Comparative evaluation of antimicrobial and healing properties of Argyreia nervosa leaf extract in excision wound model

Introduction

A wound is a disruption in anatomical structure and function, skin being a primary surface barrier for the protection of the body. They are disruptions to the body's innate mechanisms. These wounds trigger an inflammatory response, leading to tissue breakdown and the formation of debris, which in turn could cause contamination from the airborne pathogen. Platelet-fibrin complex could stop bleeding/exposure to the circulatory system. The growth of the pathogen in the wound could potentially impair the immune response. Any delay in the healing of these wounds they become highly susceptible to microbial colonisation leading to infections, particularly by air-borne pathogenic bacteria, which can rapidly invade and proliferate in the injured tissue [1 , 2] . These wounds create a direct entry portal for bacteria and pose a significant risk for the onset of systemic infections. Infected wounds tend to exhibit delayed healing due to an imbalance in microbiota and several localised factors that lead to the disruption of the normal wound healing process, which in turn leads to the persistent inflammatory response and impaired cellular regeneration [3] . Sometimes, the presence of infection often leads to the formation of odorous pus (if the exudate contains pus), which may include a variety of toxins released by microbiota, further worsening the wound. This toxic environment contributes to the necrosis of surrounding healthy cells, thereby prolonging the healing timeline and increasing the risk of chronic wound formation [4] .

Argyreia nervosa, commonly known as the railway creeper or morning glory, is a perennial, woody climber belonging to the Convolvulaceae family, native to India and Southeast Asia [5] . It is a pleiotropic plant; its roots, leaves, stems, seeds, and flowers, have been reported to exhibit a diverse spectrum of pharmacological activities. These include their hepatoprotective, aphrodisiac, anti-inflammatory, anticonvulsant, immunomodulatory, antioxidant, analgesic, antimicrobial, Antiviral, antiulcer, central nervous depressant and Antidiarrheal activities [6] . A. Nervosa is also known for its potential to manage metabolic disorders, such as hyperglycemia, and its ability to act as an antiulcer, and antidiarrhea [7] l. Due to its wide pharmacological profile, A. Nervosa has garnered significant attention in modern phytotherapy and drug development research. Recently, A. Nervosa a medicinal plant has been reported to facilitate the process of wound healing using its leaf extract [8] . Considerable evidence has shown that Silver nanoparticles of the plant's leaf extract have anti-microbial properties [6] , as well as show a maximum zone of inhibition for various types of microorganisms [9] . Leaves of A. Nervosa, mainly contain β-sitosterol, 1-tricontanol and quercetin, which implicates clinical effects such as antiinflammatory as well as anti-bacterial [10] . Leaf extract of A. Nervosa when applied topically promotes the healing of wounds more significantly as compared to oral application, in both control rats and alloxan-induced diabetic rats, where healing is otherwise delayed [8] . 50% ethanolic extract of the seeds in the preliminary biological screening shows antibacterial activity against Staphylococcus aureus [11] . The alcoholic extract of the root also exhibited statistically significant anti-inflammatory activity against the granuloma technique in albino rats [12] . The leaves of A. Nervosa are traditionally used to promote purulent wound healing, potentially reducing drug resistance emergence [8] . Rich in flavonoids, saponins, tannins, and alkaloids, these phytochemicals exhibit antibacterial properties and contribute to various stages of wound healing, including inflammation, tissue regeneration, and collagen synthesis [13,14] . It is important to note that, there are other ways in which plant materials can exert their antibacterial activity than directly inactivating the intended microorganism. The active compounds found in A. Nervosa, namely β-sitosterol, 1tricontanol and quercetin can reduce the virulence of bacterial invaders or increase their susceptibility to antibiotic treatment by altering the metabolism of the bacterial cell, regulating gene expression, or interfering with various molecular targets within the bacterial cell. One such mechanism is by interfering with antimicrobial resistance mechanisms [6,15] . The A. Nervosa plant contains various phytochemicals namely alkaloids, flavonoids, saponins, tannins, steroids, terpenoids, and phenolic acids (caffeic and chlorogenic acids) they have shown meaningful potential in the prevention and treatment of microbial infections and wounds. Phytochemicals with anti-microbial, antioxidant, and wound-healing properties promote blood coagulation, combat infections, and quicken the healing process [16] .

