In vitro anti-HSV-1 activity of crude Croton kinondoensis root, stem, leaf and twig extracts in vero E6 cells: GC–MS profiling and computational characterisation of candidate phytochemicals

Background Herpes simplex virus type 1 (HSV-1) infects an estimated 67% of the global population under 50 years of age, with pooled adult seroprevalence in Sub-Saharan Africa reported at 96.2%. Resistance to first-line nucleoside-analogue therapy, together with cost and access barriers in low-resource settings, motivates continued discovery of anti-HSV-1 leads from medicinal plants. Croton kinondoensis is a recently described species endemic to coastal Kenya that has not previously been evaluated for antiviral activity. Methods Crude dichloromethane:methanol (1:1, v/v) extracts of the roots, stems, and leaves and twigs of C. kinondoensis were evaluated for cytotoxicity (MTT assay) and anti-HSV-1 activity (time-of-addition and plaque reduction assays) in Vero E6 cells using HSV-1 strain 7401H. The leaf-and-twig extract (CKA), selected on the basis of its most favourable activity–cytotoxicity profile, was characterised by GC–MS. The thirteen putatively identified GC–MS constituents were subsequently evaluated by molecular docking and MM-GBSA binding free-energy estimation against HSV-1 thymidine kinase, DNA polymerase, and the helicase–primase complex. Results All three extracts inhibited viral plaque formation in a concentration-dependent manner. CKA achieved the most favourable activity–cytotoxicity profile (CC 50 = 324.8 ± 18.08 μg/mL; plaque reduction IC 50 = 20.00 ± 3.52 μg/mL; SI = 16.24) and maintained selectivity across pretreatment (SI 13.48), simultaneous (SI 7.95), and post-treatment (SI 8.42) conditions. GC–MS putatively identified thirteen constituents dominated by methyl eugenol (45.81%) and methyl isoeugenol (14.04%). Molecular docking and MM-GBSA screening prioritised 1, 2-dimethoxy-4-(1, 2-dimethoxyethyl) benzene as the top computationally predicted TK binder (MM-GBSA −42.8 kcal/mol; comparable to acyclovir) and stigmasterol as the top computationally predicted helicase–primase binder (−46.2 kcal/mol). Conclusion Crude leaf-and-twig extracts of C. kinondoensis demonstrate reproducible in vitro anti-HSV-1 activity in Vero E6 cells with an acceptable cytotoxicity margin. GC–MS profiling putatively identified thirteen constituents, and computational screening prioritises 1, 2-dimethoxy-4-(1, 2-dimethoxyethyl) benzene and stigmasterol as candidates for mechanistic follow-up against thymidine kinase and the helicase–primase complex, respectively. These are computationally predicted binding interactions, not demonstrated mechanisms of action, and require biochemical and bioassay-guided fractionation studies before any mechanistic or translational conclusion can be drawn.