Formulation and In Vitro Assessment of Tragacanth Gum-Based Hydrogel Loaded with Artemisia vestita Leaf Extract for Wound Healing

Abstract

Artemisia vestita Wall. ex Besser, a traditional medicinal plant with healing properties, is receiving significant attention as a potential therapeutic agent for wound healing. In this study, eight Artemisia vestita leaf extract hydrogel formulations (F1–F8) were carefully designed and studied. The hydrogel formulations contained A. vestita leaf extract, tragacanth gum, humectants, preservatives, pH stabilizers, and Milli-Q water. A preformulation study was conducted to ensure safety and efficacy. Moreover, various experiments assessed the potential application and characteristics of A. vestita leaf extract hydrogel (ALEH). Drug release and swelling studies were conducted at different pH levels and temperatures. The best drug release model was identified based on the regression coefficient (R²). Antimicrobial efficacy was assessed using the agar well diffusion method, and wound healing in HaCat cells was assessed using the scratch assay. ALEH exhibited non-Fickian diffusion, with higher drug release noted at pH 6.8 than at pH 4.5, indicating pH-responsive behavior. It exhibited significant antimicrobial activity against various strains and achieved 95% wound closure after 24 h in vitro, indicating strong wound healing properties. It also had a long shelf life; therefore, it could have pharmaceutical and medical applications. Our study is the first to report the potential applications of ALEH in skincare and wound management.

1. Introduction

Wound healing is a complex biological process. It involves a sequence of intricate events that occur in response to skin injury, followed by the actions of signaling molecules, various cell types, and extracellular matrix components [1,2,3]. The wound healing process involves three main phases. In the inflammatory phase (0–3 days), inflammatory cells migrate toward the wound site to remove debris and pathogens [4,5]. In the cellular proliferation phase (2–12 days), new tissue formation is initiated. In this phase, fibroblasts generate collagen, while epithelial cells multiply and move across the wound surface to cover it [6,7]. Between days 12 and 90, the scar continues to mature, becoming smoother, softer, and less noticeable. Although the scar tissue gains strength during this time, it may still be more susceptible to injury or trauma than intact skin. In the remodeling phase (3–6 months), the newly formed tissue undergoes maturation and remodeling [8,9]. In this phase, wound size decreases, collagen fibers are reorganized, and the healed tissue gradually gains functionality and strength. However, during the healing process, there are chances of microbial infections, which can delay the healing process and affect patients’ quality of life [10]. Therefore, in order to facilitate a speedy recovery, a moist healing environment is required for tissue regeneration to ensure adequate tissue perfusion and mitigate tissue damage [9]. Moisture can lower the risk of infection by creating an optimal environment that enhances the body’s natural defense mechanisms. In a moist environment, the skin barrier remains intact and is more effective at blocking pathogen entry. Furthermore, moisture helps maintain a balanced pH level and promotes the activity of immune cells and antimicrobial peptides, which are essential for combatting infections. A moist environment also prevents scab formation, reducing the risk of cracks and openings through which bacteria can enter [11].

Ethnopharmacology harnesses the traditional wisdom of indigenous communities to uncover and develop natural remedies for various health conditions. This interdisciplinary field focuses on medicinal plants and traditional therapeutic techniques used in various cultures by integrating anthropology, botany, chemistry, pharmacology, and medicine. It aims to identify bioactive compounds with therapeutic potential within these traditional remedies. Recent progress in ethnopharmacological research has resulted in the identification of new compounds and validation of traditional treatments using modern scientific approaches [12,13]. Through these studies, researchers have gained insights into the mechanisms of action of traditional medicines, identified bioactive compounds responsible for their effects, and explored the potential of these bioactive compounds in treating numerous human diseases. Ayurveda integrates traditional medicinal herbs with modern practices for healthcare and wound management. It can provide a holistic approach and leverage the benefits of natural remedies alongside conventional remedies to treat skin damage [14]. Documented records of Ayurveda contain multiple descriptions of various herbal formulations that are believed to possess wound healing properties. These remedies are often used topically to reduce inflammation, prevent infections, and expedite wound closure [15,16].

In general, gums are natural polymers that exhibit diverse properties resulting from their multifunctional exudates. For instance, they can thicken, stabilize, and emulsify gels; provide structure; and enhance viscosity [17]. They are water-soluble mixtures of polysaccharides derived from the drained sap of plant roots. After extraction, the collected sap is dried to obtain natural gum in powdered form [18,19,20]. On the other hand, a hydrogel is a mixture of porous and permeable solids, constituting at least 10% of interstitial fluid (primarily composed of water) by weight or volume. It is a biphasic material that forms a 3D network. It consists of either natural or synthetic polymers and can absorb a significant amount of biological fluids or water. These characteristics serve as the foundation for various applications, especially in the field of biomedicine. Some hydrogels originate from natural sources, while many are artificially created [21,22,23].

A. vestita Wall. ex Besser, commonly known as Russian wormwood, is a traditional medicinal plant belonging to the Asteraceae family. It is traditionally known as “kubsha” and is found in the upper regions of Himachal Pradesh [24] and hilly regions of the Himalayas, such as Nepal, Pakistan, Tibet, and India. This plant typically grows at an altitude of 1500–3600 m above sea level [25,26]. A. vestita has long been used as a therapeutic agent in conventional medical practices because of its outstanding therapeutic value and medicinal properties (Figure 1). This plant is used for treating itching, wounds, and dermal infections. Indigenous knowledge of this plant has been applied for treating wounds by local inhabitants and practitioners in Himachal Pradesh for a long time [26]. Although its extensive traditional use supports its efficacy, scientific evidence is lacking. Fresh leaf paste prepared from this traditional herb has been traditionally employed to facilitate rapid wound healing [26,27]. However, no research has reported its formulation to date. To bridge the gap between traditional use and scientific validation, we aimed to formulate a gel using the natural polymer tragacanth gum. Moreover, we aimed to use this hydrogel formulation to extend the shelf life of A. vestita and facilitate its future commercialization for promoting wound healing and treating skin infections.

