The Effects of Anthyllis vulneraria Hydroalcoholic Leaf Extract as an Adjuvant in Wound Healing

Featured Application

Our study serves as a potential future steppingstone for in vivo studies, as well as for pharmacological and pharmaceutical applications of Anthyllis vulneraria extract cream as a potential wound adjuvant agent.

Abstract

Anthyllis vulneraria is a traditional medicinal plant with confirmed anti-inflammatory properties, attributed to its high polyphenolic content. This study aimed to evaluate the wound-healing potential of A. vulneraria leaf extract in a rat burn model. Four groups of eight Wistar rats each received the following daily topical applications for 14 days: vehicle cream (negative control); silver sulfadiazine (positive control); or plant-based creams containing either 1 mg/cm² or 2 mg/cm² of polyphenols (experimental groups 1 and 2, respectively). On days 7 and 14, four animals per group were euthanized for histological and oxidative stress evaluations. LC-MS/MS analysis of the leaf extract identified hyperoside, ferulic acid, and p-coumaric acid as major constituents. Experimental group 1 showed significantly enhanced wound closure on days 5 and 7, while group 2 exhibited a significant effect on day 5. All oxidative stress markers, except catalase activity, differed significantly among the groups, with the most favorable results observed in group 2. IL-8 levels decreased after the extract treatment, while no significant microscopic changes were observed. These results indicate that A. vulneraria leaf extract may serve as a valuable adjuvant in burn wound healing.

1. Introduction

Recent years have seen an increased interest in finding and exploiting the anti-inflammatory, anti-microbial, and anti-oxidative properties of medicinal plants as alternatives to synthetic compounds. Naturally occurring substances, such as phenolic components, proteins, minerals, and vitamins, found in both the leaves and inflorescences of plants, are becoming attractive to the pharmaceutical, cosmetic, and food industries [1,2].

Natural compounds, particularly polyphenols and flavonoids, have been extensively documented through both in vitro and in vivo studies and have been shown to exhibit a range of beneficial biochemical activities, including anti-inflammatory, antitumoral, immunomodulatory, wound healing, and anti-aging effects. Their popularity has increased over the past few years as they have become attractive therapies for debilitating diseases such as cardiac, metabolic, neurodegenerative, hepatic, and dermatological diseases [3,4,5,6,7].

A burn is a generic term, indicating a skin or other tissue injury caused by exposure to heat, chemicals, friction, radiation, or electricity [8]. Burn injuries pose a major contemporary health problem, both because of their economic burden, as well as their effect on the quality of life of patients. They are amongst the most frequent forms of wounds and a primary cause for hospital presentation. Every year, countless patients acquire and suffer from different forms of such afflictions, which increase their overall mortality and morbidity, while also affecting and overloading the health system [9]. It is estimated by the WHO that more than 180 000 people die annually from burn or burn-related injuries, while the survivors face prolonged hospitalization, infections, as well as chronic complications such as disfigurement, disability, or stigmatization [10].

Burn pathophysiology and healing are complex processes that involve multiple cellular and molecular mechanisms in three phases: inflammation, proliferation, and remodeling. Each phase must be correctly carried out to ensure proper healing and to avoid acute or chronic complications [11].

The inflammatory phase is characterized by vasodilatation and a cascade of inflammatory factors. In the first 1–5 days post injury, neutrophils, monocytes, and macrophages are chemoactively attracted to the wounded site and secrete proinflammatory mediators and free oxygen radicals [11]. The increased quantity of substances, like oxygen radical species, in parallel with decreased quantities of antioxidant enzymes, easily create an imbalance in oxidative stress [1]. Therefore, substances that can tip the balance towards an anti-inflammatory response could help to reduce the negative effects of inflammation, providing a better environment for both molecular and structural healing [8].

The proliferative phase is characterized by the processes of angiogenesis, collagen formation and deposition, epithelization and wound contraction, which usually starts on the 3th or 4th day [8,12]. In the case of superficial burns, healing starts from the still-present epithelial structures, such as hair follicles, and it usually resolves without scarring. For deep burns, as the epithelial adjacent structures are destroyed, healing requires the migration of keratinocytes from the surrounding healthy skin, as well as the contraction of the wound site, carried out by fibroblasts, and usually leads to scarring [8,11].

Finally, the remodeling phase usually starts after 3–4 weeks, and, depending on the wound’s severity, can last for years. The key cell player in this phase is the fibroblast. Temporary structures are destroyed and replaced with permanent structures like collagen type 1. The disruption of this phase leads to scar formation or chronic wounds [8,13].

Consequently, the identification of more effective and cost-efficient therapeutic options for burn treatment remains a priority. In this context, the search for alternatives to synthetic active compounds has increasingly focused on bioactive substances derived from the plant kingdom [1,14]. Among these, polyphenols represent promising candidates due to their potential to offer sustainable, health-promoting therapies and to serve as valuable adjuvants or alternatives to conventional treatments.

Anthyllis vulneraria (A. vulneraria) is a herbaceous plant from the Fabaceae family found throughout the Mediterranean basin and most of Europe [15,16]. Its name can be translated as: Anthyllis from the Greek words “Anthos”+”ioulos”, meaning “down inflorescence”, describing the orientation of the calyx, and “vulneraria” from the Latin “vulnerus”, meaning wound, describing the plant’s healing properties [17]. Traditionally, A. vulneraria has been employed to treat skin injuries, cardiovascular diseases, inflammation, and acne [2,16]. The flowers of A. vulneraria have been most used, followed by the aerial parts and leaves. The plant has been administered in various forms, such as ointments, hot infusions, and freshly pressed juice, and is also applied directly to wounds by bathing, rinsing, or as dressings [18]. Due to its ease of cultivation and adaptability across a wide range of habitats, A. vulneraria holds promise as a viable candidate for medico-pharmaceutical use, based on the scientific validation of its therapeutic efficacy [2,19].

Several previous studies have investigated the phytochemical composition of A. vulneraria extracts, reporting high concentrations of flavonoids, saponins, tannins, and carotenoids, bioactive compounds known for their significant antioxidant activity [2,15,16,17,20,21,22]. Additionally, antimicrobial and antiviral properties have been documented [17,20,23], along with in vitro wound-healing effects demonstrated in cell culture models [20,24]. These findings support the therapeutic potential of A. vulneraria in wound care and underscore the need for further validation through in vivo studies.