It has been found that plants with high polyphenols and flavonoid content have exceptional wound-healing properties. As demonstrated by [16] water extracts of A. Nervosa leaves have a remarkable amount of total phenolic (110.8±0.19 mg GAE/g) and flavonoid contents (76.60±0.36 mg RE/g) when compared with the Gallic acid as standard. The main reasons phenolic aids in wound healing are because they are astringent, antibacterial, and scavenge free radicals. Due to their antibacterial and anti-oxidative qualities, polyphenolic components like flavonoids can aid in the excellent healing of wounds by blocking lipid peroxidation, which prevents cell damage and increases the vitality of collagen fibrils. Thus, it can be intriguing whether A. Nervosa leaf extract and its phytochemical interaction with the wound's innate mechanism endogenously play an active role in inhibiting microbial growth and promoting wound healing. The present work aims to fill this gap by comparing the effectiveness of the leaf extract in inhibiting microbial growth and promoting wound healing in a controlled rat model. By assessing these activities, the study provides a clearer understanding of the plant's therapeutic potential and its possible applications in modern healthcare. This investigation into A. Nervosa aims to validate its traditional uses and explore its dual benefits in combating infections and enhancing wound healing, potentially offering new avenues for treatment in clinical settings.

Materials and methods

Collection and Identification of Plant Materials

Leaves of the A. Nervosa were collected from the regional area of Gadarpur, Uttarakhand and authenticated by Dr. M. N. Reddy, Head of the Bioscience Department, Veer Narmad South Gujarat University, Surat, Gujarat (voucher specimen number: BVBRC206).

Animals

Adult male rats of 238-285 g were obtained from the Animal Breeding Facility, Jai Research Foundation. They were housed in an environment-controlled room, in sterile solid floor polypropylene mouse cages (size: 290 × 220 × 140 mm), under the controlled conditions (22±3 °C and 12 h light/dark cycle, light on at 06:00 am) with free access to food and water. The experimentation was approved by the "Institutional Animal Ethics Committee (IAEC)" JRF. The experiment was undertaken in compliance with the guidelines of the "Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International" and "Guidelines for Laboratory Animals Facility" issued by the Committee for the Control and Supervision of Experiments on Animals (CCSEA), India.

Preparation of extract

The freshly collected leaves were sun-dried and ground to make fine powder. The powder was extracted with water in a 1:8 ratio. The decoction was then boiled and concentrated until only one-eighth of the original volume remained. The extract was then cooled and strained, the filtrate was further air-dried and stored in airtight bottle (Fig. 1) [17] .

Excision wound creation

All subjects were experimentally novices at the beginning of the study. Excision wounds were used to study the Wound Surface Area (mm), Percentage (%) of Wound Contraction, Bacteriological Load in Wound Surface (CFU/ml), and its Microscopic Examination. All wounds were of fullthickness type, extending up to the adipose tissue. Rats were anaesthetized with xylazine (10 mg/kg, i.p.) and ketamine (100 mg/kg, i.p.). The stage of an anaesthesia was decided on the base of the tail pinch, pedal, and pupillary reflexes. Surgical procedure was initiated after the complete absence of all the reflexes and an area of about ≈500 mm 2 was marked on the back of the rat by a standard ring. The full thickness of the marked skin was then cut carefully. In addition, a new series of experiments for bacteriological load on the wound surface was carried out, pathogenic strains of Staphylococcus aureus were grown on nutrient agar medium at 37°C for 24 hours and then suspension was prepared comparing the turbidity with 0.5 Mac Farland. To induce infection, 0.1 mL of the above inoculum (1.5 x 10 8 CFU/mL) was inoculated on the wound area of each animal. The infection was allowed to be induced for 24 hr and the treatment using A. Nervosa leaf extract was topically applied thereafter [18,19] . The application site was covered by a porous gauze dressing (not more than 8 ply) and nonirritating tape. On the next day approximately 24 hours after exposure, the dressing was removed from the rat using scissors. Also following excision, the rats were individually in the cage to avoid any disturbance. Their bedding material was changed daily to avoid microbial contamination. Animals were closely observed for any unwanted infection, and those that showed any sign of infection were separated, excluded from the study and replaced. No typical postoperative care was taken as no blood loss was observed in all animals. However, to reduce surgical pain in rats, rats were treated with Meloxicam (10 mg/kg b. wt.) after completion of surgery.

