Avaliação de Reparo de DNA e Atividade Antioxidante do Extrato Etanólico e Substâncias Isoladas de Casearia sylvestris
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Data
2012-11-28
Autores
Prieto, Aline Miranda [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Trabalhos demonstraram que derivados da planta Casearia sylvestris protegem o DNA de danos. Dessa forma, este trabalho visa avaliar por quais mecanismos essa proteção ocorre. Os produtos naturais avaliados foram o extrato etanólico das folhas de C. sylvestris e os diterpenos clerodânicos obtidos deste extrato: casearinas B, D e X e caseargrewiina F. Para a avaliação dos efeitos propostos foram utilizadas como modelo as linhagens celulares HepG2, MRC5, XP4PA e Hepa 1c1c7. Primeiramente, os produtos naturais foram avaliados com relação à sua atividade citotóxica pelo teste do MTT e ensaio de sobrevivência clonogênica nos tempos de 24 e 48 h, obtendo-se o IC50 e o IC20. Em seguida, foi realizado o ensaio de genotoxicidade do cometa no tempo de 24 h utilizando-se incubação com a enzima FPG nas linhagens HepG2, MRC5 e XP4PA. Os produtos naturais se mostraram genotóxicos mesmo em pequenas concentrações. Após este ensaio, foi avaliada a antigenotoxicidade dos produtos naturais já nas concentrações pouco genotóxicas e não citotóxicas por 24 h. Para tanto, as células HepG2 foram expostas ao mutágeno peróxido de hidrogênio (H2O2 - 1mM) e pré e pós-tratadas com os produtos naturais. E, as linhagens MRC5 e XP4PA foram expostas à radiação ultravioleta C (UVC) e pré e pós-tratadas com os produtos naturais. Para a linhagem HepG2, o extrato etanólico, as casearinas B e D apresentaram inibição de danos fortes nas maiores concentrações testadas. Já para a linhagem MRC5 somente a casearina B foi capaz de produzir inibição moderada na concentração de 0,04 µM. Adicionalmente, foi verificada a resposta antiapoptótica dos produtos naturais pelo método da anexina-V-FITC/PI nas linhagens submetidas aos mesmos mutágenos do ensaio do cometa, aplicando-se o protocolo...
Studies have shown that Casearia sylvestris derivatives protect DNA from damage. Thus, the aim of this study was to evaluate the mechanisms through which this protection occurs. The samples used in this study were the ethanolic extract of C. sylvestris leaves and the clerodane diterpenes obtained from this extract, including casearins B, D and X, and caseargrewiin F. The following cell lines were used: HepG2, MRC5, XP4PA and Hepa 1c1c7. First, all samples were assessed for their cytotoxic activity by using both the MTT assay and the clonogenic survival assay after 24 and 48 h of exposure, yielding the IC50 and IC20. Afterwards, the genotoxicity of the samples was assessed within 24 h following incubation with the samples using the FPG enzyme in the cell lines HepG2, MRC5 and XP4PA. The compounds proved to be genotoxic at all concentrations tested. Subsequently, the antigenotoxicity of the samples was assessed at concentrations that were at very low levels of cytotoxic or genotoxic after 24 h. HepG2 cells were exposed to the mutagen hydrogen peroxide (H2O2, 1 mM) and either pre or post-treated with the samples. Additionally, the cell lines MRC5 and XP4PA were exposed to ultraviolet C (UVC) and either pre or post-treated with the compounds. For the ethanolic extract in the HepG2 cell line, the casearins B and D showed strong inhibition of damage at the highest concentrations tested. In the MRC5 cell line, only casearin B was able to produce moderate inhibition (at 0.04 mM). Additionally, the anti-apoptotic response of the samples was assessed by an anexinV-FITC/PI assay in cell lines that were exposed to the same mutagens used in the comet assay, followed by post-treatment with the samples. The clerodane diterpenes, at high concentrations, increased the percentage of live cells and decreased... (Complete abstract click electronic access below)
Studies have shown that Casearia sylvestris derivatives protect DNA from damage. Thus, the aim of this study was to evaluate the mechanisms through which this protection occurs. The samples used in this study were the ethanolic extract of C. sylvestris leaves and the clerodane diterpenes obtained from this extract, including casearins B, D and X, and caseargrewiin F. The following cell lines were used: HepG2, MRC5, XP4PA and Hepa 1c1c7. First, all samples were assessed for their cytotoxic activity by using both the MTT assay and the clonogenic survival assay after 24 and 48 h of exposure, yielding the IC50 and IC20. Afterwards, the genotoxicity of the samples was assessed within 24 h following incubation with the samples using the FPG enzyme in the cell lines HepG2, MRC5 and XP4PA. The compounds proved to be genotoxic at all concentrations tested. Subsequently, the antigenotoxicity of the samples was assessed at concentrations that were at very low levels of cytotoxic or genotoxic after 24 h. HepG2 cells were exposed to the mutagen hydrogen peroxide (H2O2, 1 mM) and either pre or post-treated with the samples. Additionally, the cell lines MRC5 and XP4PA were exposed to ultraviolet C (UVC) and either pre or post-treated with the compounds. For the ethanolic extract in the HepG2 cell line, the casearins B and D showed strong inhibition of damage at the highest concentrations tested. In the MRC5 cell line, only casearin B was able to produce moderate inhibition (at 0.04 mM). Additionally, the anti-apoptotic response of the samples was assessed by an anexinV-FITC/PI assay in cell lines that were exposed to the same mutagens used in the comet assay, followed by post-treatment with the samples. The clerodane diterpenes, at high concentrations, increased the percentage of live cells and decreased... (Complete abstract click electronic access below)
Descrição
Palavras-chave
DNA, Antioxidantes, Produtos naturais, Apoptose, Ensaio cometa, Radiação ultravioleta
Como citar
PRIETO, Aline Miranda. Avaliação de Reparo de DNA e Atividade Antioxidante do Extrato Etanólico e Substâncias Isoladas de Casearia sylvestris. 2012. 136 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, 2012.