Assessment of Wound Healing activity of Polyherbal Gel, by “Burn Wound Model” in Wistar rats

Yograj Mahajan¹, Vishal Gupta²

¹Faculty of Pharmacy, Mansarovar Global University, Sehore, M.P. 466111.

²Faculty of Pharmacy, Mansarovar Global University, Sehore, M.P. 466111.

*Corresponding Author E-mail:

ABSTRACT:

Aim: Burns are significant worldwide healthcare concern, often difficulty by the existence of microorganism like Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli in the wounds. Burn damages first line of defence against microorganism. Number of allopathic formulations are commonly used for the treatment of burn wound, but they cause skin irritation and delay wound healing. The aim of the present study was comparative assessment of polyherbal gel preparations for cutaneous application of healing agents at the time of burn wound to quickly skin heal. Method: In the present study, polyherbal gel formulations containing 10% hydroalcoholic (Ethanol/Methanol and Water) extract of Calendula officenalis, Turmeric, Azairachta indica and Aloe-vera at different concentration are prepared. The extraction of selected plants done by hot extraction and cold maceration methods. Gel was topically applied on wistar rats, weight between 150 gm.to 200 gm. of both sexes by using burn wound model. 5% betadine ointment are used as marketed standard preparation. The effectiveness and safety of polyherbal formulation was evaluated. Result: The result showed all the optimize gel formulations exhibited significant wound healing (P<0.0001) response. Differences in burn wound healing were observed between varied formulated gel formulations (F-9 = 84.68±3.12%, F-6 = 79.93±2.26% and F-2 = 75.63±2.96% respectively) compared with negative control (65.84±2.26%) and standard treated (86.51±2.89%) animals’ groups. Conclusion: The data obtained from the experiential study were encouraging and the data exhibited the hydroalcoholic (ethanol and water) extract significantly good wound healing response (F-6 and F-9) compared to methanol and water extract (F-2) and negative control. The wound contraction rate and epithelization rate (P<0.0001.) is excellence in hydroalcoholic preparation F-9: 25.83±1.47 and F-6: 29.5±1.87 days as compared to methanolic extract formulation F-2: 33.67±1.21 and negative controlled treated (35.67±1.63 days) animals’ groups. The result indicates that the prepared polyherbal gel formulation has accelerated the burn wound healing process and effective in skin injuries and burn.

KEYWORDS: Calendula officenalis, Curcuma longa, Burn Wound, Wistar rat, Azadirachta indica, Hydroalcoholic, Aloe-vera.

1. INTRODUCTION:

Skin, the largest and most visible part of the body primary protecting organ in the body and has a surface area of about 1.5-2 square meter in adult. It varies in thickness, being thickest on the soles of feet and the palms of the hands¹. Skin maintains a first-order physical barrier among the internal and external environment and perform as a barrier against external pathogens and protecting against external environmental hazard such as Microbial infection, incisions and various injuries like burn and radiation². Burn injury destroys the skins protective covering that generally prevents invasion of microbes^3,4. Wound can be classified according to depth, etiology of injury, location and appearance. Wound is classified on acute and chronic wound. Wound healing may be defined as a process in which body replace damaged or destroyed tissue by newly generated tissue, in which body restore their normal structure and function⁵. It is very complex process; involve various steps such as inflammation, coagulation, production of collagen and regeneration of epithelial. Collagen gives strength, fitness and structure of cell. After the injuries blood flow and platelet count increase around wound area. It contacts with collagen and release clotting and growth factor, then neutrophils emigrate to the wound area to kill and eliminate the microorganism from the infected tissue. Present of microorganism in wound site release inflammatory cytokines such as interleukin and TNF- α. Herbal plant plays vital role in the life of mankind^6,7. The greater community of the world trust on traditional medicine and use for their health care. It is mostly developing countries where the price of western medication and the consulting fees of doctor much higher than their resources of the people⁸. For more than 5000 years, Indigenous peoples of Asia, Africa, Egyptians and Romans have used herbal medicinal plant for the treatment of burns, inflammation ulcer and wound healing. Turmeric inhibits the formation of ROS. ^9,10,11.

