentes atividades leishmanicidas, no entanto
nenhum deles vem sendo avaliado em estudos clínicos ou são projetados para uma
futura aplicação clínica (MISHRA et al., 2009).
Bhakuni et al. (1972) isolaram da casca do tronco e das sementes da A.
squamosa o alcaloide anonaina (Figura 4), na qual Queiroz et al. (1996) demonstraram
atividade leishmanicida contra L. donovani e L. amazonenses. Bhakuni et al. (1979)
isolaram das folhas deste mesmo vegetal alcalóides xilopina e O-metilarmeparvina
(Figura 4). A ação leishmanicida, contra L. mexicana e L. pananmensis da xilopina foi
descrita por Montenegro et al. (2003); a ação leishmanicida contra L. chagasi do O-
metilarmeparvina foi descrito por Vila-Nova et al. (2011).
33
O
ONH
I
O
O
N
HH
MeO
R
II
III
Figura 4. Estrutura química da (I) Anonaina, (II) Xilopina e (III) O-metilarmeparvina.
O mecanismo de ação dos alcalóides não está totalmente esclarecido, porém
Fournet et al. (2000) observaram que alcalóides bisbenzilisoquinolínicos inibem uma
enzima antioxidante essencial na Leishmania, a Tripanotione redutase. Chan-Bacan e
Pena-Rodriguez (2001) propõem que o mecanismo de ação dos alcalóides indólicos se
dá pela inibição da cadeia respiratória do parasito, e Mishra et al. (2009) propõem que,
devido a estrutura dos alcalóides Naftil-isoquinolinicos ser parecida com a miltefosine,
uma morte por apoptose seria um possível mecanismo de ação.
2.4.3 – Dimorphandra gardneriana
A D. gardneriana popularmente conhecida como faveira ou fava D’anta (Figura
5) é uma planta típica do cerrado e da caatinga brasileira, é encontrado no Ceará e na
Chapada do Araripe (PIRES et al., 2010). Esta espécie é uma grande produtora de
rutina, um flavonóide com diversas atividades farmacológicas e de grande exportação
pelo Brasil (CUNHA et al., 2009).
34
Figura 5. A planta e sementes de Dimorphandra gardneriana. Fonte: Ícaro, 2003
2.4.3.1 – Quercetina
A quercetina (Figura 6) é um destacado integrante do grupo de flavonóides e por
sua ação antioxidante o seu desempenho terapêutico têm sido mencionado por vários
autores no combate ao estresse oxidativo. O seu efeito farmacológico se deve à sua ação
na inibição de certas enzimas e sua capacidade antioxidante (MILTERSTEINER et al.,
2003), esta também age interagindo com DNA topoisomerase (SEN et al., 2006). Entre
as várias atividades farmacológicas destacam-se a anti-câncer e sensibilização de células
cancerígenas resistentes aos tratamentos tradicionais (BORSKA et al., 2012); anti-
inflamatória (LIN et al., 2012) e anti-viral (SAVOV et al., 2006). Estudos in vitro têm
mostrado que a quercetina e outros flavonóides inibem fortemente a produção de Óxido
Nítrico e do Fator de Necrose Tumoral pelas células de Kupffer quando estimuladas
pela injúria. Os flavonóides, por combaterem diretamente as espécies ativas de oxigênio
ou aumentar a capacidade de reação hepática aos mesmos, poderiam contribuir para a
redução do dano oxidativo hepático e da formação de fibrose causada pela obstrução
biliar (MILTERSTEINER et al, 2003).
35
OH
OH
O
OH
O
HO
OH
Figura 6. Representação da estrutura molecular da quercetina.
A quercetina pode ser encontrada em diversas plantas, no entando ainda há
carência em estudos avaliando as atividades leishmanicida deste flavonóide isolado
ainda é carente. Muzitano et al. (2006), observaram que a quercetina isolada da
Kalanchoe pinnata, planta vulgarmente conhecida como saião, exibiu atividade
leishmanicida contra L. amazonenses. A inibição da arginase pela quercetina pode ser
um importante mecanismo de ação contra diversas espécies de Leishmania, pois a
arginase sintetiza arginina em ornitina e uréia, e a ornitina é essencial na proliferação
celular (SILVA et al., 2012).
2.4.3.2 – Rutina
A rutina (Figura 7) é um flavonoide glicosídico pertencente a uma importante
classe de flavonóides, sendo extensamente encontradas na natureza. Apresenta uma
importância terapêutica em virtude de determinar a normalização da resistência e
permeabilidade das paredes dos vasos capilares, além de inibir o processo de formação
de radicais livres em vários estágios (PATHAK et al., 1991).
Entre as importâncias terapêuticas da rutina estão: anti-alérgico (SHEN et al.,
2012); pró-carcinogênico, reduzindo os danos ao DNA em células hepáticas
(MARCARINI et al., 2011); antioxidante (KIM et al., 2011) e anti-inflamatória
(SELLOUM et al., 2003).
Este flavonóide inibe o processo de formação de radicais livre em vários
estágios, por reagir com o íon superóxido e radicais peroxilas lipídicos, e por formar um
complexo com o ferro que cataliza a formação de radicais de oxigênio ativo (PATHAK
et al., 1991), e não é tóxico (AFANASE’EV et al., 1989). A ação anti-inflamatória deste
flavonóide se dá pela capacidade de inibição de enzimas envolvidas no processo da
36
sinalização inflamatória, como a cicloxigenase e a lipoxigenase (SELLOUM et al.,
2003).
Figura 7. Representação da estrutura molecular da Rutina.
Estudos na procura de novas vacinas contra a leishmaniose visceral vêm
utilizando a rutina como adjuvante em sua composição com resultados promissores
(OLIVEIRA-FREITAS et al., 2006).
2.4.4 – Platymiscium floribundum
O P. floribundum é uma planta comum no nordeste brasileiro e popularmente
conhecido como sacambu ou jacarandá do litoral (Figura 8). Esta espécie é utilizada
pela população como anti-inflamatório e sua madeira possui valor comercial na
indústria de móveis (FALCÃO et al., 2005).
Observando a importância fitoquímica deste vegetal destaca-se a presença de
isoflavonóides com potencial farmacológico e atividades biológicas antivirais,
antifúngicas e anti-câncer (MILITÃO et al., 2006; FALCÃO et al., 2005; VALLILO et
al., 2007).
O estudo químico desta espécie revelou a presença de flavonoides, isoflavonóides
e cumarinas como principais constituintes, sendo a segunda classe com maior número
de compostos as cumarinas, com um total de dez compostos identificados (FALCÃO,
2003).
37
Figura 8. As flores e planta Platymisciym floribundum. Fonte: Falcão, 2003
2.4.4.1 Cumarinas
Cumarinas fazem parte de um grupo de fenóis naturais encontrados em diversos
vegetais como frutas cítricas, tomates, legumes e chá verde (BILGIN et al., 2011).
Centenas de cumarinas já foram isoladas de plantas e algumas delas são sintetizadas em
laboratório (HOULT; PAYÁ, 1996; MANHAS et al., 2006). Essa classe de fenóis
possui uma variedade de funções com potencial terapêutico para diversas doenças
(KONTOGIORGIS et al., 2012), entre essas propriedades se destacam as atividades
anti-câncer (KIM et al., 2012), hepatoprotetor (BILGIN et al., 2011), anti-HIV
(HUANG et al., 2005), antibacteriana (JAISWAL et al., 2012) e hipotensora (GILANI
et al., 2000).
Diversas cumarinas apresentam atividade leishmanicida, Iranshahi et al. (2007)
isolaram duas cumarinas sesquiterpênicas e comprovaram suas atividades contra L.
major, também com atividade contra esta mesma espécie de Leishmania, auraptene
(Figura 9) isolada da Esenbeckia febrífuga (NAPOLITANO et al., 2004), Ahua et al.
(2004) isolaram das raízes da Thamnosma rhodesicca, oito furanocumarinas e uma
cumarina e demonstraram sua atividade contra L. major. A atividade leshmanicida in
vivo foi evidenciada por Tiuman et al. (2012) que analisaram a ação da cumaria
mammea A/BB em tratamentos oral e tópico em camundongos BALB/c infectados com
L. amazonenses, dados semelhantes ao de Ferreira et al. (2010) que isolaram e
confirmaram a atividade de duas cumarinas da casca da Helietta apicupata contra esta
mesma espécie de Leishmania.
38
Figura 9. Estrutura química auraptene.
A escoparona (6,7-dimetoxicumarina) (Figura 10) é uma cumarina com ação
anti-alégica (CHOI; YAN, 2009), indutora de liberação de dopamina (YANG et al.,
2010), protetora de mucosa gástrica (CHOI et al., 2012), anti-asmática (FANG et al.,
2003), no entanto ocorre uma falta de pesquisas sobre a atividade leishmanicida desta
cumarina.
Figura 10. Estrutura química da escoparona.
2.4.5 - Componentes de óleos essenciais - Monoterpenóides e
fenilpropanóides
2.4.5.1 – Eugenol
O eugenol (Figura 11) é um fenol fenilpropanóide metoxilado
farmacologicamente muito ativo, abundantemente encontrado no óleo essencial de
Eugenia caryophyllus (cravo-da-índia) (JIROVETZ et al., 2006), em óleos essenciais
de algumas plantas do Nordeste brasileiro como Dicipelium cariophyllatum (o craveiro
do Maranhão ou cravinho), Croton zenhtneri (canela-da-cunha) e Ocimum gratissimum
(alfavaca) (CRAVEIRO et al., 1981; UEDA-NAKAMURA et al., 2006).
39
OH
OCH3
Figura 11. Representação da estrutura molecular do Eugenol.
Devido a sua estrutura molecular complexa, o eugenol possui diversas ações
farmacológicas comprovadas como antifúngico (NAIR et al., 2012); anti-helmíntico
(PESSOA et al., 2002); inseticida (MACIEL et al., 2010); também é utilizado em
práticas odontológicas como anti-séptico tópico, analgésico, e conferir propriedades
farmacológicas aos cimentos obturadores de canais, visto ter ação bactericida, portanto,
eficaz no tratamento de algumas enfermidades infecciosas na cavidade bucal
(MARKOWITZ et al., 1992; ESCOBAR, 2002). O óleo essencial de Ocimum
gratissimum rico em eugenol mostrou atividade leishmanicida contra L. amazonensis,
causando alterações mitocondriais como inchaço, desorganização da membrana interna
e aumento do número de cristais (UEDA-NAKAMURA et al., 2006), assim como o
eugenol puro que também possui ação leishmanicida (ARANGO et al., 2012).
O eugenol apresenta uma atividade inibitória de cicloxigenases e
prostaglandinas, enzimas envolvidas em processos inflamatórios e carcinogênicos, e
também tem a capacidade de induzir a lise celular devido ao aumento da perda de
proteínas e lipídios causados pelo dano à membrana celular (KAMATOU et al., 2012).
2.4.5.2 – Timol
O timol (figura 12) é um fenol monoterpenóide encontrado em diversas plantas
como Thymus eriocalyx e Thymus x-porlock. No nordeste brasileiro é encontrado
principalmente no óleo essencial de Lippia sidoides. É irritante da mucosa gástrica e a
gordura ou o álcool aumentam sua absorção (BOTELHO et al., 2007).
Entre suas atividades terapêuticas se destacam a ação antimicrobiana (GUARDA
et al., 2011), anti-inflamatória e cicatrizante (RIELLA et al., 2012), fungicida (AHMAD
et al., 2010), antioxidante (UNDERGER et al., 2009) e apresenta ação inotrópica
40
negativa devido a redução de cálcio no sarcoplasma do retículo devido a sua
combinação na indução de liberação de cálcio e inibição da bomba de cálcio
(SZENTANDRÁSSY et al., 2004).
OH
Figura 12. Representação da estrutura molecular do Timol.
Robledo et al. (2005) validaram a atividade leishmanicida in vitro e in vivo do
timol e de seus derivados estruturais contra Leishmania panamensis, em um outro
estudo Medeiros et al. (2011) observaram o ação inibitória do óleo de Lippia sidoides
contra L. amazonensis.
2.4.5.3 Derivados Sintéticos do Eugenol e Timol
O timol é um derivado do p-cymene onde diversos derivados possuem atividade
leishmanicida. No estudo realizado por Robledo et al. (2005), o timol teve sua estrutura
quimicamente modificada e seus derivados foram avaliados contra as formas
promastigota e amastigota de L. panamensis, comprovando a atividade inibitória dos
derivados do timol. O eugenol é um fenilproponóide com sua atividade leishmanicida
amplamente estudada. Arango et al. (2012) sintetizaram e avaliaram a ação contra L.
panamensis de seis híbridos do eugenol, encontrando atividade inibitória melhor que o
eugenol em três dos seis híbridos estudados.
Assim, supõe-se que derivados do timol e eugenol possam ter atividade
leishmanicida melhor que a substância pura, então a obtenção de derivados pelos
processos de benzoilação (Fig. 13) e acetilação (Fig. 14) poderá possibilitar melhores
atividades quimioterápicas e menor toxicidade.
41
(a)
(b)
Figura 13. Processos de (a) Acetilação Eugenol e (b) Acetilação Timol
(a)
(b)
Figura 14. Processos de (a) Benzoilação Eugenol e (b) Benzolição timol.
42
3 JUSTIFICATIVA
A leishmaniose visceral é uma zoonose que afeta principalmente cães
domésticos, silvestres e o homem, no entanto não existe um tratamento efetivo ou
vacina que cure ou previna com seguraça esta enfermidade. Além disso, o uso rotineiro
de drogas antimoniais, utilizadas no tratamento em humanos e em cães, induz à
remissão temporária dos sinais clínicos, não previne a ocorrência de recidivas, tem
efeito limitado na infectividade de flebotomíneos e leva ao risco de selecionar parasitos
resistentes às drogas utilizadas para o tratamento humano. Desta forma, o tratamento da
leishmaniose visceral canina ainda constitui um desafio para a ciência, estimulando
diversos grupos de pesquisa à descoberta de novos medicamentos mais efetivos e menos
tóxicos. Diversos estudos já validaram o efeito de produtos naturais como potenciais
fontes de novos e seletivos agentes para o tratamento de doenças tropicais causados por
protozoários e outros parasitas. Desta forma este estudo foi realizado com o intuito de
comprovar o efeito leishmanicida de alcalóides e acetogeninas isolados das sementes e
folhas espécies de A. muricata e A. squamosa, respectivamente, cumarinas do caule e
cerne da espécie P. floribundum, flavonóides das sementes da espécie D. gardneriana, e
do eugenol, timol e seus derivados sintéticos.
43
4 HIPÓTESE
Produtos naturais como alcalóides e acetogeninas isolados de espécies de
Anonna muricata e Annona squamosa, cumarinas da espécie Platymiscium floribundum,
flavonóides da espécie Dimorphandra gardneriana, bem como componentes de óleos
essenciais como eugenol, timol e derivados sintéticos possuem efeito leishmanicida.
44
5 OBJETIVOS
5.1 Objetivos Gerais
Avaliar o efeito in vitro e in vivo de compostos isolados da Annona
muricata, Annona squamosa, Platymiscium floribundum, Dimorphandra
gardneriana e monoterpenóides e fenilpropanóides como o eugenol e timol,
contra as formas infectantes de L. i. chagasi e contra as formas
promastigotas de L. major, L. mexicana e L. donovani.
5.2 Objetivos Específicos
Obter compostos das plantas A. muricata, A. squamosa, P. floribundum, D.
gardneriana
Síntetizar os derivados do timol e eugenol
Realizar screening in vitro utilizando promastigotas e amastigotas de
Leishmania spp. com compostos orgânicos naturais
Avaliar in vivo os compostos que mostraram ação in vitro
Analisar a toxicidade dos compsotos ativos e suas combinações em
macrófagos peritoniais murinos e Artemia salina
Avaliar in vivo a toxicidade aguda em camundongos Swiss.
45
6 CAPÍTULO 1
Atividade leishmanicida e citotoxicidade de constituintes químicos de duas espécies
de Annonaceae cultivadas no nordeste do Brasil
(Leishmanicidal activity and citotoxicity of compounds from two Annonacea species
cultivated in Northeastern Brazil)
Periódico: Revista da Sociedade Brasileira de Medicina Tropical, v. 44, p. 567-571,
2011.
46
RESUMO
A leishmaniose visceral e uma enfermidade endêmica em 88 países, com um total de 12
milhões de pessoas infectadas e 350 milhões em risco. Na procura de novos agentes
com ação leishmanicida, alcalóides e acetogeninas isoladas de Annona squamosa e
Annona muricata,respectivamente, foram testados contra as formas promastigotas e
amastigotas de Leishmania chagasi. Foram preparados extratos com metanol: água (80:
20) das folhas de A. squamosa e sementes de A. muricata que foram extraídos com
solução de ácido fosfórico 10% e solventes orgânicos, para obter extratos ricos em
alcalóides e acetogeninas. Estes extratos foram cromatografados em coluna de sílica gel
sendo eluídos com solventes de diferentes polaridades para o isolamento dos
constituintes, e feita a determinação estrutural por análise espectroscópica. Os
constituintes isolados foram testados contra Leishmania chagasi, responsável pela
leishmaniose visceral, utilizando o teste MTT. Testes de toxicidade foram realizados em
todos os compostos isolados, sendo utilizadas células RAW 264.7. Um alcalóide
benzilisoquinolínico, O-metilarmepavina, e uma C37-triidroxi-acetogenina com anel
bistetrahidrofurânico adjacente foram isolados da A. squamosa e duas acetogeninas
annonacinona e corossolona da A. muricata. O alcalóide mostrou um índice de
Concentração Efetiva mínima (CE50) de 23,3 μg/mL e as acetogeninas apresentaram
CE50 variando entre 25,9 a 37,6μg/mL contra promastigotas, e no ensaio de amastigotas,
o CE50 valores variaram entre 13,5 a 28,7 μg/mL. A toxicidade mostrou resultados que
variaram entre 43,5 a 79,9μg/mL. Estes resultados caracterizam A. squamosa e A.
muricata como fontes potenciais de agentes leishmanicidas.
