# The Effects of _Anthyllis vulneraria_ Hydroalcoholic Leaf Extract as an Adjuvant in Wound Healing
## 2 Materials and Methods
### Methodology of Experimental Animal Study  
#### 2.5.1 Animal In Vivo Wound Healing  
The study was approved by the Ethics Committee of the Iuliu Hatieganu University of Medicine and Pharmacy in Cluj-Napoca and the Veterinary Health Directorate of Romania, in accordance with all applicable laws. (Authorization No. 353 of 13 February 2023). A total of 32 female Wistar Albino rats, each weighing between 200 and 250 g, were included in the study. The animals were housed under standard laboratory conditions, with a controlled temperature of 21 \(\pm\) 2 \({}^{\circ}\)C and humidity maintained at 65 \(\pm\) 4%. A 12-h light/dark cycle was followed, and both food and water were provided _ad libitum_. After the randomization in the groups, the rats were housed together in spacious cages, depending on their group. Based on similar works in the literature [43; 44], an effect size between 0.47 and 0.99 was calculated based on the first recording of the studies. We chose a more conservative effect size of 0.65. Power analysis was performed using G*Power version 3.1.9.7 [45] in order to determine the minimum sample size required. With an effect size of 0.65, an \(\alpha\)-error rate of 0.05, a power of 0.8, the total minimum sample size for a four-group one-way ANOVA test was computed to be 32 (8 rats per group). Using the experimental data collected, we validated an effect size of 0.82 based on the first HA values. A sample size of \(n=8\) per group is in line with similar in vivo wound-healing studies [43; 46; 47; 48], where \(n=5\)-10 per group was sufficient to detect treatment effects. The group size represents a compromise between statistical robustness and ethical considerations according to the 3 Rs principle (Replacement, Reduction, and Refinement) [49]. Before the start of the experiment, on day 0, a skin burn was created by applying a metal heated plate with a diameter of 2.2 cm on the dorsal side of the animals, to create a standard burn with the average area of 2 cm\({}^{2}\). To minimize negative side effects, the skin was cleaned before the procedure and anesthetic was administered by intraperitoneal injection of a cocktail made from ketamine (90 mg/kg) and xylazine (10 mg/kg). The burns were deemed to be full-thickness skin burns (Figure 2). Following this procedure, the animals were randomized and divided into 4 groups, with 8 animals per group as follows:  
* Group 1 served as the negative control (NC) and received daily topical applications of base cream on the lesion surface;
* Group 2 served as the positive control (PC) and received daily topical applications of silver sulfadiazine on the lesion surface;
* Group 3, the experimental group 1 (EG1), received daily topical applications of 1 mg/cm\({}^{2}\) polyphenols cream of _A. vulneraria_;
* Group 4, the experimental group 2 (EG2), received daily topical applications of 2 mg/cm\({}^{2}\) polyphenols cream of _A. vulneraria_. The animals received daily topical application of about 0.1 g cream/cm\({}^{2}\). The procedure was carried out in the morning, for 14 days. Figure 2: Typical macroscopic aspect of the standardized burn wound. To evaluate the healing rate, the appearance, size, and progression of the wounds were monitored and photographed on days 0, 3, 5, 7, 10, and 14. On the 7 th day, four randomized rats from each group were selected and euthanized to collect skin biopsies and blood samples for the evaluation of the healing process, which was conducted by histopathological and biochemical analysis of the oxidative stress markers and IL-8 levels. The remaining rats were euthanized on the 14 th day and the same procedures were conducted. Skin biopsies used for microscopic analysis were collected from the center of the wound, which consisted of healthy, regenerated, and harmed tissue. The euthanasia of the animals was carried out by using an overdose of anesthesia with xylazine (10 mg/kg) and ketamine (90 mg/kg).