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ANDREA MAYUMI KOROISHI
ATIVIDADE ANTIFÚNGICA IN VITRO DOS EXTRATOS E NEOLIGNANAS DE Piper
regnellii CONTRA DERMATÓFITOS
Dissertação apresentada ao Programa de Pós-Graduação em
Ciências Farmacêuticas (área de concentração – Produtos
naturais e sintéticos biologicamente ativos), da
Universidade Estadual de Maringá para a obtenção do grau
de Mestre em Ciências Farmacêuticas.
Orientador: Prof. Dr. Benedito Prado Dias Filho
Co-orientador: Prof. Dr. Diógenes Aparício Garcia Cortez
Maringá
2006
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Este trabalho foi realizado no Departamento de Farmácia e
Farmacologia da Universidade Estadual de Maringá, sob
orientação do Prof. Dr. Benedito Prado Dias Filho, e contou
com o apoio financeiro parcial do Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CAPES),
Fundação Araucária e Programa de Pós-graduação em
Ciências Farmacêuticas da Universidade Estadual de
Maringá.
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Aos meus pais, Toshiharu Koroishi e Marly Mieco Ishizu Koroishi, ao meu irmão Edson
Hideki Koroishi, ao meu namorado Daniel Rodrigues Silva, aos meus avôs Munenobu Ishizu,
Siyone Koroishi e Tissato Koroishi pela paciência, incentivo, carinho e amor.
AGRADECIMENTOS
Ao professor Dr. Benedito Prado Dias Filho, meus sinceros agradecimentos, não apenas pela
orientação firme e segura demonstrada na elaboração deste trabalho, mas também pelo
incentivo, confiança e amizade nesses anos de convivência.
Ao coordenador do Programa de Pós-graduação em Ciências Farmacêuticas da Universidade
Estadual de Maringá, Prof. Dr. Celso Vataru Nakamura.
Ao Prof. Dr. Diógenes Aparício Garcia Cortez, pela co-orientação e amizade.
Aos professores Dr. Benício Alves de Abreu Filho e Dra. Tânia Ueda-Nakamura pela
amizade, pelos conhecimentos transmitidos e pela confiança depositada.
A aluna de iniciação científica e grande amiga Simone Rochtaschel Foss pelo apoio na
elaboração de todo o trabalho.
Aos meus amigos que apoiaram em todos os momentos Cecília Valente Truite, Elza
Yamaguti, Jean Colacite, Nilza de Lucas Rodrigues Bittencourt, Raíssa Bocchi Pedorso.
Aos meus colegas que me auxiliaram no Laboratório de Microbiologia Aplicada a Produtos
Naturais e Sintéticos e pela grande amizade Amanda Bortoluci da Silva, Denise de Oliveira
Scoaris, Eliana Harue Endo, Ivens Camargo Filho, Adriana Oliveira dos Santos, Érika
Ravazzi Franco Ramos, Heloísa Bressan Gonçalvezs, Kelly Ishida, Rafael Eidi Yamamoto,
Simone Evellyn Daniel Hernandes, Michele Cristina Vendrametto, Patrícia Mayumi Honda,
Thelma Onozato.
Aos técnicos do Laboratório de Microbiologia e Farmacognosia Adimir Arantes, Adriana R.
Barra Vieira, Maria Manzoti, Marcio Guilhermetti, Marinete Martinez, Prisciliana Carvalho,
Rosana Monteiro, Zelita Rodrigues pela amizade.
Às minhas grandes amigas Ângela Delly Cembranel, Patrícia Nishimura, Solange Pinoti
Primo pelo incentivo e companheirismo.
Ao Programa de Pós-graduação em Ciências Farmacêuticas desta Universidade e ao corpo
técnico/administrativo, pela oportunidade de realização deste trabalho, apoio e serviços
prestados, em especial a Helena e a Sônia, secretárias do Departamento de Farmácia e
Farmacologia.
E a todos que contribuíram de alguma forma para a realização deste trabalho, muito obrigada.
“A coisa mais bela que o homem pode experimentar é o
mistério. É essa a emoção fundamental que está na raiz de
toda a ciência e de toda a arte.”
(Albert Einstein)
RESUMO
O presente estudo foi desenvolvido para avaliar a atividade antifúngica dos extratos
hidroalcoólicos das folhas de Piper regnellii, comumente conhecida como pariparoba,
amplamente distribuída em regiões tropicais e subtropicais. As micoses superficiais estão
presentes em regiões cutâneas, pêlos, unhas, e os agentes patogênicos compreendem os
fungos dermatófitos (Microsporum, Trichophyton e Epidermophyton), Piedraia hortai,
Corynebacterium sp., Malassezia furfur (Pitiríase versicolor), Nocardia minutíssima
(Eritrasma), Cladosporium vernecki (Tinea nigra), Aspergillus peniciloides (Tinea albigena),
e além disso, as leveduras, Rhodotorula, Torulopsis, Cryptococcus, Candida, Trichosporon,
Klöckera.. Foram obtidos extratos brutos hidroalcoólicos das folhas de Piper regnellii a 50,
70 e 90%, sendo o mais ativo o extrato bruto 90% frente aos fungos dermatófitos T. rubrum,
T. mentagrophytes, M. canis e M. gypseum (CIM= 15,6, 15,6, 15,6 e 62,5 µg/mL,
respectivamente) e inativo a fungos não dermatófitos Aspergillus niger (CIM>1000 µg/mL) e
levedura, Candida albicans (CIM>1000µg/mL). O modelo biológico escolhido foi T. rubrum
por ser mais sensível as drogas. Baseado nestes resultados, o extrato hidroalcoólico 90% foi
fracionado por cromatografia em coluna com ensaios bio-dirigidos, sendo a fração
clorofórmio mais ativo com CIM=6,2 µg/mL. A fração ativa foi então submetida à
cromatografia em camada delgada preparativa, foram obtidos seis bandas, e a mais ativa F2C,
apresentou CIM de 3,1 µg/mL, esta foi caracterizada em cromatografia líquida de alta
eficiência e foram obtidas três substâncias, o conocarpano (1), eupomatenóide-5 (3) e
eupomatenóide-6 (4), ambas com CIM igual a 100 µg/mL. Para o entendimento do
mecanismo de ação foram feitos ensaios de inibição da germinação de esporos, inibição do
crescimento de hifas, invasão em unhas e de citotoxicidade. O extrato bruto 90% inibiu a
germinação de esporos e crescimentos de hifas a 7,8 µg/mL e no ensaio de citotoxicidade,
cerca de 98% das células estavam viáveis a 50 µg/mL, indicando assim que o extrato bruto
hidroalcoólico 90% é seletivo às células fúngicas. Além disso, o extrato ativo foi fungicida na
concentração de 1,2 mg/mL no ensaio de invasão em unhas. Em conclusão, os dados in vitro
podem ser úteis no uso de folhas de Piper regnellii para o tratamento de infecções por
dermatófitos, e em termos de conservação, indicando que pode ser usado sem danos à planta.
