Prospecção de Produtos Naturais de Fungos Endofíticos de Bromelia balansae Mez

Abstract

Fungi biosynthesize metabolites with high diversity and structural complexity that are potentially bioactive. The rocky outcrops occur in the region of the western edge of the Pantanal Sul-mato-grossense, Brazil, which are inhospitable places, their plant species may be adapted for these locals and the symbiosis with microorganisms can help their defense system. The present work aimed to bioprospect of bioactive metabolites from the endophytic fungi of Bromelia balansae Mez, collected in Pantanal RA. Sixty-one endophytic fungi were isolated and ten were molecularly identified, among them the genera: Aspergillus, Penicillium, Diaporthe, Daldinia and Neodeightonia. The isolates were grown in potato-dextrose agar medium (BDA) and in potato dextrose broth. The organic extracts obtained from these cultures were evaluated in vitro bioassays of antimicrobial, antibiofilm, antileishmania, and cytotoxic activity. Of these, 25 endophytes showed antimicrobial activity against Staphylococcus aureus with MIC (minimum inhibitory concentration) ≤ 500 µg/mL, and 12 strains showed MIC ≤ 125 µg/mL. None of the microorganisms isolated was promising against Pseudomonas aeruginosa. In the Leishmania infantum assay, four strains had an infection rate lower than 15% compared to the control. In the cytotoxicity evaluation against leukemic lines, Kasumi and KG-1, five isolates inhibited more than 90% of cell growth at the concentration of 100 µg/mL. The isolate 5MP2F4, identified as Penicillium sp., showed MIC of 32 µg/mL against S. aureus, and therefore, it was selected for a study using the OSMAC strategy, antibacterial and antibiofilm screening, with application of metabolomics, statistical analysis, and molecular networking to determine potential bioactive metabolites and understanding of the chemical profile of the fungus. In this experiment, the obtained extracts showed different chemical profiles and antibacterial activity. Several metabolites were annotated, among them different braziliamides, verruculogen, fumitremorgin, penicillic acid, viridicatum toxin A, and spirohexalin. An optimization study of extraction from fungal mycelia from the isolate 5MP2F4 by accelerated solvent extraction (ASE) was also performed, and viridicatum toxin A (VRA) was purified by this methodology. VRA was evaluated for the activities anti-leishmanial, antibiofilm, and cytotoxic against leukemic cell lines, and it showed to be active in all experiments. Fungal isolates of B. balansae have expanded knowledge of the endophyte biodiversity of the Pantanal. Several substances belonging to different chemical classes were found, some were produced in high levels according to the variation in the culture medium, and the bioassays suggest that they have potential as antibiotics and cytotoxics.