Pharmacological Evaluation of Wound Healing Activity of Some Medicinal Plants using Animal Model

Caesulia axillaris Roxb flourishes in wetland ecosystems i.e., marshes, riverbanks, and other saturated environments. It is essential to the local ecology and well suited to these environments [1][2]. Along the stalk, the leaves are arranged in alternating order. Their lanceolate to elliptic shape makes photosynthesis more efficient. The majority of leaf edges are entire, although they can occasionally show slight serration, which adds to their variety. In addition to providing shelter and food for a variety of species, including pollinators like bees and butterflies, C. axillaris Roxb improves the overall health of the wetland environment by stabilizing the soil and helping to purify the water [3].

C. axillaris Roxb. is rich in a number of bioactive compounds that improve its pharmacological characteristics. The plant's antioxidant, anti-inflammatory, antibacterial, analgesic, and gastro protective qualities are attributed to a variety of components, including phenolic compounds, flavonoids, alkaloids, terpenoids, saponins and essential oils. The potential therapeutic benefits of C. axillaris Roxb are being revealed by ongoing research on these bioactive compounds [4][5]. Limonene and γ-Asarone make up the majority of it. The blossoming shrub Clerodendrum Viscosum Ventenat is known for its ugly foliage. The straight, unbranched stem is 0.5–4 m (1.6–13.1 ft.) tall and has round leaves that are 15 cm (5.9 in) in diameter. The leaves are simple, opposite, elliptic, wide elliptic, ovate, or elongated oval, with a dentate edge, measuring 3.5-20 cm (1.4-7.9 in) in width and 6-25 cm (2.4-9.8 in) in length. Both surfaces are sparsely covered with villous-pubescent hairs. The inflorescence is a terminal cyme with few flowers that is peduncled [6][7].

C. viscosum Ventenat contains a number of bioactive phytochemicals. Because of their different spatial arrangement, the phytochemical profiles of the leaf, root, and flower show different bioactivities in the seed and stem. Flavonoids, terpenoids, steroids and alkaloids are the main bioactive compounds that have been extracted from C. viscosum Ventenat, according to numerous research. Numerous bioactive compounds with notable pharmacological efficacy were found in many parts of C. viscosum Ventenat, according to thorough research [8][9]. Gallic acid, methyl gallate, tannic acid, ellagic acid, reserpine, limonene, α-pinene, β-pinene and ρ-cymene are the main constituents of C. viscosum Ventenat leaves.

Allium stracheyi Baker. is a perennial herb that can grow up to 35 cm in height. The heads are globose, the pedicel is shorter than the flower, and the tepals are dark pink-reddish; the stem is glabrous, leafy at the base, and has thin, linear, flattened leaves [10]. In the Garhwal region, it is known locally as Jamboo, Dhungar, and Pharan. At elevations between 2500 and 3625 meters, this species can be found in Jammu Kashmir, Himachal Pradesh, Uttarakhand, Nepal, and Pakistan [11]. 

Herbal medicine constitutes a complex system of formulations. Its formulations for medical use comprise several types of secondary metabolites or bioactive substances. Diverse chemical and analytical methodologies facilitate the comprehension of quality control and chemical ingredients in herbal pharmaceuticals. Allium species have been documented to contain a variety of physiologically active chemicals, including phenolic acids, flavonoids, thio-sulfinates, alkaloids, fixed oils, phytosterols, sulfur-containing compounds, and others [12][13]. A. stracheyi Baker. has sulfur-rich compounds having the antioxidant, anti-inflammatory, and antibacterial properties. Compounds high in sulphur have been documented to reduce blood cholesterol [14]. The current study was based on the phytochemical screening, isolation and estimation of total phenolic contents & total flavonoids contents of some medicinal plants i.e., Caesulia axillaris Roxb, Clerodendrum Viscosum Ventenat and Allium stracheyi Baker. The aim of study was pharmacological evaluation of wound healing activity of some medicinal plants using animal model.

MATERIALS AND METHODS

Experimental requirements

Fresh leaves of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat, Allium stracheyi Baker. methanol, ethanol, distilled water, rotatory evaporator and weighing machine. 

Collection, authentication, and preparation of extract

The Fresh leaves of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat, Allium stracheyi Baker. were collected from UP East region and authenticated by a Botanist Dr. Vijay Kumar Sinhal, Department of Plant Science, MJP Rohilkhand University Bareilly, UP India, with the ref. no. MJPRU/PS/2024/01. The leaves of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat, Allium stracheyi Baker. were washed to make dust-free and dried under shade. The leaves were rendered into coarse powder and further in fine ones. The powders of each plant were weighed and soaked in aqueous, methanolic and hydroalcoholic solvent (1:1), separately for 15 days with frequent stirrings. The obtained slurry was kept for drying under partial vacuum using a rotatory evaporator [15].

Preparation of rats

Wistar albino rats (160-220 g) were utilized to determine the wound healing potential of leaves extracts of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat, and Allium stracheyi Baker. The rats were obtained from the Animal House, Department of Pharmacy, Faculty of Engineering and Technology, MJP Rohilkhand University, Bareilly with the ref. no. MJPRU/PY/IAEC/24/64. Rats were kept in plastic cages at ambient temperature and relative humidity which fed with pellet diet and water ad libitum. Institutional Animal Ethics Committee (IAEC) authorized the protocol for the animal experiment.

