Evaluation of Wound Healing Activity of Ageratum conyzoides Linn

 

Prabhat Khare*1, RB Goswami1, Smita Khare1 and AK Pathak2

1Sagar Institute of Research, Technology and Science – Pharmacy, Bhopal

2Department of Pharmacy, Barkatullah University, Bhopal

 

 

ABSTRACT

The present study was aimed to investigate the possible effect of Ageratum conyzoides leaf extract on wound healing activity on the excision would model. The wound healing property of Ageratum conyzoides appears to be due to the presence of its active principles, which accelerates the healing process and confers breaking strength to the healed wound.

 

KEYWORDS: Wound, Wound healing, Ageratum, Ethanolic Extract, Excision Model

 

INTRODUCTION

A wound may be defined as a break in the epithelial integrity of the skin or may also be defined as a loss or breaking of cellular and anatomic or functional continuity of living tissue.1 Wound healing studies are mainly aim to detect various means and factor influencing healing process, so they could be either used or avoid in clinical practice to favorably alter the healing process.2

 

Although many indigenous tribes around the world have long suspected that this ubiquitous, annual, herbaceous plant might have medicinal wound healing properties, it has not really got the attention of orthodox medical practitioners as a potential source of a healing agent which may prove to be useful in the treatment of wounds.3

 

ageratum conyzoides belonging to Asteraceae is a common weed found everywhere in India and commonly known as goat weed, white weed, in various parts of India. The leaves are applied to the wounds act as septic and heel them quickly. The juice of the fresh plant and extract of dried plant are used to cure allergic rhinitis and sinusitis.4-7

 

MATERIALS AND METHODS

The plant was selected on the bases of their antimicrobial activities and wide medicinal uses in the traditional literatures. The ease of availability of plant is also taken into consideration during selection. Leaves of Ageratum conyzoides (Ageratum) was collected and authenticated by the Department of Pharmacy, B. U. Bhopal.

 

Extraction

The leaves are collected and thoroughly dried under shed than ethanolic extract was prepared by Double maceration using alcohol (Ethanol 95% v/v). Extracts after filtration were concentrated at low pressure by distillation and finally air-dried.

 

Phytochemical Studies

Collected extract was subjected to various chemical tests for the preliminary determination of phytoconstituents. Extracts was mixed with equal proportion of alcohol and water (to get a hydro-alcoholic sample), before subjected to various chemical reagents.

 

 

TABLE-I: Chemical Tests

S. No.

Chemical test

Result

1.

Alkaloids

+

2.

Carbohydrates

-

3.

Proteins and Amino acids

-

4.

Steroids

-

5.

Terpenoids (Carotenoids)

+

6.

Saponins

+

7.

Glycosides

+

8.

Phenolic compounds (Flavanoids)

+

9.

Tannins

+

*+ = Present,                 - = Absent

 

Formulation:

After preparation of extract and phytochemical studies, the next step was to formulate a suitable formulation. An ointment with water soluble base was of first choice due to their ease of preparation and also eases of cleaning after application.

 

Polyethylene Glycol (PEG) Ointment base,8 a mixture of PEG 4000 and PEG 600 found to have sufficient consistency in ratio 3:7 respectively, thus suitable for ointment preparation with concentration of 10 % w/w of extract.

 

A formulation was prepared by Fusion method containing Ageratum leaf extract (10% w/w) in PEG ointment base. The prepared formulation was then evaluated by various parameters e.g. consistency, stability etc.

 

Wound Healing Activity:

Excision wound model as described by Mukherjee P K9 with some modifications, using Albino rats was selected for assessing the wound healing activity. This model was employed to study the rate of wound contraction and the time required for full epithelization of the wounds. These parameters were selected because of easy availability of Albino rat and simplicity in handling them.

 

Selection and Procurement of Animals:

Healthy albino rats were procured and rats weighing 150-200 gm were selected, maintained at 24-280C, housed individually with free access to food and water. They were fed with standard diet and kept in well-ventilated animal house with alternate dark-light cycle of 12 hrs throughout the studies. The study was permitted by the Institutional Animal Ethical Committee (Reg. No- CPCSEA/444).

 

For wound healing activity the animal were divided into three groups having six animals in each group.

I Group- serve as CONTROL treated with PEG ointment base.

II Group- serves as STANDARD treated with Framycetin sulphate 1%w/w ointment.

III Group- serves as TEST treated with Ageratum leaves extract formulated ointment (10 % w/w)

 

Excision Wound Model:

For the excision wound studies, eighteen albino rats were taken, divided in three groups of six each. Rats depilated by removing hairs at the dorsal thoracic region before wounding. Rats were locally anaesthetized by Lignocaine hydrochloride gel I.P., 2% w/v prior to excision.

 

Circular wound of about 2.5 cm diameter was made on depilated dorsal thoracic region of rats under aseptic conditions and were observed throughout the study. The areas of the wounds were measured (in sq. mm) immediately by placing a transparent polythene graph paper over the wound and then tracing the area of the wound on it (Approx. area 500 sq mm). This was taken as the initial wound area reading.

 

All the samples e.g. Control, Standard, and Test, were applied once daily for 16 days, starting from the day of wounding. The observations of percentage wound closure were made on 4th, 8th, 12th and 16th, post wounding days. The wound area of each animal was measured at intervals of 24-48 hrs using tracing paper method. The percentage of wound contraction was calculated from the days of measurements of wound area.

The wound contraction was calculated as percentage reduction in wound area with respect to initial wound area while the epithelization time was noted as the number of days after wounding required for scar to fall off leaving no raw wound behind.

