Prepare and Evaluate Wound Healing Herbal Gel of Aqueous Extract of Terminalia chebula and Azadirachta indica

Wounds are injuries that break the skin or other body tissues. They include cuts, scrapes, scratches and punctured skin. A wound forms when biological tissues like skin, mucous membranes, and organs are damaged. Different injuries can cause wounds; properly cleaning and dressing the wounds is essential to prevent infections and additional harm.^[1] The surgical wound classification (SWC) system was initially developed in 1964 by the National Academy of Sciences and the National Research Council.^[2] The SWC system was created to represent the bacterial load in a surgical field. The Centers for Disease Control and Prevention (CDC) later refined this system by establishing 4 different classes of wound statuses outlined below^.[3] Each class has a postoperative risk of a surgical site infection (SSI) with scores of 1% to 5%, 3% to 11%, 10% to 17%, and more than 27%, respectively.^[4]

• Class 1 wounds are categorized as clean wounds. These types of wounds are not infected, do not exhibit any signs of inflammation, and are typically closed. If drainage is required, a closed draining approach is recommended. It is worth noting that Class 1 wounds do not involve the respiratory, alimentary, genital, or urinary tracts. Examples of clean wounds include an inguinal hernia repair or a thyroidectomy.
• Class 2 wounds are categorized as clean-contaminated, which means they have a low level of contamination. These types of wounds involve entry into the respiratory, alimentary, genital, or urinary tracts but only under controlled circumstances.
• Class 3 wounds are classified as contaminated and typically result from a breach in sterile techniques or leakage from the gastrointestinal tract. Incisions resulting from acute or nonpurulent inflammation are also considered Class 3 wounds.
• Class 4 wounds are considered to be dirty or infected. These injuries usually occur from inadequate treatment of traumatic wounds, gross purulence, and evident infections. When tissues lose vitality, it can lead to Class 4 wounds. This is often caused by surgery or microorganisms found in perforated organs^.[3]

Wound Healing

Wound healing refers to a living organism's replacement of destroyed or damaged tissue by newly produced tissue.^[4]

In undamaged skin, the epidermis (surface, epithelial layer) and dermis (deeper, connective layer) form a protective barrier against the external environment. When the barrier is broken, a regulated sequence of biochemical events is set into motion to repair the damage. This process is divided into predictable phases: blood clotting (hemostasis), inflammation, tissue growth (cell proliferation), and tissue remodeling (maturation and cell differentiation). Blood clotting may be considered to be part of the inflammation stage instead of a separate stage.

Wound care encourages and speeds wound healing via cleaning and protection from reinjury or infection. Depending on each patient's needs, it can range from the simplest first aid to entire nursing specialties such as wound, ostomy, and continence nursing and burn center care.

Stages:-

• Hemostasis (blood clotting): Within the first few minutes of injury, platelets in the blood begin to stick to the injured site. They change into an amorphous shape, more suitable for clotting, and they release chemical signals to promote clotting. This results in the activation of fibrin, which forms a mesh and acts as "glue" to bind platelets to each other. This makes a clot that serves to plug the break in the blood vessel, slowing/preventing further bleeding.
• Inflammation: During this phase, damaged and dead cells are cleared out, along with bacteria and other pathogens or debris. This happens through the process of phagocytosis, where white blood cells engulf debris and destroy it. Platelet-derived growth factors are released into the wound that cause the migration and division of cells during the proliferative phase.
• Proliferation (growth of new tissue): In this phase, angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction occur. In angiogenesis, vascular endothelial cells form new blood vessels. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting collagen and fibronectin. Concurrently, re-epithelialization of the epidermis occurs, in which epithelial cells proliferate and 'crawl' atop the wound bed, providing cover for the new tissue. In wound contraction, myofibroblasts decrease the size of the wound by gripping the wound edges and contracting using a mechanism that resembles that in smooth muscle cells. When the cells' roles are close to complete, unneeded cells undergo apoptosis.^[7]
• Maturation (remodeling): During maturation and remodeling, collagen is realigned along tension lines, and cells that are no longer needed are removed by programmed cell death, or apoptosis.

