Abstract: Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.
Our previous works showed that extracts and fractions from Polymnia sonchifolia had presented an inhibitory activity on growth and aflatoxin production by Aspergillus flavus in non-cytotoxic concentrations. In this work, report results on the in vitro cytotoxicity of subfractions and of a mixture of sesquiterpenes lactones from Polymnia sonchifolia on Vero cells. The cytotoxicity was assayed through crystal violet staining (CVS) method and the 50% inhibitory concentration for cell growth (IC50) was calculated. Both subfractions and the mixture of sesquiterpenes lactones did not show in vitro cytotoxicity to Vero cells on active biologically concentrations against production of aflatoxin B1 by Aspergillus flavus.
Extratos e frações de Polymnia sonchifolia apresentaram em estudos preliminares uma atividade inibidora na produção de aflatoxinas e no crescimento de Aspergillus flavus. No presente trabalho foi avaliada a citotoxicidade das subfrações e de uma mistura de lactonas sesquiterpênicas de Polymnia sonchifolia pelo método de coloração pelo violeta cristal (CVS) em células Vero. A concentração que inibe o crescimento celular em 50% (IC50) foi determinada. Não foi observada nenhuma citotoxicidade para as células Vero nas concentrações biologicamente ativas contra a produção de aflatoxina pelo Aspergillus flavus.
The aqueous extract from Polymnia sonchifolia leaves (AE) was tested for inhibitory activity on aflatoxin B1(AFB1) production and growth of Aspergillus flavus. The cytotoxicity of AE on Vero cells was also performed. Suspensions of A. flavus spores were inoculated into 50 mL of YES medium together with different concentrations of the AE. The aflatoxin B1 was extracted, analyzed by thin layer chromatography and quantified by photodensitometry. All the concentrations of AE induced inhibition of AFB1 production. The aqueous extract showed in vitro cytotoxicity to Vero cells only at concentrations above 500 µg/mL.
Neste trabalho verificou-se a atividade do extrato aquoso de folhas de Polymnia sonchifolia no crescimento e na produção de aflatoxinas B1 por Aspergillus flavus. Suspensões de esporos de A. flavus foram inoculadas em 50 mL de meio de YES com diferentes concentrações do extrato aquoso. A aflatoxina B1 foi extraída e analisada por cromatografia de camada delgada e quantificada por fotodensitometria. Todas as concentrações testadas inibiram a produção de aflatoxina B1. O extrato aquoso apresentou citotoxicidade em células Vero somente em concentrações acima de 500 µg/mL.