INTRODUCTION:

The largest organ in the human body is the skin and defends against external threats. Injuries to the skin's epidermal layer impair its integrity, leading to the creation of wounds¹. Wounds can arise from a variety of sources, including trauma, insect or animal bites, and mechanical abrasions².

Severe infection is caused by different harmful bacteria invading the wounded tissue. The phrase "wound infection" relates to the proliferation of microorganisms that cause tissue damage, a delayed and exaggerated inflammatory reaction, a delay in collagen production, and a delay in epithelium formation³. The process of healing a wound goes through numerous phases, such as the stages of inflammation, migration, proliferation, and maturation. The duration of the homeostasis process is around 48 hours. Hemostasis and the inflammatory stage cooperate to produce results. At this point, lymphocytes, monocytes, and neutrophils have all infiltrated. Macrophages, which are created when monocytes develop into them, are in charge of phagocytosing germs from the exterior of the body. According to a recent study, wound healing is impacted by macrophage dysfunction. The inflammatory process lasts for three to four days at its height. Usually, there is erythema and irritation throughout this procedure. The process of re-epithelialization occurs at the next stage, proliferation, when the damaged layer starts to heal and develop normally again. At this point, blood vessel capillary development, or angiogenesis, will also take place. Furthermore, the development of extracellular matrix and collagen production occurs. Proliferation lasts for two to three weeks. The final phase is remodelling, which may take years to complete depending on the patient's body's overall condition. Collagen remodelling, vascular blood vessel maturation, and regression all affect how long the remodelling process takes⁴. In order to speed up wound healing, nutrients were required. During every stage, nutrients would be essential. The primary nutrients required for wound healing are fatty, amino acids as well as protein and Minerals (zinc, iron, and copper). Some are present in Natural products from plants including Bauhinia purpurea, Bauhinia variegate, Aerva tomentosa, Bridelia airyshawii, Acacia catechu, and Amaranthus spinosus, as well as from animals like Channa striatus and Monopterus albus, have been researched for their impact on wound healing^5,6. The Salicaceae family includes the significant tree Populus deltoides, which may reach heights of 20 to 40 metres and has a trunk that can reach 1.8 metres in diameter. It has enormous, triangular leaves Fig. 1 that are 12cm tall and 12cm broad with a horizontal base and a petiole that is 3 to 12 centimetre long. The leaves are very coarsely serrated; the petiole is flat. The leaves are green in the cold climate and become yellow in the falling season ⁷.

Fig. 1. Leaves of Populus deltoides

Numerous plant components have been utilized for centuries for their analgesic, antipyretic, and anti-inflammatory properties⁸. The eastern cottonwood, Populus deltoides, is also known as the poplar in India. In addition to the opposite of the northern Canadian, eastern Canada, and opposite of southern Mexico, it may be found in the opposite of western, central, and south western USA⁹.

Phytochemistry: The ethanolic extract of Populus buds is a significant hepatoprotective, analgesic, proving that it contains metabolites with such actions, according to recent studies in phytochemistry. These findings are in contrast to those of different herbs, like Clematis flammula and Fraxinus angustifolia, whose anti-oxidant capabilities have been established and whose tannins, flavonoids, and total phenols have been found in the related states. Some of the main 2ndry products of Populus include flavonoids, fatty acids, terpenoids, anthocyanins, terpenoid and phenolic glycosides etc^10,11,12.