47
Atividade leishmanicida e citotoxicidade de constituintes
químicos de duas espécies de Annonaceae cultivadas no Nordeste do
Brasil
Leishmanicidal activity and citotoxicity of compounds from two Annonacea
species cultivated in Northeastern Brazil
Leishmanicidal activity from compounds isolated from Annonacea
Nadja Soares Vila-Nova1, Selene Maia de Morais
1,2*, Maria José Cajazeiras Falcão
2,
Lyeghyna Karla Andrade Machado2, Cláudia Maria Leal Beviláqua
1, Igor Rafael Sousa
Costa2, Nilce Viana Gramosa Pompeu de Sousa Brasil
3, Heitor Franco de Andrade
Júnior4
1Programa de Pós-Graduação em Ciências Veterinárias- Faculdade de Medicina
Veterinária, Universidade Estadual do Ceará; Avenida Paranjana 1700, Campus do
Itaperi, 60740-000, Fortaleza, Ceará, Brazil
2Curso de Química – Centro de Ciências e Tecnologia, Universidade Estadual do Ceará;
Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará, Brazil
3Departamento de Química Orgânica e Inorgânica, Centro de Ciências – UFC, Caixa
Postal 12.200. 60455-760, Fortaleza, Ceará, Brazil.
4Instituto de Medicina Tropical, Laboratorio de Protozoologia, Universidade de São
Paulo; Av. Dr. E. C. Aguiar 470, 05403-000, SP, São Paulo, Brazil
Address to: Selene Maia de Morais, Rua Ana Bilhar no 601, Apto. 400, Meireles, CEP
60160-110, Fortaleza, Ceará, Brasil
Phone: 85 32426811 / 31019933
e-mail: [email protected]
48
ABSTRACT
Introduction: Visceral leishmaniasis is endemic in 88 countries, with a total of 12
million people infected and 350 million at risk. In the search for new leishmanicidal
agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds
of Annona muricata were tested against promastigote and amastigote forms of
Leishmania chagasi. Methods: Methanol-water (80:20) extracts of A. squamosa leaves
and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to
obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed
on a silica gel column and eluted with a mixture of several solvents in crescent order of
polarity. The compounds were identified by spectroscopic analysis. The isolated
compounds were tested against Leishmania chagasi, which is responsible for American
visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated
for all isolated compounds, and for this assay, RAW 264.7 cells were used. Results: O-
methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent
bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins,
annonacinone and corossolone, were isolated from A. muricata. Against promastigotes,
the alkaloid showed an IC50 of 23.3 μg/mL, and the acetogenins showed an IC50 ranging
from 25.9 to 37.6 μg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to
28.7 μg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 μg/mL.
Conclusions: These results characterize A. squamosa and A. muricata as potential
sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural
compounds and an important tool in the search for new leishmanicidal therapies.
Keywords: Leishmaniasis. Benzylisoquinolinic alkaloids. Acetogenins. Annona
squamosa. Annona muricata.
49
1. INTRODUCTION
Leishmaniasis is a tropical zoonotic disease caused by at least 17 protozoa species
of the Leishmania genus1. The forms of the disease are related to the type of parasite
and differ in geographic distribution, host and vector involved, incidence rate, and
mortality2. Visceral leishmaniasis is endemic in 88 countries, with prevalence of 12
million people, causing 500.000 cases a year, besides those cases of asymptomatic
individuals which were not diagnosed3,4
.
The chemotherapy of leishmaniasis is based on the use of toxic heavy metal-based
compounds, particularly pentavalent antimonials. However, these compounds must be
administered over prolonged periods and are often associated with serious side effects,
including cardiotoxicity, pancreatitis and musculoskeletal affections when, used at
therapeutic doses. Other treatments for leishmaniasis, such as amphotericin B and
pentamidine, are associated with multiple adverse side effects such as bone marrow
suppression, renal toxicity and glucose metabolism disturbances1,5,6
.
Plants that are traditionally used for treatment of several diseases caused by
protozoa are attracting attention in tests against different Leishmania species.
Leishmanicidal acetogenins and alkaloids from Annonaceae species have demonstrated
the great potential of this plant family as a source of leishmanicidal agents5,7
.
In Northestern Brazil two species of Annonacea are largely cultivated due to
edible characteristics of edibility and high amount of waste material for the pulp
industry and other markets. To make use of this discharged material, this study aimed to
evaluate, in vitro, the efectiveness of constituents from A. squamosa leaves and A.
muricata seeds against the promastigote and amastigote forms of Leishmania (L)
chagasi.
2. MATERIAL AND METHODS
Plant Materials
Leaves of A. squamosa and A. muricata were collected from the Ceará State University
campus in Fortaleza, Ceará State, Brazil. The aerial parts of the plants were deposited in
the Prisco Bezerra Herbarium under reference number 43,604 and 43,951, respectively.
50
Isolation of compounds and spectroscopic identification
The plant materials (2kg) were powdered, air-dried, immersed in a methanol-H2O
solution (80:20, 1.5 l), and left for 7 days at room temperature. After this period, the
solvent was eliminated using a rotative evaporator, leaving the crude extract (CE). Part
of the CE
was dissolved with 10% phosphoric acid and then the aqueous acid mixture was washed
with dichloromethane. The organic phase was evaporated to dryness to obtain an
acetogenin-rich extract (ACE, 82 g). The ACE was submitted to silica gel column
chromatography, being eluted with hexane, dichloromethane, ethyl acetate, and
methanol in mixtures of increasing polarity. The fractions were collected and compared
in thin layer chromatographic (TLC) plates sprayed with Kedde´s reagent to reveal the
acetogenins. Ammonium hydroxide was added to the aqueous acid solution until pH 9,
after which the solution was partitioned with dichloromethane. Dragendorff ’s reagent
was used until a negative reaction was seen. The dichloromethane phase was dried over
sodium sulfate and concentrated under reduced pressure until complete dryness to
obtain the total alkaloid extract (AE, 0.58g). This extract was submitted to the same
silica gel column chromatographic treatment as above, using Dragendorff´s reagent for
spraying the TLC plates. The chemical structures of the isolated compounds were
determined by spectroscopic analysis of infrared spectra, recorded on a PerkinElmer
100 FT-IR spectrophotometer; the values were expressed in cm-1
, and the nuclear
magnetic resonance spectra were recorded on a Bruker Avance DRX-500 spectrometer
in CDCl3.
Parasites
Leishmania (L.) chagasi (M6445 strain) promastigotes were cultured in M199 medium
supplemented with 10% fetal bovine serum and 5% human male urine at 24 °C. RAW
264.7 murine macrophages (ATCC TIB-71) were maintained in RPMI-1640 medium
supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 humidified incubator
and seeded for 24 h at 4.105 cells per well in 96-well plates before infection with L.
chagasi promastigotes. The amastigotes were obtained from RAW 264.7 murine
macrophage cells infected with promastigote at a ratio of 1:10
51
(macrophage/promastigote), kept in a 5% CO2 humidified incubator for 72 h at 37 °C,
and then analyzed under a light microscope to confirm infection.
Leishmanicidal activity
Test against promastigotes: to determine the 50% effective concentration (EC50 value)
of the compounds against L. chagasi promastigotes, all compounds were dissolved
previously in ethanol at a concentration of 0.2% and diluted with M199 medium in 96-
well microplates. The assay was performed at concentrations of 100, 50, 25, 12.5, and
6.25 μg/mL and controls with ethanol and without drugs were performed. Each
concentration was tested nine times. Promastigotes were counted in a Neubauer
haemocytometer and seeded at 1x106 cells per well for a final volume of 150 μl. The
plates were incubated at 24 °C for 24 h, and the viability of the promastigotes was
assessed by morphological observation under a light microscope. Diphenyltetrazolium
(MTT) assay was performed; initially, MTT (5 mg/mL) was dissolved in PBS and
sterilized through 0.22-μm membranes, and then 20μl/well was added to a 96-well plate
and left at 37 °C for 4 h. Promastigotes were incubated without compounds and used as
viability control. Formazan extraction was performed using 10% SDS (100 mL/well) at
24 °C for 18 h, and the optical density (OD) at 570 nm was determined in a Multiskan
MS spectrophotometer (UniScience). Pentamidine (Itaca Laboratorios Ltda, Rio de
Janeiro, Brazil) was used as standard drug.
Test against amastigotes: To determine the EC50 value of the compounds against L.
chagasi amastigotes, all compounds were dissolved in 0.2% ethanol at concentrations of
100, 50, 25, 12.5, and 6.2 5μg/mL and added to microplates containing a confluent layer
of cells with amastigotes for 48 h at 37 °C in a 5% CO2 humified incubator. Glucantime
was used as standard drug, and macrophages incubated without drugs were used as
control. For the in vitro assay, an adapted methodology from Piazza et al.8 was used.
The RAW 264.7 cells were incubated with 0.01% saponin in PBSA containing 1%
bovine serum albumin for 30 min. After blocking the wells for 30 min with 5% defatted
milk (Nestle) in PBSA, the cells were incubated at 37 °C for 1 h with serum from a
rabbit immunized with saline extract of L. chagasi promastigotes, collected 30 days
after the infection. The serum employed was diluted at 1:500 and pre-absorbed with
10% fetal calf serum (FCS) at 22 °C for 1 h. After washing the wells three times with
52
0.05 % Tween 20 in PBSA, peroxidase-conjugated goat anti-rabbit IgG (Sigma
Chemical Co.), diluted 1:5,000 in 5% defatted milk was added, and the mixture was left
at 37 °C for l h. The wells were washed three times, after which o-phenylenediamine
(0.4 mg/mL) and 0.05% H2O2 were added. The solution was then transferred to ELISA
microplates. The reaction was stopped by adding 1 M HC1, and the plates read at
492nm in a Titertek Multiskan ELISA reader.
Cytotoxicity assay
For the cytotoxicity assay, an adapted methodology from Tempone (2005) was used.
RAW 264.7 murine macrophage cells were seeded at 4x104 cells per well in 96-well
microplates and incubated at 37 °C for 48 h in the presence of the compounds, dissolved
previously in ethanol at a concentration of 0.2% and diluted with M199 medium to the
highest concentration of 120 μg/mL. The microplates were incubated for 48 h at 37 °C
in a 5% CO2 humidified incubator. Control cells were incubated in the presence of
DMSO,
without drugs, Glucantime, and pentamidine (standard drugs). The viability of the
macrophages was determined with the MTT assay, as described above, and was
confirmed by comparing the morphology of the control group via light microscopy.
Statistical analysis
The EC50 values at 95% confidence interval (CI) were calculated using a nonlinear
regression curve. One-way ANOVA and comparative analysis between treatments were
performed by Tukey’s parametric test using the number of living promastigotes and
amastigotes determined indirectly by the optical density (OD, 570 nm), representing the
percentage of survival and/or murine macrophage cells after normalization using the
statistical software GraphPad Prism 4.0.
3. RESULTS
The silica gel column chromatography of the alkaloid extract of A. squamosa leaves led
to the isolation of O-methylarmepavine (I), a benzylisoquinolinic alkaloid; and from the
methanol extract free from alkaloids, a C37 trihydroxy adjacent bistetrahydrofuran
53
acetogenin (II) was isolated (Figure 1). This acetogenin was shown to be identical,
when compared by thin layer chromatography (TLC) and spectroscopic data, with the
acetogenin previously identified in A. squamosa seeds, which showed anthelmintic
activity against Haemonchus contortus, the main nematode in small ruminants in
Northeastern Brazil9. The complete assignment of carbons and hydrogens for the
structure of O-methylarmepavine was performed using one- and two-dimensional NMR
spectral analysis and by comparison with data from previous studies10, 11
.
I II
III, R = H
IV, R = OH
Fig. 1. Chemical structures of leishmanicidal compounds O-methylarmepavine (I) and
C37 trihydroxy adjacent bistetrahydrofuran acetogenin (II) from A. squamosa and
Corossolone (III) and Annonacinone (IV) from A. muricata.
Compound 1 was isolated as a brown solid: m.p.: 49.9 - 50.7 oC; UV (λmax,
MeOH, nm): 225 (log ε 3.19); IR (KBr) δmax 2935, 1612, 1514, 1460, 1250, 1118, 1068,
1033, 833 cm-1
; 1H NMR (CDCl3, 500 MHz) 5.83 (H1), 6.58 (H4, s), 2.2-2.4 (H5, m),
3.10 (H6a, d, 7.0), 3.39 (H6b, t , 7.15), 4.32 (H7a, m), 2.78 (H8, t, 6.4), 7.0 (H9/H13, d,
8.4), 6.79 (H10/H12, d, 8.4), 3.48 (OCH3), 3.82 (OCH3), 3.77 (OCH3), 2.64 (N- CH3)
and 13
C NMR 65.05 (C1), 149.45 (C2), 147.49 (C3), 111.24 (C4), 22.02 (C5), 44.94
(C6), 65.45 (C7a), 40.25 (C8), 114.27 (C9/C13), 131.17 (C10/C12), 159.21 (C11), 122
(C14), 121 (C15), 122 (C16), 55.72 (OCH3), 56.06 (OCH3), 55.86 (OCH3), 40.44 (N-
CH3).
54
Compound 2 was isolated as a viscous oil: UV (λmax, MeOH, nm): 281 (log ε
max 3418, 2927, 2855, 1748, 1652, 1463, 1319, 1118, 1068, 1028, 953,
877, 756, 666 cm-1
; 1H NMR (CDCl3, 500 MHz) 2.29 (H3, t 7.8), 1.56 (H4, m), 3.62
(H5, m), 1.55 (H6, m), 1.28 (H7, m), 1.28 (H8-12, m), 1.28 (C13, m), 1.56 (H14, m),
3.34 (H15, m), 3.84 (H16, m), 1.98 (H17a, m), 1.65 (H17b, m), 1.98 (H18a, m), 1.65
(H18b, m), 3.94 (H19, m), 3.94 (H20, m), 1.98 (H21a, m), 1.65 (H21b, m), 1.98 (H22a,
m), 1.65 (H22b, m), 3.84 (H23, m), 3.43 (H24, m), 1.56 (H25, m), 1.28 (H26, m), 1.28
(H27,31, m), 1.28 (H32, m), 1.28 (H33, m), 0.90 (H34, t 7.0), 6.99 (H35, s), 5.02 (H36,
m), 1.43 (H37, d 6.7) and 13
C NMR (CDCl3, 125 MHz) 173.8 (C1), 134.2 (C2), 25.6
(C3), 37.1 (C4), 71.7 (C5), 37.3 (C6), 24.8 (C7), 28.9-29.6 (C8-12), 25.1 (C13), 33.0
(C14), 74.1 (C15), 83.2* (C16), 27.3 (C17), 28.4 (C18), 82.5
* (C19), 82.1
* (C20), 28.4
(C21), 27.3 (C22), 82.7* (C23), 71.4 (C24), 32.3 (C25), 25.6 (C26), 28.9-29.6 (27-31),
31.8 (C32), 22.5 (C33), 14.0 (C34), 148.9 (C35), 77.4 (C36), 19.1 (C37). *Values are
exchangeable.
The 1H and
13C-NMR spectral data of compounds III and IV (Table 1) indicates the
characteristics of y-lactones mono-tetrahydrofuraniques with a keto group (peak at
211.60 for compound III and at 211.55 for compound IV in 13
C-NMR), differing in
the number of hydroxyl groups. Compound III, which is less polar, shows two
hydroxyls located at C15, 74.15 and at C20, 74.00 in 13
C-NMR spectra, these data
was compared with corossolone, which was previously isolated by Cortes et al.12
Compound IV, with three hydroxyls linked to C15, 74.32; C20, 74.01 and to C4,
70.01 was compared with the structure of annonacinone13
. The structures of
corossolone (III) and annonacinone (IV) are shown in Figure 1.
55
Table 1. 1H (CDCl3, 500 MHz) and
13C-NMR (CDCl3, 125 MHz) chemical shifts
of compounds 3 and 4 isolated from Annona muricata.
Compound/C 3 3 4 4
1H
13C
1H
13C
1 - 174.84 - 174.08
2 - 131.31 - 134.45
3a 2.39 - - -
3b 2.50 33.64 2.26 25.31
4 3.83 70.01 1.52-1.58 27.55
5 1.25-1.29 37.30 1.31 28.97-29.92
6-7 1.25-1.29 29.20-29.92 1.31 28.97-29.92
8 1.55 23.99 1.52-1.58 23.97
9 2.38 42.79 2.37-2.43 42.99
10 - 211.60 - 211.55
11 2.38 42.88 2.37-2.43 42.91
12 1.55 23.84 1.52-1.58 25.35
13 1.25-1.29 25.42 1.31 25.47
14 1.38 33.64 1.38 33.45
15 3.49 74.32 3.40 74.15
16 3.80 82.75 3.79 82.79
17-18 1.64; 1.98 28.96 1.68; 1.98 29.01
19 3.80 82.89 3.79 82.89
20 3.40 74.02 3.40 74.00
21 1.39 33.59 1.38 33.70
22 1.25-1.29 25.78 1.31 25.80
23-29 1.25-1.29 29.20-29.92 1.31 28.97-29.92
30 1.25-1.29 32.11 1.31 32.12
31 1.25-1.29 22.88 1.31 22.89
32 0.87 14.32 0.89 14.32
33 7.18 152.12 6.99 149.15
34 5.05 78.21 4.99 77.62
35 1.39 19.29 1.38 19.42
56
In the search for new drugs with leishmanicidal activity, A. squamosa and A.
muricata constituents were tested against L. chagasi promastigotes. In this assay,
pentamidine was used as standard drug and showed an EC50 value of 1.63 μg/mL; the
acetogenin from A. squamosa showed an EC50 value of 26.4 μg/mL, and the alkaloid O-
methylarmepavine showed an EC50 value of 23.3 μg/mL. The assay using annonacinone
and corossolone isolated from A. muricata showed EC50 values of 37.6 and 25.9 μg/mL,
respectively (Table 2).
