Cymbopogon nardus (L.) Rendle (citronela): prospecção química-biológica do óleo essencial com destaque no estudo de biofilme e controle da candidíase vulvovaginal
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Data
2020-03-26
Autores
Toledo, Luciani Gaspar de
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Universidade Estadual Paulista (Unesp)
Resumo
A candidíase vulvovaginal (CVV) é uma infecção fúngica oportunista que ocasiona relevante problema à saúde da mulher, visto os desagradáveis sintomas desta afecção, somada às dificuldades terapêuticas dos fármacos da prática clínica e ao surgimento de cepas resistentes. Neste sentido, as plantas medicinais associadas a alternativas que melhorem o potencial antifúngico, como o emprego da nanotecnologia, despontam como uma importante fonte no controle e erradicação de infecções fúngicas. Deste modo, o objetivo deste trabalho foi avaliar a atividade antifúngica do óleo essencial (OE) de Cymbopogon nardus (L.) Rendle, em forma livre e incorporado em microemulsão lipídica (ME+OE) frente cepas ATCC e clínicas de C. albicans, C. glabrata e C. krusei. O OE foi obtido por hidrodestilação. A análise química do OE foi realizada por cromatografia gasosa acoplada a espectrofotômetro de massas (CG-EM). As microemulsões lípidicas (ME) foram desenvolvidas e um diagrama de fases foi construído. A ME e ME+OE foram caracterizados por dispersão de luz dinâmica, análise do potencial zeta, microscopia de luz polarizada, ensaios reológicos, força mucoadesiva e microscopia eletrônica de transmissão (MET). A potencialidade antifúngica do OE e ME+OE foi avaliada pela determinação da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM) por microdiluição, ensaio de interação sorbitol e ergosterol. O ensaio de inibição da formação de hifas de C. albicans em presença de probiótico e patógeno vaginal foi realizado por meio de sistema transwells®. Além disso, superfícies revestidas com OE, ME+OE e limpa foram caracterizadas por ângulo de contato (bolha cativa). O efeito do OE e ME+OE sobre processo de adesão em biofilme foi avaliado em câmara de fluxo em placas paralelas e força de adesão entres superfície revestidas e C. albicans por microscopia de força atômica (MFA). Adicionalmente, foi avaliado a ação do OE e ME+OE sobre biofilmes maduros monotípicos e heterotípicos (C. albicans + Lactobacillus acidophilus). A capacidade de toxicidade do OE e ME+OE foi analisada sobre linhagem celular humana HaCat e em modelo alternativo de Artemia salina. A avaliação da atividade terapêutica do OE e ME+OE em ensaio in vivo de CVV foi executada em camundongos fêmeas da linhagem C57BL/6. A análise química do OE apresentou como componentes químicos majoritários: citronelal, geranial, geraniol, citronelol, geranial e neral. A caracterização físico-química mostrou que ME e ME+OE apresentam tamanho de partículas e homogeneidade adequadas, potencial zeta negativo e estabilidade. Pela microscopia de luz polarizada, ME e ME+OE demonstraram um comportamento isotrópico, sugerindo a formação de sistema microemulsionado. ME e ME+OE apresentaram características não newtonianas, com comportamento pseudoplásico. A força mucoadesiva não foi alterada após a incorporação do OE na ME. As imagens de MET demonstraram estruturas esféricas para a ME, entretanto, as fotomicrografias de ME+OE mostraram possíveis gotículas de OE dentro das estruturas esféricas, indicando a incorporação do OE na ME. O OE apresentou efetiva ação inibitória para as cepas testadas. Sobretudo, frente a cepa padrão de C. albicans, quando o OE foi incorporado a microemulsão (ME+OE) exibiu melhora expressiva da atividade antifúngica com diminuição da CIM (de 500 para 31,2 µg/mL). Para as outras espécies de Candida (C. krusei e C. glabrata) ME+OE não apresentou inibição fúngica. Deste modo, os posteriores ensaios de atividade antifúngica foram realizados com a espécie C. albicans. OE e ME+OE exibem a capacidade de atuar na membrana celular de C. albicans, uma vez que a CIM se mostrou aumentada na presença de ergosterol. OE e ME+OE preveniram a formação de hifa de C. albicans, entretanto o OE foi mais efetivo. A presença de probiótico e patógeno vaginal reduz a formação de hifas de C. albicans. As superfícies revestidas com OE e ME+OE apresentaram-se menos hidrofílicas que o vidro limpo pela medida de ângulo de contato. ME+OE apresentou redução expressiva da adesão de C. albicans sobre superfície de vidro em câmara de fluxo. Sobretudo, após a passagem da interface ar-líquido (bolha de ar) foi observado efetivo destacamento das leveduras na superfície de vidro revestida com ME+OE. O OE e ME+OE inibiu o biofilme de cepa padrão e clínica de C. albicans, porém ressalta-se que o OE foi capaz de promover ausência de crescimento fúngico (UFC/mL) em ágar (plaqueamento). O biofilme monotípico de L. acidophilus exibiu alta atividade metabólica bacteriana frente ao OE e ME+OE. O OE mostrou padrão aceitável de citotoxicidade em linhagem celular HaCat. O OE e ME+OE demonstraram baixa toxicidade em modelo alternativo de Artemia salina. Em ensaio in vivo de CVV o ME+OE conferiu a cura em 100% dos animais no terceiro dia de tratamento e o OE ocasionou a cura em 50% dos animais no segundo dia de tratamento. A utilização da nanotecnologia como a ME, para a incorporação do OE, aprimora a atividade antifúngica do OE de C. nardus sobre células planctônicas e promove inibição da formação de hifas em presença de probiótico e patógeno vaginal. Além disso, o ME+OE promove a prevenção de adesão de C. albicans em câmara de fluxo em placas paralelas somada ao aumento do destacamento de leveduras aderidas em superfície de vidro revestida com ME+OE. Adicionalmente, o OE e ME+OE inibem os biofilmes monotípicos e heterotípicos. Sobretudo, o OE incorporado em ME exibe relevante efeito em ensaio in vivo de CVV com camundongos, sendo uma alternativa promissora no controle de infecções causadas por C. albicans.