2. MATERIALS AND METHODS:

2.1 Materials:

2.1.1 Selection, procurement and authentication of plant materials:

Leaves of Azadirachta Indica, flower of Calendula officenalis, and leaves of Aloe-Vera plant was collected from the local area of Bhopal. The turmeric was purchase from the local market of Bhopal city. The authentication of plants done from the Department of Botanical Science, Government Jaywanti Haksar Post Graduate College Betul Madhya Pradesh India.

2.1.2 Chemicals:

All chemicals and reagents used in the preparation of polyherbal gel were of either analytical grade or of highest purity grade available. Carbopol 934, Carbopol 940, Propyl Paraben {Sunchem industries Mumbai), Glycerin, Propylene glycol (Oxford Lab Fine Chem LLP Palghare MH), Chloroform, Sodium Hydroxide (Fizmerk Research Chemicals), Methanol, (Titan Biotech LTD), and Ethanol (Changshu Song Sheng Fine Chemical), was purchase from Indian scientific P. Ltd Bhopal.

2.1.3 Animal:

Wistar rats (150-200 gm) of either sex were selected for the present study. The experimental protocol for the proposed study was approved by Institutional Animal Ethical Committee (IAEC) TIP/IAEC/02/13 under the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA). The animals were procured from animal house of Truba Institute of pharmacy Bhopal.¹².

2.2 Methods:

2.1 Preparation of extract:

The extraction of Neem leaves and Turmeric rhizome were done by hot Soxhlet method and the extraction of Calendula officenalis was done by cold maceration. Hydroalcoholic solvent (Ethanol/Methanol and water) were used to extraction the phytochemicals compound from the natural matter.^{13,14,15,16,17.}

2.2 Burn wound model:

Thermal injuries create critical damage on the cellular and tissue level and are observe mainly challenging in the clinical routine. Burn wound model is used for the finding for wound contraction rate¹⁸. Burn wound model are divided in to tree types-based o degree of wound (1) first degree wound (2) second degree wound and (3) third degree wound. A partial burn wound model was applied on Wistar rats, weight between 150 to 185gm. of both genders are used for the experimental study. Animal was anaesthetized by using light Chloroform or other suitable local anesthetics. The hairs of the animals back were shaved with a sterilized surgical blade. Hair removed area was disinfected with ethanol 70%, for the creation of burn wound, molten wax approx. 2gm. at 80°C poured on the rat’s skin with the help of metallic cylinder 300mm²circular opening. The wax will be solidified on animals’ skin then remove it. Treatment of experimental animals was started immediately after the injury. Poly-herbal gel (F-2, F6 and F-9) formulations were applied topically till complete epithelialization done. Animal were kept separately in cages. Provide normal food and water during the study.^{19,20,21,22,23.}

Animal was randomly divided in to 3 groups and each group containing 6 animals. Poly-herbal gel (500mg/rats) was applied once daily by topical application.

Group 1: Treated with normal poly-herbal gel base applied.

Group 2: Treated with 5% Standard Betadine applied.

Group 3: Treated with poly-herbal gel formulation F-2.

Group 4: Treated with polyherbal gel formulation F-6.

Group 5: Treated with polyherbal gel formulation F-9.

Animal will be observed alternative day and monitored healing on the bases of following parameter namely:

a. Wound contraction-Wound contraction will be determined by measuring the progressive wound healing area by using transparent paper cover over the wound surface and that tracing draw over the millimeter scale graph sheet and calculate percentage wound contraction by using this formula-

Intial wound size – Specific day wound size

Wound % = -------------------------------------------- x 100

contraction Intial wound size

c. Epithelialization-Time period taken for complete epithelialization was measure by recorded days. ^{24, 25.}

3. RESULT AND DISCUSSION:

3.1 Burn Wound Model:

The results of burn wound model is shows in figure 1. On day 1^st and 3^rd absence of epithelialization and slightly thick eschar shows no burn wound closure. On the day 3^rd the groups treated with F-9 polyherbal gel formulation shows softer and quite exfoliated eschar. Also, the group that treated with povidone iodine shows softer eschar but no sign of exfoliation.

