Formulation Development and Evaluation of Indian Propolis Hydrogel for Wound Healing

Flavonoids and polyphenolic compounds play a key role in wound healing cycle modulation. Propolis, a natural bee product, has been widely reported as an enriched source of polyphenols and flavonoids as important chemical constituents and for its wound healing potential. The goal of this study was to develop and characterize a propolis-based polyvinyl alcohol (PVA) hydrogel composition with wound healing potential. To understand the impacts of critical material attributes and process parameters, formulation development was carried out using a design of experiment approach. A preliminary phytochemical analysis of Indian propolis extract showed the presence of flavonoids (23.61 ± 0.0452 mg equivalent of quercetin/g) and polyphenols (34.82 ± 0.0785 mg equivalent of gallic acid/g), both of which aid in wound healing and skin tissue regeneration. The pH, viscosity, and in vitro release of the hydrogel formulation were also studied. The burn wound healing model results revealed significant (p < 0.0001) wound contraction by propolis hydrogel (93.58 + 0.15%) with rapid re-epithelialization relative to 5% w/w povidone iodine ointment USP (Cipladine®) (95.39 + 0.16%). The excision wound healing model confirms significant (p < 0.0001) wound contraction by propolis hydrogel (91.45 + 0.29%) with accelerated re-epithelialization comparable to 5% w/w povidone iodine ointment USP (Cipladine®) (94.38 + 0.21%). The developed formulation offers promise for wound healing, which may be investigated further for clinical research.


Introduction
Propolis (bee glue) is a resinous and sticky material that bees acquire to build and alter their hives [1]. Propolis has almost 300 chemical constituents, including polyphenols, amino acids, sesquiterpenes, quinine, coumarins, steroids, and some inorganic substances. Unlike many natural remedies, propolis has a comprehensive database on its biological activity and toxicity, indicating that it has a wide range of pharmacological activities such as antibacterial, antifungal, antiviral, and anticancer activities in many traditional texts such as ayurveda, sidhha, Chinese medicine, and so on [2]. To extract the physiologically active components of propolis, several extraction procedures were applied. Extraction procedures such as conventional maceration, ultrasonic, and microwave-aided extraction were used to enrich the active components and hence increase pharmacological response [3][4][5][6].
Wound healing is a multistage tissue regeneration process that consists of inflammatory, proliferative, and remodeling stages [7]. To enhance rapid tissue regeneration, dead tissue is replaced with fresh healthy cells, which reduces the acute inflammatory reactions

Extraction and Standardization of Propolis
The propolis extract was standardized in terms of specific wound healing indicators using a developed and validated RP HPLC method [24]. Total polyphenols, total flavonoids, and pesticidal content were also evaluated in the extract. Total polyphenol content was 34.82 + 0.072 mg equivalent of gallic acid/g and 23.61 + 0.045 mg equivalent of quercetin/g, respectively. A total of 113 pesticides were confirmed to be absent or within limits in the sample [24].

Formulation Optimization
From the initial screening for polyvinyl alcohol at different concentration ranges from 5 to 15%,the concentrations from 8% to 10% were observed with desirable ranges of responses in terms of viscosity (29,000-32,000 cps) as well as in vitro drug release (93. .79%), so these concentrations were selected for the further experimental design. The incorporation of Indian propolis extract into the prepared gel bases resulted in a brownish hue with a pH of 5.4, which is within the acceptable pH range (5-5.5) for topical treatments. Tables 1 and 2 shows details of the 3 2 factorial design that was used to optimize the formulation. Effect on Drug Release and Viscosity Polymer concentration and stirring speed were identified as critical parameters needing optimization in the formulation development process to obtain a final optimized formulation with desirable characteristics.
The responses of these batches are shown in Table 2. Multiple regression analysis was conducted using Design Expert ® Version 10.0.
Positive coefficients of the main terms X1 and X2 for in vitro drug release and negative coefficients of the main term X2 for viscosity indicated a favorable effect.
The polymer concentration and stirring speed both had a linear effect on in vitro drug release and viscosity as seen in the response surface plot (Figure 1). Figure 1 also reveals that both polymer concentration and stirring speed show desirable effects with their optimized combination as shown in the solution for optimum batch selection, where the highest desirability of 1.000 was obtained at 9.564% polymer concentration and 739.21 rpm for hydrogel formulation at the desired in vitro drug release and viscosity, so F2 was selected as an optimized formulation and further evaluated for various parameters. Figure 2 shows the image of the representative optimized batch of propolis hydrogel.