In the amastigote assay, compounds I and II showed EC50 values of 25.3 and
25.4 μg/mL, respectively, and compounds III and IV showed EC50 values of 13.5 and
28.7 μg/mL, respectively, which were statistically similar. The standard drug used,
pentamidine, showed an EC50 value of 1.60 μg/mL (Table 2).
Table 2. Effect of A. squamosa and A. muricata compounds and standards on extra-
extracellular promastigote, intra-intracellular amastigote forms of Leishmania chagasi
and their cytotoxicity in mammalian cells.
Compounds
*EC50
promastigotes
(µg/mL) (95% CI)
*EC50
amastigotes
(µg/mL) (95% CI)
EC50 Citotoxicity
(µg/mL) (95% CI)
Alkaloid (1) 23.3a (12.3 – 38.7) 25.4
a (6.1 – 105.9) 79.7
a (9.3 – 61.8)
Acetogenin (2) 26.4a (22.6 – 98-5) 25.3
a (22.7 – 28.1) 43.5
b (23 – 129.1)
Acetogenin (3) 25.9a (7.6 – 88.2) 28.7
a ( 6.2 – 67.4) 54
b (28.3 – 119.7)
Acetogenin (4) 37.6a (25.8 – 54.80) 13.5
b (2.1 – 53.6) 59.5
b(9.4 – 88.4)
Pentamidine 1.6b
(0.06 – 63.4) nd** 17.9c(2.4 – 25.8)
Glucantime nd** 17.4b
(0.01 – 169.3) >100d
Different letters in the column show statistical difference between the EC50 values (p<0,05) by Tukey´s
Test
*Values indicate the effective concentration of a compound in µg/mL necessary to achieve 50% growth
inhibition (EC50).
** nd, not determined.
The cytotoxicity of the compounds was determined in RAW 264.7 macrophages
after 48-h incubation. The cytotoxicity of the alkaloid (I) and acetogenin (II) isolated
from A. squamosa, and the acetogenins corossolone (III) and annonacinone (IV) from A.
muricata against RAW 264.7 murine macrophage cells showed values ranging from
57
43.5 to 79.7 μg/mL. The standard drug glucantime showed toxicity to mammalian cells
greater than 100 μg/mL (Table 2).
4. DISCUSSION
Leishmaniasis occurs globally. In particular, visceral leishmaniasis has a major
impact in the Horn of Africa, South Asia, and Brazil, and cutaneous leishmaniasis in
Latin America, Central Asia, and southwestern Asia. The species responsible for
leishmaniasis in Latin America are divided in two taxonomic groups. The first is the
subgenus Viannia, which mainly includes the species L. braziliensis, L. panamensis,
and L. guyanesis, responsible for cutaneous or mucocutaneous lesions. The other is the
Leishmania subgenus, which includes the species L. mexicana and L. amazonensis,
responsible for localized or diffused skin lesions, and L. chagasi, which causes
American visceral leishmaniasis14
.
In this study, an alkaloid and three different acetogenins from two species of
Annonacea plants, Annona squamosa and Annona muricata, were isolated. A. squamosa
leaves contain a benzylisoquinolinic alkaloid, O-methylarmepavine (I), and a C37
trihydroxy adjacent bistetrahydrofuran acetogenin (II). From A. muricata seeds, two
different acetogenins, corossolone (III) and annonacinone (IV) were isolated. These
compounds were screened against Leishmania chagasi promastigote and amastigote
forms, and their cytotoxicities were evaluated.
The leishmanicidal tests against L. chagasi using the alkaloid isolated from A.
squamosa, O-methylarmepavine, revealed lower effectiveness when compared with the
standard drug pentamidine. Tempone et al. tested the total alkaloid and ethanol extract
from eight different Annonacea plants, which produce isoquinoline alkaloids, and
showed effective results in vitro against L. chagasi. The most effective total alkaloid
extract against promastigotes and amastigotes was that from Annona crassiflora.
The alkaloid O-methylarmepavine isolated from A. squamosa is a
benzylisoquinolinic alkaloid. Isoquinoline and benzylisoquinoline analogues are the
main leishmanicidal alkaloid types in the Annonacea family. Benzylisoquinolinic
alkaloids are widely distributed in nature and have been isolated from different plants
commonly used in traditional medicine for the treatment of parasitic diseases.
Bisbenzylisoquinolinic alkaloids isolated from the stem bark of Guatteria boliviana
have also been reported to show moderate activity when tested against promastigotes of
58
L. donovani, L. amazonensis, and L. braziliensis5. Berberine, a quaternary isoquinolinic
alkaloid, has been used in the clinical treatment of leishmaniasis, malaria, and amebiasis
for more than 50 years and has shown in vitro and in vivo response against many
species of Leishmania. This alkaloid, at a concentration of 10 μg/mL, effectively
eliminates L. major parasites in peritoneal mice macrophages15,16
. Another isoquinolinic
alkaloid, isoguattouregidine, isolated from Guatteria foliosa, caused lysis of parasitic
cell membrane when tested against L. donovani and L. amazonensis at a concentration
of 100 μg/ml. Anonaine and liriodenine obtained from the roots and trunk bark of A.
pinescens showed an IC50 value of 100 μg/mL against promastigotes of L. braziliensis,
L. amazonensis, and L. donovani17
. About 20 bisbenzylisoquinoline alkaloids were
screened for antileishmanial and antitrypanosomal activity in vitro; Fangchinoline (EC50
0.39 μM) was found to be as active as the standard drug pentamidine against
Leishmania donovani promastigotes. Based on the above results, the leishmanicidal
action of isoquinoline and benzylisoquinoline against the promastigote forms of
Leishmania spp ranged from 0.39 to 100 μg/mL18
. The mechanism of action of
alkaloids is not completely understood, but Fournet et al.19
observed that
bisbenzylisoquinolinic alkaloids inhibit an essential antioxidant enzyme in Leishmania,
trypanothione reductase.
The acetogenin isolated from seeds of A. muricata, corossolone, showed the best
activity among the three acetogenins used in this study. More than 160 different types of
acetogenins are found in the Annonacea family. From the different species of
Annonacea, 12 containing mono- and bis-THF ring acetogenins were isolated and tested
against promastigotes and amastigotes of L. donovani. The results of this study against
promastigotes showed a range between 2.5 and 47.3 μM. Rollinistatin was the most
effective against amastigotes, with an EC50 value of 2.5 μM. Some acetogenins with one
THF ring, such as senegalene, or two THF rings, such as squamocine, asimicine, and
molvizarine, isolated from seeds of A. senegalensis showed activity against
promastigotes of L. major and L. donovani at 25-100 μg/mL20
.
Nine acetogenins with one or two THF rings were isolated from the seeds of A.
glauca; their activity against L. donovani, L. braziliensis, and L. amazonensis was
evaluated. The mono-THF ring acetogenins annonacin A and goniothalamicin showed
activity against promastigotes, with EC100 values of 10 and 5 μg/mL, respectively21
.
In the amastigote assay using L. chagasi strains, the acetogenins from the two
Annonacea species in the present study showed EC50 values ranging from 13.5 to 28.7
59
μg/mL, indicating the relevance of these compounds in the search for new
leishmanicidal drugs. Regarding cytotoxicity, the alkaloid and all the acetogenins were
more toxic to mammalian cells when compared with the standard drug.
The World Health Organization recommends pentavalent antimonials as first-
choice drugs for leishmaniasis treatment. Although Glucantime® has traditionally been
used to treat leishmaniasis, its mechanisms of action and ability to induce damage in
DNA are still unclear. In the study of Lima et al.22
, the genotoxic activity of this drug
was evaluated in vitro using human lymphocytes, and in the in vivo tests, Swiss mice
received acute treatment with three doses (212.5, 425, and 850 mg/kg) of pentavalent
antimony. While no genotoxic effect was observed in the in vitro tests, the in vivo tests
showed that GlucantimeR induces DNA damage. The results of the authors indicate
Glucantime® as a pro-mutagenic compound that causes damage to DNA after the
reduction of pentavalent
antimony (SbV) into the more toxic trivalent antimony (SbIII) in the antimonial drug
meglumine antimoniate. These results encourage the search for other leishmanicidal
compounds.
The chemotherapy for visceral leishmaniasis has been a great challenge, as the
standard drugs used for treatment are toxic, and some strains are already resistant. Few
studies using natural products against L. chagasi are carried out; therefore, the alkaloid
and acetogenins isolated from A. squamosa leaves and A. muricata seeds are promising
leishmanicidal agents, as they display a similar activity to glucantime in the in vitro
assay.
These plants are largely cultivated in Northeastern Brazil, producing agro-industrial
waste material, which could be used in leishmaniasis phytotherapic treatment.
Nevertheless, the in vitro toxicity indicates the need for in vivo tests for the production
of safe phytotherapics.
ACKNOWLEDGMENTS
We are grateful to CENAUREMN (Northeastern Center for the Application and
Use of Nuclear Magnetic Resonance) of Federal University of Ceará for the NMR
spectra of the compounds. We also thank Dr. Selma M. B. Jerônimo, Dr. Daniella R. A.
Martins and Dr. Gloria R. G. Monteiro of the Biochemistry Laboratory of Federal
60
University of Rio Grande do Norte for technical support in the development of this
work.
CONFLICT OF INTERESTS
The authors declare there is no conflict of Interests in this study.
FINANCIAL SUPPORT
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico
(FUNCAP), Projeto de pesquisa financiado pelo Sistema Único de Saúde (PPSUS).
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annonacin-10-one, isoannonacin, and isoannonacin-10-one. Novel polyketides from
Annona densicoma (Annonaceae). J Org Chem 1989; 54:5418-5412.
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ao Programa de Vigilância e Controle da LVA no Estado de São Paulo. BEPA. Bol
Epidemiol Paul (Online) 2009; 6:4-13.
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Soc Trop Med Hyg 1991; 85:18-21.
16. Aniszewski T. Alkaloids – Secrets of Life. 1st ed. Elsevier. Hardbound, Finland,
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al. Aporphine alkaloids from Guatteria foliosa. J Nat Prod 1994; 57:890-895.
18. Camacho MR, Phillipson JD, Croft SL, Rock P, Marshall SJ, Schiff PL Jr. In
vitro activity of Triclisia patens and some bisbenzylisoquinoline alkaloids against
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19. Fournet A, Rojas A, Ferreira ME, Nakayama H, Torres-de-Ortiz S, Schinini A,
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20. Grandic SR, Fourneau C, Laurens A, Bories C, Hocquemiller R, Loiseau PM. In
vitro antileishmanial activity of acetogenins from Annonaceae. Biomed Pharmacother
2004; 58:388-392.
21. Waechter A, Yaluff G, Inchausti A. Leishmanicidal and trypanocidal activities
of acetogenins isolated from Annona glauca. Phytother Res 1998; 12:541-544.
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22. Lima MIS, Arruda VO, Carneiro Alves EC, Azevedo APS, Monteiro SG,
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2010; 84:327-332.
63
7 CAPÍTULO 2
Atividade inibitória Leishmanicida e Colinesterásica de Compostos Fenólicos de
Dimorphandra gardneriana e Platymiscium fliribunbum, plantas nativas do bioma
Caatinga.
(Leishmanicidal and Cholinesterase Inhibiting Activities of Phenolic Compounds of
Dimorphandra gardneriana and Platymiscium floribundum, native plants from
Caatinga biome.)
Periódico: Pesquisa Veterinária Brasileira, v. 32, p. 1164-1168, 2012.
64
RESUMO
Nos últimos anos, o Ministério da Saúde e da Organização Mundial da Saúde tem
apoiado a investigação em novas tecnologias que possam contribuir para a vigilância,
novos tratamentos e controle da leishmaniose visceral no país. Em vista disso, o
objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a
toxicidade desses compostos contra formas promastigotas e amastigota de Leishmania
infantum chagasi, o principal parasita responsável pela leishmaniose visceral na
América do Sul, e avaliar a sua capacidade de inibir a enzima acetilcolinesterase
(AChE). Um ensaio utilizando cepas luciferase dependente, e um ensaio de ELISA in
situ foram usados para avaliar a viabilidade das formas promastigotas e amastigotas,
respectivamente, após a exposição a estas substâncias. O ensaio colorimétrico MTT foi
realizado para determinar a toxicidade destes compostos na linha celular monocítica de
murino RAW 264.7. Todos os compostos foram testados in vitro para as suas
propriedades anti-colinesterase. A cumarina, escoparona, isolada a partir das hastes do
Platymiscium floribundum e os flavonóides rutina e quercetina foram isolados a partir
das sementes da Dimorphandra gardneriana. Estes compostos foram purificados
utilizando cromatografia em coluna de sílica gel, eluída com misturas de solventes
orgânicos de polaridade crescente, e identificado por análise espectral. Nos ensaios
leishmanicida, os compostos mostraram uma eficácia dose-dependente contra as formas
promastigotas extracelulares, a escoporona mostrou uma EC50 de 21,4 µg/mL, a
quercetina e rutina 26 e 30,3 µg/mL, respectivamente. Os flavonóides apresentaram
resultados comparáveis aos da droga de controle positivo, a anfotericina B, contra as
formas amastigotas com a EC50 de quercetina e rutina de 10,6 e 43,3 µg/mL,
respectivamente. Todos os compostos inibiram a AChE com zonas de inibição variando
de 0,8-0,6 cm indicando um possível mecanismo de acção para a atividade
leishmacicida.
65
Leishmanicidal and Cholinesterase Inhibiting Activities of Phenolic Compounds of
Dimorphandra gardneriana and Platymiscium floribundum, native plants from
Caatinga biome.
Nadja S. Vila-Nova2, Selene M. Morais
2,3*, Maria J.C. Falcão
3, Claudia M.L.
Bevilaqua2, Fernanda C.M. Rondon
2, Mary E. Wilson
4, Icaro G.P. Vieira
5, Heitor F.
Andrade6
2Programa de Pós-Graduação em Ciências Veterinárias- Faculdade de Medicina
Veterinária, Universidade Estadual do Ceará; Avenida Paranjana 1700, Campus do
Itaperi, 60740-000, Fortaleza, Ceará, Brazil
3Curso de Química – Centro de Ciências e Tecnologia, Universidade Estadual do Ceará;
Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará, Brazil
4 Departments of Internal Medicine and Microbiology, University of Iowa and the VA
Medical Center, Iowa City, IA 52242, USA.
5PADETEC-Parque de Desenvolvimento Tecnologico, Universidade Federal do
Ceará, Av. Humberto Monte 2977, Bairro Parquelandia Bloco 310, CEP 60.440-593,
Fortaleza, Ceará, Brazil.
6Laboratório de Protozoologia, Universidade de São Paulo; Avenida Eneas de Carvalho
Aguiar, 470, 05403-000, São Paulo-SP, Brazil
*Corresponding author e-mail: [email protected]
66
Abstract
In recent years, the Brazilian Health Ministry and the World Health
Organization has supported research into new technologies that may contribute to the
surveillance, new treatments and control of visceral leishmaniasis within the country. In
light of this, the aim of this study were to isolate compounds from plants of the Caatinga
biome, and to investigates the toxicity of these compounds against promastigote and
amastigote forms of Leishmania infantum chagasi, the main responsible parasite for
South American visceral leishmaniasis, and evaluate their ability to inhibit
acetylcholinesterase enzyme (AChE). A screen assay using luciferase-expressing
promastigote form and an in situ ELISA assay were used to measure the viability of
promastigote and amastigote forms, respectively, after exposure to these substances.
The MTT colorimetric assay was performed to determine the toxicity of these
compounds in murine monocytic RAW 264.7 cell line. All compounds were tested in
vitro for their anti-cholinesterase properties. A coumarin, scoparone, was isolated from
Platymiscium floribundum stems, and the flavonoids rutin and quercetin were isolated
from Dimorphandra gardneriana beans. These compounds were purified, using silica
gel column chromatography, eluted with organic solvents in mixtures of increasing
polarity, and identified by spectral analysis. In the leishmanicidal assays, the
compounds showed dose-dependent efficacy against the extracellular promastigote
forms, with an EC50 for scoporone of 21.4 µg/mL, quercetin and rutin 26 and 30.3
µg/mL, respectively. The flavonoids presented comparable results to the positive
control drug, amphotericin B, against the amastigote forms with EC50 for quercetin and
rutin of 10.6 and 43.3 µg/mL, respectively. All compounds inhibited AChE with
inhibition zones varying from 0.8 to 0.6, indicating a possible mechanism of action for
leishmacicidal activity.