Table 1: Wound contraction among different treatment groups on Burn wound model (Wound contraction area mean± Standard deviation.

Group	Day 1	Day 3	Day 6	Day 9	Day 12	Day15
Negative Control (Gel Base)	30.12±0.166	27.19±0.732	24.48±0.799	19.65±0.746	14.62±0.861	10.29±0.678
Standard (P. Iodine)	30.07±0.148	25.16±0.931	20.47±0.798	16.19±0.718	9.72±0.643	4.05±0.862
F-2	30.02±0.273	26.1±0.689	21.58±0.54	17.33±0.555	12.19±0.913	7.32±0.909
F-6	30.12±0.258	24.71±0.972	21.47±0.750	17.13±0.764	11.94±0.923	6.04±0.688
F-9	30.14±0.274	23.87±0.962	21.01±0.937	16.82±0.863	10.64±0.989	4.62±0.969

Table 2: Percentage Wound contraction among different treatment groups.

Days	Negative Control Gel Base	Standard (Povidone Iodine)	Test F-2	Test F-6	Test F-9
Day 1	0%	0%	0%	0%	0%
Day 3	9.74±2.123%	16.34±2.76%	13.05±2.21%	17.98±3.40%	20.8±3.26%
Day 6	18.72±2.60%	31.9±2.52%	28.1±1.37%	28.74±2.59%	30.3±3.02%
Day 9	34.7±2.37%	46.15±2.24%	42.28±1.57%	43.13±2.75%	44.21±2.64%
Day 12	51.45±2.81%	67.68±2.18%	59.4±2.88%	60.36±3.08%	64.71±3.06%
Day 15	65.84±2.26%	86.51±2.89%	75.63±2.96%	79.93±2.26%	84.68±3.12%

All values are represented as mean± standard deviation, n= 6 animal in each group. Data were analyzed by one-way ANOVA, followed by Tukey’s Multiple Comparisons Test, P<0.0001, a significant difference as compared to negative control and standard group.

3.1.1 Average mean wound area closure:

The wound area was manually drawn and recorded at 3-day interval, and the cure area was evaluated by subtracting it from the actual wound area. Wistar rats were used in the current study. The starting mean excision wound area on day 1 as follows: Group I: 30.12±0.166 mm², Group II: 30.07±0.148 mm², Group III: 30.02±0.273 mm², Group IV: 30.12±0.258 mm², and Group V: 30.14±0.274mm². Group I represent the negative control group, Group II was standard (5% Povidone-Iodine) treated group, and group III, IV, and V were the test groups treated with polyherbal gel formulations respectively. The result of each groups is displayed in the table 2. And fig. 2.

During the earlier 3 days. no remarkable wound contraction was detected in both the negative control and standard treated group as well as the polyherbal gel treated groups. On the day 6^th notable wound contraction was observed in the standard treated group and polyherbal gel treated groups. On the 9^th day, the significant wound contraction area was determined for the negative control treated group 19.65±0.746, standard treated group 16.19±0.718, polyherbal gel treated group F-2: 17.33±0.555, F-6: 17.13±0.764 and F-9: 16.82±0.86. respectively.

Figure 2: percentage wound contraction in Burn Wound model for various gels treated groups compared with Negative control and Standard formulation.

The wound contraction rate in the negative control treated groups was slower compared to polyherbal gel treated groups and standard. From the 12^th day onwards, maximum wound closure was observed in the polyherbal gel treated experimental group F-2, F-6 and F-9 formulations and the marketed standard formulation.

