Atividade antifúngica do extrato bruto e frações de Streptococcus mutans sobre Candida albicans em modelos de estudo in vivo
Carregando...
Data
2018-01-15
Autores
Santos, Jéssica Diane dos
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Estudos realizados in vitro tem demonstrado que Streptococcus mutans podem produzir metabólitos capazes de inibir Candida albicans, tornando interessante a identificação e desenvolvimento de novas substâncias para o tratamento da
candidose bucal. Assim, o objetivo desse estudo foi extrair, fracionar e identificar as substâncias produzidas por S. mutans e avaliar seus efeitos sobre a patogenicidade de C. albicans e na resposta imunológica em modelos de estudo in vivo. As
substâncias do sobrenadante da cultura de S. mutans foram extraídas com acetato de etila e posteriormente fracionadas em coluna de sílica derivatizada C-18 (150 g,
Φ = 3,5 cm) utilizando diferentes soluções de MeOH:H2O (36:64, 49:51, 60:40,
76:24, 100:0) como eluente, obtendo cinco diferentes frações (SM-F1, SM-F2, SMF3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e
frações foi realizada por cromatografia gasosa acoplada a espectrometria de
massas. Foram testados os efeitos do extrato bruto e frações do sobrenadante da cultura de S. mutans sobre a candidose experimental induzida em modelo invertebrado de Galleria mellonella e em camundongos imunossuprimidos. Para a escolha da concentração a ser testada nos modelos in vivo foi realizada a determinação da Concentração Inibitória Mínima do extrato bruto e frações sobre C. albicans. No modelo de infecção experimental com G. mellonella, os efeitos do extrato e frações foram analisados pelos testes de curva de sobrevivência, quantificação de UFC/mL de C. albicans na hemolinfa e determinação da densidade hemocitária das larvas de G. mellonella. O extrato, assim como as frações com melhores resultados em modelo de invertebrado foram selecionados para o estudo de candidose bucal em camundongos. Nesse modelo experimental, o desenvolvimento de candidose foi avaliado pelos testes de recuperação e determinação de UFC/mL de C. albicans da cavidade bucal, análise macroscópica e microscópica do dorso da língua. Os dados obtidos foram analisados
estatisticamente pelo Programa Graph Pad Prism 5.0, com nível de significância de 5%. Na determinação da concentração inibitória mínima, apenas o extrato bruto e a
fração SM-F2 demonstraram efeito sobre C. albicans, sendo 10 mg/mL e 15mg/mL, respectivamente. No modelo de G. mellonella não houve aumento da sobrevida das
larvas com a utilização profilática do extrato, ocorrendo 100% de morte nas primeiras 24 h em ambos os grupos com infecção. No entanto, ao realizarmos o tratamento pós-infecção com o extrato bruto houve um aumento da sobrevida em 18,75 a 25%. Como não houve efeito profilático para o extrato bruto, as frações foram administradas apenas terapeuticamente, verificando-se aumento da sobrevida das larvas com as fração SM-F1 e SM-F2. Na contagem de UFC/mL de C. albicans na hemolinfa das larvas, foi verificada diferença estatisticamente significante apenas com o extrato bruto e fração SM-F2 após 12 h de infecção. Em relação a densidade hemocitária, os grupos tratados com extrato bruto e frações (SM-F1 e SM-F2) apresentaram maior número de hemócitos circulantes na hemolinfa em relação ao grupo apenas infectado com C. albicans. Portanto, as frações SM-F1 e SM-F2 foram escolhidas para os testes em camundongos. Nesse modelo de estudo, verificou-se
que o extrato bruto, SM-1 e SM-F2 foram capazes de reduzir significamente o número de UFC/mL de Candida na cavidade bucal e as lesões de candidose no dorso da língua, sendo esses efeitos mais proeminentes para SM-F2. Assim, concluiu-se que as frações SM-F1 e SM-F2 do extrato de S. mutans contêm substâncias antifúngicas com ação terapêutica sobre a candidose experimental, podendo ser alvos de novas estratégias terapêuticas para a candidose bucal.
