Sistema precursor de cristal líquido associado ao terpinen-4-ol e nistatina: caracterização, ação antifúngica, sinérgica, citotoxicidade e adesão em células orais
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Data
2018-03-19
Autores
Francisconi, Renata Serignoli [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os sistemas precursores de cristal líquido associados aos extratos vegetais e compostos fitoquímicos abrem novas perspectivas para prevenção e controle eficiente de doenças infecciosas fúngicas. Os objetivos deste estudo foram: I) avaliar o efeito sinérgico da associação do terpinen-4-ol (t-4-ol) com a nistatina (nis); II) avaliar na forma de cristal líquido, a propriedade antifúngica do t-4-ol e possível sinergismo com a nis; III) avaliar a citotoxicidade do t-4-ol em células orais normais imortalizadas (NOK) e verificar se o t-4-ol interfere na adesão de Candida albicans sobre células orais. Assim, foi definida a Concentração Inibitória Mínima (CIM) e a Concentração Fungicida Mínima (CFM) do t-4-ol sobre isolados clínicos de C. albicans (Genotipagem A e B) e cepa SC 5314, empregando-se o método de microdiluição em caldo. Biofilmes foram preparados usando o modelo de placa de microtitulação estática e quantificados por metodologia colorimétrica do ensaio de redução de sal de tetrazólio (XTT). Os mesmos testes foram aplicados para avaliar o t-4-ol e nis na forma de cristal líquido. Adicionalmente, as células foram cultivadas em meio de cultura Dulbecco ́s Modified Eagle ́s Medium (DMEM) suplementado com 10% de soro fetal bovino e 1% de penicilina, estreptomicina e glutamina e mantidas em incubadora a 37o C em 5% de CO2. A citotoxicidade sobre linhagem de células NOK foi avaliada por citometria de fluxo e foi realizado o teste de co-cultura, para verificar a interferência do t-4-ol na adesão de Candida albicans sobre células orais (NOK). A adesão de Candida albicans sobre as células epiteliais foram avaliadas em unidades formadoras de colônias (UFC mL-1) e mostraram que o t-4-ol associado ou não ao SPCL, reduzem a adesão de C. albicans sobre células epiteliais. Os componentes apresentaram ação antifúngica tanto em cultura planctônicas quanto em biofilme, além disso observou-se efeito sinérgico dos componentes testados. Os componentes não foram citotóxicos nos tempos de aplicação de 4 e 8 horas. Esse estudo demonstrou que o t-4-ol isolado ou em combinação com a nis pode proporcionar uma aplicação clínica eficaz e quando associados ao sistema precursor de cristal líquido, induz melhor eficácia em menores concentrações, com controle dos microrganismos resistentes.
The liquid crystal precursor systems associated with plant extracts and phytochemical compounds open new perspectives for efficient prevention and control of infectious fungal diseases. The objectives of this study were: I) to evaluate the synergistic effect of the combination of terpinen-4-ol (t-4-ol) with nystatin (nys); II) to evaluate in liquid crystal form the antifungal property of t-4-ol and possible synergism with nys; III) to evaluate the cytotoxic potential of t-4-ol in normal immortalized oral cells (NOK) and to verify whether t-4-ol interferes with the adhesion of Candida albicans to oral cells. Thus, the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (CFM) of t-4-ol on clinical isolates of C. albicans (Genotype A and B) and strain SC 5314 were determined using the microdilution method in broth. Biofilms were prepared using the static microtiter plate model and quantified by colorimetric methodology of the tetrazolium salt reduction assay (XTT). The same tests were applied to evaluate t-4-ol and nys in liquid crystal form. In addition, the cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin and glutamine and maintained in an incubator at 37 ° C in 5% CO2 . Linear cytotoxicity of NOK cells was assessed by flow cytometry and the coculture test was performed to verify the interference of t-4-ol on Candida albicans adhesion on oral cells (NOK). Candida albicans adhesion on epithelial cells was evaluated in colony forming units (UFC mL-1) and showed that t-4-ol associated or not with SPCL reduced C. albicans adhesion on epithelial cells. The components presented antifungal action in both planktonic and biofilm culture, in addition, a synergistic effect of the tested components was observed. The components were not cytotoxic at application times of 4 and 8 hours. This study demonstrated that t-4-ol alone or in combination with nys may provide an effective clinical application and when associated with the liquid crystal precursor system induces better efficacy at lower concentrations with control of resistant microorganisms
The liquid crystal precursor systems associated with plant extracts and phytochemical compounds open new perspectives for efficient prevention and control of infectious fungal diseases. The objectives of this study were: I) to evaluate the synergistic effect of the combination of terpinen-4-ol (t-4-ol) with nystatin (nys); II) to evaluate in liquid crystal form the antifungal property of t-4-ol and possible synergism with nys; III) to evaluate the cytotoxic potential of t-4-ol in normal immortalized oral cells (NOK) and to verify whether t-4-ol interferes with the adhesion of Candida albicans to oral cells. Thus, the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (CFM) of t-4-ol on clinical isolates of C. albicans (Genotype A and B) and strain SC 5314 were determined using the microdilution method in broth. Biofilms were prepared using the static microtiter plate model and quantified by colorimetric methodology of the tetrazolium salt reduction assay (XTT). The same tests were applied to evaluate t-4-ol and nys in liquid crystal form. In addition, the cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin and glutamine and maintained in an incubator at 37 ° C in 5% CO2 . Linear cytotoxicity of NOK cells was assessed by flow cytometry and the coculture test was performed to verify the interference of t-4-ol on Candida albicans adhesion on oral cells (NOK). Candida albicans adhesion on epithelial cells was evaluated in colony forming units (UFC mL-1) and showed that t-4-ol associated or not with SPCL reduced C. albicans adhesion on epithelial cells. The components presented antifungal action in both planktonic and biofilm culture, in addition, a synergistic effect of the tested components was observed. The components were not cytotoxic at application times of 4 and 8 hours. This study demonstrated that t-4-ol alone or in combination with nys may provide an effective clinical application and when associated with the liquid crystal precursor system induces better efficacy at lower concentrations with control of resistant microorganisms
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Palavras-chave
Biofilmes, Nistatina, Candida albicans, Cristais líquido, Biofilm, Nystatin, Candida albicans, Liquid Crystal