In this study, we developed a gel using A. vestita extract and tragacanth gum to harness its traditional healing properties and assessed its drug release profile for commercial applications. We assessed the drug kinetics to optimize efficacy and create a cost-effective formulation using the locally available plant A. vestita in order to ensure accessibility for the local population. By achieving these objectives, we aimed to scientifically validate the traditional use of A. vestita for wound therapy. In the future, we intend to develop a commercially viable, cost-effective formulation that could promote healing and treat dermal infections.

2. Materials and Methods

• Collection of Plant and Extract Preparation

Fresh A. vestita leaves were collected from the forest of Kotgarh, Himachal Pradesh, India (31.31° N 77.47° E), during the flowering stage. The authentication of the plant material was performed at Nauni University, Solan, Himachal Pradesh. A herbarium number was assigned for documentation and identification purposes (UHF-Herbarium no. 13916). The leaves were stored and shade-dried for 15 days to remove moisture.

The dried leaves were pulverized into coarse powder using a grinder. The powder was then sieved through a fine mesh to achieve a fine powder, which was stored in a clean glass jar for further use. Extraction was performed using the Soxhlet extraction method, as described by Dogra (2024) [28]. Phytochemical analysis of the extract using GC–MS revealed a total of 22 compounds, accounting for 94.89% of the extract. The identified compounds exhibited various peaks; these compounds included grandisol, camphor, 1,8-cineol, limonene, thujone, β-caryophyllene, isofraxidin, naringenin, myrcene, camphene, apigenin, santolina triene, α-pinene, germacrene-D, yomogin, artemisia alcohol, cirsilineol, borneol, quercetin, copaene, artemisia ketone, and amyrin. These compounds were identified by comparing the mass spectra and retention times with those mentioned in the NIST library and available literature [23].

• Gel Preparation

The specified quantity (

Table 1. Artemisia vestita leaf extract formulations containing different concentrations of tragacanth gum.

Composition	F1	F2	F3	F4	F5	F6	F7	F8
Aqueous extract (g)	1	1	1	1	1	1	1	1
Tragacanth gum (g)	0.25	0.5	0.75	1.0	1.25	1.5	1.75	2.0
Milli-Q water (mL)	100	100	100	100	100	100	100	100
Methylparaben (mL)	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Propylparaben (mL)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Propylene glycol (mL)	10	10	10	10	10	10	10	10
Triethanolamine (mL)	Adjust pH to 6–6.5

F: formulation.

) of tragacanth gum (Sigma-Aldrich (Merck), St. Louis, MO, USA) was immersed in distilled water and left undisturbed to swell for 30 min at room temperature. Thereafter, the mixture was stirred until the gum was uniformly dispersed. In another beaker, propylene glycol (mol. wt.- 76.09 g/mol, Sigma-Aldrich (Merck), St. Louis, MO, USA), methyl paraben, and propyl paraben (Sigma-Aldrich (Merck), St. Louis, MO, USA) were weighed and dissolved to prepare the preservative solution (Table 1). The solution was stirred until the preservatives were fully dissolved. Thereafter, this solution was added to the tragacanth gum mixture and mixed with the specified amount of extract. The volume was adjusted to 100 mL, and the pH was set to 6.8–7 by using triethanolamine to achieve the desired gel consistency. The mixture was stirred to ensure uniformity and later stored in a closed container at −20 °C until further use [29,30].

• Drug–Excipient Compatibility Study

Drug–excipient interactions were assessed based on physical compatibility. Each excipient was blended with the drug at the levels used in the gel formulation to reflect realistic drug dosage forms. To maximize molecular contact between the drug and excipients, the drug extract was thoroughly mixed with each excipient. The mixtures were placed in separate vials and stored at a temperature of 40 °C and relative humidity of 75% for 15 days to observe any changes. Additionally, the drug–excipient mixtures were kept at room temperature in different ratios for one month to observe any physical alterations. Assessing color, appearance, and clarity aids in identifying any irregularities or changes in the formulation that could impact its efficacy or visual appeal.

• Preformulation Studies

Preformulation studies were conducted to ensure the safety of the hydrogel composition. These studies are necessary to guarantee the development of safe, efficacious, and stable dosage formulations [31,32,33,34]. They provide valuable insights into the stability, compatibility, and physiochemical properties of formulations, in addition to development feasibility, thereby laying the foundation for successful formulation development. The primary aim of our study was to establish drug–excipient compatibility and assess the physicochemical properties of the newly developed formulation.

Physiochemical Characterization

• Physical appearance: The physical appearance of a formulated gel is an essential aspect with regard to its overall quality and user acceptance. Evaluation of color, appearance, and clarity helps detect any abnormalities or changes in the formulation that may affect its effectiveness or esthetics. To evaluate the color, appearance, and clarity of the hydrogel formed by incorporating A. vestita leaf extract (ALE) into tragacanth gum, the following procedures were systematically and reliably performed. Samples were prepared in identical containers to ensure uniformity. Color assessment was performed by trained observers in triplicate. Standardized color charts were used for precise measurement. Assessments were performed under standardized lighting conditions. The same trained observers evaluated the hydrogel thrice to observe visible particulates, uniformity, and texture. Clarity was measured using a transparent grid behind the gel, and a numerical scale (ranging from 1 to 5) with detailed criteria was used for scoring. Turbidity or transmittance was spectrophotometrically measured at 600 nm, with lower turbidity or higher transmittance indicating higher clarity.
• pH: The pH of the A. vestita leaf extract hydrogel (ALEH) was measured thrice using a calibrated Seven Excellence S400 pH meter (Mettler Toledo, India) to ensure accuracy. To determine the pH of the formulations using the calibrated pH meter, an optimal pH range was required (e.g., for skin compatibility). Hence, 1 g of the gel formulation was dispersed in 100 mL of distilled water and allowed to stand for 2 h. The pH of the resultant solution was then measured. Data were presented as the mean ± SEM.
• Spreadability: In total, 1 mL of the prepared ALEH was transferred onto a glass plate using a sterile syringe. A calibrated plate was placed on top of the hydrogel. Weights of increasing mass (25, 50, 100, 200, 300, 400, and 500 g) were then sequentially placed on the plate at 20 s time intervals to enable ALEH to stabilize and spread under the applied weight. After placing each weight, the radii of the ALEH formulation were measured. To ensure the reliability and variability of the results, spreadability was assessed in triplicate at ambient temperature. The area covered by the prepared ALEH was calculated using the following formula:

