Formulation and Evaluation of Wound Healing Cream Using Tridax Procumbens Linn

Wound healing is a biological process that occurs in a complex series of steps including inflammation, proliferation, and remodeling. Although many treatments are provided by modern medicine, the quest for effective and safe alternatives continues. The therapeutic potential of various plants has been recognized by traditional medicine for wound care. This study intends to explore the wound healing potential of a novel cream formulation containing three powerful medicinal herbs: Tridax Procumbens Linn, Withania somnifera (Ashwagandha), and Centella asiatica. Tridax Procumbens Linn has been applied traditionally for wound healing and inflammatory conditions. Withania somnifera exhibits in vitro anti-inflammatory, antioxidant and immunomodulatory activities. In the literature, Centella asiatica is recognized in stimulating collagen synthesis and facilitating wound repair. These herbs, if added together in the form of topical cream, synergistically could aid in enhancing healing of wounds in the present protocol. This research is to prepare and characterize the herbal cream and to conduct in vitro and/or in vivo to assess its efficacy for promoting wound closure and inhibiting inflammation and rapid regeneration of tissue. The results of this study will open doors to the development of a safe herbal wound healing cream that will supplement or even supplant conventional treatments. Asiatica, also known as Gotu kola, is a herb that holds much promise in wound healing with its bioactive compounds like asiatic acid, madecassic acid, asiaticoside, and madecassoside that induce collagen synthesis, proliferation of fibroblasts, angiogenesis, and inflammation reduction, thereby promoting the healing of wounds across various stages including re-epithelialization and tissue remodeling; making it a potential natural remedy for a variety of wounds, burns, and scars.

MECHANISM OF ACTION:

• Collagen production: The respective protein quantity of tissue repair is found to be stimulated by centella asiatica by activating fibroblasts.
• Angiogenesis: It encourages the growth of new blood vessels, which is essential for the delivery of oxygen and nutrients to the wound site, thereby healing faster.
• Anti-inflammatory effects: Bioactive compounds present in the plant can result in inflammation reduction that may further retard healing.
• Cell proliferation: Centella asiatica enhances the cell division rate, so the rate of regenerating tissues is faster.

Clinical uses:

• Skin lesions: Topical use of Centella asiatica extracts is useful for the treatment of minor cuts, abrasions, and surgical wounds.
• Burns: Centella asiatica has been reported to be useful in the healing of burns by enhancing re-epithelialization and reducing scar formation.
• Diabetic ulcers: Centella asiatica may be useful in the management of diabetic foot ulcers due to its enhanced blood circulation and wound healing activity.
• Keloids and hypertrophic scars: Topical application may decrease the size of the raised scars.
• Many in-vitro and animal experiments have established the healing activity of Centella asiatica in wounds.
• Clinical studies have also revealed excellent results on the application of Centella asiatica as topical to different forms of wounds.

Physiology of Skin:

The skin is the largest organ of the body, playing a critical role in protecting the internal organs, regulating temperature, and providing sensory information.

 It is made of three layers:

1. Epidermis: It is composed of stratified squamous epithelium and is made up of dead, keratinized cells that form a strong, impermeable barrier. Only thick skin (palms, soles) has stratum lucidum, a thin, translucent layer. The stratum granulosum is made up of cells that help the skin's barrier function by producing lipids and keratin. The Langerhans cells, which mediate immunity, are found in the stratum spinosum. The lowest layer, the stratum base, contains stem cells to replace the epidermis and melanocytes, which produce melanin for coloration.
2. Dermis: A thicker layer of connective tissue, providing strength and flexibility. It contains blood vessels, nerves, sweat glands, hair follicles and sebaceous glands.  

Two layers:

Papillary layer: Contains dermal papillae that interlock with the epidermis, increasing surface area for nutrient exchange.  

Reticular layer: Contains dense collagen and elastin fibers, providing strength and elasticity.

3. Hypodermis: It is the deepest layer, primarily composed of adipose tissue. It provides insulation, cushioning and energy storage.