Experimental groups

For the accomplishment of the study, 18 infected male rats with S. aureus were randomly divided into three groups:  Group I: Negative control group: the animals underwent surgery but did not receive any treatment but received sterile water for injection (SWFI) applied topically as a vehicle.  Group II: positive control group: This animal underwent surgery and received standard treatment 2% w/w Mupirocin Ointment applied topically.  Group II: low dose: The animals underwent surgery and were treated with A. Nervosa leaf extract at a dosage of 100 mg/kg body weight per day.  Group IV: high dose: The animals underwent surgery and were treated with A. Nervosa leaf extract at a dosage of 1000 mg/kg body weight per day.

The selection of doses of A. Nervosa leaf extract was selected by considering the minimum inhibitory concentration (MIC) as per a previously published in-vitro study for microbial strain (Staphylococcus aureus) i.e., 100 mg/kg b. wt./day [20,21] . The dose of positive control was selected as 10 mg/kg b. wt. /day based on a previously published in-vivo study [22] . A. Nervosa leaf extract at the dose of 100 and 1000 mg/kg bwt/day was applied topically [18] for a consecutive period of 21days [17 , 19] .

The means of wound surface area measurement and percentage of wound contraction between groups at different time intervals were measured by the Vernier calliper (Mitutoyo Corporation, Japan) on the day of excision and subsequently on an alternative day (Days 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22). The area of the wound on the day of wound excision was considered 100%. The rate of wound contraction is calculated as given in the formula below [23] .

Percentage (%) of Wound Contraction= Initial day wound size-Specific day wound size Initial day wound size X 100

Anti-microbial rate

The severity of the bacterial infection was quantitatively assessed every alternate day (Days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21) by collecting swab samples. Each swab was then rinsed in 1 mL of sterile nutrient broth, and colonyforming units per millilitre (CFU/mL) were determined using the spread plate method. Serial dilutions were performed up to a 10⁻⁶ dilution to reduce the microbial load. Three selected dilutions, spaced alternately, were plated onto the surface of sterile nutrient agar plates. All plates were incubated in a bacteriological incubator at 37°C for approximately 24 hours. After incubation, the CFU/mL was calculated [24] .

Histopathological examination

The rats were euthanized on day 22 followed by hematoxylin and eosin (H&E) staining is most commonly used for histopathological examinations. Hence, its integrity in the quality of staining has paramount importance [25] . The materials required for the procedure included reagents such as Eosin Y solution, ethanol (100%, 95%, and 70%), Mayer's hematoxylin solution, and Permount mounting medium. The tissue samples were properly embedded in paraffin, sectioned, and mounted onto glass slides. The staining process involved several key steps. First, paraffin was removed from the slides by soaking them in xylene.

The slides were then hydrated by sequential immersion in ethanol solutions of decreasing concentrations (100%, 95%, and 70%) and subsequently rinsed with tap water. Following this, the tissue sections were stained with hematoxylin and rinsed with water. Eosin Y was then applied to stain the tissues, after which the samples were dehydrated through graded ethanol and cleared with xylene. Finally, a drop of Permount was placed on each tissue section, and a coverslip was applied [26] . The stained tissues were examined under a light microscope (1000× magnification) and graded subjectively as mild (+), moderate (++), or severe (+++) for various parameters including epidermal fibroplasia, hypertrophy/hyperplasia, squamous epithelium, inflammatory infiltrates, and the presence of wounds or ulcers, in order to evaluate epidermal and dermal remodelling.

Statistical analysis

Data were analysed with Graph Pad Prism Version 10.4.1 (627), November 18, 2024, for Windows-64 bit (Graph Pad Software Inc., San Diego, CA, United States) statistical package program. The results are presented as means±S.E.M. The effects of A. Nervosa leaf extracts treatment at the dose of 100 mg/kg (G3) and 1000 mg/kg (G4) and 2% w/w Mupirocin ointment at the dose of 10 mg/kg (G2), the data obtained from these treatment groups were compared with a control group of dose 0 mg/kg (G1) and were statistically analysed by two-way analysis of variance (ANOVA) followed by post hoc Dunnett's test. A value of p<0.05 was considered significant.