  P = πr²,

  where P is the surface area covered by ALEH (cm²) and r is the radius of ALEH (cm).
• Rheological properties: The viscosity of the formulation was assessed using a Brookfield viscometer. Viscosity affects spreadability, product handling, and release kinetics. The digital viscometer was loaded with the hydrogel and placed into the flow jacket of the viscometer. The flow jacket is a component of the apparatus used to regulate temperature or create specific conditions during the measurement process. An adapter was used to measure the viscosity of the formulated gel at a rotation speed of 20 rotations per minute (rpm). The temperature was kept constant at 24.8 °C by circulating water through a thermostated water jacket. Before taking readings, the sample was allowed to settle for 5–6 min. The viscosity of the hydrogels was determined by increasing the shear rate.
• Extrudability: The formulated gel (10 g) was filled into standard caps containing eight collapsible aluminum tubes. They were sealed at the end by crimping. To assess extrudability, the weights of the tubes were recorded. Subsequently, the tubes were securely positioned between two glass slides and firmly clamped in place. A 500 g weight was placed on top of the slides, following which the cap was removed to allow the extrusion of the gel. The gel extruded from the tubes was gathered and weighed. Based on the amount of gel extruded, the percentage of extruded gel was calculated.

$$\mathrm{Extrudability} (\%)=\frac{\mathrm{Weight\; of\; extruded\; gel}}{\mathrm{Total\; weight\; of\; gel\; in\; the\; tube}}\times 100$$

Extrudability was categorized as follows:

• Excellent: more than 90% extrudability;
• Good: more than 80% extrudability;
• Fair: more than 70% extrudability.
• Drug Release Study

An in vitro drug release analysis was conducted using a customized Franz diffusion cell setup. A dialysis membrane with a molecular weight cutoff of 5000 Dalton was placed between the donor and receptor compartments. In the donor compartment, 1 g of ALEH was added. Phosphate buffer solutions at different pH levels (pH 4.5 and 6.8) were added to the receptor compartment. The cells were agitated at 50 rpm.

Three sets of experiments were performed corresponding to different temperatures: 25 °C (room temperature), 37 °C (body temperature), and 40 °C (elevated temperature). For each temperature condition, the samples were divided into three groups (one for each pH level). Hydrogel samples were placed in the respective pH buffer solutions. At predetermined time intervals (0, 1, 2, 4, 6, 8, 10, and 12 h), a specific volume of release medium (1 mL) was withdrawn and replaced with an equal volume of fresh buffer to maintain sink conditions. Withdrawn samples were filtered if necessary to remove particulate matter. By maintaining consistent temperatures using incubators, 3 mL samples were extracted from the receptor compartment through a lateral tube at predetermined time points. Drug concentrations in these samples were assessed using a UV–visible spectrophotometer at a wavelength of 276 nm. The cumulative amount of drug released at each time point was calculated. The cumulative drug release amount was plotted against time for each pH and temperature condition [35].

To investigate the release mechanism, drug release data from F1–F8 formulations were analyzed using three different mathematical models: zero-order, first-order, and Higuchi models. This protocol enabled the systematic evaluation of the drug release profile of ALEH across different pH and temperature conditions, offering valuable insights into its potential performance in diverse physiological settings.

In the zero-order model, Q_t = K₀ × t. Here, Q_t is the amount of drug released at a specified time, t. The total amount of drug released is not influenced by the initial drug concentration. K₀ represents the rate constant for zero-order drug release. In the first-order model, ln Q_t = ln Q₀ − K₁t. Here, Qt denotes the amount of drug remaining at a specified time, t. On plotting the logarithm of the remaining drug concentration against time, a linear relationship is observed. The equation is typically represented as Q_t = Q₀ × e^−K1t, where Q₀ is the initial amount of drug in the gel and K₁ is the rate constant for first-order drug release.

In the Higuchi model, Q_t = K_H × t^1/2. Here, Qt is the amount of drug released at a specified time, t. On plotting the cumulative percentage of the released drug against the square root of time, a linear graph is generated. The equation is typically represented as Q_t = K_H √t, where K_H, i.e., the Higuchi release rate, is constant. These equations elucidate the kinetics of drug release from formulations through different mechanisms, providing valuable insights into the drug release behavior over time [36].

The Korsmeyer–Peppas equation is commonly used to assess the mechanism of drug diffusion from pharmaceutical formulations [37]. This equation is especially valuable for examining drug release from polymeric systems, such as hydrogels, because of its versatility in describing various release mechanisms. The equation is often expressed as follows

M_t/M_∞ = K × tⁿ

Here, M_t/M denotes the proportion of drug released at time t relative to the total drug released at infinite time (M_∞). K is the release rate constant. Moreover, n, the release exponent, offers insights into the mechanism of drug release. The value of n can signify various release mechanisms:

• Fickian diffusion or case I transport: n = 0.45;
• Non-Fickian or anomalous transport: 0.45 < n < 0.89;
• Case II transport or relaxation-controlled release: n = 0.89;
• Super case II transport: n > 0.89.

All experiments were conducted in triplicate for every formulation, and the optimal formulation was identified based on statistical data.

• Antimicrobial Activity Assay

The bacterial cultures used in this study included Escherichia coli (MTCC 443), Bacillus subtilis (MTCC 2395), Staphylococcus aureus (MTCC 3160), Streptococcus pyogenes (MTCC 1927), Aspergillus flavus (MTCC 9390), Aspergillus niger (MTCC 281), and Candida albicans (MTCC 183). These cultures were procured from the Microbial Type Culture Collection and Gene Bank (IMTECH, Chandigarh). The strains were cultured overnight before assessing antimicrobial activity. Subsequently, microbial cell suspensions were prepared in 0.5% NaCl and standardized to a 0.5 McFarland standard, resulting in a final concentration of 10⁶ colony-forming units per milliliter (CFU/mL).