Palavras-chave: Piper regnellii, Piperaceae, eupomatenóide, antidermatófito, neolignanas
LISTA DE ILUSTRAÇÕES
Figure 1 Effect of different concentrations of the hydroalcoholic (90%) extract of P. regnelli
leaves on growth of M. canis (A) and T. rubrum (B). The spore suspensions (5 l)
were mixed with 20 l of various concentrations of crude extract (0.03, 0.07, 0.15,
0.32, 0.64, 1.28, 2.5 and 5.0g/ml) and added on petri dishes containing SDA, as
described in Materials and methodos. The minimal concentration of crude extract
that resulted in inhibition of both dermatophytes was 1.28 g. Data correspond to
one representative experiment out of three………………………………………52
Figure 2 Spore germination inhibition of hydroalcoholic (90%) extract against T. rubrum: (A),
Control; (B) and (C) treated with 3.9 and 7.8 g/ml of hydroalcoholic (90%)
extract, respectively. Magnification x 20. Similar results were obtained in the
different
analyses…………………………………………………………………………..53
Figure 3 Growth of T. rubrum on cover slips after treatment with different concentrations of
P. regnellii leaves extracts. (A) 7.8µg/mL 50% hydroalcoholic extract; (B)
15.62µg/mL 70% hydroalcoholic extract; (C) 7.8 g/ml 90% hydroalcoholic
extract; (D) 0.3 g/ml Nystatin. Data correspond to one representative experiment
out of three………………………………………………………………………54
Figure 4 Light microscopy (A-B) and scanning electron microscopy (C-D) of nail fragments
treated (A and C) and untreated (B and D) with hydroalcoholic (90%) extract at a
concentration of 1.2 g/ml. Similar results were obtained in the different
analyses……………………………………………………………………………55
Figure 5 Cytotoxic assay. The hydroalcoholic (90%) extract was also evaluated for its potential
toxic effects on Vero cell monolayer. After 48 h of incubation with 50 g/ml of
crude extract (A) and 5 g/ml of Nystatin (B) 98 and 100 % of the cells were still
viable, respectively. (C) Control. These results indicate that hydroalcoholic (90%)
extract is selectively toxic to the fungal cells. Similar results were obtained in the
different analyses…………………………………………………………………56
Figure 6 (A) Thin layer chromatography plates of hydroalcoholic extracts (50, 70 and 90%,
v/v) and fractions (F1 to F9) obtained from active hydroalcoholic (90%) extract.
(B). Preparative thin layer chromatography of the chloroform fraction being
visualized, 6 bands were scraped from the other set, fractionated by thin-layer
chromatography (F2A-F2F), assayed for antifungal activity and used for bioassay-
guided isolation. Similar results were obtained in the different
analyses……………………………………………………………………………57
Figure 7 HPLC on reverse-phase resin Microsorb C-18. (A) The sub-fraction F2C with
antifungal activity was applied in a reverse-phase column Microsorb- MV 100-5
C-18 (250 x 4.6) equilibrated and eluted with. Acetonitrile:water (60:40, v/v)
containing 2% acetic acid: flow-rate of 1.0 ml/min; temperature: 30 °C; detection:
280 nm. (B) The standard mixture of neolignans: conocarpan (1), eupomatenoid-6
(2) and eupomatenoid-5 (3). Data correspond to one representative experiment out
of three…………………………………………………………….…………...…58
Figure 8 Structures of the neolignans isolated from P. regnellii. (1) conocarpan, (2)
eupomatenoid-3, (3) eupomatenoid-5, (4) eupomatenoid-6..…………………………….......59
LISTA DE TABELAS
TABELA 1 Minimal inhibitory concentration (MIC) of hydroalcoholic crude extracts of
leaves from P. regnellii………………………………………………….….60
TABELA 2 Antifungal activity of fraction obtained from hydroalcoholic extract (90%) of
leaves from P. regnellii against dermatophyte T. rubrum…………………..61
TABELA 3 Minimal concentration of sub-fractions obtained from fraction F2 (chloroform)
required for inhibition of spore germination of dermatophyte Trichophyton
rubum………….……………………………………………………………62
SUMÁRIO
1 REVISÃO BIBLIOGRÁFICA............................................................................................14
1.1 PLANTAS MEDICINAIS..................................................................................................14
1.2 FAMÍLIA PIPERACEAE...................................................................................................15
1.3 FUNGOS DERMATÓFITOS.............................................................................................19
1.4 MICOSES SUPERFICIAIS................................................................................................19
1.5 AGENTES ANTIFÚNGICOS............................................................................................20
2 OBJETIVOS.........................................................................................................................25
REFERÊNCIAS...................... ……………………………………………………………26
3 ARTIGO: IN VITRO ANTIFUNGAL ACTIVITY OF EXTRACTS AND
NEOLIGNANS FROM Piper regnellii AGAINST
DERMATOPHYTES……………………….…………………………...33
4 ANEXO……………………………….………………………………………………….52
1- REVISÃO BIBLIOGRÁFICA
1.1 - PLANTAS MEDICINAIS
No planeta existe uma grande variedade de plantas, cerca de 250 – 500 mil espécies,
contudo a maioria ainda desconhecida cientificamente e pouco mais de 5% estudada
fitoquimicamente, e uma porcentagem menor avaliada sob os aspectos biológicos
(CECHINEL FILHO e YUNES, 1998). Até o início do século XIX, a atividade terapêutica
das plantas medicinais utilizadas na época pouco se diferenciava dos medicamentos
(SCHENKEL et al, 2004).
As plantas utilizadas na medicina popular em todo o mundo, e em especial na América
Latina, têm uma grande importância no atendimento primário à saúde. Entretanto, poucas
plantas foram validadas do ponto de vista farmacológico, fitoquímico, biológico ou clínico
(YUNES e CECHINEL FILHO, 2001).
É importante ressaltar que plantas utilizadas na medicina popular com finalidades
terapêuticas, bem como os estudos de plantas com fins de preservação ambiental, têm
contribuído, e muito, para a descoberta de novos fármacos que recentemente são utilizados em
tratamentos clínicos.
Espécies nativas dos gêneros Baccharis sp. (carqueja), Bauhinia sp. (pata-de-vaca),
Cecropia sp.(embaúba), Maytenus sp. (espinheira santa), Mikania sp. (guaco) e Passiflora sp.
(maracujazeiro) são de uso popular, além disso, outras espécies nativas do Brasil foram
inicialmente usadas com finalidade terapêutica por comunidades indígenas e caboclas (REIS
et al, 2004). Nos séculos IX e X, descobriram compostos ativos de Valeriana officinalis com
ação sedativa, entre os compostos ativos estão os ésteres do ácido isovalérico. Em 1763, Salix
alba foi estudada devido ao uso das cascas para combater a febre e a dor, descobriu-se que
tinha efeito analgésico. Em 1828, Buchner isolou a salicilina (glicosídeo do álcool salicílico).
Em 1860, Kolbe e Lauteman sintetizaram o ácido salicílico e seu sal sódico a partir do fenol, e
assim, ocorreu a primeira produção de salicilatos em 1874. Felix Hofman, em 1898, acetilou o
grupo hidroxila em posição orto, e descobriu o ácido acetil salicílico, este foi o primeiro
fármaco sintético a partir de uma molécula derivada de uma planta (YUNES e CECHINEL
FILHO, 2001).
Portanto se faz necessário à ampliação dos estudos com plantas que sejam tanto de uso
popular ou não, pesquisando assim, novas moléculas com mais seletividade, baixa toxicidade
e alto potencial farmacológico.
1.2 – FAMÍLIA PIPERACEAE
A família Piperaceae pertence à classe Magnolipsida, subclasse Magnoliidae, subordem
Nymphaeiflorae e ordem Piperales (MAcRAE; TOWERS, 1984; SANTOS et al., 2001 apud
PESSINI, 2003). Está presente desde o México até o sudeste da Argentina (FIGUEIREDO,
2000). A família Piperaceae é constituída pelos gêneros Peperomia, Ottonia, Pothomorphe e
Piper, e é um exemplo por possuir propriedades medicinais largamente empregadas pela
população (COSTA, 1972).
Arrigoni-Blank et al (2004) verificou que o extrato aquoso de partes aéreas de
Peperomia pellucida inibiu o edema induzido por carragenina em animais, e que isso, poderia
ser pela inibição de diferentes efeitos e mediadores químicos da inflamação. Verificaram
também que esta espécie tem um efeito analgésico, provavelmente envolvido no mecanismo
de síntese de prostaglandinas.
