Avaliação do potencial genotóxico e antigenotóxico de Lycium barbarum (goji berry), nutracêutico usado como citoprotetor e antioxidante
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Data
2018-02-23
Autores
Gonçalves, Letícia Cristina [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O L. barbarum, popularmente conhecido goji berry, tem despertado o interesse da comunidade científica ocidental, devido a sua riqueza nutritiva e a ação antioxidante que promovem efeitos benéficos para a saúde humana. Entretanto, há poucos relatos na literatura sobre sua possível ação tóxica ou comprovação do seu potencial protetor. Sendo assim, o presente trabalho teve como objetivo avaliar os efeitos citotóxicos (pelo MMT), genotóxicos e antigenotóxicos (pelo ensaio do cometa), mutagênicos, e antimutagênicos (pelo teste do micronúcleo com bloqueio de citocinese – MN), de estresse oxidativo (pela quantificação dos níveis de GSH e TBARS e pela atividade das enzimas SOD e GST), de potencial antioxidante, do teor de polifenóis totais e da fitoquímica qualitativa de 3 diferentes concentrações recomendadas para o uso diário do extrato de L. barbarum (0,2; 0,4 e 0,6 g/L), por meio de ensaios realizados com sistemas-teste in vitro e in vivo. Neste estudo foram utilizados o organismo teste Allium cepa e culturas de duas linhagens celulares: células de hepatocarcinoma humano (HepG2) e adenocarcinoma de mama humano (MCF-7). Para A. cepa, foram aplicados os testes de aberrações cromossômicas e de micronúcleos em células meristemáticas e dos micronúcleos em células F1. As análises de antigenotoxicidade e antimutagenicidade realizada com esse organismo seguiram protocolos convencionais de pré-tratamento, pós-tratamento e tratamento simultâneo simples. Os resultados destes testes mostraram que o L. barbarum não apresentou potencial genotóxico ou mutagênico para A.cepa, mas apresentou respostas positivas para ações antigenotóxica e antimutagênica. Pela aplicação da fórmula de porcentagem de redução dos danos, pode-se verificar que a taxa de redução, promovida pelo L. barbarum variou significativamente (41,7% a 64% para as células meristemáticas e de 65% a 100% para as células F1). Assim, inferimos que o extrato de L.barbarum apresentou um efeito protetor sobre as células de A. cepa. Em relação ao teste do MTT, realizado com cultura celulares das linhagens HepG2 e MCF-7, foi possível observar que o L. barbarum não foi citotóxico para nenhuma das linhagens testadas. As avaliações realizadas pelo ensaio do cometa, nas mesmas linhagens celulares, mostraram que o extrato não foi genotóxico para HepG2, mas foi genotóxico para a todas as concentrações testadas (0,2; 0,4 e 0,6 g/L), para MCF-7. No entanto, na avaliação de genotoxicidade, por meio do teste do micronúcleo com bloqueio de citocinese, foi possível observar uma diminuição significativa na frequência de brotos, pontes e MN para as três concentrações testadas (0,2; 0,4 e 0,6 g/L) na linhagem MCF-7, indicando um possível reparo dos danos genotóxicos observados para o ensaio do cometa. Já no teste do MN (avaliação de mutagenicidade), não foram observadas mutagenicidade para nenhuma das linhagens testadas. As avaliações de antigenotoxicidade e antimutagenicidade do L. barbarum foram realizadas apenas com a linhagem HepG2, devido a ausência de potencial genotóxico e mutagênico registrado para essas células. Nesta avaliação foram seguidos os protocolos de pré-tratamento, pós-tratamento, tratamento simultâneo e tratamento simultâneo com incubação. Na avalição de antigenotoxicidade e antimutagenicidade, os protocolos de pré e pós-tratamento com as concentrações 0,4 g/L e 0,6 g/L se mostraram mais eficazes, para essas ações, o que permite inferir que o extrato estudado age tanto por desmutagênese como bioantimutagênese. Pela análise de porcentagem de redução dos danos, verificamos que a taxa de redução, promovida pelo L. barbarum, teve uma eficácia de 100%, para todos os pré-tratamentos e para os pós-tratamentos nas concentrações 0,4 g/L e 0,6 g/L, enquanto que para a concentração 0,2 g/L, a eficiência variou de 40% a 70%. Foi observado ainda que, todos os tratamentos simultâneos induziram morte celular, indicando uma possível interação entre o MMS (substância utilizada como controle positivo) com algum composto do extrato de L. barbarum, como as quinonas. Os efeitos protetivos do L. barbarum observados no sistema teste A. cepa e nas células HepG2 podem estar associados à presença de flavonóides, saponinas e alcalóides presentes em todas as concentrações testadas (0,2; 0,4 e 0,6 g/L), conforme verificado nas análises fitoquímicas qualitativas. Este efeito protetivo também pode ser associado ao teor de polifenóis totais, quantificados em 6,03 mg equivalente (eq.) de ác. gálico/mL, e a maior atividade antioxidante, dada pela concentração 0,6 g/L de L. barbarum, determinada pelo método do DPPH. Por fim, foi avaliado a capacidade do L. barbarum induzir alterações no equilíbrio redox em ambas as linhagens celulares (HepG2 e MCF-7). Foram quantificados os níveis de GSH, TBARS e a atividade das enzimas SOD, GST. Foi possível observar um aumento significativo apenas para os níveis de GSH na linhagem HepG2, o que confirma a proteção do material genético sugerida, anteriormente, nas demais análises realizadas com esta linhagem. Já para as células MCF-7, não foram observadas alterações significativas para nenhuma das análises realizadas, indicando ausência de indução de estresse oxidativo. Os resultados desta pesquisa sugerem que o L. barbarum pode apresentar efeitos protetivos sobre a molécula do DNA, pois mostrou ter ação antigenotóxica e antimutagênica, além de promissiores potenciais de redução de danos, tanto em testes in vivo como in vitro.