Vulvovaginal candidiasis (VVC) is an opportunistic fungal infection that causes relevant problem to woman’s health, due to unpleasant symptoms, difficulties of the therapy and the emergence of resistant strains. In this context, the medicinal plants associated to alternatives that improve the antifungal potential, such as the nanotechnology, is an important source in the eradication and control of fungal infections. Thus, the objective of this study was to evaluate the antifungal activity of the essential oil (EO) of Cymbopogon nardus (L.) Rendle, free form and loaded into a lipid microemulsion (ME+EO) against ATCC and clinical strains of C. albicans, C. glabrata and C. krusei. The EO was obtained by hydrodistillation. The chemical analysis of the EO was performed by gas chromatography-mass spectrometry (GC-MS). The lipid microemulsions (ME) were developed and a phase diagram was constructed. The ME and ME+EO were characterized by scattering, zeta potential, polarized light microscopy, rheological assays, bioadhesiveness and transmission electronic microscopy (TEM). The antifungal activity of the EO and ME+EO were evaluated by determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) by microdilution, sorbitol and ergosterol assay. The inhibition of C. albicans hyphae formation in presence of probiotic and vaginal pathogen was carried out by transwells® system. Moreover, the coated and clean surfaces were characterized by contact angle (captive bubble). The effect of EO and ME+EO was evaluated on biofilm adhesion process by flow chamber in parallel plates and adhesion force between coating surfaces and C. albicans by atomic force microscopy (AFM). Additionally, the action of EO and ME+EO was performed against monotypic and heterotypic mature antibiofilm (C. albicans + Lactobacillus acidophilus). The toxicity of EO and ME+EO was analyzed on human cell line HaCat and using alternative model assay with A. salina. The experimental in vivo VVC was carried out in female mice (C57BL/6). The chemical analysis of the EO showed as main compounds the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The characterization of ME and ME+EO exhibited suitable size and homogeneity, negative charge and stability. By polarized light microscopy, ME and ME + OE demonstrated an isotropic behavior, suggesting the formation of a microemulsified system. ME and ME+EO showed non-Newtonian characteristics, with pseudoplastic behavior. The bioadhesiveness was not altered after the incorporation of the EO in the ME. TEM images showed spherical structures for ME, however, the ME+EO photomicrographs showed possible droplets of OE within the spherical structures, indicating the incorporation of OE in the ME. The EO showed effective inhibitory action for all tested strains. The EO when loaded in the ME (ME+EO) exhibited expressive improvement of the antifungal action with reduction of the MIC (from 500 to 31, 2 µg/mL). For all Candida species (C. krusei e C. glabrata) the ME+EO did not cause fungal inhibition. Thus, the subsequent antifungal activity tests were carried out with C. albicans. The EO and ME+EO have the action in the fungal cell membrane, since the MIC was increased in presence of ergosterol. EO and ME+EO prevented the C. albicans hyphae formation, however, the EO was more active. The presence of probiotic and vaginal pathogen decreased C. albicans hyphae formation. ME+EO showed a significant reduction on C. albicans adhesion to the glass surface in flow chamber. Above all, after the passage of the air-liquid interface (air bubble) effective detachment was observed detachment on the ME+EO coated glass surface. EO and ME+EO coated surfaces were less hydrophilic than the control by contact angle measurement The EO and ME+EO inhibited the biofilm of standard and clinical strain of C. albicans, however, the EO was able to promote the absence of fungal growth on agar (CFU/mL). The L. acidophilus monotypic biofilm exhibited high bacterial metabolic activity in presence of EO and ME+EO. The EO showed acceptable standard of toxicity on cell line HaCat. The EO and ME+EO demonstrated low toxicity in alternative model assay with A. salina. In vivo VVC assay the ME+EO was able to promote the cure of the 100% of the animals in the third day of the treatment and the EO caused the cure in 50% of the animals in the second day of treatment. The use of nanotechnology as a lipid microemulsion, for the incorporation of EO, improves the antifungal activity of OE from C. nardus on planktonic cells, inhibiting the formation of hyphae in the presence of probiotic and vaginal pathogen. Moreover, ME+EO promotes the prevention of adherence of C. albicans in a parallel chamber flow chamber and increased the detachment of adhered yeasts to a ME+EO coated glass surface. Additionally, OE and ME + OE inhibit monotypic and heterotypic biofilms. Above all, the EO incorporated in the ME shows a relevant effect in in vivo CVV assay with mice, being a promising alternative to control of infections caused by C. albicans.
