Efeito leishmanicida do óleo essencial de Vernonia brasiliana (L.) Druce (Asteraceae) em Leishmania infantum

Abstract

The aim of this work was to evaluate the leishmanicidal effect of Vernonia brasiliana essential oil (VBEO) in promastigote forms of Leishmania infantum, its mechanisms of action and the effect of its association with the standard drug miltefosine. V. brasiliana leaves were collected in the city of Santa Luzia, Maranhão, followed by botanical identification, and voucher specimen deposit. Leaves were dried and submitted to hydrodistillation process, for the obtention of the essential oil. The chemical composition was determined through gas chromatography-mass spectrometry (CG-MS). The relative amounts of individual compounds were computed from GC peak areas. The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 macrophages were evaluated by MTT colorimetric assay. To identify ultrastructural changes, promastigote forms of L. infantum were treated with VBEO IC50 and evaluated by transmission electron microscopy. Variations in the mitochondrial membrane potential, in the production of reactive oxygen species, and analyses of apoptosis were determined by flow cytometry. Parasites treated with VBEO at IC50 concentration were incubated with TMRE, H2DCFDA and Annexin/Propidium iodide (PI), respectively. The combination between VBEO-miltefosine was evaluated in L. infantum promastigote forms by the modified isobologram method. Different proportions of both drugs were tested to calculate the fractional inhibitory concentration index and determine the nature of interaction. The results were analyzed statistically by Kruskal-Wallis test followed by Dunn's posttest. Analyses were performed with the software GraphPad Prism 7 and differences were considered significant when p<0.05. The yield of the essential oil was 0.1%. The major compound of the essential oil was the sesquiterpene 􀈕-caryophyllene (21.47%). Anti-Leishmania assays indicated an IC50 against promatigote forms of 39.01±1.080 μg/mL. The CC50 was 63.13±1.211 μg/mL, showing that VBEO is less toxic to cells than to parasites. The antileishmanial action of the essential oil was evidenced by ultrastructural alterations found in promastigote forms, as kinetoplast swelling, occurrence of vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of cytoplasmic organelles. VBEO decreased mitochondrial membrane potential and increased the production of reactive oxygen species in L. infantum promastigotes, with consequent cell death due to late apoptosis. The combination of the essential oil and miltefosine showed an antagonistic effect, with a fractional inhibitory concentration index equal to 4.779. This study demonstrates the anti-Leishmania activity of VBEO against L. infantum promastigotes.