Morphologically, the species forms basal leaf rosettes, while its inflorescences consist of compact flowerheads containing between four and over twenty individual flowers [24]. A. vulneraria typically begins flowering in its second year of growth, although it may be delayed for up to eight years under certain ecological conditions, which limit the timely availability of floral biomass [25]. Moreover, most plants are monocarpic; however, some of them may produce flowers more than once [24]. Therefore, the traditional use of leaves, together with the plant’s flowering cycle, makes the leaf material more favorable for use.

The objectives of this study were to evaluate the wound-healing potential of an A. vulneraria leaf extract using a rat burn model, by quantifying wound surface area reduction, assessing histopathological changes, and analyzing oxidative stress markers and IL-8 levels in both serum and skin tissue. In parallel, the phytochemical composition of the leaf extract was characterized and a topical cream formulation was developed for use in in vivo experiments.

2. Materials and Methods

2.1. Chemicals

Folin–Ciocâlteu reagent, calcium carbonate, ethanol, petrolatum, paraffin oil, cetyl alcohol, and sodium laurylsulfate were obtained from Merck (Darmstadt, Germany). All solvents were of HPLC grade and each of the used reagents was of analytical grade. The quality of the water was Milli-Q-quality. Reagents included Trolox (250.29 g/mol) as the antioxidant standard, ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidized with H₂O₂ in acetate buffer (30 mM, pH 3.6; 0.4 M, pH 5.8), and DPPH (2,2-diphenyl-1-picrylhydrazyl) prepared in methanol (1 mg/mL, diluted to 40 µg/mL For the flavonoid analysis, a solution of AlCl₃ (20 mg/mL) in 5% acetic acid was prepared and mixed with ethanol in a 3:1 (v/v) ratio. A 50:50 ethanol–water solvent was used for plant extraction. Silver sulfadiazine was used as a dermatological positive control. The excipient cream was created using petrolatum, cetyl alcohol, and paraffin oil.

2.2. Plant Source

A. vulneraria seeds were acquired from an Internet gardening shop in Germany (https://www.jelitto.com/Seed/Perennials/ANTHYLLIS+vulneraria+var+coccinea+Portion+s.html, accessed on 24 October 2022).

Both indoor and outdoor plantings were used to grow the plant material. Seeds were planted in February–March of 2023. Indoor plants were grown in 20 × 50 cm planter boxes with drainage, with 25 cm of soil, watered at a regular interval, under natural light. The soil used was peat-based potting soil (black peat, sphagnum peat, composted fertilizer of bovine origin, clay) with 40% minimum organic material with an NPK of 0.3:0.1:0.1. Watering was conducted when the topsoil was dry, approximately once a week during the spring months, and once every three days in the summer months.

The outdoor plants were cultivated in open-field conditions in the southern region of Romania. They were exposed to natural light and watered at regular intervals to prevent excessive soil dry-out. For the soil, based on the geographic location (approximately 45.14° latitude, 24.38° longitude) and soil characteristics, the soil was determined to be eutric Cambisol (palebrown, ochrA) based on the SRCS–extended WRB description [26]. Watering was conducted on a similar schedule to the indoor conditions.

During the first year of vegetative growth, the plants developed only foliar structures, with no evidence of inflorescence formation. Plant material, namely leaves, was collected at regular intervals (every 3–4 weeks, depending on the growth rate) from both indoor and outdoor-grown plants. The harvested plant material was weighed and air-dried in a cool, dry environment, protected from direct light, until a constant mass was achieved. Once dried, the material was transferred to dry paper bags, labeled according to lot type (indoor or outdoor) and collection date, and stored under appropriate conditions until the beginning of the study.

All plant material used in this study was certified as A. vulneraria by Ana-Maria Vlase, Assistant Professor PhD, from the Department of Pharmaceutical Botany, University of Medine and Pharmacy “Iuliu Hațieganu” Cluj-Napoca, Romania. A voucher specimen (58.16.1.1.2) was deposited in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy at “Iuliu Hațieganu” University of Medicine and Pharmacy.

It is noteworthy to mention the marked difference in leaf size observed between the A. vulneraria specimens cultivated under controlled indoor and outdoor conditions and wild populations collected from local mountain regions, where the species spontaneously grows. Both the indoor and outdoor plants displayed more well-developed leaves compared to their wild counterparts. As illustrated in Figure 1, these differences can probably be attributed to increased nutrient availability and warmer growing conditions in the cultivation environments than in the natural habitat.

2.3. Obtaining and Characterization of the Vegetal Extract

2.3.1. Leaf Extract Preparation

To prepare the leaf extract, an equal mixture of dried leaves collected from both indoor- and outdoor-grown plants was used. These leaves were finely ground into powder using a mechanical grinder (Argis, RC-21, Electroargeş SA, Curtea de Argeş, Romania) to produce a uniform dried leaf powder. The resulting material was then stored in an opaque, airtight container until needed for further extraction.

For extracts intended for use in the animal study, preparation methods were adapted from established protocols for A. vulneraria described in previous studies in the literature [2,16,20]. Specifically, 5 g of the powdered leaves were combined with 50 mL of a 1:1 ethanol–water solution (50% v/v). The mixture was subjected to homogenization using an Ultra-Turrax device (T 18; IKA Labortechnik, Staufen, Germany) operated at 4000 rpm for a duration of 5 min. Following homogenization, the mixture underwent centrifugation at 10,000 rpm for 10 min using a bench-top centrifuge (Hettich, Micro 22R, Andreas Hettich GmbH & Co., Tuttlingen, Germany). The resulting supernatant was collected and used for various analyses, including spectrophotometric measurements of total polyphenol content (TPC), total flavonoid content (TFC), antioxidant capacity (ABTS and DPPH assays), and phytochemical profiling via LC-MS/MS.

Ethanol was subsequently removed from the hydroalcoholic extract using a rotary evaporator maintained at 50 °C (Hei-VAP, Heidolph Instruments GmbH & Co., Schwabach, Germany). The remaining aqueous extract was transferred into amber vials and subjected to freeze-drying. The samples were initially frozen at −55 °C for 24 h, followed by lyophilization under 200 mTorr pressure at a set temperature of −25 °C for an additional 48 h using a freeze dryer (SP Scientific Virtis AdVantage 2.0 BenchTop Freeze Dryer/Lyophilizer, Model Advantage Plus EL-85, American Laboratory Trading Inc., East Lyme, CT, USA). The final lyophilized plant extract was stored at ambient room temperature until further analysis.