Pothomorphe umbellatta tem sido usada como agente analgésico, diurético e
antiespasmódico, antinflamatório, antimalárico, asma e distúrbios gastrointestinais. Estudos
têm mostrado que uma concentração de 500mg/Kg de extrato hidroetanólico de P. umbellatta
inibiu o edema de pata induzido por carragenina durante quatro horas, efeito similar ao da
indometacina, comprovando assim, o uso popular no tratamento de distúrbios inflamatórios
(PERAZZO, 2005).
Estudos anteriores mostraram que a piperovatina, uma amida encontrada em Ottonia
frutescens, apresentou atividade biológica de anestesia, além de outras características como
um leve efeito de salivação e queimação na língua (MAKAPUGAY et al, 1983). A
piperovatina também foi encontrada em Piper alatabaccum (FACUNDO, 2005)
O gênero Piper tem cerca de 700 espécies amplamente distribuído em regiões tropicais e
subtropicais, tanto no hemisfério norte quanto no sul, além disso, é usado popularmente como
medicinal (PARMAR et al, 1997). P. sarmentosum, conhecida popularmente como “Cha-plu”
na Indonésia, Malásia e Tailândia, possui amidas ativas contra a Mycobacterium tuberculosis
e Plasmodium falciparum, a sarmentina e 1-piperetil pirrolidina (RUKACHAISIRIKUL,
2004).
A presença de metil-éster ácido lancefólico e chalcona pinocembrina de P.
lanceaefolium foram ativos contra Candida albicans, comprovando o uso popular na
Colômbia (LÓPEZ et al, 2002).
P. dilatatum, usado popularmente pelos índios Kuna no Panamá, apresentou compostos
isolados a partir do extrato diclorometano fracionado em cromatografia em coluna de sílica
gel e purificado em coluna de Sephadex gel LH 20. Os compostos obtidos foram ativos contra
um fungo patogênico de plantas, o Cladosporium cucumerinum (TERREAUX et al, 1998).
Foi relatado também a presença de diversas classes de substâncias em outras espécies de
Piper como neolignanas e lignanas em P. clarkii (PRASAD et al, 1995), ciclohexanos
oxigenados em P. cubeb (KOUL et al, 1996), fenilpropanóides e neolignanas em P. regnellii
(BENEVIDES et al, 1999), neolignanas em P. aequale (MAXWELL et al, 1999).
Holetz et al (2002) em um estudo de avaliação de plantas utilizadas popularmente para o
tratamento de doenças infecciosas, verificaram que P. regnellii apresenta CIM de 7,8µg/mL
para S. aureaus, 15,6µg/mL para B. subtilis, 250µg/mL para P. aeruginosa, 125µg/mL para
Candida krusei.
N
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MeO
N
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4
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O
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O
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3
C
H
H
H
OAc
OAc
AcO
H
AcO
H
H
H
OBr
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OMe
OMe
O
H
3
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O
H
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Figura 1 (1) Piperovatina (Ottonia frutescens), (2) sarmentina e (3) 1-piperetil pirrolidina (P.
sarmentosum), (4) 4’-metóxi flavona (P. clarkii), (5) piperenol C (P. cubeb), (6) dilapiol (P.
regnellii), (7) conocarpano, (8) eupomatenóide-6, (9) eupomatenóide-5 (P. aequale).
1
2
3
4
5
6
7
8
9
1.3. FUNGOS DERMATÓFITOS
Os fungos dermatófitos pertencem ao reino Fungi, divisão Deuteromicetes, classe
Coelomicetes, ordem Moniliales, família Moniliaceae e gêneros Trichophyton, Microsporum
e Epidermophyton. Os fungos dermatófitos se reproduzem assexuadamente pelo processo de
gemulação, ou seja, as células se desprendem das hifas, sofrem diferenciação e disseminam
pelo meio ambiente, essas células são chamadas de conídios que são geneticamente iguais às
células parentais (TORTORA, 2000). Em locais com nutrientes apropriados, presença de água
e oxigênio, os conídios se hidratam rapidamente e ocorrem alterações nas propriedades da
superfície como o aumento na adesão com outros conídios e ao substrato (OSHEROV; MAY,
2001).
Quando cultivados in vitro, as condições de crescimento são a temperatura de 28°C,
umidade e meio de cultura apropriado. Os dermatófitos além de consumir carboidratos,
proteínas, polipeptídeos, aminoácidos, uréia, são capazes também de hidrolisar a gelatina e a
queratina. Outros nutrientes são essenciais como o fósforo, cloro, enxofre, sódio, potássio,
cálcio e magnésio que estão presentes em sais como o fosfato monopotássico, fosfato
dipotássico, cloreto de cálcio, sulfato de magnésio, cloreto de sódio e cloreto de potássio
(ESTEVES, 1990).
1.4 - MICOSES SUPERFICIAIS
As micoses superficiais se localizam em regiões cutâneas e seus anexos, como pêlos,
unhas e cabelos. Entre os agentes que causam esta patologia estão os dermatófitos, além disso,
Piedraia hortai, Corynebacterium sp., Malassezia furfur (Pitiríase versicolor), Nocardia
minutíssima (Eritrasma), Cladosporium vernecki (Tinea nigra), Aspergillus peniciloides
(Tinea albigena), e além disso, as leveduras, Rhodotorula, Torulopsis, Cryptococcus,
Candida, Trichosporon, Klöckera. Os fungos dermatófitos parasitam a pele e seus anexos
para consumir a queratina, incluem as espécies dos gêneros Microsporum, Trichophyton e
Epidermophyton (LACAZ, 1984). De acordo com Costa et al (1999), um estudo feito com
indivíduos da cidade de Goiânia, o pé foi o local mais acometido pela infecção (30%), seguida
da região crural (17%), corpo (16%), couro cabeludo (13%), unha do pé (12%) e unha da mão
(12%). Nesse mesmo estudo, os fungos mais freqüentemente isolados foram T. rubrum e T.
mentagrophytes, sendo a espécie M. canis o maior agente etiológico das lesões de couro
cabeludo.
Segundo Esteves (1990), T. rubrum pode ser transmitido através de várias gerações e em
vários membros da mesma família. Este microorganismo também pode estar presente em
vários locais, como roupas, calçados, toalhas, escovas de cabelo, sabonete e piscinas.
Entre as micoses superficiais está a onicomicose, e os fungos patogênicos mais comuns
são o T. rubrum e T. mentagrophytes. Este tipo de micose, caracteriza-se como unhas friáveis,
corroídas, secas, escamosas, com estrias longitudinais, podendo haver separação do limbo e
do leito da unha. Outros fungos das espécies Microscporum, Epidermophyton, Candida
albicans, Rhodotorula sp., Aspergillus sp., Penicillium sp., Fusarium oxysporum, entre
outros, também podem causar esta infecção de unha (LACAZ, 1984). Além disso, a micose
superficial pode ocorrer em várias partes do corpo, como região inguinal (tinea cruris),
pescoço, tronco e abdômen (tinea versicolor), perigenital e axilar (eritrasma), no couro
cabeludo (tinea capitis e tinea tonsurans), barba (tinea barbae), entre outros.
1.5 - AGENTES ANTIFÚNGICOS
Os agentes antifúngicos atuais são classificados em fármacos sistêmicos (orais ou
parenterais), fármacos orais para infecções mucocutâneas, e fármacos tópicos para infecções
mucocutâneas (SHEPPARD et al, 2001).