• Agar Well Diffusion Technique

The agar well diffusion technique outlined by Bauer et al. (1966) was employed to evaluate the antimicrobial activity of ALEH [38]. Nutrient agar and potato dextrose agar (Sigma Aldrich, St. Louis, MO, USA) were sterilized by autoclaving and subsequently poured into Petri dishes within a laminar airflow hood. The agars were then allowed to solidify overnight. The next day, 500 μL of overnight bacterial culture was evenly distributed onto the solidified nutrient agar using an L-shaped spreader. Subsequently, 6 mm diameter wells were created in the agar medium using a cork borer. A small fraction (100 μL) of ALEH (25, 50, and 100 µg/mL) was added to the first three wells using a sterile pipette. Furthermore, 10 µg/mL of the positive control containing fluconazole (for fungi) or azithromycin (for bacteria) was added to the last well. Subsequently, the plates were incubated for 24 h at 28 °C for fungi and 37 °C for bacteria. The antimicrobial efficacy of ALEH was assessed by measuring the zone of inhibition (ZOI) using a ruler, and the results were expressed in millimeters (mm). To assess efficacy, the size of ZOI produced by ALEH was compared with that produced by the control against the tested microorganisms. The experiments were performed in triplicate.

• Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration (MIC) Assays

The MIC and MBC of ALEH were determined using the serial dilution technique following the protocol established by the Clinical and Laboratory Standards Institute (CLSI), as described previously [39]. Nutrient broth (HiMedia, Mumbai, India) was used to culture S. aureus, B. subtilis, E. coli, and S. pyogenes, while potato dextrose broth (HiMedia, Mumbai, India) was used to culture C. albicans, A. niger, and A. flavus. In the initial phase, ALEH concentrations ranging from 10 to 40 µg/mL were tested; however, no turbidity was observed. In the second phase, the ALEH concentrations were increased to 100, 150, 200, 250, and 300 µg/mL. A negative control that did not contain ALEH was included to confirm the presence of adequate fungal or bacterial growth and the sterility of the media.

The dilutions were incubated for 24 h and then observed. Turbidity in the broth cultures indicated microbial growth; it was observed by visual inspection. MIC was considered to be the minimum concentration of ALEH at which no turbidity was visually observed. MBC was considered to be the minimum concentration of ALEH required to kill 99.9% of the initially inoculated microbes. The experiments were conducted in triplicate. Both MIC and MBC help determine the effectiveness of formulations and select the appropriate concentrations of formulations for therapeutic use.

• Cell Culture

The HaCaT cell line sourced from the National Centre for Cell Science (NCCS) (Pune, India) and stored at Lovely Professional University (Punjab, India) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Waltham, MA, USA). The medium was supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (100 U/mL penicillin and 100 U/mL streptomycin). Cultures were maintained in a humidified atmosphere at 37 °C with 5% CO₂. Cell passage was performed upon reaching 70–80% confluence [40].

• Wound Healing Assay

The wound healing potential of ALEH was evaluated by performing a wound healing assay in HaCaT cells, as described previously [41]. In brief, HaCaT cells were plated at a density of 10,000 cells/well in a six-well culture plate and cultured for 24 h in DMEM containing 10% FBS and 1% antibiotic solution (penicillin and streptomycin) at 37 °C with 5% CO₂. After 24 h, 70–80% confluent cells were rinsed with phosphate-buffered saline (PBS) and then scratched using a 200 µL pipette tip. Subsequently, the cells were exposed to the designated concentrations of ALEH (125 μg/mL) without disrupting neighboring cells. Cipladine (5 μg/mL), a standard drug frequently employed for wound repair purposes [42,43], was utilized as the positive control, while untreated cells served as the negative control. The cell culture plates were placed in an incubator under optimum conditions (37 °C with 5% CO₂). Photographs were captured at specific time intervals of 0 and 24 h after scratching to assess the rate of cell migration and wound closure in the presence of ALEH. The gap area was assessed using Image J software version 1.54 (developed by Wayne Rasband at the National Institutes of Health (NIH), Bethesda, MD, USA). Wound closure in the untreated, positive control, and hydrogel-treated groups was compared to evaluate the potential effects of the formulation on wound closure.

• Statistical Analysis

The experiments were conducted in triplicate, and the outcomes were averaged and visualized using Microsoft Excel and Graph Pad Prism version 6.0 (Graph Pad, San Diego, CA, USA). The data are depicted as average values with their corresponding standard deviations.

3. Results and Discussion

The primary aim of this study was to improve the stability of ALE and to augment its antimicrobial and wound healing properties through the development of gel formulations. Previous research on A. vestita [23] has examined the pharmacological properties of ALE. However, our study aimed to enhance the shelf life and stability of ALE by incorporating it into tragacanth gum to form a hydrogel (ALEH). To achieve this goal, the prepared formulations were subjected to comprehensive assessments, including the assessment of physical appearance, pH, spreadability, extrudability, viscosity, stability, in vitro drug release profiles, antimicrobial activity, and wound healing activity. Ultimately, the aim was to develop gel formulations with improved stability, better antimicrobial activity, improved wound healing properties, and controlled drug release, laying the groundwork for their potential application in dermatological or wound management settings.

3.1. ALEH Preparation

Tragacanth gum, serving as a gelling agent, was used at various concentrations during the formulation process to prepare eight batches of gels containing A. vestita extract. Gel formulations were prepared according to the standardized composition specified in Table 1.

Physical Assessment of Compatibility

To evaluate the physical appearance, pH, spreadability, and extrudability of the eight formulations, samples were withdrawn initially and after a month to monitor the quality and stability of the formulations over time. After storing the excipients with the drug extract for a month, no noticeable physical changes were observed. No discoloration, caking, or liquefaction was noted initially (day 0). The results remained consistent after 30 days (Figure 2 and

Table 2. Compatibility study of drugs and excipients used for ALEH preparation.