2.3.2. Total Phenolic and Total Flavonoid Content of A. vulneraria Leaves

TPC was determined using the Folin–Ciocâlteu spectrophotometric method, as previously described [27,28]. A total of 5 g of dried leaf powder was mixed with the extraction solvent (50 mL, 50% ethanol—50% water), followed by ultra-turrax extraction at 4000 rpm for 5 min and centrifugation at 10,000 RPM for 10 min.

Sample analysis was carried out using a Synergy HT multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) and 96-well microplates. For each reaction, 60 μL of the extract was mixed with 270 μL of a 6% (w/v) sodium carbonate solution. Subsequently, 270 μL of diluted Folin–Ciocâlteu reagent (1:10 dilution) was added. The mixtures were incubated at ambient temperature in the absence of light for 30 min. Absorbance measurements were taken at 760 nm, using a solvent blank as reference. The total polyphenol content (TPC) was expressed in milligrams of gallic acid equivalents (GAE) per milliliter of extract. A standard calibration curve was generated with gallic acid across concentrations ranging from 10 to 100 μg/mL (equation: y = 0.0053x + 0.059, R² = 0.9901).

TFC was determined through a modified version of Pinacho et al.’s method [29]. The leaf extract was made using the same parameters as previously described, with 5 g of dried leaf powder mixed into 50 mL of extraction solvent (50% ethanol—50% water), followed by ultra-turrax extraction at 4000 rpm for 5 min and centrifugation at 10,000 RPM for 10 min.

A volume of 400 mL of aluminum chloride solution (AlCl₃, 20 mg/mL) prepared in 5% acetic acid and ethanol (3:1, v/v) was added to 200 mL of plant extract. The absorbance was measured at 420 nm using a microplate reader. Quercetin was used as the reference standard for calibration. The flavonoid content was initially calculated as millimolar quercetin equivalents (mM QE) and later converted to milligrams of quercetin equivalents per gram of dry plant material (mg QE/g). The calibration curve was established using quercetin across a concentration range of 20–150 μM (y = 0.0015x + 0.0666, R² = 0.9894).

2.3.3. Total Antioxidant Capacity (TAC)

The plant extract was made using the parameters previously described, with 5 g dried leaf powder mixed into 50 mL extraction solvent (50% ethanol—50% water), followed by ultra-turrax extraction at 4000 rpm for 5 min and centrifugation at 10,000 RPM for 10 min. The results were expressed as mM Trolox/g of vegetal product.

Assessment of ABTS Radical Scavenging Activity

TAC levels were measured according to a validated method previously described by Erel [30]. The assay is based on the capacity of antioxidants in the solution to decolorize the blue green ABTS according to their concentrations and antioxidant capacities. For the calibration curve, Trolox, a water-soluble analogue of vitamin E widely used as a traditional standard for TAC measurement assays, was used.

The plant extract was diluted to 1:200 before use. To prepare the reaction mixture, 200 µL of 0.4 M acetate buffer (pH 5.8) was combined with 20 µL of ABTS⁺ solution (30 mM in acetate buffer, pH 3.6), followed by the addition of 12.5 µL of the diluted plant extract. The calibration curve was conducted using Trolox as the reference standard, with a concentration range of 0.05–1 μM (y = 0.264x + 0.0048, R² = 0.9972).

Evaluation of DPPH Radical-Scavenging Capacity

The plant extract was diluted to 1:100 before testing. A DPPH stock solution (1 mg/mL) was prepared in methanol and then diluted to a working concentration of 40 µg/mL. For the assay, 0.8 mL of the DPPH working solution was mixed with 0.2 mL of the diluted extract. The mixture was incubated at 40 °C in the dark for 30 min, after which the absorbance was recorded at 517 nm. The antioxidant activity was assessed by comparing the absorbance difference relative to the DPPH control solution (A = A_control − A_sample). A calibration curve was constructed using DPPH standards within a concentration range of 2.68 to 67 μg/mL (equation: y = 0.0032x + 0.2271, R² = 0.9654).

2.3.4. Methodology of Phytochemical Analysis by LC-MS/MS

The chemical composition of the plant extracts was analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), based on two validated protocols described in previous studies [31,32]. All measurements were performed on an Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA), connected to an Ion Trap SL 1100 mass spectrometer (LC/MSD Ion Trap VL) [33]. The setup included a binary pump, autosampler, column oven, UV detector, and degasser.

The first method targeted 23 polyphenolic compounds. Chromatographic separation was carried out using a Zorbax SB-C18 column (100 mm × 3.0 mm, 3.5 µm particle size, Agilent). The mobile phase consisted of methanol and 0.1% acetic acid (v/v) and was run in gradient mode starting at 5% methanol, ramping up to 42% over 35 min, held for 3 min, then brought back to 5% over 7 min [31,32,33]. The column temperature was maintained at 48 °C, flow rate at 1 mL/min, and injection volume was 5 µL.

Detection was conducted in both UV and MS modes. The UV detector was set at 330 nm for the phenolic acids (first 17 min), and at 370 nm for the flavonoids and aglycones (up to 38 min). The MS operated in negative electrospray ionization mode (ESI) with a capillary voltage of +3000 V, nitrogen at 60 psi as the nebulizer gas, and a drying gas flow of 12 L/min at 360 °C [34,35].

The second method focused on eight specific polyphenols: catechin, epicatechin, gallic acid, syringic acid, vanillic acid, protocatechuic acid, epigallocatechin, and epigallocatechin gallate [36]. The same hardware and column were used with a different gradient, starting at 3% methanol, increased to 8% at 3 min, and to 20% at 8.5 min, held until minute 10, and then returned to 3% [37]. All compounds were monitored using extracted ion chromatograms (EICs) under the same ESI conditions. In order to keep the manuscript concise, we have not displayed all EIC traces, following common reporting practices.

Identification of the compounds was based on matching retention times and MS spectra with those of the reference standards. Quantification was performed via UV absorbance using calibration curves prepared from the corresponding standards. Data were processed using ChemStation (vB01.03) and DataAnalysis (v5.3) software (Agilent Technologies, Santa Clara, CA, USA) [31,32,33,35,36]. Results are reported as micrograms of compound per milliliter of extract.