Para o tratamento de micoses superficiais utiliza-se a griseofulvina cujo mecanismo de
ação é interferir nos microtúbulos fúngicos. No grupo dos azóis, o clotrimazol e miconazol de
uso tópico são os mais comuns para tratar micoses superficiais. A terbinafina tem ação
fungicida, porém, interfere na síntese de ergosterol agindo sobre a enzima esqualeno
epoxidase, ocorre o acúmulo de esterol esqualeno que é tóxico ao microorganismo
(SHEPPARD et al, 2001). Um outro azol, o 1-amino-6-metil-4-fenilpirazol-[3,4,-d]-1,2,3-
triazol promoveu alterações extra e intracelulares no fungo (MARES et al, 1999). De acordo
com Rashid et al (1995), a infecção de unhas pelo T. rubrum foi baixo quando tratadas com
terbinafina. Segundo Zaug (1995), verificou que o uso de esmalte contendo amorolfina em
pacientes com onicomicose apresentou cura ou melhora clínica. Gupta (2000) verificou
também o uso de esmalte para tratamento de onicomicose, porém, em sua formulação
continha ciclopirox 8% confirmou a eficácia deste antifúngico tópico.
Diterpenos, derivados do ácido p-cumárico e flavonas de Baccharis grisebachii
(Asteraceae) apresentaram atividade antifúngica entre 100 e 125µg/mL contra T. rubrum, 50 -
250µg/mL contra T. mentagrophytes (FERESIN et al, 2003)
Segundo Apisariyakul et al (1995), o óleo turmérico de Curcuma longa (Zingiberaceae)
inibiu o crescimento de Microsporum gypseum com CIM de 459,6µg/mL, T. mentagrophytes
e T. rubrum com CIM variando de 229,8 – 919,2µg/mL e Epidermophyton floccosum com
CIM entre 114,9 – 229,8µg/mL.
Aljabre et al (2005) através do método de difusão em ágar consideraram a porcentagem
do crescimento fúngico como sendo controle 100%, e o CIM foi considerado quando a
inibição estiver na faixa de 80 a 100%. A porcentagem de inibição para T. rubrum foi de 32,5
a 48 para o extrato de Nigella sativa (Ranunculaceae) (2,5mg/mL), 0 – 32,8 para timoquinona
(0,062mg/mL) e 39,6 – 52,1 para griseofulvina (0,00095mg/mL).
A partir de folhas de Hyptis ovalifolia (Lamiaceae) foram extraídos o óleo essecial,
frações aquosas, metanólicas e hexânico. O extrato metanólico teve amplo espectro de
atividade, o qual inibiu 90% dos dermatófitos isolados, a concentração que inibiu foi menor
ou igual a 1000µg/mL (SOUZA et al, 2003).
Ghahfarokhi et al (2004) utilizaram extrato aquoso de cebola (Allium cepa L.) para
verificar a inibição do crescimento que foi de 78,12 e 53,19% na concentração de 3,12% de
extrato, respectivamente para T. mentagrophytes e T. rubrum. A inibição foi determinada pela
pesagem do peso micelial através dos filamentos retidos durante a filtragem.
A seiva de Croton urucuran (Euphorbiaceae) foi testada através do ensaio de difusão em
disco de papel e o método de diluição em caldo. No teste de difusão, produziu zonas de
inibição na concentração de 3mg/mL e com diâmetro variando entre 21,8 – 26,9mm contra T.
tonsurans, T. rubrum, M. canis e E. floccossum. Enquanto no método de diluição, a CIM foi
de 2,5mg/mL, com exceção de T. tonsurans que foi de 1,25mg/mL (GURGEL, 2005).
Além disso, a família Fabaceae também tem atividade antidermatofítica, como exemplo,
a Psoralea corylifolia. Extratos obtidos a partir da utilização de solventes com polaridades
diferentes, como o éter de petróleo, éter dietílico, benzeno, clorofórmio, acetona, metanol,
etanol e água em soxlet a 50 – 55°C. O extrato metanólico teve atividade de 250µg como
atividade máxima, apresentando um halo de inibição de 28mm de diâmetro. Posteriormente,
este extrato, foi submetido à cromatagrafia em camada delgada, e apresentou seis diferentes
bandas (PRASAD et al, 2004).
A pesquisa de novos agentes antifúngicos a partir de plantas usadas popularmente com a
finalidade de se tratar algum tipo de enfermidade, como cicatrização, doenças de pele,
tumores, inflamação, entre outras, tem aumentado devido à toxicidade dos antifúngicos
sintéticos. Schmourlo et al (2005) através de um screening de plantas pelo método da
decocção ou caldo, verificaram que a Xanthosoma sagittifolitum L. Schott apresentou
atividade inibitória de 100ng/mL contra T. rubrum.
O
O
O
CH
3
OCH
3
Cl
OCH
3
H
3
CO
N
N
C
Cl
N
N
CH
2
Cl
Cl
CHO
CH
2
Cl
Cl
N
CH
3
CH
3
CH
3
CH
3
N O
CH
3
OH
N C(CH
3
)
3
CH
3
Figura 2 Antifúngicos sintéticos. (1) Griseofulvina, (2) Clotrimazol, (3) Miconazol, (4)
Amorolfina, (5) Ciclopirox, (6) Terbinafina
2
1
3
4
5
6
COOH
OCH
3
O
O
OH
OH
H
3
CO
O O
Figura 3 Antifúngicos naturais (1) Ácido O-hexano-3-onil-éter trans-ferúlico (Baccharis
grisebachii), (2) Metil-éster ácido lancefólico (Piper lanceaefolium)
1
2
2- OBJETIVOS
Com base no uso de diversas plantas pela população com finalidade terapêutica, o
presente trabalho teve como objetivo:
- Pesquisar extratos brutos em diversas alcoolaturas de Piper regnellii, conhecida
popularmente como pariparoba, para a atividade antifúngica contra fungos filamentosos e
leveduras;
- Purificação das frações ativas;
- Isolamento e identificação das substâncias isoladas;
- Determinar os mecanismos de ação e alterações morfológicas causadas pelas ações
dos extratos, frações e substâncias isoladas, através de ensaios de inibição de germinação de
esporos, crescimento de hifas, microscopia eletrônica de varredura e microscopia de
fluorescência;
- Verificar a invasão em unhas e tratamento, por microscopia óptica e microscopia
eletrônica de varredura;
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In vitro antifungal activity of extracts and neolignans from Piper regnellii against
dermatophytes
Antidermatophyte activity of extracts and neolignans from Piper regnellii
Andrea M. Koroishi
a
, Simone R. Foss
b
, Diógenes A. G. Cortez
c
, Tânia Ueda Nakamura
d
,
Celso Vataru Nakamura
d
, Benedito P. Dias Filho
d
*
a
Programa de Pós-graduação em Ciências Farmacêuticas.
b
CNPq fellowship
c
Departamento de Farmácia e Farmacologia.
d
Departamento de Análises Clínicas, Universidade Estadual de Maringá, Av. Colombo, 5790,
87020-900 Maringá, PR
Corresponding author
Phone: + 55 44 3261 4955. Fax: + 55-44-32614860, e-mail address: [email protected]
ABSTRACT
The present study was done to evaluate the antifungal activity of the hydro alcoholic leaves
extracts of Piper regnellii, popularly known as pariparoba, which is widely distributed at
tropical and subtropical regions. Superficial mycosis are present at cutaneous regions, nails
and hair, and the pathogenic agents comprehend the dermatophytes fungi (Microsporum,
Trichophyton and Epidermophyton), Piedraia hortai, Corynebacterium sp., Malassezia furfur
(Pityriasis varicolored), Nocardia minutíssima (Erythrasma), Cladosporium vernecki (Tinea
nigra), Aspergillus peniciloides (Tinea albigena), besides that, the yeasts, Rhodotorula,
Torulopsis, Cryptococcus, Candida, Trichosporon, Klöckera. 50, 70 and 90% crude
hydroalcoholic extracts of Piper regnellii leaves were obtained, being the last one the most
active against the dermatophytes fungi T. rubrum, T. mentagrophytes, M. canis and M.
