Phytochemical Validation and Wound Healing Efficacy of a Polyherbal Gel using HPLC and Zebrafish Models

Background: Wound healing is a multifaceted physiological process involving inflammation, tissue regeneration and remodeling. Silver sulfadiazine remains a conventional topical treatment; its clinical use is often limited by delayed epithelialization and cytotoxicity. Medicinal plants, rich in diverse bioa ctive compounds, are recognized as safer alternatives for wound management. Hence, this study aimed to formulate a standardized polyherbal gel using Trigonella foenum-graecum, Lawsonia inermis, Basella alba, Peristrophe paniculata and Portulaca oleracea and to evaluate its phytochemical composition and wound-healing efficacy. Methods: A Carbopol-based gel incorporating hydroalcoholic extracts of the selected plants was developed. Highperformance liquid chromatography (HPLC) was employed to identify and quantify key phytoconstituents, protocatechuic acid, syringic acid, kaempferol, apigenin and diosmetin. In vivo evaluation was conducted using zebrafish (Danio rerio) wound models, including superficial mechanical injury and tail fin transection, with wound closure assessed for seven days. Histopathological analyses were also performed. Results: HPLC analysis confirmed the stability and quantifiable presence of bioactive phytochemicals, with excellent linearity (R² > 0.999). The polyherbal gel demonstrated significant wound-healing activity, with wound closure rates of 88.7% ± 2.7 in mechanical injury and 94.5% ± 2.1 in tail fin transection by Day 7, outperforming untreated controls and exhibiting superior efficacy to 1% silver sulfadiazine. Histopathological assessment revealed organized re-epithelialization, restored fin structure and reduced inflammation in gel-treated groups. Conclusion: The formulated polyherbal gel offers a safe, biocompatible and efficacious topical therapy for wound healing. Its synergistic phytoconstituents contribute to enhanced regeneration, supporting its potential for further preclinical and clinical translation in wound care.