In vitro antiplasmodial activity and cytotoxicity of three Ziziphus (Rhamnaceae) species from South Africa

The Zulu, Swazi, Tsonga, and Venda people of South Africa utilize various parts of Ziziphus mucronata Willd., Z. rivularis Codd., and Z. zeyheriana Sond. to treat different ailments, including malaria. However, despite their use in traditional medicine, Z. rivularis and Z. zeyheriana remain underexplored for their antiplasmodial activity and cytotoxicity.

This study assessed the in vitro antiplasmodial activity and cytotoxicity of Z. mucronata, Z. rivularis, and Z. zeyheriana. It further identified antiplasmodial constituents using ¹H NMR-based metabolomics and GC-MS analyses.

Ground stem bark, leaf, and root bark samples from each plant species were weighed separately at 40 g before sequential extraction using n-hexane, dichloromethane, ethyl acetate, a mixture of ethyl acetate and methanol (1:1; v/v), and methanol. Each extraction was performed three times before concentrating the resulting extracts by evaporating the excess organic solvent using a rotary evaporator (Buchi, R-200, Switzerland). Decoctions were also prepared to replicate the traditional preparation method for comparative analysis. Forty-eight successive extracts were obtained and subjected to [³H]hypoxanthine incorporation assay using P. falciparum NF54 and cytotoxicity using rat skeletal (L6) myoblast cells. Furthermore, ¹H NMR-based metabolomics was used to identify classes of compounds associated with the observed antiplasmodial activity, while GC-MS was employed to identify specific phytoconstituents potentially contributing to this activity.

Five of the 48 tested extracts exhibited high antiplasmodial activity (IC₅₀ < 5 microg/ml), while 12 and 17 extracts demonstrated promising (5 microg/ml < IC₅₀ ≤ 20 microg/ml) and moderate (20 microg/ml < IC₅₀ ≤ 50 microg/ml) activity, respectively. The remaining extracts were inactive (IC₅₀ > 50 microg/ml). Notably, the dichloromethane stem bark extract of Z. mucronata and the ethyl acetate root extract of Z. zeyheriana had the highest antiplasmodial activity, with IC₅₀ values of 3.04 and 3.6 microg/ml, respectively. Only the dichloromethane and ethyl acetate stem bark extracts of Z. zeyheriana exhibited selectivity, with indices of 10 and 12, respectively. Principal Component Analysis (PCA) did not discriminate the training set based on the observed antiplasmodial activity. However, upon applying the Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), the samples clustered according to the observed antiplasmodial activity, with R² and Q² values of 0.8 and 0.7, respectively. Statistically, the OPLS-DA model was significant, with a P-value of 0.05. Antiplasmodial activity is linked to aliphatic, allylic, methyl ketone, and carboxylic acid-based classes of constituents. Further GC-MS analysis revealed lupeol, palmitic acid, and friedelin as contributors to the observed antiplasmodial activity in Z. mucronata, Z. rivularis, and Z. zeyheriana.

The study confirmed the significant antiplasmodial activity of Z. mucronata, and for the first time, it reported the antiplasmodial activity of Z. rivularis and Z. zeyheriana. It further demonstrated that the tested samples have no apparent cytotoxicity. The integration of ¹H NMR-based metabolomics and GC-MS analysis allowed for the identification of bioactive classes of compounds and the specific constituents contributing to the observed antiplasmodial activity. Lupeol, palmitic acid, and friedelin, previously recognized for their antiplasmodial activity, are partly attributed to the observed antiplasmodial activity in Z. rivularis, Z. zeyheriana, and Z. mucronata.