Abstract
Malaria remains a major public health concern, with over 249 million cases and 608,000 deaths reported in 2022, primarily due to Plasmodium falciparum infections in Sub-Saharan Africa. The rise of artemisinin-resistant strains highlights the urgent need for new antimalarial agents with novel mechanisms of action. This study explored the antimalarial potential of Ximenia. Caffra var., a Southern African medicinal plant traditionally used for treating malaria like illnesses, through a multidisciplinary approach combining phytochemistry, bioassays, metabolomics, and molecular target analysis. Sequential solvent extraction of X. caffra bark yielded four crude extracts with varying activity. Hexane and methanol extracts exhibited the most potent antiplasmodial effects (IC₅₀ = 5.96 μg/mL and 7.48 μg/mL), while bioassay guided fractionation led to the isolation of lupeol, betulinic acid, ursolic acid, and palmitoleic acid. Among these, ursolic acid and lupeol showed strong activity against P. falciparum (IC₅₀ < 10 μg/mL) with low cytotoxicity toward mammalian cells (IC₅₀ > 50 μg/mL), confirming excellent selectivity and safety potential. Untargeted LC-MS/MS metabolomic profiling and Global Natural Products Social Molecular Networking (GNPS) revealed distinct chemical signatures among extracts, distinguishing polar flavonoids and phenolics in methanol extracts from non-polar triterpenoids and fatty acids in hexane extracts. Pathway enrichment analysis identified key biosynthetic routes, including flavonoid, phenylpropanoid, and fatty acid biosynthesis, elucidating the plant’s secondary metabolic framework responsible for its medicinal properties. Molecular target studies established P. falciparum heat shock protein 70-1 (PfHsp70-1) as a key target. The recombinant protein was expressed and purified for inhibition assays, where ursolic acid and palmitoleic acid significantly suppressed PfHsp70-1 chaperone activity, comparable to the control polymyxin B (p < 0.0001). Fluorescence spectroscopy confirmed direct binding and conformational modulation of the protein, with ursolic acid showing the strongest interaction, while palmitoleic acid acted as an allosteric modulator stabilizing an inactive state. Overall, the study scientifically validates the traditional use of X. Caffra as an antimalarial remedy. Its multi-target mechanism combining triterpenoid-mediated chaperone inhibition, fatty acid-induced membrane disruption, and flavonoid antioxidant support represents a promising network pharmacology model for natural product-based drug discovery. The identification of PfHsp70-1 as a novel drug target, along with potent, selective lead compounds, lays the foundation for future in vivo efficacy, pharmacokinetic, and structure activity optimization studies. The study also highlights the power of integrating ethnobotanical wisdom with modern metabolomics and molecular biology to accelerate the discovery of next-generation antimalarial agents capable of addressing drug resistance.