Batch	Discoloration		Caking		Liquefaction
	Initial	After 30 Days	Initial	After 30 Days	Initial	After 30 Days
Aqueous extract	No	Consistent	No	Consistent	No	Consistent
Tragacanth gum	No	Consistent	No	Consistent	No	Consistent
Aqueous extract + Tragacanth gum	No	Consistent	No	Consistent	No	Consistent

).

3.2. Physiochemical Characterization

Preformulation studies are vital for the development of hydrogel formulations, as they offer valuable insights into the physicochemical properties, stability, and compatibility of the formulation. These studies are crucial for ensuring the safety and efficacy of the hydrogel for therapeutic applications. By thoroughly investigating various factors, such as solubility, pH, viscosity, and compatibility with other ingredients or excipients, preformulation studies help optimize the formulation to meet the desired requirements and performance criteria. Adopting this proactive approach early in the development process can help identify potential issues and pave the way for a successful formulation with enhanced therapeutic outcomes.

3.2.1. Physical Appearance

None of the formulations contained clumps or particles. Tragacanth gum was found to have a translucent appearance. Good homogeneity was observed in all formulations (F1–F8) with the absence of lumps. Moreover, they appeared clear and transparent. All formulations were found to be homogeneous light-yellow gel preparations. The homogeneous appearance indicated the uniform distribution of ingredients throughout the gel, which is crucial for ensuring consistent dosage and efficacy. The light-yellow color suggested that the formulations were visually consistent and met the intended specifications. The findings of this visual inspection provide assurance regarding the quality and uniformity of the gel formulations, which are preferred characteristics in the formulation development process.

3.2.2. pH

Skin pH is influenced by various factors, including external factors (such as deter- gents, dermatological drugs, and cosmetics) and internal factors (such as age, sex, sweat composition, and sebum secretion intensity) [44,45]. Maintaining an acidic pH on the skin surface is advantageous for the physiology of the epidermis (i.e., the outer layer of the skin) and the skin microflora (i.e., the community of microorganisms that naturally inhabit the skin). It helps preserve the integrity of the stratum corneum, which is the outermost layer of the epidermis, and the lipid barrier, both of which play essential roles in skin hydration and protection. The acidic pH of the skin contributes to the antimicrobial response and the process of skin barrier regeneration [46].

Table 3. Characteristics of formulated hydrogels.

Formulation Code	pH	Spreadability (g/cm2)	Viscosity (cP)
F1	6.86 ± 0.1	15.08 ± 0.085	2318 ± 3.78
F2	6.93 ± 0.15	14.75 ± 0.16	2540 ± 2.51
F3	6.83 ± 0.05	13.03 ± 0.09	2463 ± 5.13
F4	7.03 ± 0.11	15.75 ± 0.19	2450 ± 2.08
F5	7.16 ± 0.05	12.84 ± 0.08	2637 ± 2.08
F6	6.90 ± 1.08	11.63 ± 0.07	2429 ± 3.21
F7	6.83 ± 0.05	12.83 ± 0.11	2318 ± 2.08
F8	6.9 ± 0.10	14.31 ± 0.11	2411 ± 1.15

cP—centipoise.

presents the pH levels of different formulation batches; all batches exhibited satisfactory results. In this study, the highest pH levels were those of F2, F4 and F5—of 6.93 ± 0.15, 7.03 ± 0.11, and 7.16 ± 0.05, respectively—while the lowest pH levels recorded for the ALEH formulations were those of F3, F7, F1, F8 and F6—of 6.83 ± 0.05, 6.83 ± 0.05, 6.86 ± 0.1, 6.9 ± 0.10, and 6.90 ± 1.08, respectively. Overall, the pH levels of the ALEH formulations suggest that they can be applied to the skin with out causing irritation. The closeness of these pH levels to the natural skin pH, typically ranging from 4.5 to 5.5, indicates that these formulations are compatible with the physiology of the skin [47].

3.2.3. Spreadability

Spreadability plays an important role in ensuring the uniform distribution of a product on the skin. It is determined by various factors, such as the expansion of the surface area of a hydrogel when pressure is applied. The smoother the spread, the more likely are patients to apply it consistently and evenly. Thus, ease of spread enhances patient compliance and promotes uniform application across the skin. The spreadability of ALEH was evaluated in this study. All formulations exhibited acceptable spreadability (Table 3). The highest spreadability was 15.75 ± 0.19 g/cm², while the lowest was 11.63 ± 0.07 g/cm². Based on spreadability, the ALEH formulations could be grouped in ascending order as follows: F6 < F7 < F5 < F3 < F8 < F2 < F1 < F4.

3.2.4. Rheological Properties

The viscosity of ALEH was assessed in triplicate using a Brookfield viscometer (LV-61 spindle); the data are depicted as the average of three measurements (Table 3 and Figure 3).

3.2.5. Extrudability

Achieving the right consistency of gel formulations is critical for ensuring easy extrusion from collapsible aluminum tubes, particularly during application. Gels with low consistency tend to flow too quickly, potentially leading to wastage. On the other hand, highly viscous gels may be difficult to extrude from the tube, causing inconvenience to the user. In this context, the extrudability of tragacanth gum was found to be satisfactory. Thus, tragacanth gum, likely used as a thickening or gelling agent in formulations, can effectively balance viscosity to enable smooth extrusion from the tube without being too thin or too thick. This finding is significant because it contributes to the usability and practicality of the gel formulation, ensuring a seamless application experience for the end user. F4, F6, and F7 demonstrated excellent extrudability, with a high percentage of the gel being successfully extruded from the tubes. F2, F3, and F8 showed good extrudability, with a substantial amount of the gel being extruded; however, this amount was slightly lower than that in formulations with excellent extrudability. F1 and F5 exhibited fair extrudability, with a moderate amount of the gel being extruded; therefore, these formulations need improvement in terms of ease of extrusion.

3.2.6. Optimization

According to the ICH guidelines, gels were prepared and packaged in collapsible (aluminum) tubes [48,49]. After a 1-month interval, samples of the gels were collected to evaluate pH, spreadability, extrudability, and physical appearance. Among the formulations, F4 exhibited favorable results (in terms of spreadability, pH, and viscosity) following batch optimization, indicating its suitability for further development or use. F4 was therefore used for further evaluations of antimicrobial activity and in vitro wound healing activity.

