Introduction

Physical harm that causes the skin to split or open up are called wounds. For the repair of broken anatomical continuity and compromised functional status of the skin, proper wound healing is crucial. It is the end result of a coordinated response to harm from various cell types. The contraction and closure of the wound as well as the restoration of a functional barrier are the consequences of the multifaceted, complex process of wound healing (Umachigi, Kumar, Jayaveera, & Dhanapal, 2007). A series of actions, including inflammation, cell proliferation, and cell type migration, result in the repair of damaged tissues (Sidhu et al., 1999). The physiologic process of wound healing is divided into three phases: the substrate phase, the proliferative phase, and the remodeling phase (Umachigi, Jayaveera, Kumar, & Kumar, 2007). A number of cytokines, including growth factors, govern how all these actions are carried out (Pierce, Berg, Rudolph, Tarpley, & Mustoe, 1991). Herbal medicine is a crucial component of traditional medicine, which is often the only accessible or cheap form of healthcare in underdeveloped nations. Although many people think herbal medications are harmless, they are frequently combined and derived from plant sources, each of which has a unique variety of species, growing conditions, and biologically active ingredients. The availability of numerous active components in botanicals that combined can produce a potentiating effect that may not be possible with any one compound is a significant potential benefit above traditional single-component medications. We created a polyherbal emulgel formulation combining manjistha, guduchi, moringa, neem, and aloe and tested its ability to treat wounds against extracts while keeping in mind the benefits of herbal medications.

Methodology

Material

The authentic plants extracts and oils including Manjistha, neem extract, guduchi, moringa oil, aloe vera were procured from Amsar Pvt. Ltd. Colvale Goa.

All the other chemicals were purchased from S. G. pharma.

Method

Method of Preparation and Composition of Emulgel

The emulgel EG1 was prepared by weighing all the powdered actives including 0.4 g of manjishta extract, 0.1 g of neem, 0.2 g of guduchi extract and aloe extract. Add one by one actives in the vortex in the beaker containing ¾ th ml of water (q.s. 100 ml) and kept on a magnetic stirrer rotated at 300 rpm with continuous stirring for 10 min. Dissolved 0.1 g moringa oil in the 5 ml of ethanol. The oil- ethanol mixture was then added beaker containing powder- water mixture. Weighed 2 g of carbopol and then added in it. Continue the stirring for 20 mins, until the mixture was free from carbopol lumps and resulting into the emulgel. The mixture of triethanolamine (1 ml) and water (2.5 ml) was added dropwise to the gel in order to neutralize and thickened the emulgel (Shrikhande, 2013; Yadav, Mishra, Tiwari, & Shukla, 2016).

Evaluation of Formulations

Determination of Surface pH

The pH of the formulation was determined using a pH paper by referring to standard pH scale.

Determination of the pH of 10% Solution of the Formulation

1g of the formulation was weighed and dissolved 10ml of water to produce 10% solution. The pH of the formulation was determined using a pH meter.

Spreadability

The glass slide was used to calculate spreadability. 0.1g of the formulation was weighed and placed on one slide and then the second slide was placed on top of that slide and then 100g of weight was placed on it. The weight was kept for 1 minute and measured the diameter of the formulation.

Extrudability

The formulation was filled completely in the 5g tubes and crimped. The tube was placed on the table at a height. The weighing machine was placed on the floor holding a petri plate. The tube was pierced and the specific weight was placed on it and the amount of the formulation that falls in the petri plate in 1min was recorded by recording its weight.

Viscosity

The viscosity of approx. 50g of formulation was measured using a Brookfield viscometer with LV spindle having low viscosity and 64 spindle number (Badi & Khan, 2014; Gyawali, Gupta, Shrestha, Joshi, & Paudel, 2020).

Wound Healing Activity: Excision/ burnt model

Grouping of Animals

For excision, incision and burn wound model, animals will be divided into one positive control and others depending upon stable topical dosage form is formulated.

Excision Wound Model

Anesthetic ether was used to anaesthetize each set of animals, which included six rats. The rats' backs were depilated, and a predefined area of 500 mm² full thickness skins in the dorsal interscapular region was excised. Rats were exposed to the open environment while undressed. The formulas were used on a daily basis for a total of 14 days. The wound contraction and epithelialization time were tracked in this model. Wound contraction was calculated as a percentage of the initial wound size every two days. Each rat had a specimen sample of tissue taken from the recovered lesion for histopathological analysis (Nagar et al., 2016).

Burn Wound Model

Concentrated sulfuric acid was used to make burn wounds on the dorsal region of a shaved rat's skin surface, which were exposed for 10 seconds. Dead tissues were removed 24 hours later using a sterile surgical blade and a template designed to generate a third-degree burn. All groups were given the same treatment as in the excision model. The wound contraction and epithelialization time were tracked in this model. Each rat had a specimen sample taken from the healed wound of tissues for histological evaluation (Vaghardoost, Ghavami, & Sobouti, 2019).

