Abstract
Plasmodium falciparum is a unicellular protozoan parasite that is the causative agent of malaria. Despite the significant progress in malaria response, it remains a major global health burden, killing hundreds of thousands annually, especially in sub-Saharan Africa. Therapeutic agents to treat this disease have numerous side effects and have been compromised by mutations in the parasite genome, causing resistance. Thus, new therapeutics with novel mechanisms of action need to be developed. Since ancient times, plants have been used as a primary source of pharmacologically active compounds to make drugs. More than 80% of the rural population relies on traditional medicine for their primary healthcare needs. Therefore, this study aims to explore the antimalarial properties of three South African plants used in traditional medicine to treat malaria. The study focuses on scientifically validating the use of Ziziphus mucronata, Ximenia caffra, and Ricinus communis by investigating their antiplasmodial properties and the lupane-type pentacyclic triterpenes isolated from them, as well as their interaction with the protein drug target, Plasmodium falciparum hypoxanthine-guanine- xanthine phosphoribosyltransferase (PfHGXPRT). The methodologies followed in conducting the study included sequential extraction of plants using hexane, ethyl acetate (EA), dichloromethane (DCM), and methanol to obtain crude extracts that were subjected to asexual antiplasmodial activity screening with SYBR Green I Assay on chloroquine-sensitive strain NF54 strain of P. falciparum parasites. DCM and EA extracts of the bark of Z. mucronata displayed promising antimalarial activities. These crude extracts were further subjected to column chromatography to isolate the pure compound(s), which were characterized using Nuclear magnetic spectroscopy (NMR) spectroscopy. PfHGXPRT expression in BL21 E. coli
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cells was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside, and the protein was purified with the Nickel-charged nitrilotriacetic acid column and further analyzed on 15% SDS-PAGE. UV-vis absorption spectroscopy and molecular docking simulations analyzed the structural interactions between the protein and compounds. DCM and EA extracts of the bark of Z. mucronata displayed promising antimalarial activities with IC50 values of 5.494 μM and
7.227 μM, respectively. These were further subjected to column chromatography for the isolation of pure compound(s), which were later characterized to be betulinic acid (BA), methyl betulinate (MA), and lupeol (LU). The size of the purified PfHGXPRT was approximately 28 kDa. The intensity of the absorption peak of PfHGXPRT obtained using UV was observed to increase consecutively as the concentration of BA increased; the opposite was true for MA and LU. Molecular docking showed that PfHGXPRT amino acid residues formed hydrophobic interactions with all compounds. LU showed the highest binding energy (-7.6 kcal/mol), followed by MA & BA (-7.2 kcal/mol). The antimalarial activity of the crude extracts and the isolated compounds confirm these medicinal plants could serve as effective agents for treating malaria.