Investigação dos efeitos citotóxico e genotóxico do extrato de salix alba L.: análises in vitro, in vivo e histológicas
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Data
2017-10-30
Autores
Terrazas, Peterson Menezes [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A Salix alba L. (SA), popularmente conhecida como Salgueiro Branco, é uma planta utilizada na medicina popular para o tratamento de inflamações crônicas e agudas, infecções, dores, febre, entre outros. A caracterização fitoquímica do extrato da casca desta planta revelou que seu principal componente é a salicina, com uma concentração de 4,94 mg/mL, um precursor do anti-inflamatório ácido acetilsalicílico. Considerando que existem poucos estudos que avaliam a ação tóxica e citotóxica do extrato da SA, o presente estudo foi elaborado visando investigar o potencial citotóxico, genotóxico e mutagênico da SA em células mononucleares do sangue periférico humano e de hepatocarcinoma humano HepG2 in vitro, e em diferentes células de camundongos in vivo, utilizando alguns dos testes tradicionais na área de mutagênese, como o teste do MTT, o Ensaio do Cometa e o Teste do Micronúcleo, bem como a verificação de potencial citotoxicidade por meio de análises histológicas e histoquímicas. Os testes de viabilidade celular e citotoxicidade (azul de tripan e MTT) permitiram a escolha de 3 concentrações do extrato da SA para serem analisadas nos ensaios de genotoxicidade in vitro: 5, 50 e 100 µg/mL. Pelo ensaio cometa com as células mononucleares de sangue periférico, pôde-se observar que as concentrações de 50 e 100 µg/mL acarretaram um aumento estatisticamente significativo de danos no DNA, em comparação ao controle negativo. Já no teste do micronúcleo, as 3 concentrações avaliadas (5, 50 e 100 µg/mL) não produziram aumentos significativos de células binucleadas micronucleadas, nem alterações no índice de divisão nuclear, em comparação ao controle negativo, indicando ausência de efeitos clastogênico/aneugênico e citoxicidade da SA. Já nas culturas de células HepG2, expostas as 3 diferentes concentrações do extrato, não foram encontrados efeitos citotóxico, genotóxico e clastogênico/aneugênico da SA. Nos experimentos in vivo, após tratamento dos camundongos durante 7 dias consecutivos (24 horas de intervalo) com as doses de 500, 1000 e 2000 mg/Kg de SA, o ensaio cometa evidenciou que não houve aumento significativo de danos ao DNA em nenhuma das diferentes células somáticas e germinativas analisadas, em comparação ao controle negativo. O mesmo resultado foi observado no teste do micronúcleo, onde não foi encontrado um aumento significativo no número de eritrócitos policromáticos micronucleados e nem alteração na razão entre eritrócitos policromáticos e normocromáticos, indicando ausência de efeitos clastogênico/aneugênico e citoxicidade da substância teste. Nas células hepáticas dos camundongos, também não foram encontradas alterações histológicas e histoquímicas. Frente as condições experimentais, os resultados obtidos permitem concluir que, o extrato de Salix alba, sem a metabolização por enzimas hepáticas, produziu efeitos genotóxicos em células mononucleares de sangue periférico humano em cultura. Por outro lado, o extrato, após metabolização por enzimas hepáticas das células HepG2 em cultura, e administrado via oral aos animais, não acarretou efeitos citotóxicos e/ou genotóxicos.