Visual Appearance
The prepared formulation was inspected visually and the results of same were observed as given below in Table 3. for various parameters. Figure 2 shows the image of the representative optimized batch of propolis hydrogel.

Visual Appearance
The prepared formulation was inspected visually and the results of same were observed as given below in Table 3. The viscosity of developed formulation batches was analyzed and observed in the range of 24,200 to 34,200 cps. Viscosity was evaluated as a dependent variable to optimize the formulation batch. The pH of the gel formulation was determined using a digital pH meter and it was found to be 5.4 ± 0.246 pH; a formulation in the slight acidic range favors intact drug delivery through the skin as it remains acidic at the surface as well as in different skin layers.  for various parameters. Figure 2 shows the image of the representative optimized batch of propolis hydrogel.

Visual Appearance
The prepared formulation was inspected visually and the results of same were observed as given below in Table 3. The viscosity of developed formulation batches was analyzed and observed in the range of 24,200 to 34,200 cps. Viscosity was evaluated as a dependent variable to optimize the formulation batch. The pH of the gel formulation was determined using a digital pH meter and it was found to be 5.4 ± 0.246 pH; a formulation in the slight acidic range favors intact drug delivery through the skin as it remains acidic at the surface as well as in different skin layers.  The viscosity of developed formulation batches was analyzed and observed in the range of 24,200 to 34,200 cps. Viscosity was evaluated as a dependent variable to optimize the formulation batch. The pH of the gel formulation was determined using a digital pH meter and it was found to be 5.4 ± 0.246 pH; a formulation in the slight acidic range favors intact drug delivery through the skin as it remains acidic at the surface as well as in different skin layers.

FT-IR Study
The FTIR spectrum of propolis extract was recorded in the range of 400-4000 cm −1 . Various modes of vibrations were identified and assigned to determine the different functional groups present in the propolis extract.
As shown in Figure 3, the FT-IR spectrum of propolis extract showed various peaks at 3571.52 cm −1 , 3541.63 cm −1 , 3485.7 cm −1 , and 3333 cm −1 showing -OH stretching. The band obtained at 3083.62 cm −1 was assigned to C-H stretching. The strong and narrow peaks at 1639.2 cm −1 , 1594.8 cm −1 , and 1164.7 cm −1 were attributed to C=O, C=C, and C-O stretching, respectively.          The FTIR spectrum of the propolis hydrogel formulation shows peaks of -OH stretching at 3571.52 cm −1 and 3541.63 cm −1 ; C-H stretching at 3083.62 cm −1 , 1639.2 cm −1 , 1594.8 cm −1 , and 1164.7 cm −1 with decreased intensity; and prominent peaks at 3325 cm −1 due to the O-H stretching vibration, 2937 cm −1 due to the C-H stretching vibration, 1419 cm −1 due to C-O carbonyl stretching, and 1245 cm −1 due to the C-H bending vibration of CH 2 , confirming the presence and distribution of propolis in the hydrogel formulation.

