LUCIANI GASPAR DE TOLEDO
Extrato etanólico e óleo essencial de Cymbopogon nardus (L.) Rendle: caracterização química e
avaliação in vitro do potencial antifúngico
São José do Rio Preto
2016
LUCIANI GASPAR DE TOLEDO
Extrato etanólico e óleo essencial de Cymbopogon nardus (L.) Rendle: caracterização química e
avaliação in vitro do potencial antifúngico
Dissertação apresentada à Faculdade de Medicina de São José do Rio Preto para a obtenção do título de Mestre no curso de Pós-graduação em Ciências da Saúde. Eixo temático: Medicina e Ciências Correlatas.
Orientadora: Profa. Dra. Margarete Teresa Gottardo de Almeida
SÃO JOSÉ DO RIO PRETO
2016
LUCIANI GASPAR DE TOLEDO
Extrato etanólico e óleo essencial de Cymbopogon nardus (L.) Rendle: caracterização química e
avaliação in vitro do potencial antifúngico
BANCA EXAMINADORA
DISSERTAÇÃO PARA OBTENÇÃO DO TÍTULO DE MESTRE
Presidente e Orientador: Profa. Dra. Margarete Teresa Gottardo de Almeida
2º Examinador: Profa. Dra. Tais Maria Bauab
3º Examinador: Profa. Dra. Mara Corrêa Lelles Nogueira
Suplente: Prof. Dr. Luis Octavio Regasini
Suplente: Profa. Dra. Cinara de Cássia Brandão de Mattos
São José do Rio Preto, 16/02/2016
SUMÁRIO
Dedicatória ... i
Agradecimentos ...ii
Epígrafe ... v
Lista de Figuras ... vi
Lista de Tabelas ... vii
Lista de abreviaturas e siglas... viii
Resumo... ix
Abstract ... x
1. INTRODUÇÃO ... 1
2. OBJETIVOS ... 7
2.1. Objetivo Geral ... 7
2.2. Objetivos específicos ... 7
3. ARTIGOS CIENTÍFICOS ... 9
ARTIGO 1... 11
1. INTRODUCTION ... 13
2. MATERIALS AND METHODS ... 15
2.1. Plant Material ... 15
2.2. Essential oil (EO) extraction ... 15
2.3. Citronellal ... 16
2.4. Gas chromatography (GC) analysis of EO ... 16
2.4.1. Gas chromatography-mass spectrometry (GC–MS) ... 16
2.4.2. Gas chromatography (GC-FID) ... 17
2.5. Antifungal activity ... 17
2.5.1. Fungal strains ... 17
2.5.2. Determination of minimum inhibitory concentration (MIC) ... 18
2.5.3. Determination of minimum fungicidal concentration (MFC) ... 19
2.6. Inhibition on Candida albicans hyphae growth ... 19
2.7. Time-kill assay ... 19
2.8. Biofilm assay ... 20
2.9. Cytotoxic activity ... 21
2.9.1. Cell lines ... 21
2.9.2. Cytotoxic assay ... 21
3. RESULTS AND DISCUSION ... 22
3.1. Chemical composition of essential oil ... 22
3.2. MIC and MFC determination of EO ... 25
3.3. MIC and MFC determination of citronellal ... 27
3.4. Inhibition on Candida albicans hyphae growth ... 28
3.5. Time-kill assay ... 29
3.6. Biofilm ... 31
4. CONCLUSION ... 33
5. Acknowledgments ... 33
6. REFERENCES ... 33
ARTIGO 2... 38
Abstract ... 39
Introduction ... 40
Material and Methods ... 42
Plant Material ... 42
Extract preparation ... 42
UPLC-ESI-QTOF-MS(/MS) analysis of extract ... 42
Solid phase extraction (SPE) ... 43
Thin layer chromatography (TLC) ... 44
Antifungal activity ... 44
Fungal strains ... 44
Determination of minimum inhibitory concentration (MIC) ... 44
Determination of minimum fungicidal concentration (MFC) ... 45
Inhibition on Candida albicans hyphae growth ... 46
Time-kill assay ... 46
Biofilm assay... 47
Cytotoxic activity ... 48
Cell lines ... 48
Cytotoxic assay ... 48
Results and Discussion ... 49
Solid phase extraction ... 49
Chemical analysis ... 49
MIC and MFC determination of ethanolic extract ... 55
MIC and MFC determination of fractions ... 57
Inhibition on Candida albicans hyphae growth ... 59
Time-kill assay ... 60
Effect of EE on mature biofilm ... 62
Cytotoxic evaluation ... 63
Conclusions ... 64
Acknowledgments ... 64
References ... 64
4. CONCLUSÕES ... 70
5. REFERÊNCIAS ... 72
Dedicatória
Aos meus queridos pais,
Valdomiro e Maria, e aos
meus irmãos, Luciano e
Luciomar, por me darem o
privilégio de conhecer o amor
nesta vida.
Agradecimentos
A Deus por me guiar em meus sonhos e ao meu anjo da guarda por sempre me fazer persistir.
À minha família por todo o apoio, em especial aos meus pais, Valdomiro e Maria, pelo amor infinito.
Aos meus irmãos, Luciano e Luciomar, por todo o companheirismo e por ser o meu elo único entre meu passado e meu futuro.
Às minhas cunhadas, Camila e Maria Emília por toda sensibilidade diante das realizações dos meus sonhos e por toda a nossa amizade.
Aos meus sobrinhos, Maria Carolina, Ana Julia e Felipe por darem sentido a minha vida e por me fazerem conhecer a pureza do amor.
À Profa. Margarete Teresa Gottardo de Almeida pela orientação, por todo o conhecimento científico e pessoal e pela amizade tão verdadeira. Sobretudo, pela orientação espiritual sempre presente nos momentos de dificuldades e por ser uma fonte inspiração na minha caminhada, me iluminado com a sua luz intrínseca.
Ao Matheus Aparecido dos Santos Ramos, pela parceria profissional abençoada por Deus. Sobretudo, por ser um irmão de alma, seu anjo da guarda se encontrou com o meu e desde então nunca mais se separaram e a partir dai conheci a verdadeira cumplicidade e confiança que possa existir em uma amizade. Além disso, agradeço por ser uma inspiração me fortalecendo em seguir em frente e ainda pela motivação profissional e pessoal, sendo o meu porto-seguro.
À Larissa Sposito pelo companheirismo profissional e acima de tudo por toda a amizade verdadeira e companheirismo em todos os momentos do desenvolvimento deste trabalho.
Aos amigos Felipe Guiotti e Lucio Barros pela amizade verdadeira e por me sempre me acolherem como grandes amigos.
À vó Nilde por me acolher em sua casa como uma filha, por todos os momentos de alegria, pela jovialidade e paz de espirito que ela transmite e por todos os ensinamentos de vida.
Aos meus tios, Pedro, Fatima, Rui e Celso, aos meus primos, Jociene, Fabiano, Pedro Manoel, Ricardo, Lidia e Lucas por todo o carinho e companheirismo durante esta vida e por constituírem a minha segunda família.
Aos meus avós Benedito Amâncio Soares, Leontina Dias Soares, Francisco Gaspar de Toledo, Antônia de Souza e à tia Anita pela força espiritual recebida e por me guiarem em todas as etapas da minha vida.