2.4. Cream Preparation

A hydrophilic cream with a 70% water content was prepared as a negative control, referred to as the base cream. The lipophilic components were petrolatum, cetyl alcohol, and paraffin oil. In this cream, in the aqueous phase, a lyophilized extract powder was incorporated. This lyophilized extract powder was created by using 100 g of dried leaf powder and applying the above-mentioned extraction method. The extract was concentrated by evaporation of the solvent in a rotary evaporator coupled with a vacuum pump, thus resulting in the lyophilized extract.

Two formulations of active creams were prepared by incorporating the lyophilized A. vulneraria leaf extract into a hydrophilic cream base to achieve final concentrations of 1% and 2% total polyphenols, corresponding to 1 mg and 2 mg of total polyphenols per 0.1 g of cream, respectively. These concentrations were determined based on the total polyphenol content (TPC) of the extract prior to lyophilization and were calculated to deliver 1 mg/cm² and 2 mg/cm² of total polyphenols upon topical application. A commercially available silver sulfadiazine cream (Dermazin^®, Lek Pharmaceutical and Chemical Company in Ljubljana, Slovenia), obtained from a local pharmacy, was used as the positive control in the animal experimental model.

The two extract concentrations (1 mg/cm² and 2 mg/cm²) were selected based on prior studies in the literature and the experience of our research group. Previous experiments showed that low concentrations of polyphenols tend to exert antioxidant and anti-inflammatory effects, while higher concentrations may exhibit pro-oxidant and potentially cytotoxic properties [38,39]. Our choice was therefore aimed at balancing efficacy with safety, which was also supported by findings from other groups, further reinforcing the therapeutic window of polyphenolic doses in skin models [40,41,42]. Moreover, A. vulneraria extract has been shown to exert dose-dependent cytotoxicity in vitro [20], supporting the need for cautious dose selection. We acknowledge that a dose–response curve experiment would have brought more information; however, due to ethical and logistical concerns, we chose only two concentrations.

2.5. Methodology of Experimental Animal Study

2.5.1. Animal In Vivo Wound Healing

The study was approved by the Ethics Committee of the Iuliu Hațieganu 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 ± 2 °C and humidity maintained at 65 ± 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 α-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 3Rs 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 cm². 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:

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Group 1 served as the negative control (NC) and received daily topical applications of base cream on the lesion surface;

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Group 2 served as the positive control (PC) and received daily topical applications of silver sulfadiazine on the lesion surface;

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Group 3, the experimental group 1 (EG1), received daily topical applications of 1 mg/cm² polyphenols cream of A. vulneraria;

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Group 4, the experimental group 2 (EG2), received daily topical applications of 2 mg/cm² polyphenols cream of A. vulneraria.

The animals received daily topical application of about 0.1 g cream/cm². The procedure was carried out in the morning, for 14 days.

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 7th 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 14th 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).

Exclusion criteria for the animals were as follows: visible wound infections, allergic reactions to the cream, self-inflicted wound damage, signs of distress or suffering, excessive weight loss, death before endpoint, or housing condition violations.

Throughout the experiment, there were no side effects, as the experimental extract cream was well tolerated by the animals, with no signs of restlessness, aggressiveness or irritation. Therefore, there were no excluded animals throughout the experiment.

Group allocation and treatment administration during the animal experiment were carried out by Mr. Remus Moldovan, the animal care technician. Separate researchers from our group, who were not involved in the experimental procedures, performed the outcome assessment and data analysis. This approach was used to reduce bias in interpreting the results.

2.5.2. Methodology of Wound Contraction Assessment

Assessment of Wound Contraction

On days 0, 3, 5, 7, 10, and 14 of the experiment, top-down pictures of the wounds were taken, utilizing a 12.1 effective megapixels camera affixed above the animal handling area; a tripod was used for stability and to ensure similar fields of view for all pictures.

Wound areas were calculated by two independent investigators using ImajeJ software, version 1.54g, by automatic color analysis of the images, with manual intervention and demarcation of the areas in cases of aberrant detection, with a precision of ±0.01 cm².

Given the imprecision of the burn induction process, in order to ensure consistency between the animals, a healed area (HA) was calculated for each assessment by subtracting from the actual area (AA) on each day the actual area from day 0 for each animal, according to the following formula:

HAx = AAx − AA0, where x represents one of the assessment days (3, 5, 7, 10, 14); AA0 stands for the initial area of the wound created on day 0; and AAx stands for the respective area of the wound on day x. Therefore, their difference stands for the healed area. This value was used in the statistical tests performed (one-way ANOVA test).

2.5.3. Methodology of Oxidative Stress Markers Evaluation

To assess redox imbalance, both serum and tissue samples were collected from the experimental animals. Blood was drawn via tail vein puncture under anesthesia; serum was separated by centrifugation. Tissue specimens were excised from the central region of the wound site, carefully minced, and subsequently processed for analysis.

Oxidative stress markers (reduced (GSH) and oxidized glutathione (GSSG) as well as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities) were assessed in the tissue sample, while the malondialdehyde (MDA) level was quantified in the tissue sample and blood. We also assessed the level of interleukin 8 (IL-8) from the tissue samples. These markers describe the oxidative stress (MDA, GSSG, and GSH/GSSG) and the antioxidant capacities (GSH, SOD, GPX, CAT) of the tissue microenvironment [35]. IL-8 is an early marker of inflammation, its coding gene being among the first upregulated during acute processes, which regulates neutrophil and monocyte–macrophage growth and differentiation [50].

The concentrations of GSH, a key endogenous enzyme, as well as GSSG, a marker of oxidative stress, were measured in skin homogenates using a spectrofluorometric technique involving a technique with o-phthalaldehyde and were expressed as nmol/mg protein [51], as well as the GSH/GSSG ratio, an indicator of redox status in the tissue [35,52,53].

SOD and CAT activities, two enzymes involved in the elimination of superoxide radicals and hydrogen peroxide specifically, were measured in skin tissue homogenates using the nitroblue tetrazolium Assay, and Pippenger’s method, respectively, with results presented as U/g protein [35,54,55,56].

GPX, a peroxidase involved in the conversion of hydrogen peroxide into water, was determined using the Flohé method, with results presented as U/g protein [55,57].

MDA, a marker of lipid peroxidation, was analyzed in serum and skin homogenates using a fluorometric method based on the method described by Conti, with modifications [35,58]. The results were reported as nmol/mL for serum samples and nmol/mg protein for tissue homogenates.