InVitro Drug Release Study
The drug release pattern from the hydrogel formulation is closely related to the drug interaction and matrix structure. The propolis release pattern obtained from the hydrogel formulation is presented in Figure 5. The release study reveals a biphasic drug release pattern where an initial burst release pattern of the drug was observed during first 40 min, with about 40% of the drug being released, followed by a comparatively slow release up to 4 h. The pattern might be due to the dense hydrogel matrix with strong physical drug-hydrogel interactions which reduces drug release through hydrogel as compared with pure drug release, which releases in 70 min with a 98.22% release. The

In Vitro Drug Release Study
The drug release pattern from the hydrogel formulation is closely related to the drug interaction and matrix structure. The propolis release pattern obtained from the hydrogel formulation is presented in Figure 5. The release study reveals a biphasic drug release pattern where an initial burst release pattern of the drug was observed during first 40 min, Gels 2023, 9, 375 9 of 19 with about 40% of the drug being released, followed by a comparatively slow release up to 4 h. The pattern might be due to the dense hydrogel matrix with strong physical drughydrogel interactions which reduces drug release through hydrogel as compared with pure drug release, which releases in 70 min with a 98.22% release. The detailed mechanism with effects of pore size and water absorption capacity can be further investigated.
Gels 2023, 9, x FOR PEER REVIEW 10 of 20 detailed mechanism with effects of pore size and water absorption capacity can be further investigated.

Acute Dermal Toxicity Study
Study observations showed no death or clinical alterations in terms of various parameters observed as mentioned in the methods section in terms of skin, respiratory, circulatory, and autonomic and central nervous systems. Hair shedding, tremors, seizures, salivation, sedation, and drowsiness were not observed. Study results reveal the extract was found to be not toxic at a dose of 2000 mg/kg. The significant difference (p < 0.05) in percent body weight (weight gain) observed upto day 14 suggests no systemic toxic effects or any organic damages.

Burn Wound Model
Wound healing activity of the developed propolis-loaded hydrogel was carried out with the burn wound model in comparison with standard 5% w/w povidone iodine ointment USP (Cipladine ® ). The developed formulation-treated group showed significant wound contraction compared with the untreated group as shown in Table 4 and Figure 6. The study results demonstrated the role of polyphenol, flavonoids, and oleic acid in down regulating COX 2, inducing collagen type II expression and thereby accelerating the wound healing process as suggested in the literature.
At the end of the treatment period of 21 days, significant marked improvement in contraction of wound size was observed in the animals treated with propolis hydrogel (G3 93.58% ± 0.15% p < 0.0001) and 5% w/w povidone iodine ointment USP (Cipladine ® ) (95.39% ± 0.16%) as compared with blank hydrogel G2 58.38% ± 0.19% and disease control G1 52.37% ± 0.14%.The hydrogel formulation-treated group (G3) was found to be efficient in reducing the wound size and comparable to the standard group receiving 5% w/w povidone iodine ointment USP (Cipladine ® ).

Acute Dermal Toxicity Study
Study observations showed no death or clinical alterations in terms of various parameters observed as mentioned in the methods section in terms of skin, respiratory, circulatory, and autonomic and central nervous systems. Hair shedding, tremors, seizures, salivation, sedation, and drowsiness were not observed. Study results reveal the extract was found to be not toxic at a dose of 2000 mg/kg. The significant difference (p < 0.05) in percent body weight (weight gain) observed upto day 14 suggests no systemic toxic effects or any organic damages.

