Prospecção químico-biológica de extratos hidroetanólicos das folhas de Astronium urundeuva na investigação do potencial antifúngico
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Data
2018-01-25
Autores
Bonifácio, Bruna Vidal
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Universidade Estadual Paulista (Unesp)
Resumo
Apesar do grande avanço tecnológico direcionado à indústria farmacêutica, os micro-organismos continuam adquirindo cada vez mais resistência aos fármacos convencionalmente utilizados e, pesquisadores de diversas áreas buscam novas fontes antimicrobianas de origem natural, a exemplo das plantas medicinais. Dentre as infecções fúngicas, destacam-se aquelas causadas por leveduras do gênero Candida sp. a exemplo da a candidíase vulvovaginal (CVV). A planta Astronium urundeuva (Allemao) Engl. apresenta uma série de propriedades biológicas, incluindo a atividade antifúngica. Este trabalho teve como objetivo aprofundar os estudos direcionados a atividade antifúngica dessa planta medicinal de modo a investigar as substâncias responsáveis por esta propriedade biológica através do fracionamento biomonitorado. Dentre os componentes majoritários identificados no extrato (ácido gálico, galato de metila e galato de etila), pode-se observar que as substâncias galato de etila e galato de metila são responsáveis por grande parte da atividade antifúngica contra a espécie C. glabrata enquanto que a atividade contra a espécie C. albicans parece estar relacionada a um efeito sinérgico dos componentes do extrato, incluindo os minoritários. Os resultados do ensaio in vivo confirmaram a atividade antifúngica exibida pelo extrato, mas, além disso, mostrou uma melhor atividade do extrato incorporado na microemulsão (ME), confirmando o efeito fungicida in vitro apresentado por este. Os ensaios de citotoxicidade (IC50), realizados frente às linhagens celulares VERO, MRC5 e HaCaT mostraram que o extrato não apresenta-se citotóxico quando comparado com o controle positivo utilizado (doxorrubicina). Além disso, após a incorporação do extrato em uma ME, o índice de seletividade (IS) aumentou significativamente principalmente quando analisado para a linhagem VERO. Quando testado contra os isolados clínicos de ambas as espécies de Candida sp., o extrato livre apresentou marcante atividade, mesmo contra aqueles considerados resistentes aos fármacos convencionalmente utilizados na terapia clínica (fluconazol, anfotericina B e caspofungina). Além do ensaio de susceptibilidade para células planctônicas, a amostra vegetal também foi avaliada quanto a sua atividade anti-biofilme e, embora não tenha apresentado atividade em biofilmes pré-formados nas concentrações testadas, o extrato livre foi capaz de inibir a formação de biofilme de C. albicans SC5314 na mesma concentração efetiva para células planctônicas. O estudo de combinação, avaliado pelo ensaio checkerboard, apresentou sinergismo quando o extrato livre foi combinado com fluconazol contra as duas espécies de Candida sp. Além disso, o efeito sinérgico também foi observado quando a anfotericina B foi combinada com o extrato livre (contra C. albicans) e o padrão de galato de etila (contra C. glabrata). Após o ensaio de passagem seriada, foi observado que as cepas desenvolveram resistência apenas ao fluconazol, mas não para o extrato livre. Além do gênero Candida sp., a amostra vegetal também foi testada contra o gênero Cryptococcus sp., destacando-se os resultados apresentados para a espécie C. neoformans WSA e, dentre alguns fungos filamentosos testados, a amostra vegetal mostrou-se efetiva apenas contra Rhizopus arrhizus. Os resultados mostraram que o extrato das folhas de A. urundeuva apresenta atividade antifúngica promissora contra as espécies de Candida, principalmente contra a espécie de C. glabrata.
