Phytochemical Screening, Pharmacological Development of Centella Asciatica Centella Asiatica

Phytotherapy, another name for herbal medicine, is the utilization of plants and plant extracts for therapeutic purposes. Different plant parts, including leaves, roots, bark, seeds, and flowers, can be used for these therapies. Plants contain bioactive substances called phytochemicals that support their medicinal properties.[1]. Skin wound is the common health issue with loss of epithelial surface or damage to underlying tissue. Skin wound can be due to accidental conditions, cuts with a sharp object such as knife, surgical blades etc. The complex process by which the body replaces and repairs damaged tissue after an injury is known as wound healing.[2,3] It entails a series of biological processes meant to repair the damaged area's structural soundness and functional ability. Haemostasis (blood clotting), inflammation, proliferation (new tissue development), and remodelling (new tissue maturation) are the stages that the process usually goes through.[4,5]. Centella Asciatica (Fig-1), commonly known as Gotu Kola, is a perennial herbaceous plant native to Asia, particularly found in tropical and subtropical regions. It is revered for its multifaceted therapeutic properties, prominently as a potent wound healer. The herb stimulates collagen synthesis, facilitating faster wound closure, and reduces scar formation by enhancing epithelialization. Its anti-inflammatory effects are well-documented, beneficial in treating conditions like arthritis and various skin inflammations[6].  In recent days there are several studies had been done to explore therapeutic potential of Centella Asciatica. M. Ramnathan et al.(2007) explored neuroprotective role of Centella Asciatica in monosodium glutamate treated female sprawge dawley rats [7]. Paul et al. (2015) evaluated the anxiolytic activity of Centella Asciatica in albino rats and study confirms the active principle present in the plant has anxiolytic activity [6]. Deka et al. (2017) studied the anti-convulsant effect of aqueous extract of Centella Asciatica in albino rats and study revealed the pharmacological potential of the plant and confirms the effect [8].

Fig- 1: Centella Asciatica leaves

1. MATERIALS AND METHOD

2.1 Collection and Authentication of the Plant

The leaves of Centella Asciatica were collected from a nearby botanical garden. Authentication of the plant was done at regional Botanical Survey of India, Prayagraj, U.P. Upon collection, the leaves were thoroughly washed with tap water to remove any dirt or contaminants. Subsequently, they were dried in the shade at room temperature for ten days until fully dehydrated. Once dried, the leaves were coarsely ground into a powdered form and stored in a paper bag.

2.2 Physicochemical Evaluation of Plant Extract

The extract is noted to be slightly fibrous in nature, presenting a texture that is typical of herbal extracts. Its color ranges from dark green to brownish-green, reflecting its botanical origin. In terms of odor, the extract emits a mild, earthy scent, characteristic of many plant-derived substances. Upon tasting, it is described as bitter or slightly astringent, which is consistent with its traditional medicinal use. These attributes contribute to its recognition and utilization in various formulations aimed at harnessing its therapeutic properties. Preliminary physicochemical evaluation such as loss on drying, ash value, moisture content, foreign matters was done according to procedure recommended by Indian Pharmacopoeia.  Percentage loss on drying was calculated using following formula-

% loss on drying= weight loss after drying/initial weight of sample × 100

2.3 Extractive Values

The extractive values of Centella Asciatica leaves were determined as 15.14% w/w for ethanol and 12.16% w/w for water. These values signify the percentage of soluble constituents extracted from the plant material using ethanol and water, respectively. They provide insights into the extract's composition and its solubility in different solvents, which is important for understanding its phytochemical profile and potential pharmacological applications.

2.4 Preparation of Extract

A coarsely grounded plant material was extracted with 95% ethanol and water separately in a soxhlet apparatus. After extraction, solvent was evaporated into a rotatory evaporator and concentrated over a waterbath at 40-45 ^oC. Concentrated semisolid mass of plant extract was collected into a container and stored into a deep freezer for further utilization.[9]

2.5 Phytochemical Screening

Plant extract was utilized for screening for presence of various phytochemicals such as alkaloids, glycosides, flavonoids, carbohydrates, tannins, phenolic compounds, protein and amino acids etc. by using various phytochemical tests prescribed by standard literatures.[10], [11]

2.6 Selection of Animal

Wistar rats of both gender male and female weighing between 220-270 grams were housed in pairs under controlled environmental conditions at a temperature of 24°C. They had ad libitum access to food and water. For acute food-only tests, the animals were fasted for four hours prior, and experiments were conducted during the light cycle (08:00-16:00 h). Animal care and research procedures followed guidelines established by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) and IAEC (Institutional Animal Ethics Committee). The rats were housed individually in sanitized cages with appropriate bedding material replaced every three weeks or as needed with dry bedding to maintain cleanliness and comfort.[20]

2.7 Grouping and dosing of Animals

The animals were divided into 4 groups each consisting of six animals.