Aos queridos amigos do Laboratório de Microbiologia da Faculdade de Medicina de São José do Rio Preto- FAMERP:
À Luceli Ferreira de Souza, pela disposição e por ser sempre tão prestativa no desenvolvimento deste trabalho. À Natália Seron Brizzotti, Mayara Gambellini, Luis Paulo Teixeira, Maira Gazzola Arroyo, João Paulo Zen, Mariela Ribeiro, Maicon Henrique Caetano, Thiago Henrique Lemes, Beatriz Gomes Ricardo, Diego Maximiano, Fabio Buscariolo, Maísa Guimarães Sartim, Lorena Galete, Bianca Gottardo Almeida por todo o companheirismo e amizade.
Ao Eduardo José de Carvalho Reis e à Emília Cristina Gianizella Amorim por tornarem os meus dias de trabalho mais felizes na companhia de um café.
Às queridas amigas, Crislene Barbosa Almeida, à Iara Thais Dias Mendes e à Renata Jorge Tiossi pela amizade verdadeira e confiança, todas sempre me emprestando o colo e os ouvidos diante das minhas dificuldades.
À Profa. Elza Maria Castilho por ter me acolhido em seu laboratório e pela disposição e atenção durante todos esses anos. Além disso, por toda a amizade e apoio espiritual na minha caminhada.
À Profa. Cleuzenir por também ter me acolhido em seu laboratório e pela amizade e companheirismo sempre presente durante todos esses anos, e que certamente se eternizarão.
À Profa. Vanderli Marchiori por todo o apoio, pela amizade infinita e pelos ombros sempre dispostos a me acolher. Sobretudo, por mexer seu caldeirão perfumando nossos dias sendo uma fonte de inspiração.
À Profa. Cinara Cassia Brandão de Mattos por todo atenção durante a solicitação de bolsa deste projeto, pelas contribuições científicas e pela amizade.
À Profa. Mara Correa Lelles Nogueira por todo apoio e amizade durante a realização deste projeto.
À Profa. Tais Maria Bauab, por ser parte do desenvolvimento deste trabalho me abrindo caminhos sempre de forma tão disposta, com muito carinho e atenção.
Sobretudo, por toda a disposição e trabalho em meu processo seletivo de doutorado, possibilitando novos caminhos.
A todos do Laboratório de Fisiologia de Micro-organismos da Faculdade de Ciências Farmacêuticas de Araraquara- UNESP/FCFAR, por todo auxilio.
Ao Prof. André Gonzaga dos Santos por me acolher em seu laboratório e por ter sido tão atencioso e prestativo.
A todos do Laboratório de Farmacognosia da Faculdade de Ciências Farmacêuticas de Araraquara- UNESP/FCFAR, por todo auxilio.
Ao Prof. Fernando Rogério Pavan por me receber em seu laboratório e por todo auxílio.
A todos do Laboratório de Micobacteriologia da Faculdade de Ciências Farmacêuticas de Araraquara- UNESP/FCFAR, por todo auxilio.
Ao Prof. David Hewitt por todo ensinamento e pelos momentos de psicoterapia em suas aulas de inglês.
À Luciana Lemos Palma por me fazer acreditar nas possibilidades da alma e do coração, e por todo companheirismo e cumplicidade, me fortalecendo na realização deste trabalho.
Às minhas queridas amigas de infância, adolescência e de toda a vida, Karla Green, Natália Bernardo, Mariele Simon, Elisama Brito, Roberta Green, Ana Paula Fais, Renata Ribeiro e Maria Claudia Piccolo por serem uma fonte de confiança e de amizade verdadeira durante toda a minha vida.
Às queridas amigas de pós-graduação lato sensu, Ana Carolina Garcia, Gabriele Comachio, Simone Penha, Renata Ferracioli, Rosana Loiola, Sandra Giacomelli, Rosana Bugati, Ellen Vieira, Regiane Nobre por sempre vibrarem com as minhas conquistas e por toda a amizade durante os dois anos de pós-graduação.
A todos os meus queridos alunos que me fizeram acreditar em meus sonhos dando sentido a minha vida profissional.
A todos que contribuíram direta e indiretamente para a realização desta dissertação Mestrado.
Epígrafe
“A felicidade só é verdadeira se for compartilhada”
(Christopher McCandless)
Lista de Figuras
INTRODUÇÃO
Figura 1: Cymbopogon nardus (L.) Rendle...4
ARTIGO 1
Figure 1. Inhibitory effect of EO on transition of C. albicans from yeast to hyphal form.
... 28 Figure 2. Time-kill curves of Candida spp following exposure essential oil (EO) and amphotericin-B. Control represents the untreated Candida cells. ... 30 Figure 3. Percentage of inhibition of essential oil (EO) against biofilms of Candida species. ... 31
ARTIGO 2
Figure 1. Total ion chromatogram (TIC) of EE obtained in the positive mode. ... 49 Figure 2. Total ion chromatogram (TIC) of EE obtained in the negative mode. ... 51 Figure 3. Inhibitory effect of EE on transition of C. albicans from yeast to hyphal form.
... 59 Figure 4. Time-kill curves of Candida spp following exposure ethanolic extract and amphotericin-B. Control represents the untreated Candida cells. ... 61
Lista de Tabelas
ARTIGO 1
Table 1. Composition of essential oil from the leaves of C. nardus. ... 23 Table 2. MIC values (µg/mL) and MFC values (µg/mL) of essential oil (EO) from Cymbopogon nardus against Candida species. ... 25 Table 3. MIC values (µg/mL) and MFC values (µg/mL) of citronellal against Candida species. ... 28 Table 4. Anti-biofilm effect of EO against C. albicans, C. krusei and C. parapsilosis. 31 Table 5. Cytotoxic activity of essential oil of Cymbopogon nardus and citronellal. ... 32
ARTIGO 2
Table 1. Data of SPE fractions. ... 49 Table 2. Proposed phenolic compounds detected in C. nardus extract by UPLC-ESI- QTOF-MS(/MS). ... 53 Table 3. MIC and MFC values of C. nardus extract against Candida species. ... 55 Table 4. MIC values of fractions (A-G) from Cymbopogon nardus against Candida species. ... 58 Table 5. Concentration of EE against mature biofilm of Candida species ... 63 Table 6. Cytotoxic activity of ethanolic extract of Cymbopogon nardus. ... 63
Lista de abreviaturas e siglas
AmB- Amphotericin-B
ATCC- American Type Culture Collection
CEP-FAMERP- Comitê de Ética em Pesquisa- Faculdade de Medicina de São José do Rio Preto
CFU- Colony-forming unit
DMEM- Dulbecco's Modified Eagle Medium DMSO- Dimetilsulfoxide
EDTA- Ethylenediamine tetraacetic acid EE- Ethanolic extract
EO- Essential oil
ELISA- Enzyme Linked Immuno Sorbent Assay FLU- Fluconazole
GC-MS- Gas chromatography-mass spectrometry GC-FID- Gas chromatography-flame ionization detector IC50- Half maximal inhibitory concentration
MIC- Minimal inhibition concentration MFC- Minimal fungicidal concentration
MOPS- 3-(N-morpholino) propanesulfonic acid MS- mass spectra
SDA- Sabouraud Dextrose Agar SDB- Sabouraud Dextrose Broth SPE- Solid phase extraction TIC- Total ion chromatogram TLC- Thin layer chromatography tR- Time retention
TTC- 2,3,5-triphenyltetrazolium chloride
UPLC-MS- Ultra-performance liquid chromatography-mass-spectrometry
UPLC-ESI-QTOF-MS(/MS)- Ultra-performance liquid chromatography-electrospray ionization quadruple time-of-flight/mass spectrometry
XTT-2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H- tetrazolium hydroxide
Resumo
