Evaluation of Wound Healing Activity of Coscinium fenestratum (Gaertn.) Colebr in Albino Rats

A. Thangathirupathi¹*^, and Bhuvaneswari Santharam²

¹Department of Pharmacology, S.B. College of Pharmacy, Sivakasi-626130, Tamil Nadu, India

²Department of Biochemistry, S.B. College of Pharmacy, Sivakasi-626130, Tamil Nadu, India

ABSTRACT:

The objective of this investigation was to ascertain the scientific basis for the use of the ethanolic extract of Coscinium fenestratum for the treatment of wound. Coscinium fenestratum are used traditionally for the treatment of various diseases. The wound healing activity of Coscinium fenestratum was evaluated for its wound healing potential in two types of experimentally induced in wound models in albino rats excision wound model and incision wound model. Povidone iodine ointment (5% w/w) was used as standard wound healing agent for comparison. In the excision wound model treatment with ethanolic extract ointment at 5% and 10%w/w concentration, enhanced significantly the rate of wound contraction. In the incision wound model the ethanolic extract ointment at 5% and 10%w/w concentration, facilitated the healing process as evidenced by increase in tensile strength. The results suggested that Coscinium fenestratum extract ointment applied possessed significant wound healing activity. Ethanol extract of Coscinium fenestratum was examined in the form of 5% w/w extract and 10% w/w extract in the excision wound extracted on the dorsal side of the experimental animals, the 10% w/w extract ointment showed considerable difference in wound models and the result were compatible to that of the standard drug povidone iodine (5% w/w) in terms of wound contracting ability, epithelisation period and tensile strength.

KEYWORDS: Coscinium fenestratum, wound healing.

INTRODUCTION:

Wound may be defined as a loss or breaking of cellular and anatomic or functional continuity of living tissues¹. Wound healing is the body’s natural process of regenerating dermal and epidermal tissue. The healing cascade is activated when platelets come in contact with exposed collagen leading to platelet aggregation and the release of clotting factors resulting in the deposition of a fibrin clot at the site of injury. The fibrin clots serve as a provisional matrix and sets the stage for the subsequent events of healing². The process of wound healing occurs in different phases such as coagulation, epithelisation, granulation, collegenation and tissue remodeling³. Several factors delay or reduce wound healing including bacterial infection, necrotic tissue, and inference with blood supply, lymphatic blockage and diabetes mellitus. Generally if the above factors could be altered by any agent, an increased healing rate could be achieved^4,5. Wound healing is a process, which is fundamentally a connective tissue response; initial stage of this process involves an acute inflammatory phase followed by the synthesis of collagen and other extracellular macromolecules which are later remodeled to form a scar⁶.

The Indian system of medium today uses across various systems i.e., folk and codified around 8000 plant species out of which maximum number of plants are utilized by folk tradition⁷.

The plant Coscinium fenestratum commonly known as Maramancal belongs to the family Menispermaceae and is an ethano botanically important medicinal plant. The plant has been considered as a woody climbing shrub with cylindrical stem, distributed in Western Ghats in Tamilnadu and Kerala. The stem parts of this plant are being used in traditional medicine to treat various diseases⁸. Coscinium fenestratum has been heavily promoted for a wide range of uses; including in vitiated conditions of kapha and vata, opthalmopathy, inflammations, wounds, ulcers, skin diseases, abdominal disorders, jaundice, diabetes and general debility⁹. The antibacterial ability of Coscinium fenestratum colebr against Clostridium tetani was carried out¹⁰. However, very little information has been published on this plant and there is no scientifically proven data to show whether the Coscinium fenestratum ethanol extract has wound healing activity or not. Therefore, we have undertaken the present study to explore the effects of the above extract on wound healing.

MATERIALS AND METHODS:

Plant material used:

The plant Coscinium fenestratam was purchased from a local crude drug shop at Madurai and it is authentically verified by Mrs. M. Shanthi, Botanist, Department of Botany, SFR College of Arts and Science, Sivakasi.