3.3. Drug Release Study

An in vitro drug release study was conducted on batches F1–F8 using Franz diffusion cells. This analysis provided valuable information regarding the release kinetics and mechanisms of the formulations. The results are presented in

Table 4. Drug release study of eight distinct formulations at 25 °C.

Time (h)			Cumulative Percentage of Drug Release (25 °C)
	F1	F2	F3	F4	F5	F6	F7	F8
0	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
0.25	7.982 ± 0	6.390 ± 0	12.58 ± 0	3.878 ± 0	3.291 ± 0	13.593 ± 0	5.553 ± 0	10.578 ± 0
0.5	12.664 ± 0	9.282 ± 0	17.362 ± 0	11.995 ± 0	8.969 ± 0	23.831 ± 0	10.940 ± 0	22.096 ± 0
1	29.078 ± 0	24.540 ± 0	25.912 ± 0	17.259 ± 0	16.349 ± 0	31.840 ± 0	25.395 ± 0	32.333 ± 0
2	46.067 ± 0	32.814 ± 0	38.144 ± 0	24.633 ± 0	21.613 ± 0	42.010 ± 0	39.883 ± 0	40.337 ± 0
4	60.369 ± 0	50.540 ± 0	52.285 ± 0	33.069 ± 0	42.721 ± 0	57.061 ± 0	53.896 ± 0	56.109 ± 0
6	68.155 ± 0	61.488 ± 0	63.093 ± 0	47.778 ± 0	52.014 ± 0	72.312 ± 0	69.515 ± 0	75.699 ± 0
8	80.596 ± 0	75.400 ± 0	78.201 ± 0	61.512 ± 0	70.020 ± 0	84.246 ± 0	77.050 ± 0	82.176 ± 0
10	88.988 ± 0	84.709 ± 0	86.468 ± 0	76.508 ± 0	75.468 ± 0	87.848 ± 0	83.787 ± 0	88.752 ± 0
12	98.440 ± 0	93.254 ± 0	94.200 ± 0	86.587 ± 0	84.431 ± 0	92.244 ± 0	91.636 ± 0	96.267 ± 0

and Figure 4 as a cumulative percentage of drug release over time. The time required for the release of 50% of the total drug content from each formulation (F1–F8) was also determined.

An in vitro drug release study was conducted to determine the percentage of the drug released from the developed hydrogels at two different pH levels: pH 4.5 and 6.8. The maximum drug release percentage was observed at pH 6.8 (

Table 5. Drug release study of F4 at different pH levels and temperatures.

Time (h)	Cumulative Percentage of Drug Release (F4)
	pH 4.5 Temperature 37 °C	pH 4.5 Temperature 40 °C	pH 6.8 Temperature 37 °C	pH 6.8 Temperature 40 °C
0	0.000	0.000	0.000	0.000
0.25	4.811	1.604	1.815	11.028
0.5	11.248	9.510	5.961	15.542
1	18.702	14.781	15.818	22.267
2	23.784	23.031	22.083	33.138
4	32.819	29.448	29.361	40.785
6	42.532	34.260	43.272	50.274
8	49.760	40.792	47.879	65.198
10	64.329	51.677	68.147	74.411
12	74.380	54.656	81.044	79.110

and Figure 5).

Drug release and swelling of the hydrogel were observed to be greater at pH 6.8 than at pH 4.5 at 37 °C and 40 °C, indicating the pH-responsive nature of the hydrogel. The polymer volume fractions were lower at high pH, indicating the maximum swelling capacity of the hydrogel at higher pH levels. Thus, the prepared hydrogel could be applied externally for the controlled delivery of drugs.

The drug release data from batches F1–F8 were analyzed by fitting them to different kinetic models, including zero-order, first-order, and Higuchi models, as well as the Korsmeyer–Peppas equation (Figure 6). This enabled the identification of the most suitable model that best describes the release behavior of the formulations. The most suitable drug release model and mechanism were confirmed based on the regression coefficient (R²). Drug release data revealed an R² value of 0.9143 for the zero-order model, 0.9902 for the first-order model, and 0.9926 for the Higuchi model. To assess the mechanism of drug release from the gels, in vitro drug release data were fitted into the Korsmeyer–Peppas equation. All formulations exhibited non-Fickian diffusion, indicating a complex release mechanism involving both diffusion and polymer relaxation.

• Antimicrobial Activity

The antimicrobial activity of ALEH was evaluated against microbial strains by using the agar well diffusion method. These pathogens are commonly associated with skin infections and wound environments [50,51]. These strains included S. pyogenes, S. aureus, E. coli, B. subtilis, A. flavus, A. niger, and C. albicans. The potency of the hydrogel was investigated by comparing its ZOI with that of the standard drug azithromycin for bacteria or fluconazole for fungi. Moreover, MIC and MBC values were determined. ALEH exhibited significant antimicrobial activity against the tested microbial strains, showing notable effectiveness in comparison with the standard drugs. In particular, ALEH demonstrated high antimicrobial activity against E. coli, S. pyogenes, and C. albicans, showing the maximum ZOIs (19.6, 20.3, and 19.4 mm, respectively). On the other hand, intermediate ZOIs were observed against S. aureus, B. subtilis, A. niger, and A. flavus (15, 15.1, 14.8, and 17.5 mm, respectively) at an ALEH concentration of 100 µg/mL. The ZOI data are presented in

Table 6. Antimicrobial activity of A. vestita leaf extract hydrogel (ALEH).