Salix alba L. (SA), popularly known as White Willow, is a plant used in folk medicine for the treatment of chronic and acute inflammations, infections, pains, fever, among others. The phytochemical characterization of the bark extract of this plant revealed that its main component is salicin, with a concentration of 4.94 mg/mL, a precursor of the antiinflammatory acetylsalicylic acid. Considering that there are few studies evaluating the toxic and cytotoxic action of SA extract, the present study was designed to investigate the cytotoxic, genotoxic and mutagenic potential of SA bark wood extract in human peripheral blood mononuclear cells and human hepatoma cell line HepG2 in vitro and in different mouse cells in vivo using some of the traditional mutagenesis tests such as the MTT test, the Comet assay and the Micronucleus test, and cytotoxicity in the liver of mice also by histological and histochemical analysis. Cell viability and cytotoxicity tests (trypan blue and MTT) allowed the choice of 3 concentrations of the SA extract to be analyzed in the in vitro genotoxicity assays: 5, 50 and 100 μg/mL. By the comet assay with the peripheral blood mononuclear cells, it was observed that concentrations of 50 and 100 μg/ml resulted in a statistically significant increase in DNA damage, as compared to the negative control. In the micronucleus test, the 3 concentrations evaluated (5, 50 and 100 μg/mL) did not produce significant increases of micronucleated binucleate cells, as well as alterations in the nuclear division index, in comparison to the negative control, indicating absence of clastogenic/aneugenic effects and cytotoxicity of SA. In the cultures of HepG2 cells exposed to the 3 different concentrations of the extract, no cytotoxic, genotoxic and clastogenic/aneugenic effects of SA were found. In the in vivo experiments, after treatment of the mice for 7 consecutive days (24 hour intervals) at doses of 500, 1000 and 2000 mg/kg of SA, the comet assay showed that there was no significant increase of DNA damage in any of the somatic and germ cells analyzed, compared to the negative control. The same result was observed in the micronucleus test, where there was no significant increase in the number of micronucleated polychromatic erythrocytes and no change in the ratio between polychromatic and normochromatic erythrocytes, indicating absence of clastogenic/aneugenic effects and cytotoxicity of the test substance. In the hepatic cells of mice, histological and histochemical changes also were not found. Under the experimental conditions employed in the present study, the results obtained allow us to conclude that Salix alba bark wood extract, without the metabolism by hepatic enzymes, produced genotoxic effects in human peripheral blood mononuclear cells in culture. On the other hand, the extract, after being metabolized by hepatic enzymes from HepG2 cells in culture, and administered orally to the animals, did not cause cytotoxic and/or genotoxic effects.
Salix alba L. (SA), popularly known as White Willow, is a plant used in folk medicine for the treatment of chronic and acute inflammations, infections, pains, fever, among others. The phytochemical characterization of the bark extract of this plant revealed that its main component is salicin, with a concentration of 4.94 mg/mL, a precursor of the antiinflammatory acetylsalicylic acid. Considering that there are few studies evaluating the toxic and cytotoxic action of SA extract, the present study was designed to investigate the cytotoxic, genotoxic and mutagenic potential of SA bark wood extract in human peripheral blood mononuclear cells and human hepatoma cell line HepG2 in vitro and in different mouse cells in vivo using some of the traditional mutagenesis tests such as the MTT test, the Comet assay and the Micronucleus test, and cytotoxicity in the liver of mice also by histological and histochemical analysis. Cell viability and cytotoxicity tests (trypan blue and MTT) allowed the choice of 3 concentrations of the SA extract to be analyzed in the in vitro genotoxicity assays: 5, 50 and 100 μg/mL. By the comet assay with the peripheral blood mononuclear cells, it was observed that concentrations of 50 and 100 μg/ml resulted in a statistically significant increase in DNA damage, as compared to the negative control. In the micronucleus test, the 3 concentrations evaluated (5, 50 and 100 μg/mL) did not produce significant increases of micronucleated binucleate cells, as well as alterations in the nuclear division index, in comparison to the negative control, indicating absence of clastogenic/aneugenic effects and cytotoxicity of SA. In the cultures of HepG2 cells exposed to the 3 different concentrations of the extract, no cytotoxic, genotoxic and clastogenic/aneugenic effects of SA were found. In the in vivo experiments, after treatment of the mice for 7 consecutive days (24 hour intervals) at doses of 500, 1000 and 2000 mg/kg of SA, the comet assay showed that there was no significant increase of DNA damage in any of the somatic and germ cells analyzed, compared to the negative control. The same result was observed in the micronucleus test, where there was no significant increase in the number of micronucleated polychromatic erythrocytes and no change in the ratio between polychromatic and normochromatic erythrocytes, indicating absence of clastogenic/aneugenic effects and cytotoxicity of the test substance. In the hepatic cells of mice, histological and histochemical changes also were not found. Under the experimental conditions employed in the present study, the results obtained allow us to conclude that Salix alba bark wood extract, without the metabolism by hepatic enzymes, produced genotoxic effects in human peripheral blood mononuclear cells in culture. On the other hand, the extract, after being metabolized by hepatic enzymes from HepG2 cells in culture, and administered orally to the animals, did not cause cytotoxic and/or genotoxic effects.
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Palavras-chave
Salix alba L., Salgueiro Branco, Ensaio cometa., Teste do micronúcleo., White Willow, Comet assay, Micronucleus test