Annickia affinis (Exell) Versteegh & Sosef methanol stem bark extract, potent fractions and isolated Berberine alkaloid target both blood and liver stages of malaria parasites

Abstract

Ethnopharmacological relevanceHaving identified Annickia affinis as the most potent antiplasmodial plant constituent in a hepta-herbal Agbo-iba (HHA) formula commonly used to manage malaria in Benin city, Nigeria, we have in this study attempted to identify the specialized metabolites responsible for antiplasmodial activity of A. affinis through anti-blood stage malaria parasite activity guided isolation of potent molecules from its stem bark methanol extract. After that, phenotypic effects, including stage-specific kill kinetics, were investigated. Further, the crude extract, its potent fractions, and specialized metabolites were also tested against the liver-stage malaria parasite. Materials and methodsA. affinis was subjected to molecular PCR-based analysis to confirm its identity. Thereafter, extraction of its stem bark with methanol was carried out. Alkaloid enriched fractions from this stem bark extract were obtained using the acid-base-solvent extraction method. These alkaloid-enriched fractions were subjected to various chromatographic techniques that led to the isolation of two protoberberine alkaloids identified as berberine and palmatine based on NMR and mass spectrometry analysis. The efficacy of crude extract, fractions and purified alkaloids was tested against the malaria parasite's blood and liver stages, respectively. Results and DiscussionAnnickia affinis methanol extract, fractions, and the isolated protoberberine alkaloids showed excellent antiplasmodial activity with good selectivity against blood-stage malaria parasite. Thus, their IC50 against various strains of the parasite ranged from 0.95 to 18.65 μg/ml, while CC50 against Human embryonic kidney (HEK) and the human hepatoma (HUH-7) cell lines ranged between 10 and > 100 μg/ml. Interestingly, the crude extract and the alkaloid enriched fractions showed promising activity against the liver-stage malaria parasite. Between berberine and palmatine isolated from the potent fractions, only the former showed ∼100% and 90% inhibitions of liver stage parasite at 5 μg/ml and 1 μg/ml, respectively, while the latter showed no inhibition even at 20 μg/ml. ConclusionThis study reports that the ethnomedicinal use of HHA to manage malaria can be attributed to the presence of promising antiplasmodial protoberberine alkaloids together with synergistic effects via either enhancement of bioavailability or improved pharmacokinetics by other phytoconstituent(s) coming from other HHA constituent plants. The protoberberine alkaloids isolated have been identified as fast-acting antiplasmodial agents, with activity against all erythrocytic stages of the malaria parasite. Further, A. affinis methanol stembark extract and the protoberberine alkaloid berberine isolated from it also displayed excellent activity (>90% inhibition at 1 μg/ml) against the liver-stage malaria parasite. A. affinis and HHA can thus be useful as both liver-stage prophylactic and blood-stage curative agents.