In vitro studies have shown that Streptococcus mutans can produce metabolites capable of inhibiting Candida albicans, becoming interesting the identification and development of new substances for the treatment of oral candidiasis. Thus, the objective of this study was to extract, fractionate and identify the substances produced by S. mutans and evaluate their effects on the pathogenicity of C. albicans and on the immune response in in vivo study models. Substances from the S. mutans culture supernatant were extracted with ethyl acetate and subsequently fractionated on a C-18 derivatized silica column (150 g, Φ = 3.5 cm) using different solutions of MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) as eluent, obtaining five different fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The crude extract products and the fractions of the supernatant of the S. mutans culture were assessed on experimental candidiasis induced in invertebrate model of Galleria mellonella and in immunosuppressed mice. For a choice of the concentration to be tested in the in vivo models, a determination of the Minimum Inhibitory Concentration (MIC) of the crude extract and fractions on C. albicans was performed. In the model of experimental infection with G. mellonella, the effects of extract and fractions were analyzed by the survival curve test, quantification of CFU/mL of C. albicans in hemolymph and determination of haemocyte density of G. mellonella larvae. The extract, as well as fractions with better results in invertebrate model were selected for the study of oral candidiasis in mice. In this experimental model, the development of candidiasis was evaluated by the tests of recovery and determination of CFU/mL of C. albicans from the mice’s oral cavity, macroscopic and microscopic examination of the tongue dorsum. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. In the determination of the minimal inhibitory concentration, only the crude extract and the SM-F2 fraction showed effect on C. albicans, with 10 mg/mL and 15 mg/mL, respectively. In the model of G. mellonella there was no increase in the larvae survival with the prophylactic use of the extract, occurring 100% of death in the first 24 h in both groups with infection. However, when we performed the post infection treatment with the crude extract there was an increase in survival from 18.75 to 25%. As there was no prophylactic effect for the crude extract, the fractions were administered only therapeutically, verifying increased survival of the larvae with the SM-F1 and SM-F2 fraction. In the CFU/mL count of C. albicans on larval hemolymph, a statistically significant difference was observed only with crude extract and SM-F2 fraction after 12 h of infection. In relation to hemocyte density, the groups treated with crude extract and fractions (SM-F1 and SM-F2) had a higher number of hemocytes circulating in the hemolymph compared to the group only infected with C. albicans. Therefore, the fractions SM-F1 and SMF2 were chosen for the tests in mice. In this study model, it was verified that the crude extract, SM-F1 and SM-F2 were able to significantly reduce the number of CFU/mL of Candida in oral cavity and lesions of candidiasis of the tongue dorsum, these effects were more prominent for SM-F2. Thus, it was concluded that the SM-F1 and SM-F2 fractions of the S. mutans extract contain antifungal substances with therapeutic action on experimental candidiasis, being able to be targets of new therapeutic strategies for oral candidiasis.
In vitro studies have shown that Streptococcus mutans can produce metabolites capable of inhibiting Candida albicans, becoming interesting the identification and development of new substances for the treatment of oral candidiasis. Thus, the objective of this study was to extract, fractionate and identify the substances produced by S. mutans and evaluate their effects on the pathogenicity of C. albicans and on the immune response in in vivo study models. Substances from the S. mutans culture supernatant were extracted with ethyl acetate and subsequently fractionated on a C-18 derivatized silica column (150 g, Φ = 3.5 cm) using different solutions of MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) as eluent, obtaining five different fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The crude extract products and the fractions of the supernatant of the S. mutans culture were assessed on experimental candidiasis induced in invertebrate model of Galleria mellonella and in immunosuppressed mice. For a choice of the concentration to be tested in the in vivo models, a determination of the Minimum Inhibitory Concentration (MIC) of the crude extract and fractions on C. albicans was performed. In the model of experimental infection with G. mellonella, the effects of extract and fractions were analyzed by the survival curve test, quantification of CFU/mL of C. albicans in hemolymph and determination of haemocyte density of G. mellonella larvae. The extract, as well as fractions with better results in invertebrate model were selected for the study of oral candidiasis in mice. In this experimental model, the development of candidiasis was evaluated by the tests of recovery and determination of CFU/mL of C. albicans from the mice’s oral cavity, macroscopic and microscopic examination of the tongue dorsum. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. In the determination of the minimal inhibitory concentration, only the crude extract and the SM-F2 fraction showed effect on C. albicans, with 10 mg/mL and 15 mg/mL, respectively. In the model of G. mellonella there was no increase in the larvae survival with the prophylactic use of the extract, occurring 100% of death in the first 24 h in both groups with infection. However, when we performed the post infection treatment with the crude extract there was an increase in survival from 18.75 to 25%. As there was no prophylactic effect for the crude extract, the fractions were administered only therapeutically, verifying increased survival of the larvae with the SM-F1 and SM-F2 fraction. In the CFU/mL count of C. albicans on larval hemolymph, a statistically significant difference was observed only with crude extract and SM-F2 fraction after 12 h of infection. In relation to hemocyte density, the groups treated with crude extract and fractions (SM-F1 and SM-F2) had a higher number of hemocytes circulating in the hemolymph compared to the group only infected with C. albicans. Therefore, the fractions SM-F1 and SMF2 were chosen for the tests in mice. In this study model, it was verified that the crude extract, SM-F1 and SM-F2 were able to significantly reduce the number of CFU/mL of Candida in oral cavity and lesions of candidiasis of the tongue dorsum, these effects were more prominent for SM-F2. Thus, it was concluded that the SM-F1 and SM-F2 fractions of the S. mutans extract contain antifungal substances with therapeutic action on experimental candidiasis, being able to be targets of new therapeutic strategies for oral candidiasis.
Descrição
Palavras-chave
Streptococcus mutans, Candida albicans, Candidose experimental, Galleria mellonella, Camundongos imunossuprimidos, Experimental candidiasis, Immunocompromised mice, Streptococcus mutans, Candida albicans, Experimental candidiasis, Galleria mellonella, Immunocompromised mice