Efeitos carrapaticida e anti-helmíntico in vitro de alcaloides sobre parasitos de ruminantes e citotoxicidade

Abstract

The inappropriate use of antiparasitic agents has favored the selection of parasites resistant to commercial chemical products, which has encouraged research on the investigation of compounds obtained from plants for the control of animal’s parasites. The aim of this study was to evaluate in vitro the antiparasitic activity and cytotoxicity of berberine, harmaline and piperine alkaloids on ruminant’s parasites. The alkaloids used in this study were obtained commercially, and their acaricidal and anthelmintic activities were investigated against Rhipicephalus microplus larvae and gastrointestinal nematodes (GIN) of goats, respectively. For the acaricidal evaluation of the alkaloids, the larval immersion test was used, while in the anthelmintic evaluation, the egg hatch (EHA) and larval motility (LMA) assay were used. The relationship between the acaricidal effect and the in vitro (spectrophotometry) and in silico (docking and molecular dynamics) inhibition of R. microplus acetylcholinesterase (RmAChE) and the possible cytotoxic effects of these alkaloids in Vero cell (MTT test) were also investigated. In the acaricidal evaluation, both berberine (97%) and piperine (98%) showed activity greater than 95%, not differing from the positive control (fipronil, 100%, p > 0.05). At the same concentration (24 mM), only treatment with harmaline resulted in low larvicidal activity (32.75%). The highest anticholinesterase activity in vitro was observed for berberine (IC50 = 88.13 µM, inhibitory concentration 50%), while piperine showed low inhibitory activity of RmAChE (IC50 > 200 µM). Evidence obtained in the in silico study with berberine and RmAChE 1 suggests that this alkaloid fills preferentially the peripheral anionic site (PAS) of the enzyme. The results of the anthelmintic evaluation revealed that berberine, harmaline and piperine showed ovicidal activity with values effective concentration 50% (EC50) equal to 1.32, 1.6 and 0.0074 mM, respectively. In LMA, only the berberine alkaloid showed larvicidal efficacy with a percentage of larval motility inhibition equal to 98.17% (2.69 mM). In cytotoxic evaluation, the three alkaloids have reduced toxicity to Vero cells, with a cytotoxic effect of less than 50%. These results suggest that the alkaloids berberine and piperine have antiparasitic potential with low toxicity for mammalian cells, and the likely mechanism of acaricidal action of berberine is related to the inhibition of the acetylcholinesterase enzyme of R. microplus.