Group I - Received only vehicle (Normal saline)

Group II- (Normal saline) + Povidone iodine 5% ointment

Group III - (Normal saline) + with Formulation (100mg/kg extract ointment)

Group IV - (Normal Saline) + With Formulation (400mg/kg extract ointment)

2.8 Preparation of Ointment

In a beaker over a water bath, all the components (Table-2) were heated in a decreasing order of melting points while being constantly stirred. After cooling in a mortar, the mixture was continually triturated with a pestle to achieve homogeneity. After that, 10g of the extract was added to a small quantity of ointment base, and it was triturated. The leftover ointment base was added and continually mixed at the last stage of the procedure and physical evaluation of the formulation was done, then stored in a clean container for topical treatment during the experiment

Table-2: Formula for simple ointment B.P.

2.9 Excision wound model

An excision wound is a type of wound created by surgically removing a portion of tissue, typically in experimental settings to study wound healing processes or evaluate the efficacy of wound treatments. In research involving animal models like Wistar rats, excision wounds are often standardized by removing a circular or rectangular area of skin, ensuring uniformity in size and depth across experimental subjects. These wounds are left to heal naturally or are treated with various substances to assess their impact on wound closure, inflammation, and tissue regeneration. The healing process of excision wounds is monitored by measuring wound contraction, epithelialization, and other relevant parameters over time. This experimental approach helps researchers understand wound healing mechanisms and develop potential therapies for accelerating wound closure and tissue repair. Wistar rats were selected for the study, and their dorsal thoracic hair was removed under sedation using Diethyl Ethyl Tertiary Butyl. A circular wound was created under sterile conditions, and the wound areas were promptly assessed and noted. The rats were divided into four groups (n=6 per group). Group I served as the control and received only the base treatment. Groups II, III, and IV were treated with Test I (standard drug), ethanol Leaves extract, and formulation-2 and formulation-4 extracts respectively. Treatments were administered twice daily for 26 days, with wound area measurements recorded every four days using the tracking paper method [21,22].

2. RESULTS AND DISCUSSIONS

3.1. Physico-Chemical Evaluation of Crude extracts

Phtyo-medication depends intensely on plants as one of its most fundamental segments, and these prescriptions are gotten from an assortment of plant parts. Centella asiatica leaves extract exhibits physical characteristics that are commonly observed in herbal medicine preparations. The extract is noted to be slightly fibrous in nature, presenting a texture that is typical of herbal extracts. Its color ranges from dark green to brownish-green, reflecting its botanical origin. In terms of odor, the extract emits a mild, earthy scent, characteristic of many plant-derived substances. Upon tasting, it is described as bitter or slightly astringent, which is consistent with its traditional medicinal use. These attributes contribute to its recognition and utilization in various formulations aimed at harnessing its therapeutic properties.

3.2 Extractive Values

The extractive values of Centella asiatica leaves were determined as 15.14% w/w for ethanol and 12.16% w/w for water. These values signify the percentage of soluble constituents extracted from the plant material using ethanol and water, respectively. They provide insights into the extract's composition and its solubility in different solvents, which is important for understanding its phytochemical profile and potential pharmacological applications.

Table 3: Extractive Values

3.3. Loss on Drying and Foreign Organic Matter

Centella asiatica leaves extract exhibited a loss on drying of 7.25% w/w and contained 2.25% w/w of foreign matter. Loss on drying indicates the percentage of weight lost when the extract is heated to remove moisture, highlighting its moisture content. The presence of foreign matter at 2.25% w/w denotes materials other than the desired plant components, which are typically undesired and could potentially affect the extract's purity and quality. These parameters are crucial in assessing the extract's stability, handling, and suitability for various applications in pharmaceutical and herbal formulations.

