Evaluation of Wound Healing Activity of Ethanolic Extract of

Azadirachta indica Leaves in Male Albino Rats

S.K. Purohit*, R. Solanki, R. Soni, V. Mathur

Ex-Director, College of Veterinary and Animal Science, Rajasthan

*Corresponding Author E-mail: solankirenu@yahoo.com

ABSTRACT:

Skin is the important organ of the human body as it plays a vital role in maintaining health of human being. Most of the diseases are caused by pathogenic microorganisms. Some of the most common causative microorganisms related with infections include Staphylococcus aureus, Streptococcus pyogenes, Enterococci, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Wounds are major cause of physical disabilities. Wound healing consists of orderly progression of series of events that establish the integrity of the damaged tissues. Wound disease spread by the invasion of bacteria inside the body through skin. Bacterial infections on skin are the common ailment for generation of other diseases in the body. Bacterial diseases are type of infectious diseases caused by pathogenic bacteria. Wound and skin infections represent the invasion of tissues by one or more species of microorganisms. These infections disturb the body immune system and causes inflammation, tissue damage and thus resulting in delayed wound healing process. Leaves of Azadirachta indica (Neem) are used for various medicinal purposes. It is useful in tiredness, cough, fever, loss of appetite, worm infestation. It heals wound, vomiting, skin diseases, heat-rash, boils, jaundice, leprosy, skin disorders, eye disorders, stomach ulcers, chicken pox, excessive thirst, diabetes and insect poisons. It act as anti-leprotic, antimalarial, anti-hemorrhoidal and anthelmintic agent. The present study was undertaken on evaluation of Azadirachta indica (Neem) leaves ethanolic extract for wound healing activity through topical route on excision wound model. The activity was compared with standard drug Povidone Iodine ointment (5% w/w). Azadirachta indica leaves ethanolic extract was found to have better and faster wound healing effect than standard drug Povidone Iodine ointment on excision wound model.

This article throws light on wound healing activity of ethanolic extract of Azadirachta indica leaves.

KEY WORDS: Azadirachta indica, povidone iodine ointment, wound healing activity, wound contraction, excision wound model

INTRODUCTION:

Wounds are major cause of physical disabilities. They arise due to physical, chemical or microbial agents. Healing is a survival mechanism and represents an attempt to maintain normal anatomical structure and junction¹. Many immunosuppressant, cytotoxins and non-steroidal anti-inflammatory drugs suppress the wound healing. Management of wound healing is a complicated and expensive step². Restoration of damaged tissue (wound) is an important process which plays vital role in survival of life. It is imminent for the basis of all surgical manipulations³.

Many plants have proved to possess significant healing properties in different types of wounds. Using certain plants, possessing antiseptic, astringent, anti-inflammatory, antimicrobial property the rate of wound healing can be enhanced⁴. Such plant can increase the rate of tissue healing by providing different essential substances, required at various steps of wound healing. Plants being cheaper and safer than allopathic drugs, so treatment by natural ways may be useful in veterinary practice, especially in India where these are found in plenty⁵. Azadirachta indica is a plant commonly known as Neem, belongs to the family Meliaceae^6-7. Azadirachta indica (Neem) leaves are used for medicinal purposes. It is useful in tiredness, cough, fever, loss of appetite, worm infestation. It heals wound, vomiting, skin diseases, heat-rash, boils, jaundice, leprosy, skin disorders, eye disorders, stomach ulcers, chicken pox, excessive thirst, diabetes and insect poisons. It acts as anti-leprotic, antimalarial, anti-hemorrhoidal and anthelmintic agent etc^8-10. Based on the above source of information, the present study aimed to evaluate the wound healing activity of Azadirachta indica (Neem) leaves.

MATERIALS AND METHODS:

Collection of plant material:

The fresh leaves of Azadirachta indica (Neem) were purchased from local nursery garden during the month of July 2006. The plant material was identified and authenticated at Botanical Survey of India, Jodhpur by Dr. P. J. Parmar. The fresh plant material was dried under shade. Dried plant material was powdered using mechanical grinder and passed through sieve no.60 to get the powder of desired coarseness. Powdered material was preserved in an air tight container.

Extraction and drug formulation:

750 g of the coarsely powdered, dried leaves of Azadirachta indica (Neem) were mixed with 4500 ml of ethanol, allowed to stand for 7 days. It was filtered and distilled under vacuum to get concentrated ethanolic extract. The ethanolic extract of Azadirachta indica (Neem) leaves were stored under desiccators for further phytochemical and pharmacological screening. A 5% w/w ointment formulation was made by incorporating the Azadirachta indica leaves ethanolic extract with simple ointment base I.P10 for external application of the drug in the excision wound model.