Burn Wound Model
Wound healing activity of the developed propolis-loaded hydrogel was carried out with the burn wound model in comparison with standard 5% w/w povidone iodine ointment USP (Cipladine ® ). The developed formulation-treated group showed significant wound contraction compared with the untreated group as shown in Table 4 and Figure 6. The study results demonstrated the role of polyphenol, flavonoids, and oleic acid in down regulating COX 2, inducing collagen type II expression and thereby accelerating the wound healing process as suggested in the literature.
Propolis hydrogel G3 exhibited significant differences in wound contraction (p < 0.0001) as compared to blank hydrogel and 5% w/w povidone iodine ointment USP (Cipladine ® ).    All values are represented as mean ± SEM, n = 6 animals in each group. Data we one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test. a: signifi compared to Group 1. b: significant difference compared to Group 2. c: significanc Group 4. **** p < 0.0001.  Figure 7 shows the histopathological changes in the healing process o in various treatments. In all groups, the formation of collagen coir, re-epit and fat tissues was observed in the burn area. Wound healing has indicat clude collagen deposition, fibrosis, angiogenesis, and PMN infiltration. Thes were revealed in varying levels in all four groups. Disease control (G1) sho tifocal severe necrosis of the epidermis and dermis with infiltration of inflam and fibrous connective tissue. As compared to disease control and blank propolis hydrogel and standard 5% w/w povidone iodine ointment USP showed a better response in terms of collagen deposition, development of Figure 6. Percent wound contraction at time intervals of the 3rd, 9th, 18th, and 21st days (Data were analyzed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test *** p < 0.001, **** p < 0.0001.) At the end of the treatment period of 21 days, significant marked improvement in contraction of wound size was observed in the animals treated with propolis hydrogel (G3 93.58% ± 0.15% p < 0.0001) and 5% w/w povidone iodine ointment USP (Cipladine ® ) (95.39% ± 0.16%) as compared with blank hydrogel G2 58.38% ± 0.19% and disease control G1 52.37% ± 0.14%.The hydrogel formulation-treated group (G3) was found to be efficient in reducing the wound size and comparable to the standard group receiving 5% w/w povidone iodine ointment USP (Cipladine ® ). Figure 7 shows the histopathological changes in the healing process of burned skin in various treatments. In all groups, the formation of collagen coir, re-epithelialization, and fat tissues was observed in the burn area. Wound healing has indicators which include collagen deposition, fibrosis, angiogenesis, and PMN infiltration. These parameters were revealed in varying levels in all four groups. Disease control (G1) showed the multifocal severe necrosis of the epidermis and dermis with infiltration of inflammatory cells and fibrous connective tissue. As compared to disease control and blank hydrogel, the propolis hydrogel and standard 5% w/w povidone iodine ointment USP (Cipladine ® ) showed a better response in terms of collagen deposition, development of epithelial lining, and neovascularization or angiogenesis, indicating an efficient wound healing process. ing, and neovascularization or angiogenesis, indicating an efficient wound healing p cess.

Histopathological Studies
The histopathological study of the wounded animal tissue is shown in the images below ( Figure 9A-D).

Histopathological Studies
The histopathological study of the wounded animal tissue is shown in the imag below ( Figure 9A-D).

Histopathological Studies
The histopathological study of the wounded animal tissue is shown in the images below ( Figure 9A-D). The histopathological study reveals that Group 1 (A) has a lower presence of collagen fibers and blood vessels. In the image, some necrotic cells and inflammatory cells can be seen as well. In Group 2 (B), less collagen is visible compared to Group 4. The image of Group 3 (C) displays an increased number of fibroblastic cells, collagen fibers, and no necrotic changes. Images of Group 4 (D) show a significant amount of collagen deposition and a completely developed epithelial cell lining, which symbolizes wound healing.

Incision Wound Healing Measurement of Tensile Strength
The tensile strength of the wound represents the effectiveness of wound healing. It is a measure of the completeness of wound healing and thus is an important parameter. From the results obtained, it was evident that the propolis hydrogel formulation showed significant tensile strength when compared against standard and control groups. Results of the tensile strength of the wound are represented in Table 6 and Figure 10. Group 4: Propolis hydrogel 198.18 ± 0.22 a ****, b ****, c **** All values are represented as ±SEM, n = 6 animals in each group. Data were analyzed by using one-way ANOVA, followed by Tukey-Kramer Multiple Comparison Test. a: significant difference compared to Group 1. b: significant difference compared to Group 2. c: significant difference compared to Group 3. **** p < 0.0001.