INDEX TERMS: Leishmaniasis; scoparone; flavonoids; acetylcholinestarase inhibition,
treatment
67
INTRODUCTION
Leishmaniasis is still one of the most neglected diseases in the world. During the
last 10 years, many scientific studies involving this disease has been related to treatment
strategies and led to a reduction in drug prices; however, the morbidity and mortality of
this disease has continued to increase worldwide (WHO 2010). For more than 50 years,
the traditional chemotherapy used to treat leishmaniasis has been based on the use of
pentavalent antimonial drugs (Chan-Bacab & Pena-Rodriguez 2001). However, the
toxicity of these agents and their side effects, along with the development of resistance
and differences in strain sensitivity to these drugs, are challenges that must be overcome
(Carvalho, Arribas & Ferreira 2000, Osório et al. 2007).
Antileishmanial drugs have different mechanisms of action. Antimonial drugs
interfere with bioenergetic process of Leishmania amastigotes, by inhibition of several
proteins (Chan-Bacab & Pena-Rodriguez 2001). Miltefosine
(hexadecylphosphocholine) is thought to perturb the microorganism’s cell membrane by
interfering with the biosynthesis of the glycosyl phosphatidylinositol (GPI) membrane
anchor. Miltefosine may also interfere with leishmania signal transduction (Croft,
Seifert & Duchene 2003). Amphotericin B interacts with sterols present in the
Leishmania membrane, the drug binds with ergosterol damaging the cell permeability,
permitting ions such as K+ to scape (Ordonez-Gutierrez et al. 2007, Filippin & Souza
2006).
Choline is the precursor of phosphatidylcholine, a main component of
Leishmania promastigote membranes (Wassef, Fioretti & Dwyer 1985). Therefore,
inhibition of choline formation may decrease Leishmania survival. This hypothesis can
be tested by using inhibitors of the acetylcholinesterase enzyme (AChE), which
catalyzes the hydrolysis of acetylcholine to choline and acetic acid, as leishmanicidal
compounds. This may identify another mechanism of action for leishmanicidal activity.
As part of the search for new and better drugs with high feasibility and low
toxicity, the Special Programme for Research and Training in Tropical Diseases of the
World Health Organization (WHO) encourages research into plants for the treatment
leishmaniasis (WHO 2009). In recent years, the Brazilian Health Ministry has supported
research into laboratory diagnosis of leishmaniasis in humans and dogs, patient
treatment, evaluation of the effectiveness of control strategies, and the development of
new technologies that may contribute to the surveillance and control of visceral
68
leishmaniasis in the country (Maia-Elkhoury et al. 2008). Several compounds from
plants that have activity against Leishmania species have been reported in the literature
(Chan-Bacab & Pena-Rodriguez 2001). These results encourage the investigation into
other compounds present in the Brazilian flora.
Two plants native to the Caatinga biome of Northeastern Brazil, Platymiscium
floribundum and Dimorphandra gardneriana, were selected for study, contain phenolic
compounds, such as coumarins and flavonoids, with potential leishmanicidal and
anticholinesterase activities, respectively. The aim of this work was to test the
compound, scoparone, a coumarin isolated from P. floribundum, and the two
flavonoids, rutin and quercetin, isolated from D. gardneriana, against L. infantum
chagasi promastigotes and amastigotes and evaluate their ability to inhibit
acetylcholinesterase.
MATERIALS AND METHODS
The heartwood and sapwood from the stems of P. floribundum, and the beans of
D. gardneriana, were collected in the city of Acarape and city of Crato, respectively, in
the state of Ceará, Brazil. The aerial parts from both plants were deposited in the Prisco
Bezerra Herbarium under the numbers 31052 and 32339, respectively.
For column chromatography, silica gel ( 0.063 - 0.200 mm; 70 - 230 mesh) and
Sephadex LH – 20 were used. Thin layer chromatography (TLC) was performed using
silica gel 60 G-F-254 on glass plates. TLC plates were observed under an ultraviolet
lamp (Vilbert Loumart, CN-15 LM model), with two wavelengths (312 and 365 nm).
Then, the plates were sprayed with 2.5% vanilin in perchloric acid in ethanol (1:1),
heated in a 100ºC oven, and, if necessary, saturated in an iodine chamber.
Plant heartwood (800 g) was subjected to cold extraction using the organic
solvents hexane, chloroform and ethanol. The solvents were eliminated using a rotary
evaporator to obtain the, respective extracts. The chloroform fraction was subjected to
silica gel column chromatography and was then eluted with mixtures of hexane,
dichloromethane, ethyl acetate and methanol with increasing polarity. The fractions
were collected and compared by TLC for the presence of a blue spot under UV light.
The chemical structures of the isolated compounds were determined by spectroscopic
analysis of the infrared spectra, recorded on a FT-IR PerkinElmer 1000
69
spectrophotometer. The values are expressed in cm-1
. Nuclear magnetic resonance
spectra were recorded on a Bruker Avance DRX-500 spectrometer, in CDCl3.
The beans of D. gardneriana (150 g) were added to a Soxhlet extractor and were
extracted with hexane, ethyl acetate, methanol and water. The solvents were
concentrated in a rotary evaporator, and hexane (0.38 g), ethyl acetate (4.47
g), methanol (47.53 g), and aqueous (8.49 g) extracts were obtained. After analysis of
the extracts by TLC, the ethyl acetate and methanol extracts were combined, and
dispersed in 200 mL of cold water. After stirring, the mixture was filtered, the resulting
residue was washed with additional 100 mL of water, and after filtration, and the
residue was dried in a 100 º C oven, yielding 18.0 g of a yellow powder. This material
was subjected to column chromatography on silica gel column, and was eluted with
mixtures of hexane, dichloromethane, ethyl acetate and methanol with increasing
polarity. The fractions were collected and compared by TLC. This method resulted in
the purification of 12.42 g of rutin. Spectral data were compared to those found in the
literature (Agrawal & Bansal 1989).
The method of Pulley & Loesecke 1939, adapted by Kratkay & Tandy 1991, was
used to prepare quercetin. A mixture of 0.9 g of rutin and 9.0 mL of a 3% sulfuric
acid solution was added to a 25 mL flask adapted with a reflux condenser. The reaction
mixture was refluxed for 5 hours, cooled to 60 ºC and filtered. The resulting
quercetin was washed with hot water until a neutral pH was reached. The crude
quercetin was dried, and then dissolved in 7.2 mL of hot ethanol solution (78 ºC) at 75
ºGL. After the quercetin was completely dissolved, the solution was filtered and cooled
to 25 ºC for subsequent crystallization. The spectral data were compared to those found
in the literature (Agrawal & Bansal 1989).
A strain of L. infantum chagasi expressing luciferase, called Lic-luc, was
generated with the integrating vector pIR1SAT, provided by Dr. Steve Beverley, as
described (Thalhofer et al. 2010). The promastigote form of Lic-Luc was cultured in
hemoflagellate-modified MEM (HOMEM) medium, supplemented with 10% fetal
bovine serum at 26 °C. For the leishmanicidal assay, an adapted methodology from
Tempone et al. 2005 was used. The compounds were dissolved in DMSO at a
concentration of 0.2% and diluted with HOMEM medium in 96-well microplates. The
assay was performed at concentrations of 100, 50, 25, 12.5 and 6.25 µg/mL. Control
wells contained DMSO or no additives. Each concentration was tested in triplicate in
replicate experiments. Promastigotes were counted in a Neubauer hemocytometer and
70
seeded at 1x106/well. The plates were incubated at 26 °C for 24 h, and the viability of
the promastigotes, based on their morphology, was observed under a light microscope.
Pentamidine (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control
drug. The optical density (OD) was determined in a Fluostar Omega (BMG Labtech) at
620 nm.
The drug activity against amastigote was measured by a modified ELISA
measuring the viability of L. i. chagasi infecting the murine macrophage RAW 264.7
cell line (ATCC TIB-71). Four x 105 RAW 264.7 cells in RPMI-1640, 10% fetal bovine
serum, were added to each well of a 96-well plate, and incubated with L. i. chagasi
promastigotes at a 1:10 (macrophage/promastigote) ratio at 37ºC, 5% CO2 in an
incubator for 72 h. An adapted methodology from Piazza et al. 1994 was used for the in
vitro assay. Stock solutions of all of the compounds were prepared at a concentration of
0.2% in ethanol. An additional five concentrations were obtained by two-fold serial-
dilution. The compound solutions were added to microplates containing the confluent
layer of cells with amastigotes for 48 h at 37°C in a humidified 5% CO2 incubator.
Macrophages incubated without drugs were used as the control and, and amphotericin B
(Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control drug at same
concentrations as the other compounds. Murine macrophage RAW 264.7 cells were
incubated with 0.01% saponin in PBSA, containing 1% bovine serum albumin, for 30
min. The wells were then blocked for 30 min with 5% nonfat milk (Nestlé) in PBS. In
the next step, the cells were incubated in serum from a rabbit immunized with a saline
extract of L. i. chagasi promastigotes, collected 30 days after the infection, at 37°C for 1
h. The serum employed was diluted 1:500 and pre-absorbed with 10% fetal calf serum
(FCS) at 22°C for 1 h. The wells were washed three times with 0.05% Tween-20 in
PBSA, and peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co.) diluted
1:5000 in 5% nonfat milk was added and incubated at 37°C for l h. After the wells were
washed, o-phenylenediamine (0.4 mg/mL) and 0.05% H202 were added. The reaction
was stopped by the addition of 1 M HC1. The plates were read at 492 nm in a Multiskan
MS (UniScience) ELISA reader.
To test the toxicity of compounds for mammalian cells, murine RAW 264.7
macrophages were seeded at 4x104/well in 96-well microplates and incubated at 37 °C
for 48 h in the presence of the compounds at a concentration of 120 µg/mL. These
compounds had previously been dissolved in 0.2% ethanol. The microplates were
incubated for 48 h at 37 °C in a humidified 5% CO2 incubator. Control cells were
71
incubated in the presence of DMSO, without drugs, and with the positive control drugs,
amphotericin B and Pentamidine, at a concentration of 40 µg/mL and 100 µg/mL,
respectively. The viability of the murine RAW 264.7 macrophages cells was determined
using the MTT assay, as described previously by Vila-Nova et al. 2011. MTT at a
concentration of 5mg/mL were added at 20 µg/mL in 96-well microplates and incubated
at 37 ºC for 4 h. Formazan extraction was performed using 10% SDS incubated at 24 ºC
for 18 h. The OD was determined in a Multiskan MS (UniScience) at 570 nm. Light
microscopy was used to observe the morphology of the cells and confirm the viability
of the control group.
Inhibition of the AChE was evaluated by TLC in accordance with the methodology
described by Ellman et al. 1961, later adapted by Rhee et al. 2001. The following
solutions were prepared for this test: (1) 50 mM Tris / HCl, pH 8 (buffer); (2) 1 mM
Ellman's reagent, which was prepared by mixing 5,5'-dithiobis-2-nitrobenzoic acid
(DTNB) and a buffer solution of acetylthiocholine iodide (ATCI); and (3) 1 mM ACTI.
The lyophilized AChE enzyme was diluted in buffer solution (1) to obtain a 1000 U/mL
enzyme solution. The compounds, in 5 μL aliquots dissolved in CHCl3 (4 mg/mL), were
applied to the TLC plates (DC-Alufolien, silica gel 60 F254, 0.2 mm Merck). The plate
was then sprayed with solutions (2) and (3). After 3 min, which is the time necessary for
the solution to completely dry, the plate was sprayed with AChE (3 U/mL). After
approximately 10 min, the appearance of white spots was observed, and the spot
diameters were immediately measured. Physostigmine (Sigma-Aldrich, St. Louis, MO,
USA) was used as a positive control.
The 50% effective concentration (EC50) values at the 95% confidence interval
(95% CI) were calculated using a nonlinear regression curve. One-way ANOVA and
comparative analysis between treatments was performed with Tukey’s parametric post-
test using the numbers of living promastigotes, amastigotes and/or murine RAW 264.7
cells, which were determined indirectly by optical density (OD), to represent the
percentage of surviving cells after normalization. GrhaphPad Prism 4.0 statistical
software was used for analyses.
RESULTS
The 13
C-NMR spectroscopic data of the compound isolated from P. floribundum
revealed the presence of nine sp2 carbons, among them one carbonyl (161.4 δ) and two
methoxyl groups. In the 1H-NMR spectrum, a cis-double bond at 6.23 δ and 7.59 δ (J =
72
9.5 Hz) conjugated with a carbonyl is characteristic of a lactone ring of a coumarin.
Comparison with literature data (Ma et al. 2006) confirmed the structure of 6,7-
dimethoxycoumarin, named scoparone, for the compound isolated from P. floribundum.
The NMR spectral data are shown in Tab. 1.
Table 1 - NMR 1H and
13C data from 6,7-dimethoxycoumarin.
C C H
2( C=O) 161,4 _
4ª 111,4 _
6 146,3 _
7 152,8 _
8ª 150,0 _
CH
3 113,4 6,23 (d, J= 9,5 Hz)
4 143,3 7,59 (d, J= 9,5 Hz)
5 108,0 6,83 (s)
8 100,0 6,78 (s)
CH3
OCH3 56,4 3,90 (s)
OCH3 56,4 3,88 (s)
To determine the 50% effective concentration (EC50) of the phenolic compounds,
promastigotes of L. i. chagasi were incubated with the compound for 24 hours at 24 °C.
Leishmanicidal activity for the extracellular cells was observed for all compounds.
Pentamidine was used as the control and was suitable for comparison with the
compounds. Scoparone had no activity in the amastigote assay, and the flavonoids, rutin
and quercetin, were not significantly different compared to the control, amphotericin B
(Tab. 2).
73
Table 2 - Leishmanicidal activity against L. i. chagasi and acetylcholinesterase
inhibition activities of phenolic compounds scoparone, rutin and quercetin and standard
drugs amphotericin B and Pentamidine.
Compound
(µg/mL)
*EC50 Promastigote
(µg/mL)
*EC50 Amastigote
(µg/mL)
AChE
Inhibition (cm)
Scoparone
21.4b
(16.9 – 27.2)
>100d 0.8
Rutin 30.3a (24.5 – 37.4) 43.35c (16.04 – 117.1) 0.6
Quercetin 26.0a (16.6 – 40.8) 10.64c (1.72 – 65.56) 0.6
Pentamidine 5.2a (0.06 – 403.8) Nd -
Amphotericin B Nd 19.75c (6.3 – 62) -
Physostigmine Nd Nd 0.9
The different letters in the columns show the statistical difference between the EC50
values (p<0.05) by Tukey´s test. 95% CI: 95% confidence interval; Nd: not
determined. *Values indicate the effective concentration of a compound in μg/mL
necessary to achieve 50% growth inhibition (EC50).
The toxicity of compounds for mammalian cells was tested at a drug
concentration that was higher than all EC50 levels for sensitive drugs in Table 2.
Viability was measured using the MTT assay, which measures mitochondrial
respiration. None of the compounds caused a significant decrease from control was
measured with the MTT assay; i.e. none of compounds demonstrated toxicity at the
concentration tested (Fig. 1).
74
No d
rugs
Am
photerici
n B
Pen
tam
idin
e
Sco
parone
Rutin
Quer
cetin
0
20
40
60
80
10087%
80%
100%90%
100% 100%
Via
ble
Cell
s R
AW
264.7
(%
)
Figure - 1. Toxicity of compounds at 120 µg/mL on murine RAW 264.7 macrophage
cells comparing with Anphotericin B and Pentamidine (40 µg/mL and 100 µg/mL,
respectively). P < 0,05.
Scoparone, quercetin, and rutin, at a concentration of 2mg/mL, were tested for
AChE inhibitory activity using Ellman's colorimetric method adapted by Reed on TLC
plates. Scoparone had an inhibition zone of 0.8 cm, which was similar to the inhibition
zone of the control physostigmine (0.9 cm). Quercetin and rutin were less active, with
inhibition zones of 0.6 cm (Tab. 2).
DISCUSSION
The list of compounds available to treat leishmaniasis is short and the expanding
problems of parasite resistance and difficulty of delivering drugs to afflicted
populations, demonstrate a critical need for efficacious and nontoxic drugs against
leishmaniasis. Although plant extracts have been used by traditional healers, the active
compounds are often unknown. In this study screened phenolic compounds as a
coumarin (scoporone) isolated from P. floribundum and two flavonoids, rutin and
quercetin, isolated from D. gardneriana, showed potential leishmanicidal activities (Fig.
1). The data suggested that all 3 compounds exhibit activity against the extracellular
promastigote form of L. i. chagasi, and two of three had activity against the intracellular
amastigote form.
75
The lactone groups present in coumarins are also present in the structures of
Annonaceous acetogenins that show leishmanicidal activity (Vila-Nova et al. 2011).
Annonaceous acetogenins have chains ranging from 37 to 35 carbon atoms, depending
on the species from which they are derived. In this study, scoparone had an EC50 of 21.4
µg/mL (95% CI = 16.9 – 27.2) in the promastigote assay. In the AChE assay, the
inhibition zone of scoparone was 0.8 cm, and was similar to the control, physostigmine
(0.9 cm), implying similar acetylcholinesterase inhibition characteristics. In a previous
study, scoporone, was isolated from Helietta apiculata and tested against three other
Leishmania promastigotes strains (L. amazonensis, L. infantum and L. braziliensis), and
the results showed and EC50 greater than 50 µg/mL (Ferreira et al. 2010). AChE
inhibition was demonstrated for nine coumarins isolated from Ferula szowitsiana, with
inhibition zones ranging from 6-8 mm at a concentration of 4 mg/mL (Santos et al.
