Evaluation of NTD-O2, a Ghanaian herbal medicine, for onchocerciasis and animal African trypanosomiasis

Background: Herbal medicine remains central to primary healthcare in Ghana due to its accessibility and perceived safety. NTD-O2 is an aqueous herbal formulation derived from Xylopia aethiopica fruits and Bambusa vulgaris leaves, traditionally used for managing onchocerciasis, a neglected tropical disease (NTD) endemic to the country. This study evaluated the antionchocercal activity of NTD-O2 and assessed isolated compounds from the formulation and its source plants for activity against animal African trypanosomiasis, another prevalent NTD. Methods: Dichloromethane (DCM) and butanol (BuOH) extracts of NTD-O2 were screened in vitro against Onchocerca ochengi and Trypanosoma brucei brucei. Bioassay-guided fractionation, coupled with spectroscopic and spectrometric techniques, facilitated structural elucidation of isolated compounds. Potential mechanisms of action were explored through in silico molecular docking. Results: Both NTD-O2 extracts achieved 100% inhibition of adult male O. ochengi motility. Activity against female worms was moderate (NTD-DCM: 61.0 ± 1.8%; NTD-BuOH: 56.6 ± 4.4%), and weaker against microfilariae (NTD-DCM: 50 ± 0%; NTD-BuOH: 0%). Antitrypanosomal activity was more pronounced, with IC₅₀ of 10.68 µg/mL (NTD-DCM) and 9.44 µg/mL (NTD-BuOH), compared to diminazene aceturate (IC₅₀ = 0.13 ± 0.02 µg/mL). Cytotoxicity testing on Monkey Kidney Epithelial (LLC-MK2) cells indicated no toxicity. Column chromatography of NTD-BuOH yielded bis(4-methylheptyl) phthalate (1) (IC₅₀ = 1.1 ± 0.3 µg/mL) and O2-F3-S (IC₅₀ = 100 ± 0.46 µg/mL). By contrast, kaurene diterpenoids from X. aethiopica [ent-kaur-16-en-19-oic acid (2), xylopic acid (3), and ent-kaur-16-en-15-one-19-oic acid (4)], along with long-chain carbonyl compounds (5, 6) from B. vulgaris were inactive (IC₅₀ = > 100 ± 0.46 µg/mL). Molecular modelling results consistently revealed weak binding scores for compounds 2–4 across all three targets (glutathione S-transferase and glutamate-gated chloride channel for onchocerciasis and ornithine decarboxylase for trypanosomiasis). Conclusion: NTD-O2 demonstrated selective antionchocercal and moderate antitrypanosomal activities, validating aspects of its traditional use. However, the presence of phthalate, a compound of known toxicological risks, and the absence of key plant-derived constituents raise concerns about formulation consistency and safety. These findings underscore the need for routine quality control and safety monitoring of herbal medicines to ensure efficacy and public health protection.