Figure 1

Physiological function of skin:

1. Protection
2. Thermoregulation
3. Sensation
4. Excretion
5. Vitamin D Secretion                                                                                   

AIM AND OBJECTIVE:

The aim of the present research work is to formulate, develop, and evaluate a herbal wound healing cream consisting of Tridax Procumbens Linn as the chief constituent, Centella Asiatica , and Withania Somnifera. Traditionally these plants are medicinally used to treat various ailments. Therefore, this research aims to utilize their natural medicinal properties to produce an effective, natural, and inexpensive wound healing cream.

Specifically, the objectives are to:

• Extract active constituents from Tridax Procumbens Linn, Centella asiatica and Withania somnifera.
• Combine these extracts to formulate a cream base suitable for topical application.
• Evaluate the wound healing efficacy of the developed cream through in vitro (laboratory-based) and in vivo (living organism) studies.
• Compare the healing effects of the herbal cream with conventional wound healing treatments.
• Assess the cream for any potential side effects or skin irritation through dermatological testing.

This study aims at providing scientific support for the application of these traditional herbal remedies in healing of wounds, which may develop into a potential commercially viable herbal wound-healing product.

1. Drug Profile

Tridax Procumbens Linn:

Figure 2

• Family: Asteraceae
• Genus: Tridax
• Species: Tridax Procumbens Linn Linn Linn
• Common Name: Coat Buttons, Ghamara and Kansari
• Active Components: Flavonoids, Alkaloids, Carotenoids, Saponins, Tannins, Steroids, Triterpenoids

Centella Asiatica:

Figure 3

• Family: Apiaceae
• Genus: Centella
• Species: Centella asiatica
• Common Name: Gotu Kola, Indian Pennywort, Mandukaparni
• Active Components: Triterpenoids, Flavonoids, Alkaloids, Essential Oils

Withania Somnifera:

Figure 4

• Family: Solanaceae
• Genus: Withania
• Species: Withania somnifera (L.) Dunal
• Common Name: Ashwagandha, Indian Ginseng, Winter Cherry
• Active Components: Alkaloids, Flavonoids, Withanolides

Tridax Procumbens Linn Leaves:

Tridax procumbens Linn is a common medicinal plant that is a member of the Steraceae family and is used in yurvedic medicine because of its pharmacological properties. Known as "Coat buttons" or "ridax daisy" in English, this annual or perennial plant is native to Central and South America and is found as a weed throughout India. Its leaves have insecticidal, antiparasitic, and antiseptic qualities. Therefore, it might be applied to organic farming in the future to promote sustainable agriculture. Wound healing involves a complex interaction between epidermal and dermal cells, the extracellular matrix, regulated angiogenesis, and plasma-derived proteins interact intricately during wound healing, all of which are regulated by a variety of growth factors and cytokines. Tridax counteracted the known healing suppressant agent dexamethasone's anti-epithelialization and tensile strength depressing effects without compromising the drug's anticontraction and anti-granulation properties.

Though not as much as whole plant extract, aqueous extract was also successful in raising lysyl oxidase. Additionally, in a dead space wound healing model, it has been demonstrated that an extract from the leaves of this plant also promotes wound healing in both normal and immunocompromised (steroid-treated) rats. The plant increases the amount of protein and nucleic acids in the granulation tissue in addition to lysyl oxidase, most likely due to an increase in glycosaminoglycan content ^[1-8].

Centell Asiatica:

The herbaceous plant Centella asiatica, popularly referred to as gotu kola, has a lengthy history of traditional use in Ayurvedic medicine. Known as "Brahmi" or "Mandookaparni" in India, it has long been used as an adaptogen. The leaves and stem are used for the wound healing activity.

Phytochemistry: Triterpenoids make up the majority of the bioactive substances found in C. asiatica. These consist of triterpene acids like asiatic acid and madecassic acid, as well as triterpene glycosides like asiaticoside, madecassoside, and centelloside. These substances add to the plant's varied pharmacological characteristics.