Factors Effecting Wounds Healing:-

Many factors controlling the efficacy, speed, and manner of wound healing fall under two types: local and systemic factors.

Local Factors:-

• Moisture; keeping a wound moist rather than dry makes wound healing more rapid and with less pain and less scarring^[8]
• Mechanical factors
• Oedema
• Ionizing radiation
• Faulty technique of wound closure
• Low oxygen tension

Systemic factors :-

• Inflammation
• Diabetes – Individuals with diabetes demonstrate reduced capability in the healing of acute wounds. Additionally, diabetic individuals are susceptible to developing chronic diabetic foot ulcers, a serious complication of diabetes which affects 15% of people with diabetes and accounts for 84% of all diabetes-related lower leg amputations.^[9] The impaired healing abilities of diabetics with diabetic foot ulcers and/or acute wounds involves multiple pathophysiological mechanisms. This impaired healing involves hypoxia, fibroblast and epidermal cell dysfunction, impaired angiogenesis and neovascularization, high levels of metalloproteases, damage from reactive oxygen species and AGEs (advanced glycation end- products), decreased host immune resistance, and neuropathy.^[9]
• Metabolic diseases
• Immunosuppression
• Connective tissue disorders
• Smoking – Smoking causes a delay in the speed of wound repair notably in the proliferative and inflammatory phases. It also increases the likelihood of certain complications such as wound rupture, wound and flap necrosis, decrease in wound tensile strength and infection. Passive smoking also impairs a proper wound healing process.^[10]
• Age – Increased age (over 60 years) is a risk factor for impaired wound healing. It is recognized that, in older adults of otherwise overall good health, the effects of aging causes a temporal delay in healing, but no major impairment with regard to the quality of healing. Delayed wound healing in patients of increasing age is associated with altered inflammatory response; for example delayed T-cell infiltration of the wound with alterations in the production of chemokines, and reduced macrophage phagocytic capacity.^[11]
• Alcohol – Alcohol consumption impairs wound healing and also increases the chances of infection. Alcohol affects the proliferative phase of healing. A single unit of alcohol causes a negative effect on re-epithelialization, wound closure, collagen production and angiogenesis.^[9]

MATERIALS & METHODS:-

Materials

1. Terminalia chebula

Chemical constituents:-

A number of glycosides have been isolated from haritaki, including the triterpenes arjunglucoside I, arjungenin, and the chebulosides I and II. Other constituents include a coumarin conjugated with gallic acids called chebulin, as well as other phenolic compounds including ellagic acid, 2,4-chebulyl-β-D-glucopyranose, chebulinic acid, gallic acid,ethyl gallate, punicalagin, terflavin A, terchebin, luteolin, and tannic acid. ^[12] Chebulic acid is a phenolic acid compound isolated from the ripe fruits. ^[13] Luteic acid can be isolated from the barkh.

Pharmacological activity:-

• Anti- inflammatory activity
• Antimicrobial activity
• Antibacterial activity
• Antifungal activity
• Antiviral activity
• Wound healing properties

2. Neem

Chemical constituents:-

There is various chemical constituent which have been isolated from the neem some of them are: Azadirachta, b-sitosterol, kaempferol, myricetin, nimbidinine, nimbiol, quercetin, stigmasterol, tannins, glycosides, terpenoids and alkaloids are present.

Pharmacological activity:-

• Analgesic agent
• Antipyretic agent
• Antimicrobial activity
• Antibacterial activity
• Antifungal activity
• Antiviral activity
• Anti-hyper glycaemic age

Method

Collection of Terminalia chebula  fruits:- Collect the fruits of the plant Terminalia chebula  from the shop of the local market. Then reduce the size of the fruits through grinder and then passes the powder from the sieves 20 and 40. To get rid of the waxy elements, to make a fine powder.

Extraction of the fruits of Terminalia chebula :- Weigh 25g of the dry powder were constantly extracted using methanol in a Soxhlet apparatus for 24 hours. Then, extract was filtered and the extract was used for formulating the wound healing herbal gel.

Terminalia chebula  Extraction

Collection of Azadirachta indica leaves

Azadirachta indica leaves were collected from Himachal pharmacy college campus, maganpura situated in state Himachal Pradesh. The collected leaves were washed with the water and shade dried at room temperature for 5 to 10 days.

