Avaliação do potencial bioativo do produto de fermentação de actinomicetos isolados de terra preta antropogênica da Amazônia

Abstract

The terra preta de índio (TPI) has a great microbial diversity. In view of the increasing resistance of pathogenic microorganisms to the known antibiotics and the importance of antioxidant compounds in the prevention of cellular oxidative damage, the objective of this study was to evaluate the bioactive potential of extracts from the fermentation product of actinomycetes isolated from TPI. Unidentified five actinomycetes (FL1, FL2, FL3, FL4 and FL5), isolated from TPI of Central Amazonia, were undergone to fermentation, and their extracts were obtained with ethyl acetate. Minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) assays were performed with five extracts against three microbial strains: Staphylococcus aureus, Escherichia coli and Candida albicans. The extracts that had the best results of antimicrobial activity were characterized by thin-layer chromatography (TLC), and further fractionated by the Sephadex LH-20 column. The fractions were evaluated by TLC with different developers and submitted to MIC and MMC tests against the strain that showed the highest sensitivity in the tests with the crude extracts. The most promising fraction was submitted to assays of quantification phenolic compounds and antioxidant activity, and was also analyzed by high performance liquid chromatography and ultraviolet spectroscopy. Fractions that showed antimicrobial activity were evaluated about your potential of action synergistic in association with the amoxicillin and its cytotoxicity against Vero cells. Experiments to identify the actinomycetes considered promising were initiated with the sequencing of the 16S rRNA gene. The microbial lines FL3 and FL4 stood out for the antimicrobial potential against S. aureus; thus, these actinobacteria and their extracts were selected to continue the work. The extract FL3 presented MIC of 100-200 μg/mL and MMC > 400 μg/mL, and the extract FL4 presented MIC of 25-50 μg/mL and MMC of 100-200 μg/mL. Fractionation of the FL3 extract resulted in four fractions, two of which, B3 and C3, presented the best MIC values, between 100-200 μg/mL. From the FL4 extract, six fractions were obtained, being relevant to mention the best values of MIC observed for B4 and D4: between 50 and 100 μg/mL. Among these, only fraction B4 presented microbicidal activity, with CMM of 100-200 μg/mL. In the preliminary chemical tests, using TLC, it was possible to suggest the presence of chromophore groups, primary amines, flavonoids, terpenes and antioxidant substances. The D4 fraction, submitted to phenolic quantification and antioxidant activity assays, presented 140.71 ± 4.0105 mg of GAE/g of fraction, and obtained promising results of antioxidant activity in the ORAC method: 5581.85 ± 515.11450 μmol of Trolox/g of fraction. The association between the fractions A3, B3 and D4 and the antimicrobial amoxicillin potentiated the action of the drug by up to four times. In the evaluation of cytotoxicity, it was observed that fractions B3, B4, C4 and D4 were considered to be more effective than toxic to Vero cells (IS > 1). Sequencing of the 16S gene, together with the phylogenetic position of the FL3 and FL4 isolates, showed that both belong to the family Streptomycetaceae, being probable species of the genus Streptomyces.