Extraction procedure:

The stem of Coscinium fenestratum was collected, shade dried and then size reduced to small pieces by manual and mechanical crushing. The dried materials were coarsely powdered before extraction. The ethanolic extract was prepared by cold maceration process with occasional shaking, continued for 72 hours. Extraction procedure was followed according to standard methods^{2, 11}.

Identification tests for plants:

Preliminary phyto chemical tests were performed to detect the presence of phyto constituents.

Preparation of ointments:

A control ointments base was formulated without any drug content. The ethanol extract of Coscinium fenestratam was used for wound healing activity studies. Two ointments were formulated by using 5% and 10% extract, where 5gm and 10gm of dried ethanol extracts were incorporated in 100gm of ointment base BP (British Pharmacopoeia, 1993). The standard drug for screening wound healing activity is povidone iodine ointment (5% w/w) which was bought commercially.

Experimental animals:

Wistar strain albino rats (150- 250gm) of either sex were procured from animal house of S.B. College of Pharmacy, (Regd. No. 622/02/C/CPCSEA) used for the present study. The rats were housed at ambient temperature (21±1°C) and relative humidity (55±5°C) with fixed 12 hours light/ dark cycles and free access to food and water. The rats were kept in experimental room for one week for acclimatization.

Wound healing activity:

Wound healing activity of the herbal drug Coscinium fenestratum was done by two reported methods. For present study the ointments of ethanol extract of Coscinium fenestratum was prepared by incorporating the extract into the melted simple ointment base by the fusion method^12-15.

Excision wound model:

Excision wound model was employed to study the rate of wound contraction and epithelization. Four groups with six animals in each group were anaesthetized with anesthetic ether. The rats were depilated on the back. One excision wound was inflicted by cutting away a 500 mm² full thickness of skin from the depilated area. Then this chirurgical wound was left undressed to open environment. The wounds were traced on 1 mm² graph paper on the day of wounding. Then the various formulations, i.e. simple ointment B.P, reference standard (povidone iodine ointment 5% w/w) and various concentration of ethanol extract ointment of Coscinium fenestratum (5% and 10%w/w) ointment were applied daily till the wound was completely healed. Changes in wound area were calculated on alternative days, which give an indication of the rate of wound contraction. The progressive changes in wound area were monitored planimetrically by tracing the wound margin on graph paper every alternate day. Number of days required for falling of the eschar without any residual raw wound gave the period of epithelization.

Incision wound model:

Four groups with six animals in each group were anesthetized and two para vertebral long incisions were made through the skin and cutaneous muscles at a distance of about 1.5 cm from the middle on each side of the depilated back of the rat. Full aseptic measures were not taken and no local or systemic antimicrobials were used throughout the experiment. No ligature was used for stitching. After incision was made the parted skin was kept together and stitched with black silk at 0.5 cm intervals. Surgical threads (No: 000) and a curved needle (No: 11) were used for stitching. The continuous threads on both wound edges were tightened for good closure of the wound model. The ointments were applied once daily for 9 days. When the wounds were cared thoroughly the surfaces were removed as the ninth day and tensile strength was measured with a tensiometer¹⁶.

Measurement of healing:

Tensile strength is the force required to open a healing skin wound which was used to measure healing. The instrument for this measurement is called as tensiometer. It consist of a 6 X 12 inch board with one post of 4 inch long fixed on each sided the longer ends. The board was placed at the end of a table. A pulley with bearing was mounted on the top of one of the post. An alligator clamp with 1cm width was tied on the tip of post without pulley by a piece of fishing line, so that the clamp could reach the middle of board. Another alligator clamp was tied on the piece of fishery line with minimum weight on the other end. Before testing, the animal was anaesthetized with ether in an open mask. The animal was then placed on a stock of paper towels on the middle of the board. The amount of the towels could be adjusted so that the wound was on the same level of tips of the posts. The clamps were then carefully clamped on the skin of opposite sides of the wound at a distance of 0.5 cm away from wound. The longer piece of fishing line was placed onto pulley and the position of the board was adjusted so that the weights should freely hanging in air. The weights were added at a rapid by constant rate until the wound began to open up. The total weights are considered as tensile strength of wound.