Introdução: Leveduras do gênero Candida são patógenos oportunistas isolados da biota humana no trato gastrointestinal, mucosa oral e vaginal, que podem levar ao desenvolvimento de lesões superficiais até infecções disseminadas, especialmente em situações de imunossupressão. O alto custo para tratamento de infecções, a elevada toxicidade, e o surgimento de cepas resistentes justificam a busca de novos agentes terapêuticos. A biodiversidade vegetal é rica em princípios ativos que têm contribuído com o desenvolvimento de novos e efetivos medicamentos, mais econômicos e de fácil acesso populacional. Cymbopogon nardus (L.) Rendle, é uma planta popularmente conhecida como citronela e cultivada em áreas subtropicais e tropicais da Ásia, África e América, incluindo o Brasil. Os óleos essenciais presentes em plantas do gênero Cymbopogon têm sido amplamente estudados, porém, a analise química e microbiológica do extrato etanólico de C. Nardus,é pouco explorada Objetivo: O objetivo deste estudo foi avaliar o potencial antifúngico, in vitro, do extrato etanólico (EE) e do óleo essencial (OE) das folhas de Cymbopogon nardus (L.) Rendle (citronela) frente isolados clínicos de Candida spp. Material e Métodos: Foram consideradas as espécies: Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis sensu stricto e C. orthopsilosis. O EE foi obtido por extração em banho ultrassônico e analisado por cromatografia liquida de ultra performance (UPLC- ESI-QTOF-MS). O OE foi extraído por hidrodestilação e analisado por cromatografia gasosa acoplada a espectrômetro de massa (CG-EM). A atividade antifúngica do EE e do OE foi realizada através da determinação da concentração inibitória mínima (CIM), ensaio time-kill, inibição do crescimento hifal de C. albicans e inibição de biofilme maduro. Adicionalmente, a avaliação citotóxica (determinação de IC50) foi estabelecida em linhagens celulares HepG-2 (hepática) e MRC-5 (fibroblasto). Resultados: Os resultados da análise química do EE evidenciaram presença de flavonas e fenilpropanoides glicosilados. De acordo com a análise química do OE, os principais compostos observados foram monoterpenos contendo oxigênio: citronelal, geranial, geraniol, citronelol e neral. Os ensaios biológicos mostraram importante atividade antifúngica para o EE (CIM de 1000 a 125 µg/mL) e, para o OE (CIM de 1000 a 250 µg/mL). A inibição do crescimento ocorreu para todas as espécies avaliadas frente aos produtos, EE e OE. O EE (1000 até 31 µg/mL) e OE (1000 até 15,8 µg/mL) inibiram o desenvolvimento da hifa de C. albicans, durante 12 e 24 horas, além de inibir o biofilme maduro das espécies de C. albicans, C. krusei e C. parapsilosis, nas concentrações de 50xCIM e 10xCIM, respectivamente. O EE apresentou os menores valores de IC50 para HepG-2 (322 µg/mL) e MRC-5 (181,1 µg/mL) em comparação com o óleo essencial- IC50 para HepG-2 (96,6 µg/mL) e MRC-5 (33,1 µg/mL). Conclusões: O EE e OE de C.
nardus apresentam-se como uma fonte promissora de novas moléculas com atividade antifúngica com destaque à inibição dos principais fatores de virulência: formação de hifa e biofilme.
Palavras-chave: Cymbopogon nardus; óleo essencial; extrato etanólico; atividade antifúngica; Candida spp; análise química;
Abstract
Introduction: Candida spp are opportunistic pathogens isolated from human biota in the gastrointestinal tract, oral and vaginal mucosa, which can lead to the development of superficial lesions to disseminated infections, especially in immunosuppression. The high toxicity, the high cost of treatment and the emergence of resistant strains justify the search for new therapeutic agents. The plant biodiversity is rich in active ingredients that have contributed to the development of new and effective drugs, less expensive treatments and population access. Cymbopogon nardus (L.) Rendle is a plant popularly known as citronella and cultivated in subtropical and tropical areas of Asia, Africa and America, including Brazil. Essential oils present in the Cymbopogon genus plants have been widely studied, but there are few studies involving chemical analysis and microbiological ethanol extract of C. nardus. Objective: The objective of this study was to evaluate the antifungal potential, in vitro, of ethanol extract (EE) and essential oil (EO) from the leaves of Cymbopogon nardus (L.) Rendle (citronella) clinical isolates against of Candida spp. Material and Methods: In this study the species Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis sensu stricto and C. orthopsilosis were selected. EE was obtained by extraction ultrasonic bath and analyzed by ultra-performance liquid chromatography (UPLC-ESI- QTOF-MS). The EO was extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometer (GC-MS). The antifungal activity of EE and EO was performed by determination of the minimum inhibitory concentration (MIC), time- kill assay inhibition of hyphal growth of C. albicans and inhibit mature biofilm.
Additionally, the cytotoxic evaluation (determination of IC50) was assessed in HepG-2 cell lines (hepatic) and MRC-5 (fibroblast). Results: The results of the chemical analysis of EE showed presence of glycosylated flavones and glycosylated phenylpropanoids. According to the EO chemical analysis, the main compounds observed were monoterpenes containing-oxygen: citronellal, geranial, geraniol, citronellol and neral. Biological assays showed effective antifungal activity of EE (MIC 1000 to 125 µg / ml) and of EO (MIC 1000 the 250 µg/ml). In the time-kill assay was observed inhibition of growth of the species tested for EE and EO. The hyphal growth of C. albicans was inhibited by EE (1000 to 31 µg/ml) and the EO (1000 to 15.8 µg/ml) for 12 and 24 hours. The EE and EO inhibit mature biofilm species C. albicans, C.
krusei, and C. parapsilosis at concentrations of 50xCIM and 10xCIM, respectively. EE exhibited the lower IC50 values for HepG-2 (322 µg/ml) and MRC-5 (181.1 µg/ml) than essential oil that showed IC50 values for HepG-2 (96.6 µg/ml) and MRC-5 (33.1 µg/ml).
Conclusions: The EE and EO from C. nardus present as a promising source of new molecules with antifungal activity especially to the inhibition of the main virulence factors such as formation of hyphae and biofilm.
Key-words: Cymbopogon nardus; essential oil; ethanolic extract; antifungal activity;
Candida spp; chemical analysis;
INTRODUÇÃO
1. INTRODUÇÃO
Leveduras do gênero Candida são componentes normais da biota humana no trato gastrointestinal, mucosa oral e vaginal. Entretanto, na vigência de fatores de risco como, imunodeficiência, terapia antineoplásica, transplante de órgãos, disfunções endócrinas, tratamento prolongado com antibióticos, estas leveduras tornam-se clinicamente relevantes (SCORZONI et al., 2013; PRASAD, RAWAL, 2014; LUM et al., 2015).
As infecções por espécies de Candida são amplas, incluindo acometimentos superficiais, subcutâneos e sistêmicos. Candida albicans é a espécie prevalente em infecções sistêmicas, embora, espécies não albicans como C. glabrata, C. krusei, C.
parapsilosis e C. tropicalis, estão sendo associadas ao padrão clinico descrito (LOCKHART et al., 2011; STORM et al., 2014). Este evento pode estar relacionado aos padrões emergentes de resistência aos antimicrobianos, bem como a melhoria dos métodos diagnósticos (SILVA et al., 2012).