Evaluation of Wound Healing Parameters

Measurement of Wound Contraction

After the wound was created, an excision wound area was traced with transparent paper and the area recorded using graph paper. Wound contraction was measured every two days until the wound was completely healed, and the results were represented as a percentage of the healed wound area. Using the evaluated surface area, the percentage of wound contraction was computed using the standard equation, assuming the original size of the wound, 300 mm², as 100%:

$\% Wound contraction=\frac{Initial wound size-Specificday woundsize}{Initial woundsize}\times 100$

Histopathological Studies

Following complete healing of the excision and dead burn wounds, wound tissue specimens from the control, test, and standard groups were obtained, and after routine processing, 6-mm thick sections were cut and stained with hematoxylin and eosin. Under a light microscope, sections were evaluated qualitatively for fibroblast proliferation, collagen production, angiogenesis, and epithelialization.

RESULT AND DISCUSSION

Evaluation of Formulations

The prepared formulation emulgel EG1 was evaluated for various parameters including colour, appearance, texture, pH, surface pH 10% pH solution, spreadability (diameter), extrudability (weight in a min), viscosity. The prepared emulgel was reddish brown, slightly glossy and smooth in texture. The pH of formulation was 7 and surface pH was found to be 5.14. The spreadability, extrudability and viscosity were also within the stated range. The evaluation concluded that formulation EG1 showed better results further evaluated for its wound healing potential [Table 1].

Wound Healing Study

The polyherbal formulation EG1 composed of moringa oil, carbopol 940, ethanol as an emulsifier, triethanolamine, and water. The prepared formulation was found to be easy to formulate and exhibited all the criteria required for better and stable emulgel.

Effect of Emulgel on Excision Wound

Figure 1 illustrates the images of excision wound after 0 and 14 days of application of the preparations (EG1). The results concluded that the wound of each group healed with time. The excision wound treated emulgel was mostly healed.

Effect of Emulgel on Burn Wound

Figure 2 illustrates the images of excision wound after 0 and 28 days of application of preparations (EG1). The results concluded that the wound of each group healed with time. The wound treated with emulgel began to heal noticeably after 14 days. After 28 days the complete healing of wound was observed.

Histopathological Evaluation of Excision Wound and Burnt Wound

Histopathological staining analysis was performed to evaluate the effect of emulgel formulation on the wound given in Figure 3. After 14 days of completion of treatment for excision wound and 28 days of completion of treatment for burnt wound, the rats were sacrificed to obtain their skin samples. Observing these images revealed that reduction in the thickness of skin indicating the healing of skin. Re-epithelialisation of cells present in epidermal as well as sub-epidermal layer found in newly generated tissue of nanofibers in a group of animals treated with drug. The fibroblastic procreation was also seen at the site of healing. The epidermal layer of adjacent skin in the test group of animals treated with drug was normal, with an improved quantity of collagen. In test animals, fibroblastic procreation and vascular procreation coated in granulation tissue were also detected after the wound had healed entirely. The appearance of scar tissue, inflammatory cells, decreased collagen fibres, fibroblast cells, and blood vessels were seen in control group of mice. The prepared emulgel exhibited the procreation of epithelial tissue along with keratinization. The emulgel formulations efficiently elevate the epithelialization rate, collagen viability all over the wound area with no any microorganisms. It also induce the wound strength. The results concluded that the prepared formulations (EG1) was more effective than extract.

In Figure 3,

$\updownarrow$ Orange double arrowed line indicates the thickness of the skin in control and treated group.

$\uparrow$ Green arrowed line indicates the hair follicles, in treated skin they are near to surface but in other groups moved down.

$\uparrow$ Blue arrowed line indicates the blood vessels which are seen more in treated skin but not in other group.

Conclusion

The term "wound" refers to physical harm that causes the skin to split or open. For the repair of broken anatomical continuity and compromised functional status of the skin, proper wound healing is crucial. People are now concentrating on the traditional medical system because it does not result in serious bad responses. The effectiveness of using a single herb to treat the infection, however, was lower. By using several medicinal plants at once, these restrictions can be avoided. This type of composition is called polyherbal. In the current study, the effectiveness of a polyherbal emulgel formulation against wound healing was assessed. Excision and burn wounds were created for the study on wound healing. According to the findings, a burn wound required 28 days of treatment to fully heal, whereas an excision wound treated with emulgel was substantially healed after 14 days. According to histopathological examinations, wound remodelling occurred more quickly in the treated groups of animals when compared to the control groups of animals. These parameters included collagen, fibroblasts, and neovascularization. The aforementioned observations lead us to the conclusion that these topical formulations with herbal extract have potent wound-healing properties. The active ingredients in the created formulations have a variety of functions, including antimicrobial, antioxidant, anti-inflammatory, antibacterial, and antifungal, as well as the ability to keep moisture in the skin.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that there is no conflict of interest.