Skin Health Benefits: C. asiatica has drawn a lot of interest due to its possible advantages for skin health. Research has indicated that it might. Encourage wound healing: C. asiatica may hasten wound closure and enhance tissue regeneration by inducing the synthesis of collagen and fibronectin. Increase skin elasticity: According to certain studies, it may increase skin elasticity and lessen the visibility of wrinkles. Reduce inflammation: C. asiatica may have antioxidant and anti-inflammatory qualities that can help reduce inflammation in skin conditions like psoriasis and eczema ^[9-16].

Withania Somnifera Roots:

"Indian ginseng," or Withania somnifera, is a significant medicinal plant found on the Indian subcontinent. Since ancient times, Indian systems of medicine have used it extensively, either alone or in conjunction with other herbs, to treat a wide range of illnesses. It has a wide range of biological implications because it contains a variety of phytochemicals. It has been shown to lower reactive oxygen species, improve endothelial function, control apoptosis, modify mitochondrial activity, and lessen inflammation. W. somnifera is a promising drug candidate to treat a variety of clinical conditions, especially those pertaining to the nervous system, because of these pharmacologic characteristics. The pharmacologic properties of the plant and its active ingredients are outlined in this review, along with their mechanisms of action and possible therapeutic uses. In a number of disease models, Withania somnifera has demonstrated notable anti-inflammatory properties. By eliminating neutrophil infiltration, edema, and necrosis in inflammatory bowel disease caused by trinitro-benzyl-sulfonic acid (TNBS), its root extract demonstrated anti-inflammatory and muco-restorative properties. In a mouse model of lupus, powdered root extract was found to have a strong inhibitory effect on inflammatory markers like cytokines like interleukin (IL)-6 and tumor necrosis factor (TNF)-α, nitric oxide (NO), and ROS, as well as proteinuria and nephritis ^[19-21].

2. Experimental Studies

METHODOLOGY:

Materials Required:

• Centella Asiatica Extract
• Tridax Procumbens Extract
• Withania Somnifera Extract
• Beeswax
• Liquid Paraffin
• Borax
• Methyl Paraben
• Rose Oil

1. Preparation of Extract:

• Cold maceration method was used for the extraction.
• First, the powdered plant material, Tridax Procumbens Linn, Centella Asiatica and Withania Somnifera was macerated for 24 hours, shaking periodically, in a 70:30 mixture of alcohol and water in a round-bottom flask (RBF).
• Following a 24-hour period, the solvents underwent filtration, following which the powdered tridax procumbens extracts were gathered.

2. Preparation of Herbal Cream:

• Every ingredient was precisely weighed.
• Heat beeswax and liquid paraffin in a borosilicate glass beaker at 75 ? and maintain that heating temperature. (Oil phase).
• In another beaker, dissolve borax, methylparaben in distilled water and heat this beaker to 75 ? to dissolve borax and methylparaben and to get a clear solution. (Aqueous phase).
• Then slowly add aqueous phase in heated oily phase.
• Then add a measured amount of tridax procumbins extract, ashwagandha roots extract and centella asiatica and stir vigorously until it forms a smooth cream. Rose oil is added as a fragrance.
• Put this cream on the slab and add few drops of distilled water and mix the cream on the slab to give a smooth texture to the cream and to mix all the ingredients properly ^[22].

VI. Formulation Table for preparation of cream: Table No :1

3. Evaluation of The Cream

A number of quality control tests, such as physiochemical, visual evaluation and conditioning performance tests, were carried out to assess the prepared formulation's quality.

• Organoleptic Properties

Table No: 2

• Screening of Cream Formulation Phytochemically

Table No: 3

• Procedure of Phytochemical Test:

1. Test for Carbohydrate:

Table No: 4

2. Test for Protein:

1. Solubility Test:

Add 0.5 grams of casein to 2 milli-litters of 0.1 N NaOH, 2 milli-litters of 0.1 N HCL, and 2 milli-litters of distilled water separately to test the solubility of the protein. Casein dissolves entirely in 0.1N NaoH, after soaking the tubes in water for ten minutes, remove them.