IL-8 levels were assessed using a commercially available Elisa kit, following their protocol (Abbexa, abx576575 Rat IL8 (CXCL8) ELISA Kit,, Abbexa, Cambridge, United Kingdom). The results were expressed as pg/mg protein.

2.5.4. Methodology of Histopathological Analysis

Skin biopsies were collected from the central area of the wound and fixed in 4% formaldehyde for 48 h. The tissue samples were subsequently dehydrated using a graded ethanol series (80%, 90%, 95%, and 100%), with each step lasting between 1 and 6 h. After dehydration, the specimens were cleared in three consecutive xylene baths, embedded in paraffin, and sectioned into 5 μm slices using a microtome. Sections were deparaffinized, rehydrated, and stained using Mayer’s hematoxylin and eosin (H and E) method. Images were acquired with a Leica ICC50 HD camera using LAS EZ software version 2.4 and examined under a Leica DM750 microscope.

The histopathological evaluation was carried out independently by two blinded investigators. The analysis focused on five parameters: re-epithelialization, suppuration, acanthosis, fibrin crust formation, and calcification. Statistical interpretation of the data was conducted using Pearson’s chi-squared test and the independent samples Kruskal–Wallis test.

2.6. Statistical Analysis

Statistical analysis was performed using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA), version 26.0.0. Graphical representation of the data was conducted using the ggplot 3.5.2 package in R 4.4.3 software and Modde software, version 11.0 (Sartorius Stedim Data Analytics AB, Umeå, Sweden). The Shapiro–Wilk test of normality was used to check if the quantitative variables follow a normal distribution. One-way ANOVA tests were used to compare quantitative variables (HA, oxidative stress markers) between the four groups. Post hoc tests using LSD correction were performed to assess differences between the group pairs after the ANOVA tests. Pearson’s chi-squared test was used to compare Boolean variables between the groups. The independent samples Kruskal–Wallis (k samples) test was used to compare ordinal variables between the groups. The results of the tests have been reported as p values. The significant threshold was set at less than 0.05.

G*Power software version 3.1.9.7 was used to determine the minimum necessary sample size [45].

3. Results

3.1. Characterization of the Vegetal Extract

3.1.1. Leaf Extract

Following the extraction protocol described above, from 100 g of dried leaf powder mixed into 1000 mL of solvent mix (ethanol: distilled water—50:50 ratio by volume), 700 mL of solution was obtained after centrifugation; 150 mL was obtained after subsequent use of the rotavapor. This final volume was later lyophilized and incorporated into the experimental creams.

3.1.2. Total Phenolic Content and Total Flavonoid Content Determination

Initially, an extract from the leaf powder was prepared using 5 g of the dried leaf powder mixed into 50 mL of the extraction solvent (50% ethanol—50% water), followed by ultra-turrax extraction at 4000 rpm for 5 min and centrifugation at 10,000 RPM for 10 min. The TPC and TFC were determined according to the already detailed methodology. The results are provided in

Table 1. Total phenolic content and total flavonoid content of A. vulneraria hydroalcoholic leaf extract.

TPC (mg gallic acid equivalent/mL extract)	2.058 ± 0.034
TFC (mg quercetin equivalents/g vegetal material)	31.030 ± 4.029

.

3.1.3. Total Antioxidant Capacity Determination in the Leaf Extract

The determination of the total antioxidant capacity (TAC) was carried out through two distinct methods: ABTS and DPPH assays. The results can be seen in

Table 2. Total antioxidant activity of A. vulneraria hydroalcoholic leaf extract.

	ABTS	DPPH
TAC (mM Trolox/g vegetal product)	0.693 ± 0.026	0.15 ± 0.014

, expressed as mM Trolox/g vegetal product.

The TAC of A. vulneraria leaves, measured using the ABTS and DPPH assays, revealed higher activity in the ABTS assay compared to the DPPH assay. This suggests that the extract contains predominantly hydrophilic antioxidants or compounds more effective at scavenging the ABTS⁺ radical. The results indicate that A. vulneraria leaves possess moderate antioxidant potential, with ABTS providing a more responsive assessment of their antioxidant profile.

3.1.4. Phytochemical Analysis by LC-MS/MS

The quantification of individual polyphenolic compounds in the A. vulneraria leaf extract was performed using both validated LC-MS/MS analytical methods, with detection based on the UV signal for the first analytical method. Figure 3 presents the UV chromatogram for the polyphenolic compounds identified and quantified in the leaf extract. The phytochemical composition of the extracts can be seen in

Table 3. Phytochemical analysis results—identification and quantification of bioactive compounds using both validated LC-MS/MS analytical methods in A. vulneraria hydroalcoholic leaf extracts (expressed as µg/mL).

Compound	A. vulneraria Leaf Extract
Gentisic acid	0.655 ± 0.052
p-coumaric acid	0.762 ± 0.053
Ferulic acid	0.784 ± 0.023
Hyperoside	10.775 ± 1.185
Isoquercitrin	<LOQ
Luteolin	<LOQ
Kaempferol	<LOQ
Apigenin	<LOQ
Protocatechuic acid	0.101 ± 0.013
Epigallocatechin	0.144 ± 0.011
Epigallocatechin gallate	0.342 ± 0.051

<LOQ—below the quantification limit of the analytical method.

. The results are presented as µg/mL.

The phytochemical characterization of A. vulneraria leaf extract, assessed using two validated LC-MS/MS analytical methods, revealed the presence of several polyphenolic compounds.

Among the identified phenolic acids, gentisic, p-coumaric, and ferulic acids were identified and quantified, although in low concentrations. These compounds are known for their antioxidant and anti-inflammatory properties and may contribute to the observed biological effects of the leaf extract.

Regarding flavonoid content, the leaf extract was characterized by the presence of hyperoside (10.775 ± 1.185 µg/mL) as the predominant compound, while other flavonoid glycosides and aglycones were not detected at quantifiable levels. Additionally, luteolin, kaempferol, and apigenin were only detected in trace amounts, suggesting a limited flavonoid diversity in the leaves. These results support previous research, in which it was concluded that the inflorescences serve as primary sites of flavonoid accumulation in A. vulneraria [59].

The second LC-MS/MS analytical method employed for phytochemical analysis revealed the presence of epigallocatechin (0.144 ± 0.011 µg/mL) and epigallocatechin gallate (0.342 ± 0.051 µg/mL) in the A. vulneraria leaf extract. The identification of these catechin derivatives, commonly found in Fabaceae species, supports the antioxidant profile of the extract and underscores the relevance of the leaf matrix as a source of bioactive compounds [60,61].