Statistical analysis:

The differences between experimental groups are compared using ANOVA followed by Dunnet’s t -test. Results are expressed as mean ± SE.

RESULTS AND DISCUSSION:

Preliminary phytochemical analysis:

The dried powder of stem of Coscinium fenestratum was subjected to successive solvent extraction by cold maceration technique. The results were shown in Table.1. The ethanolic extract contains the phytoconstituents such as alkaloids and phytosteroids. So during the present study, this extract was alone chosen for wound healing activities. The extract was dried and finally percentage yield was calculated as 12.38 % (w/w). Plant products have been shown to possess good therapeutic potential as wound healing agent due to the presence of alkaloids, active terpenes and flavonoids¹⁷ (Suguna et al., 1996).

Excision wound studies:

The progress of the wound healing induced by ethanolic extract ointment (5% and 10% w/w) treated groups, simple ointment (control) treated group and povidone iodine ointment (standard drug) treated group of animals are shown in Table.2. Out of the two best extract ointment used, extract ointment (10% w/w) appears to be the best in promoting the wound healing in terms of wound contraction and epithelization period. The percentage of wound contraction was much more with 10% w/w extract ointment treated group (20 days for 100% contractions) which was almost similar to that of the povidone iodine ointment treated group. The epithelization period was found to be decreased in extract ointment treated group, which was comparable with povidone treated group are shown in Figure 1-3.

Fig 1: Wound healing activity of Coscinium fenestratum against excision wound model in albino rats on the initial day

a) Simple ointment treated (Control)

b) Povidone iodine ointment treated (Standard)

c) 5% ethanolic extract ointment of Coscinium fenestratum treated

d) 10% ethanolic extract ointment of Coscinium fenestratum treated

Fig 2: Wound healing activity of Coscinium fenestratum against excision wound model in albino rats on the 10^th day

a) Simple ointment treated (Control)

b) Povidone iodine ointment treated (Standard)

c) 5% ethanolic extract ointment of Coscinium fenestratum treated

d) 10% ethanolic extract ointment of Coscinium fenestratum treated

Fig 3: Wound healing activity of Coscinium fenestratum against excision wound model in albino rats on the final day

a) Simple ointment treated (Control)

b) Povidone iodine ointment treated (Standard)

c) 5% ethanolic extract ointment of Coscinium fenestratum treated

d) 10% ethanolic extract ointment of Coscinium fenestratum treated

Table 1: Presence of phyto-constituents of various extracts of Coscinium fenestratum

Phyto-constituent	Petroleum ether extract	Benzene extract	Chloroform extract	Acetone extract	Ethanol extract	Aqueous extract	Total Aqueous extract
Alkaloids	-	-	+	+	+	+	+
Carbohydrates	-	-	-	-	-	-	-
Glycosides	-	-	-	-	-	-	-
Phytosterol	-	-	-	-	+	-	-
Fixed oil and fats	-	-	-	-	-	-	-
Saponin	-	-	-	-	-	-	-
Phenolic compounds	-	-	-	-	-	-	-
Proteins and aminoacids	-	-	-	-	-	-	-
Gums and mucilage	-	-	-	-	-	-	-
Volatile oils	-	-	-	-	-	-	-

Table 2: Effects of ethanolic extract ointment of Coscinium fenestratum on excision wound model in albino rats

Drug

Treatment

Days

Period of Epithelisation

Control

535+ 1.291

505.5+ 1.707 (5.5)

465+ 1.301 (13.0)

415+ 1.291 (22.4)

375+ 1.301 (29.9)

345+ 1.291 (35.5)

295 + 1.301 (44.85)

195 + 1.301 (63.5)

155+ 1.290 (71.0)

125+ 1.2909 (76.6)

22.5+ 1.2909

(0)

Povidone Iodine Ointment

516.5+ 1.707

486.5 **

+ 1.707 (5.8)