2. Preparation of sample solution:

To create a solution, breakdown 100 milliliters of 0.1N NaoH and add 1 gram of casein.

3. Biuret Test:

 Mix with 2 ml of protein solution, add 2 ml of 10% NaoH solution, and add three to four drops of 1% copper sulfate solution.  Purple is created when a bond of peptides is present and the sample being analyzed is a protein.

3. Test for Alkaloids

Every single extract (0.5 g) was shaken in a steam bath with 5 mL of 1% HCL.  After filtering the resulting solution, one milliliter of the filtrate was mixed with a few drops of Mayer's reagent.  The turbidity of the filtrate following the addition of Mayer's reagent indicated the existence of alkaloids in the extract. 

4. Test for Glycosides

After adding a milliliter of pyridine and a few drops of sodium nitropruside solution, the hydrolysate was made alkaline by adding sodium hydroxide solution.  Glycosides are indicated by a pink to red appearance.

4. Extraction of Herbal Drug

For ages, maceration has been employed as a technique for compound extraction. The fundamental idea is to soak a solid in a liquid to extract its soluble components. In order to enhance the surface area between the sample and the liquid solvent, the sample is typically prepared by drying and grinding it. Using a rotavapor to continuously extract the desired components of a mixture is an other method. A spinning flask immersed in a heated water bath and under vacuum is used in the rotary evaporator. A thin layer of the mixture is produced by the rotation, and heat causes it to evaporate. In contrast to my pressure cooker, the vacuum has the opposite effect. The vacuum lowers the boiling point rather than raising it as a result of increased pressure. As a result, the solvent can evaporate at lower temperatures. Even more intricate tasks, such continuous extractions, can be completed by contemporary rotary evaporators. In a continuous extraction procedure, the vaporized solvent condenses and is gathered in a different container. The following extraction cycle then makes use of the condensed solvent. Because the solvent can be used again, this approach enables an effective and continuous procedure. Heat-sensitive materials do not deteriorate due to the low temperatures.

5. Beneficial Properties of “Powder of The Drugs”

An extractive soluble in alcohol:

100 milli-litters of 90% alcohol were added to a Stoppard conical flask containing five grams of precisely measured powdered medication. After being continuously in an electrical shaker, the mixture was allowed to macerate overnight. The weight and percentage of the extractive were then calculated after the filter was cautiously evaporated until it was dry.

Alcohol Soluble Extractive: Extractive Weight/ Drug Weight X 100

Extractive Soluble in Water

One hundred milli-litters of chloroform water were added to a Stoppard conical flask that held five grams of accurately measured powdered medication. After six hours of constant shaking in an electrical shaker, the flask was kept overnight to macerate. After that, the extractive was meticulously filtered and dried completely by evaporation. After determining the extractive's weight, the percentage was calculated using

Water-Soluble Extractive: Weight of extractive / Weight of drug X 100 equals

Total Ash

A China dish was used to weigh three grams of the drug, burn it at a temperature of no more than 450 degrees Celsius until the carbon was gone, let it cool, and then weigh it again until it stayed the same for three readings. To calculate the percentage of ash the air-dried drug was used.

Total Ash = Wt. of ash /Wt. of drug x 100

Acid Insoluble Ash

The ash was obtained after boiling it for five minutes with 25 milli-litters of diluted hydrochloric acid in it. After that, the insoluble substance was collected in a Gooch Crucible, cleaned with hot water, and burned until its weight remained constant. The amount of acid-insoluble ash was calculated with respect to the drug that had been allowed to air dry.

