Aspidosperma species (Apocynaceae) as sources of antimalarials: from the in vitro antiplasmodial activity of extracts to pre-clinical toxicological studies for the development of efficient and safe antimalarial phytomedicines

We report here our investigations on plants belonging to the botanical genus Aspidosperma (Apocynaceae) that are used to treat malaria and/or fevers in Brazil, aiming to prepare standardized alkaloid extracts for phytomedicines development. Ethanol and alkaloid extracts of A. parvifolium and A. subincanum barks were prepared and their in vitro antiplasmodial activity was evaluated against Plasmodium falciparum (W2 strain) by the pLDH method. Bioguided fractionation of the active alkaloid extracts was undertaken. A study on antiplasmodial activity versus alkaloids content variation from three A. subincanum specimens that were collected in different regions was carried on by HPLC-UV. Ethanol and alkaloid extracts from A. parvifolium and A. subincanu m were evaluated in toxicological studies in male and female Swiss mice and male Wistar rats (n = 10); a control group was included in the experiments. At the end of treatment, animals were sacrificed, hematology and serum biochemical analyses were performed. Fractionation of alkaloid extracts from A. parvifolium and A. subincanum guided by in vitro assays against P. falciparum (W2 strain) led to the isolation of the indole alkaloids uleine (Oliveira et al., 2010) and N-demethyluleine. HPLC-UV and LC-UV-MS/ESI analyses of the extracts disclosed uleine as the major constituent, its content reaching 70.7% in the alkaloid extract from A. subincanum (ASALB) which was more potent than uleine and N-demethyluleine themselves in the in vitro assays, as can be inferred from the selectivity indexes in relation to HepG2 cells (SI = CC₅₀/IC₅₀) of ASALB (100), uleine (44) and N-demethyluleine (21). Moreover, a significative variation on the contents of these two alkaloids was observed for the same plant species when collected in different regions (Paula, 2014). Both ethanol and alkaloid extracts from A. parvifolium bark were relatively safe, producing minor changes in the parameters evaluated, especially in smaller doses, thus making feasible their future use in phase I clinical trials. On the other hand, high toxicity of both extracts from A. subincanum bark was observed in doses >200 mg/kg. The present results point to the necessity of rigorous investigation of traditional antimalarial plants including in vitro studies for identification of active natural products, standardization of extracts to be submitted to pre-clinical toxicological studies and controlled clinical trials. Our results disclose that efficient and safe phytomedicines might be developed from standardized crude or semi-purified extracts of Aspidosperma species containing the active chemical entities.