436.5 n.s + 1.707 (15.5)

383 n.s + 1.291 (25.8)

295 ** + 1.301 (42.88)

225 ** + 1.291 (56.4)

165 ** + 1.301 (68.0)

85 * + 1.291 (83.5)

25 ** + 1.301 (95.1)

12 * + 1.291 (97.6)

(100)

17 * + 0.576

(24.4)

5% ethanolic extract ointment

525+ 1.301

495** + 1.2909 (5.71)

455 ** + 1.301 (13.3)

413.5 n.s + 1.707 (21.2)

345 ** + 1.291 (34.28)

255 ** + 1.301 (51.4)

185 ** + 1.291 (64.7)

95 ** + 1.301 (81.9)

34.5 ** + 1.705 (93.4)

13 * + 1.301 (97.5)

(100)

18 * + 0.816

10% ethanolic extract ointment

545+ 1.291

505.5 n.s + 1.301 (7.2)

445 ** + 1.291 (14.6)

405 **+ 1.301

(25.6)

355 ** + 1.291

(34.8)

245 ** + 1301

(55.0)

175 ** + 1.291

(67.8)

85 ** + 1.30 (84.4)

30 ** + 1.825

(94.4)

9 n.s. + 1.291

(98.5)

(100)

17.5 * + 0.9013

(22.2)

One way Anova

35.856

76.88

14.088

20.019

520.52

1256.25

1054. 46

54.47

7.81

9378. 75

8.63

(3,12)

I. Values are mean + SEM; II. ** P<0.001, * P<0.01, n.s (non-significant) when compared to control.

Table 3: Effect of ethanolic extract ointment of Coscinium fenestratum on

incision wound model in albino rats

Drug Treatment		Tensile Strength (gm)
Simple ointment (Control)		230+2.580
Povidone Iodine ointment (Standard)		570*+3.162
5% Ethanolic extract ointment		546*+2.581
10% Ethanolic extract ointment		571*+2.885
One way ANOVA	F	3505.35
	DF	(3,12)

I. Values are Mean + SEM

II. *Indicates significant different at P<0.001 when compared to control.

Incision wound studies:

In the incision wound activity there was a significant increase in tensile strength of the 10 days old wound due to treatment with various test extract ointments (5% and 10% w/w) and the reference standard povidone ointment (5% w/w) when compared with the control group. The increased tensile strength of treated wounds may be due to increased collagen formation/unit area and stabilization of the fibers¹⁸. The measurement of tensile strength was shown in Table.3. Significant increase in tensile strength of incision wound suggests that the ethanolic test extract ointment promotes collagen formation¹⁹. It is observed that 10% w/w extract ointment showed faster healing than 5%w/w extract ointment and control ointment are shown in Figure 4-5.

Fig 4: Wound healing activity of Coscinium fenestratum against incision wound model in albino rats on the initial day

a) Simple ointment treated (Control)

b) Povidone iodine ointment treated (Standard)

c) 5% ethanolic extract ointment of Coscinium fenestratum treated

d) 10% ethanolic extract ointment of Coscinium fenestratum treated

Fig 5: Wound healing activity of Coscinium fenestratum against incision wound model in albino rats on the final day

a) Simple ointment treated (Control)

b) Povidone iodine ointment treated (Standard)

c) 5% ethanolic extract ointment of Coscinium fenestratum treated

d) 10% ethanolic extract ointment of Coscinium fenestratum treated

CONCLUSION:

Based on our study, we conclude that the 10% ethanolic extract of Coscinium fenestratum has potent wound healing activity.

ACKNOWLEDGEMENT:

The authors express their gratitude to Shri. S. Sriram Ashok BE, correspondent and Shri. P. Dhramar, Secretary of S.B. College of Pharmacy, Sivakasi, India for their constant support to accomplish the study.

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Received on 07.12.2010

Accepted on 25.12.2010

Research J. Pharmacology and Pharmacodynamics. 3(2): March –April, 2011, 81-87