Table No: 5

6. Therapeutic Uses

Tridax Procumbens:

Coat Buttons, or Tridax procumbens, is a medicinal plant that has a variety of therapeutic uses. It can help lessen inflammation in diseases like arthritis because of its strong anti-inflammatory qualities. Antioxidants, which are abundant in the plant, help scavenge dangerous free radicals, lowering oxidative stress and avoiding cell damage. Tridax procumbens is frequently used to promote quicker tissue regeneration and prevent infection in wound healing. Additionally, it has antimicrobial qualities that make it effective against a variety of bacteria and fungi, which makes it beneficial for treating digestive problems and skin infections. Because of its hemostatic qualities, the plant helps stop bleeding from wounds and cuts. It has also been used historically to treat fever and malaria, with some success against these illnesses.  Its potential anti-diabetic qualities can help control blood sugar levels, and its hepatoprotective effects aid in liver detoxification. According to certain research, it might also have anticancer properties by preventing the growth of cancer cells. Tridax procumbens has anti-epileptic properties that help control seizures and is used for respiratory health, alleviating the symptoms of coughing and asthma. Lastly, it promotes digestive health by curing ailments like diarrhea and indigestion. Although some scientific studies and traditional uses support these therapeutic benefits, more research is required to fully confirm their efficacy ^[1-8].

Centella Asiatica and Withania Somnifera :

Withania somnifera also known as ashwagandha, and Centella asiatica known as gotu kola, are well-known medicinal plants with a range of therapeutic benefits. Because of its neuroprotective qualities, Centella asiatica is well known for its capacity to support brain health, improve memory, and improve cognitive function. By increasing skin elasticity, decreasing scars, and boosting collagen synthesis, it is also used to aid in wound healing. The plant's potent antioxidant and anti-inflammatory qualities aid in the body's fight against inflammation and oxidative stress. Centella asiatica is also frequently used to reduce stress and anxiety; research has indicated that it may also lessen the symptoms of anxiety and depression, making it a mental health adaptogen. Ashwagandha, or Withania somnifera, on the other hand, is mainly recognized for its adaptogenic qualities, which support general vitality and assist the body in managing stress. It helps the body reduce oxidative stress and inflammation by having anti-inflammatory and antioxidant properties. Because it increases muscle mass and stamina, ashwagandha is frequently used to improve physical strength and endurance. By promoting immune function and preventing the growth of cancer cells, it has also been demonstrated to have anti-cancer effects. Furthermore, because of its calming effects on the nervous system, Withania somnifera is useful for promoting better sleep patterns, lowering anxiety, and elevating mood. It is also helpful for maintaining hormonal balance and controlling thyroid function, which makes it an important herb for treating ailments.  Both plants have been used for many years in traditional medicine to treat a variety of ailments, and research is still being done to fully understand their therapeutic benefits ^[9-21.

7. Toxicity:

• Tridax Procumbens

Traditional medicine makes extensive use of the plant Tridax procumbens. Like many medicinal plants, its toxicity can change based on how much is taken, how it is prepared, and how it is consumed.

Toxic Components: Depending on the quantity ingested, the flavonoids, alkaloids, and saponins found in Tridax procumbens may have both positive and negative effects. When taken in excess, these substances can cause liver damage and gastrointestinal issues.

Hepatotoxicity: According to certain research, high or prolonged dosages of Tridax procumbens can result in liver toxicity, which can raise liver enzyme levels and possibly cause hepatomegaly, or liver enlargement.

Toxicological Studies: Although more research is required to determine the precise human toxicity thresholds, some animal models exhibit both reproductive toxicity and renal damage.

Allergic Reaction: It has occasionally been connected to allergic reactions or skin irritation, especially when applied topically.

Contraindication: People who already have liver or kidney disease, as well as those who are pregnant or nursing, should use it with caution^[1-8].

• Withania Somnifera:

One of the most widely used herbs in Ayurvedic medicine is Withania somnifera, also referred to as ashwagandha. Its adaptogenic qualities make it a popular choice for managing stress, anxiety, and overall health. However, when used improperly, it can also be toxic.