Results

A. Nervosa leaf extract exhibits a faster reduction in wound surface area over time, indicating a natural wound-healing process

As shown in Fig. 2 and Table 1, p<0.05] at the dose of 100 mg/kg (G3) and 1000 mg/kg (G4) showed a significant effect on reducing the mean wound surface area (mm) and as compared to the control (G1) group. Post hoc Dunnett's multiple comparisons test analysis showed a significant reduction on day 6 [at 100 mg/kg (p<0.05)], day 8 [at 100 mg/kg (p<0.05)], day 10 [at 100 mg/kg (p<0.05) and at 1000 mg/kg (p<0.01)], day 12 [at 100 mg/kg (p<0.001) and at 1000 mg/kg (p<0.05)], day 16 [at 100 mg/kg (p<0.001) and at 1000 mg/kg (p<0.01)], day 18 [at 100 mg/kg (p<0.05)]. Conversely, the positive control (G2) group of 2% w/w Mupirocin ointment did not show statistically significant data [at 10 mg/kg (p>0.05) but, a noticeable improvement in wound surface area reduction, particularly during the early stages (days 2-10) can be seen. However, the rate of healing differed significantly on some days but the A. Nervosa leaf extract accelerates healing and highlights the potential effectiveness in enhancing wound repair. Nervosa was applied topically at the dose of 100 mg/kg and 1000 mg/kg and 2% w/w Mupirocin ointment was applied as a positive control at 10 mg/kg and means of wound surface area (mm) was measured using Vernier Calliper on the day of excision and subsequently on the alternate days. Each data represents means±SEM, n = 6. *** And $$$ p<0.001; ** and $$ p<0.01; * and $ p<0.05 compared to the control group (Two-way ANOVA followed by Dunnet's post hoc test).

As depicted in Fig. 3 Fig. 3 and Table 2. Effect of A. Nervosa leaf extract (100 mg/kg and 1000mg/kg) and 2% w/w Mupirocin ointment (10 mg/kg) on the means of percent wound contraction. An excision wound was created in rats in addition with S. aureus infection (0.5 MacFarland standard/1.5 x 10 8 CFU/mL) induced for 24 hours. Thereafter, leaf extract of A. Nervosa was applied topically at the dose of 100 mg/kg and 1000 mg/kg and 2% w/w Mupirocin ointment was applied as a positive control at 10 mg/kg and means of percent wound contraction was measured using Vernier Calliper on the day of excision and subsequently on the alternate days. Each data represents means±SEM, n = 6. ***p<0.001; ** and $$ p<0.01; * and $ p<0.05 compared to the control group (Two-way ANOVA followed by Dunnet's post hoc test).

A. Nervosa leaf extract illustrates a dose-dependent reduction in bacterial loadpossessing anti-microbial properties

Fig. 4 and Table 3 Fig. 4 and Table 3. Effect of A. Nervosa leaf extract (100 mg/kg and 1000mg/kg) and 2% w/w Mupirocin ointment (10 mg/kg) on the means of Bacteriological Load on Wound Surface (CFU/mL). An excision wound was created in rats in addition with S. aureus infection (0.5 McFarland standard/1.5 x 10 8 CFU/mL) induced for 24 hours. Thereafter, leaf extract of A. Nervosa was applied topically at the dose of 100 mg/kg and 1000 mg/kg and 2% w/w Mupirocin ointment was applied as a positive control at 10 mg/kg and the severity of bacterial infection was quantitatively assessed by collecting swab samples from the wound, serially diluted, placed on agar plates and incubated for 24 hr and means of bacteriological load on the wound surface and assessed subsequently on the alternate days. Each data represents means±SEM, n = 6. *** , $$$, ### p<0.001; ** , $$, and ## p<0.01; * , $, and # p<0.05 compared to the control group (Two-way ANOVA followed by Dunnet's post hoc test).