In 2020, Mission Regional Medical Centre released a number of study studies and statistics that indicated an estimated 6.7 million people worldwide were expected to be suffering from chronic wounds. With a compound annual growth rate of 6.7%, by 2029, the global market for chronic wound care is projected to grow from USD 12.36 billion in 2022 to USD 19.52 billion¹³. On a partially separate topic, healing from wounds that result in scarring is just as important as healing from chronic wounds. This is because of the enormous number of patients—roughly 100 million in the industrialised world annually—who have scars from 25 million operations linked to trauma and 55 million surgeries connected to elective procedures¹⁴. There has been a "silent epidemic"-like increase in the number of patients with chronic wounds. In Western countries, 2–4% of health expenditures are related to chronic wounds¹⁵. This has frequently resulted in the development and execution of management, treatment, and prevention strategies that are insufficient. As a result, wound research is a significant yet underappreciated field of . There are several allopathic drugs on the market that have wound-curing properties. However, they have several negative effects, and the expenses of such products are also quite costly. The average person cannot afford such high charges. Therefore, the demand for substitute agents with both antibacterial and wound-healing properties is constant. Povidone iodine ointment is an antiseptic agent and may have some delayed wound healing activity owing to its cytotoxic impact. Additionally, the antiseptic agent needs to be the least cytotoxic. There is a need to design efficient wound-curing dosage forms that can limit the occurrence of issues and complications caused by allopathic or synthetic medications. Populus deltoides was selected for gel formulations because of their well-documented pharmacological (wound-healing, antioxidant, and anti-inflammatory) qualities and traditional usage, especially in the different phases of wound healing (tissue regeneration and repair). In order to provide scientific support for curative value of Populus deltoides as a good wound curative formulation, an attempt was made in this work to manufacture and examine the wound curative activity of freshly developed gel formulation which will be inexpensive, safe, sound and have wound cure activity to study wound healing process using excision (to measure wound Surface Area, % wound closure) and incision (to measure tensile strength) models respectively.

MATERIALS AND METHODS:

Experimental animals:

According to the requirements of the CPCSEA, India, the Institutional Animal Ethics Committee accepted the study protocol (approval no. MIET/ IAEC/CPCSEA/02/2023/119). Young, healthy adult female wistar Albino rats were collected in similar numbers each group (n = 6). Rats typically weighed 150–200 gm. The experimental animal room was kept at a persistent temp. of 22°C (+3°C). A 50–60% relative humidity was maintained.

Authentication and extraction of plant:

The plants of Populus deltoides (Salicaceae) were collected at the time of April from Suraj Nursery Sardhana, Meerut U.P. India. The leaves of Populus deltoides were dried under normal environmental conditions and were authenticated by Prof. Vijay Malik (Head of Botany Department Chaudhary Charan Singh University). The leaves of Populus deltoides were dried in the shelter, ground into a relatively fine particles, and placed into a soxhlet extractor where they underwent two separate extraction processes using petroleum ether(60°-80°C) for defatting and Ethanol (70°-80°C) for extract^16,17.

Formulation of gels:

In a 100ml beaker, 2gm of carbopol-934 were diffused in 50ml of distilled water while being constantly stirred. The container was set aside so that the carbopol 934 might grow over night. On a water bath, 0.2ml of 0.5% methyl paraben and 0.1ml of 0.2% propyl paraben were heated with 4-5ml of saline water. After cooling, the necessary amounts of extracts (2g for 2% gel, 4g for 4% gel and 6g for 6% gel), 0.2gm of sodium metabisulphide, and 5ml of 5% propylene glycol-400 were combined¹⁸. Triethanolamine was added drop by drop to the finished mixture while it was being continuously stirred in order to achieve the desired skin pH. The finalised mixture received sufficient water additions to form the gel of the necessary consistency¹⁹.

Albino wistar rats were separated into groups of five for the excision model, as follows:

· Group 1 treated as negative control (placebo).

· Group 2 served as standard treated with 2% povidone iodine ointment topically.

· Group 3 treated as test group treated with 2% Populus deltoides ethenolic extract gel A topically.

· Group 4 treated as test group treated with 4% Populus deltoides ethenolic extract gel B topically.

· Group 5 treated as test group treated with 6% Populus deltoides ethenolic extract gel C topically.

Excision wound model:

The excision wound model is one of the most widely used models for wound healing²⁰. They are thought to mimic acute clinical wounds that need to heal by secondary intention, meaning that the skin's margins do not need to be sutured together. These lesions are caused by the mechanically removal of the animal's whole skin structure, including the fat, dermis, and epidermis. With the use of this procedure, research can be done on pain, bleeding, granulation tissue, angiogenesis preparation, and remodelling.