Espécies de Candida se destacam pela presença de diversos fatores de virulência, que incluem a transição morfológica entre levedura e hifa, aderência, formação de biofilme, produção de enzimas hidrolíticas, como proteases, fosfolipases e hemolisinas (SARDI et al., 2013; HOLLAND et al., 2014, DA SILVA-ROCHA et al., 2015).
A formação de hifa destaca-se como evento biológico importante para invasão de tecidos. Esta estrutura filamentosa (hifa e pseudohifa) apresenta maior resistência à fagocitose quando comparadas à levedura (TSANG; BANDARE; FONG, 2012; SILVA
et al., 2012; VYLKOVA, LORENZ, 2014), o que potencializa sua manutenção em células hospedeiras.
O biofilme é definido como uma comunidade de micro-organismos que apresenta variações estruturais de espécie para espécie conferindo proteção contra mecanismos de defesa do hospedeiro, proteção ao ambiente externo, resistência à tensão física e química, cooperação metabólica, entre outras (RAMAGE et al., 2012).
O biofilme de C. albicans é formado por uma camada basal compacta de células leveduriformes e uma camada mais espessa e menos compacta de hifa, envolvidos por uma matriz extracelular composta principalmente de carboidratos, proteínas, fósforo e hexosamina. Por outro lado, C. parapsilosis não tem a capacidade de formar hifas verdadeiras e o biofilme é composto somente de células leveduriformes e pseudohifas, altos níveis de carboidrato e quantidade baixa de proteína MAYER; WILSON; HUBE, 2013; SILVA et al, 2012). Todavia, a matriz extracelular do biofilme de C. glabrata apresenta altos níveis de carboidratos e proteínas, enquanto para C. tropicalis as quantidades de carboidratos e proteínas são baixas (SILVA et al, 2012).
O processo de formação do biofilme é sequencial e inclui a aderência do micro- organismo, proliferação das células, formação de hifas, acúmulo de matriz extracelular (MAYER; WILSON; HUBE, 2013), atividades expressivas para espécies de Candida (PANNANUSSORM et al., 2013). A baixa penetração dos fármacos em resposta ao estresse adaptativo, variabilidade fenotípica e a proliferação de células, são fatores que dificultam a sua erradicação (JAFRI, HUSAIN, AHMAD, 2014).
A terapia antifúngica, com destaque aos compostos azólicos, poliênicos e equinocandinas, apresenta limitações, como o alto custo, toxicidade elevada, interações medicamentosas, biodisponibilidade insuficiente do princípio ativo (ENDO et al., 2010;
FERREIRA et al., 2015). Além disso, o seu uso indiscriminado promove, muitas vezes, a emergência de cepas resistentes em decorrência às variações genéticas quando da exposição a estes fármacos (CALABRESE et al., 2013).
Os poliênicos (anfotericina-B e nistatina) agem lesando a membrana citoplasmática da célula do fungo e combina-se com os esteróis da membrana, interferindo na sua permeabilidade (KARIMZADEH, KHALILI, SAGHEB, 2015).
Estes fármacos apresentam amplo espectro de ação antifúngica, mas os efeitos colaterais são graves, como nefrotoxicidade, condição limitante para sua utilização (CALABRESE et al., 2013).
Os derivados azólicos inibem a 14-α-desmetilase, enzima do citocromo P450, responsável pela conversão do lanosterol em ergosterol, o principal esterol na membrana celular fúngica (RANG et al., 2007; BRUNTON et al., 2007). Ocorre um comprometimento da biossíntese do ergosterol na membrana citoplasmática, o que leva ao acúmulo de 14-α-metilesteróis. Estes podem comprometer as funções de determinados sistemas enzimáticos ligados à membrana, inibindo assim o crescimento dos fungos (BRUNTON et al., 2007).
As equinocandinas surgem como recente alternativa terapêutica, e inibem a síntese da β (1,3)-D-glucana, um dos componentes da parede celular dos fungos (RANG et al., 2007; GLOCKNER, 2011). Estes fármacos demonstram potente atividade contra espécies de Candida e apresentam um perfil de segurança melhor do que os derivados azólicos e poliênicos. No entanto, a biodisponibilidade oral baixa e alta ligação às proteínas, aliadas ao custo elevado e concentração reduzida no liquido cefalorraquidiano e urina, limitam seu emprego (CALABRESE et al., 2013).
O aumento da incidência de patógenos resistentes aos antifúngicos, o pequeno número de antifúngicos disponíveis, estimula a pesquisa por novas alternativas de tratamento (SARDI et al., 2013). Neste contexto, as propriedades bioativas das plantas, justificada pela presença de metabólitos secundários como taninos, terpenos, alcaloides, flavonoides, constituem-se relevantes quanto às propriedades antimicrobianas (MATU et al, 2012; PICCINELLI et al, 2014).
Pesquisas atuais têm documentado efetiva ação antimicrobiana de extratos alcóolicos (ALSHAMI, ALHARBI, 2014; COSTA et al., 2015) e óleos essenciais (FREIRES et al., 2014; AZZIMONTI et al., 2015) obtidos de plantas.
A família Poaceae, incluindo a planta Cymbopogon nardus (L.) Rendle (Figura 1), tem sido investigada pelo potencial farmacológico, com destaque, a ação contra mosquitos Aedes aegypti, Culex quinquefasciatus e Anopheles dirus (NERIO, VERBEL, STASHENKO, 2010), atividade antibacteriana (DUARTE et al, 2007) e antifúngica (TRINDADE et al., 2015). A ação antifúngica descrita por Trindade et al.
(2015) demonstrou a capacidade do óleo essencial de C. nardus em inibir a aderência de cepas clínicas e ATCC de C. albicans em superfícies. Entretanto, há poucos estudos envolvendo o extrato etanólico (fracionamento e análise química) de C. nardus frente cepas clínicas de espécies de Candida.
Figura 1: Cymbopogon nardus (L.) Rendle
Fonte: a autora
C. nardus, popularmente conhecida como citronela, é nativa do Sri Lanka e cultivada em áreas subtropicais e tropicais da Ásia, África e América, incluindo o Brasil (CHANTHAI et al., 2012; MAN et al., 2012). O óleo essencial de citronela é utilizado pelas propriedades antissépticas, em cosméticos e perfumaria devido aos seus maiores constituintes químicos (geraniol, citral, citronelal e citronelol) (CHANTHAI et al., 2012).
Na Tailândia, a infusão das folhas de citronela é indicada para flatulência, indigestão e cólicas abdominais (CHANTHAI et al., 2012;). Outros estudos têm demonstrado a atividade desta planta como antioxidante e antiviral (NAKAHARA et al., 2003; AINI et al., 2006; LERTSATITTHANAKORN et al., 2006; INNSAN et al., 2011).
Considerando-se que o conhecimento sobre o potencial terapêutico dos vegetais tem despertado o interesse científico, indicando novas alternativas para o controle e tratamento de diversas doenças fúngicas. Na tentativa de ampliar o espectro de agentes antifúngicos, a partir de extratos de plantas, este estudo avaliou o potencial antimicrobiano do extrato etanólico e do óleo essencial das folhas de Cymbopogon nardus.
