Bioassay-guided isolation and characterization of antiplasmodial compounds from Ximenia caffra bark and their interaction with PfHsp70-1

Malaria continues to pose a major global health challenge, particularly in sub-Saharan Africa, where increasing resistance to frontline antiplasmodial therapies threatens disease control efforts. The emergence of artemisinin-resistant Plasmodium falciparum strains necessitates the discovery of novel antiplasmodial agents. Ximenia caffra , traditionally used across sub-Saharan Africa for treating various ailments, represents an underexplored source of antiplasmodial compounds. Bark samples of Ximenia caffra were sequentially extracted using solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and methanol). Extracts were screened for antiplasmodial activity using the parasite lactate dehydrogenase (pLDH) assay. Active extracts underwent bioassay-guided fractionation. Isolated compounds were characterized using LC-MS and NMR spectroscopy and evaluated for cytotoxicity against CHO cells. Mechanism of action studies were conducted using recombinant Pf Hsp70-1, including malate dehydrogenase aggregation assays and intrinsic tryptophan fluorescence spectroscopy. Hexane and methanol extracts demonstrated potent antiplasmodial activity (IC₅₀ = 5.96 and 7.48 µg/mL, respectively). Four compounds were isolated and identified: lupeol, betulinic acid, ursolic acid, and palmitoleic acid. Lupeol and ursolic acid showed moderate antiplasmodial activity (IC₅₀ = 7.56 and 8.39 µg/mL; 17.7 and 18.4 µM, respectively). While betulinic acid and palmitoleic acid showed weaker antiplasmodial activity, all four isolated compounds showed low cytotoxicity against CHO cells (IC₅₀ > 50 µg/mL), indicating a favourable selectivity margin, particularly for lupeol and ursolic acid. Mechanistic studies revealed that ursolic acid and palmitoleic acid potently inhibited Pf Hsp70-1 chaperone activity, while intrinsic tryptophan fluorescence demonstrated distinct binding modes for each compound. X. caffra bark contains antiplasmodial compounds warranting further investigation. The identified triterpenoids represent candidates for antiplasmodial drug development, with ursolic acid of particular interest for combining direct antiplasmodial activity with candidate inhibitory activity against the essential Pf Hsp70-1 chaperone, lending in vitro support to the plant’s traditional medicinal use.