The Wound healing, anti inflammatory and haemostatic effect of Eupatorium odoratum

R. Suresh*, D. Benito Johnson, Appalaraju Gorle, Ashok Kumar Javvadi, Tamil Selvan A.

Department of Pharmacology, R.V.S. College Pharmaceutical Science, Sulur, Coimbatore-640 402

ABSTRACT:

Wound is perhaps an inescapable event in the life of an organism. Healing process begins during the early phase of inflammation, but reaches completion usually after the injurious influence has been neutralized. It should be emphasized that without inflammation wounds would never heal. Eupatorium odoratum Linn. (Family: Compositae) is a perennial herb growing widely in the south of the country. The wound healing effect of ethanol extract of E. odoratum was studied using, re sutured incision wound, excision wound model, dead space wound model and the results obtained by the three models were compared and found that excision wound model is statistically significant (p<0.01). The anti inflammatory activity of ethanol- leaf extract of E. odoratum was evaluated using carregeenan induced paw edema in rats, there was no statistically significant change in the drug treated compared to the control and the haemostatic activity assessed by capillary tube method, there was a statically significant reduction in the blood coagulation time

KEY WORDS: wound healing, anti inflammatory, haemostatic, Eupatorium odoratum

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. Eupatorium odoratum, belonging to the compositae family, is a perennial herb growing widely in various parts of South America and tropical countries. The leaf juice of this plant has been used to apply on wounds by village people. It has been reported that leaf juice of this plant has been used to stop bleeding and has also anti microbial activity. The present study is undertaken mainly to evaluate the effect of the ethanol extract of E.odoratum like wound healing on various types of wounds, anti inflammatory action, blood coagulation⁷. The greatest contribution of plant kingdom to mankind is that it has provided a large variety of potent drugs to alleviate suffering from diseases.

In spite of spectacular advances in the synthetic drugs in recent years some of the drugs of plant origin have still retain their importance. In recent years there is a great deal of use of plant based drugs in the western countries.

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, Tamilnadu, 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 2% Tween 80 orally, since the E.odoratum ethanol extract suspension was made with the help of Tween 80

Plant material and extraction

The E. odoratum 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 E.odoratum 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 two 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 Eupatorium odoratum 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 Eupatorium odoratum showed the presence of Phytoconstituents viz., steroids, triterpene, alkaloids and flavonoids

Acute toxicity studies

Healthy adult albino rats starved overnight were divided into four groups (n=5) and were given different doses of the extract. Group I 0.25g/kg orally, Group II 0.50g/kg orally, Group III 1g/kg orally, Group IV 2g/kg orally. The animals were observed continuously every five minute, every ten minutes for one hour and at the end of twenty four hours. Since no animal died even in the maximum dose of 2g/kg of body weight, 1/10^th of it i.e. 200mg/kg of body weight was selected for the study.

WOUND HEALING MODELS

a) Resuture dincision wound:

This wound model was utilized to study the breaking strength of the incision wound in rats. Duration of study and treatment is 10 days⁸. The animals divided into two groups each group containing five animals. Group-I: control 2% Tween 80, dose-2ml/kg orally. Group-II: Ethanol extract of E. odoratum, dose-200mg/kg orally. Six centimeters long paravertiberal straight incisions were made, one centimeter lateral to the vertebral column on either side through the entire thickness of skin⁹. Intermittent sutures were placed one centimeter apart with the help of black silk thread. The wounds were then mopped with cotton swab soaked in the 70% alcohol. The animals received the drugs once every day throughout the period of study sutures were removed on the 7^th post operative day. Wound breaking strength was measured on the 10^th day by continuous constant water flow technique as described by Lee. The anaesthetized animals were placed on the operation table, to allis forceps were firmly applied on either side of the wound at a distance of 5mm from the wound margin. One forceps was hooked to the fixed metal rod and the other forceps was connected to a light plastic gratitude bottle, through a string run over a pulley. The bottle was connected to a constant running water stream which had stop cock to arrest the flow.