Procedure^21,22

Rats were anesthetized by injecting intraperitonially the ketamine (50mg/kg). it gives about 20 minutes of surgical anaesthesia. To get rid of the hair, the rat's dorsal skin was shaved with a trimmer and 70% alcohol was used to sterilize the region after shaving. The region of the lesion to be formed was indicated, and Using dental forceps, surgical blade, and scissors, a typical ring was used to outline a 500 mm² region on the rat's dorsal flap. A full-thickness circle of skin was removed. After dressing, the animal was maintained in a warm environment and watched until it was awake again. The animal was returned to its regular habitat and was placed in a cage after it was awake from the anaesthesia.

Wound closure monitoring:

The wound area was observed every day until the full wound closure. The wound was measured by Vernier Callipers/micrometre. The following formula²³ was used to get the percentage of wound closure:

Healed region

% Wound contraction = ----------------------------- × 100

Entire wound region

(Healed region = actual wound region – present wound region)

Wound analysis by images (Photographic documentation):

Photography is a useful technique in research, particularly in dermatology since it is non-invasive, may offer information that is frequently equivalent to diagnosis, and can support with case documentation and aftercare²⁴. Pictures can be useful for tracking morphological changes, colour variations, and other aspects of wound healing in both clinical and experimental settings. Digital photos can also be electronically transferred, making them perfect for remote wound management²⁵.

Incision wound model (Procedure)²⁶

Ketamine hydrochloride (50mg/kg, i.p.) was used to anaesthetize all albino rats. On dorsal area of rat on either side of the vertebral column, a 3 cm long paravertebral cut (incision) was created by the whole thickness of the shaved skin with the use of a sharp scalpel, of the rats. Dark surgical silk thread and a curve needle (no. 11) were used to perform interrupted suture on the incisions. After stitching, the incision was kept exposed for 10 days while the rats accepted daily medical treatment. On the seventh post-injury day, the stitches were eliminated. On the tenth post-wounding day, rats under anaesthesia had their wound breaking strength (WBS) tested.

Tensile strength of skin:

Skin tensile strength in an incision model was assessed on a 10-day basis using a tensiometer. The amount of force necessary for splitting an animal's skin in an incision wound model is known as tensile strength. Two Allis forceps were securely fastened to the line, facing each other. The 2^nd forceps were applied to a weighing scale, while one was fixed. Conventional weights was carefully added, and the weight's gradual increase conveyed to the wound site pulled the edges of the wound apart. The weight was stopped as soon as the wound first appeared and reported.

Histopathological analysis:

To understand origin of a disease, to track healing progress during therapy, to clear understand the epidemiology of non-healing wounds, to evaluate physical changes, and to assist with diagnosis, histopathology of wounds is a highly useful tool²⁷. Clinically, the outermost layer of the wound is the ideal location for a biopsy because it allows comparison of the ulcerated region with the surrounding skin²⁸. The whole wound, including the margins, will be sampled for histological study in the laboratory²⁹. To preserve their integrity and prevent alterations to the cellular structure, tissue samples should be immersed in certain solutions as soon as they are harvested, such as 10% buffered formalin, one of the most used fixatives. Following that, the tissue will go through a number of histological processes, including embedding, sectioning, and staining.

Dry and wet tissue study³⁰

· Estimation of Hydroxyproline- 250 mg of wet tissue was drained at 50°C for 24 hours. Then the samples were homogenized by using 6N HCL. After homogenization samples were incubated for 4hrs at 130 °C so that hydrolysis can be increased. The std. solution of hydroxyproline were prepared in concentration of 1.0-100µg/ml using L-Hydoxyproline. 10µl of sample and std. solutions were taken and the 90µl of 0.05M chloramine T was added into it. These solutions were kept at room temp. for 20 min. Ehrlich’s reagent (100µl) was added and solutions were incubated for 20 min in water bath at 65°C and reading taken at 550nm.