Microbes	Zone of Inhibition (mm)				MIC and MBC/MFC (µg/mL)
	25 µg/mL	50 µg/mL	100 µg/mL	Standard Drug (Azithromycin/Fluconazole)	MIC	MBC/MFC
S. aureus	-	12.14 ±0.52	15.0 ± 0.26	20.4 ± 0.22	110	220
E. coli	6.4 ± 0.84	14.6 ± 0.28	19.6 ± 0.51	22.2 ± 0.31	200	240
B. subtilis	-	13.46 ± 0.40	15.1 ± 0.17	17.4 ± 0.23	160	200
S. pyogenes	12.12 ± 0.9	18.1 ±0.70	20.3 ± 0.60	23.1± 0.20	200	250
A. flavus	12.3 ± 0.22	15.2 ± 0.31	17.5 ± 0.21	20.3 ± 0.23	100	260
A. niger	-	10.7 ± 0.42	14.8 ± 0.50	16.8 ± 0.14	100	220
C. albicans	10.2 ± 0.36	12.1 ± 0.32	19.4 ± 0.01	22.1 ± 0.24	200	250

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; MFC: minimum fungicidal concentration; and ALEH: A. vestita leaf extract hydrogel.

.

The antibacterial activity based on the ZOI values was as follows in descending order: S. pyogenes (20.3 mm) > E. coli (19.6 mm) > B. subtilis (15.1 mm) > S. aureus (15 mm). The antifungal activity was as follows: C. albicans (19.4 mm) > A. flavus (17.5 mm) > A. niger (14.8 mm). No ZOI was observed for A. niger, B. subtilis, and S. aureus at a concentration of µg/mL. Moreover, the MBC and MIC values fell within the range of 100–250 µg/mL for bacteria, while the minimum fungicidal concentration (MFC) and MIC values fell within the range of 100–300 µg/mL for fungi. These values were deemed satisfactory in comparison with those of the standard drugs fluconazole and azithromycin. Overall, these findings reveal the promising antimicrobial potential of ALEH in the treatment of dermal infections. In prior research, a combination of tragacanth gum, acrylic acid, and amphotericin B has been shown to reduce swelling in mice infected with C. albicans [52].

3.4. Wound Healing Activity

At present, the in vitro scratch assay is widely adopted to assess wound closure and cell migration dynamics. In this study, HaCaT cells were exposed to ALEH for 24 h. Cell migration was observed at the start (0 h) and end (24 h) of the experiment, and the extent of wound closure was measured using NCBI Image J software version 1.54. ALEH at a concentration of 125 μg/mL resulted in approximately 95% wound closure within 24 h. Figure 7 presents the percentage of wound closure at the end (24 h) relative to that at the start (0 h); both cipladine-treated (positive control) cells and ALEH-treated cells were compared.

As shown in Figure 8, 94% wound closure was achieved with ALE treatment (to Figure 7F), while 99.05% closure was achieved on treatment with the standard drug cipladine (to Figure 7D) within a 24 h period, yielding satisfactory outcomes. At 24 h, wound healing in the ALE-treated group (p = 0.05) was significantly higher than that in the control group (Figure 8).

A previous study demonstrated that the combination of tragacanth gum and silver nanoparticles for enhancing antibacterial activity is particularly intriguing. Silver nanoparticles have been studied for their antimicrobial properties, and combining them with tragacanth gum could potentially lead to innovative biomedical applications, especially for combating bacterial infections [53,54]. Anti-inflammatory, antibacterial, antioxidant, and anticancer agents [55] are the most delivered therapeutics using tragacanth gum. In a prior study, the use of tragacanth gum dressing bandages for treating burns led to swift and complete wound recovery; the outcomes were superior to those in the control group. In that study, a rat with full thickness was treated with a solution of tragacanth gum twice a day for 10 days [56]. It exhibited beneficial effects in topical treatments by accelerating both wound healing and wound contraction due to the presence of active constituents, i.e., tragacanthin and bassorin, which were found to be responsible for its healing properties. The entire data mentioned in that manuscript have also been disclosed in the patents.

The disclosed information pertains to a hydrogel formulation designed for topical application and drug delivery. It consists of the natural polymer tragacanth gum and an extract from the medicinal herb A. vestita. This hydrogel is intended for treating wounds. Moreover, there is a recognized need to develop a novel composition with extended shelf life. A. vestita has long been used in traditional medicine by gathering it from wild forests. It has also been used to produce therapeutic agents for treating fever, inflammation, and skin conditions. Its therapeutic applications have been documented in China and Tibet, where it has been used to treat various ailments, such as sepsis and contact dermatitis. It can also treat abdominal pain [24,25,26,27,28]. Moreover, the paste of crushed A. vestita leaves can be applied externally to control bleeding on the skin. The incorporation of natural gums, such as tragacanth gum, into the formulation adds various functionalities, such as emulsification, stabilization, and thickening, as they are polysaccharides derived from plant exudates. Apart from tragacanth gum (Astragalus gummifer), other natural gums, such as guar gum (Cyamopsis tetragonoloba), gum acacia (Acacia arabica), xanthan gum (Xanthomonas campestris), psyllium polysaccharides (Plantago ovata), gum ghatti (Anogeissus latifolia), and locust bean gum (Ceratonia siliqua), can be utilized for gel formulations [57,58,59,60,61,62,63,64]. Various preservatives, such as methylparaben, ethylparaben, and butylparaben, and humectants, such as propylene glycol and glycerine, can also be added to the formulation. Moreover, pH stabilizers, such as triethanolamine, sodium hydroxide, and potassium hydroxide, can be used to maintain the pH of the formulation [65,66]. Tragacanth gum plays various roles in hydrogel formulations, contributing to their structure, viscosity, hydration, biocompatibility, and controlled drug release. Slight alterations observed in the viscosity, extrudability, and spreadability of the hydrogel despite a significant increase in the concentration of tragacanth polysaccharide could be attributed to the distinctive characteristics of this polysaccharide and the inherent behavior of the hydrogel [52,67,68]. These include the formation of a gel network with an increase in concentration, which imparts stability and viscosity to the hydrogel. Additionally, the rheological properties facilitate easy extrusion and spreadability even at higher concentrations. Furthermore, the high water-holding capacity of the polysaccharide ensures that the gel remains hydrated and flexible. Intermolecular interactions, such as hydrogen bonding and electrostatic interactions, play a role in stabilizing the gel network [69]. Moreover, synergistic effects help enhance the stability and functionality of the hydrogel, compensating for any variations in concentration. In ALEH formulations, the porous nature of hydrogels containing tragacanth gum enables the controlled release of ALE incorporated into the gel matrix. To characterize hydrogels based on tragacanth gum, a range of physical and chemical characterization techniques can be utilized. Physical characterization methods include rheological studies, which evaluate the mechanical properties and viscoelastic behavior of hydrogels, and swelling studies, which assess water absorption capacity and hydrogel stability [70].