2008), presenting results similar to those in this study.
Acetylcholinesterase inactivates acetylcholine by hydrolyzing it, into the acetyl
and choline groups. This enzyme provides a mean of regulating the concentration of the
active neurotransmitters (Soreq & Seidman 2001). Phosphatidylcholine (PC) is the most
abundant phospholipid both in the surface membranes (14.9%) and the whole cell
cytoplasm (51.6%) of Leishmania; however PC synthesis requires acquisition of the
choline precursor from the host (Wassef, Fioretti & Dwyer 1985, Zuffrey & Mamoun
2002). Without sufficient host-derived choline, it is very likely that PC synthesis would
be impaired and the Leishmania plasma membrane would be compromised. The AChE
inhibitory activity of scoporone (0.8 mm) indicates a possible action mechanism by
disrupting the viability of leishmania’s cell membranes. The metabolic pathways
leading to the PC uptake and biosynthesis are likely to play critical roles in parasite
development and survival, and may be a viable target for antileishmanial chemotherapy
(Zufferey & Mamoun 2002). The uptake of choline is highly specific and is inhibited by
choline carrier inhibitors, including the antileishmanial phosphocholine analogs
miltefosine and edelfosine and other choline analogs that have antimalarial and anti-
cancer activities (Zuffrey & Mamoum 2002, Croft, Seifert & Duchene 2003). We
hypothesize that the coumarin and scoparone has mechanism of action acting on the
same pathway as above compounds, resulting in a net negative effects on choline uptake
by the parasite. Since the coumarin may act by the decreasing the amount of available
choline for transport, one would hypothesize that these agents would have synergistic
activity. It is possible that a combination of our compounds with miltefosine would
76
enable lowering the dose, and consequent toxicities, of both drugs.
Quercetin and rutin demonstrated leishmanicidal activity in both the promastigote
and amastigote stages. The activity of these flavonoids was similar to that of
Pentamidine and Amphotericin B, established agents used to treat leishmaniasis. These
results correlate with another study that tested the activity of quercetin isolated from
Galphimia glauca against L. donovani promastigotes, where the activity was evaluated
by counting microscopically the number of live parasites. In that study, quercetin had an
EC50 29.5 µg/mL against L. donovani promastigotes (Camacho et al. 2002). However,
the leishmanicidal activity of 3-O-methyl-quercetin isolated from Chromolaena hirsuta
against L. amazonensis had an EC50 of 87.8 µg/mL (Taleb-Contini et al. 2004).
Furthermore, in a third study, using quercetrin (glicosyl-quercetin) with is an
aminoglycoside, the amastigote form of L. donovani, had an EC50 of 1.0 µg/mL
(Tasdemir et al. 2006).
These flavonoids are able to inhibit acetylcholine hydrolysis and interfere in the
PC due to their low concentration of choline precursor from the host. Nevertheless, the
leishmanicidal activity of these compounds may be related to their ability to chelate iron
(Fe), depriving this essential nutrient from the intracellular amastigote forms (Sen et al.
2008, Fonseca-Silva et al. 2011).
CONCLUSIONS
The data provide evidence that Brazilian Caatinga biome is a rich source of
leishmanicidal compounds. In this study, scoparone, rutin and quercetin have shown
activity Leishmania infantum chagasi promastigote and amastigote forms, and act as
AChE inhibitor. This suggests a new mechanism of action for these leishmanicidal
natural compounds. These observations raise the possibility of further studies using
these compounds in combination with existing clinically approved antileishmanial
compounds to evaluate the synergism.
Acknowledgments - This research was supported by the Brazilian Ministry of Health,
SPU Number 09100213-3.
77
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8 CAPÍTULO 3
Diferentes susceptibilidades de espécies de Leishmania spp. a acetogeninas
anonacinona e corossolona isoladas da Annona muricata, e cumarina escoporona
isolada do Platymiscium floribundum.
(Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata
acetogenins annonacinone and corossolone, and the Platymiscium floribundum
coumarin scoparone.)
Periódico: Experimental Parasitology in press
81
RESUMO
A leishmaniose é uma zoonose que pode se manifestar de forma visceral e cutânea. O
objetivo deste estudo foi buscar novos compostos leishmanicidas de duas plantas
encontradas na região Nordeste do Brasil, Platymiscium floribundum e Annona
muricata, contra três cepas de Leishmania (L. donovani, L. mexicana e L. major). Uma
cumarina, escoparona, e duas acetogeninas, anonacinona e corossolona foram isolados
das plantas estudadas. Todos os compostos indicaram atividade leishmanicida em todas
as cepas testadas. As diferentes espécies de Leishmania indicam sensibilidade distinta
em relação aos compostos testados: L. mexicana foi mais sensível a escoparona seguido
de L. major e L. donovani. As três espécies apresentaram inibição semelhante ao
corossolona e anonacinona. Acetogenina anonacinona indicou atividade leishmanicida
elevada (EC50 = 6,72-8,00 g/mL); corossolone (EC50 = 16,14-18,73 g/mL) e
scoparone (EC50 = 9,11-27,51 g/mL) demonstraram atividade moderada. A toxicidade
foi avaliada usando o ensaio em Artemia salina, e corossolona foi o composto mais
tóxico. Em conclusão, as atividades leishmanicida demonstradas pela cumarina e pelas
acetogeninas neste estudo indicam que estes compostos são excelentes candidatos para
o desenvolvimento de novas drogas leishmanicida.
82
Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata
acetogenins annonacinone and corossolone, and the Platymiscium floribundum
coumarin scoparone.
Short communication
Nadja Soares Vila-Nova1, Selene Maia de Morais
1,2*, Maria José Cajazeiras Falcão
2,
Terezinha Thaize Negreiros Alcantara2, Pablito Augusto Travassos Ferreira
2, Eveline
Solon Barreira Cavalcanti2, Icaro Gusmão Pinto Vieira
3, Cláudio Cabral Campello
1,
Mary Wilson4
1Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do
Ceará; Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará,
Brazil. 2Curso de Química – Centro de Ciências e Tecnologia, Universidade Estadual do Ceara;
Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará, Brazil.
3PADETEC - Parque de Desenvolvimento Tecnologico, Universidade Federal do
Ceará, Av. Humberto Monte 2977, Bairro Parquelandia Bloco 310, CEP 60.440-593,
Fortaleza, Ceará, Brazil.
4Departments of Internal Medicine and Microbiology, University of Iowa and the VA
Medical Center, Iowa City, IA 52242, USA.
*Corresponding author: Tel. +55 85 3101 9789; Fax: +55 85 3101 9933. E-mail
address: [email protected]
83
Abstract
Leishmaniasis is a zoonotic disease that can manifest itself in visceral and
cutaneous form. The aim of this study was to search for new leishmanicidal compounds.
Preliminarily, Artemia salina assay was applied to compounds from two plants found in
Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these
compounds were tested against three Leishmania species (L. donovani, L. mexicana and
L. major). A screening assay using luciferase-expressing promastigote form were used
to measure the viability of promastigote One coumarin, scoparone, isolated from P.
floribundum and two acetogenins, annonacinone and corossolone isolated from A.
muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania
species indicated different susceptibilities in relation to the tested compounds: L.
mexicana was more sensitive to scoparone followed by L. major and L. donovani. The
three species presented similar inhibition to corossolone and annonacinone. Acetogenin
annonacinone (EC50= 6.72 – 8.00 g/mL) indicated high leishmanicidal activity;
corossolone (EC50= 16.14 – 18.73 g/mL) and scoparone (EC50= 9.11 – 27.51 g/mL)
moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and
acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity
demonstrated by the coumarin and acetogenins indicate these compounds for further
studies aiming the development of new leishmanicidal agents.
Keywords: Leishmanicidal, acetogenin, coumarin, Leishmania, Artemia salina
84
1. Introduction
Leishmaniasis is a tropical zoonotic disease caused by more than 20 protozoa
species of Leishmania genus (Singh et al., 2012) manifesting itself in visceral and
cutaneous form. The geographical distribution of leishmaniasis relate to the growth of
sandfly vector, which are dominant in tropical and temperate regions (Roy et al., 2012).
The chemotherapy used in the treatment of leishmaniasis is based on the use of drugs
that are toxic heavy metals, known as antimoniates, among which the most used are
meglumine antimoniate (Glucantime®
) and sodium stibogluconate (Pentostan®). When
this type of treatment is not effective, other medications such as pentamidine and
amphotericin B and its lipossomal formulation, are also used. All these medications are
administrated intravenously and/or intramuscular require clinical supervision or
hospitalization due to the severity of side effects (Chan-Bacab and Pena-Rodriguez,
2001; Rath et al., 2003). In dogs, one of the main reservoirs, treatment is not a
recommended measure in Brazil, since it does not diminish the importance of these
animals as reservoir of the parasite. It only provides clinical remission and not a
parasitological cure (Sessa et al., 1994, Alves and Bevilacqua, 2004; Ait-Oudhia et al.,
2012).
In the early 1990s, the World Health Organization (WHO) reported that 65-80%
of the population of developing countries depended on medicinal plants as the only
form of access to basic health care (Veiga et al., 2005). A vast number of Brazilian
plants are able to produce secondary metabolites with antiparasitic activity; many of
these metabolites have not been properly isolated and chemically or biologically
evaluated. The Annonaceae family is a great producer of acetogenins that has shown
promising results in the search for new drugs against various protozoa, revealing good
potential as a source of agents for the treatment of leishmaniasis (Rocha et al., 2005).
Annona muricata is a well-known plant in Brazil, the compounds there are most found
in this plant are acetogenins and alkaloids (Vila-Nova et al., 2011, Tempone et al.,
2005), and has antiprotozoal, antioxidant and anticancer properties (Rondon et al., 2011;
Boyon et al., 2009; Lima et al., 2011; Chen et al., 2012a). Platymiscium floribundum is
a regional plant in Ceará state used by the population as anti-inflammatory and the
isoflavonoids isolated from this plant have demonstrated activity against cancer cells
(Militão et al., 2006, Falcão et al., 2005), and the crude extract demonstrated antifungal
and acetylcholinesterase activity (Cardoso-Lopes et al., 2008), pterocarpans isolated
from the heartwood demonstrated antimitotic effect on sea urchin eggs (Militão et al.,
85
2005), but a few of studies have been developed in the search of others
chemotherapeutic values as well as isolating others compounds from this plant.
Due to the shortcomings described above, there is a pressing need for new
leishmanicidal treatments, and compounds from Northeastern Brazilian plants can be
promising sources for future drugs.
2. Material and Methods
2.1 Plants
The heartwood and sapwood of the stem of P. floribundum and leaves of A.
muricata were collected in the state of Ceará, Brazil. Aerial parts of the two plants were
deposited in the Prisco Bezerra Herbarium under the reference number 31052 and
43951, respectively.
2.2 Chromatographic procedures
The active chemical constituents were isolated from plant extracts by silica gel
( 0,063 – 0,200 mm; 70-230 mesh) and Sephadex (LH - 20) column chromatography
and solvents for elution were petroleum ether, hexane, chloroform, ethyl acetate,
acetone and methanol from VETEC (Brazil). The fractions collected in the columns
were compared by thin layer chromatography (TLC), using silica gel (60 G F 254) on
glass plates (3 cm x 8 cm). The TLC plates were analyzed in a iodine chamber, under
UV Light (at 312 and 365 nm), Vilbert Loumart, CN-15 LM model, and by spraying the
reagent 2.5 % vaniline in sulfuric acid diluted in ethanol (1:1), followed by heating in an
oven at 100 °C.
2.3. Isolation of compounds and spectroscopic identification
A. muricata seeds (2 kg) were triturated and left in contact with methanol for one
week, then the solvent was filtered, evaporated to dryness and a light yellow solid was
obtained (402 g). This material was chromatographed on a filtering silica gel column
yielding two main compounds. The trunk heartwood of P. floribundum (800 g) was cold
extracted using hexane, chloroform and ethanol successively. Respective extracts were
obtained by elimination of solvents in a rotatory evaporator. The chloroform extract was
introduced on the top of a glass chromatographic column, filled with silica gel, and the
solvents hexane and ethyl acetate, in mixtures of increasing polarity, were used to elute
the column. Fifty-six 10 mL fractions were collected and compared by TLC. This
86
procedure conducted to the isolation of a unique compound, revealed by a simple spot
on TLC plate.
The structures of compounds were determined by spectroscopic analysis of
infrared spectra, recorded on a FT-IR PerkinElmer 1000 spectrophotometer, values
expressed in cm-1
, and nuclear magnetic resonance spectra, recorded on a Bruker
Avance DRX-500 spectrometer in CDCl3.
2.4 Leishmanicidal assay
L. major, L. mexicana and L. donovani mCherry-Luciferase-expressing (mCherry-
Luc) species were generated with the integrating vector pIR1SAT, provided by Dr.
Steve Beverley, as described (Thalhofer et al., 2010). The promastigote forms were
cultured in hemoflagellate-modified MEM (HOMEM) medium, supplemented with
10% fetal bovine serum at 26 °C. For the leishmanicidal assay, an adapted methodology
from Tempone et al. (2005) was used. The compounds were dissolved in DMSO at a
concentration of 0.2 % and diluted with HOMEM medium in 96-well microplates. The
assay was performed at concentrations of 100, 50, 25, 12.5 and 6.25 µg/mL. Control
wells contained DMSO or no additives. Each concentration was tested in triplicate in
replicate experiments. Promastigotes at a logarithmic phase were counted in a Neubauer
hemocytometer and seeded at 1x106/well. The plates were incubated at 26 °C for 24 h,
and the viability of the promastigotes, based on their morphology, was observed under a
light microscope. Pentamidine (Sigma-Aldrich, St. Louis, MO, USA) was used as the
positive control drug. The optical density (OD) was determined in a Fluostar Omega
(BMG Labtech) at 620 nm.
The number of living promastigotes was determined indirectly by the optical
density (OD – 620 nm) representing the percentage of survival. The EC50 values (±SD)
were calculated using a nonlinear regression curve using the statistical software
GhaphPad Prism 4.0.
2.5 Toxicity tests using Artemia salina
A. salina (Artemiidae), also known as brine shrimp larva, is an invertebrate
normally used as a safe, practical and economic substitute assay to determine toxicity of
compounds from natural products (Barbosa et al., 2009). The toxicity tests were
developed using A. salina assay by Vanhaecke et al. (1981) with modifications. Dried
cysts of A. salina (15-20 g in 50 mL of seawater) were incubated in a hatcher (a mini-
87
aquarium with two compartments, one at darkness and another illuminated) at 28–30 °C
with strong aeration, under a continuous light regime during twenty-four hours. A.
salina cysts were placed for hatching in the dark compartment of the aquarium. The
phototropic nauplii that migrated to the illuminated compartment were collected with a
Pasteur pipette and placed in another flask, when then they were used in the toxicity
bioassay. A solution is prepared containing the chemical compound and DMSO (1%
v/v) in seawater. Each test consisted of exposing groups of 30 larvae aged 24 h to
various concentrations of the toxic compound (10000, 1000, 100, 10 and 1 ppm). The
toxicity was determined after 24 h (nauplii in instar II/III) of exposure. Each
concentration was tested in triplicate in replicate experiments. The numbers of survivors
were counted, larvae were considered dead if they did not exhibit any internal or
external movement during several seconds of observation.
3. Statistical analysis
The data were tested for compliance with the requirements for carrying out the
analysis of variance and normal distribution and homogeneity of variances among
treatments. The ANOVA was then performed using the GLM procedure of SAS (2002)
and Tukey's test was used to compare means. For the toxicity assay using A. salina, the
EC50 and its confidence intervals were calculated by Probit analysis using the software
StatPlus 2009 Professional 5.8.4.
4. Results and discussion
The assay of acute toxicity against A. salina has been used in a great diversity of
medicinal plants research and its chemical compounds in order to determine their
pharmacological assessment, including pursuing anticancer agents, which are those with
high toxicity. Barbosa et al. (2009) demonstrated that the A. salina assay was efficient
in the search for antileishmanial substances. The evaluation of plant extract toxicity by
the brine shrimp bioassay, an LC50 value lower than 1000 µg/mL is considered
bioactive (Meyer et al., 1982). This test was used to find active compounds from plants
in our laboratory, as well as to a toxicity screening of the isolated compounds. The
assay demonstrated that A. saline larvae were less sensitive to the coumarin scoparone
and acetogenin corossolone was the most toxic compound of all three (Table 1).
88
Table 1. Leishmania spp. response to secondary metabolites isolated from Brazilian
Northeastern plants and A. salina toxicity.
Compounds
(µg/mL)
IC50 (µg/mL) ±SD
L. donovani
L. mexicana
L. major
Toxicity
A. salina
Scoparone 27.51 ± 0.97Aa
9.11 ± 0.25Cc
14.37 ± 0.98Bb
59.4a ± 8.34
Corossolone 18.73 ± 0.82Ab
18.64 ± 0.79Ab
16.14 ± 1.13Ab
7.09c ± 2.17
Annonacinone 7.66 ± 0.77Ac
8.00 ± 1Ac
6.72 ± 0.37Ac
17.05b ± 3.46
Pentamidine 1,41 ± 0.24Ad
1,75 ± 0.34Ad
1,46 ± 0.5Ad
-
Different capital letters indicate statistical differences among columns (species) and
different lower case letters indicate significant statistical differences among lines (p <
0.001).