Table-2 showed excision wound model

Day	2	4	6	8	10	12	14	16	18	20	22
Control	1.18± 0.747	3.1± 1.16	18.8± 7.05	26.3± 8.40	55.8± 6.60	67.8± 6.60	74.4± 1.15	74.4± 1.15	78.8± 3.40	86.9± 1.6	90.9± 1.78
Drug treated	10.9± 5.58	26.7^**± 6.86	36.0± 7.38	38.2± 7.13	68.7± 4.25	76.9^*± 3.40	80.1± 2.93	83.7± 3.01	87.8± 2.52	90.5± 2.47	96.8± 2.66

*p < 0.02 **p < 0.01

Tensile strength was measured by allowing the water into the bottle and thus gradually increasing its weight which causes disruption of the wound. As soon as gapping of wound was observed, the flow was stopped and the volume of water in the bottle was noted. For such readings were taken on either side of the wound to give eight readings. The average of such 8 readings in each animal of any group was taken to calculate the average tensile strength of each animal averages, mean value for group was calculated and showed in table-1

b) 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 period¹⁰. Duration of treatment and study is twenty two days. The animals divided into two groups, each group containing six animals. Group-I: Control-2% Tween 80, dose-2ml/kg orally. Group-II: Ethanol extract of E. odoratum, dose-200mg/kg orally. 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 molone⁵⁴ 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 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 method¹¹, the W.C-50 in days was estimated and its S.E calculated by the method described by Ghosh¹². 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-2.

c) Dead space wound:

This method is usually employed for assessing the extent of collagenation¹³. The dead space wounds were created to harvest granulation by implanting the subcutaneously, 2.5×0.5cm polypropylene tubes¹⁴ in the dorsal region, through a small (0.5cm) transverse incision about four to five centimeters cephaloid to the site of implantation. Drugs were administered once daily from day’0’ to day’9’. On day ‘10’, the granulation tissue harvested on the implanted tube was carefully dissected out along with the tube after anaesthetizing the rat.

This is used to monitor breaking strength and dry weight of granuloma. Duration oftreatment and study is 10 days. The animals were divided into two groups, each group containing eight animals. Group-I: control 2% Tween 80, dose-2ml/kg orally. Group-II: ethanol extract of E. odoratum, dose-200mg/kg orally. The tubular granulation was cut along its length, to obtain a sheet of granulation tissue, which was further cut into two approximately equal pieces. The breaking/tensile strength of each piece was measured as by the method of Lee¹⁵, and the mean was calculated. After measuring the breaking strength, the pieces of the granulation tissues were collected, dried at 60^oc for 24 hours and weighed. The dried granuloma weight was taken expressed as a mg/100g of rat and calculations showed in table-3.

ANTI INFLAMMATORY STUDY

There are various models acute and chronic for screening of anti inflammatory agents.

Carrageenan induced paw edema in rats:

The procedure adopted is that of winter et al¹⁶ .one hour after drug administration, a sub plantar injection of 0.05 ml of 1%solution of carrageenan was injected into the left hind paw. The paw volume was determined immediately according to the method of Bhatt et al¹⁷ .This volume was designated as ‘0’ hours volume. The volume of injected paw was again measured affter three hours and designated as ‘3’ hours volume; difference between these two was taken as actual increase in the paw volume was showed in table no 4, 5. Mean increase in paw volume was calculated, and then the percent inhibition of edema was calculated by using the formula.

Percentage of inhibition=100(1-Vt/Vc)

Where Vt = Increase in paw volume of treated group.

Vc =Increase in paw volume of control group.

