INTRODUCTION:

One of the important attributes of life is its capacity for self repair. Wound healing represents a restoration of continuity of living cells and overlaps the inflammatory process^1-2. The process of wound healing has been one of the earliest medical problems. Healing is essential for survival mechanisms and represents an attempt to maintain normal anatomical structure and function³. Healing of a wound is an important biological process involving tissue repairs and regeneration. It involves the activity of an intricate network of blood cells, cytokines and growth factors which ultimately leads to the restoration to the normal condition of the injured skin or tissue⁴. The aim of wound care is to promote wound healing in the shortest time possible, with minimal pain, discomfort and scarring to the patient and must occur in a physiologic environment conducive to tissue repair and regeneration⁵. Wound healing processes are known to be influenced by among other factors by infections, nutritional status, drugs and hormones, types and sites of wound and wasting diseases like diabetes⁶. Tridax procumbens Linn belongs to Asteraceae family.

It’s a hispid, procumbent herb with woody base, sometimes rooting at the nodes up to 60 cm high, found as a weed up to an altitude of 2,400m, leaves are ovate, flower in small, long pedeculed heads, ray florets strap sharpen white, disc florets yellow. The leaves are reported to be employed in bronchial catarrhs, dysentery, and diarrhea and for restoring hair. The leaf juice possesses antiseptic, insecticidal and parasiticidal properties and issued to check haemorrhage from cuts, bruises and wounds. The present investigation effect of Tridax procumbens which is used in the traditional system of Indian medicine was evaluated for its possible wound healing activity in the experimental animals.

MATERIALS AND METHODS:

Animals:

Healthy male albino rats weighing between 150-250g were used. They were individually housed and maintained on animal chow and water ad libitum. The protocol of the experiments was approved by the Institutional Animal Ethical Committee (IAEC) of R.V.S. College of Pharmaceutical Sciences, Coimbatore, Tamil Nadu, and according with the guidelines of the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ethical guidelines were strictly followed during all the experiments. The rats were starved for 36 hours with free access to water, before infliction of wounds. The anesthetic administered was Phenobarbitone (dose-20mg/kg weight of rat), supplemented with ether whenever necessary. No local or systemic chemotherapeutic agents were used. The surgical materials were stabilized and skin was prepared by clipping the fur and cleaning with 70% alcohol before wounding.

Materials:

The general surgical materials including anaesthetics were obtained from R.V.S. Hospital Pharmacy.

Drugs:

Control:

Animals were given only 0.5% CMC orally, since the ethanolic extract Tridax procumbens suspension was made with the help of 0.5% CMC.

Plant material and extraction:

The Tridax procumbens Linn was collected from south Coimbatore in the month of April and was authenticated by Professor Dr. R. Venkatanarayan, Department of Pharmacognosy, R.V.S College of pharmaceutical sciences. The air dried leaves of Tridax procumbens was used for the study. The coarse powder of the leaves (2kg) was extracted with 95% ethanol under reflux in a round bottom flask in batches of 500g each. Each batch was extracted for twenty hours. The extracts were then concentrated under reduced pressure till it acquires semisolid consistency. This was then allowed to evaporate to dryness.

Preliminary phytochemical analysis:

The ethanol extract of Tridax procumbens was subjected to preliminary phytochemical Screening for detection of major chemical groups. In each case test 10% w/v solution of the extract in ethanol was used and unless otherwise mentioned in individual test. Results of different chemical tests on the ethanol extract of Tridax procumbens showed the presence of Phytoconstituents Viz., steroids, triterpene, alkaloids, protein, fibers and flavonoids.

Pharmacological Studies:

The Pharmacological screening of Tridax procumbens extract were carried out the following parameters.

1. Gross behavioural studies

2. Wound healing activity

3. Leucocytes count.

Gross Behavioural and Toxicity studies:

This was studied as suggested by Turner (1965).⁷Albino mice from either sex weighing from 28 to 30 g were divided into seven groups of six animals each. Group 1 to 5 received the various doses of Tridax procumbens extract each wound. Extract was applied twice daily for twenty days.