Measurement of Tensile Strength
The tensile strength of the wound represents the effectiveness of wou a measure of the completeness of wound healing and thus is an impor From the results obtained, it was evident that the propolis hydrogel form significant tensile strength when compared against standard and control of the tensile strength of the wound are represented in Table 6 and Figure   Table 6. Effect of propolis hydrogel on tensile strength.

Stability Studies
Accelerated stability studies of the formulations carried out at 40 ± 2 for a period of 6 months showed that the developed hydrogel does not e siderable changes in pH, viscosity, and drug content. Visual appearance sh Figure 10. Effect of propolis hydrogel on tensile strength (Data were analyzed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test **** p < 0.0001).

Stability Studies
Accelerated stability studies of the formulations carried out at 40 ± 2 • C/75 ± 5% RH for a period of 6 months showed that the developed hydrogel does not exhibit any considerable changes in pH, viscosity, and drug content. Visual appearance showedno phase separation and it was found intact with no leakage during the period of six weeks at specified temperature ranges as shown in Table 7.

Conclusions
Indian propolis-loaded PVA hydrogel was developed, optimized, and evaluated for wound healing. A design of experiment approach was used to explore how critical formulation parameters affected dependent formulation variables. Propolis samples were extracted utilizing literature-based extraction methods to increase polyphenols and flavonoids for wound healing. A validated RP HPLC method standardized the extract for specified constituents. The extract has 34.82 + 0.072 mg equivalent of gallic acid/g and 23.61 + 0.045 mg equivalent of quercetin/g, which may act synergistically. The extract was also free of 113 pesticides, proving its safety for drug delivery. Formulation development was conducted using a 3 2 factorial design to determine how independent variables (critical material attributes and process parameters) impact dependent variables (in vitro drug release and viscosity). Design Expert ® Version 10.0 was used for multiple regression analysis for batch optimization. The formulation batch (F2) was optimized utilizing the software's highest desirability of 1.000 at the desirable in vitro drug release and viscosity. FTIR and DSC analysis showed uniform distribution of propolis in hydrogel composition. Acute toxicity studies and burn, excision, and incision wound healing models show safety and substantial wound healing (p < 0.0001). Overall research results indicate promising finished product specifications of a developed formulation that could be studied for therapeutic effect in clinical trials.

Chemicals
Crude propolis was purchased from Bharatpur, Rajasthan, India. Polyvinyl alcohol LR was purchased from Research Lab Fine Chem Industries (Mumbai, India). Dialysis bags with a molecular weight cut off of 12,000 Dalton were purchased from Sigma-Aldrich Chemical Private Ltd. (Bangalore, India). All other chemical reagents used were of analytical grade.

Extraction and Standardization of Propolis Extract
The propolis sample was first treated with hexane to remove wax and other debris. The sample was then extracted with ethanol to obtain an extract high in polyphenols and flavonoids and desired groups of chemical constituents and standardized with the developed and validated RP HPLC method as described in our previously published work [24].
The extract was also standardized with the developed and validated RP-HPLC method with respect to specific marker compounds caffeic acid, quercetin, apigenin, and caffeic acid phenethyl ester [24]. The extract was also evaluated for its polyphenol, flavonoid, and pesticide contents.

FTIR Study
FTIR studies were carried out with the KBR dispersion technique for identification of specific functional groups from propolis extract and formulation. The spectrum was recorded and the spectral analysis was conducted (Shimadzu Model-8400S) [25].

DSC
The differential scanning calorimetry (DSC) thermograms of propolis extract and formulation were conducted on a DSC 821e (Mettler-Toledo, Greifensee, Switzerland). Samples were (5 mg) heated in a hermetically sealed aluminum pan with a heating rate of 10 • C/min under a nitrogen atmosphere (flow rate 50 mL/min) [26].

