INTRODUCTION:

Wound can be defined as a cut or break in the continuity of any tissue which may arise due to physical, chemical or microbial agents¹. Wound a clinical entity contemporary to mankind is a common clinical problem till today. Wound healing or wound repair is an intricate process in which the skin or some other organ repairs after injury². Healing is essentially a survival mechanism and represents an attempt to maintain normal anatomical structure and function. All wound heal following a specific sequence of phases which may overlap^3,4. The healing depends on the type of tissue which has been damaged and the nature of tissue disruption. The treatment is aimed to shortening the duration required for healing or minimizing the undesirable consequences.

The basic principles of optimal wound healing which include minimizing tissue damage, maximsing tissue perfusion and oxygenation, proper nutrition and moist wound healing environment have been recognized for many years⁵.A number of drugs ranging from simple analgesics to complex and expensive chemotherapeutic agents administered in the management of wound affect healing either positively or negatively.⁶Aspirin, indomethcin, cytotoxic agents and immunosuppressant have been proved experimentally to affect healing negatively^.7-10.

Management of wound healing, particularly the under healing is complicated and expensive programme. Research on wound healing drugs is a developing area in modern biomedical sciences, Several drugs from plants have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients, but the potential of most remains unexplored. Hence there is a dearth of safe, economic and effective prohealing agents for wound management programme, which can enhance healing as well as control infection.

Commelina benghalensis is a weed of the tropic and sub tropic, widely distributed in throughout India, West Africa, Japan, Australia, Ceylon, and China. It is naturally propogated by seeds Traditionally Commelina benghalensis commonly called as Benghal dayflower reported to possess number of medicinal properties ^11-14

According to ayurveda, it is bitter and useful in the treatment of leprosy, nervous system related disorders^{15, 16} “vata”, haemorrhage, dysuria and mastititis. The sap is used for treatment of eye ailments¹⁷, hair care,¹⁸ sore throat, burns and tropical application for thrush in infants¹⁹.

In La Reunion, the plant is considered emollient and mucilaginous, demulcent, laxative ,diuretic, antiseptic, deobstruent, antirheumatic, astrigent cooling fever, scorpion sting, wounds, boils, constipation , digestive, liver complaints²⁰ etc. The Sutos prepare a medicine from the plant for treating barrenness in women.

In the Ayurvedic system of medicine, herbal extracts but not purified compounds have been used from centuries because many constituents with more than one mechanism of action are considered to be beneficial but no scientific reports were available for its wound healing activity. Hence the present study is designed to fill up the lacunae in the literature for wound healing activity with a view to provide scientific evidence on wound healing.

MATERIALS AND METHODS:

Plant material and preparation of Extracts:

In the present study, the roots of Commelina benghalensis Linn (Commelinaceae) were collected from Jamboti forest Dist. Belgaum, Karnataka in the month of July. The plant Commelina bengalensis was authenticated from the Scientist Mr. Harsha Hegade of ICMR (Regional Medical Research centre, Belgaum.) Accession No.RMRC-486.

Preparation of alcoholic and aqueous extracts of Commelina benghalensis Linn:

Around 1 Kg of fresh shade dried powdered roots of Commelina benghalensis was subjected to hot continuous extraction (soxhlet) with alcohol for 48h in batches of 250 g each. The extract was filtered, cooled and solvent was recovered under reduced pressure at 40±5^oC by rotary flash evaporator. The aqueous extract was prepared by maceration process by using chloroform water. The yield of alcoholic and aqueous extract was 5.78 g. and 8.6 g. respectively. The extract was stored in a cool place for further study.

PHARMACOLOGICAL ACTIVITY:

Experimental animals:²¹

Healthy young albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were used for assessing Wound healing and Swiss albino mice of either sex weighing between 25-30 gms for acute toxicity study to determine LD₅₀ of various extracts. Animals were procured from Venkateshwara Enterprises, Bangalore. The animals were randomly selected, marked to permit individual identification, divided into different groups comprising of six animals in each group and kept in polypropylene cages for 5 days prior to dosing at 23±1⁰C in 12:12 dark: light cycle with free accession to standard pellet feed (Amrut Sangli) and water ad libitum.This project was cleared by Institutional Animal Ethical Committee. (Resolution No 1/2/2007, dated 23-11-2007).

Acute Oral Toxicity study:²²

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co–operation and Development (OECD), received draft guidelines 425, received from Committee for the Purpose of Control and Supervision of Experiments on Animals ( CPCSEA) , Ministry of Social Justice and Empowerment, Government of India. Swiss albino mice weighing between 20-30 gms were fasted over night prior to the acute experimental procedure. The principle, which is based on a stepwise procedure with the use of a minimum number of animals per step. The LD₅₀ of different extracts were determined. The therapeutic dose was calculated as1/10^th of the lethal dose for further investigation

WOUND HEALING ACTIVITY:

Healthy young albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were selected divided into different groups comprising of six animals in each group. The animals were depilated at the desired site before wounding. They were housed individually with free access to food and water, the basal food intake and body weights to the nearest gram were noted. The animals were starved for 12 h prior to wounding. Under light ether anesthesia wounding was performed semi-asceptically. The first group served as control, second received alcoholic extract and third group aqueous extract by oral route.

