Investigation of Ximenia afra Sond. var. afra L. as a source of antiplasmodial and trypanocidal phytochemicals through accelerated bioactivity guided isolation

Ethnopharmacological relevance: Ximenia afra has been shown to be a potential source for antiprotozoal lead compounds based on its tentatively assigned phytochemistry or purely based on ethnomedicinal applications, where infusions of root, stem, and leaf mixtures, are often used for treating a wide range of parasitic ailments, including malaria and human African trypanosomiasis (HAT). Previous phytochemical analyses of extracts from the leaves showed high abundances of flavonoids and proanthocyanidins, and exhibited moderate to good activity against infections of Plasmodium sp. Aim of the study: This study was designed to (i) Rapidly determine the bioactivity of the extract and seven corresponding fractions, from the crude extract of X. afra leaves and twigs, against both P. falciparum and Trypanosoma brucei; (ii) isolate and characterise the bioactive compound from these fractions; (iii) confirm the bioactivity of the isolated compound against P. falciparum and T. b. brucei. Materials and methods: An extract and fractions were produced from the mixed stems and leaves of X. afra. A set of seven polarity-based fractions were produced from the extract and evaluated in an in vitro phenotypic screen against single strains of bloodstream form P. falciparum (3D7) and T. b. brucei (Lister strain 427). High resolution mass spectrometry analysis was done on the bioactive fractions, to tentatively identify the constituents. Guided by these phytochemical profiles and the antiprotozoal activity of the fractions, a single compound was selected for targeted isolation. Quercitrin was isolated by semi-preparative high performance liquid chromatography (HPLC) and fully characterised by accurate mass analysis and nuclear magnetic resonance spectroscopy (NMR). Biological assays were done to determine the antiplasmodial and trypanocidal activities of quercitrin. Results: In vitro phenotypic screening of the extract and fractions showed the mid-polar fractions exhibited significant antiplasmodial and trypanocidal activity. Tentative chemical profiling showed one of the highly bioactive fractions contained one major compound, which was then isolated. Accurate mass and NMR analyses confirmed the structure of the isolated compound as quercitrin. It was found to be active against P. falciparum (3D7), with IC₅₀ = 2.76 μM (n = 2). However, the activity of the compound was poor against T. b. brucei (strain 427) (19 % inhibition at 25 μM; n = 1) and showed no inhibitory activity in the P. falciparum lysyl tRNAse synthetase (PfKRS1) assay. Conclusion: Further scientific evidence for the effectiveness of X. afra, in the treatment of malaria and HAT, was obtained. Quercitrin, for the first time, was isolated from the leaves and stems of X. afra and its activity against P. falciparum (3D7) was confirmed in vitro by phenotypic assay. Its activity against this strain was, until now, unknown. Although quercitrin alone was not a potent trypanocidal compound, the combination of it with other flavonoids and proanthocyanidins provided good activity (%-inhibition > 80 %).