Formulation Development of Hydrogel and Optimization by DOE
A 10% w/v PVA aqueous solution was prepared (60-90 • C) under mechanical stirring for 6 h, and this solution was kept under stirring until it reached room temperature. For the PVA-propolis hydrogel formulation, when the PVA solution reached room temperature, 4 gm of ethanolic extract of propolis was added under mechanical stirring. Further formulation was then freeze-thawed for 24 h at −18 • C followed by 5-7 cycles of 30~60 min at room temperature and 1 h at −18 • C [19,20,27].
Then, 3 2 factorial designs were used to carry out experimental runs to understand the impact of variables on responses. The concentration of PVA and stirring speed were selected as independent variables, whereas drug release and viscosity were selected as dependent variables. The resulting data were fitted into design expert software (DOE version 10) and statistically analyzed using analysis of variance (ANOVA). The data were also subjected to 3D response surface methodology to determine the influence of material and process attributes on responses. The designs of the experimental runs are shown in Table 1.
4.6. Characterization of Hydrogel 4.6.1. Physical Appearance, pH, and Viscosity The prepared gel formulation was observed for a period of 6 months (0, 1, 2, 3, 6 months) for physical appearances such as homogeneity, color, consistency, grittiness and separation, etc. The pH of the gel formulation was determined by using a digital pH meter (Systronics pH meter, Type 335) [28].Viscosity was determined with a Brook field RVDV-II + Pro viscometer with a small volume adaptor spindle (S96) and T-bar spindle. All experimentation was carried out in triplicate and average values were calculated [29,30].

In Vitro Drug Release Study
The release of Indian propolis from the hydrogel formulation was assessed by the dialysis bag diffusion technique with phosphate buffer at pH 7.4 as a release medium at 37 ± 0.5 • C with the continuous stirring process at 100 rpm. A propolis hydrogel sample equivalent to 80 mg was filled in the dialysis bag membrane (molecular weight cut off of 12,000 Da) and the bag was immersed in release medium. Then, 2 mL of the aliquots were withdrawn at different time points: 0, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h. Thereafter, aliquots were filtered through Whatman no.1 filter paper, and the obtained solution was diluted and the propolis concentration was estimated. The pure Indian propolis release pattern was analyzed in a similar manner, taking Indian propolis solution (1 mg/mL in 50% w/w mixture of PEG 400 and water) as a control [31].

Acute Dermal Toxicity
The study was conducted according to the OECD guideline 434 (11). The extract at a dose of 2000 mg/kg of body weight was administered dermally as a single dose to Albino Wistar rats (male and females; 180-200 gm body weight) and allowed to keep in contact with the skin for 24 h. Animals were carefully observed for the first 30 min followed by 24 h periodically, and monitoring continued until14 days. Animals were observed for any rashes on skin, changes in skin, eye changes, respiratory changes, nervous system changes, behavioral changes, locomotor activity, convulsions, tremors, comas, etc. Percent body weight changes in the animals were recorded [32].

Wound Healing Activity Burn Wound Model
Male Wistar rats weighing 150-250ginthe age range of three to four months were used in the study. Animals were procured from the National Toxicological Centre (NTC), Pune, India, and were housed in an animal house at room temperature, being maintained at 37 ± 5 • C. Animals were fed on a standard diet. Animals were given free access to food and water. All the experiments were performed in accordance with guidelines laid by the Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and approved by the Institutional Animal Ethics committee as per protocol number DYPIPSR/IAEC/14-15/P-34.
The animals were randomly divided into 4 groups (n = 6). The animals were anesthetized using ketamine hydrochloride (50 mg/kg, i.p., body weight) and xylazine (5 mg/kg, i.p., body weight). Group 1 (disease control) animals were animals that were wounded but did not receive treatment, Group2 (vehicle control group) was treated with blank PVA hydrogel without drugs, Group 3 was treated with the propolis hydrogel formulation, and Group 4 animals received treatment of standard 5% w/w povidone iodine ointment USP(Cipladine ® ). Groups 1-4 were treated once daily for a period of 21 days. The animals were kept in separate cages and were provided a standard diet and water adlibitum throughout the study. The rate of wound contraction was noted. At the end of the study, the animals were sacrificed and wound tissue mass was obtained from the wound area by sharp dissection [21,27].
Percent wound contraction was measured with a digital vernier caliper by calculation with the formula mentioned below and taking the initial size of the wound as 100%.
Histopathological Studies were carried out to confirm healing improvement. The wound tissue specimens were processed in 5% formalin solution embedded in paraffin. A section of 3-5 cm of skin epidermis tissue was stained with hematoxylin and eosin (H & E) for routine histological processing. The prepared slides were examined qualitatively under a light microscope, for examining hair follicles, inflammation, blood vessels, fibroblast, necrosis, bacterial colonies, neutrophils, edema, and collagen [21,27].

