Avaliação da atividade antifúngica in vitro das folhas de Xylosma ciliatifolia (Clos) Eichler (Salicaceae) frente a linhagens de Cryptococcus gattii e Cryptococcus neoformans

Abstract

The steady increase in the incidence of opportunistic fungal diseases has become a major public health problem, especially after the advent of AIDS when the number of immunocompromised individuals increased dramatically. Cryptococcosis is an important systemic mycosis caused by fungi of the genus Cryptococcus, with a global estimate of one million new cases and approximately 600,000 deaths per year. The current therapeutic options of cryptococcosis are restricted, both by the increase of the resistance of these yeasts to the drugs used and by the high toxicity of the antifungal to the human organism being necessary the continuous search for new chemical entities for the treatment of infections. In this context, natural products and derivatives are an invaluable source of substances with biological potential. Xylosma ciliatifolia, popularly known as Sucará, is a semi-deciduous tree, native to Brazil, and can also be found in other countries. The present work evaluated the antifungal activity of X. ciliatifolia leaves, in vitro, against C. gattii and C. neoformans. The MIC of the ethanolic extract and the fractions hexane, dichloromethane, ethyl acetate, butanol and aqueous residue were determined. The ethyl acetate fraction (FAXC) showed promising activity and was selected for subsequent trials. Through the fungistatic / fungistatic activity assay it was observed that FAXC has fungicidal action. In the interaction test with fluconazole and amphotericin B, in vitro, no interference was observed in the action of the drugs and the death curve assay demonstrated that FAXC shows a dose-dependent behavior. FAXC didn't change the MIC value in the presence of sorbitol, indicating that its action possibly don´t occur directly through the cell wall. Through the quantification of ergosterol it was possible to observe that FAXC interferes in the biosynthesis of the steroid, being this one of the mechanisms of action. The production of ROS and RNS in fungal cells was evaluated by the emission of fluorescence and after treatment with FAXC there was an increase in peroxynitrite production, which causes cell damage. Lipid peroxidation products were measured as thiobarbituric acid reactive substances (TBARS), where lipid peroxidation was evidenced in fungal cell membranes in a higher concentration. Cell size and capsule size of Cryptococcus was evaluated by comparing the count of treated and untreated cells stained with the India ink. After exposure to the concentration equivalent to half of the MIC, the cell diameter decreased suggesting a defense attempt, but there wasn't change in capsule size. Cell surface loading was evaluated by zeta potential, which also showed no change. The interaction between FAXC and fungi, as assessed by ITC, is probably weak as non-polar interactions. Finally, it was verified by the MTT colorimetric assay that FAXC didn't present cytotoxicity in the active concentrations, being this dose dependent.