Ethnopharmacological Investigation and Mechanistic Characterisation of Anti-Trypanosomiasis Plants in Tsetse-Endemic Communities of Benue State, North Central Nigeria

Human African trypanosomiasis (sleeping sickness) and animal trypanosomiasis (nagana) continue to threaten lives and livestock in the tsetse fly-endemic vegetation zones of Benue State, North Central Nigeria. Existing trypanocidal drugs are associated with severe adverse effects and emerging resistance. This study combines primary ethnopharmacological data collection with systematic phytochemical and mechanistic biological investigation to characterise anti-trypanosomiasis plants from Benue State. Ethnopharmacological surveys in eight tsetse-affected LGAs using informant-based interview methods documented 45 plant species used for trypanosomiasis management across human and veterinary contexts. The five most-cited species, including Khaya senegalensis, Vernonia amygdalina, and Combretum molle, were subjected to bioassay-guided fractionation targeting Trypanosoma brucei brucei growth inhibition in in vitro bloodstream form assays. Mechanism of action studies investigated mitochondrial membrane potential disruption (JC-1 assay), reactive oxygen species induction, kinetoplast ultrastructure by TEM, and cell cycle arrest by flow cytometry. Khaya senegalensis limonoid-rich fraction (IC50 0.84 ug/mL) and a Combretum molle tannin-terpenoid fraction (IC50 2.1 ug/mL) were identified as the most active. Mechanism studies confirmed mitochondrial dysfunction and kinetoplast disruption as primary modes of action. Selectivity indices above 30 were demonstrated against Vero cells. A novel limonoid analogue was isolated and characterised by 2D NMR and HRMS from Khaya senegalensis. Keywords: trypanosomiasis, Benue State, Khaya senegalensis, limonoid, mechanism of action