The L. barbarum, popularly known as goji berry, has aroused the interest of the Western scientific community because of its nutritional richness and antioxidant action that promote beneficial effects on human health. However, there are few reports in the literature about its possible toxic action or proof of its protective potential. The present work aimed to evaluate the cytotoxic (by MMT test), genotoxic and antigenotoxic (by comet assay), mutagenic and antimutagenic effects (by micronucleus test with cytokinesis-block - MN), oxidative stress (quantification of GSH and TBARS levels and the activity of SOD and GST enzymes), antioxidant potential, total polyphenol content and qualitative phytochemical of 3 different concentrations recommended for daily use of L. barbarum extract (0.2; 0.4 and 0.6 g / L) by assays performed with in vitro and in vivo test systems. In this study, the test organism Allium cepa and cultures of two cell lines were used: human hepatocarcinoma cells (HepG2) and human breast adenocarcinoma (MCF-7). For A. cepa, the chromosomal aberrations and micronucleus tests wereapplied in meristematic cells and the micronuclei in F1 cells. The antigenotoxicity and antimutagenicity analyzes performed with this organism followed conventional protocols for pretreatment, post-treatment and simple simultaneous treatment. The results of these tests showed that L. barbarum did not present genotoxic or mutagenic potential for A.cepa, but presented positive responses to antigenotoxic and antimutagenic actions. By applying the harm reduction percentage formula, it can be seen that the rate of reduction promoted by L. barbarum varied significantly (41.7% to 64% for meristematic cells and 65% to 100% for the cells F1). Thus, we infer that L.barbarum extract had a protective effect on A. cepa cells. In relation to the MTT test, carried out with HepG2 and MCF-7 lines cell culture, it was possible to observe that L. barbarum was not cytotoxic to any of the tested strains. Evaluations carried out by the comet assay in the same cell lines showed that the extract was not genotoxic for HepG2 but genotoxic at all concentrations (0.2, 0.4 and 0.6 g / L), for MCF-7. However, in the genotoxicity evaluation, by the micronucleus test with cytokinesis-block, it was possible to observe a significant decrease in the frequency of nuclear buds, nucleoplasmatic bridges and MN for the three tested concentrations (0.2, 0.4 and 0.6 g / L) in the MCF-7 line, indicating a possible repair of the genotoxic damages observed for the comet assay. In the MN test (mutagenicity evaluation), no mutagenic effect was observed for any of the tested cell lines. The antigenotoxicity and antimutagenicity evaluations of L. barbarum were performed only with the HepG2 lineage, due to the absence of genotoxic and mutagenic potential registered for these cells. In this evaluation the protocols of pre-treatment, post-treatment, simultaneous treatment and simultaneous treatment with incubation were followed. In the evaluation of antigenotoxicity and antimutagenicity, the pre and post treatment protocols with concentrations of 0.4 g / L and 0.6 g / L were more effective for these actions, which allows to infer that the studied extract acts both as demutagenesis as bioantimutagenesis. By the analysis of percentage of reduction of damages, we verified that the reduction rate, promoted by L. barbarum, had an efficacy of 100%, for all pre-treatments and for post-treatments in concentrations of 0.4 g / L and 0.6 g / L, while for the concentration 0.2 g / L, the efficiency ranged from 40% to 70%. It was also observed that all the simultaneous treatments induced cell death, indicating a possible interaction between the MMS (substance used as positive control) and some compound of L. barbarum extract, such as quinones. The protective effects of L. barbarum observed in the A. cepa test system and HepG2 cells may be associated with the presence of flavonoids, saponins and alkaloids present in all tested concentrations (0.2, 0.4 and 0.6 g / L), as verified in qualitative phytochemical analyzes. This protective effect can also be associated with the total polyphenol content, measured in 6.03 mg equivalent (eq.) of aq. gallic acid / mL, and the highest antioxidant activity, given by the concentration 0.6 g / L of L. barbarum, determined by the DPPH method. xi Finally, the ability of L. barbarum to induce changes in the redox balance in both cell lines (HepG2 and MCF-7) was evaluated. The levels of GSH, TBARS and the activity of the enzymes SOD, GST were quantified. It was possible to observe a significant increase only for the GSH levels in the HepG2 line, which confirms the protection of the genetic material previously suggested in the other analyzes performed with this lineage. As for MCF-7 cells, no significant changes were observed in any of the analyzes performed, indicating absence of oxidative stress induction. The results of this research suggest that L. barbarum may have protective effects on the DNA molecule, as it showed antigenotoxic and antimutagenic action, as well as promising harm reduction potentials, both in vivo and in vitro.