The spectral data of the compounds isolated from A. muricata were the same of
previous reported data (Vila-Nova et al., 2011) for the acetogenins, corossolone, and
annonacinone. The compound isolated from P. floribundum was characterized as
scoparone (6,7-dimethoxycoumarin) by comparison of 1H and
13C-nuclear magnetic
ressonance data with earlier described by Ma et al. (2006).
Corossolone: 13
C NMR (CDCl3, 125 MHz): 174.84 (C1), 131.31 (C2), 33.64 (C3),
70.01 (C4), 37.30 (C5), 29.20-29.92 (C6-7), 23.99 (C8), 42.79 (C9), 211.60 (C10),
42.88 (C11), 23.84 (C12), 25.42 (C13), 33.64 (C14), 74.32 (C15), 82.75 (C16), 28.96
(C17-18), 82.89 (C19), 74.02 (C20), 33.59 (C21), 25.78 (C22), 29.20-29.92 (C23-29),
32.11 (C30), 22.88 (C31), 14.32 (C32), 152.12 (C33), 78.21 (C34), 19.29 (C35). 1H
NMR (CDCl3, 500 MHz): 2.39 (H3a), 2.50 H3b), 3.83 (H4), 1.25-1.29 (H5), 1.25-1.29
(H6-7), 1.55 (H8), 2.38 (H9), 2.38 (H11), 1.55 (H12), 1.25-1.29 (H13), 1.38 (H14), 3.49
(H15), 3.80 (H16), 1.64; 1.98 2 H18), 3.80 (H19), 3.40 (H20), 1.39 (H-21), 1.25-1.29
(H-22), 1.25-1.29 (H23-29), 1.25-1.29 (H30), 1.25-1.29 (H31), 0.87 (H32), 7.18 (H33),
5.05 (H34), 1.39 (H35).
Annonacinone. 13
C NMR (CDCl3, 125 MHz): 174.08 (C1), 134.45 (C2), 25.31 (C3),
27.55 (C4), 28.97-29.92 (C5), 28.97-29.92 (C6-7), 23.97 (C8), 42.99 (C9), 211.55
(C10), 42.91 (C11), 25.35 (C12), 25.47 (C13), 33.45 (C14), 74.15 (C15), 82.79 (C16),
29.01 (C17-18), 82.89 (C19), 74.00 (C20), 33.70 (C21), 25.80 (C22), 28.97-29.92 (23-
29), 32.12 (C30), 22.89 (C31), 14.32 (C32), 149.15 (C33), 77.62 (C34), 19.42 (C35). 1H
89
NMR (CDCl3, 500 MHz): 2.26 (H3), 1.52-1.58 (H4), 1.31 (H5), 1.31 (H6-7), 1.52-1.58
(H8), 2.37-2.43 (H9), 2.37-2.43 (H11), 1.52-1.58 (H12), 1.31 (H13), 1.38 (H14), 3.40
(H15), 3.79 (H16), 1.68; 1.98 (H17-18), 3.79 (H19), 3.40 (H20), 1.38 (H-21), 1.31 (H-
21), 1.31 (H23-29), 1.31 (H30), 1.31 (H31), 0.89 (H32), 6.99 (H33), 4.99 (H34), 1.38
(H35).
Scoparone: 13
C NMR (CDCl3, 125 MHz): 161.4 (C2, C=O), 113.4 (C3), 111.4 (C4ª),
143.3 (C4), 108.0 (C5), 146.3 (C6), 152.8 (C7), 100.0 (C8), 150,0 (C8a) 56,4 (OCH3),
56.4 (OCH3). 1H NMR (CDCl3, 500 MHz): 6.23 ( H3, d, J= 9.5 Hz), 7.59 (H4, d, J= 9.5
Hz), 6.83 (H5), 6.78 (H8), 3.90 (OCH3), 3.88 (OCH3).
In preliminary evaluation, all compounds inhibited the promastigote growth in all
Leishmania species in this study (Table 1). The results revealed that the activity
response differs for each species, observing that each species of Leishmania tested with
the studied compounds, presented different inhibition. In the evaluation of results, it was
observed that the leishmanicidal activity was based in a dose-dependent response, the
lower dose (3.123 µg/mL) of all compounds induced lower inhibitions and the higher
dose (100 µg/mL) demonstrated the greater inhibitions in all species (Fig. 1).
Pentamidine used as positive control in this study, control wells with DMSO did not
affect the growth of parasites.
90
Fig 1. Leishmanicidal activity of corossolone (I), annonacinone (II) and scoporone (III),
against promastigotes of L. donovani, L. major and L. mexicana. Promastigotes in a
logarithimic phase were seeded at 1x106/well and incubated for 24 h with the isolated
compounds, the number of living promastigotes was determined indirectly by optical
density (OD-620nm) and correlated to the perncentage of survival. Control wells
contained DMSO or no additives, and Pentamidine was used as positive control. Each
concentration was tested in triplicate in replicate experiments.
(I)
(II)
(III)
91
The wide ranges of pharmacological properties of scoparone are evident in many
previous works, such as anti-allergic effect, inhibiting anaphylaxis in rats (Choi et al.,
2009), as a potent hepatoprotective (Bilgin et al., 2011) and inducing the release of
dopamine (Yang et al., 2010). The leishmanicidal activity of several coumarins has been
reported to inhibit parasite growth in vitro. Ferreira et al. (2010) reported the activity
against L. amazonensis, L. infantum and L. braziliensis of nine coumarins; a second
study showed a significant inhibition of L. major promatigotes in the presence of
aurapten (Napolitano et al., 2004); a third study indicated significant inhibition against
using coumarins such aurapten and umbelliprenin (Iranshahi et al, 2007). A lack of
studies showing the antiparasitic activities of scoparone was found.
Acetogenins are widely studied due to its anticancer (Chen et al., 2012b;
D’Ambrosio et al., 2011; Tantithanaporn et al., 2011), antimicrobial (Parellada et al.,
2011; Lima et al., 2011) and antiprotozoal (Boyom et al., 2009; Camacho et al., 2003;
Grandic et al., 2004) properties. Vila-Nova et al. (2011) reported the effect of
annonacinone and corossolone isolated from A. muricata against L. chagasi
promastigote and amastigote forms, and analyzed the toxicity against murine
macrophages cells.
In table 1, promastigotes of Leishmania species demonstrated different
susceptibilities in relation to tested compounds; L. mexicana was more sensitive to
scoparone followed by L. major and L. donovani (EC50= 9.11±0.25, 14.37±0.98 and
27.51±0.97 g/mL, respectively). Comparing the three species within the same line, a
similar resistance to corossolone and annonacinone was observed. The drug control
Pentamidine was statically different from all the compounds tested. Comparing each
species in relation to each compound (within the same column), L. donovani and L.
major were more susceptible to annonacinone (EC50= 7.66±0.77 and 6.72±0.37 g/mL,
respectively), and L. mexicana to annonacinone and scoparone (EC50= 8±1 and
9.11±0.25 g/mL, respectively). In general, annonacinone was the most active
substance. Pentamidine was statically similar to each other in all Leishmania species.
Compounds with anti-Leishmania-activity presenting an EC50 < 10 g/mL demonstrates
high inhibition activity, EC50 > 10 g/mL < 50 g/mL moderate and EC50 > 50 g/mL
weak (Kaiser et al., 2000). Then, acetogenin annonacinone (EC50= 6.72 – 8.00 g/mL)
indicated high leishmanicidal activity, corossolone (EC50= 16.14 – 18.73 g/mL), and
scoparone (EC50= 9.11 – 27.51 g/mL) presented moderate actions. Another study
92
using A. muricata seed extracts against L. amazonensis, L. brasiliensis and L. donovani
(Osorio et al., 2007) presented lower activity (EC50= 63.2 – 98.6 g/mL) than the
acetogenins in this work. It can be explained by the fact that the extract can contain
inferior concentrations of acetogenins.
The emergence of drug resistance in some countries led to development of
strategies to prevent it, and the introduction of new therapies are essential to prevent this
resistance to overcome. Drugs developed from medicinal plants can be a great source of
new compounds for protozoa diseases treatments with lower protozoa resistance.
Researches for new drugs are encouraged by WHO as part of search for new and better
drugs for leishmaniasis treatment (WHO, 2009).
Herbal remedies are an ancient source of new drugs but many compounds are still
unknown. Leishmaniasis is a neglected disease and its treatment has been the same for
over 60 years, which induced resistance in this protozoa. The need of new drugs with
lack of resistance from the parasite and available to the population lead us to the
research for leishmanicidal compounds from plants such as A. muricata and P.
floribundum. A coumarin, scoparone, and two acetogenins, annonacinone, and
corossolone, were isolated and its leishmanicidal activity was tested in three different
species of Leishmania (L. donovani, L. mexicana and L. major), confirming the
antiparasitic potential of these compounds. Nevertheless different species present
different susceptibilities in relation to the tested compounds. Further studies are
necessary to reveal the pharmacological significance of antiparasitic compounds from
A. muricata and P. floribundum.
Acknowledgments:
We are grateful to the CENAUREMN (Northeastern Center for the Application and Use
of Nuclear Magnetic Resonance) of the Federal University of Ceará for the NMR
spectra of the compounds, and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Fundação Cearense de Apoio ao Desenvolvimento Científico e
Tecnológico (FUNCAP), and Sistema Único de Saúde (PPSUS) for their financial
support.
93
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9 CAPÍTULO 4
Derivados do timol e eugenol como potenciais agentes anti-Leishmania
(Thymol and Eugenol Derivatives as Potential anti-Leishmania Agents)
Periódico: Enviada para Jounal of Antimicrobial Agents
98
RESUMO
No Nordeste brasileiro, a leishmaniose é uma doença endêmica, com vários casos letais
entre os seres humanos e cães. O objetivo deste trabalho foi realizar um levantamento
de compostos naturais ativos para encontrar novas moléculas com atividade contra
Leishmania infantum chagasi e com baixa toxicidade. O timol e eugenol foram
escolhidos para serem compostos de partida para a síntese de derivados acetilados e
benzoladios e utilizados para testar a sua ação leishmanicida in vitro e in vivo contra L.
i. chagasi. Screening utilizando promastigotas luciferase dependente, e ensaio ELISA in
situ utilizando amastigotas, foram realizados para avaliar a viabilidade destas formas.
Para o ensaio in vivo foram utilizados camundongos BALB/c, e os derivados de timol e
eugenol foram administrados intraperitonialmente na dose de 100 mg/kg/dia durante 30
dias. No ensaio com promastigota os derivados do timol demonstraram as melhores
antividades leishmanicida, o benzoil-timol apresentou a melhor atividade inibitória
(8,67 ± 0,28). No ensaio com amastigotas, todos os derivados foram semelhantes, no
entanto o acetil-timol demosntrou o efeito mais significativo sendo inclusive mais ativo
que o timol e o controle, anfotericina B. A imuno-histoquímica demonstrou a presença
de amastigota de Leishmania apenas no baço do grupo tratado com a acetil-timol. Em
conclusão, estes derivados de fácil obtenção constituem compostos úteis para estudos
com o intuito de encontrar novos agentes para o tratamento de leishmaniose.
99
Thymol and Eugenol Derivatives as Potential anti-Leishmania Agents
Leishmanicidal activity of phenolic derivatives
Nadja Soares Vila-Nova1, Selene Maia de Morais
1,2*, Davi Varela Magalhães
2, Claudia
Maria Leal Bevilaqua1,3
, Fernanda Cristina Rondon3, Carlos Henrrique Lobo
4, Adriana
da Rocha Tomé5, Claudio Cabral Campello
1,5, Mary Wilson
6, Heitor Franco de Andrade
Jr7
1. Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do
Ceará; Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará,
Brazil.
2. Departamento de Quimica, Centro de Ciências e Tecnologia, Universidade Estadual
do Ceara; Avenida Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará,
Brazil.
3. Laboratório de Doenças Parasitárias, Universidade Estadual do Ceará; Avenida
Paranjana 1700, Campus do Itaperi, 60740-000, Fortaleza, Ceará, Brazil.
4. Laboratório de Biologia da Reprodução Universidade Federal do Ceará; Av. Mister
Hull s/n, Campus do Pici, 60021-970, Fortaleza, Ceará, Brazil
5. Faculdade de Veterinária – FAVET, Avenida Paranjana 1700, Campus do Itaperi,
60740-000, Fortaleza, Ceará, Brazil.
6. Departments of Internal Medicine and Microbiology, University of Iowa and the VA
Medical Center, Iowa City, IA 52242, USA.
7. Laboratório de Protozoologia, Universidade de São Paulo; Avenida Eneas de
Carvalho Aguiar, 470, 05403-000, São Paulo-SP, Brazil.
*Universidade Estadual do Ceará, Av. Paranjana, 1700 Tel: +55+85 3101-9933.
100
Abstract
In Northeastern Brazil there are many places where Leishmaniasis is endemic with
several lethal cases among humans and dogs. The aim of this work was to survey
natural active compounds to find new molecules with more activity against Leishmania
infantum chagasi and with low toxicity. The compounds thymol and eugenol were
chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test
their leishmanicidal action in vitro and in vivo against L. i. chagasi. A screening assay
using luciferase-expressing promastigote form was used to measure the viability of
promastigote, and an ELISA in situ was performed to evaluate the viability of
amastigote. For the in vivo assay, thymol and eugenol derivatives were given intra
peritonially to BALB/c at 100 mg/Kg/day during 30 days. In the promastigote assay the
thymol derivatives demonstrated the greater activity rates, being benzoyl-thymol the
best inhibitor (8.67 ± 0.28). In the amastigote assay all compounds were similar, and
acetyl-thymol was more active then thymol and control amphotericin.
Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the
spleen of the group treated with acetyl-thymol. In conclusion, these easy-making
derivatives constitute useful compounds for further studies to find new agents for
Leishmaniasis treatment.
101
Introduction:
For over sixty years, the treatment of leishmaniasis has been carried out with
pentavalent antimonials such as antimoniate N-methyl glucamine (Glucantime ®) and
sodium stibogluconate (Pentostan ®), which are the drugs of first choice for the
treatment of viscel leishmaniasis. These drugs are toxic, not always effective, are used
in prolonged schemes1. Alternative drugs like pentamidine, amphotericin B, and its
lipossomal formulation are used in the treatment of resistant forms2. The leishmanicial
drug development follows three lines, the first explores the parasite metabolic pathways
to find targets and develop synthetic compounds, the second is the study of other drugs
that are already on the market with leishmanicidal activity hitherto unknown (e.g.
cancer drugs) and the third is focused on the use of medicinal plants as a source of anti-
protozoan molecules3. As part of the search for new and better drugs with high viability
and low toxicity, the Programme for Tropical Diseases of the World Health
Organization (WHO) is considering research on using plants to treat leishmaniasis
essential and high priority4.
Plants are important sources of drug discovery, especially in regard to
antiparasitic drugs due to the coexistence in the environment of parasites and medicinal
plants5. Research into herbal resources can lead to the identification of secondary
metabolites that can either serve as valuable drugs or lead to the development of new
therapeutic substances6. Several studies have validated the effect of natural products as
potential sources of new and selective agents for the treatment of tropical diseases
caused by protozoa and other parasites7.
Thymol and eugenol are largely used as flavouring agents for food since they are
the main constituents of oregano and clove respectively and display many
pharmacological actions including antimicrobial8,9
, anti-inflammatory10,11
,
fungicidal12,13
, antioxidant14,15
, besides others. Common medicinal plants used by local
populations include Ocimum gratissimum, whose essential oil and main constituent
eugenol displays anthelmintic and antimicrobial activities16,17
, and Lippia sidoides rich
in thymol, which is used as a general antiseptic18
.
The cytotoxicities and antileishmanial activities of thymol and some
hemisynthetic derivatives and also the synthesis and the leishmanicidal activity of
quinoline-triclosan and quinoline-eugenol hybrids have been demonstrated both in vitro
and in vivo19,20
. Lippia sidoides Cham. essential oil was shown to display in vitro
102
cytotoxicity and antileishmanial activity21
, and the antileishmanial activity of Eugenol-
rich essential oil from Ocimum gratissimum was also reported22
.
In Northeastern Brazil there are many places where Leishmaniasis is endemic
and there have been several lethal cases among humans and dogs. In our concern about
the lack of leishmanicidal efficient drugs, we surveyed natural active compounds to find
new molecules with more activity against Leishmania spp which also had low toxicity.
Thus, thymol and eugenol were chosen to be the first compounds to synthesize acetyl
and benzoyl derivatives and to have their leishmanicidal action in vitro and in vivo
against Leishmania chagasi promastigotes and amastigotes tested.
Materials and Methods
Acetylation reaction
Eugenol and thymol were purchased from VETEC, in Fortaleza, Brazil. A
mixture of acetic anhydride (6 g) and pyridine (2 g) was added to eugenol or thymol (3
g). The mixture was left stirring for 24 h at room temperature, and then cold water was
added (20 mL). The solution was neutralized to pH 7.0. The reaction mixture was
transferred to a separating funnel and washed three times with chloroform (20 mL). The
chloroform layer containing acetylated material was washed with water and then dried
with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure.
Benzoylation reaction
Eugenol (8.2 g or 0.05 mol) or thymol (7.5 g or 0.05 mol) was dissolved in 40
mL of 5% NaOH in the cold, and added benzoyl chloride (7g) (5.8 ml, 0.05 mol). The
mixture was vigorously mixed until the odor of benzoyl chloride disappeared (20 to 25
minutes). The solid product was filtered on a Buchner funnel and wash with cold water,
recrystallize with 60 mL of rectified spirit, and the crystals were filtered23
.