· Estimation of (Glutathione) GSH- Homogenate (w/v) and 10% TCA were mixed, and the mixture was centrifuged at 3000 rpm for 15 minutes in order to separate the proteins. 0.01 ml of supernatant will be mixed with 2 ml of phosphate 31 buffer (pH 7.5), 0.5 ml of DTNB, and 0.4 ml of distilled water. The absorbance at 412 nm was measured in 15 minutes.

Statistical analysis:

ANOVA was used in the statistical analysis using Dunnet's multiple comparisons. The control group was cmpared to standard grp, group 3, group 4 and group 5. Statistically significant values were defined as ***p≤0.001, and **p≤0.005 as significant.

RESULTS:

Excision wound model (Wound closure monitoring)

From the observation Table. 1, we have found that gel C and gel B shows the significant activity when compared to negative control group. Values are mean±SEM of 6 albino rats in every group. p values: ^ap≤0.001 and ^bp≤0.005 were compared to the respective group (Dunnet's test for multiple comparisons was used after two-way analysis of variance for statistical analysis). The efficacy of the gel C and gel B was found good because the wound contraction of treated animal was 100% while the negative control animal was 89% and Fig. 2 showed time dependent increase in percent wound contraction of all groups. Gel C and Gel B were shown to serve an effect in increasing the frequency of epithelialization and needed less time to complete the process than the control during epithelialization.

Fig. 2. Dunnet's multiple comparisons were used in the two-way ANOVA statistical analysis. p values: a= p≤0.001 and b= p≤0.005 were compared to the negative control group.

Photographic documentation:

Change in the wound area Fig. 3 of animal treated with negative control (placebo), standard (povidone iodine solution), Gel A, Gel B and Gel C at 0 day, 4^th day, 16^th day and 20^th day.

Fig. 3. Pictures showing wound area contraction in different animals’ groups

Incision model (Tensile strength of skin):

The best healing drug must have the activity to improve the credibility of collagen fibrils surrounding the lesion site, which raises the wound's evaluated tensile strength by comparing it to the wound's healed tensile strength.

Fig. 4. Mean and SEM are used to represent values (N = 6). For the statistical analysis, Dunnet's multiple comparisons were combined with one-way ANOVA. ***p≤0.001 and **p≤0.005 represented the p-values in relation to the negative control group.

From the observation it was found that gel C and gel B showed the significant activity when compared to negative control group. The efficacy of the gel C was found good because the tensile strength of treated animal was 788gm while the control animal was 483gm shown in Fig. 4.

Histopathological analysis:

Fig. 5. Histopathological evaluation of wound healing in skin tissue of albino wistar rats

Histopathology of the skin tissue at 10^th day after staining with haematoxylin and eosin dye in Fig. 5. [A] Skin tissue of the negative control group rat showing edema and ulceration (black arrow). [B] Skin of standard group rat showing decrease number of inflammatory cells (black arrow) and hair follicle (blue arrow). [C] Skin of treated gel A rat showing earlier epithelization and ulceration (black arrow) and hair follicle (blue arrow). [D] Skin of treated Gel B rat showing subcutaneous tissue (black arrow) and large amount of granulation tissue. [E] Skin of treated Gel C rat showing large amount of granulation tissue (blue arrow) and near to normal epidermis (black arrow).

Dry and wet tissue study:

The enhanced strength of collagen fibres surrounding the wound region was confirmed by the enhanced hydroxyproline amount, which was responsible for boosting the collagen level. In treatment group gel C rat, the hydroxyproline level was observed in to be considerably higher (p≤0.001) Fig. 6 than in negative control group. In addition, their GSH levels were higher than those of the negative control group. Gel B demonstrated a statistically significant rise in GSH levels (p≤0.001) in comparison to the negative control group Fig. 7.

Fig. 6,7 Mean and SEM are used to represent values (N = 6). One-way ANOVA was used for the statistical analysis along with Dunnet's multiple comparisons. ***p≤0.001 and **p≤0.005 were the p values when compared to the negative control group.