Table-4 showed acute inflammation (paw volume at third hour)

No of animals	1	2	3	4	5	6	7	Mean± S.E. ml
Control	0.8	0.4	0.4	0.2	0.26	0.52	0.38	0.42± 0.074
Drug treated	0.64	0.72	0.12	0.68	0.00	0.58	0.28	0.43± 0.11

Table-5 showed chronic inflammation(weight of granulation tissue in mg/100g of body weight)

No of animals	1	2	3	4	5	6	7	8	Mean± S.E
Control	0.03	0.04	0.04	0.04	0.04	0.042	0.031	0.049	0.039± 0.002
Drug treated	0.036	0.031	0.035	0.046	0.023	0.031	0.067	0.076	0.043± 0.006

Table-6 blood coagulation (in minute/sec):

No of animals	1	2	3	4	5	6	mean± S.E
Control	13	13	8.5	9.5	10	11	10.8± 0.76
Drug treated	4.5	2	2.5	3.0	2	2	2.66± 0.40

P < 0.001

HAEMOSTATIC EFFECT

Effect of blood coagulation was determined by capillary tube method. Twelve albino male rats were selected; six rats were treated with ethanol extract 200mg/kg orally once daily for 10 days. The six rats were for control, i.e. 1ml of 2%Tween 80 per kg of rat for 10 days. The initial blood clotting time was noted by the capillary tube of about 10-15 cm length. A part of the capillary tube was broken each minute. The elapsing from the moment the wound was made to that when fine thread of fibrin appeared between the ends of the broken sections of the tube was taken as the clotting time. The results were showed in table-6.

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 remodeling of damaged tissue¹⁸. In spite of tremendous advances in the chemical drug industry, the availability of substances capable of stimulating the process of wound repair is still limited¹⁹. Moreover the management of chronic wounds is another major problem due to high cost therapy and presence of side effects²⁰. 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 phases²¹. 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 phase²³. During epithelization, the epithelial cells crawl across the wound bed to cover it²³. 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 remodeling phase, Collagen is remolded and realigned along tension lines and cells that are no longer needed are removed by apoptosis²⁴

In the present study, all three wound models were employed to evaluate the action of the indigenous drug E.odoratum, since no single model can monitor all the phases of healing. From the study, it was observed that the drug had no significant effect on any phase of healing, as evidenced by tensile strength, wound contraction rate and epithelization period. Epithelization time was prolonged (control: 21 days, drug treated: 22 days). Wound contraction rate on the fourth post wounding day was 26.7±6.8% as compared to the

controls(3.1±1.2,p≤0.01) where as by the 12^th day the rates were comparable(control:67.8±6.6; drug treated:76.94±3.0, p≤0.02). Hence the drug seems to be effective in space dening contraction only in the early stage of healing. The tensile strength of skin (incision wound) and granuloma tissue (dead space wound) of drug treated animals were also comparable to controls. Skin tissue: control 501±10.59; dug treated: 475±23.7; granuloma tissue: control 521±10.9; drug treated: 457±40.4. In Incision wound model and Dead space wound model are there is no statistically significant change in the tensile strength of the given drug as compared to the control. In Excision wound model there is a statistically significant increase in the rate of wound contraction in drug treated animals on 4^th and 12^th day as compared to the normal control. Inflammation there is no statistically significant change in the drug treated group as compared with control. However E.odoratum showed significant haemostatic effect evidenced by reduction in clotting time in the capillary tube method.

CONCLUSION:

In conclusion, it can be reported that the indigenous drug E. odoratum was found to be ineffective as prohealer. However, since it has antimicrobial action associated with haemostatic effect, it has potential to be used locally to arrest bleeding or hemorrhages of this drug. Earlier reports of this drug being useful in the process of wound healing these may be due to its antiseptic action in keeping the wound germ free rather than enhancing the healing process.

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Received on 29.10.2011

Modified on 27.11.2011

Accepted on 29.11.2011

Research Journal of Pharmacognosy and Phytochemistry. 4(2): March-April 2012, 75-79

No of Animal	1	2	3	4	5	Mean± S.E
Control	503.3	531.6	470.0	510.0	492.0	501.38±10.15
Drug treated	548.3	436.7	421.6	460.0	511.6	475.64±23.72

No. of animals	1	2	3	4	5	6	7	8	Mean± S.E
Control	525	485	535	490	545	495	575	520	521.25± 10.89
Drug treated	565	485	265	395	365	433	550	600	457.10± 40.38