OBJETIVOS
2. OBJETIVOS 2.1. Objetivo Geral
Avaliar o potencial antifúngico do extrato etanólico (EE) e do óleo essencial (OE) das folhas de Cymbopogon nardus (L.) Rendle (citronela) frente cepas clínicas e ATCC de espécies Candida.
2.2. Objetivos específicos
Ø Avaliar a atividade antifúngica (concentração inibitória mínima e concentração fungicida mínima) do EE e do OE de folhas de citronela sobre as cepas clínicas e ATCC do gênero Candida;
Ø Fracionar o extrato etanólico (3-5 frações) e avaliar sua atividade antifúngica;
Ø Realizar análise química do EE, frações e do óleo essencial e correlacionar cada uma à atividade antifúngica - substância ou classe vegetal;
Ø Caracterizar a curva de morte microbiana (ensaio de cinética), viabilidade celular e interferência ao biofilme frente EE, frações ou OE que melhor apresentou atividade antifúngica.
ARTIGOS CIENTÍFICOS
3. ARTIGOS CIENTÍFICOS
Nesta seção estão demonstrados dois artigos científicos originais, os respectivos periódicos e classificação Qualis.
Artigo 1.
Título: Essential oil of Cymbopogon nardus (L.) Rendle: a strategy to combat fungal infections caused by Candida species.
Periódico de submissão: International Journal Antimicrobial Agents Qualis: A1
Artigo 2.
Título: Chemical composition and antifungal potential of ethanolic extract of Cymbopogon nardus (L.) Rendle against Candida species with different patterns of resistance
Periódico de submissão: PlosOne Qualis: A2
ARTIGO 1
ARTIGO 1
Essential oil of Cymbopogon nardus (L.) Rendle: a strategy to combat fungal infections caused by Candida species.
Luciani Gaspar de Toledoa, Matheus Aparecido dos Santos Ramosb, Larissa Spósitob, Elza Maria Castilhoa, Fernando Rogério Pavanb, Érica de Oliveira Lopesb, Guilherme Julião Zocoloc, Francisca Aliny Nunes Silvac, Tigressa Helena Soaresc, André Gonzaga dos Santosd, Taís Maria Bauabb, Margarete Teresa Gottardo de Almeidaa*
a Department of Infectious Diseases, Faculty of Medicine of São José do Rio Preto, São José do Rio Preto, São Paulo, Brazil.
b Department of Biological Sciences, School of Pharmaceutical Sciences, Univ Estadual Paulista, Araraquara, São Paulo, Brazil.
c Brazilian Agricultural Research Corporation, Embrapa Tropical Agribusiness, Fortaleza, Ceará, Brazil.
d Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, Univ Estadual Paulista, Araraquara, São Paulo, Brazil.
*Correspondence and reprint requests:
Margarete Teresa Gottardo de Almeida (Ph.D.)
Department of Infectious Diseases, School of Medicine of São José do Rio Preto.
Av. Brig. Faria Lima, 5416, 15090-000, São José do Rio Preto, São Paulo, Brazil.
Phone: +55 17 3201 6955; +55 17 3201 5843 Email: [email protected]
Abstract
The incidence of infections caused by Candida yeasts has increased in the last two decades. However, the antifungal therapy has limitations. Essential oils of medicinal plants emerge as an alternative in the search for new antifungal agents. The essential oil (EO) of Cymbopogon nardus (L.) Rendle presents antitumor profile, antinociceptive and antibacterial activity, thus the study of this compound against pathogenic fungi is interesting. The aim of this study was to evaluate the chemical composition and biological potential of the essential oil from C. nardus against Candida species. The EO was obtained by hydrodistillation process and analyzed by gas chromatography-mass spectrometry. The main compounds of the EO were: citronellal, geranial, geraniol, citronellal and neral. The antifungal potential was performed by determination of minimal inhibitory concentration (MIC), time kill assay, inhibition on Candida albicans hyphae growth and inhibition of mature biofilms. Additionally, the cytotoxic evaluation (IC50 determination) was investigated in HepG-2 (hepatic) and MRC-5 (fibroblast) cell lines. The MIC values ranging from 1000 to 250 µg/mL, except to two clinical isolate of C. tropicalis (MIC >1000 µg/mL). The time-kill assay showed that the EO inhibited the growth of the yeasts and hyphal formation of C. albicans. The inhibition of the mature biofilms of the C. albicans, C. krusei and C. parapsilosis strains occurred at concentration of 10X MIC. The values of IC50 of EO were 96.6 ug/mL (HepG-2) and 33.1 ug/mL (MRC-5). EO is an important compound to inhibition of the Candida species, especially considering the action against biofilm.
Keywords: Cymbopogon nardus; essential oil; gas chromatography; Candida;
antifungal activity.
1. INTRODUCTION
Candida species have been a problem in human clinical practice due to the significant increase in cases of infection, especially in immunocompromised patients.
The immune status of the host, the use of broad-spectrum antibiotics and corticosteroids, transplants, long-term intravascular and urethral catheters, parenteral nutrition, are mentioned as risk factors for the development and increased incidence of fungal infections [1].
Candida species can develop on mucous membranes of the human body and this is associated with various types of diseases ranging from mucocutaneous overgrowth to disseminate infections [2]. Although the C. albicans is the prevalent specie in candidemia, other species has been observed, such as C. krusei, C. glabrata, C.
tropicalis and C. parapsilosis [3].
The ability of Candida species to cause disease is mainly related to mechanisms involving several virulence factors that include the morphological transition between yeast and hyphae, ability to defend themselves against the immune system of the host, adhesion, biofilm formation in the host tissue or in medical devices, and production of harmful enzymes such as hydrolytic proteases, phospholipases and hemolysin [4].
Several antifungals have been indicated for the treatment of these infections, including those belonging to the polyenic, azole, and echinocandin class; however, due to the indiscriminate use of these antimicrobials and physiological characteristics of the fungus, there has been a significant increase in the profile of resistance. Furthermore, the high toxicity, drug interactions, insufficient bioavailability of the active ingredient contributes to fail therapeutic [5].
Plants essential oils may be alternatives bioactive compounds with antifungal properties, justified by the presence of secondary metabolites such as tannins, terpenes, alkaloids, flavonoids, etc [6,7].
The genus Cymbopogon of the Poaceae family has been investigated by its pharmacological potential. Cymbopogon nardus (L.) Rendle is popularly known as
“citronella” and cultivated in subtropical and tropical areas of Asia, Africa and America, including Brazil [8]. The essential oil of leaves of the C. nardus is very used in perfumery, production of cosmetics and as an insect repellent. The major constituent chemicals are geraniol, citral, citronellal and citronellol [9]. Studies have demonstrated the activity of this plant as antibacterial [10], antiviral [11] and antioxidant [12].
The essential oil is a complex mixture of monoterpene and sesquiterpenes hydrocarbons (10 and 15 carbon atoms, respectively), their oxygenated derivatives such as alcohols, aldehydes and ketones, phenylpropanoids, and other minor compounds [13]. They are also called volatile oils or ethereal oils, as they have a high degree of evaporation when exposed to air at room temperature; it is this feature that confers the significant odour to plants, both for attraction of pollinators and as insect and herbivore repellents [14].
Essential oil is important in several areas of sciences, especially in the antimicrobial field against pathogenic or opportunistic microorganisms, [15] [16]. The presence of terpenes, as one of the chemical compounds, contributes to the complex constitution, and the action against microorganisms is directly related to this feature [17].
