Medicinal plants are considered as rich resources of ingredients which can be used in drug development and synthesis of medicines. Plants play a vital role in the development of human cultures around the whole world. Medicinal plants have a promising future because there are about half million plants around the world, and most of them their medical activities have not investigate yet, and their medical activities could be decisive in the treatment of future studies. However, the blind dependence on synthetics is over and people are returning to the naturals with hope of safety and security. Over three-quarters of the world population relies mainly on plants and plant extracts for health care. More than 30% of the entire plant species, at one time or other was used for medicinal purposes [1]. Wound healing is a complex and dynamic process, with the wound environment changing with the shifting health status of an individual. Knowledge of the physiology of the normal wound healing trajectory through the phases of hemostasis, inflammation, granulation and maturation provides a framework for understanding the basic principles of wound healing. Through this understanding, the health care professional can develop the skills required to care for a wound and the patient can be helped with the complex task of tissue repair [2]. Wound healing in mammalian tissues is a natural process of restoring the cellular structures and tissue layers, comprising three distinct phases: the inflammatory phase, the proliferation phase, and the remodeling phase; the main events being chemo-taxis, phagocytosis, neocollagenesis, collagen deposition, angiogenesis and re-epithelialization. These phases and their bio physiological functions must occur at the right time in proper sequence and continue for a specific duration at an optimal intensity. However, angiogenesis and re-epithelialization play a major role in the successful completion of the process. In spite of the fact that many mammalian animal models have been widely used in wound healing studies, several limitations for routine use of such models render them non-viable. Most important being, ethical constraints, cost and time consumption for preliminary screening and quantitative assessments of the crude extracts and phytoconstituents. Also, the molecular mechanism behind wound healing process is difficult to explore due to the lack of proper models. These issues can be overcome by using models like chick embryo, zebra fish, and drosophila, among others which serve as alternatives to the animal models. In the present study chick embryo has been used as a model to screen the wound healing potential of Dipteracanthus patulus leaf extracts [3, 4].

MATERIAL AND METHODS

Collection and authentication of plant and the plant parts:

The plant materials used in this study were leaves of Dipteracanthus patulus  (Jacq). (Family - Acanthaceae) is collected from the sengundrapuram, (Virudhunagar Dist, Tamil Nadu, India.). The plant was authenticated by Dr.Stephen, Department of  Botany, American College, Madurai, India.

Preparation extracts:          

D.patulus leaf collected was dried at room temperature under shade 15 days and coarsely powdered. The powdered materials were extracted with ethanol and chloroform. The last traces of the solvent were removed and concentrated to dryness under vacuum using a rotary evaporator. The dried extract was weighed and then kept at -4ºC until ready for use. The yield of the extract was 66.42 % (w/w) and 33.68 %  (w/w). In each experiment, the extract was diluted with water to desired concentration [5].

Preliminary Phytochemical Screening

 D.patulus leaves extracts were subjected to qualitative testing to determine the presence of various plant bioactive ingredients such as alkaloids, flavonoids, tannins, saponins, polysaccharides, glycosides, and other compounds by using the following standard methods [6, 7].

Embryo collection

Fertilized white shell eggs were purchased from Tamilnadu Veterinary College And Research Institute, Tirunelveli. The outer surface of the embryos were cleaned with 75% ethanol and incubated at 37°C throughout the study.

Preparation of saturated filter disk for wound assay [8]

Whatman No.1 filter paper was Small disks were generated using a standard 5 mm whole puncher, sterilized by auto-claving and stored for further use. The pre-sterilized filter disks were saturated with add D.patulus extracts, from 500 ?g/ml,  Diclofenac sodium 50 ?g/ml and sterile saline were used as positive and negative controls respectively. Embryonated chicken eggs (9 days old) were selected then divided into four groups and a small window, (1cm2) was made in the shell follows

GROUP I        :           Negative Control saline.

GROUP II       :           Positive Control Diclofenac sodium (50 ?g/ml)

GROUP III      :           Ethanolic extract of D.patulus. (500 ?g/ml)

GROUP IV     :           Chloroform extract of D.patulus. (500 ?g/ml)

Wound assay

Egg shell window was closed and incubated at 370 C for 72 hrs. All dissection tools used in the assay were sterilized using 75% ethanol before use. The embryos were incubated for  to allow good maturation of the chorioallantoic membrane.The window was then opened and the growth of new capillary blood vessels were observed and finally compared with the positive and negative control.

RESULTS AND DISCUSSION

Preliminary phytochemical screening of the ethanolic and chloroform extract of D.patulus (Jacq).

Chorioallantoic membrane assay model (CAM)

The result was tabulated by counting the number of blood vessels in various treatments. Ethanolic and chloroform extract, positive control promoted an increase in number of blood vessels compared to negative control saline. Treated 500 ?g/ml Ethanolic and chloroform extract group and 50 ?g/ml Diclofenac sodium positive control treated group was observed formation of new blood vessels.(Fig A, B & C)  The negative control does not show the formation of blood vessels. (Fig D)

A) Treated 500 ?g/ml of Ethanolic extract of D.patulus.

B) Treated 500 ?g/ml of Chloroform extract of D.patulus.

C) Positive Control Diclofenac sodium-50 ?g/ml.

D) Negative Control saline.