Estudo da atividade biológica do produto da fermentação do fungo endofítico Gibberella moniliformis, proveniente do mangue brasileiro

Abstract

The search for new compounds to use on medicine, agriculture and industry is continuously growing, and natural products are the most successful strategy for discovery of new molecules. Among the various existing natural products, endophytic fungi of mangrove are considered a valuable source for discovery of new substances with therapeutic and biotechnological interest, since they offer an uncountable diversity of chemical structures. Considering the bioactive potential of these microorganisms, the aimed of this work was to evaluate the biological activity and chemical composition of the butanolic fraction of the extract from the fermentation of the endophytic fungus Gibberella moniliformis 99(3) in order to identify new bioactive metabolites. The endophyte isolated from of mangrove plant Laguncularia racemosa, was submitted to the fermentation process in Czapek broth and later the fermented broth was partitioned with different solvents resulting in the butanolic fraction (FBuOH). The chemical composition of the FBuOH fraction was analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC-DAD), as well as its total phenol content (15.85 mg GAE/g). Chromatographic study of the FBuOH fraction suggested the presence of chromophore groups (revealed in UV light 254 nm), terpenes, saponins and flavoinoids (positive for sulfuric anisaldehyde), alkaloids (reactive Dragendorff) and primary amines (ninhydrin positive). The biological potential of the FBuOH fraction was investigated by different in vitro assays. The minimum inhibitory concentration (MIC) and minimum microbicide (MMC) against different microorganisms were evaluated in the antimicrobial assay, in which FBuOH fraction showed strong antifungal potential against Candida albicans ATCC 10231, with MIC of 25-50 μg/mL and MMC of 50 μg/mL, moderate activity against Escherichia coli ATCC 25922 (MIC: 100-200 μg/mL and MMC > 400 μg/mL) and was inactive against Staphylococcus aureus ATCC 6538 at the concentrations tested. Synergistic effect was observed in the combination of the FBuOH fraction with fluconazole (ICIF = 0.375), with eight times potentiation of the action of the drug against C. albicans. The antioxidant activity of the FBuOH fraction was evaluated by the DPPH, ABTS, ORAC and FRAP methods, with results of 39.03; 152.16; 213.72 μmol Trolox/g of fraction and 182.28 μmol Fe2+/g respectively, and also by HPLC-DAD-ABTS on-line, showing no compounds with expressive activity. The antiproliferative activity was tested by the sulforhodamine B (SRB) method against ten human tumor cell lines and one non-tumoral line and it was found that FBuOH did not inhibit cellular proliferation of any of the tested lines at the tested concentrations (TGI > 250 μg/mL), being relevant to highlight that presented TGI > 250 μg/mL also for the non-tumoral HaCaT (keratinocyte) line. The FBuOH fraction did not show activity against Leishmania amazonensis promastigotes in the tested concentrations (IC50 > 100 μg/mL). In the cytotoxicity assay the cytotoxic concentration of the FBuOH fraction which inhibited 50% Vero cells was 449 μg/mL, with selectivity index of 8.98. It is concluded that the fungus G. moniliformis 99 (3) is promising in the production of compounds antifungal, encouraging the continuity of studies with the FBuOH fraction both for a possible isolation of the compound responsible for the activity and for future in vivo tests.