Papain enzyme possesses wound healing, anti-inflammatory, and anti-fungal activity. Chitosan has topical wound healing activity, and its activity would be improved when combined with papain enzyme. In the presented study, the chitosan hydrogel containing papain was prepared and applied to wounded skin to check its anti-inflammatory activity. The hydrogel was prepared using chitosan in variable concentrations (1-5%), and papain (0.5%) was added. Glacial acetic acid was used to solubilize the chitosan. The topical gel was evaluated for appearance/clarity, pH, viscosity, spreadability, bio adhesive strength, and tube extrudability. Batch CP8 containing 5% chitosan and 0.5% papain showed the highest spreadability, adhesive strength, and extrudability and hence was selected for in vivo study. A wound healing study was carried out on albino mice by creating wounds by excision, incision, and burn. The treatment of mice was started by applying topical gel till the wound healed. The progressive changes in wound area were measured in mm2 by tracing wound boundaries on transparent paper at every 4-day interval. It was observed that when the concentration of polymer was increased, spreadability and adhesive strength were increased. Prepared hydrogels showed pseudoplastic behavior during viscosity determination using Brookfield viscometer. Amongst all the studied formulations, batch CP8 showed good gelling ability, pH in range, excellent spreadability, good adhesive strength, extrudability and was stable at temperatures 5±3 °C and 25±3 °C during three months stability study. After applying the gel over the wound at the end of 16 days of study, it was observed that batch CP8 healed 99.77% wound, which was higher as compared to a positive control (standard drug- salicylic acid), which covered 98.8% of the wound, Plain Gel covered 87.10% wound, and negative control (without gel) covered 84.70% wound. From the experimental evidence of in-vitro studies, it was observed that papain enzyme is a promising alternative to conventional drug therapy; it showed significant wound healing activity. The developed herbal formulation was stable, safe, and effective over synthetic formulations for the treatment of wound healing activity.
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