3.2. Experimental Animal Study

3.2.1. Assessment of Wound Contraction

The Shapiro–Wilk test indicated that the data were normally distributed amongst the groups (p > 0.05).

After analyzing the HA between the four groups using the one-way ANOVA test, significant differences between the groups were observed for HA on day 5 (HA5) (p = 0.001) and HA on day 7 (HA7) (p = 0.018), with no significant differences for HA3, HA10, and HA14 (p = 0.127; 0.392; respectively, 0.125).

For the measurements on day 5, significant differences were seen between the negative control and EG1 (p < 0.001) and EG2 groups (p = 0.002), as well as between the positive control and EG1 (p = 0.007) and EG2 groups (p = 0.038). Thus, EG1 showed the highest healed area, followed by EG2, with both values being significantly higher than the control groups. Although relatively widespread, the data showed a substantial benefit of the EG1 and EG2 cream on the healing speed on the 5th day of the experiment. The graphic representation can be seen in Figure 4a.

For the measurements on day 7, significant differences were seen between the EG1 and negative control (p = 0.003) and positive control groups (p = 0.012), while there were no differences between EG2 and either control groups (p = 0.352 and p = 0.155). The highest value was seen in the EG1 group, suggesting a positive influence on the healing process. For the EG2 group, however, the healing capacities declined and there was no significant difference between the EG2 and the controls. The graphic representation can be seen in Figure 4b.

In Figure 5, the macroscopic aspect of the wound can be seen. We chose one representative photo from each group on day 7, because on day 7 there were significant differences between the groups. The photographs were correlated with the microscopic images on day 7.

The measurements taken on subsequent days revealed no significant differences between the experimental groups and the control group. These findings suggest that A. vulneraria may have adjuvant properties during the initial stages of the healing process; however, no additional benefits were observed during the later phases of healing.

In our study, we assessed wound-healing progress based on changes in the wound area. However, some rats exhibited a negative HA, due to the unseen necrosis, which appeared in the first days after the burn wound was created, thus increasing the total area of the wound and rendering the HA as negative.

3.2.2. Oxidative Stress Markers Evaluation

The Shapiro–Wilk test indicated that the data were normally distributed amongst the groups (p > 0.05).

The analysis of oxidative stress markers in the tissue samples revealed significant differences between the experimental groups, while the MDA levels in blood showed no significant variation (p = 0.966). Detailed levels of the investigated markers are illustrated in Figure 6.

Notably, MDA levels in the tissue samples were significantly lower in the EG2 group compared to the NC (p < 0.001). Additionally, a trend toward significance was observed between EG1 and the NC (p = 0.066).

GSH levels were significantly elevated in both experimental groups compared to the NC (both p < 0.001), with EG2 displaying the highest values among all groups. Similarly, GSSG levels were significantly reduced in both EG1 and EG2 relative to the NC (p ≤ 0.001), with EG2 also showing a significant decrease compared to the PC (p = 0.035). Moreover, GSSG levels differed significantly between EG1 and EG2 (p = 0.017).

The GSH/GSSG ratio followed a consistent pattern, with both experimental groups differing significantly from the NC (both p < 0.001). Furthermore, EG2 exhibited a significantly higher ratio compared to both the positive control and EG1 (both p < 0.001). The highest ratio was recorded in EG2, followed by EG1, the PC, and the NC.

Among the oxidative stress markers analyzed, CAT activity remained unchanged across all groups except for the PC, which exhibited an increase compared to the others.

SOD activity was significantly elevated in both experimental groups compared to the NC (both p ≤ 0.001), with the highest levels observed in EG2, followed closely by EG1 and then by PC. A similar trend was noted for GPX activity, which reached its peak in the experimental groups, particularly EG2, with significantly higher values than the NC (p = 0.011 for EG1 and p = 0.006 for EG2).

IL-8 values were significantly different between the groups, presenting smaller levels in the experimental groups. There were differences between both experimental groups compared to the NC (p = 0.002 and 0.017, respectively) and PC (p = 0.004 and 0.35, respectively). The lowest value was in EG1 followed by EG2.

These findings suggest that the experimental treatments effectively modulated oxidative stress markers and IL-8 secretion, particularly in the EG2 group, which consistently displayed the most pronounced differences across multiple parameters.

In all the parameters tested, EG2 showed either the highest or the lowest value, except for CAT, whose value in the EG2 group was not statistically different from the controls, and for IL-8, where EG1 had the lowest value. The highest values of EG2 were determined for GSH, GSH/GSSG, SOD, and GPX, while the lowest values of EG2 were for MDA and GSSG. EG1 followed the same pattern as EG2, but with slightly lower or higher values, except for IL-8, where the lowest levels were found in EG1.

EG2 exhibited an ability to increase the antioxidant capacity in wound tissue, which was demonstrated by low values of MDA and GSSG and increased levels for endogen antioxidants—both non enzymatic and enzymatic—such as GSH, GSH/GSSG, SOD, and GPX.

3.2.3. Histopathological Analysis

Histopathological analysis was conducted on skin biopsies collected from the wound center on days 7 and 14. The parameters assessed included reepithelization, suppuration, epithelial acanthosis, fibrin crust presence, and calcification (recorded as absent [0] or present [1]), as well as granulation tissue, fibrosis, congestion, chronic inflammation, and the presence of skin adnexa (scored on a 0–3 scale: 0 = absent, 1 = mild, 2 = moderate, 3 = severe).

Statistical analysis revealed no significant differences between the four groups for any histopathological parameter. The p-values for each parameter are presented in

Table 4. Histological evaluated parameters and the respective p values between the groups.

Evaluated Parameter	p Value
Reepithelization	0.917 *
Suppuration	0.801 *
Epithelial acanthosis	0.943 *
Fibrin crust presence	0.710 *
Calcification	0.456 *
Granulation tissue	0.796 **
Fibrosis	0.537 **
Congestion	0.542 **
Chronic inflammation	0.292 **
Presence of skin adnexa	0.486 **

* Pearson chi-squared test; ** independent samples Kruskal–Wallis (k samples).

, with all p-values > 0.05, indicating no statistically significant differences.