Preparation of ointment base and extract-based ointment

Six different formulations were prepared using leaves extracts of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat and Allium stracheyi Baker. The ointment was carried out with individual plant extracts, in which methanolic and hydroalcoholic extracts were separately used. Paraffin wax (1.5 g) was melted using water bath, and further lanolin, cetostearyl alcohol (1.5 g each) and petroleum jelly (24.5 g) were mixed to make a homogenous mixture. Thus, ointment base was prepared which utilized in control (group I). To this base mixture, 1g of leaves extracts of Caesulia axillaris Roxb, Clerodendrum viscosum Ventenat, and Allium stracheyi Baker. (methanolic and hydroalcoholic) were separately added, and thus 6 different ointments (30g each) were made through triturating by using pestle mortar [16].

Table 1. Composition table for ointment base

Table 4. Wound contraction (%) of excision wound model

Group	Wound contraction (%)
	Day 5	Day 10	Day 15	Day 20
Group I	31.64±0.19	36.21±0.57	62.11±0.43	65.32±0.11
Group II	59.26±0.43	82.43±0.12	92.27±0.64	100±0.32
Group III	54.43±0.14	68.14±0.51	84.26±0.10	96.81±0.11
Group IV	56.32±0.11	71.29±0.46	87.14±0.53	100±0.23
Group V	52.12±0.34	64.12±0.47	81.27±0.32	92.16±0.31
Group VI	55.24±0.64	68.37±0.13	83.20±0.33	95.24±0.52
Group VII	51.27±0.43	62.14±0.57	79.12±0.31	93.42±0.16
Group VIII	54.39±0.12	66.32±0.24	81.27±0.40	96.10±0.37

n=6,  P<0.05; values denoted as Mean ± Std Deviation

Fig 4. Graphical data of Wound contraction (%) of excision wound model

Fig 5. Graphical data of Tensile strength of excision wound model

Fig 6. Graphical data of Epithelisation time of excision wound model

Fig 7. Graphical data of Tensile strength of incision wound model

Many plants and/or their derivatives were used as wound-healing agents by people all over the world, even though there was conflicting evidence on their efficacy and safety. Conventional wound-healing agents are unable to stop all phases of wound healing, which highlights the need for the development of innovative, safe, and effective wound-healing medications [22].

The topical administration of the hydroalcoholic extracts increased the rate of wound reduction in the excision wound model, which may be explained by improved wound healing progression and observable hydration of the wound boundary as a result of tissue regeneration. The percentage closure of all extracts fell between 97.7% to 99.9% on the final day of treatment, compared to 89.9% for the group that received a basic ointment. A decrease in the proportion of the initial wound size is an example of wound contraction that aids in wound closure [23]. Coagulation, inflammation, macrophages, fibroplasias, collagenation, contraction, and epithelization are all directly related to the healing process. Treatment may influence the healing process by mediating at least one stage of the healing process. The major extracellular protein in the granulation tissue of wounds, collagen, is significantly increased in mice treated with WE ointment. Therefore, the beginning of many factors connected to the wound healing process and inflammatory pathways, which may be attributable to the capability of plant materials in GE, is most likely what causes the wound healing activity of the WE ointment [24].

The role of wound contraction is critical because it reduces the size of the wound, increases the amount of extracellular matrix needed to repair the faulty cell, and promotes re-epithelization. The extracts' ability to reduce wounds may be attributed to their mitogenic activity, which increases fibroblast motility and cellular proliferation, inhibition of microbial growth, especially during the inflammatory phase, and subsequent conversion to myofibroblasts during the healing process, which is primarily dermal. One way that plant extracts promote wound healing is by stimulating fibroblasts, which migrate from the wound edge to the site, proliferate, and eventually create collagen, the primary component of extracellular matrix [25]. Restrain the oxidative reactive species developed by phagocytes is important aspect of wound healing [26]. By reducing or delaying the start of cell necrosis and enhancing vascularity, another metabolite known as flavonoids lowers lipid peroxidation. Circulation promotes the viability of collagen fibrils thus preventing cell damage, and promotes DNA synthesis. Due to their antimicrobial and antioxidant properties, which appear to be the cause of wound contraction and an accelerated rate of epithelization, these substances, along with triterpenoids, are also aiding in the healing of wounds. According to a different study, certain flavonoids have astringent properties and inhibit enzymes like phospholipase A2, which are crucial for wound contraction and the pace of epithelization [27].

CONCLUSION

In conclusion, % yield and phytochemical constituents were observed in this order: hydroalcoholic > methanolic > aqueous. Plants species demonstrated as a rich source of phytochemicals including alkaloids, cardiac glycosides, tannins, saponins, coumarins, steroids and flavonoids. They exhibited an excellent quantity of total flavonoids and total phenolic contents. In excision and incision wound models, medicinal plants showed the wound healing potential in this order: Caesulia axillaris Roxb > Clerodendrum viscosum Ventenat > Allium stracheyi Baker., when evaluated for wound contraction (%) measurement, Tensile strength measurement and Epithelisation time.

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