Eficácia dos extratos de diferentes espécies de Cryptocarya (Lauraceae) sobre biofilme de Candida albicans e biocompatibilidade: estudos in vitro e in vivo
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2021-08-06
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Universidade Estadual Paulista (Unesp)
Resumo
Candidíase é uma infecção oportunista da mucosa causada por espécies de Candida, sendo a Candida albicans o agente etiológico mais comum. O uso de terapias antifúngicas deve ser limitado por causa da sua toxicidade, das baixas taxas de efetividade, da indução de resistência fúngica e baixa solubilidade. O objetivo do presente estudo foi avaliar a eficácia dos extratos de diferentes espécies de Cryptocarya sobre biofilme de Candida albicans e sua biocompatibilidade por meio de estudos in vitro e in vivo. Em um primeiro estudo, os extratos foram avaliados em relação à Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM) em células planctônicas de C. albicans bem como a Concentração Fungicida Mínima dessas células em biofilme (CFMb). Tais análises foram realizadas por meio de contagem de unidades formadoras de colônias (UFC/mL). Posteriormente, um biofilme maduro (48 horas) foi formado sobre amostras de resina acrílica para bases de próteses e os efeitos fungicidas dos extratos sobre esse biofilme também foram avaliados por meio dos testes AlamarBlue® e contagem das UFC/mL. Além disso, foi realizada a microscopia de varredura confocal a laser para análise qualitativa e quantitativa do biofilme após o tratamento proposto. Em um segundo estudo, a citotoxicidade de extratos das espécies do gênero Cryptocarya, que apresentaram os melhores resultados nos testes anteriores, foi avaliada por meio do teste AlamarBlue®, utilizando-se queratinócitos orais normais (NOK). Por fim, foi determinada a eficácia do extrato selecionado nos estudos in vitro na inativação de biofilme de C. albicans presente no dorso lingual de camundongos com candidose induzida. Para isso, após os tratamentos com a planta medicinal, os fungos presentes no dorso lingual de camundongos foram coletados e analisados por meio dos testes AlamarBlue® e UFC/mL. Foi realizada a análise histológica do tecido da língua do camundongo após os diferentes tratamentos. Para cada variável, os dados foram submetidos à análise estatística mais adequada, com nível de significância de 5%. Os resultados mostraram que os extratos obtidos das folhas e frutos de C. moschata e C. mandioccana foram capazes de reduzir o número de unidades formadoras de colônias e o metabolismo celular do biofilme de C. albicans formado sobre amostras de resina acrílica para base de próteses, comprovando seu efeito antimicrobiano. Esses extratos também reduziram a viabilidade das células epiteliais quando avaliados por meio do teste AlamarBlue® E no estudo in vivo, em que foi utilizado o extrato de folha de C. moschata observamos histologicamente que não houve dano celular na língua dos camundongos, e houve diminuição do biofilme de Candida semelhante ao uso de nistatina. Podemos concluir que o extrato foi eficaz contra o biofilme de C. albicans e foi biocompatível no estudo in vivo
Candidiasis is an opportunistic mucosal infection caused by Candida species, Candida albicans being the most common etiological agent. The use of antifungal therapies should be limited because of their toxicity, low effectiveness rates, induction of fungal resistance, and low solubility. The present study aims to evaluate the efficiency of the extracts from different Cryptocarya species on Candida albicans biofilm and its biocompatibility by in vitro and in vivo studies. In a first study, the plants were evaluated in relation to the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of planktonic cells of C. albicans as well as the Minimum Fungicidal Concentration of these cells in biofilm (CFMb). These analyzes were carried out counting the number of colony-forming units (CFU/mL). Subsequently, a mature biofilm (48 hours) was formed on acrylic resin samples for denture bases and the fungicidal effects of the extracts on this biofilm were also evaluated. These analyzes were performed using AlamarBlue® tests and counting of CFU/mL. Moreover, confocal laser scanning microscopy was performed for qualitative and quantitative analysis of the biofilm after the proposed treatment. In a second study, the cytotoxicity of extracts from species of the Cryptocarya genus, which categories showed the best results in previous tests, was evaluated using the AlamarBlue® test, using normal oral keratinocytes (NOK). Finally, the efficiency of the selected extract in inactivating C. albicans biofilm presents on the dorsal lingual surface of mice with induced candidosis was determined. For this, after treatments with the medicinal plant, the fungi present on the dorsal lingual surface of mice were collected and analyzed using the AlamarBlue® and UFC/mL tests. Histological analysis of the mice epithelial tongue cells was performed after the different treatments. For each variable, the data were subjected to the most appropriate statistical analysis, with a significance level of 5%. The results showed that the extracts obtained from the leaves and fruits of C. moschata and C. mandioccana were able to reduce the number of colony-forming units and the cellular metabolism of the C. albicans biofilm formed on acrylic resin samples for denture base, proving its antimicrobial effect. These extracts also reduced the viability of epithelial cells when evaluated by the AlamarBlue® test. The in vivo study, in which C. moschata leaf extract was used, we observed that histologically there was no cell damage in the mice's tongue, and there was a decrease in Candida biofilm similar to the use of nystatin. It therefor has to be concluded that the extract was effective against C. albicans biofilm and was biocompatible in the in vivo study
Candidiasis is an opportunistic mucosal infection caused by Candida species, Candida albicans being the most common etiological agent. The use of antifungal therapies should be limited because of their toxicity, low effectiveness rates, induction of fungal resistance, and low solubility. The present study aims to evaluate the efficiency of the extracts from different Cryptocarya species on Candida albicans biofilm and its biocompatibility by in vitro and in vivo studies. In a first study, the plants were evaluated in relation to the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of planktonic cells of C. albicans as well as the Minimum Fungicidal Concentration of these cells in biofilm (CFMb). These analyzes were carried out counting the number of colony-forming units (CFU/mL). Subsequently, a mature biofilm (48 hours) was formed on acrylic resin samples for denture bases and the fungicidal effects of the extracts on this biofilm were also evaluated. These analyzes were performed using AlamarBlue® tests and counting of CFU/mL. Moreover, confocal laser scanning microscopy was performed for qualitative and quantitative analysis of the biofilm after the proposed treatment. In a second study, the cytotoxicity of extracts from species of the Cryptocarya genus, which categories showed the best results in previous tests, was evaluated using the AlamarBlue® test, using normal oral keratinocytes (NOK). Finally, the efficiency of the selected extract in inactivating C. albicans biofilm presents on the dorsal lingual surface of mice with induced candidosis was determined. For this, after treatments with the medicinal plant, the fungi present on the dorsal lingual surface of mice were collected and analyzed using the AlamarBlue® and UFC/mL tests. Histological analysis of the mice epithelial tongue cells was performed after the different treatments. For each variable, the data were subjected to the most appropriate statistical analysis, with a significance level of 5%. The results showed that the extracts obtained from the leaves and fruits of C. moschata and C. mandioccana were able to reduce the number of colony-forming units and the cellular metabolism of the C. albicans biofilm formed on acrylic resin samples for denture base, proving its antimicrobial effect. These extracts also reduced the viability of epithelial cells when evaluated by the AlamarBlue® test. The in vivo study, in which C. moschata leaf extract was used, we observed that histologically there was no cell damage in the mice's tongue, and there was a decrease in Candida biofilm similar to the use of nystatin. It therefor has to be concluded that the extract was effective against C. albicans biofilm and was biocompatible in the in vivo study
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Palavras-chave
Cryptocarya, Candida albicans, Biofilmes, Cryptocarya, Biofilms, Candida albicans