The antimicrobial potential demonstrated by terpenes (e.g. monoterpenes) is attributed to their interference with the integrity and functioning of the cell membrane
through induction of changes in membrane potential, loss of cytoplasmic material and inhibition of the respiratory chain, thus, these characteristics of essential oil is a relevant profile to the discovery of new antifungal agents [18].
Considering the fungal aetiology of several diseases with large impacts on public health, and the use of plants of the Cymbopogon genus in medical literature, the aim of this study was to evaluate the chemical composition and biological potential of the essential oil of C. nardus, focusing on the exploration of the antifungal profile against Candida species and presenting this compound as a possible antifungal or adjuvant agent.
2. MATERIALS AND METHODS 2.1. Plant Material
The leaves of Cymbopogon nardus (L.) Rendle were collected in July 2013, during in the morning, from Garden of Toxic and Medicinal Plants: “Profa. Dra. “Célia Cebrian de Araújo Reis” (Univ Estadual Paulista, Araraquara, São Paulo, Brazil). A voucher specimen (HRCB-60752) was deposited at Herbarium Rioclarense of the Institute of Biosciences (Univ Estadual Paulista, Rio Claro, São Paulo, Brazil).
2.2. Essential oil (EO) extraction
Fresh leaves of C. nardus (150 g) were submitted to hydrodistillation process using a Clevenger type apparatus attached to a round bottom flask (3 L) with 1500 mL of deionized water. The obtained EO was stored under refrigeration until chemical analysis and biological tests.
2.3. Citronellal
The commercial (+/-)- citronellal standard (≥ 95 % purity) used was purchased from Sigma-Aldrich.Co. (St Louis, MO, USA).
2.4. Gas chromatography (GC) analysis of EO
2.4.1. Gas chromatography-mass spectrometry (GC–MS)
GC-MS analysis was performed on Agilent® GC-7890B/MSD-5977A gas chromatograph (mass detector: electron impact ionization; mass quadrupole analyzer) fitted with a HP-5ms capillary column (5%-diphenyl)-polydimethylsiloxane (30 m x 0.25 mm, film thickness 0.25 µm, Agilent®) using helium as carrier gas adjusted to a flow rate of 1,00 mL/min (8.2 psi) and linear velocity of 36.6 cm.s-1. Injector temperature: 250° C; injection volume: 1 μL; splitting ratio: 1:100; oven temperature program: 60-246° C (3° C.min-1, 62 min); transfer line temperature: 280°C; detector temperature: 150° C; ionization energy: 70 eV. EO was solubilized in hexane (chromatographic grade; Merck®) 1:100 (v/v). The identification of the EO components was based on the comparison of acquired mass spectra (from chromatogram peaks) with reference spectra of the NIST mass-spectral library version 2.0 2012 (243,893 compounds) and literature data. Furthermore, arithmetic retention indices [20] were calculated as described in reference [19] by linear interpolation relative to the retention times (tR) of a series of n-alkanes (C7–C30) and the obtained values were compared with literature retention index values [19]. Relative amounts of EO components were calculated based on chromatogram peak area normalization method.
2.4.2. Gas chromatography (GC-FID)
GC-FID analysis was performed on Shimadzu® GC-2010 Plus gas chromatograph (flame ionization detector) fitted with a RTX-5MS capillary column (5%-diphenyl)-polydimethylsiloxane (30 m x 0.25 mm, film thickness 0.25 µm, Restek®) using nitrogen as carrier gas adjusted to a flow rate of 1,00 mL/min (8.2 psi) and linear velocity of 36.6 cm.s-1. Injector temperature: 250° C; injection volume: 1 μL;
splitting ratio: 1:30; oven temperature program: 70-180° C (4° C.min-1) and 180-250° C (10° C.min-1); total analysis time: 34.5 min; detector temperature: 280° C. For the analytical curve, (+/-)- citronellal standard (Sigma-Aldrich®; ≥ 95 % purity) solutions were prepared in hexane (chromatographic grade; Merck®): 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 e 5.0 mg.mL-1. EO was solubilized in hexane (chromatographic grade; Merck®) 1:100 (v/v). All analysis was performed in triplicate. Citronellal identification in the EO was based on retention time (tR) comparison and its quantification was realized according external standard method using an analytical curve.
2.5. Antifungal activity 2.5.1. Fungal strains
The strains - 20 samples of Candida spp - were obtained from Laboratory of Microbiology, Department of Infectious Diseases, Faculty of Medicine of Sao José do Rio Preto, São Paulo, Brazil; three clinical isolates and one ATCC for each species: C.
albicans (CA-ATCC 90028, CA2, CA3, CA4); C. krusei (CK-ATCC 6258, CK2, CK3, CK4); C. glabrata (CG-ATCC 2001, CG2, CG3, CG4); C. tropicalis (CT-ATCC 13803, CT2, CT3, CT4), parapsilosis complex - C. parapsilosis (CP-ATCC 22019,
CP1) and C.orthopsilosis (CO-ATCC 96141, CO1). C. albicans ATCC 10231 was used for inhibition on hyphae growth assay.
The clinical strains were donated to the Microbiology Laboratory of the Faculty of Medicine of Sao Jose do Rio Preto for purposes of scientific research through a written consent of the donors. The use of these strains was approved by the Human Research Ethics Committee CEP-FAMERP (Comitê de Ética em Pesquisa- Faculdade de Medicina de São José do Rio Preto) and received the protocol number 152/2006.
2.5.2. Determination of minimum inhibitory concentration (MIC)
The evaluation of the antifungal activity for MIC determination was performed by the dilution in microplate technique according to the protocol described by M27-A3 document [21], with modifications. The concentration of EO and citronellal was 1000 to 7.8 µg/mL. The EO was dissolved in 10% methanol and 2% Tween 80. 0.1 mL was placed in a 96-well microtiter plate containing RPMI 1640 medium. Each well was inoculated with 0.1 mL of a suspension containing 2.5x103 cfu/mL of yeast.
Amphotericin B (AmB) (Sigma-Aldrich®) and fluconazole (FLU) (Sigma- Aldrich®) were used as the positive controls. Additional controls also included the culture medium, yeast growth, EO and solvent. The microplates were incubated at 37
°C for 48 hours. After incubation, 20 µL of an aqueous 2% solution of 2,3,5- triphenyltetrazolium chloride (TTC) was added and the plates were incubated at 37 °C for 2 hours [22] and absorbance of the samples was measured by spectrophotometer (ELISA- Labtech-LT-4000). All tests were performed in triplicate.
According to obtained results from EO MIC determination, the more sensitive strains (1 ATCC and 1 clinical strain of each specie) were selected for evaluation of antifungal activity of citronellal.
2.5.3. Determination of minimum fungicidal concentration (MFC)
An aliquot from each well that showed antifungal activity was plated on Petri dish containing Sabouraud Dextrose Agar (SDA) - DIFCO, for to determination of minimal fungicidal concentration (MFC). The assays were carried out in triplicate. MFC was defined as the lowest concentration of the EO and citronellal that allowed no visible growth on the solid medium [22].
2.6. Inhibition on Candida albicans hyphae growth
A microassay was developed for evaluating the inhibition effect on the growth of fungal strains. Growth of C. albicans (ATCC 10231) from a 48 h culture were transferred to microplate with RPMI 1640 medium supplemented with fetal bovine serum to obtain a final concentration of 2.5x103 yeast/mL. EO was added to the growth medium to concentrations ranging from 1000 µg/mL to 7, 5 ug/mL, and the cultures were incubated for 12 and 24 h at 37 °C. The hyphal formation of C. albicans was observed through an inverted light microscope. Amphotericin B (16 ug/mL) was used as a positive control [22].