A. Nervosa leaf extract ameliorates tissue regeneration and the healing process

As depicted in Fig. 5 and Table 4. Specimens of wound tissue were collected on day 21 for histopathology examination. The histopathological assessment mainly in the dose of 100 mg/kg (G3) and 1000 mg/kg (G4) groups revealed marked re-epithelization/hyperplasia of the squamous epithelium, hair follicles, collagen deposition, neovascularization, and fibrous tissue proliferation (Fig. 5 -C, D). And, all the groups showed distinct patterns in fibroplasia, epithelial changes, inflammatory cell infiltration, and wound severity (Fig. B,C,D). Histological sections of wound surface tissue taken from the Control group of dose 0 mg/kg (G1) exhibited marked (n=1), mild (n=2), minimal (n=2) and, moderate fibroplasia (n=1). Furthermore, G1 showed mild (n=4) and, minimal hypertrophy/hyperplasia of squamous epithelium (n=2) and, minimal inflammatory cell infiltrate (n=4), marked (n=1) and, severe wound/ulcer presence (n=3). The overall findings suggest impaired and suboptimal wound healing in the control group. Animals from the Positive control group 10 mg/kg (G2) displayed moderate (n=4), with mild fibroplasia (n=2). Hypertrophy/ Hyperplasia of squamous epithelium was predominantly noted mild (n=4) with few minimal cases (n=2). Inflammatory cell infiltrate was marked (n=3), and mild (n=2), with marked (n=2), moderate (n=1) and, severe wound/ulcer presence (n=2). These observations indicate partial improvement in wound healing with persistent inflammation and epithelial changes. The 100 mg/kg (G3) showed balanced fibroplasia with cases of mild, moderate, and minimal (n=2 each). The prominent characteristics of were the minimal Hypertrophy/ Hyperplasia of squamous epithelium (n=4) and, mild (n=4), as well as the presence of moderate (n=1), minimal (n=4), and mild (n=1) inflammatory cell infiltrate, with balanced wound or ulcer presence with cases of marked, mild, moderate and, severe (n=1 each). The findings suggest a more regulated healing response with controlled tissue regeneration and inflammation. The 1000 mg/kg group (G4) was predominated by mild (n=5) and moderate fibroplasia (n=1) while minimal hypertrophy (n=4), marked (n=4), and mild hypertrophy/ hyperplasia of squamous epithelium (n=2), mild (n=4) to minimal (n=2) inflammatory cell infiltrate and, marked to moderate presence of wound/ulcer (n=2 each) were observed. So, the changes exhibited in G4 shows significant healing efficacy, characterized by efficient fibroplasia, controlled epithelial changes, minimal inflammatory response, and reduced ulcer severity.

In conclusion, the 100 mg/kg (G3) and 1000 mg/kg (G4) treatment groups demonstrated significant advancements in wound healing, characterized by improved tissue regeneration, controlled inflammation, and reduced wound severity. In contrast, the control (G1) and positive control (G2) groups exhibited suboptimal healing with persistent inflammatory responses. Specifically, G1 showed higher fibroplasia and severe inflammatory infiltration, while G4 exhibited more pronounced mild fibroplasia, marked hyperplasia, and moderate squamous epithelial changes. The variability in inflammatory infiltrates and wound severity across the groups highlights the heterogeneity of tissue responses in wound healing and chronic ulcer pathology. These findings align with established descriptions of chronic wound histopathology, where persistent inflammation, fibroplasia, and epithelial changes are central features.

Table 4: Histopathological analysis of tissue responses across four experimental groups. The table presents the incidence of fibroplasia (marked, mild, minimal, and moderate), hypertrophy/hyperplasia of squamous epithelium (mild, and minimal), inflammatory cell infiltrate (mild, Minimal, and moderate), and wound/ulcer presence (marked, mild, moderate, and severe) in Groups G1-G4.

Values represent the number of samples exhibiting each condition within each group (n=6 per group). MacFarland standard/1.5 x 10 8 CFU/mL) induced for 24 hours. The 100 mg/kg (G3) and 1000 mg/kg (G4) groups revealed marked re-epithelization/hyperplasia of the squamous epithelium, hair follicles, collagen deposition, neovascularization, and fibrous tissue proliferation.

Discussion

The present study investigated the Antimicrobial and Healing properties of A. Nervosa Leaf extract using an excision wound model. An excision wound was created in rats in addition with S. aureus infection (0.5 MacFarland standard/1.5 x 10 8 CFU/mL) induced for 24 hours. Thereafter, leaf extract of A. Nervosa was applied topically at the dose of 100 mg/kg and 1000 mg/kg and 2% w/w Mupirocin ointment was applied as a positive control at 10 mg/kg and rats were subjected to evaluation of wound surface area (mm), percentage (%) of wound contraction, bacteriological load in wound surface (CFU/mL), and its microscopic examination.