Preliminary phytochemical analysis:

A preliminary phytochemical screening was carried out for the extract employing the standard procedure to reveal the presence of alkaloids, steroids, terpenoids, flavonoids, saponins, tannins, glycosides, carbohydrates, phytosterols and proteins¹¹.

Animals:

Adult albino rats (wistar strain) of either sex weighing 180-200 g were taken. They were placed in polypropylene cages in a controlled room environment (25°C±2°C) at a natural day night cycle and they were provided with standard laboratory food and water ad libitum. Approval for the study was obtained from the institutional animal by the Institutional Animal Ethical Committee (IAEC), Reg No. 990, U.D.P.S.

Wound healing activity:

Animals were assigned into 3 groups containing 6 animals in each group. All the wounds (control, standard and test groups) were cleaned with the sterilized normal saline solution, then dried gently with sterilized gauze before every dressing. Group I was called as control group, was

kept untreated, group II was called as standard group, was treated with market formulation (Povidone Iodine ointment), group III was called as test group, was treated with the ethanolic extract ointment of Azadirachta indica leaves. Excision wound model was selected for assessing wound healing activity of Azadirachta indica leaves on topical route of administration for 15 days. Animals were anaesthetized with ether and shaved on part to be exposed. A circular piece (500 mm² area) was impressed on the dorsal thoracic region 5cm away from ears and 1 cm away from the vertebral column. The animals were individually housed in separate cages. The test and standard preparation were topically applied once a day till epithelialisation was complete, starting from the day of the operation. The wounds were traced on 1 mm² graph paper on the day of wounding and then subsequently on the 4th, 8th, 12th and 16th post wound days and thereafter daily until healing was complete. The parameters studied were percentage of wound contraction or wound closure and period of epithelialisation¹².

RESULTS AND DISCUSSION:

The yield of the ethanolic extract of Azadirachta indica leaves was found to be as 8.56%w/w. The Azadirachta indica leaves extract showed the presence of phytoconstituents like azadirachtin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, quercetin and salannin etc.Wound healing involves a highly dynamic integrated series of cellular physiological and biochemical processes that occurs in living organisms^13-14. The majority of world population relies on traditional medicine for their health care¹⁵. This is also the case in the treatment of wounds. Many research proposed that wound healing can be improved by herbal drugs having antiseptic, antibacterial, antioxidant and anti-inflammatory properties^16-17. In the present study, the excision wound model is considered for evaluation of wound healing activity of fresh leaves of Azadirachta indica (Neem) as topical application. The rate of wound contraction in mm² was taken as a measure of wound healing. During initiation of the study from the day 0 there was not much difference in the healing of wounds in all 3 groups. But after day 9, the healing process was faster in the groups treated with ethanolic extract ointment of Azadirachta indica leaves as compared to the standard group which was treated with Povidone Iodine ointment as shown in table 1. Statistical analysis was done by ANOVA and Dunnet’s multiple comparision tests. Results are expressed as mean ± SE, n=6 in each group. Significant difference was compared to control group at p<0.01. From the results, it was observed that in the topical route, test group III shows faster wound closure and wound contraction and the results are significant (P<0.01) when compared with standard drug Povidone Iodine ointment.

Table 1: Effect of topical administration of ethanolic extract ointment of Azadirachta indica leaves on excision wound model

Comparative mean wound area of different groups (in mm^2)
Post wounding days	Group I (control)	Group II (standard)	Group III (test group)
0	232.65 ± 1.78	231.86 ± 1.92	231.96 ± 1.03
3	161.62 ± 1.96	162.86 ± 1.66	101.06 ± 1.45
6	101.04 ± 1.10	73.01 ± 1.74	49.01 ± 1.62
9	82.78 ± 1.07	51.58 ± 1.56	06.45 ± 0.23
12	52.58 ± 1.66	6.08 ± 1.56	0.00 ± 0.00
15	12.66± 1.22	0.00 ± 0.00	0.00 ± 0.00

CONCLUSION:

It can be concluded that when the ethanolic extracts of Azadirachta indica (Neem) leaves was separately applied externally on male albino rats by topical route through excision wound model then it showed faster as well as better wound closure and wound contraction as compared to standard marketed formulation called as Povidone Iodine ointment. Therefore, we may interpret that the observation of such response may be due to the presence of various phytoconstituents which were found to be present in the leaves of Azadirachta indica (Neem). However further studies can be performed to isolate the particular component responsible for generating wound healing activity.

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Received on 31.03.2013 Accepted on 24.04.2013

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Asian J. Pharm. Tech. 3(2): April-June. 2013; Page 73-75