Excision wound model:

This model was employed to study the rate of wound contraction and epithelization. The parameter monitored was median wound contraction time and epithelization period. Duration of treatment and study is twenty two days. The animals divided into two groups, each group containing six animals. Group-I: Control, Group-II: Nimesulide, Group-III: Ethanol extract of Tridax procumbens. Round seal of 2.5cm in diameter (500mm) was impressed on the dorsal thoracic region 5 cm away from the ears, as described by Morton and molone⁸ under light anesthesia. The entire full thickness of skin from the marked area was excised Wound were cleaned with cotton swab soaked in 70% alcohol. Animals received drugs daily from “0” day till wounds completely healed or up to 21^st post operative day, which ever was earlier. Contraction which mainly contributes for wound closure was studied by tracing the raw wound area on polythene paper on wounding day followed by 5^th , 10^th , 15^th , 20^th days and subsequently on every alternate day till wounds were completely covered with epithelium. These wound tracings were retraced on a millimeter scale graph paper, to determine the wound area. Then wound closure was expressed as a percent of original wound size (500 mm²) for a group and group mean on particular day was taken for final analysis of the results. Employing Litchfield and Wilcoxon analytical method⁹, the W.C-50 in days was estimated and its S.E calculated by the method described by Ghosh¹⁰. Epithelization period was monitored by noting the number of days required for eschar to fall away leaving no raw wound behind and calculations showed in table no: 1-4

The degree of wound healing was calculated as percentage closure in wound from original wound area using the formula.

%Closure= (1-A_d/A _o)

Where

A _o = the wound area on day zero,

A _d = the wound area on corresponding days.

W.B.C:

The WBC count was done with the use of Haemocytometer. First the cover slip, counting chamber and lens of microscope was cleaned then the counting chamber was adjusted under high power of microscope. The tail end of the rats were cleaned, sterilized and blood withdrawn from the tail vein and transferred to clean sterile test tube. The WBC pipette was cleaned and blood from the test tube was sucked into it till the mark 0.5 then it was diluted with the WBC dilution fluid (Truck’s fluid) till mark 11. The blood and fluid was thoroughly mixed by rotating it in between the hands then the first 2-3 drops of blood and fluid mixture was discarded and next drop was charged into counting chamber without air bubbles and WBCs were counted in four big squares, the results were noted and tabulated.

RESULTS AND DISCUSSION:

Wounds are the physical injuries that results in an opening or break of the skin. Proper healing of wounds is essential for the restoration of disrupted anatomical continuity and disrupted functional response of the several cell types to injury. Cutaneous wound repair is accompanied by an ordered and definable sequence of biological events starting with wound closure and progressing to the repair and remodelling of damaged tissue¹¹. In spite of tremendous advances in the chemical drug industry, the availability of substances capable of stimulating the process of wound repair is still limited¹². Moreover the management of chronic wounds is another major problem due to high cost therapy and presence of side effects¹³.Wound healing is a natural process of regenerating dermal and epidermal tissues. Whenever there is wound, a set of overlapping events takes place to repair the damage. These processes have been categorized into phases which include the inflammatory, proliferative and remodelling phases¹⁴. In the inflammatory phase, bacteria and debris are phagocytosed and removed and cytokines and mediator are released that cause the migration and division of cells involved in the proliferative phase. Angiogenesis, collagen deposition, granulation tissue formation, epithelization and wound contraction occur in the proliferative phase¹⁵. During epithelization, the epithelial cells crawl across the wound bed to cover it¹⁶. The wound is eventually closed by a combination of all these and by the process of wound contracture. During wound contraction, the wound is made smaller by the action of myofibroblasts, which establish a grip on the wound edges and contract themselves using a mechanism similar to that in smooth muscle cells. In the maturation and remodelling phase, Collagen is remolded and realigned along tension lines and cells that are no longer needed are removed by apoptosis.¹⁷

Ethanolic Extract of Tridax procumbens did not produce any toxic symptoms or mortality up to the dose level of 2000 mg/kg body weight. There was neither change in behavioural pattern nor any sign of toxicity during the observations up to 24hrs for mortality. Thus the extract was considered to be safe for pharmacological evaluation.