Extraction of the leaves of Azadirachta indica

After being dried leaves the leaves were size-reduced, the shade-dried leaves were put through sieves no. 20 and 40. To get rid of the waxy elements, 25g of the dry powder were constantly extracted using methanol in a Soxhlet apparatus for 24 hours. Then, extract was filtered and the extract was used for formulating the patches.

Extraction of the Azadirachta indica

Formulation

2g of carbopol940 was dispersed in 100 ml of hot purified water with moderate stirring and the beaker was kept aside for overnight to swell the HPMC to form gel. Take 5ml of distilled water and required quantity of methylparaben were dissolved by heating on water bath and solution was cooled and 5ml glycerin was added. Then 2ml of each extract was mixed to the above mixture. Then this mixture was added into the carbopol940 gel with continuous stirring. Triethanolamine was added dropwise at last to adjust the pH of gel according to the skin pH and to obtain the gel at required consistency. Prepared formulations were filled in a suitable container and labeled accordingly.

EVALUATION OF HERBAL GEL FORMULATION

Physical Evaluation

Physical parameters such as color and appearance were checked.

Measurement of Ph

The pH of various gel formulations were determined by using digital pH meter. 2.5gm of gel  was accurately weighed and dispersed in 25ml of distilled water and stored for two hours. The measurement of pH of each formulation was done.

Spreadibility^[17]

Spreadibility was determined by the apparatus which consists of a wooden block, which was provided by a pulley at one end. By this method spreadibility was measured on the basis of slip and drag characteristics of gels. An excess of gel (about 2g) under study was placed on this ground slide. The gel was then sandwiched between this slide and another glass slide having the dimension of fixed ground slide and provided with the hook. A. one kg weighted was placed on the top of the two slides for 5 minutes to expel air and to provide a uniform film of the gel between the slides. Excess of the gel was scrapped off from the edges. The top plate was then subjected to pull of 80 gm. With the help of string attached to the hook and the time (in seconds) required by the top slide to cover a distance of 7.5 cm be noted. A shorter interval Indicate better spreadibility.

Spreadibility was calculated using the following formula:

S = M × L / T

Where,

S = Spreadibility

M= Weight in the pan (tied to the upper slide)

L = Length moved by the glass slide

T = Time (in sec.) taken to separate the slide completely each other.

Extrudabilty^[18]

The gel formulation were filled in standard capped collapsible aluminium tubes and sealed by crimping to the end. The weight of tubes were recorded and the tubes were placed between two glass slides and were clamped. 500gm was placed over the slides and then the cap was removed. The amount of extruded gel was collected and weighed. The percent of extruded gel was calculated as

1. When it is greater than 90% then extrudability is excellent.
2. When it is greater than 80% then extrudability is good.
3. When it is 70% then extrudability is fair

Viscosity^[19]

Viscosities of gels were determined using Brookfield viscometer. Gels were tested for their rheological characteristics at 25oC using Brookfield viscometer (DV-III programmable Rheometer). The measurement was made over the whole range of speed settings from 10rpm to 100rpm with 30seconds between 2 successive speeds and then in a descending order.

RESULT AND DISCUSSIONS

The herbal gel was prepared and subjected to evaluation of the various parameters. The herbal Gel was light brown in color and translucent in appearance and had a cool and smooth feeling on application. pH also maintained constant throughout the study which was found to be 6.9 to 7.0 and the gel was non-irritant upon application on the skin. Spreadibility were also measured and found to be less variant than the initially prepared gel after performing stability study.

CONCLUSION

Formulation of the herbal neem and Terminalia chebula  herbal gel were successfully developed that met the relevant pharmaceutical characteristics. The prepared formulation F1 shown good results as comparison to F2, F3. The formulation showed proper pH range that was approximately 6.85, it mainly confirms that the herbal gel are mainly compatible to the skin secretions. From the present study it can be concluded that it is possible to develop gel containing herbal extracts and can be used as the provision of a barrier to heal wound heals. Plants are more potent healers because they promote the repair mechanism in the natural way. The wound healing property of the formulated herbal gel has been done and experimented and will be effective throughout the skin without any side effects.

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