gypseum (MIC= 15.6, 15.6, 15.6 and 62.5 µg/mL, respectively) and inactive against the non
dermatophytes fungi Aspergillus niger (MIC>1000 µg/mL) and the yeast Candida albicans
(MIC>1000µg/mL). The biological model chosen was T. rubrum because it is more sensible
to drugs. Based on the results, the 90 % hydroalcoholic extract was fractionated by column
chromatography with bioassays-guided, being the chloroform fraction the most active with a
MIC=6.2 µg/mL. The active fraction was than submitted to preparative thin-layer
chromatography and six bands were obtained, being F2C the most active with a MIC of 3.1
µg/mL, this one was characterized by high performance liquid chromatography and three
compounds were obtained, conocarpan (1), eupomatenoid-5 (3) and eupomatenoid-6 (4), both
with MIC equal to 100 µg/mL. For a better understanding of the mechanism of action, spore
germination inhibition, hyphal growth inhibition, inhibitory effect on the invasion of nails and
cytotoxicity assays were done. The 90 % crude extract inhibited the spore germination and
hyphal growth at 7.8 µg/mL and at the cytotoxicity assay 98 % of the cells were viable at 50
µg/mL, indicating that 90 % hydroalcoholic crude extract is selective for fungal cells. Besides
that, the active extract was fungicidal at 1.2 mg/mL at the nails invasion assay. In conclusion,
the in vitro data can be helpful to the use of Piper regnellii leaves for the treatment of
dermatophytes infections and in terms of conservation; it could be used without any
detrimental effect for the plant.
Key words: Piper regnellii, Piperaceae, eupomatenoid, conocarpan, antidermatophyte,
neolignans.
1. Introduction
The dermatophytes belonging to three genera, Trichophyton, Microsporum and
Epidermophyton, have the ability to invade keratinized tissues, such as hair, skin or nails, of
humans and other animals (Weitzman and Summerbell, 1995). As a result of the tissue
invasion, these fungi cause dermatitis named dermatophytosis. Forms of the disease include
tinea corporis, tinea pedis and onychomycosis. The advent of HIV infection and
immunosuppression induced by organ transplants or cancer chemotherapy lead to increased
predisposition to fungal infections (Walsh et al., 2004). Many antifungal drugs including
imidazoles, butenafine and terbinafine, have been used clinically for the topical treatment of
dermathophytosis (Watanabe, 1999). In addition, triazoles, griseofulvin and terbinafine are
used as oral antifungal drugs for systemic therapy of severe dermatophytosis (Lesher, 1999).
The prolonged duration of treatment, drug toxicity and interactions, fungal resistance and high
costs are the major reasons for discontinue (de Pauw, 2000; Bennett et al., 2000). These
factors render the development of new more efficient and safe antifungal drugs a requirement.
The advent of synthetic antimicrobials, in the mid of the last century, lead to lack of
interest in plants as a natural source for antimicrobial drugs (Cowan, 1999). In the recent
years the situation has changed and the field of ethnobotanical research is raising
(McCutcheon et al., 1992).
Plants which have been used as medicines over hundreds of years, constitute an obvious
choice for study. Piper regnellii of Piperaceae family is an herbaceous plant found in tropical
and subtropical regions of the world (Cronquist, 1981). Leaf and root are used as crude
extracts, infusions or plasters to treat wounds, reduction of swellings and skin irritations
(Yuncker, 1972, 1973; Corrêa 1984). The search for active constituents from different Piper
species has been intensified in recent years, especially due to the finding that several species
have been shown to have a number of biological activities. Phytochemical study of P.
regnellii roots has shown the accumulation of several phenylpropanoids and
dihydrobenzofuran neolignans including (+)-conocarpan as major compound. This compound
displays a variety of biological activities including anti-PAF (Pan et al., 1987), antifungical
(Nair and Burke, 1990) and insecticidal activity (Boll et al., 1994; Chauret et al., 1996). In a
screening of Brazilian medicinal plants, we reported the antimicrobial activity of the
hydroethanolic extract obtained fromthe leaves of P. regnellii against the bacteria
Staphylococcus aureus and Bacillus subtilis and against the yeasts Candida krusei and
Candida tropicalis (Holetz et al., 2002). More recently, we reported the chemical composition
and the antibacterial activity of ethanolic extracts fractions from leaves of P.regnellii as well
as of the bioactivity-directed isolates identified as conocarpan (1), eupomatenoid-3 (2),
eupomatenoid-5 (3) and eupomatenoid-3 (4) by spectroscopic analysis and by comparison
with literature data (Pessini et al., 2003).
In the present study the in vitro antidermatophyte activity of hydroalcoholic extracts and
fractions of crude extract from the leaves of P regnellii and the bioassay-guided isolation of
active compounds are described.
2. Material and methods
2.1. Plant material
The leaves of Piper regnellii (Miq.) C. CD. var. pallescens (C. DC.) Yunck. were
collected in August 2001 in Horto of Medicinal Plants “Profª. Irenice Silva" in the Campus of
Universidade Estadual de Maringá. The plant material was identified by Marilia Borgo of the
Botanical Department of Universidade Federal do Paraná, and a voucher specimen (no. HUM
8392) is deposited at the Herbarium of Universidade Estadual de Maringá, Paraná, Brazil.
2.2. Preparation of extracts
The air-dried and powdered leaves of plant (210 g) were extracted with hydroalcoholic
(50, 70 and 90%, v/v) by maceration method at room temperature for 5 days at dark room.
The ethanol-water extracts were filtered, evaporated under vaccum at 40 ºC, lyophilized and
kept in a freezer at about -10 °C. The extracts were assayed against yeast, dermatophyte and
non-dermatophyte fungi as described below.
2.2.1. Isolation of the active compound
The active hydroalcoholic extract (90%) from leaves of P. regnellii was submitted to
vacuum chromatography over on silica gel eluted with hexane (F1) (1000 mL), chloroform
(F2) (1400 mL), chloroform/ethyl acetate 19:1 v/v (F3)(1000 mL), chloroform/ethyl acetate
9:1 v/v (F4) (700 mL), chloroform/ethyl acetate 1:1 v/v (F5)(500 mL), ethyl acetate (F6) (500
mL), acetone (F7) (700 mL), methanol (F8) (1400 mL) and methanol/water 9:1 v/v (F9)
(1800 mL). The resulting fractions were assayed for antifungal activity (Table 1). The active
chloroform fraction was lyophilized and fractionated by preparative thin-layer
chromatography (TLC) on 0.25 mm layers on silica gel G (Merck) using hexane-ethyl acetate
(65:35, v/v) as solvent. Preparative TLC plates were run in duplicate and one set was used as
the reference chromatogram. Spots and bands were visualized by UV irradiation (254 and 365
nm) and vanillin/sulphuric acid (2%) spray reagent. After being visualized, 6 bands were
scraped from the preparative TLC plates and assayed for antifungal activity. The active band
was dissolved in methanol and characterized by HPLC and spectroscopic methods (UV,
NMR) and by comparison with literature data. The HPLC was carried out in using a
Shimadzu LC-10 liquid chromatograph equipped with quaternary pump (LC-10 AD), manual
injection valve (Caplucs) with loop of 20 µl, degasser (DGU – 14A), thermostatted column
compartment (CTO-10ASvp) and a UV-Vis detector (SPD-10Avp), controlled by CLASS
LC-10 Software. In the chromatographic analysis was used MetaSil ODS column, 5 µm, 250
x 4.6 mm, maintained at 30°C. The separation was carried out in isocratic system, using as
mobile phase a mixture of acetonitrile-water (60:40, v/v) containing 2% acetic acid, with flow
rate of 1.0 ml/min. The detection was carried out at 280 nm and the running time was 25 min.