By the end of the 15^th day, the average mean wound contraction area measured for the negative control treated group and standard (5% povidone-iodine ointment) treated group animals were 10.29±0.678 mm² and 4.05±0.862 mm² respectively. On the same 15^th day the experimental groups showed maximum wound contraction area for F-2: 7.32±0.909 mm², F-6: 6.04±0.688 mm² and for F-9: 4.62±0.969 mm² respectively from the beginning of the wound. The analysis of that data indicates these all three polyherbal gel formulations having good ability to heal wound when applied topically and demonstrate remarkable effect on wound closure. It has observed that the polyherbal gel formulation F-9 treated group animals exhibited similar effect to the marketed formulation.

3.1.2 Evaluation of Epithelialization period:

The result of the epithelialization periods for the various treatment groups are showed in the table 3 and 4. it was observed that the periods of epithelialization for group V, treated with polyherbal gel formulation F-9: 25.83±1.47, as well as group II treated with marketed standard formulation (5% povidone-iodine ointment) were found almost similar with duration 25.17±1.83 days. in case of group I treated with negative control (gel base) and group III, treated with polyherbal gel formulation F-2, the epithelialization time was 35.67±1.63 and 33.67±1.21 days respectively, was found to be longer compared to other treatment groups. Fig. 4.

Table 3: Epithelialization Days among different treatment groups in Days.

Animals	Negative Control	Standard (Povidone Iodine)	F-2	F-6	F-9
1	34	25	35	29	25
2	35	27	32	32	27
3	38	23	33	30	24
4	36	26	35	28	26
5	37	27	34	27	25
6	34	23	33	31	28

Table 4: Mean Epithelialization periods of various treatment groups.

Group No.	Treated Groups	Epithelialization periods (Days)
1	Negative Control (Gel Base)	35.67±1.63
2	Standard (P. Iodine)	25.17±1.83
3	F-2	33.67±1.21
4	F-6	29.5±1.87
5	F-9	25.83±1.47

All values are represented as mean± standard deviation, n= 6 animals in each group. Data were analyzed by one-way ANOVA, followed by Tukey’s Multiple Comparision Test. A significant difference as compared to negative control treated and standard control treated groups. P<0.0001.

Figure 4: Epithelialization periods among different treatment groups (in Days)

The analysis of these value indicates that the polyherbal gel formulation, especially formulation F-9, showed comparable effective in elevate epithelialization as the reference standard drug. However, group IV treated with F-6 gel formulation exhibited slightly longer epithelialization duration (29.5±1.87) compared with standard and F-9 treated groups respectively. The result of this study suggest that the experimental groups treated with polyherbal gel formulations had favorable and accelerated epithelialization activity compared with negative control group.

CONCLUSION:

In this study, we developed polyherbal wound healing gel formulation and evaluated for its therapeutic efficacy and angiogenic activity using burn wound model. Fron the result of experimental analysis, it was concluded that optimize gel formulation could achieved significant burn wound healing and maintained higher cellular action in burn wounds. The topical application of polyherbal gel formulation to burn wound significantly promote the healing rate and angiogenesis in wound area. F-6 and F-9 gel formulations of hydroalcoholic fraction (ethanol and water) showed significantly higher wound contraction rate as compared to F-2 gel formulation (methanol and water) and negative controlled groups. The duration of epithelialization periods is shorter in F-9 gel formulation compared with F-2 and negative controlled groups. The tested polyherbal gel formulations demonstrated their efficacy primarily during the succeeding phases of wound healing. All optimize polyherbal gel formulation promote the proliferation and migration of epithelial cells, enhance granulation tissue generation and significantly enhanced wound contraction. These findings suggest that both polyherbal gel formulation F-6 and F-9 can be showed significant wound healing activity. Wound repair is well synchronized operation that include a series of overlapping phases. Fundamental repair is a complicated dynamics stage of event involved clotting, inflammation, formation of granulation tissue, epithelialization, angiogenesis, collagen matrix synthesis and wound contraction.

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Received on 25.06.2025 Revised on 28.07.2025

Accepted on 23.08.2025 Published on 11.10.2025

Available online from October 25, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(4):257-262.

DOI: 10.52711/2321-5836.2025.00041

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