Excision wounds heal by contraction and epithelization. The parameters studied were wound closure, time for complete epithelization, scar, size, and shape or complete epithelization. The measurements on 5^th day showed that the percentage closure of original excision wound area in solvent control group was 12.99 ± 1.342 sq.mm% in Nimesulide treated group was 52.35 ± 1.259sq.mm% and Tridax procumbens extract 20µl/wound treated group was 45.91 ± 1.017sq.mm%. the Tridax procumbens and Nimesulide have significantly promoted the wound healing when compared with solvent control group. The results were showed in table no: 1

Table no: 1 showed percentage closure of original Excision wound area on 5^th post wound day

Animal no	Solvent control	Nimesulide	*Tridax procumbens* extract
1.	8.72	58.52	39.94
2.	15.14	55.34	39.78
3.	11.62	50.49	48.34
4.	13.48	51.38	47.48
5.	14.82	48.53	49.58
6.	14.14	49.84	50.31
Mean±SEM	12.99±1.342	52.35±1.259	45.91±1.017

The measurements on 10^th day showed that the percentage closure of original excision wound area in solvent control group was 44.01 ± 1.294 sq.mm% in Nimesulide treated group was 59.85 ± 1.432 sq.mm% and Tridax procumbens extract 20µl/wound treated group was 67.91 ± 1.394 sq.mm%. The Tridax procumbens and Nimesulide have significantly (P<0.001) promoted the wound healing when compared with solvent control group. The results were showed in table no: 2

Table no: 2 showed percentage closure of original Excision wound area on 10^th post wound day

Animal no	Solvent control	Nimesulide	*Tridax procumbens* extract
1.	42.38	60.21	67.42
2.	41.49	61.34	67.48
3.	40.37	59.82	68.93
4.	42.78	58.54	68.78
5.	47.84	59.78	69.38
6.	49.21	59.43	65.48
Mean±SEM	44.01±1.477	59.80±0.413	67.91±0.586

The measurements on 15^th day excision wound area in solvent control group was 57.47 ± 2.989 sq.mm% in Nimesulide treated group was 74.02 ±1.048 sq.mm% and Tridax procumbens extract 20µl/wound treated group was 83.77 ± 1.302 sq.mm%. The Tridax procumbens and Nimesulide have significantly (P<0.001) promoted the wound healing when compared with solvent control group. The results were showed in table no: 3

Table no: 3 showed percentage closure of original Excision wound area on 15^th post wound day

Animal no	Solvent control	Nimesulide	*Tridax procumbens* extract
1.	52.89	70.48	80.14
2.	58.62	72.84	82.34
3.	60.14	74.89	85.68
4.	58.34	76.38	84.92
5.	59.38	75.12	84.16
6.	55.42	74.39	85.37
Mean±SEM	57.46±1.126	74.01±0.848	83.76±0.874

The extract wound closure on 20^th day was 73.97±1.493 sq.mm% in solvent control, 87.99±0.129 sq.mm% Nimesulide, 99.95±1.742 sq.mm% in Tridax procumbens extract. When compared to solvent control, Nimesulide, Tridax procumbens extract were significantly (P<0.001) promoted the wound closure. The closure was almost complete on 20^th day. The results were showed in table no: 4

Table no: 4 showed percentage closure of original Excision wound area on 20^th post wound day

Animal no	Solvent control	Nimesulide	*Tridax procumbens* extract
1.	70.18	85.43	99.94
2.	74.34	89.68	100
3.	75.34	87.35	99.97
4.	76.12	89.18	99.82
5.	75.18	88.17	99.97
6.	72.64	88.14	100
Mean±SEM	73.96±0.899	87.99±0.613	99.95±1.742