Reepithelization was observed in 25 out of 32 samples across all groups, with no significant variation (p = 0.917), indicating that epithelial regeneration occurred similarly, regardless of treatment. Similarly, granulation tissue scores ranged from 0 to 3 across samples; however, the distribution showed no significant difference between groups (p = 0.796). Chronic inflammation, scored as mild (1) in most samples, also showed no significant variation (p = 0.292), suggesting that the inflammatory response was not markedly altered by the extract. The presence of skin adnexa, which varied widely (scores of 0 to 3), also did not differ significantly between groups (p = 0.486), indicating that the regeneration of skin appendages was not influenced by the treatment.

In Figure 7, the microscopic aspect of the wounds can be seen. We chose one representative photo from each group on day 7, because on day 7 there were significant macroscopic differences between the groups.

4. Discussion

Our study explored the healing potential of A. vulneraria extract in a burn injury animal model and analyzed the phytochemical profile of the extract. The results demonstrated that creams containing A. vulneraria extract significantly accelerated the macroscopic healing of burn wounds, enhanced antioxidant enzyme activity, and reduced oxidative stress markers in the treated tissues. The histological analysis revealed no structural alterations in the skin following treatment. Phytochemical screening revealed a rich composition of polyphenolic compounds, known for their potent antioxidant properties.

A. vulneraria has not yet been reported in any other in vivo experiments. However, numerous studies have shown its potential as a healing adjuvant, due to its significant quantities of polyphenols and long-time use in traditional folk medicine [2,18,21]. A. vulneraria has already been documented in a previous in vitro study to possess pro-migration properties on both mouse cell fibroblasts and human keratinocytes; a dose-dependent effect was found, with the lowest-concentration extract exerting the most efficient migration stimulation. The extract also showed a dose-dependent cytotoxic effect by reducing the ATP and protein content of both cell lines [20]. This could explain some of the results observed regarding the EG2 effects.

Numerous previous studies have documented the polyphenolic profile of A. vulneraria, both quantitatively and qualitatively. Although there are differences between reports concerning the exact polyphenol composition, all studies agreed on the impressive quantity and potential antioxidant capacity of the plant. Through LC-MS/MS methods, Lorenz et al. determined that kaempferol, quercetin, isorhamnetin, rhamnocitrin, and ferulic acid were the most abundant phenolic components [22], with similar results obtained by Csepregi et al. [20]. Ouerfelli et al. obtained slightly different results, reporting ferulic acid, cinnamic acid, kaempferol, caffeic acid, and sinapinic acid as the most prevalent phenolic components [24]. Our findings confirm that certain polyphenols are still found in cultivated A. vulneraria, with hyperoside as a predominant flavonoid in the leaf extract, followed by ferulic acid, p-coumaric acid, gentisic acid, epigallocatechin gallate, and protocatechuic acid, with traces of isoquercitrin, luteolin, kaempferol, and apigenin. Regarding the notably high concentration of hyperoside, which was approximately 10 times higher than that of other compounds, this is consistent with both the species-specific metabolic profile of A. vulneraria and the biological role of hyperoside in plant physiology. Hyperoside (quercetin-3-O-galactoside) is known to be a major flavonol glycoside in many Fabaceae species, where it serves as a protective antioxidant against oxidative stress and UV radiation, particularly in aerial organs. Its high abundance likely reflects an evolved defense strategy and active flavonoid biosynthesis, especially under environmental stimuli. From a biochemical perspective, hyperoside results from a highly efficient metabolic pathway involving the glycosylation of quercetin by UDP-galactosyltransferase, a reaction that enhances its solubility, stability, and accumulation in vacuoles. Finally, the elevated concentration detected in our study is supported by a validated LC-MS/MS method using a high-purity analytical standard, ensuring both specificity and reliability.

However, the absence of certain flavonoids, such as rutin, which has been widely reported in other studies [22,24], suggests potential variations due to environmental factors, extraction methods, or plant physiology, as well as due to the controlled growth of A. vulneraria specimens in non-native conditions as opposed to wild-type specimens, which were used in the other studies. More studies are needed to determine the origin of these discrepancies, as well as to determine the possible in vitro and in vivo interactions between the active substances of the extract.

Interestingly, previous reports have rarely documented the presence of epigallocatechin gallate in A. vulneraria leaves, thereby making our findings novel. The leaf extract, despite its modest overall polyphenol content, still contains quantifiable amounts of gentisic acid and epigallocatechin derivatives, which may provide anti-inflammatory benefits. The presence of catechins in the leaf extract suggests a possible role in plant defense mechanisms, as these compounds exhibit strong radical-scavenging activity and contribute to wound-healing effects through collagen stabilization [62].

The values we obtained for TPC and TFC can be compared to past findings in the literature. Ouerfelli et al. obtained values of 82.86 mg gallic acid equivalent/g vegetal material for TPC, and 20.14 mg quercetin equivalent/g vegetal material for TFC [2], while the same group determined different values in another study, namely, 93.27 mg gallic acid equivalent/g vegetal material for TPC and 37.88 mg quercetin equivalent/g vegetal material for TFC [17]. When compared to our values (TPC 20.58 mg gallic acid equivalent/g vegetal material and TFC 31.03 mg quercetin equivalent/g vegetal material), we can see that while TFC has similar values, TPC is significantly diminished, possibly due to different growing and geographic conditions.

Considering the antioxidant capacity of the leaf extract, the results we obtained can be compared to other data in the literature. Papp et al. obtained values of 0.026 mM Trolox/g vegetal product for DPPH [63]. Gođevac et al. obtained an ABTS value of 0.44 mM Trolox/g vegetal product [64], while Ouerfelli et al. obtained an ABTS value of 0.82 mM Trolox/g vegetal product, while also finding a higher DPPH value of 0.33 mM Trolox/g vegetal product [2]; this is compared to our values for ABTS of 0.693 mM Trolox/g vegetal product and DPPH of 0.15 mM Trolox/g vegetal product. Overall, our results confirm the antioxidant capacity of the A. vulneraria leaves.

Other studies have found that hyperoside [65], ferulic acid [66] and p-coumaric acid [67] have promising antioxidant, anti-inflammatory, and antimicrobial properties, while ferulic acid [66,68], gentisic acid [69], and protocatechuic acid [70,71] have shown good reepithelization and collagen-deposition capabilities, including in in vivo studies.

