Repeated-Dose Intravaginal Local Mucosal Tolerability and Characterization of Methanolic Fraction of Flueggea virosa Root-Bark in Female Rabbits

Background Flueggea virosa (Roxb. ex Willd.) Royle is used in East Africa for traditional fertility regulation. Its methanolic root-bark fraction (KBLM) has demonstrated sperm-disrupting and contraceptive activity. This study aimed to evaluate the intravaginal tolerability of KBLM in female rabbits following 21 days repeated-dose and delineate its chemical composition to inform the viability of its development as a non-hormonal contraceptive. Materials and methods Twenty-five female New Zealand White rabbits were randomized (n = 5/group) to saline, nonoxynol-9 (20 mg/mL), and KBLM (3.9, 7.8, or 15.6 mg/mL) and dosed intravaginally once daily for 21 days. Clinical observations and histopathology of the cervicovaginal tissue, uterus, ovaries, liver, and spleen were determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data for KBLM and the ethyl acetate fraction (KBLE) were utilized for molecular networking and in silico annotation. Results Nonoxynol-9 caused marked epithelial injury, whereas KBLM triggered predominantly focal, minimal-to-mild inflammatory changes. No definitive KBLM-related destructive lesion pattern was identified in the uterus, ovaries, liver, or spleen. Median cervicovaginal composite lesion scores were 1 (range, 0–3) for saline, 9 (6–11) for nonoxynol-9, and 3 (1–6), 3 (2–5), and 4 (4–7) for KBLM at 3.9, 7.8, and 15.6 mg/mL, respectively (p = 0.002). LC-MS/MS yielded 183 blank-corrected features and identified five flavonol-glycoside-rich profile chemical markers. Conclusions KBLM demonstrated less mucosal integrity damage than nonoxynol-9 but low-grade cervicovaginal inflammation at all tested concentrations precluded establishment of a local no-observed-effect concentration. The 3.9 mg/mL concentration represents the most conservative starting point for confirmatory studies. The chemical profile provides candidate markers for standardization and further toxicological evaluation.