DISCUSSION:

A significant health issue in terms of morbidity and death is wound. The controlled production, deposition, and maturity of new collagens are key components of the healing process. In order to heal damaged tissue, inflammatory cells promote endothelial cell migration and proliferation. This causes connective tissue cells to neovascularize and creates extracellular matrices like collagen and keratinocytes, which reepithelializes the damaged tissue³¹. Scar formation, collagen maturation, and inflammation are a few of the several stages of wound healing. These phases take place consecutively but apart from one another. The ability of this plant to cure wounds was assessed using excision and incision models. The significant wound healing activity in both models was seen when contrasted with the negative control. In excision model, the assessment criteria were % of wound contraction; whereas, in the incision model, the evaluation criterion was skin tensile strength³². Gel C(6%), gel B(4%), and gel A(2%) of Populus deltoides ethenolic extract give results that are comparable to the negative control. The skin didn’t show any sign of change in colour or morphology. All gel formulations were discovered to be irritant-free. The effectiveness of the gels C and B was deemed good since the treated animal's wound contraction was 100% whereas the negative control animal's wound contraction was 89%. In incision model, the tensile strength of the treated animal was 788gm whereas the control animal's was 483gm, demonstrating the gel C's effectiveness, which was deemed to be good. The greatest stress a material can bear when stretched or pulled before breaking is referred to as its tensile strength. A higher tensile strength indicates that the collagen fibres in the healed wound are more robust and well-organized. Higher tensile strength is associated with better wound closure and a lower chance of problems like infection or sluggish healing. The best healing drug must have the activity to improve the credibility of collagen fibrils surrounding the lesion site, which raises the wound's evaluated tensile strength by comparing it to the wound's healed tensile strength. The main phytochemicals in Populus deltoides that may be involved in their ability to promote wound healing are flavonoids and alkaloids³³. Flavonoids are promoting the wound healing process due to astringent and antimicrobial property that is responsible for increase rate of wound contraction. Alkaloids increased rate of formation of epithelial cells thus speeding up the epithelialization process. Flavonoids and alkaloids have the potential to affect the migration and proliferation of several cell types, such as fibroblasts, endothelial cells, and keratinocytes, which are involved in wound healing. These substances have the ability to quicken the healing process and the production of new tissue by encouraging cell migration and proliferation. Therefore, we looked into the capability of the plant Populus deltoides to cure wounds. Our research supported the claims made in the literature that Populus deltoides had wound-healing properties. The specific mechanism of this activity requires extensive investigation that might provide information about the key components and mode of action of this plant in the treatment of wounds.

ACKNOWLEDGMENTS:

This work was supported by Meerut Institute of Engineering and Technology, Meerut.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.03.2024 Revised on 13.07.2024

Accepted on 16.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1590-1596.

DOI: 10.52711/0974-360X.2025.00228

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Groups	04^th (day)	08^th (day)	12^th (day)	16^th (day)	20^th (day)	Epithelization period (days)
1.	Negative Control	368 ±3.5 (9)	291 ±3.1 (28)	194 ±2.6 (52)	104 ±3 (74)	43 ±2.8 (89)	21
2.	Standard	360 ±3.4 (12)^a	235 ±2.5 (42)^a	141 ±4.6 (65)^a	50 ±3.2 (87)^a	5.2 ±0.8 (99)^a	18
3.	Gel A	372 ±3.8 (7)	247 ±3.9 (38)	171 ±5.7 (57)	76 ±4.2 (81)	20 ±3.7 (95)	20
4.	Gel B	325 ±2.5 (20)^b	216 ±4.2 (46)^b	142 ±4.5 (65)^b	24 ±1.7 (94)^b	1.2 ±0.4 (99)^b	19
5.	Gel C	304 ±4.1 (24)^a	200 ±4.2 (50)^a	71 ±3.5 (82)^a	0.2 ±0.1 (99)^a	0 ±0 (100)^a	16