Wound models:

Albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were used for assessing wound healing activity.

Excision wound:²³

An excision wound was inflicted by cutting away 500 mm2 of a pre-determined area on the depilated back of the rat. Epithilization period was noted as the number of days after wounding required for the scar to fall off leaving no raw wound behind. Wound contraction rate was monitored by planimetric measurement of the wound on a graph paper on4th, 8th, 12^th and 16th post wounding days and there after daily until healing was complete. Reduction in wound area was expressed as percentage of the original wound size on zero days.

Incision wound:²⁴

Under light ether anesthesia on the depilated backs of the animal was two paravertebral straight incisions of 6 cm. were made through entire thickness of the skin, on either side of the vertebral column with the help of a sharp blade as described by Ehrlich and Hunt, care was taken to see that the incisions were at least 1 cm apart, using four zero silk thread and straight round bodies needle. Wounds were then mopped with cotton swabs soaked in 70% alcohol. The animals were caged individually. Removal of sutures was done on 8^th post wounding day. Tensile strength was determined on 10^th post wounding day by continuous, constant water flow technique of Lee.

STATISTICAL ANALYSIS:

All the results were analyzed by ANOVA followed by Dennett’s test. The level of significance was set at p < 0.05.

RESULTS:

The powdered crude drug was subjected to determination of extractive value, total ash, water soluble ash, acid insoluble ash, loss on drying, heavy metals and microorganisms. These values are useful in determining the quality and purity of crude drug. Further these values indicate the nature of the constituents present in a crude drug. The results of physiochemical characterization are presented in Table-1

Table-1: Physiochemical characterization of Commelina benghalensis

Sl. No.	Parameters	As per IP/BP Standards	Results
1	Loss on Drying	NMT 10% w/w	9.05%
2	Total Ash	-	6%
3	Acid insoluble Ash	-	1.2%
4	Water soluble Ash	-	7.%
6	Total Microbial count	NMT 10,000	Confirms
7	Heavy metals
	Mercury	NMT 10 ppm	Confirms
	Lead	NMT 10 ppm	Confirms
	Lithium	NMT 10 ppm	Confirms
	Cadmium	NMT 10 ppm	Confirms

The phytochemical tests revealed that the roots of Commelina benghalensis possess presence of various secondary metabolites like steroids, tannin, saponins, resins, carbohydrate and Flavonoids in the alcoholic and aqueous extract as shown in table-2

Significant wound healing activity was observed in both the groups of animals treated with the alcoholic and aqueous extract of Commelina benghalensis. The percentage of closure of wound was significant (p < 0.001) in the animals treated with alcoholic extract 54.52 ± 0.79 and 81.45 ± 0.51 on day 12^th and 16^th days and in the aqueous extract treated group it was 41.35 ± 0.31 and 68.98 ± 0.70 on day 12^th and 16^th days respectively(p<0.01) compare to control group.

The time required for complete epithelization of the excision wound is an important parameter to assess the wound healing process. It was also found that the Mean time taken for complete epithelization of the excision wound in alcoholic and aqueous extract group was less than control animals. The data was shown in the Table-3.

The alcoholic and aqueous extract showed a scar area of 09.38 ± 0.23 mm² and 10.37 ± 0.16 mm²respectively as compared to scar area of control group 16.65 ± 0.28 mm²).The results are shown in Table-3

In incision wound model the alcoholic and aqueous extract of Commelina benghalensis showed significant increase in wound breaking strength compared to control group of animals. The data was shown in the Table-4.

DISCUSSION:

Plants are the storehouses for the array of phytochemical constituents. Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its natural state. Granulation, collagen, maturation and scar formation are some of the phases of wound healing.

The healing process depends upon the reparative abilities of the tissue, the type and extent of damage and general state of the tissue. Wound contracture is a process that occurs throughout the healing process, commencing in the fibroblastic stage whereby the area of wound undergoes shrinkage. It has three phases; inflammatory, proliferative and maturational and is dependent upon the type and extent of damage, the general state of the host’s health and the ability of the tissue to repair. The inflammatory phase is characterized by hemostasis and inflammation, followed by epithelization, angiogenesis and collagen deposition in the proliferative phase. In the maturational phase, the final phase of wound healing undergoes contraction resulting in a smaller amount of apparent scar tissue. Granulation tissue formed in the final part of the proliferative phase is primarily composed of fibroblasts, collagen, edema, and small blood vessels. The increase in dry granulation tissue weight in the test treated animals suggests high protein content.