A. Nervosa, a perennial climbing plant. It is known for its diverse health benefits, including antimicrobial, antiinflammatory, and tissue-regenerating properties. The plant's leaves, rich in bioactive compounds like β-sitosterol and quercetin, are particularly effective in promoting wound healing. Topical application of its leaf extract accelerates tissue regeneration, reduces inflammation, and inhibits microbial growth, making it a promising agent for faster and more efficient wound recovery [5,6] . The present study demonstrated the effects of A. Nervosa leaf extract on wound surface area over time. Consistent with previous findings, [8] , showed that topical application of ethanolic A. Nervosa leaf extract led to a significantly faster reduction in wound surface area compared to controls. The effect was more pronounced in topically treated groups than orally treated ones, and especially notable in diabetic rats where healing is typically delayed. The study concluded that A. Nervosa leaf extract accelerates wound contraction and promotes faster epithelization, supporting its traditional use in wound healing. [27] Concluded that the wound healing activity is attributed to the presence of alkaloids, flavonoids, triterpenoids, saponins, tannins, and steroids in the extract, which contributes to anti-inflammatory and antimicrobial effects, further supporting rapid wound closure and tissue regeneration. In the current study, A. Nervosa leaf extract demonstrated a significant acceleration of wound healing, as evidenced by the progressive reduction in mean wound surface area over 22 days. Treatment with both 100 mg/kg (G3) and 1000 mg/kg (G4) doses resulted in statistically significant wound contraction compared to the control group of dose 0 mg/kg (G1) at multiple time points, this consistent performance not only hastened wound contraction but also sustained its improvement. Interestingly, while the 10 mg/kg group (G2) (2% w/w Mupirocin ointment) showed observable improvements in wound surface area, especially during the initial phase (days 2-10), its effects were less consistent and did not achieve the same degree of statistical or biological significance as the A. Nervosa leaf extract. These findings suggest that A. Nervosa leaf extract may possess some potent bioactive compounds capable of promoting wound closure more effectively than conventional treatment in certain conditions. The extract's ability to enhance healing may be attributed to anti-inflammatory, antimicrobial, and proangiogenic properties, thereby collectively accelerating tissue repair and regeneration. We have also investigated the effect of A. Nervosa leaf on the percent wound contraction and its healing potential. As per the previously conducted studies, [8] also concluded that topical application of the extract significantly increased wound contraction percentage compared to controls, especially in diabetic rats where healing is delayed. The extract promoted faster wound closure and epithelization, confirming its dose-dependent wound-healing efficacy. Histological improvements correlated with enhanced contraction rates [28] . Research also showed that A. Nervosa extracts significantly increased wound contraction rate, tensile strength, and hydroxyproline content (a collagen marker), indicating enhanced wound healing quality and speed. In our study, in the earliest phase of treatment, a slight increase in wound size was observed in some groups, which reflects the natural inflammatory stage that is essential for initiating wound healing. However, as the days progressed, particularly from day 4 onward, a clear pattern of improved wound contraction was seen in animals treated with A. Nervosa. Both the 100 mg/kg (G3) and 1000 mg/kg (G4) doses promoted a faster and more consistent closure of wounds compared to the control group (G1). This steady progress highlights the extract's ability to guide the healing process through critical early and middle phases, where timely regulation is most important. While the standard 10 mg/kg group (G2) (2% w/w Mupirocin ointment) provided some early improvement, it did not match the consistent wound closure achieved by A. Nervosa. As healing advanced toward day 22, all groups naturally showed a slowing down of wound contraction, marking the final stage of repair. These findings suggest that A. Nervosa does more than simply close wounds, it supports and harmonizes the body's own healing mechanisms, helping to balance inflammation, encourage tissue growth, and strengthen repair.

We further investigated on the controlling bacterial contamination at the wound site. As found in the earlier findings, [29] , investigated alcoholic and aqueous extracts of A. Nervosa leaves against multiple bacterial strains, including S. aureus, P. aeruginosa, and E. coli. Their results showed clear antibacterial activity, with inhibition zones increasing with higher extract concentrations, indicating a dose-dependent effect [30] , compared the antimicrobial activity of ethanolic extracts of A. Nervosa leaves with other medicinal plants. The study found that all ethanolic extracts, including that of A. Nervosa, produced significant inhibition zones against tested bacteria, with activity comparable to standard antibiotics. The results were consistent across repeated experiments and reflected a concentrationdependent inhibition [31] , reported that different solvent extracts of A. Nervosa leaves possess antibacterial activity, and the degree of inhibition varied with extract concentration and solvent used, further supporting dosedependency [32] . Revealed that the antimicrobial activity is likely due to the presence of flavonoids, alkaloids, saponins, and tannins, which are known for their bactericidal properties. In the current study, on the first day of observation, all groups showed a similarly high level of bacterial load, confirming that the starting conditions were uniform. As healing progressed, a remarkable reduction in bacterial count was observed in the groups treated with A. Nervosa leaf extract, particularly at the doses of 100 mg/kg (G3) and 1000 mg/kg (G4). This decline was both steady and dose-dependent, with a significant decrease appearing as early as day 3 and continuing consistently through the later days of the experiment. While the standard 10 mg/kg group (G2) (2% w/w Mupirocin ointment) also helped reduce bacterial numbers, A. Nervosa leaf extract demonstrated a strong and sustained antimicrobial effect across most of the study period. Although a slight variation was noted on day 17 at the higher dose, the overall trend clearly supports the role of A. Nervosa leaf extract in suppressing bacterial growth. These results suggest that the leaf extract either acts directly by inhibiting microbial proliferation or indirectly by creating a healthier wound environment that resists secondary infections. Through this antimicrobial action, A. Nervosa leaf extract not only accelerates wound healing but also preserves the integrity of newly forming tissue.