Despite the great technological advances directed to the pharmaceutical industry, microorganisms continue to acquire more and more resistance to the drugs conventionally used in the therapy, and researchers from several areas keep searching new natural antimicrobial sources, such as medicinal plants. Among fungal infections, we highlight those caused by yeasts from the genus Candida sp., for example vulvovaginal candidiasis (VVC). Astronium urundeuva (Allemao) Engl. plant has various biological properties, including antifungal activity. This work aimed to improve the researches directed to the antifungal activity of this medicinal plant in order to investigate the substances responsible for this biological property using bioassay-guided fractionation technique. Among the major components identified in the extract (gallic acid, methyl gallate and ethyl gallate), we could observe that the substances ethyl gallate and methyl gallate are responsible a large part of the antifungal activity against C. glabrata species whereas the activity against C. albicans species seems to be related to a synergistic effect of the components of the extract, including the minority ones. The in vivo results confirmed the antifungal activity exhibited by the extract, but, in addition, showed a better activity of the extract loaded into a microemulsion (ME), confirming the in vitro fungicidal effect presented by it. The cytotoxicity assays (IC50) performed for VERO, MRC5 and HaCaT cell lines showed that the extract were not cytotoxic when compared to the positive control (doxorubicin). In addition, after loaded into the ME, the selectivity index (SI) significantly increased, especially when analyzed for the VERO cell line. When tested against clinical isolates of both species of Candida sp., the free extract showed really effective activity, even against those considered resistant to the drugs conventionally used in clinical therapy (fluconazole, amphotericin B and caspofungin). In addition to the susceptibility test for planktonic cells, the plant sample was also evaluated for its anti-biofilm activity and, although it did not show activity in preformed biofilms at the concentrations that were tested, the free extract was able to inhibit the biofilm formation of C. albicans SC5314 at the same effective concentration for planktonic cells. The combination study, evaluated by the checkerboard assay, showed synergism when the free extract was combined with fluconazole against both species of Candida sp. In addition, the synergistic effect was also observed when amphotericin B was combined with the free extract (against C. albicans) and the ethyl gallate (against C. glabrata). After the serial passage assay, it was observed that the strains developed resistance only to fluconazole, but not to the free extract. In addition to the genus Candida sp., the plant sample was also tested against the genus Cryptococcus sp., highlighting the results presented for C. neoformans WSA and, among some filamentous fungi that were tested, the plant sample was only effective against Rhizopus arrhizus. The results showed that A. urundeuva leaves extract presents promising antifungal activity against Candida species, especially for C. glabrata.
Despite the great technological advances directed to the pharmaceutical industry, microorganisms continue to acquire more and more resistance to the drugs conventionally used in the therapy, and researchers from several areas keep searching new natural antimicrobial sources, such as medicinal plants. Among fungal infections, we highlight those caused by yeasts from the genus Candida sp., for example vulvovaginal candidiasis (VVC). Astronium urundeuva (Allemao) Engl. plant has various biological properties, including antifungal activity. This work aimed to improve the researches directed to the antifungal activity of this medicinal plant in order to investigate the substances responsible for this biological property using bioassay-guided fractionation technique. Among the major components identified in the extract (gallic acid, methyl gallate and ethyl gallate), we could observe that the substances ethyl gallate and methyl gallate are responsible a large part of the antifungal activity against C. glabrata species whereas the activity against C. albicans species seems to be related to a synergistic effect of the components of the extract, including the minority ones. The in vivo results confirmed the antifungal activity exhibited by the extract, but, in addition, showed a better activity of the extract loaded into a microemulsion (ME), confirming the in vitro fungicidal effect presented by it. The cytotoxicity assays (IC50) performed for VERO, MRC5 and HaCaT cell lines showed that the extract were not cytotoxic when compared to the positive control (doxorubicin). In addition, after loaded into the ME, the selectivity index (SI) significantly increased, especially when analyzed for the VERO cell line. When tested against clinical isolates of both species of Candida sp., the free extract showed really effective activity, even against those considered resistant to the drugs conventionally used in clinical therapy (fluconazole, amphotericin B and caspofungin). In addition to the susceptibility test for planktonic cells, the plant sample was also evaluated for its anti-biofilm activity and, although it did not show activity in preformed biofilms at the concentrations that were tested, the free extract was able to inhibit the biofilm formation of C. albicans SC5314 at the same effective concentration for planktonic cells. The combination study, evaluated by the checkerboard assay, showed synergism when the free extract was combined with fluconazole against both species of Candida sp. In addition, the synergistic effect was also observed when amphotericin B was combined with the free extract (against C. albicans) and the ethyl gallate (against C. glabrata). After the serial passage assay, it was observed that the strains developed resistance only to fluconazole, but not to the free extract. In addition to the genus Candida sp., the plant sample was also tested against the genus Cryptococcus sp., highlighting the results presented for C. neoformans WSA and, among some filamentous fungi that were tested, the plant sample was only effective against Rhizopus arrhizus. The results showed that A. urundeuva leaves extract presents promising antifungal activity against Candida species, especially for C. glabrata.
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Palavras-chave
fitoquímica, atividade antifúngica, Candida sp., candidíase vulvovaginal, biofilme, checkerboard, experimento de passagem seriada, phytochemistry, antifungal activty, vulvovaginal candidiasis, biofilm, checkerboard, serial passage assay