The L. barbarum, popularly known as goji berry, has aroused the interest of the Western scientific community because of its nutritional richness and antioxidant action that promote beneficial effects on human health. However, there are few reports in the literature about its possible toxic action or proof of its protective potential. The present work aimed to evaluate the cytotoxic (by MMT test), genotoxic and antigenotoxic (by comet assay), mutagenic and antimutagenic effects (by micronucleus test with cytokinesis-block - MN), oxidative stress (quantification of GSH and TBARS levels and the activity of SOD and GST enzymes), antioxidant potential, total polyphenol content and qualitative phytochemical of 3 different concentrations recommended for daily use of L. barbarum extract (0.2; 0.4 and 0.6 g / L) by assays performed with in vitro and in vivo test systems. In this study, the test organism Allium cepa and cultures of two cell lines were used: human hepatocarcinoma cells (HepG2) and human breast adenocarcinoma (MCF-7). For A. cepa, the chromosomal aberrations and micronucleus tests wereapplied in meristematic cells and the micronuclei in F1 cells. The antigenotoxicity and antimutagenicity analyzes performed with this organism followed conventional protocols for pretreatment, post-treatment and simple simultaneous treatment. The results of these tests showed that L. barbarum did not present genotoxic or mutagenic potential for A.cepa, but presented positive responses to antigenotoxic and antimutagenic actions. By applying the harm reduction percentage formula, it can be seen that the rate of reduction promoted by L. barbarum varied significantly (41.7% to 64% for meristematic cells and 65% to 100% for the cells F1). Thus, we infer that L.barbarum extract had a protective effect on A. cepa cells. In relation to the MTT test, carried out with HepG2 and MCF-7 lines cell culture, it was possible to observe that L. barbarum was not cytotoxic to any of the tested strains. Evaluations carried out by the comet assay in the same cell lines showed that the extract was not genotoxic for HepG2 but genotoxic at all concentrations (0.2, 0.4 and 0.6 g / L), for MCF-7. However, in the genotoxicity evaluation, by the micronucleus test with cytokinesis-block, it was possible to observe a significant decrease in the frequency of nuclear buds, nucleoplasmatic bridges and MN for the three tested concentrations (0.2, 0.4 and 0.6 g / L) in the MCF-7 line, indicating a possible repair of the genotoxic damages observed for the comet assay. In the MN test (mutagenicity evaluation), no mutagenic effect was observed for any of the tested cell lines. The antigenotoxicity and antimutagenicity evaluations of L. barbarum were performed only with the HepG2 lineage, due to the absence of genotoxic and mutagenic potential registered for these cells. In this evaluation the protocols of pre-treatment, post-treatment, simultaneous treatment and simultaneous treatment with incubation were followed. In the evaluation of antigenotoxicity and antimutagenicity, the pre and post treatment protocols with concentrations of 0.4 g / L and 0.6 g / L were more effective for these actions, which allows to infer that the studied extract acts both as demutagenesis as bioantimutagenesis. By the analysis of percentage of reduction of damages, we verified that the reduction rate, promoted by L. barbarum, had an efficacy of 100%, for all pre-treatments and for post-treatments in concentrations of 0.4 g / L and 0.6 g / L, while for the concentration 0.2 g / L, the efficiency ranged from 40% to 70%. It was also observed that all the simultaneous treatments induced cell death, indicating a possible interaction between the MMS (substance used as positive control) and some compound of L. barbarum extract, such as quinones. The protective effects of L. barbarum observed in the A. cepa test system and HepG2 cells may be associated with the presence of flavonoids, saponins and alkaloids present in all tested concentrations (0.2, 0.4 and 0.6 g / L), as verified in qualitative phytochemical analyzes. This protective effect can also be associated with the total polyphenol content, measured in 6.03 mg equivalent (eq.) of aq. gallic acid / mL, and the highest antioxidant activity, given by the concentration 0.6 g / L of L. barbarum, determined by the DPPH method. xi Finally, the ability of L. barbarum to induce changes in the redox balance in both cell lines (HepG2 and MCF-7) was evaluated. The levels of GSH, TBARS and the activity of the enzymes SOD, GST were quantified. It was possible to observe a significant increase only for the GSH levels in the HepG2 line, which confirms the protection of the genetic material previously suggested in the other analyzes performed with this lineage. As for MCF-7 cells, no significant changes were observed in any of the analyzes performed, indicating absence of oxidative stress induction. The results of this research suggest that L. barbarum may have protective effects on the DNA molecule, as it showed antigenotoxic and antimutagenic action, as well as promising harm reduction potentials, both in vivo and in vitro.
Descrição
Palavras-chave
Allium cepa, Cultura de células, Antigenotoxicidade, Antimutagênicidade, Estresse oxidativo, Goji berry, Cell culture, Antigenotoxicity, Antimutagenicity, Oxidative stress