Investigation of the effects of rosmarinic acid on wound healing

Objective: This study aimed to investigate the effects of a bioactive composite gel system, formed by combining rosmarinic acid and chitosan in a specific formulation, on wound healing. Materials and Methods: The study material consisted of twenty-seven female Sprague Dawley rats, aged 20–24 weeks and weighing between 210–380 g, obtained from the Experimental Animal Unit of Aydın Adnan Menderes University Faculty of Veterinary Medicine. Two full-thickness wounds were created on the dorsal region of each rat, resulting in a total of 54 wounds examined. Considering the physiological process of wound healing, the study duration was determined as 6 days. The experimental animals were divided into three groups. In the first group, rosmarinic acid – chitosan mixture (RAC) was applied to the left wound area, while chitosan was applied to the right wound area. In the second group, RAC gel was applied to the left wound area, and the right wound area was left untreated as the control. In the third group, chitosan was applied to the left wound area, while the right wound area served as the control. All rats were anesthetized intraperitoneally (IP) with a combination of 10 mg/kg xylazine and 70 mg/kg ketamine. Following anesthesia, the dorsal area was shaved and prepared under aseptic conditions, and symmetrical full-thickness wounds were created using a 6 mm punch biopsy instrument. To prevent measurement errors caused by skin contraction, fixation sutures were placed around the wound margins. Photographs of the wound areas were taken immediately after wound creation and at predetermined time points. The obtained images were transferred to the ImageJ software, and wound areas were measured using the polygon method and recorded in mm². For histopathological evaluation, skin samples collected from the wound regions on days 3 and 6 were fixed in formaldehyde, embedded in paraffin, sectioned at 4–5 µm thickness, stained with Hematoxylin–Eosin (H&E), and examined under a light microscope. Statistical analyses were performed using repeated measures analysis of variance to evaluate intergroup and time-dependent changes; assumptions of normal distribution and sphericity were verified with appropriate tests, and post-hoc analyses were conducted to determine significant differences. Results: Minimal erythema and edema were observed in the wound areas during the early period, which were considered consistent with the physiological inflammatory phase of wound healing. Throughout the topical treatment period, no behavioral changes indicating significant discomfort, pain, or severe complications were observed in any experimental animal. Macroscopic evaluations revealed distinct differences among the groups during the inflammatory and proliferative phases of wound healing. In the control group, exudation, discoloration, and enlargement of the wound area were observed. In the chitosan gel-treated group, limited wound contraction and a decrease in closure rate during the later stages were identified. In contrast, no signs of infection or significant exudation were detected in the RAC gel-treated group; furthermore, early and organized granulation tissue formation was observed, and wound areas showed a tendency for faster closure beginning from the second day of the study. Conclusion: Rosmarinic acid was observed to accelerate wound healing and positively influence the stages of the healing process. The use of rosmarinic acid in gel form may represent a promising therapeutic option for wound healing. Keywords: Rosemary, Biopolymer, Chitin, Regeneration