Preformulation studies are essential for developing effective, stable, and safe dosage forms. Their primary objective is to examine the physicochemical properties of a newly synthesized drug molecule and assess its compatibility with the excipients intended for the formulation. Hydrogel matrices, typically comprising over 90% of water, create an ideal environment for maintaining moisture at the wound site [71]. They accelerate various processes, such as granulation hyperplasia, epidermal restoration, and excess dead tissue removal. Hydrogels can exhibit in situ crosslinking, prevent wound desiccation, and facilitate the autolytic debridement of necrotic areas [72]. Moreover, they can accommodate various active wound healing agents and antimicrobial agents, facilitating cell encapsulation. Their minimal adhesion to wounds simplifies dressing removal, reducing discomfort and the risk of infection [73].

The antimicrobial activity of ALEH aligns with previously published data on A. vestita extract (ALE). The A. vestita extract exhibited significant antimicrobial effects against E. coli, S. pyogenes, and C. albicans, with the largest ZOIs measuring 17.6, 17.3, and 17.6 mm, respectively. Intermediate ZOIs were observed against S. aureus, B. subtilis, A. niger, and A. flavus (14.2, 13.1, 15.3, and 12.7 mm, respectively). Similarly, ALEH exhibited strong antimicrobial activity against E. coli, S. pyogenes, and C. albicans, with the maximum ZOIs being 19.6, 20.3, and 19.4 mm, respectively. Intermediate ZOIs of 15, 15.1, 14.8, and 17.5 mm were observed against S. aureus, B. subtilis, A. niger, and A. flavus, respectively, at an ALEH concentration of 100 µg/mL. The wound healing activity of ALEH closely resembles that of ALE, indicating that tragacanth gum supports the extract without diminishing its wound healing efficacy. Initially, ALE treatment resulted in 94.625% wound closure within 24 h at a concentration of 125 μg/mL. After forming a hydrogel, treatment with the resulting ALEH led to approximately 95% wound closure under the same conditions.

Topical drug delivery is vital for treating different dermatological conditions and localized skin disorders. Tragacanth gum-based hydrogels offer significant advantages in topical drug delivery. These hydrogels possess exceptional mucoadhesive properties, allowing them to stick to the skin surface, improve drug retention, and facilitate effective drug penetration through the skin barrier [52]. Moreover, tragacanth gum-based hydrogels exhibit favorable biocompatibility, biodegradability, and water absorption capacity, making them suitable for sustained drug release and prolonged therapeutic effects [74]. In topical hydrogels, controlled drug release strategies involve the incorporation of drug-loaded nanoparticles, lipid-based vesicles, or microparticles into the hydrogel matrix. These methods help enhance drug stability, regulate release kinetics, and improve drug penetration into the skin layers. Recent advancements in topical drug delivery using tragacanth gum-based hydrogels have shown promising effectiveness across various applications [53]. For example, their application in wound healing [75] has led to faster healing rates, reduced infection risks, and improved tissue regeneration [76]. Furthermore, integrating growth factors or plant-derived extracts into these hydrogels has resulted in favorable outcomes in treating skin conditions, such as psoriasis and atopic dermatitis [77]. Recent research has focused on improving oral drug delivery through the use of tragacanth gum-based hydrogels. For example, incorporating nanoparticles or utilizing stimuli-responsive hydrogels enables site-specific drug release and increases therapeutic efficacy [78].

The distinctive properties of tragacanth gum-based hydrogels, including their high water absorption capacity, biocompatibility, and adjustable gelation kinetics, make them ideal for drug delivery applications [52]. These hydrogels can safeguard sensitive drugs from degradation, offer sustained release profiles, and enable localized delivery to specific target areas [79]. Moreover, the integration of bioactive molecules or nanoparticles into the hydrogel matrix can further improve drug-loading efficiency and therapeutic effectiveness.

The pH-responsive properties of hydrogels prepared from tragacanth gel enable various applications. In drug delivery systems, the pH-dependent swelling behavior can be utilized to achieve controlled release of therapeutic agents [80]. In our study, the hydrogel exhibited greater drug release and swelling at pH 6.8 than at pH 4.5 at both 37 °C and 40 °C, demonstrating its pH-responsive nature. The polymer volume fractions were lower at higher pH levels, indicating the maximum swelling capacity of the hydrogel in more alkaline conditions. Therefore, it can be concluded that the prepared hydrogel is suitable for external use in controlled drug delivery.

Overall, our research unravels a novel hydrogel composition containing A. vestita extract and tragacanth gum for various therapeutic applications, particularly in skincare and wound healing. The hydrogel formulation can effectively accelerate wound healing and facilitate gap closure, promoting tissue regeneration and repair. Qualitative assessment of cell alignment, morphology, and organization at the wound edge can provide additional insights into the impact of the formulation on cell behavior.

4. Conclusions

This study highlights the antimicrobial and wound healing benefits of a hydrogel containing ALE, making it well-suited for both skincare and cosmetic applications. The ALE-based hydrogel (ALEH) shows promise as a topical treatment that can accelerate wound healing and fight off pathogens responsible for skin infections. The hydrogel is stable, environmentally friendly, and cost-effective, making it an appealing alternative to synthetic treatments in both the cosmetics and pharmaceutical sectors.

In vitro testing showed that the hydrogel achieved 95% wound closure within 24 h, demonstrating its rapid healing potential. Additionally, with a 92.26% drug release at pH 6.8 and 40 °C, it shows promise for pH-responsive drug delivery. While the study confirms significant results in wound care, it acknowledges the need for further in vivo testing to confirm clinical effectiveness. The combination of A. vestita extract and tragacanth gum in the hydrogel provides strong antimicrobial action, making it a natural and cost-efficient solution for wound care, potentially replacing synthetic alternatives.