Excision and Incision Wound Model
The excision wound model was used to measure the wound contraction and for histopathological study, while the incision wound model was used to find out the tensile strength of the cured tissue of rats.
The animals were grouped as follows: Group 1, disease control; Group 2, vehicle control; Group 3, standard control, treated with 5% w/w povidone iodine ointment USP (Cipladine ® ); Group 4, treated with propolis hydrogel. Each group contained a total of 6 animals.
Excision Wound Model was studied with four groups containing 3 animals each were made. The animals were anesthetized using ketamine hydrochloride (50 mg/kg, i.p., body weight) and xylazine (5 mg/kg, i.p., body weight). The dorsal portion of the rats was shaved using depilatory cream. The area to be excised was marked and a full-thickness excision of 1 cm diameter was made [32,33]. The wounds were left undressed. Vehicle control (blank PVA hydrogel), standard 5% w/w povidone iodine ointment USP (Cipladine ® ), and propolis hydrogel were applied once daily until the wound had completely healed. Wound contraction was measured on the 3rd, 9th, 18th, and 21st days after the formation of the wound. After the study had been completed, i.e., on the 21st day after the wound creation, the rats were anesthetized. Tissues of rat wounds were excised and stored in 10% formalin solution for histopathological study [33,34].
Incision Wound Model was studied with 4 groups of animals containing 3 animals each were made. All the animals were anesthetized (using the same anesthetic agent used above) and depilated using depilatory cream. An incision of 3 cm length was made according to the method described by Mukherjee et al., 2000. The parted skin was kept together by stitching using a curved needle (No. 9) and black surgical thread (No.000). Vehicle control (blank PVA hydrogel), standard 5% w/w povidone iodine ointment USP (Cipladine ® ), and propolis hydrogel were applied to the wound area once daily for 9 continuous days. On the 10th day, when the wound had healed, the animals were anesthetized again and the sutures were removed. The tensile strength of the healed wound was measured using a tensiometer [35].
The tensile strength of the wound is an indication of the effectiveness of wound healing. Tensile strength, i.e., the force required to open the healing wound, is used as an important evaluation parameter. On the 10th day, the rats of the incision model were anesthetized, the sutures were removed, and healed tissue was excised from all animals. The tensile strength of this excised tissue was measured with the help of a tensiometer [35].

Statistical Analysis
The results obtained from the excision and incision models were expressed as mean ± standard error of the mean (SEM) and were compared with the vehicle control, disease control, and standard control groups. The statistical significance was analyzed by one-way ANOVA and the Tukey-Kramer Multiple Comparison Test, using statistical software Graph Pad Prism, version 5.

Stability Testing of Hydrogel Formulation
Accelerated stability studies of the propolis hydrogel formulation were carried out as per ICH guidelines at conditions of 40 • C ± 2 • C/75 ± 5% RH for a period of 6 months. The stability samples (n = 3) were analyzed for drug content, pH, and viscosity at 0, 30, 60, 90, and 180 days [36].