Triagem, isolamento de substâncias antifúngicas a partir do extrato do fungo aspergillus felis e avaliação da ação antifúngica em paracoccidioides brasiliensis

Abstract

The paracoccidioidomycosis (PCM) caused by fungi of the genus Paracoccidioides is an endemic mycosis in Latin America. The PCM is the leading cause of death among the systemic mycoses and the tenth among infectious and parasitic diseases. Drug treatment of mycosis is prolonged and can cause severe side effects and hypersensitivity reactions. Thus it is important to search for new antifungal agents that are more effective and have less serious side effects. In order to identify sources of bioactive substances against pathogenic fungus P. brasiliensis (Pb18), seventy-eight ethanolic extracts of fungi isolated from Atacama were tested in microdilution assays at concentrations from 500 to 0.9 g/ml. About 18% had lower MICs than or equal to 125 g/ml, especially isolated UFMGCB 8030 (MIC 15.6 g/ml). This isolate was identified as Aspergillus felis from morphological data and analysis of the sequencing of the internal transcribed regions, -tubulin and polymerase II rRNA gene. In order to potentiate the antifungal activity of this extract, the fungus was grown in different culture media and extraction of secondary metabolites was carried out with different solvents. The medium potato dextrose agar and extraction with dichloromethane solvent provided the extract with the best value of MIC (1.9 g/ml, 3.6 M). The identification of potential antifungal agents that could be produced by the fungus A. felis UFMGCB8030 was performed using analytical methods for dereplication, wherein the high-performance liquid chromatography coupled to spectrometry, high resolution mass and comparison with data from literature indicated the presence of the group of substances cytochalasins. After this chemical prior study, the fungus was grown on a larger scale to obtain the substances responsible for the antifungal activity. Chromatographic analysis and purification process resulted seven substances, of which two were classified as novel and named 5-N-acetyl-8--isopropyl ardeemin and cytochalasin Z15E. Besides these substances were obtained five other natural products already described in the literature, rosellichalasin, cytochalasin E, gancidin, pseurotin A1 and 2,4 dihydroxiacetophenone. Six of the substances showed activity against isolated Pb 18 P. brasiliensis in the tested concentrations (0.9 to 500 g/ml), highlighting the citosalasina E (MIC 1.9 g/ml). Treatment of yeast with this compound resulted in preventing the release of buds, generation of reactive oxygen species and damage to the plasma membrane and the cell wall. The results of this study showed that fungi isolated from extreme regions are sources of metabolites with antifungal properties against P. brasiliensis Pb 18 and x represent a source of new active componds. To our knowledge, this is the first report on the isolation of substances from the fungus A. felis and is also the first report of antifungal activity against P. brasiliensis compounds isolated in this work.