Analysis of synthesized derivatives
The crude synthesized derivatives were subjected to silica gel column
chromatography and eluted with mixtures of hexane, dichloromethane, ethyl acetate and
methanol with increasing polarity. The fractions were collected and compared by TLC.
The chemical structures of the purified compounds were confirmed by spectroscopic
analysis of the nuclear magnetic resonance spectra recorded on a Bruker Avance DRX-
500 spectrometer, in CDCl3.
103
Leishmanicidal activity
A strain of Leishmania infantum chagasi expressing luciferase, called Lic-luc,
was generated with the integrating vector pIR1SAT, provided by Dr. Steve Beverley, as
described by Thalhofer et al. (2010). The promastigote form of Lic-Luc was cultured in
a hemoflagellate-modified MEM (HOMEM) medium, supplemented with 10% fetal
bovine serum at 26 °C. For the leishmanicidal assay, an adapted methodology from
Tempone et al. 2005 was used. Thymol, acetyl-thymol, benzoyl-thymol, eugenol,
acetyl-eugenol and benzoyl-eugenol were dissolved in DMSO at a concentration of
0.2% and diluted with HOMEM medium in 96-well microplates. The assay was
performed at concentrations of 100, 50, 25, 12.5 and 6.25 µg/mL. Control wells
contained DMSO or no additives. Each concentration was tested in triplicate in replicate
experiments. Promastigotes were counted in a Neubauer hemocytometer and seeded at
1x106/well. The plates were incubated at 26 °C for 24 h, and the viability of the
promastigotes, based on their morphology, was observed under a light microscope.
Pentamidine (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control
drug. The optical density (OD) was determined in a Fluostar Omega (BMG Labtech) at
620 nm.
The drug activity against amastigote was measured by a modified ELISA
measuring the viability of L. i. chagasi infecting the murine macrophage RAW 264.7
cell line (ATCC TIB-71). Four x 105 RAW 264.7 cells in RPMI-1640, 10% foetal
bovine serum, were added to each well of a 96-well plate, and incubated with L. i.
chagasi promastigotes at a 1:10 (macrophage/promastigote) ratio at 37ºC, 5% CO2 in an
incubator for 72 h. An adapted methodology from Piazza et al. 1994 was used for the in
vitro assay. Stock solutions of all of the compounds were prepared at a concentration of
0.2% in ethanol. An additional five concentrations were obtained by two-fold serial-
dilution. The compound solutions were added to microplates containing the confluent
layer of cells with amastigotes for 48 h at 37°C in a humidified 5% CO2 incubator.
Macrophages incubated without drugs were used as the control, and amphotericin B
(Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control drug at the same
concentration as the other compounds. Murine macrophage RAW 264.7 cells were
incubated with 0.01% saponin in PBSA, containing 1% bovine serum albumin, for 30
minutes. The wells were then blocked for 30 minutes with 5% nonfat milk (Nestlé) in
PBS. In the next step, the cells were incubated in serum from a rabbit immunized with a
104
saline extract of L. i. chagasi promastigotes, collected 30 days after the infection, at
37°C for 1 h. The serum employed was diluted 1:500 and pre-absorbed with 10% foetal
calf serum (FCS) at 22°C for 1 h. The wells were washed three times with 0.05%
Tween-20 in PBSA, and peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical
Co.) diluted 1:5000 in 5% nonfat milk was added and incubated at 37°C for l h. After
the wells were washed, o-phenylenediamine (0.4 mg/mL) and 0.05% H202 were added.
The reaction was stopped by the addition of 1 M HC1. The plates were read at 492 nm
in a Multiskan MS (UniScience) ELISA reader.
Cytotoxicity assay
A methodology adapted from Tempone (2005) was used. Murine macrophages-
RAW 264.7 cells were seeded at 4x104/well in 96-well microplates and incubated at 37
°C for 48 h in the presence of the compounds, dissolved previously in ethanol at a
concentration of 0.2% and diluted with M199 medium to the highest concentration of
120 µg/mL. The microplates were incubated for 48 h at 37 °C in a 5% CO2 humidified
incubator. Control cells were incubated in the presence of DMSO, without the
drugs,Glucantime and Pentamidine (standard drugs). The viability of the macrophages
was determined with the MTT assay, as described above, and was confirmed by
comparing the morphology of the control group via light microscopy.
Animals and infection
This study involved 30 male BALB/c mice, 21-day-old, kept according to
institutional guidelines, which were inoculated intraperitoneally with 107
infective
promastigotes of L. i. chagasi. Promastigotes were cultured in M199 medium,
supplemented with 10% foetal bovine serum and 5% human male urine at 24 °C.
Metacyclic promatigotes were obtained from cultured stationary phase promastigotes,
stationary cultures were centrifuged, the supernatant was discarded and the pellet was
resuspended in M199 medium then used immediately for the animal infection. Thirty
five days post infection one animal from the group was euthanised and the liver and
spleen were imprinted to confirm infection and verify the parasite burden, and then the
treatment was started.
The animals were treated for 30 days and divided into four groups (n=5), and
treated intra peritoneally with acetyl-thymol, benzoyl-thymol, acetil-eugenol and
105
benzoyl-eugenol all with the dose of 100 mg/Kg, the control groups were given
glucantime with the dose of 80mg/Kg intra muscular, and infected not treated.
All procedures involving animals in this study were reviewed and approved by
the Ceará State University Ethics Committee (CEUA-UECE).
Qualitative Immunohistochemistry
The presence or absence of Leishmania after treatment (qualitative histological
events) was determined by the presence of amastigote forms, Leishmania antigens
detected by immunohistochemistry.
Silanized slides containing sections of fragments from BALB/c liver and spleen
obtained after the treatment of leishmaniasis were submitted to immunohistochemistry
for the detection of amastigotes forms. The tissues were deparaffinised in histological
sections (4 mm) with xylene, rehydration in a decreasing ethanol series.
Immunohistochemistry was performed with anti-Leishmania antibodies produced in
rabbits reacted with peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co.).
All reactions were developed in the same way using a diaminobenzidine chromogen
solution (Sigma Chemical Co., MO, USA-D5637) which precipitates a brown product
and counterstaining was performed with Harris hematoxylin. Next, the slides were
dehydrated in a growing ethanol series and mounted with Permount resin (Fisher
Chemicals, NJ, USA).
Histopathology analysis
Fragments of spleen and liver were removed and fixed in neutral buffered formalin
for subsequent paraffin embedding. Sections (5 mm thick) were stained with
HematoxylineEosin (HE).
Statistical analysis
The number of living promastigotes was determined indirectly by the optical
density (OD – 620 nm) representing the percentage of survival. The EC50 values (±SD)
were calculated using a nonlinear regression curve using the statistical software
GhaphPad Prism 4.0. The data were tested for compliance with the requirements for
carrying out the analysis of variance and normal distribution and homogeneity of
variances among treatments. The ANOVA was then performed using the GLM
procedure of SAS (2002) and Tukey's test was used to compare means. For the toxicity
106
assay using A. salina, the EC50 and its confidence intervals were calculated by Probit
analysis using the software StatPlus 2009 Professional 5.8.4.
Results and discussion
In the search for new compounds with lesser toxicity and greater activity, the
structure of thymol and eugenol were chemically modified by an acetylation and
benzoylation process, in this study. At the end of the process four derivates were
obtained, acetyl-thymol, benzoyl-thymol, acetyl-eugenol and benzoyl-eugenol (Fig. 1).
The spectroscopic data is shown in Table 1.
Table 1. 1H and
13C NMR Spectroscopic data of O-acetyl-thymol (AT), O-benzoyl-
thymol (BT), O-acetyl-eugenol (AE) and O-benzoyl-eugenol (BE) (CDCl3, 400Mhz).
AT BT AE BE
Carbon C H C H C H C H
1 148.8 - 135.5 - 137.98 - 129.44 -
2 116.5 - 138.6 - 150.83 - 151.03 -
3 126.0 7.25 126.3 7.28 112.70 6.9 112.78 7.1
4 126.0 7.02 126.0 7.12 138.95 - 138.93 -
5 134.9 - 134.9 - 120.62 6.7 120.62 6.8
6 122.1 6.85 122.1 7.0 122.47 6.9 122.55 7.1
7 34.4 3.02 34.4 - 40.03 3.4 40.00 3.4
8 24.1 1.29 24.1 - 136.99 5.9 137.00 6.0
9 24.1 1.29 24.1 - 116.09 5.1 116.02 5.2
10 21.3 2.34 21.3 - 55.76 3.8 55.74 3.8
11 170.3 - 166.8 - 169.13 - 164.75 -
12 20.0 - 148.5 - 20.60 2.3
138.14 -
13 - - 129.7 8.29 - - 130.16 8.2
14 - - 128.4 7.59 - - 128.38 7.5
15 - - 133.3 7.68 - - 133.28 7.6
16 - - 128.4 7.59 - - 128.38 7.5
17 - - 129.7 8.29 - - 130.16 8.2
107
Thymol is a p-cymene compound with many pharmaceutical properties
including antimicrobial8, anti-inflammatory and healing
10, fungicidal
12, and
antioxidant14
. It has been found that p-cymene derivatives have leishmanicidal activity
with low toxicity and greater inhibiton than thymol19,27
.
O-Methyl-thymol O-Benzoyl-thymol
O-Methyl-eugenol O-Benzoyl-eugenol
Fig 1. Representation of chemical structures of thymol and eugenol derivatives
In the promastigote assay the thymol derivatives demonstrated the greater
activity rates, benzoyl-thymol was the higher inhibitor with an EC50 of 8.67 µg/mL (SD
± 0.28), and acetyl-eugenol the lower with an EC50 of 23.21 µg/mL (SD ± 3.46). All
compounds, when compared to the control Pentamidine, were statistically different.
When compared to the pure substances, acetyl-thymol demonstrated a greater
leishmanicidal activity against promastigotes than thymol, and eugenol presented an
activity lower than both derivatives (Table 2).
108
Table 2. Leishmanicidal activity of thymol and eugenol derivatives and toxicity against
L. i. chagasi and RAW 264.7 cells.
Compounds Promastigote
LC50
(µg/mL)
Amastigote
LC50
(µg/mL)
Toxicity % RAW 264.7
cells survival at 100
µg/mL (±SD)
Acetyl Eugenol 23.21 ± 3.46*Ab 18.53 ± 4.79 Aab > 100
Acetyl Thymol 9.07 ± 0.06*Ae 10.95 ± 3.00*Ab > 100
Benzoyl Eugenol 10.58 ± 0.18*Ad 14.93 ± 4.50 Aab 97.7 ± 5.6
Benzoyl Thymol 8.67 ± 0.28*Af 15.09 ± 3.65 Bab 63.6 ± 5.3
Eugenol 56.13 ± 2.09*Aa 20.81 ± 1.59 Ba 29 ± 1.3
Thymol 12.85 ± 1.61*Ac 23.93 ± 6.29Ba 36.5 ± 0.5
Amphotericin B Nd 20.44 ± 0.98 98.5 ± 7.5
Pentamidine 5.30 ± 0.81 Nd 85.6 ± 6.9
*Represents significative difference in relation to the control. Different capital letters
represent differences between columns (action of each drug on different forms).
Lowercase letters represent significant differences between different lines (action of
different drugs within each form).
Nd, not determined
The thymol derivatives, acetyl-thymol and benzoyl-thymol presented an EC50 =
9.07 µg/mL (SD± 0.06) and EC50 = 8.67 µg/mL (SD± 0.28) against promastigote form,
respectively. Acetyl-thymol demonstrated greater activity in amastigote form (EC50 =
10.95 µg/mL, SD ± 3.0) followed by benzoyl-thymol (EC50 = 14.93 µg/mL, SD± 4.5)
(Table 2). Medeiros et al. (2011) presented the leishmanicidal activity of thymol-rich
essential oil from Lippia sidoides and pure thymol against promastigote of L.
amazonensis, demonstrating an EC50 of 44.3 and 19.4 µg/mL, respectively; these
inhibitions were lower than the ones presented by the benzoyl-thymol and acetyl-thymol
in this study. Another study demonstrated an inhibition from thymol and derivates
against promastigotes of L. panamensis where five of the eight derivatives obtained
were significantly more active than the thymol19
. The difference between the activity of
109
essential oil and the pure substance can be explained by the fact that the essential oil has
other elements and the concentration of the constituents is not equivalent to the pure
substance, and the greater results of the thymol derivatives is an indication that the
acetylation and benzoylation process can improve the growth inhibition and lower the
toxicity.
Eugenol is a phenylpropanoid with antifungal13
, anthelmintic16
, insecticide28
,
and leishmanicidalactivity22
. This compound is the main constituent of essential oil of
Ocimum gratissimum and Eugenia caryophyllus29,30
. Many phenylpropanoids have
leishmanicidal activity31,32
but a lack of studies on derivatives of eugenol have been
found, so, as well as the thymol derivatives, we believe that the eugenol derivatives
could also provide greater leishmanicidal activity with lower toxicity.
Comparing the two eugenol derivatives, benzoyl-eugenol had the greater
leshmanicidal activity against promastigotes (EC50 = 10.58 µg/mL, SD± 0.18), followed
by acetyl-eugenol (EC50 =23.21 µg/mL, SD± 3.46), and in the amastigote form all
compounds presented similar inhibition. Ueda-Nakamura et al. (2006) tested the
eugenol-rich essential oil of Ocimum gratissimum and its main constituent against L.
amazonensis amastigote and promastigote and observed that the pure eugenol had a
greater inhibition than the essential oil; Pessoa et al. (2002) measured the percentage of
the constituents in O. gratissimum essential oil and found that only 43.7 % is eugenol.
Comparing the activity of O. gratissimum essential oil and pure eugenol, the eugenol
derivatives tested in this study revealed a greater activity than the eugenol-rich essential
oil and the pure compound. Arango et al. (2012) evaluated the leishmanicidal activity
of six quinolone-eugenol hybrids and pure eugenol and demonstrated that the hybrids
had a greater inhibition than eugenol, indicating that the compounds derivatives can
potentiate the inhibition activity. Other studies demonstrate that the parasitic activity of
pure eugenol is superior to eugenol-rich essential oil. Santoro et al. (2007) demonstrated
that the tryponocidal activity of O. basilicum essential oil was lower than its main
constituent, eugenol; another study measured the anti-giardia activity of Syzygium
aromaticum and its major compound eugenol, and observed that the eugenol itself had a
greater growth inhibition of Giardia lamblia34
, and these activities might improve with
the use of compound derivates.
The amastigote is the parasitic form responsible for the disease and it should be
the chemotherapeutic target in studies of new antileishmanial agents19
. All the
compounds presented statistic similarity to the control Amphotericin B except acetyl-
110
thymol which had a greater activity than the control. In this study only the acetyl-
eugenol had significant difference between the activity of promastigote and amastigote
forms, the inhibition on promastigote being greater than amastigote. The other
compounds demonstrated statistic similarity between both promastigote and amastigote
activity, indicating that these compounds can act both intra and extra cellular (Table 2).
The toxicity using RAW 264.7 cells at 100 µg/mL revealed that at this
concentration acetyl-thymol was not toxic and benzoyl-thymol had a 63.6 % survival
rate, demonstrating a low toxicity. A lack of toxicity was found in both eugenol
derivatives in this study. Comparing to the pure substances, all derivatives had lower
toxicity (Table 2). This can demonstrate that the acetylation and benzoylation process
might decrease the toxicity of compounds that goes through the process.
Thymol and eugenol and its phenolic derivatives have potent antimicrobial and
antioxidant properties12
, the leishmanicidal mechanism of these compounds could be
explained by the ability of interaction with ergosterol of the Leishmania membrane,
allowing the increase of cell permeability and loss of small cations such as K+. Eugenol
also induces apoptosis, inhibiting both stages of Leishmania parasites7.
A few published studies have demonstrated the anti-parasitic activity of essential
oils rich in thymol and eugenol and the pure substances as well as their toxicity in
mammalian cells, but no studies were found in the literature which used their
derivatives such as acetyl-thymol, benzoyl-thymol, acetyl-eugenol and benzoyl-eugenol.
Most reports measure in vitro leishmanicidal activity and toxicity of essential
oils and their major compounds, lacking a follow up in vivo study. The reports that have
a follow up report evaluate the skin damage caused by Leishmania species which leads
to cutaneous disease. In this study we evaluated the activity of thymol and eugenol
derivatives in vivo in BALB/c mice infected with L. i. chagasi the main cause of
visceral leishmaniasis. We also estimate damage in the spleen and liver caused by the
treatment, since the in vitro response can differ significantly from the in vivo response.
At first all animals were infected with 107 promastigotes intra peritoneal, and the
treatment started 30 days post-infection. Thymol and eugenol derivatives were tested in
BALB/c mice, using a 100 mg/Kg dose, once a day, for 30 days. To reach this dose an
acute toxicity was previously conducted (data not shown). After the treatment all
animals were euthanasied and spleen and liver tissue samples were collected and used
for histopathology and immunohistochemistry analyses.
111
The histopathological findings of the liver and spleen samples stained with HE
showed no significant alterations in the spleen, and all the livers presented moderate
hydropic degeneration. The immunohistochemistry indicated the presence or absence of
Leishmania amastigote forms stained in brown. The spleen of the group treated with
acetyl-thymol was the only one which was positive besides the control groups treated
with Glucantime and not treated; the groups treated with acetyl-eugenol, benzoyl-
eugenol and benzoyl-thymol did not show the presence of Leishmania amastigotes in
the spleen. No parasite was stained in all livers (Fig 2).
