Alcaloides indólicos monoterpenoídicos com atividade antimalárica de carapanaúba (Aspidosperma excelsum Benth)

Abstract

The emergence and spread of Plasmodium strains resistant to current antimalarials have threatened malaria control measures. In the search for more effective substances in the fight against this disease, medicinal plants of traditional use are promising sources for the discovery of new antimalarial agents. Previous work by LAPAAM has demonstrated antimalarial activity of extracts and fractions obtained from the bark of Aspidosperma excelsum (Benth), a plant used in the treatment of malaria by traditional Amazonian communities. The present work is a study of the chemical composition of the bark of this species, in order to discover natural substances with antimalarial potential and low toxicity. The phytochemical procedure began with fractionation of the MeOH extract from the LAPAAM extract bank, partitioned by pH increase (8, 10 and 12), generating fractions later evaluated in an initial screening against Plasmodium falciparum K1 strain. A new collection of bark was made at INPA's Adolpho Ducke Forest Reserve. The dry material was ground and extracted in two different methods: continuous solid-liquid extraction in a soxhlet apparatus, using MeOH as solvent, and by room temperature maceration in MeOH and EtOH. The extracts were subjected to acid-base partitioned, to concentrate the alkaloids and to evaluate the ideal techniques for a better yield. The obtained fractions were submitted to several frations: column chromatography (CC), preparative thin - layer chromatography (PTLC) and thin-layer chromatography (TLC). The structural elucidation of the substances was performed using nuclear magnetic resonance (1D and 2D NMR) and high resolution mass spectrometry (LC-HRMS). The antiplasmodial activity was evaluated by the microtest in vitro using the F. falciparum strain K1. In vitro cytotoxicity was assessed against the normal human fibroblast lineage MRC-5 by the Alamar Blue test. It was possible to isolate 9 monoterpene indole alkaloids: isoreserpilin (C), reserpilin (D), 3α, 20β-corinan-17-ol (B), E-15S, 16S-isositsirikine (A), N-oxide-15S, 16S-E-isositsirikine (E), 10-methoxycorinan-17-ol (F), 10-methoxy-15S, 16S-E-isositsyquinine (H), 15α-10-methoxy-geissosquizol (I) and substance G (not yet reported in the literature, having a new carbonskeleton). No cytotoxic activity for the alkaloids (CI> 156 µM) against the MRC-5 lineage was observed. They were more selectively toxic to the parasites than to the normal cells. In the antimalarial tests, alkaloids had a median inhibitory concentration (IC50) of 10.3 μM (10-methoxy-15S, 16S-E-isositsyquinine (H)) to 78.9 μM (N-oxide-15S, 16S-E-isositsirikine (E)) against P. falciparum.