 

Statistical Analysis:

The results were analyzed by one-way anova and a P-value less than 0.01 was considered significant.

 

 

TABLE-II: Effect of Topical Application of leaf extract of A. conyzoides and Formulation on Excision Wound Model

Area of wound closure (sq mm ± S.E.M)

Groups (n)

4th day

8th day

12th day

16th day

Epithelization period (Days)

I Control

395.45±1.52

(20.91%)

318.14±0.63

(36.42%)

176.10±0.69

(64.78%)

124.30±0.60

(75.14%)

22.27±0.67

II Standard

262.30±0.60

(47.54%)

168.10±0.67

(66.38%)

34.20±0.65

(93.21%)

00

(100%)

14.20±0.69

III TEST

341.33±0.65*

(31.74%)

245.81±0.68*

(50.84%)

130.32±0.69*

(73.94%)

45.50±0.70*

(90.90%)

18.66±0.70*

# Initial wound area approx. 500 sq mm, ≈ n = 6 animals in each groups. ≠ Result expressed as Mean Area ± S.E.M.

* P≤ 0.01 indicates significant when compared with control. Ψ Figure in parenthesis indicate percent wound contraction.

§ Group I-Control (PEG ointment base), Group II- Standard (Framycetin sulphate 1%w/w), Group III- Test (Ageratum extract formulated ointment)

 

 

  INITIAL WOUND                               4th DAY POST WOUNDING

 

RESULT AND DISCUSSION:

Phytochemical investigation of different extract showed the presence of alkaloids, and tannins in Ageratum sample only. Also no sample shows the presence of proteins and amino acids. The details of qualitative chemical tests and phytoconstituents present in the extracts are shown in Table-I.

 

In all formulations there was no considerable change in characters like color, odor, and consistency and there was no phase separation observed during the course of study. Also, no patches on rat skin were observed during skin irritant test. No mortality was noticed amongst the animals in all the treated groups. The cases of wound infection were also negligible and of mild severity in the groups. There was noticeable homogeneity in the wound contraction observed for animals in the experimental groups compared with the control group. The end scar formed was a fine linear white scar that was visible on the flank of the animals.

 

The studies on excision wound healing model reveals that all groups showed decreased wound area from day to day. However, on 16th post wounding day, Group-I animals showed 75.14% of healing (which may be due to self immunity of the animals) where as Group-II (Standard) treated animals showed 100.00% healing. On the other hand, the Ageratum treated group showed 90.90% of wound healing. Also, all readings are found to be statistically significant and comparable with control. The epithelization time i.e. time at which complete scar formation occur, also suggest that both ageratum treated group and were found to be significant and comparable with control (Table-II).

 

On the basis of the results obtained in the present investigation, it is possible to conclude that the ointment of the leaf extract of Ageratum conyzoides has significant wound healing activity. The above findings justify the wound healing properties of the leaves of ageratum as suggested in the traditional literatures was comparable with control. It may be attributed to antimicrobial and haemostatic action of ageratum.

 

12th DAY POST WOUNDING           16th DAY POST WOUNDING

 

The wound healing property of Ageratum conyzoides appears to be due to the presence of its active principles, which accelerates the healing process and confers breaking strength to the healed wound. Several phytoconstituents like alkaloids, saponins etc. 10 are known to promote wound healing process due to their antioxidant and antimicrobial activities. The study reveals that ageratum treated group shows good wound healing properties due to presence of phytoconstituents like alkaloids, saponins and terpenoids present in it. Further investigations are necessary to determine the bioactive constituents present in the extracts used for studies.

 

ACKNOWLEDGEMENT:

We are thankful to Dr. A. K. Pathak, Dean and Head of Department of Pharmacy, Barkatullah Vishwavidyalaya, Bhopal for authentication of plant, providing valuable advices for completing research and allowing us to carry out the present work in his Laboratory.

 

Reference:

1.       Ramzi, S. C., Vinay, K. and Stanley. R. “Pathologic Basis of Diseases”, WB Saunders Company, Philadelphia, Vol. V, 1994, 86.

2.       Stuart Enoch, Patricia Price, “Pathophysiology of Healing”, 1st Ed., 2004, 234. (2)

3.       Kurian, J. C., “Plants that Heal”, Oriental Watchman Publishing House, Pune, 1995, 296.

4.       Anonymous, “The wealth of India, A dictionary of Indian Raw Materials and Industrial products”, Vol.-I, council of scientific and industrial research, New Delhi, 1995, 286-295.

5.       Kritikar and Basu, “Indian medicinal plants”, International book distribution, Dehradune, vol. II, 1996, 1330-31.

6.       Rastogi and Mehrotra, “Compendium of Indian Medicinal Plants”, CDRI, Lakhnow and NSIC, Delhi, 1990, vol. I, 188.

7.       Prajapati, Purohit, Sharma and Kumar, “A handbook of medicinal plants”, Agrobis India publication, 2003, sec.-II, 22.

8.       Ansel’s, “Pharmaceutical Dosage Form and Drug Delivery System”, Lippincort, 8th Ed., 2005, 278-281.

9.       Mukherjee, P. K., “Quality Control of Herbal Drugs”, Business Horizons, New Delhi, 1st Ed., 2002, 546-549.

10.     Marjorie M. C, Clinical Microbiology Reviews, Plant Products as Antimicrobial Agents, 1992: 12 (4): 564.

 

 

Received on 03.10.2009

Accepted on 01.11.2009     

© A & V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(3): Nov. – Dec. 2009, 217-219