The sample injection volume was 20 µl. Three determinations were carried out for each
sample. Conocarpan (1), eupomatenoid-5 (3) and eupomatenoid-6 (4) were used as reference
to the corresponding peak in the sample extracts. For determination of the UV spectra the
absorbance was recorded in the range of 200-300 mm in a Beckman DB-DC
spectrophotometer. The NMR spectra were obtained in a Bruker ARX400 (9.4 T) and Varian
Gemini 300 (7.05T), using deuterated solvent, TMS as internal standard and constant
temperature of 298K.
2.3. Microorganisms used and growth conditions
The test species used for this investigation were: Microsporum canis, Microsporum
gypseum, Trichophyton mentagrophytes, Trichophyton rubrun, Aspergillus niger and Candida
albicans. The fungi were maintained on Sabouraud dextrose agar (SDA) slants at 10 °C and
subcultured monthly throughout this study.
2.4. Antifungal activity assay
The antifungal activity of extracts, fractions and active compounds of P. regnellii was
studied by microbroth dilution, spore germination inhibition, hyphal growth inhibition and
inhibitory effect on the invasion of nails.
2.4.1. Microbroth dilution assay
The antifungal assay was performed by microdilution techniques in sterile flat botton
microplates (NCCSL 1999, 2000). Each well contained appropriate test samples, RPMI and
approximately 10
4
spores or 10
5
yeasts in a total volume of 100 µl. The plates were incubated
at 28ºC, for 24, 48 and 72 h, depending on the period of incubation time required for a visible
growth; 24 h for Candida albicans, 48 h for Aspergillus niger and 72 h for the dermatophytes.
Two susceptibility endpoints were recorded for each isolated. The MIC was defined as the
lowest concentration of compounds at which the microorganism tested did not demonstrate
visible growth. Minimum fungicidal concentration (MFC) was defined as the lowest
concentration yielding negative subcultures or only one colony.
2.4.2. Conidium germination inhibition
Fungi were grown on Sabouraud dextrose agar (SDA, Difco Laboratories, Detroit, MI)
plates for 7-14 days, after which time spores were harvested from sporulating colonies and
suspended in sterile ion solution. The concentrations of conidium in suspension were
determined using a hemacytometer and adjusted to 1.0 x 10
5
spores/ml. Various
concentrations of the test samples in 90 l were prepared in 96-well flat bottom micro-culture
plates by double dilution method. The wells were prepared in duplicates for each
concentration. The wells were inoculated with 10 l of spore suspension containing 2000-
3000 spores. The plates were incubated at 28 C for 20-30 h and then examined for spore
germination under inverted microscope. For quantification, spores were considered
germinated if they had a germ tube at least twice the length of the spore. For comparative
purposes in some experiments, the spore suspension (5 l) was mixed with 20 l of various
concentrations of extracts, fractions or active compounds and added on petri dishes containing
SDA. After incubation at 28 C for 48h, the plates were photographed for the examination of
inhibitory results
2.4.3. Hyphal growth inhibition
Sub-inhibitory concentrations of crude extracts in 500 l were prepared in 24-well flat
bottom micro-culture plates by double dilution method on which round cover slips were
placed. The wells were prepared in duplicates for each concentration. The wells were
inoculated with 100 l of spore suspension containing 2000-3000 spores. The plate were
incubated at 28 C for 20-30 h. Cover slips were carefully removed and washed in PBS, pH
7.2, with light manual shaking. The cover slips with the adhered cells were fixed in absolute
methanol and air-dried. The cells were stained with 5 mg of Calcofluor White M2R (Sigma,
St. Louis, Mo.) in 50mL of H
2
0 for 5 min, rinsed in H
2
O and mounted on a slide with
synthetic resin (Araldite 502™). Slides were observed by using a Zeiss-fluorescent
microscope.
2.4.4. Inhibitory effect on the invasion of nails
Distal fragments of normal human fingernails were collected from a healthy volunteer
who was not receiving antifungal therapy (Macura et al., 2003). The nails fragments were
crumbled into pieces approximately 2 x 2 mm
2
in diameter and autoclaved at 121 C for 15
min and place into sterile test tube. Then, nails fragments were saturated with various
concentrations of the test samples for 1 hour, inoculated on the surface with 50 l of spore
suspension, placed in a humidified atmosphere, and then incubated at 28 C for 7-14 days.
Two methods were used to assess nail invasion. For light microscopy, nails fragments were
examined by placing then on a glass microscope slide and clarified with DMSO prepared as
follows: DMSO 40.0, KOH 20.0, H
2
O 60.0. The nail fragments used to prepare the
microscope slide were washed thoroughly (twice in saline) to wash out the fungi growing on
the surface, if present. Then, the preparations were inspected under a light microscope and
searched for a presence or a lack of hyphae ingrown into the nails. For electron microscopy,
nail fragments were inoculated; after growth had proceeded for the desired time, they were
processed for scanning electron microscopy as indicated by Tanaka (1989), with the following
modifications. After fixation, small drops of the sample were placed on a specimen support
with poly-L-lysine. Postfixation was carried out with 1% osmium tetroxide in cacodylate
buffer containing 0.8% potassium ferrocyanide and 5 mM CaCl
2
for 30 min, with 1% tannic
acid in cacodylate buffer for 30 min and with 1% osmium tetroxide for 30 min. Subsequently,
the samples were dehydrated in graded ethanol, critical-point-dried in CO
2
, coated with
chromium in a Penning sputter system in a high-vacuum chamber (Gatan-Model 681), and
observed in a JEOL-JSM-6340F field-emission scanning electron microscope. Images were
obtained using secondary electrons.
2.5 Cytotoxicity assay.
The cytotoxicity assay was carried out, with some modifications, as previously
described (Skehan et al., 1990; Skehan, 1995). Briefly, confluent Vero cell monolayers grown
in 96-well cell culture plates were incubated with a ten-fold serial dilution of extracts,
fractions and active compounds – starting with a concentration of 1000 g/ml – for 48 h at
37C and 5% CO
2
. At that time, cultures fixed with 10% trichloroacetic acid for 1 h at 4C
were stained for 30 min with 0.4% sulforhodamine B (SRB) in 1% acetic acid, and
subsequently washed 5 times with deionised water. Bound SRB was solubilised with a 200 l
10 mM unbuffered Tris-base solution. Absorbance was read in a 96-well plate reader. The dye
was removed by four washes with 1% acetic acid. Protein-bound was extracted with 10 mM
Tris. The cytotoxicity was expressed as a percentage of the optical density of the control.
3. Results and discussion
The present study was designated to evaluate the antifungal activities of hydroalcoholic extracts
(50, 70 and 90%) from P. regnellii leaves. The results of antifungal activities of the extracts by using
both microdilution assays are summarised in Table 1. It was considered that if the extract displayed a
MIC equal or less than 100 g/ml, the antifungal activity was strong; from 100 to 500 g/ml the
antifungal activity was moderate; from 500 to 1000 g/ml the antifungal activity was weak; over 1000
g/ml they were considered inactive. Different results were obtained for the studied extracts against
yeast, dermatophyte and non-dermatophyte fungi. The 90% hydroalcoholic extract of P. regnellii
leaves presented a strong activity against the dermatophyte fungi T. mentagrophytes, T. rubrum, M.
canis and M. gypseum with MICs of 15.6 g/ml, 15.6µg/mL, 15.6 g/ml and 62.2µg/mL, respectively.
Both 50 and 70% hydroalcoholic extracts showed a moderated activity against the standard
dermatophytes (MICs=125 and 250 g/ml). The minimal fungicidal concentrations were within one
twofold dilution of the MICs for these organisms. Under the conditions employed here, all extracts
were virtually inactive against the yeast C. albicans and non-dermatophyte fungus A.niger (MIC>
1000 g/ml).