2.7. Time-kill assay
The time kill assay was performed according to Santos-Filho and co-workers [23], with modifications. This assay was used one ATCC strain and one clinical strain for each species of Candida (CA ATCC 90028, CA3, CK ATCC 6258, CK4, CG
ATCC 2001, CG3, CT ATCC 13803, CT3, CP ATCC 22019, CP1, CO ATCC and CO1). In brief, Sabouraud Dextrose broth (SDB)-DIFCO, containing 2.5 x 103 cfu/mL of Candida spp. and 2 x MIC of EO were incubated at 37 ºC and aliquots of 100 µL were removed at different time intervals (0, 1, 2, 4, 8, 12, 24, 36 and 48 hours) and the aliquots were diluted in buffer solution in sterile PBS 1:100, twice. Each EO-cell suspension was spread onto Sabouraud plates and colonies were counted after 48 h incubation at 37 ºC. Amphotericin B was used as positive control. Negative controls were established with cell suspensions without the addition of EO.
2.8. Biofilm assay
The biofilm adhesion method was performed as described by Pitangui and co- workers [24], with modifications. The strains, CA ATCC 90028, CA3, CK ATCC 6258, CK4, CP ATCC 22019 and CP1 were selected to biofilm assay. Initially, 100 μL of inoculum (5.0 x 108 cells/mL) suspended in saline (0.9%) was added to the wells in the microplates (96 wells) and were incubated in a shaker at 80 rpm at 37 °C for two hours.
After the pre-adhesion period, the supernatant was removed and 100 μL of RPMI medium was added to each microplate well; then, the incubation proceeded at 37 °C for 48 hours with RPMI renewed after 24 hours. After all of the incubation periods, the supernatant was removed, and the wells were washed with 100 μL of (0.9%) saline.
Next, 100 μL of EO (10xMIC) were added to each microplate well. The microplates were re-incubated for 24 hours at 37 °C. After, the EO was removed and the wells were washed with sterile saline (to eliminate the drug carryover effect). Solvent, medium culture and yeast growth were established as control. As colorimetric indicator was
employed 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H- tetrazolium hydroxide (XTT®).
2.9. Cytotoxic activity 2.9.1. Cell lines
HepG-2 (hepatic) (ATCC® HB-8065™) and MRC-5 (fibroblast) (ATCC® CCl- 171™) was used to determine cytotoxicity (IC50). The cells were maintained in flasks with a 12.50 cm2 surface area containing 10 mL of culture medium incubated at 37°C in 5% CO2. The culture medium consisted of DMEM (Vitrocell®) medium supplemented with 10% fetal bovine serum, gentamicin sulfate (50 mg/L-Sigma-Aldrich®), and Amphotericin B (2 mg/L- Sigma-Aldrich®).
2.9.2. Cytotoxic assay
The cytotoxic assay [25] consists of collecting the cells using a solution of trypsin/EDTA (Vitrocell®), centrifuging (2,000 rpm for 5 minutes) and counting the number of cells in a Neubauer chamber followed by adjustment of the cell concentration to 7,5 x 104 cells/mL in DMEM. Then, 200 μL of this suspension was deposited in each well of a 96-well microplate to obtain a concentration of 1,5 x 104 cells/well and the microplates were then incubated at 37°C with 5% CO2 for 24 hours to facilitate cell attachment to the plate. The serial dilutions of EO were prepared to obtain concentrations from 1,000 to 3. 90 μg/mL. These dilutions were added to the cells after the removal of the medium and the non-adherent cells. Then, the cells were incubated for an additional 24 hours. The cytotoxicity of the compounds was determined by adding 30 μL of resazurin and reading on a Spectrafluor Plus (TECAN®) reader after 6
hours of incubation using a microplate and excitation and emission filters at wavelengths of 530 nm and 590 nm, respectively. The IC50 was defined as the highest concentration of compound that allowed the viability of at least 50% of the cells. All experiments were performed in triplicate. As control, it was used 5% DMSO.
3. RESULTS AND DISCUSION
3.1. Chemical composition of essential oil
The qualitative and quantitative composition (GC-MS) of the EO is showed in Table 1. Oxygen-containing monoterpenes were the major constituents (90.61 %), with citronellal (27.87 %), geraniol (22.77 %), geranial (14.54 %), citronellol (11.85 %) and neral (11.21 %) as main compounds. These monoterpenes are derived from geranyl diphosphate and biosynthetically related [26].
In the GC-FID analysis the retention time (tR) of citronellal was 11.2 min. The equation and R2 value obtained from analytical curve for citronellal were y=
57,1529.9016 . x - 102,555.3281 and 0.99976. The content of citronellal in the EO (GC- FID) was determined by means of external standardization method as 282.5 mg/mL.
Considering also the relative density at 25o C of commercial C. nardus EO – 0.897 g/mL (Sigma-Aldrich, 2016) – the concentration of citronellal in the EO can be expressed as 31 % (m/m). This value is consistent with the value obtained in GC-MS analysis (28 %).
Citronellal was also identified in EO using thin layer chromatography analysis (silica gel, toluene:ethyl acetate 93:7, sulfuric anysaldheyde spray reagent) by colour and retention factor comparison.
According to study performed by Wei and Wee [27] the content of citronellal was similar to this work, representing the major compound (29.6%). Koba and co- workers [28] and Trindade and co-workers [29], founded high content of citronellal with 35.5% and 37, 75% respectively. In Thailand the contents were different [13] with geraniol (35.7%), trans-citral (22.7%), cis-citral (14.2%), geranyl acetate (5.8%), citronellal (5.8%) and citronellol (4.6%). In other recent study, authors also obtained a different chemical composition of EO with major compounds geraniol (25.9%), citronellal (3.7%) and citronellol (3.1%) [30].
Table 1. Composition of essential oil from the leaves of C. nardus.
Retention time Compound name AI1
(calculated)
AI2 (literature)
Content (%)
6.60 not identified ---- ---- 0.09
6.74 β-myrcene 992 988 0.09
7.08 n-octanal 1004 998 0.09
7.92 D-limonene 1029 1024 2.47
8.21 cis-ocimene 1037 1032 0.27
8.56 trans-ocimene 1048 1044 0.17
8.75 Bergamal 1053 1051 0.37
9.40 not identified ---- ---- 0.17
10.40 Linalool 1101 1095 0.53
10.57 α-pinene oxide 1105 1099 0.11
11.51 trans-rose oxide 1129 1122 0.14
12.16 neo-isopulegol 1145 1144 0.41
12.34 not identified ---- ---- 0.27
12.54 Citronellal 1155 1148 27.87
12.95 not identified ---- ---- 0.25
13.69 not identified ---- ---- 0.33
14.16 cis-4-decenal 1195 1193 0.09
14.63 Decanal 1207 1201 0.46
15.60 β-citronellol 1230 1223 11.85
16.12 Neral 1242 1235 11.21
16.73 Geraniol 1257 1249 22.77
17.38 Geranial 1273 1264 14.54
20.82 citronellol acetate 1355 1350 0.31
22.08 geranyl acetate 1385 1379 0.26
23.45 β-cariophyllene 1419 1417 1.28
24.81 α-humulene 1453 1452 0.12
27.25 γ-cadinene 1514 1513 1.60
27.63 δ-cadinene 1524 1522 0.36
27.87 citronellyl butyrate 1530 1530 0.24
28.60 Elemol 1550 1548 0.11
29.11 not identified ---- ---- 0.16
29.85 cariophyllene oxide 1582 1582 0.55
32.08 trans-cadinol 1642 1638 0.16
32.54 α-muurolol 1654 1644 0.30
Monoterpene hydrocarbons 3.00
Oxygen containing monoterpenes 90.61
Sesquiterpene hydrocarbons 3.36
Oxygen containing sesquiterpenes 1.12
Other compounds 0.64
Total identified 98.73
1Arithmetic retention indices [20] relative to C7-C30 n-alkanes calculated [19].