The investigation was further taken on, histopathological evaluation of wound healing efficacy of A. Nervosa leaf extract. Previous findings have demonstrated that, [8] , demonstrated that topical application of ethanolic A. Nervosa leaf extract significantly promoted wound healing in both normal and alloxan-induced diabetic rats. Histopathological analysis revealed enhanced epithelialization, increased collagen deposition, and improved granulation tissue formation in treated wounds compared to controls. The extract showed superior effects when applied topically versus orally. The presence of alkaloids, flavonoids, triterpenoids, saponins, and tannins contributed to these effects by modulating inflammation and promoting tissue regeneration [28] , showed that A. Nervosa extracts significantly increased wound contraction rate, tensile strength, and hydroxyproline content (a marker of collagen), indicating improved histological wound repair. In the current study, the histopathological analysis revealed that A. Nervosa leaf extract, particularly at doses of 100 mg/kg (G3) and 1000 mg/kg (G4), promoted efficient wound healing through several key biological activities. The anti-inflammatory properties of the extract likely played a central role in controlling inflammatory responses, which were evident in the reduced inflammatory cell infiltration observed in G3 and G4 groups. This control of inflammation allowed for more regulated tissue regeneration, as indicated by clear re-epithelialization, collagen deposition, and the growth of hair follicles. The observed neovascularization, or formation of new blood vessels, suggests that A. Nervosa leaf extract enhanced angiogenesis, providing essential nutrients and oxygen to the healing tissue. Additionally, the extract's ability to stimulate fibroplasia and collagen synthesis contributed to a more organized extracellular matrix, aiding in wound closure and strengthening tissue integrity. Moreover, the antimicrobial activity of the extract likely reduced bacterial load, preventing infections that could otherwise complicate healing and lead to severe ulceration. All this collectively promoted a balanced healing process, with minimal epithelial disruption and controlled wound severity. In contrast, the control group of dose 0 mg/kg (G1) group exhibited impaired healing, with persistent inflammation, irregular fibroplasia, and severe ulceration, highlighting the superior healing potential of A. Nervosa leaf extract.

Conclusion

In conclusion, the present study demonstrates the potential of A. Nervosa leaf extract in promoting wound healing through its antimicrobial and healing properties. The results show that topical application of the leaf extract significantly reduced wound surface area, enhanced wound contraction, and improved tissue regeneration in comparison to controls. The extract's effectiveness was notably more pronounced in higher doses, where it supported accelerated epithelization, increased collagen deposition, and reduced bacterial contamination at the wound site. Additionally, histopathological analyses confirmed the extract's role in regulating inflammation, promoting fibroplasia, and stimulating angiogenesis, contributing to more efficient and structured wound healing. However, some limitations of the study include the relatively small sample size and the focus on animal models, which may not fully reflect the complexities of human wound healing. Future studies should include a broader range of doses, and clinical trials to further validate these findings in human subjects. Additionally, the specific molecular mechanisms through which A. Nervosa exerts its therapeutic effects require deeper investigation to enhance our understanding of its full potential. Concisely, A. Nervosa leaf extract holds promise as an effective alternative in wound care, offering both antimicrobial and tissue-regenerating benefits. Further research is necessary to optimize its clinical applications and explore its long-term effects in diverse healing conditions.

Acknowledgement

This study was conducted by Jai Research Foundation, Valvada, and Gujarat, India and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.

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