For comparative purposes, the spore suspension of T. rubrum and M. canis were mixed
with various concentrations of 90% hydroalcoholic extract and added on petri dishes
containing SDA. After incubation, the plates were examined and photographed for the
examination of inhibitory results. A representative view of petri dishes in this assay is show in
Fig. 1. The minimal concentration of hydroalcoholic extract that resulted in inhibition of both
dermatophytes growth was 1.28 µg/mL. This assay was designed to achieve maximal
sensitivity with mininal consumption of reagents and it could be used in a bioassay-guided
fractionation.
In order to determine potential action sites in fungal cells which might explain the inhibitory
activity of the crude extract, we studied the effect of the hydroalcoholic extracts on the spore
germination, hyphal growth and inhibitory effect on the invasion of nails.
The cells were analyzed microscopically in order to determine the minimum concentration of the
90% hydroalcoholic extract which inhibits spore germination of T. rubrum (Fig. 2). The percent spore
germination inhibition increased with the increase in concentration of the extract. The minimum
concentration of the hydroalcoholic extract required to completely inhibit (100%) spore germination of
T. rubrum after 12 h incubation was 7.8 g/ml. This is particularly noteworthy because the MIC of
90% hydroalcoholic extract was found to be 15.6 µg/mL.
During the course of this work, T. rubrum growth inhibition was routinely checked. The
growth forms were directly evaluated on the glass surface by fluorescence staining with the
optical brightener Calcofluor. Difference in the intensity of colonization or adhesion of T.
rubrum on the cover slips can be demonstrated by the hydroalcoholic group by Figs 3A-C and
for the positive control by Fig 3D. Difference in the morphology of inhibited hyphae was
apparent between fungi treated with hydroalcoholic extracts and those treated with Nystatin.
This difference may be of great importance for understanding the mechanism by which
hydroalcoholic crude extract and other antifungal plant extracts inhibit fungal growth.
The penetration of antifungal drugs through the nail plate is a crucial requirement for
successful topical treatment of dermathophytosis. The thickness of the nails and its relatively
compact construction make it a formidable barrier to the entry of topically applied drugs.
Futhermore, the nail plate barrier property appears to remains intact after long periods of
aqueous immersion (Waters et al., 1981). For this purpose, nails fragments were saturated
with hydroalcoholic extracts and then inoculated with spore suspension of T. rubrum. On light
microscopy and scanning electron microscopy of nail fragments not exposed to
hydroalcoholic extract of P. reginelli leaves, well-formed and extensive mycelial growth was
seen (Fig 4 B and D). On nail fragments exposed to hydroalcoholic extract at concentrations
more than 1.2 mg/ml and then inoculated with spore suspension, growth was not seen.
Hydroalcoholic extract of P. regnellii leaves appeared to diffuse rapidly through the nail and
remained active as seen by its fungicidal activity. Studies of alcohol permeability patterns in
general reflect nail plate behavior and show how other organic substances of low molecular
weight might penetrate the nail (Walters et al., 1983). To ensure successful topical therapy, an
important characteristic of antifungal agent is its fungicidal potency even against non-growing
cells. This property is very important because fungal nail infections occur under conditions
that do not promote optimal growth for the pathogen.
Under certain conditions, dermatophytosis can be complicated by secondary bacterial
infections. Therefore, we investigated whether the hydroalcoholic extract exerts, in addition to
its antifungal effects, a significant antibacterial activity against Gram-negative and Gram-
positive bacteria. The hydroalcoholic extract showed moderate activity on both
Staphylococcus aureus and Bacillus subtilis with MIC of 180 g/ml (data not shown). In
contrast to the relatively low MIC for gram-positive bacteria, gram-negative bacteria were not
inhibited by hydroalcoholic extract at concentrations 1000 µg/ml. This was to be expected,
because the outer membrane of gram-negative bacteria is known to present a barrier to the
penetration of numerous antibiotic molecules, and the periplasmic space contains enzymes
that are able of breaking down molecules introduced from outside.
The hydroalcoholic extract was also evaluated for its potential toxic effects on human
cells. After 48 h of incubation with 50 g/ml of crude extract 98 % of the cells were still
viable, respectively. These results indicate that hydroalcoholic extract is selectively toxic to
the fungal cells (Fig. 5).
To obtain some information on the active components, the hydroalcoholic extract was
fractionated on silica gel in to nine fractions. In this bioassay-guided fractionation, when the
MIC was over 100 g/ml the extracts or isolated compound were considered inactive. The
hexane (F1), chloroform (F2), chloroform:ethyl acetate (19:1) (F3) and chloroform:ethyl
acetate (9:1) (F4) fractions were activity against T. rubrum with MIC ranged from 6.2 g/ml
to 50 g/ml (Table 2). The minimal concentrations required for inhibition of spore
germination ranged from 3.1 g/ml to 50 g/ml. The chloroform:ethyl acetate (4:1) (F5),
chloroform:ethyl acetate (1:1) (F6), acetone (F7), methanol (F8) and methanol:water (1:1)
(F9) showed no activity against the organisms tested (MIC >100 g/ml). The active
chloroform fraction was lyophilized and fractionated by thin-layer chromatography. TLC
plates were run in duplicate and one set was used as the reference chromatogram (Fig.6-A).
After being visualized, 6 bands were scraped from the TLC plates (Fig. 6-B) and assayed for
antifungal activity (Table 3). The active band was dissolved in methanol and characterized by
HPLC (Fig 7) and spectroscopic methods. Structures were established by comparison with
literature data. (Chauret et al., 1996; Achenbach et al., 1987; Snider et al., 1997) and
identified as conocarpna (1), eupomatenoid-5 (3), and eupomatenóide-6 (4) (Figure 8),
respectively. The pure compounds showed moderate activity on T. rubrum with MIC of 100
g/ml (data not shown)
Benevides et al. (1999) related the first phytochemical investigation carried out on the
specie P. regnellii on the chemistry of lignans/neolignans. Among the isolated compounds
from the ethyl acetate extracts of the roots of P. regnellii, are the neolignans conocarpan (1),
eupomatenoid-3 (2), eupomatenoid-5 (3) and eupomatenoid-6 (4). Benzofuran neolignans
represent a sub-class with a variety of biological activities including anti-PAF, antifungal and
insecticidal activity. Several compounds of this class have been isolated from Piperaceae
species and in case of P. regnellii, phytochemical studies of its roots showed the accumulation
of several phenylpropanoids and benzofuran neolignans including conocarpan as the major
compound (Sartorelli et al., 2001)
In recent years, a number of plants of Piper species have been found to possess
antifungal activity (Candida albicans, Cryptococcus neoformans, Saccharomyce cerevisae,
Caldosporium sphaerospermum, C. cladiosporiodes, Microsporum gypseum and Tricophyton
mentagrophytes) which has allowed the isolation of the active principles followed by their
characterization as benzofuran neolignans in leaves of P. fulvescens (Freixa et al., 2001),
benzoic acid derivatives in leaves of P. dilatatum, and pyrrolidyne and piperidine amides in
leaves and stems of P. hispidum (Alécio et al., 1998; Navickiene et al., 2000), seeds and
leaves of P. tuberculatum (Navickiene et al., 2000; Silva et al., 2002) and of the leaves of P.
arboretum (Silva et al., 2002).
Pessini et al (2003) isolated and identified the neolignans conocarpan (1),
eupomatenoid-3 (2), eupomatenoid-5 (3) and eupomatenoid-6 (4) from the hydroethanolic
extract of the leaves of P. regnellii. Moreover, it was evaluated the antimicrobial activity of
the compounds, that demonstrated potential activity, with exception of the compound
eupomatenoid-3, that was inactive against bacteria and yeast. The compounds eupomatenoid-
5 (3) and eupomatenoid-6 (4) were active only against bacteria.