2Arithmetic retention indices [19, 20].
3.2. MIC and MFC determination of EO
The antifungal activity of EO is exhibited in Table 2. The obtained data showed that EO had effective antifungal activity with MIC range of 250-1000 µg/mL, including resistant isolates to Fluconazole and Amphotericin-B. The lowest MIC value (250 µg/mL) of EO was seen against C. krusei. Besides, the EO showed fungicidal profile against all fungi, except two clinical isolates of C. tropicalis that were resistant to EO with MIC > 1000 µg/mL.
Unlike to conventional antimicrobial drugs, the literature does not present a standard to the MIC values (sensitive and resistant) to natural products against species of Candida. A study performed by Webster and co-workers [31] aimed to evaluate antifungal activity of 14 medicinal plants extracts, which found MIC values equal or lower than 1000 μg/mL, and considered as sensitive.
The values observed in this present study showed satisfactory, since the EO exhibited inhibitory action against 90% of strains tested. Although some strains were inhibited with the highest concentration evaluated (1000 ug/mL), this data are relevant, since the most of strains are resistant to fluconazole (MIC >64 ug/mL), the main drug used in medical practice [32].
Table 2. MIC values (µg/mL) and MFC values (µg/mL) of essential oil (EO) from Cymbopogon nardus against Candida species.
Candida strains MIC EO* MFC EO* MIC AmB* MIC FLU*
CA-ATCC 90028 1000 1000 1 1
CA2 1000 1000 4 16
CA3 1000 1000 1 >64
CA4 1000 1000 4 8
CK-ATCC 6258 250 500 8 >64
CK2 500 500 8 >64
CK3 500 500 8 >64
CK4 250 250 4 >64
CG-ATCC 2001 500 1000 1 >64
CG2 500 1000 4 >64
CG3 500 1000 2 >64
CG4 1000 1000 2 >64
CT-ATCC 13803 500 1000 8 >64
CT2 >1000 >1000 8 >64
CT3 1000 >1000 4 >64
CT4 >1000 >1000 4 >64
CP-ATCC 22019 500 1000 4 8
CP1 1000 1000 4 32
CO-ATCC 96141 500 1000 8 32
CO1 1000 1000 8 64
* values in µg/mL
Interesting MIC values (500 a 250 ug/mL) of EO against C. krusei ATCC and clinical isolate were shown as promising, due to the fact that C. krusei presents intrinsic resistant profile to azoles [33].
The study carried out by Nakahara and co-workers [12] demonstrated that EO inhibited filamentous fungal from environment, however, the methodology used for determination of MIC was different from this research.
The recent search performed by Trindade and co-workers [29] showed the antifungal activity of EO of C. nardus against ATCC and clinical strains of C. albicans and C. tropicalis with MIC values ranging from 64 to 32 ug/mL. The differences found are expected because factors such as climate, region and harvest time of EO, in addition to the extraction method, directly could affected the characteristics and content of chemical compounds [34].
The assay used for determination of MFC showed a fungicidal profile of EO, against Candida species, capable to kill the fungal cells under concentrations evaluated.
The antifungal activity of terpenoids, one of the major groups of volatile secondary metabolite, is known in the pharmaceutic field [17]. Thus, the antifungal activity of EO in this present may be related to monoterpenes showed in GC-MS assay.
Anti-Candida potential of terpene geraniol and citronellol was considered before, with effective inhibitory activity against C. albicans [18] and filamentous fungi Aspergillus species [35]. In addition, Mesa-Arango and co-workers [36] showed oxygenated monoterpenes in the citral chemotype, such as geraniol, citral and citronellal all with antifungal activity against C. parapsilosis, C. krusei, Aspergillus flavus and Aspergillus fumigatus.
3.3. MIC and MFC determination of citronellal
The MIC and MFC of citronellal are demonstrated in Table 3. The citronellal showed antifungal activity against C. albicans ATCC, C. krusei (ATCC and clinical strain) and C. glabrata (ATCC and clinical strain). The species C. tropicalis, C.
parapsilosis, C. orthopsilosis and C. albicans clinical strain were resistant to citronellal, with MIC > 1000 µg/mL. Thus, in this present investigation, C. nardus EO showed
better antifungal activity compared to citronellal likely owing to the synergisms among the chemical compounds presents in the EO.
Table 3. MIC values (µg/mL) and MFC values (µg/mL) of citronellal against Candida species.
Strains MIC citronellal CFM citronellal
CA-ATCC 90028 1000 1000
CA3 >1000 >1000
CK-ATCC 6258 500 1000
CK4 500 500
CG-ATCC 2001 500 1000
CG3 500 >1000
CT-ATCC 13803 >1000 >1000
CT3 >1000 >1000
CP-ATCC 22019 >1000 >1000
CP1 >1000 >1000
3.4. Inhibition on Candida albicans hyphae growth
The results exhibited that EO was able to inhibit the transition of C. albicans from yeast to hyphal form. Microscopic observation of EO-treated fungal cells revealed an absence of filamentous cells in concentrations ranging from 1000 ug/mL to 15 ug/mL (after 12 h and 24 h) (Figure 1).
Figure 1. Inhibitory effect of EO on transition of C. albicans from yeast to hyphal form.
Some therapeutic approaches are used to combat C. albicans, including the ability to block the change between yeast cells to filaments. This morphological change is considered as a virulence factor, and, the biological mechanism has been explored for several active ingredients against this fungal species [22].
The hyphal formation ability of C. albicans is a risk factor in infections because the hyphae play a role in further tissue invasion due to the ability to adhere to host epithelial and endothelial cells [1]. Therefore, the results from this present work are promising, since the EO was able to inhibit this morphological transition.
Leite and co-workers [37] showed the action of citral, a mixture of two geometric isomers known as neral and geranial, very common in the essential oil used in this study. These authors found that these components were able to promote inhibition of pseudohyphae, clamidoconides and blastoconidia at the concentration of 128 ug/mL during 48 hours.
The ability to EO of C. nardus to interfere on hyphae was demonstrated for different species of fungi. Thus, Chen and co-workers [8] proved that the oil was able to promote deformities in hyphal structure of the fungi Alternaria.
3.5. Time-kill assay
The results showed that EO inhibited the fungal growth of Candida species (Figure 2). The cell growth was constant until 24h. After this period, it was noted an exponential growth - CFU / ml - which remained proportional for long time. The strains CK-ATCC 6258, CK4, CG-ATCC 2001, GG3, CP1, CO-ATCC 96141 and CO1 presented, during 48 hours, a superior inhibition behaviour than amphotericin-B during 48 hours.