Table-2: Phytochemical Screening of Commelina benghalensis

Extracts	Steroid	Flavonoid	Carbohydrate	Saponin	Resin	Tannin
Alcohol	++	+++	-	++	+	++
Aqueous	-	++	+	+	_	++

Note: +++ = High concentration, ++ = medium concentration, + = low concentration, - = absent.

Table-3: Influence of different extracts on Excision wound model

Group	Mean ± SEM of % of wound closure on				Epithilization Time (Days)	Scar Area (mm²)
Group	4^th day	8^th day	12^th day	16^th day	Epithilization Time (Days)	Scar Area (mm²)
Control	9.41 ± 0.71	21.67 ± 1.01	33.18 ± 0.58	52.85 ± 0.45	26.50 ± 0.32	16.65 ± 0.28
Alcohol extract	15.13 ± 0.32***	34.13 ± 0.31***	54.52 ± 0.79***	81.45 ± 0.51***	18.07 ± 0.23***	9.38 ± 0.23***
Aqueous extract	11.70 ± 0.51	24.57 ± 0.49**	41.35 ± 0.31**	68.98 ± 0.70**	23.67 ± 0.29**	10.37 ± 0.16**

Note: Data analysed by ANOVA followed by Dunnett’s test. ***= p < 0.001, **=P < 0.01.

Table-4: Influence of different extracts on Incision wound model

Group	Dose (oral) mg/kg b.w.	Wound breaking strength(g)
Control	0.1% Tween 80 1ml v/v	175.90 ± 1.54
Alcohol extract	200	174.80 ± 1.70***
Aqueous extract	200	147.10 ± 1.96**

Note: Data analysed by ANOVA followed by Dunnett’s test. ***= p < 0.001, **=P < 0.01.

The two different models were used in the study to assess the wound healing effect of the alcoholic and aqueous extracts of Commelina benghalensis on various phases of wound healing, which run concurrently but independent of each other.

In excision wound model significant wound healing was observed in the animals treated with alcoholic and aqueous extract of Commelina benghalensis. Significant decrease in epithelization increase in wound contraction rate was observed in these groups of animals. In both the extract treated animals epithelization was completed in 16 days. While in control group rate of contraction and epithelization was slow.

Scar replaces the damaged tissue, scar formation is a biological process by which an excision wound shrinks. Whereas the term contracture specifically implies to loss of normal stretching as a result of excessive scar formation. Scar undergoes certain changes indicating continuation of healing process.

Breaking strength is the strength of healing wound and is measured experimentally by amount of force required to disrupt it. In the beginning wound will be having little breaking strength because clot will alone be holding the edges together. Thereafter breaking strength increases rapidly as collagen deposition increases and cross linkages are formed between the collagen fibers. In present study significant increase in breaking strength was observed in the animals treated with alcoholic and aqueous extract on 10^th post wound healing day.

Wound healing is the physiological response to the tissue injury that results in the replacement of destroyed tissue by living tissue and thus the restoration of tissue integrity. Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its natural state. Wound contracture is a process that occurs throughout the healing process, commencing in the fibroblastic stage whereby the area of wound undergoes shrinkage. It has three phases; inflammatory, proliferative and maturational and is dependent upon the type and extent of damage, the general state of the host’s health and the ability of the tissue to repair. The inflammatory phase is characterized by hemostasis and inflammation, followed by epithelization, angiogenesis and collagen deposition in the proliferative phase. In the maturational phase, the final phase of wound healing undergoes contraction resulting in a smaller amount of apparent scar tissue. The lymphatics develop new nerve fibers and also formation of scar tissue in which collagen turn over increases. Granulation tissue formed in the final part of the proliferative phase is primarily composed of fibroblasts, collagen, edema, and small blood vessels. The increase in dry granulation tissue weight in the test treated animals suggests high protein content.

The alcoholic extract of Commelina benghalensis revealed presence of secondary metabolites like steroids, tannin, saponins, carbohydrate and flavonoids. Flavonoids are known to reduce lipid peroxidation not only by preventing or slowing cell necrosis but also by improving vascularity. Hence any drug that inhibits lipid peroxidation is belived to increase the viability of collagen fibrils by increasing the circulation, strength of collagen fibres, preventing the cell damage and by promoting the DNA synthesis.²⁵ Flavonoids are also known to promote the wound healing process mainly due to their antimicrobial property which is responsible for wound contraction and increased rate of epithelization.

The recent syudies of wound healing activity claims that the flavonoids promote significant wound healing property.^26,27 The well known property of almost every group of flavonoids in their capacity to act as antioxidant. flavonoids protects the body against reactive oxygen species. Body cells and tissue are continuously threatened by the damage caused by free radicals and reactive oxygen species which are produced during normal oxygen metabolism or induced by exogenous damage.^28,29

CONCLUSION:

The use of roots of Commelina benghalensis in folk medicine for the treatment of wounds has been justified by this work, as it showed remarkable wound healing property. These findings clearly justify for the inclusion of roots of Commelina benghalensis the management of wound healing.

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Received on 19.08.2011 Modified on 02.09.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1772-1776