In the present work, comparing the activity of the active chloroform fraction obtained
from hydroalcoholic crude extract with that of isolated compounds, it is clear that the former
had greater antifungal activity against T. rubrum. Synergy is a popular concept in the field of
herbal medicine, suggesting that plant containing compounds potentiating each other’s action.
Possible synergy would explain many failed attempts to isolate single, active compounds
from medicinal plants. Solid, mechanistically supported evidence for this concept, however,
has been lacking. It is hoped that this study will stimulated investigations at the molecular
level of possible medicinal plant synergisms.
Although the present study investigated the in vitro antidermatophyte activity, the
results substatiate the ethnobotanical use of the hydroalcoholic crude extract for the treatment
of various fungi-related disease. However, in vitro data may be helpful in determining the
potential usefulness of the plants for treatment of dermatophyte infections. In terms of
conservation, the results show that leaf material is useful for antifungal uses, and could be
used without any detrimental effect on the plant.
Acknowledgements
This study was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Capacitação e Aperfeiçoamento de Pessoal de Nível Superior, (Capes),
Fundação Araucária, and Programa de Pós-graduação em Ciências Farmacêuticas da
Universidade Estadual de Maringá. The authors would like to thank Marinete Martinez
Vicentin for skillful technical assistance.
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LEGEND AND FIGURES
Figure 1. Effect of different concentrations of the hydroalcoholic (90%) extract of P. regnelli
leaves on growth of M. canis (A) and T. rubrum (B). The spore suspensions (5 l) were mixed
with 20 l of various concentrations of crude extract (0.03, 0.07, 0.15, 0.32, 0.64, 1.28, 2.5
and 5.0g/ml) and added on petri dishes containing SDA, as described in Materials and
methodos. The minimal concentration of crude extract that resulted in inhibition of both
dermatophytes was 1.28 g. Data correspond to one representative experiment out of three.
0.03
0.07
0.15
0.32
5
1.28
2.5
0.64
0.03
0.07
0.15
0.32
0.64
2.5
1.28
5
A
B
Figure 2. Spore germination inhibition of hydroalcoholic (90%) extract against T. rubrum:
(A) Control; (B) and (C) treated with 3.9 and 7.8 g/ml of hydroalcoholic (90%) extract,
respectively. Magnification x 20. Similar results were obtained in the different analyses
A
C
B
Figure 3. Growth of T. rubrum on cover slips after treatment with different concentrations of
P. regnellii leaves extracts. (A) 7.8µg/mL 50% hydroalcoholic extract; (B) 15.62µg/mL 70%
hydroalcoholic extract; (C) 7.8 g/ml 90% hydroalcoholic extract; (D) 0.3 g/ml Nystatin.
Data correspond to one representative experiment out of three.
A
B
C
D
Figure 4. Light microscopy (A-B) and scanning electron microscopy (C-D) of nail fragments
treated (A and C) and untreated (B and D) with hydroalcoholic (90%) extract at a
concentration of 1.2 g/ml. Similar results were obtained in the different analyses
A
B
D
C
Figure 5. Cytotoxic assay. The hydroalcoholic (90%) extract was also evaluated for its potential toxic effects on Vero cell monolayer. After 48 h
of incubation with 50 g/ml of crude extract (A) and 5 g/ml of Nystatin (B) 98 and 100 % of the cells were still viable, respectively. (C) Control.
These results indicate that hydroalcoholic (90%) extract is selectively toxic to the fungal cells. Similar results were obtained in the different
analyses.
A
C
B
Figure 6. (A) Thin layer chromatography plates of hydroalcoholic extracts (50, 70 and 90%, v/v) and fractions (F1 to F9) obtained from active
hydroalcoholic (90%) extract. (B). Preparative thin layer chromatography of the chloroform fraction being visualized, 6 bands were scraped from
the other set, fractionated by thin-layer chromatography (F2A-F2F), assayed for antifungal activity and used for bioassay-guided isolation. Similar
results were obtained in the different analyses
F2B
50
%
70
%
90
%
F1
F3
F4
F5
F6
F7
F8
F9
F2
F2A
F2C
F2D
F2E
F2F
A
B
Figure 7. HPLC on reverse-phase resin Microsorb C-18. (A) The sub-fraction F2C with
antifungal activity was applied in a reverse-phase column Microsorb- MV 100-5 C-18 (250 x
4.6) equilibrated and eluted with. acetonitrile/water (60:40, v/v) containing 2% acetic acid:
flow-rate of 1.0 ml/min; temperature: 30 °C; detection: 280 nm. (B) The standard mixture of
neolignans: conocarpan (1), eupomatenoid-6 (2) and eupomatenoid-5 (3). Data correspond to
one representative experiment out of three.
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12
mAU
0 250 500 750 1000 1250 1500
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12
mAU
0 500 1000 1500 2000 2500
A
B
1
2
3
1
2
3
O
H
3
C
OH
O
H
3
C
O
O
O
H
3
C
OH
H
3
CO
O
H
3
C
OH
Figure 8. Structures of the neolignans isolated from P. regnellii. (1) conocarpan, (2)
eupomatenoid-3, (3) eupomatenoid-5, (4) eupomatenoid-6.
1
4
2
3
Table 1. Minimal inhibitory concentration (MIC) of hydroalcoholic crude extracts of leaves
from P. regnellii.
MIC (g/ml)
Fungi
Hydroalcoholic extracts (%, v/v)
Positive
control
50
70
90
Nistatin
Yeast
Candida albicans
>1000
>1000
>1000
5
Non-dermatophytre
Aspegillus niger
>1000
>1000
>1000
2.5
Dermatophyte
Trichophyton mentagrophytes
250
250
15.62
0.30
Trichophyton rubrum
15.62
62.5
15.62
0.30
Microsporum canis
250
125
15.62
0.30
Microscorum gypseum
62.5
250
62.5
0.30
Table 2 Antifungal activity of fraction obtained from hydroalcoholic extract (90%) of leaves
from P. regnellii against dermatophyte T. rubrum
Antifungal activity (µg/ml) against T. rubrum
Fractions (solvent)
Minimal inhibitory
concentration
a
Minimal concentration required for
inhibition of spore germination
b
F1 Hexane
6.2
6.2
F2 Chloroform
6.2
3.1
F3 Chloroform:ethyl acetate (19:1)
50
50
F4 Chloroform:ethyl acetate (9:1)
50
12.5
F5 Chloroform:ethyl acetate (4:1)
>100
>100
F6 Chloroform:ethyl acetate (1:1)
>100
100
F7 Acetone
>100
>100
F8 Methanol
>100
>100
F9 Methanol:water (1:1)
>100
>100
Table 3. Minimal concentration of sub-fractions obtained from fraction F2 (chloroform)
required for inhibition of spore germination of dermatophyte Trichophyton rubum
Sub-fractions
Minimal concentration (µg/ml)
required for inhibition of spore
germination
a
F2A
12.5
F2B
50.0
F2C
3.1
F2D
12.5
F2E
25.0
F2F
50
Positive control (Nystatin)
0.2
a
For quantification, cells were considered germinated if they had a germ tube at least twice the length
of the cell.
Espectro de RMN
1
H do conocarpano (300MHz, CDCl
3
)
2
O
H
3
C
OH
Espectro de RMN
1
H do eupomatenóide-6 (300MHz, CDCl
3
)
O
H
3
C
OH
Espectro de RMN
1
H do eupomatenóide-5 (300MHz,CDCl
3
)
O
H
3
C
OH
H
3
CO
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