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The effects of Anthyllis Vulneraria extract as an adjuvant in wound healing First author: Iova Olga-Maria (1) Co-Authors: Marin Gheorghe Eduard (2), Pintilie Sebastian Romeo (1) Scientific Coordinator: Prof. Filip Adriana Gabriela MD PhD (3) 1: 5th medical student, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania 2: 6th medical student, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania 3: University Professor, Department of Anatomy and Embryology, University of Medicine and Pharmacy “Iuliu Hatieganu” ABSTRACT INTRODUCTION AND AIMS Anthyllis vulneraria, a traditional medicinal plant, was evaluated for its burn Anthyllis Vulneraria (AV) is a native Romanian plant with traditional use in wound-healing potential and antioxidant properties in a rat model. Thirty-two treating various conditions (e.g., internal diseases, acne, skin wounds). Wistar rats were treated with topical creams containing either 1 mg/cm² or 2 Previous research suggests AV has antioxidant and anti-inflammatory mg/cm² polyphenols extracted from A. vulneraria leaves. Wound progression, effects in vitro, but no in vivo studies have been conducted. oxidative stress markers, and histological samples were analyzed over 14 Study objective: Evaluate the healing capacity of AV extract in an animal days. The 1 mg/cm² cream significantly improved healing by day 5, while the (burn) model. 2 mg/cm² dose showed enhanced effects by day 7, correlating with increased Also aimed to measure antioxidant capacity and analyze the polyphenolic antioxidant enzyme activity and reduced oxidative stress. LC-MS/MS analysis composition of AV leaf extract. confirmed the presence of polyphenols such as hyperoside, epigallocatechin Investigation included macroscopic, microscopic, and biochemical gallate, and ferulic acid. These findings suggest A. vulneraria extract changes in the burn model treated with AV cream. promotes wound healing primarily through its antioxidant effects and may The phytochemical composition of the plant was reassessed during the serve as a natural therapeutic adjuvant in burn treatment. study. MATERIALS AND METHODS Wound images captured on days 0, 3, 5, 7, 10, and 14 Sample collection: Subjects: 32 female Wistar albino rats randomly divided into 4 groups (n=8): Group 1: Negative control (NC) – topical vehicle cream On day 14, rats were euthanized under general anesthesia Group 2: Positive control (PC) – silver sulfadiazine cream Skin samples collected for oxidative stress evaluation Biochemical markers analyzed: Group 3: Experimental Group 1 (EG1) – 1 mg polyphenol/cm² AV cream Group 4: Experimental Group 2 (EG2) – 2 mg polyphenol/cm² AV cream Antioxidant enzymes: GSH, SOD, GPX, CAT Procedure: Oxidative stress markers: GSSG, MDA Chemical composition analysis: Day 0: Standardized full-thickness burn wounds created Topical treatments applied daily for 14 days Performed LC-MS/MS analysis on AV leaf extract RESUL TS # # ** # * * Healed area (HAx = AAx - AA0) compared across groups using One-Way ANOVA Significant differences in healing observed on: Day 5 (p = 0.001)and day 7 (p = 0.018) No significance on days 3, 10, or 14 Day 5 findings: EG1 showed greatest healing, outperforming both controls ^ Figure 2. Wound healing activity of AV extract creams, measured by the wound surface area reduction (HA value), on the 5th and 7th vs. NC (p < 0.001) / PC (p = 0.007) days of treatment. The box plot compares four groups: NC (negative control), PC (positive control), EG1 (experimental group 1), and EG2 EG2 also outperformed controls (experimental group 2). * p < 0.05 and ** p < 0.001, all groups vs. NC; # p < 0.05 vs. PC vs. NC (p=0.002) / PC (p = 0.038) Day 7 findings: > Figure 3. Oxidative stress markers levels in EG1 remained significantly better than both different treatment ** ** controls groups: NC (Negative ** vs. NC (p= 0.003) / PC (p = 0.012) Control),PC (Positive Control), EG1 No group differences after day 7, indicating AV (Experimental Group 1), cream’s main benefit is in early (inflammatory and EG2 (Experimental phase) healing Group 2) for the following Oxidative stress markers: parameters: MDA, CAT, Significant improvement in EG2: GSH, GSSG, GPX, and Highest levels of GSH, SOD, GPX SOD . * p < 0.05 and ** p < * 0.001, all groups vs. NC * Lowest levels of MDA, GSSG AV’s healing effect likely due to polyphenolic compounds, identified through LC-MS/MS method ** ** ** ** ^ Figure 1. The UV chromatogram showing the quantification of individual polyphenols in AV leaves, obtained using the LC-MS analytical method. Legend: 1 – gentisic acid; 2 – p-coumaric acid; 3 – ferulic acid; 4 – hyperoside. CONCLUSION REFERENCES In summary, AV extract possesses in vivo healing capacities, confirming possible supposition from previous in vitro studies as well as from traditional medical practices. Its capacities could possibly stem from it’s particular antioxidant capacity, proved through biochemical and LC/MS analysis. This research direction can surely be further explored, as bigger studies are necessary to fully assess the potential pharmaceutical uses of A. Vulneraria as a healing adjuvant. With the dire need for natural alternatives rising, AV could prove to be an attractive potential candidate for medical care, being accessible, easy to cultivate and grow, and remarkably polyphenol rich.