Avaliação in vitro da atividade leishmanicida de fungos isolados de amostras de solo da região amazônica

Abstract

American Cutaneous Leishmaniasis (ACL) is an infectious-parasitic disease widespread in the American Continent. Thus, the bioprospecting of new leishmanicidal agents is fundamental for the development of more effective and with less side effects that those currently used. This study is testing the action of 6 fungal species (Aspergillus calidoustus, A. fumigatus, Fusarium solanim, Penicillium citrinum, P. sclerotiorum and P. purpurogenum) isolated from the Amazonian soil against promastigotes and amastigotes of Leishmania amazonensis and L. guyanensis. In vitro assays were performed against promastigotes and amastigotes incubated with culture supernatants of 6 fungal species at concentrations 125, 250, 500 and 750 µg / mL. These supernatants were also evaluated for their cytotoxicity under MRC-5 strain human cells and for NO production. At the beginning of the experiments a growth curve was made where the third day was chosen for the bioassays for L. amazonensis and L.guyanensis promastigotes. As negative control culture medium (RPMI and Dmen) and positive control Pentamidine (30 mg / mL) were used. After evaluation of anti promastigote activity it was observed that A. calidoustus (IC50 = 638.2 µg / mL), F.solani (IC50 = 739.2 µg / mL) and P.purpurogenum (IC50 = 503.67 µg / mL) activity. The concentration of 750 µg / mL showed activity against L. amazonensis promastigotes within 72 hours (p <0.05, two-way ANOVA; post-hoc Tukey). A. fumigatus was the only supernatant acting against L. guyanensis promastigotes (IC50 = 654.6 µg / mL). P. citrinum and P. esclerotiorum showed no action against promastigotes of any species. Subsequently, cytotoxicity tests were performed against human fibroblasts, where only A. fumigatus was cytotoxic and excluded in subsequent tests. Regarding the experiments with amastigotes, only A. calidoustus and F. solani showed activity against L. guyanensis amastigotes reducing the number of macrophage parasites (RAW 264.7) after 24 hours of incubation with 750 µg / mL compared to negative control. (p <0.05, ANOVA: one criterion; post hoc Tukey). When evaluated for NO production Regarding NO dosage in macrophages previously stimulated by LPS, all 6 species exhibited a significant increase (p = 0.01, ANOVA) in NO production at low concentrations, but the effect was not observed at higher concentrations. However, this same effect was not seen in F. solani and P. purpurogenum supernatants during NO dosing in macrophages infected with amastigotes of L. amazonensis and L. guyanensis and treated with supernatants at concentrations of 500 and 750 µg / mL. Only, only A. calidoustus culture supernatants at the 750 µg / mL concentration had any effect, significantly decreasing NO production in response to L. amazonensis amastigotes