The thymol and eugenol benzoylation and acetylation processes modify the
chemical structure of these compounds, which may decrease the toxicity and increase its
leishmanicidal action. In this study the leishmanicidal action of thymol and eugenol
derivatives was tested in vivo and in vitro, and their ability to reduce the parasite in the
liver and spleen was shown. Quantitative assays should be made in order to indicate
their real action in the liver and spleen as well as other organs.
112
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2 Immunohistochemistry from spleen samples of mouse BALB/c infected with L. i.
chagasi during 30 days and treated for 20 days, (a) Infected not trated, (b) Glucantime,
(c) acetyl-eugenol, (d) Benzoyl-eugenol, (e) Acetyl-thymol, (f) Benzoyl-thymol. Arrows
pointing to Leishmania amastigotes stained in brown.
113
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10 CAPÍTULO 5
Atividade leishmanicida In vivo da Corossolona e Annonacinona, acetogeninas
isoladas da Annona muricata.
(In vivo leishmanicidal activity of Corossolone and Annonacinone, acetogenins isolated
from Annona muricata.)
Periódico: Enviado para Revista da Sociedade Brasileira de Medicina Tropical
117
RESUMO
Vários aspectos limitam a busca por produtos naturais e a falta de continuidade nas
pesquisas é um deles. A maioria dos pesquisadores não validam in vivo a atividade in
vitro. Assim o objetivo deste trabalho é validar in vivo o efeito leishmanicida já estudado
in vitro de produtos naturais. O extrato metanol-água (80:20) das folhas de A. muricata
fori extraídas utilizando ácido fosfórico a 10% e de solventes orgânicos, para obter os
extratos ricos em alcalóide e acetogenina. Estes extratos foram cromatografados em
coluna de sílica gel e eluídos com uma mistura de vários solventes em ordem crescente de
polaridade. Os compostos foram identificados por análise espectroscópica. Este estudo
envolveu 20 camundongos BALB/c machos, de 21 dias de idade, mantidos de acordo com
as diretrizes institucionais. Estes foram inoculados intraperitonealmente com 107
promastigotas infectantes de L. i. chagasi e, em seguida divididos em grupos e tratados
com os compostos isolados e com a droga controle Glucantime. Após 30 dias de
tratamento os animais foram eutanaziados e o fígado e baço recolhidas para imuno-
histoquímica e qPCR. A imuno-histoquímica não mostrou a presença de parasitos nos
tecidos hepáticos dos animais de todos os grupos, no entanto detectou-se a presença de
amastigotas no baço. O ensaio de qPCR demonstrou no grupo corossolone uma carga
parasitária semelhante no fígado e no baço, no grupo annonacinone a carga parasitária no
baço foi cinco vezes mais baixo do que o fígado. Os resultados caracterizam a A. muricata
como um provedor de composto que pode ser usado como fitoterápicos.
118
In vivo leishmanicidal activity of Corossolone and Annonacinone, acetogenins
isolated from Annona muricata.
Nadja Soares Vila-Nova1, Selene Maia de Morais
1,2*, Maria Jose Cajazeiras Falcão
2, ,
Carlos Henrrique Lobo3, Arlindo de Alencar Araripe Moura
3Antônia Débora Sales
4, Ana
Paula Ribeiro Rodrigues4, José Ricardo de Figuereido
4, Heitor Franco de Andrade Jr
5
Communication/Comunicação
1. Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do
Ceará
2. Departamento de Química, Universidade Estadual do Ceará
3. Laboratório de Biologia da Reprodução, Universidade Federal do Ceará
4. Laboratório de Manipulação de Oócitos e Folículos pré-antrais, Universidade
Estadual do Ceará,
5. Instituto de Protozoologia, Universidade de São Paulo.
*Universidade Estadual do Ceará, Av. Paranjana, 1700 Tel: +55+85 3101-9933.
119
Abstract:
Introduction: several aspects which limit the search for natural products and lack of
continuity in the researches is one on them. Most researches do not validate in vivo the in
vitro activity. Methods: Methanol-water (80:20) extract of A. muricata seeds were
extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and
acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and
eluted with a mixture of several solvents in crescent order of polarity. The compounds
were identified by spectroscopic analysis. This study involved 20 21-day-old male
BALB/c mice, kept according to institutional guidelines, were inoculated intraperitoneally
with 107
infective promastigotes of L. i. chagasi and then treated with the isolated
compounds and control drug glucantime. After 30 days of treatment the animals were
euthanized and the liver and spleen were collected to immunohistochemistry and qPCR.
Results: The immunohistochemistry did not show the parasite presence in all livers but
were well marked in the spleen. The qPCR assay demonstrated in the corossolone group a
similar parasite burden in both liver and spleen, in the annonacinone group the parasite
burden in the spleen was five times lower than the liver. Conclusion: The results
characterized the Annona muricata as a compound provider that can be used as
phytotherapic agents.
120
Introdução
Plants are an important source of drug discovery, especially in regards to
antiparasitic drugs because of the association between the coexistence of parasites, living
beings, and medicinal plants (Anthony et al., 2005). Natural products offers molecules
with profound impact on human health, nature produces infinite secondary metabolites
with distinct biological properties. Several studies have validated the effect of natural
products as potential sources of new and selective agents for the tropical diseases
treatment caused by protozoa and other parasites (Mishra et al, 2009).
Drug development follows three lines, the first explores the parasite metabolic
pathways to find targets and develop synthetic compounds, the second is the study of other
drugs that are already on the market with leishmanicidal activity hitherto unknown (e.g.
cancer drugs) and the third focuses on the use of medicinal plants as a source of anti-
protozoan molecules (Lindoso et al., 2012). Due to the limited viability of effective
leishmanicidal chemotherapeutic in endemic areas, a large proportion of the population
living in these places depends on medicinal plants that are used in popular treatments, to
treat and relieve the symptoms of leishmaniasis (Chan-Bacab and Pena-Rodriguez, 2001).
These plants have secondary metabolites that can act and destroy invaders, however often
these substances are unknown and may offer alternative treatment for leishmaniasis.
Nevertheless, there is several aspects which limit the search for natural products:
(1) low viability of the compounds, in general substances extracted from plants has low
quantity and a difficult extraction, (2) high structural complexity, various stereoisomers;
(3) lack of continuity in the researches, most studies are not part of the development of
new drug programs, (4) the isolated compounds often show no activity and requires
monitoring to improve these activities (Mishra et al., 2009). In the present study we
decided to validate in vivo the activity already proven in vitro by our group (Vila-Nova et
al., 2011) using acetogenins corossolonae and annonacinone isolated from the leaves of
Annona muricata.
Materials and Methods
2.2 Plants
The leaves of A. muricata were collected in the state of Ceará, Brazil. Aerial parts
of the two plants were deposited in the Prisco Bezerra Herbarium under the reference
number 43951.
121
2.2 Chromatographic procedures
The active chemical constituents were isolated from plant extracts by silica gel (
0,063 – 0,200 mm; 70-230 mesh) and Sephadex (LH - 20) column chromatography and
solvents for elution were petroleum ether, hexane, chloroform, ethyl acetate, acetone and
methanol from VETEC (Brazil). The solvents were used in mixtures of increasing polarity
starting with hexane and finishing with methanol. The fractions collected in the columns
were compared by thin layer chromatography (TLC), using silica gel (60 G F 254) on
glass plates (3 cm x 8 cm). The TLC plates were analyzed in a iodine chamber, under UV
Light (at 312 and 365 nm), Vilbert Loumart, CN-15 LM model, and by spraying the
reagent 2.5 % vaniline in perchloric acid diluted in ethanol (1:1), followed by heating in an
oven at 100 °C.
2.3. Isolation of compounds and spectroscopic identification
A. muricata seeds (2 kg) were triturated and left in contact with methanol for one
week, then the solvent was filtered, evaporated to dryness and a light yellow solid was
obtained (402 g). This material was chromatographed on a filtering silica gel column with
the solvents hexane, chloroform and methanol in mixtures of increasing polarity, yielding
the 76 fractions. Thin layer chromatography of fractions conducted to two main fractions,
which were purified by successive chromatographic columns, furnishing two main
compounds.
The structures of compounds were determined by spectroscopic analysis of
infrared spectra, recorded on a FT-IR PerkinElmer 1000 spectrophotometer, values
expressed in cm-1
, and nuclear magnetic resonance spectra, recorded on a Bruker Avance
DRX-500 spectrometer in CDCl3.
Animals and infection
This study included 20 male BALB/c mice, with the age of 21 days, maintained
according to institucional guide lines and inoculated intraperitoneal with 107
infective
promastigotes of L. i. chagasi. Promastigotes were cultured in M199 medium,
supplemented with 10% fetal bovine serum and 5% human male urine at 24 °C.
Metacyclic promatigotes were obtained from cultured stationary phase promastigotes,
stationary cultures were centrifuged the sobrenadand was discarded and the pellet was
resuspended in M199 medium then used immediately for the animal infection. Thirty five
days post infection one animal was euthanized and the liver and spleen were inprinted to
confirm infection and verify parasite burden, and then start the treatment.
122
The animals were treated during 30 days, four groups were treated with
annonacinonde, corossolone all in the dose of 100 mg/Kg, and the control groups were
glucantime in the dose of 80mg/Kg, and infected not treated (n = 5). After the treatment all
animal were euthanized and the liver and spleen collected for qPCR.
All procedures involving animal in this study were reviewed and approved by the
Ceará State University Ethics Committee (CEUA-UECE).
RNA extractions, primer and probes.
For this procedure, total RNA was extracted from a fragment (approximately 100
mg) of spleen and liver samples of BALB/c mice. Total RNA was isolated with Trizol
Plus Purification kit (Invitrogen Life Technologies, São Paulo, Brazil). The RNA
preparations were treated with DNAse I and subjected to the RNeasy Micro kit (Invitrogen
Life Technologies, São Paulo, Brazil). Complementary DNA (cDNA) was synthesized
from RNA (0.15 µg from each sample) using Superscript™ II RNase H-Reverse
Transcriptase (Invitrogen Life Technologies, São Paulo, Brazil). The qPCR reactions were
performed in a final volume of 20 µL containing the following components: 1 µL of each
cDNA, 10 µL of 1x Power SYBR® Green PCR Master Mix, 7.4 µL of ultra-pure water
and 0.4 µM (final concentration) of both sense the and antisense primers. The gene-
specific primers used to amplify the kDNA mRNA were the same as described by
Bezerra-Vasconcelos (2011) namely forward 5’-CTCCGGGTAGGGGCGTTC-3’ and
reverse 5’-GCCCTATTTTACACCAACCCC-3’. The reference gene glyceraldehyde-3-
phosphate-dehydrogenase (GADPH) (forward 5’-TGTTTGTGATGGGCGTGAACCA-3’;
reverse 5’-ATGGCGTGGACAGTGGTCATAA-3’) was selected as an endogenous
control for normalization and to study the expression stability in all samples. Primer
specificity and amplification efficiency were verified for each gene. The cycle profile for
the first PCR step consisted of initial denaturation and polymerase activation for 15 min at
94 ºC, which was followed by 40 cycles of 15 sec at 94 ºC, 30 sec at 60 ºC and 45 sec at
72 ºC. A final extension was performed for 10 min at 72 ºC. The specificity for each
primer set was tested using a melting curve, which was performed between 60 and 95 °C
for all genes. All amplifications were performed using a Bio-Rad iQ5 system. The delta-
delta-CT method was used to transform threshold cycle values into normalized relative
expression levels (Livak and Schmittgen 2010).
123
Immunohistochemistry
The presence or absence of Leishmania after treatment (qualitative histological
events) was determined by the presence of amastigote forms, Leishmania antigens
detected by immunohistochemistry the alterations were scored as 0, absent; 1, mild; 2,
moderate; and 3, intense.
Silanized slides containing sections of fragments from BALB/c liver and spleen
obtained after the treatment of leishmaniasis were submitted to immunohistochemistry for
the detection amastigotes forms. The tissues were deparaffinizated in histological sections
(4 mm) with xylene, rehydration in a decreasing ethanol series. Immunohistochemistry
was performed with anti-Leishmania antibodies produced in rabbits reacted with
peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co.). All reactions were
developed in the same way using a diaminobenzidine chromogen solution (Sigma
Chemical Co., MO, USA-D5637) which precipitates a brown product and counterstaining
was performed with Harris hematoxylin. Next, the slides were dehydrated in a growing
ethanol series and mounted with Permount resin (Fisher Chemicals, NJ, USA).
Statistics
All gene expression profiles were generated from three independent biological
replicates, which were performed using technical triplicate. The relative gene expression
was estimated using the 2-ΔΔCt
-method (Pfaffl, 2001). Threshold and Ct values were
automatically determined by Bio-Rad iQ5 software, using internal parameters. The Ct was
expressed as means of three measurements ± SEM and then subjected to Shapiro-Wilk
normality test using the UNIVARIATE procedure of SAS 9.0 software package. The
statistical significance of the differences between the control and treatments was assessed
with the Mann–Whitney test (P<0.05).
Results
The qPCR assay demonstrated in the corossolone treatment group a statistically
similar parasite burden in both liver and spleen was found, in the annonacinone group the
parasite burden in the spleen was five times lower when compared to the liver and the
control group treated with glucantime demonstrated a parasite burden statistic lower than
spleen. When compared to the control group the parasite burden was statistically similar
among all livers and corossolone spleen. The kDNA mRNA expression was statistically
similar between annonacinone and glucantime spleen (Fig. 1).
124
The immunohistochemistry just did not demonstrated the presence of Leishmania
amastigote form stained in brown in the spleen in all groups, and any liver shown the
presence of the parasite stained (Fig. 2).
The immunohistochemistry did not show the parasite presence in all livers but
were well marked in the spleen, this result agrees with Moreira et al. (2007) that the liver
demonstrated a lower sensitivity to the immunohistochemistry than the spleen, and the
PCR assay is more precise than the immunohistochemistry. Amato et al. (2009) also
confirmed the PCR greater sensibility compared to the immunohistochemistry. These
results support our data showing that the absence of Leishmania amastigote stained in the
liver is due to the low sensitivity that the immunohistochemistry assay has in this organ,
and the qPCR is a greater way to determine the parasite burden.
The search for new phytherapics is limited by many aspects, especially by the lack
of continually of researches. In this work we tested in vitro compounds isolated from A.
muricata leaves already with in vitro leishmanicidal activity. The results showed that this
plant can provide compounds that can be used as phytotherapic agents.
Fig 1. Relative kDNA mRNA expression detected by qPCR in the liver and spleen
of BALB/c groups infected with L. i. chagasi and treated with annonacinone, corossolone
and glucantime.
125
(a)(
(a)
(b)
(c)
(d)
Fig. 2 Parasite burden by anti-Leishmania immunohistochemistry (a) Infected not treated,
(b) Glucantime, (c) annonacinone, and (d) corossolone.
Support
This research is part of the program for development of new phytotherapic against
leishmaniasis in South America supported by PPSUS, CNPq and FUNCAP.
Referências
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Amazon region. Int J Dermatol 2009; 48:1091-1095.
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Trends Parasitol 2005; 21: 462-468.
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Chan-Bacab MJ, Pena-Rodríguez LM. Plant natural products with leishmanial activity.
Roy Soc Chem 2001; 18: 674-688.
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127
11 CONCLUSÕES
Visando a obtenção de produtos naturais com atividades leishmanicidas foram
realizados procedimentos químicos para isolamento e obtenção de derivados de produtos
naturais abundantes seguidos da avaliação das suas atividades leishmanicida contra as
formas promastigota e amastigota, determinação de toxicidade e testes in vivo. As
principais conclusões deste estudo foram:
O estudo químico das folhas na Annona squamosa forneceu um alcaloide
denominado O-metilameparvina e uma acetogenina bistetrahidrofuranica. Das folhas
da Annona muricata foram isoladas duas acetogeninas, annonacinona e corossolona.
Do caule e cerne do Platymiscium floribundum obteve-se uma cumarina, escoporona, e
das sementes da Dimorphandra gardneriana foi isolado o flavonóides rutina e desta
preparou-se a quercetina.
Para obtenção dos derivados do timol e eugenol foram realizados os processos de
acetilação e benzoilação, obtendo-se: acetil-timol, benzoil-timol, acetil-eugenol e
benzoil-eugenol.
Os compostos isolados e os derivados do timol e eugenol demonstraram relevante
ação leishmanicida contra as formas amastigota e promastigota das espécies de
Leishmania: L. i. chagasi, L. major, L. mexicana e L. donovani.
A toxicidade foi avaliada utilizando-se camundongos Swiss, determinando-se a
concentração de 100 µg/Kg dos compostos para serem utilizados na avaliação da
atividade leishmanicida in vivo.
As acetogeninas corossolona e annonacina e os derivados to timol e eugenol
demonstraram sua atividade leishmanicida in vivo em camundongos BALB/c
infectados com L. i. chagasi.
128
12 PERSPECTIVAS
A leishmaniose visceral é um problema de saúde pública que afeta diversas
regiões no país, os cães acometidos pela doença são os principais reservatórios e o
tratamento destes com terapias tradicionalmente utilizadas em humanos não é eficaz e a
recidiva dos sintomas é frequente, além do animal continuar portador e permitir que o
ciclo transmissivo continue.
A ação leishmanicida de alcaloides, acetogeninas, cumarinas, flavonoides,
arilpropanóides e fenilpropanóides abordados neste trabalho abrem possibilidades que
levam ao desenvolvimento de um novo fitoterápico capaz de diminuir a parasitemia do
animal ou até mesmo a cura, o que poderá acarretar na redução da transmissão da
leishmaniose visceral.
129
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