Abstract
Introduction: Malaria is a persistent killer disease that has resulted in over 247 million clinical cases and 619 000 deaths globally, with 95 % of the reported cases occurring in Africa. Due to the increasingly reported incidences of the evolution and emergence of resistance by the parasite, most of the currently available antimalarials are challenged and undermined. Therefore, there is an urgent need to find new strategies that can help in this regard. Medicinal plants are regarded as an appealing avenue due to their deep historical usage, rich biodiversity, and complex therapeutic phytochemicals. Further, what makes medicinal plants even more appealing is that some of the widely known antimalarials such as chloroquine and artemisinin are plant-derived. Aims and objectives: The study sought to explore, harness, and validate the use of three selected plants (Ziziphus mucronata, Xysmalobium undulutum, and Ximenia caffra var) in South African traditional medicine for their antimalarial potential. Methodology: The plants were collected and sequentially extracted using hexane, ethyl acetate, dichloromethane, and methanol. The resulting crude extracts were then screened and studied for their antimalarial and cytotoxic potential using the parasite lactate dehydrogenase (pLDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The plant crude extract with the most prolific percentage yield and antimalarial activity was then subjected to isolation using silica gel and thin layer chromatography (TLC). The isolated compounds were then evaluated for their antimalarial and cytotoxic activity and structurally elucidated using spectroscopic techniques (Nuclear Magnetic Resonance: 1H, 12C, and DEPT). The compounds were then subsequently subjected to molecular docking using AutoDock Vina to target the Plasmodium falciparum heat shock protein 70-1 (PfHsp70-1). Validation of the in-silico results was performed by expression, purifying, and characterizing PfHsp70-1 in the presence of compounds. Results: All the crude extracts except the methanol extract from all three plants revealed exceptional antimalarial activity characterized by 70% parasite inhibition at 20 μg/ml and 50% parasite inhibition at 10 μg/ml of crude extract. The determination of the IC50 antimalarial activity revealed intriguing patterns of antimalarial potency, for Z. mucronata the hexane extract, had an IC50 value of 11.69 ± 3.84 μg/ml, ethyl acetate (7.25 ± 1.41 μg/ml), and dichloromethane (5.49 ± 0.03 μg/ml), X. undulutum hexane extract (4.9 ± 0.037 μg/ml), ethyl acetate (17.46 μg/ml ) and dichloromethane (19.27 μg/ml), for X. caffra var the hexane extract had an IC50 value of 18.92 ± 0.93 μg/ml, ethyl acetate (18.99 ± 0.16 μg/ml) and dichloromethane had an IC50 value of 6.92 ± 2.56 μg/ml. For the cytotoxicity, all the crude extracts except the ethyl acetate (CC50 = 10.96 μg/ml) and dichloromethane (CC50 = 10.01
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μg/ml) extracts of Z. mucronata exhibited minimal cytotoxicity characterized by CC50 values
>
50 μg/ml. The isolated compounds exhibited minimal cytotoxicity (CC50 > 50 μg/ml) and promising antimalarial activity with IC50 values of 19.95 μg/ml and 7.76 μg/ml, respectively. Structural elucidation revealed that the compounds responsible for the observed antimalarial activity of Z. mucronata were betulinic acid and lupeol. Molecular docking demonstrated exceptional binding affinity between betulinic acid, lupeol, and PfHsp70-1 with docking scores of -6.9 kcal/mol and -6.8 kcal/mol, respectively. When these computational outputs were validated in vitro, following the successful expression and purification of PfHsp70-1, Ultraviolet-visible (UV-Vis) spectrophotometry revealed that betulinic acid and lupeol disrupt the secondary structural elements of PfHsp70-1 by exposing hydrophobic amino acids and promoting the unfolding state of the protein. Similarly, the malate dehydrogenase (MDH) assay showed that the compounds disrupt the chaperone ability of PfHsp70-1 to refold client proteins. Conclusion: Betulinic acid and lupeol can serve as prospective antimalarial agents, however, structural modification to increase their antimalarial potency is warranted. Further, given these findings, the use of these plants in folk medicine is substantiated and supports the synergy between traditional healing wisdom and modern science as an appealing approach to the development of novel antimalarial agents.
Keywords: Malaria, Medicinal plants, Ziziphus mucronata, Xysmalobium undulutum, Ximenia caffra var, PfHsp70-1, Betulinic acid, and Lupeol.