Invariably in all the groups, the WBC count was increased in the 5^th post wounding day; however in solvent control the count was further increased. In Nimesulide treated group count back to the normal 10^th post wound day and all other groups the count decreased. In the Tridax procumbens extract treated group increased in the count of WBC is significantly (0.05) differing from the solvent control. The increased counts of WBC was maintained in similar fashion for the extract which indicates that the Tridax procumbens extract heals or prevent formation of collagenation with the help of our body immune system. The results were showed in table no:5

CONCLUSION:

The present study indicates the potential wound healing property of the plant Tridax procumbens extract. As a result of pharmacological screening the Tridax extract and Nimesulide shows a similar effect on wound healing property on experimentally induced wounds in rats. The literature evidences shows the plant contains crude proteins, crude fibers those were involved in the contraction of tissues. From the results concluded that Tridax procumbens extract possess significant wound healing action which was compared to synthetic anti-inflammatory agent. Further clinical studies are needed to establish its safety and usefulness in wound patients.

REFERENCES:

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3. Bhat S, Shankrappa J, Shivkumar HG. Formulation and evaluation of polyherbal wound treatments. Asian Journal of Pharmaceutical Sciences 2007; 2 (1): 11-7.

4. Clark RAF Cutaneous wound repairs. In: Goldsmith LA editors. Physiology: Biochemistry and Molecular Biology of skin. New York: Oxford University Press; 1991. 576.

5. Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated approaches to wound management. Clin. Microbial Rev 2001; 14: 244-69.

6. Karl M, Lacrix PV, Peterson HH, editors. Canine Surgery. American Veterinary Publication., California; 4th ed: pp 42-5.

7. Turner R.A. Screening methods in pharmacology, New York, Academic press, (1965): 4^th edition: pp 152-163.

8. Morton JJ, Malone MH. Evaluation of vulnerary activity by an open wound procedure in rats. Arch Int Pharmacodyn Ther. 1972; 196: 117-26.

9. Litchfield J.T., Wilcoxon F.A. J Pharmacol Exp Therapy.1949; 95; 99-100.

10. Ghosh M.N. Fundaments of experimental pharmacology. Toxicities studies. Ghosh M.N., Scientific Book Agency Calcutta. 1984a; 2^nd edn: pp 153-158.

11. Odimegwu DC, Ibezim EC, Esimone CO, Nworu CS, Okoye F B. Wound healing and antibacterial activities of the extract Dissotis theifolia (Melastomataceae) stem formulated in a simple ointment base. J. Medicinal Plant Res. 2003; 2(1): 011-16.

12. Rathi B, Pathi PA, Baheti AM. Evaluation of aqueous extract of pulp and seeds of Moringo oliefera for wound healing in albino rats. J Natural Remedies. 4: 145-49.

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17. Esimone CO, Nworu CS, Jackson CL. Cutaneous wound healing activity of a herbal ointment containing the leaf extract of Jatropha curcas L. (Euphorbiaceae). International Journal of Applied Research in Natural Products 2009; 1(4):1-4.

Received on 05.12.2011 Modified on 02.01.2011

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 239-242

Days	Animal no	Solvent Control	Nimesulide	*Tridax procumbens* extract
5^th day	1	8.1	11.8	3.2
	2	16.4	-1.3	4.7
	3	14.3	13.4	14.2
	4	1.2	-1.7	16.4
	5	13.4	5.2	14.2
	6	9.5	4.2	-1.2
Mean±SEM		10.48±2.626	5.26±2.593	8.58±2.968
10^th day	1	14.3	-1.8	-1.8
	2	8.7	3.2	13.4
	3	20.2	1.4	4.1
	4	4.5	1.7	4.6
	5	19.8	2.8	4.2
	6	8.9	2.2	4.7
Mean±SEM		12.73±2.626	1.58±0.73	4.86±1.982