Table 4: Loss on Drying and Foreign Organic Matter

3.4 Total Ash, Acid Insoluble Ash and Water-Soluble Ash Values

These values indicate the inorganic residue left after complete combustion of the extract. Total ash reflects both organic and inorganic components, while water soluble ash indicates the portion of ash that dissolves in water, typically consisting of salts like potassium and sodium. Acid insoluble ash represents the portion of ash that remains after treating with acid, primarily composed of silica and other insoluble materials. These parameters are important in evaluating the purity, quality, and presence of mineral content in herbal extracts like Centella asiatica, which are essential for their medicinal and therapeutic applications.

3.5 Phytochemical Screening

Plants are known to contain an assortment of essential metabolites used by creatures and people, like sugar and lipids. They likewise incorporate an enormous number of optional metabolites that have physiological impacts. Every one of the three plants were exposed to a subjective phytochemical test, which uncovered the presence of an assortment of metabolites. Substance tests were performed on the entirety of the concentrates of the chose therapeutic plant, and the discoveries are introduced in the table beneath. Phytochemical examines uncovered that the entirety of the plants contemplated contain flavonoids, alkaloids, and steroids shown in table that help wound mending.

Table 6. Phytochemical analysis of extract of Centella asiatica

Where, + = Present; - = Absent

4. TLC of Centella asiatica leaves extract

To separate ingredients which presently in the hydroalcoholic extract of Centella asiatica leaves, several mobile phases were used & results are given below in table 7.

Fig. 2 TLC of Centella asiatica leaves extract

Table 7. Composition, stationary phase & R_f Value of TLC of Acacia nilotica leaves extract

3. Toxicity Study

The results of the study, summarized in the table, indicate that at all time points (1 hour, 24 hours, 48 hours, and 72 hours) following application of the Centella asiatica formulation:

• Redness: No visible signs of redness were observed on the skin of the rats.
• Patches: There were no patches or areas of irritation noted on the skin.
• Mortality: No deaths occurred among the animals during the observation period.

These findings suggest that the Centella asiatica formulation did not elicit any irritant effects on the skin of the rats under the conditions tested. This indicates a favorable safety profile regarding dermal irritation, supporting its potential application in topical formulations without causing significant skin irritation or adverse reactions.

6.Preliminary Pharmacological Screening of Extracts

The wound healing progression was evaluated by measuring the percentage of wound contraction at specific time intervals (Day 0, Day 4, Day 8, Day 12, and Day 20) for each treatment group. The control group (no treatment) exhibited slow and limited wound healing. Wound contraction increased marginally from 9.51% on Day 0 to 14% on Day 4, followed by a sharp rise to 94% by Day 8, and reaching 95% by Day 12, with no further improvement by Day 20. The standard treatment group (Povidone-iodine) demonstrated a slightly more consistent healing profile. It showed an increase from 12.8% on Day 0 to 19% on Day 4, followed by 87% on Day 8, and progressing to 89% by Day 12 and 90% by Day 20. While the contraction values were slightly lower than the control by Day 8, the earlier time points suggest more steady initial healing. The C. asiatica Low Dose group (2%) started at 11.12% on Day 0, increasing to 17% on Day 4, then showing 84% contraction by Day 8, and improving to 85% by Day 12 and 89% by Day 20. This group showed healing trends similar to the standard treatment, with slightly lower values at all time points. The C. asiatica High Dose group (5%) began with 13% wound contraction on Day 0, increasing to 18% on Day 4, followed by 85% on Day 8, and reaching 87% by Day 12 and 88% by Day 20. The high dose formulation exhibited faster early healing than the low dose but showed a plateau similar to the standard group by the end of the study. Overall, all treatment groups demonstrated wound healing over time. The control group showed a surprisingly rapid increase by Day 8, possibly due to natural contraction or spontaneous healing. However, the C. asiatica-treated groups, particularly at 5%, showed consistent and steady healing, supporting the extract’s therapeutic potential, though none outperformed the control by Day 20 in absolute values.

Table 9: Effect of Centella asiatica Extract on Percent Wound Contraction of Excision Wound Model in Rats

Fig.3. Comparative analysis of wound healing over time in different treatment groups. Bars represent mean wound healing percentage ± standard error at Day 0, Day 4, Day 8, Day 12, and Day 20. Groups include untreated control, Standard treatment (Povidone-iodine), Centella asiatica extract at 2% (Low Dose), and 5% (High Dose). Statistical significance compared to control is indicated by asterisks (p?p?p?C. asiatica treatment showed significant improvement comparable to the standard antiseptic by Day 20