Hyperoside, a common flavonoid found in numerous plants, was found to inhibit the formation of several key inflammatory factors. An in silico study showed that hyperoside has the capacity to bind to substances, such as nuclear factor-κB (NF-κB) and interleukins 17 and 36, thus inactivating their activity [72]. An in vivo/in vitro mouse model study found that hyperoside inhibits TNF-α, IL-6 and the high-mobility group box 1 (HMGB1), a nuclear protein closely involved in the innate immune response that leads to the activation of NF-κB [73].

Additionally, on a rat wound model, an extract from Eugenia pruniformis, a plant rich in hyperoside, quercetin, and kaempferol, aided in reepithelization and wound architecture when compared to the controls, which was quantified microscopically through collagen deposition [65].

Ferulic acid, another substance identified in A. vulneraria leaves, exerts anti-inflammatory effects by decreasing the levels of interleukins 1β and 6 (IL-1β, IL-6), along with tumor necrosis factor α (TNF-α). It also inhibited NLRP3 inflammasome activation, a structure closely associated with innate immunity, which leads to the release and activation of various caspases and IL-1β [74]. In one in vivo study on a rat model of radiotherapy skin wounds, ferulic acid reduced inflammation and oxygen radical species, and improved skin blood flow, tissue reconstruction, and collagen deposition [68].

Based on macroscopic findings, the EG1 and EG2 creams showed beneficial effects for the healing process on day 5, while on day 7, only the EG1 cream demonstrated accelerated healing, probably due to different concentrations of polyphenols. Therefore, we could argue that A. vulneraria extract has a beneficial effect on wound healing in the first and early second stages of healing by providing an anti-inflammatory and antioxidation response, as well as encouraging epithelization. This is also confirmed by the increased levels of antioxidant enzymes and reduced levels of oxidative stress markers and IL-8 amounts detected in the skin samples. After the early healing stages, the effects of A. vulneraria no longer exceed those of the NC group, suggesting that A. vulneraria can be an adjuvant of early healing.

The possible loss of effect of EG2 cream on day 7 compared to EG1 cream could be explained by the detrimental effects of polyphenols, if present in a great amount. It may be that high levels of polyphenols can interfere with fibroblast migration and have cytotoxic effects [20,75,76], or could diminish the anti-inflammatory response in the wound, a crucial step in healing. This information can serve as an argument for calculating and using optimal concentrations of polyphenols for healing, as other studies have also indicated [20,40,41].

Considering the tested oxidative stress markers, for GSH, GSH/GSSG, SOD, and GPX a higher value is indicative of a better healing process, as these enzymes have protective antioxidant effects. For MDA and GSSG, a lower level is indicative of the healing response, as these two are markers of oxidative stress. We concluded that the EG2 group showed the best biochemical healing process, correlated with the high content of polyphenols including hyperoside, ferulic acid, p-coumaric acid, gentisic acid, epigallocatechin gallate, and protocatechuic acid. The EG1 groups showed similar results. Although these presented a weaker antioxidant response, the results were still comparable to EG2 and sometimes even better than the PC.

Regarding IL-8, this cytokine is an indicator of early inflammation and a potent chemoattractant for neutrophils, while also influencing key healing aspects such as angiogenesis and cell migration [77,78,79]. One previous study in burn patients determined that high levels of IL-8 correlated with increased inflammation, sepsis, multiorgan failure, and mortality [80]. IL-8 also induced the formation of neutrophil extracellular traps, which can lead to thrombosis and intravascular disseminated coagulation during sepsis, as another study showed [81]. Thus, high levels of IL-8 could be indicators of impaired healing and overall increased mortality and morbidity.

CAT catalyzes the conversion of H₂O₂ into H₂O; similar values in samples of different groups suggest a possible high activity of SOD and GPX, antioxidant enzymes that work in tandem to neutralize oxidative stress by reducing hydrogen peroxide, superoxide radicals, and organic hydroperoxides, thereby preventing cellular damage and maintaining redox balance [54].

Regarding the histopathological findings, the lack of significant differences between the four groups across all parameters shows that in our experiment, the A. vulneraria extract did not significantly alter the structural aspects of wound healing at the microscopic level under the tested conditions.

Several factors may explain the absence of histological differences. First, the small sample size (n = 8 per group, with only four rats analyzed at each time point) may have limited the statistical power. Second, basic hematoxylin–eosin (H and E) staining may have been too crude to detect subtle changes in tissue structure or cell populations. More sensitive methods, such as immunohistochemistry for proliferation (Ki-67), angiogenesis (CD31), or collagen deposition (Masson’s trichrome), could reveal changes missed by H and E. Third, the selected time points (days 7 and 14) may not align with the extract’s peak activity, which likely occurs earlier (days 5–7), as suggested by the macroscopic data. By day 14, the remodeling phase may mask earlier differences. Finally, the extract appears to act mainly through biochemical pathways by reducing oxidative stress and inflammation, rather than by inducing structural changes. This is supported by the significant improvements in markers like MDA, GSH, SOD, and GPX. Thus, A. vulneraria may promote healing at a molecular level, creating favorable conditions for repair. Future studies should increase the sample size and incorporate advanced histological and gene expression analyses (e.g., TGF-β, VEGF, Ki-67) to better elucidate the mechanisms of action and capture potential long-term effects on tissue remodeling.

We acknowledge the limitations of the study. The sample size was reduced due to ethical constraints; the microscopic measurement methods were limited. As for the oxidative stress quantification, although many markers were quantified, many more remain to be measured to better characterize the exact healing capacities of A. vulneraria, including more cytokine profiling, as well as multi-omics analysis. Additionally, the lack of significant histopathological findings underscores the need for further investigation into the structural effects of A. vulneraria extract, potentially using more sensitive techniques and larger sample sizes to detect subtle differences in tissue-repair processes.

5. Conclusions

In conclusion, we demonstrated that the A. vulneraria leaf extract possesses promising in vivo wound-healing activity, supporting previous in vitro findings and traditional medicinal use. These results underscore the plant’s potential as a valuable therapeutic adjuvant in wound care and fulfill the goals of this pilot study. Additionally, this study confirms that A. vulneraria grown in controlled, commercial-like settings, still possesses impressive quantities of polyphenolic compounds, and could be used as an adjuvant for wound healing.

Further research, including larger-scale studies, is needed to comprehensively evaluate its pharmaceutical applications. Given the growing demand for effective and sustainable natural alternatives, A. vulneraria emerges as a promising candidate, being readily accessible, easy to cultivate, and characterized by a diverse polyphenolic profile.