Vulvovaginal candidiasis (VVC) is an opportunistic fungal infection that causes relevant problem to woman’s health, due to unpleasant symptoms, difficulties of the therapy and the emergence of resistant strains. In this context, the medicinal plants associated to alternatives that improve the antifungal potential, such as the nanotechnology, is an important source in the eradication and control of fungal infections. Thus, the objective of this study was to evaluate the antifungal activity of the essential oil (EO) of Cymbopogon nardus (L.) Rendle, free form and loaded into a lipid microemulsion (ME+EO) against ATCC and clinical strains of C. albicans, C. glabrata and C. krusei. The EO was obtained by hydrodistillation. The chemical analysis of the EO was performed by gas chromatography-mass spectrometry (GC-MS). The lipid microemulsions (ME) were developed and a phase diagram was constructed. The ME and ME+EO were characterized by scattering, zeta potential, polarized light microscopy, rheological assays, bioadhesiveness and transmission electronic microscopy (TEM). The antifungal activity of the EO and ME+EO were evaluated by determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) by microdilution, sorbitol and ergosterol assay. The inhibition of C. albicans hyphae formation in presence of probiotic and vaginal pathogen was carried out by transwells® system. Moreover, the coated and clean surfaces were characterized by contact angle (captive bubble). The effect of EO and ME+EO was evaluated on biofilm adhesion process by flow chamber in parallel plates and adhesion force between coating surfaces and C. albicans by atomic force microscopy (AFM). Additionally, the action of EO and ME+EO was performed against monotypic and heterotypic mature antibiofilm (C. albicans + Lactobacillus acidophilus). The toxicity of EO and ME+EO was analyzed on human cell line HaCat and using alternative model assay with A. salina. The experimental in vivo VVC was carried out in female mice (C57BL/6). The chemical analysis of the EO showed as main compounds the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The characterization of ME and ME+EO exhibited suitable size and homogeneity, negative charge and stability. By polarized light microscopy, ME and ME + OE demonstrated an isotropic behavior, suggesting the formation of a microemulsified system. ME and ME+EO showed non-Newtonian characteristics, with pseudoplastic behavior. The bioadhesiveness was not altered after the incorporation of the EO in the ME. TEM images showed spherical structures for ME, however, the ME+EO photomicrographs showed possible droplets of OE within the spherical structures, indicating the incorporation of OE in the ME. The EO showed effective inhibitory action for all tested strains. The EO when loaded in the ME (ME+EO) exhibited expressive improvement of the antifungal action with reduction of the MIC (from 500 to 31, 2 µg/mL). For all Candida species (C. krusei e C. glabrata) the ME+EO did not cause fungal inhibition. Thus, the subsequent antifungal activity tests were carried out with C. albicans. The EO and ME+EO have the action in the fungal cell membrane, since the MIC was increased in presence of ergosterol. EO and ME+EO prevented the C. albicans hyphae formation, however, the EO was more active. The presence of probiotic and vaginal pathogen decreased C. albicans hyphae formation. ME+EO showed a significant reduction on C. albicans adhesion to the glass surface in flow chamber. Above all, after the passage of the air-liquid interface (air bubble) effective detachment was observed detachment on the ME+EO coated glass surface. EO and ME+EO coated surfaces were less hydrophilic than the control by contact angle measurement The EO and ME+EO inhibited the biofilm of standard and clinical strain of C. albicans, however, the EO was able to promote the absence of fungal growth on agar (CFU/mL). The L. acidophilus monotypic biofilm exhibited high bacterial metabolic activity in presence of EO and ME+EO. The EO showed acceptable standard of toxicity on cell line HaCat. The EO and ME+EO demonstrated low toxicity in alternative model assay with A. salina. In vivo VVC assay the ME+EO was able to promote the cure of the 100% of the animals in the third day of the treatment and the EO caused the cure in 50% of the animals in the second day of treatment. The use of nanotechnology as a lipid microemulsion, for the incorporation of EO, improves the antifungal activity of OE from C. nardus on planktonic cells, inhibiting the formation of hyphae in the presence of probiotic and vaginal pathogen. Moreover, ME+EO promotes the prevention of adherence of C. albicans in a parallel chamber flow chamber and increased the detachment of adhered yeasts to a ME+EO coated glass surface. Additionally, OE and ME + OE inhibit monotypic and heterotypic biofilms. Above all, the EO incorporated in the ME shows a relevant effect in in vivo CVV assay with mice, being a promising alternative to control of infections caused by C. albicans.
Descrição
Palavras-chave
Cymbopogon nardus, óleo essencial, microemulsão lipídica, Candida albicans, candidíase vulvovaginal, essential oil, lipid microemulsion, vulvovaginal candidiasis