Toxic Components: Steroids, alkaloids, and withanolides are found in Withania somnifera; these substances are believed to have therapeutic properties but can have negative side effects if taken in excess.

Liver Toxicity: Although usually thought to be safe at standard therapeutic dosages (300–500 mg/day), prolonged or excessive use has occasionally been linked to hepatotoxicity. Liver function may be impacted by withanolides.

Gastrointestinal Problems: Excessive dosages may cause nausea, vomiting, and diarrhea. Particularly if taken with other herbs or supplements or on an empty stomach, these symptoms may appear.

Endocrine Disruption: Thyroid hormone levels are significantly impacted by ashwagandha. If taken excessively, it can cause hyperthyroidism, particularly in people who already have a thyroid condition.

Effects on Sleep: Ashwagandha is an adaptogen that helps lower stress and anxiety, but it can also make you feel sleepy. Excessive sleepiness, lethargy, and cognitive impairment can result from overuse.

Contraindications: Due to the possibility of complications, it should be used with caution in patients with thyroid issues, autoimmune diseases, and pregnant or lactating women. Overdosing may also affect a man's ability to conceive ^[9-16].

• Centella Asiatica

Gotu kola, also known as Centella Asiatica, is frequently used for its ability to improve cognition and heal skin. When taken in moderation, it is generally considered safe; however, excessive or inappropriate use can pose certain risks.

Toxic Components: Triterpenoid saponins, which are found in Centella Asiatica, are advantageous in moderation but may be toxic in excess.

Liver and Kidney Toxicity: Excessive or long-term use of gotu kola has been associated with the possibility of kidney damage and liver toxicity, particularly when high doses are taken over an extended period of time.

Gastrointestinal Problems: Excessive use may result in headaches, nausea, and light headedness, as well as diarrhea in certain cases. Those who are sensitive to the herb are more likely to experience these effects.

Skin Reactions: Although it has a reputation for healing skin, sensitive people may experience allergic reactions or skin irritation from overuse, especially when applied topically.

Neurotoxic Effects: A mild sedative effect at high doses is suggested by the sporadic reports of drowsiness, lethargy, or confusion with prolonged use.

Contraindications: Avoid the herb, especially at high doses, if you have liver disease, kidney problems, or are pregnant or nursing. Additionally, using gotu kola with other sedatives or substances that affect the liver should be done with caution ^[17-21].

General Points to Remember About All Three Plants:

Dosage and Purity: The dose has an enormous effect on the risk of toxicity. The concentration of herbal supplements, particularly those derived from whole plants, can vary greatly. To prevent accidental overdosing, the herbal products must be pure and standardized.

Drug Interactions: Sedatives, blood pressure medications, and immunosuppressants are among the medications that these plants may interact with. For example, Centella Asiatica and Withania somnifera may interact with anticoagulants and thyroid medications, respectively, raising the risk of bleeding.

Long-term Effects: prolonged consumption of any herbal remedy should be examined by a healthcare professional, specifically when used over prolonged periods.

RESULT AND DISCUSSION

Evaluation studies of prepared formulations:

The prepared three formulations were evaluated by observing the appearance and consistency. Further formulations were evaluated by conducting pH measurement, spreadability, viscosity, washability and foamability

1. Physical Appearance:

The formulated wound healing cream was visually inspected for their colour, odour and consistency.

2. Determination of pH:

The ph. of prepared formulation was determined using ph. paper and the ph value are shown below

The ph. of formulated cream that is F1, F2 and F3 was found to be 6.8, 6.9 and 7 respectively and the ph. of the formulated cream was almost equal to the ph. of the skin which prevents the irritation of the skin.

3. Determination of Spredability:

Spredability plays an important role in patient compliance and ensures uniform application of the cream. Good spredability can ensure the distribution of the cream when applied to the skin.

 The spredability of the F3 formulation is 5.9 gm.cm/sec which is more than the other formulation. This shows that formulation F3 has better spredability than the other formulations.

4. Antimicrobial Studies: