WOUND HEALING ACTIVITY OF JATROPHA TANJORENSIS LEAVES

The term “wound” is defined as a disruption of normal anatomical structure. Therefore, “healing” is the complex and dynamic process that results in the restoration of anatomical continuity and function. Albino Wistar rats (150-180 g) of both sexes were selected. The experiment of Wound Healing Activity by Excision Wound Model and Incision Wound Model by the simple ointment B.P., reference standard drug (0.2% w/w nitrofurazone ointment), stigmasterol ointment (0.2% w/w), hexane, chloroform and methanol extract ointments of 3%, 4% and 5% w/w of leaves in Jatropha tanjorensis in simple ointment base (where 3g, 4g and 5 g of the extracts was incorporated in 100 g of simple ointment separately). The time for wound closure to methanol extract ointment (5% w/w) and stigmasterol (0.2% w/w) was similar to that of standard drug, nitrofurazone ointment (0.2% w/w) 16±2 days in Excision Wound Model. The significant tensile strength at 3%: 4%: 5% w/w methanol extract ointments (p<0.001), followed by chloroform extract and hexane extract. Stigmasterol ointment at 0.2% w/w produced tensile strength comparable with Standard drug, nitrofurazone ointment (0.2% w/w) (p<0.001) in Incision Wound Model. Juice of the Jatropha plant and the pounded leaves are applied to wounds and refractory ulcers. The juice is very successfully used to treat scabies, eczema and ringworm. The present study proved that the leaves have wound healing activity.


INTRODUCTION
The genus Jatropha belongs to tribe Joanneasiae of Crotonoideae in the Euphorbiaceae family and contains approximately 175 species, cultivated throughout the tropical to temperate regions of the world. The name is derived from the Greek words ἰατρός (iatros), meaning "physician," and τροφή (trophe), meaning "nutrition" as food. Homeopathically it is used for cold sweats, colic, collapse cramps, cyanosis, diarrhea, and leg cramps. The root, stem, leaves, fruit, seed, bark and latex of the plant are largely used for the treatment of many diseases in different parts of the world 1 . The juice of the plant and the pounded leaves are applied to wounds and refractory ulcers. The juice is very successfully used to treat scabies, eczema and ringworm 2 . Leaves are regarded as antiparasitic, applied to scabies, rubefacient for paralysis, rheumatism; also applied to hard tumors 3 . According to Ochse 4 , "The young leaves may be safely eaten, steamed or stewed". They are favored for cooking with goat meat, said to counteract the peculiar smell. It is reported to be abortifacient, anodyne, antiseptic, cicatrizant, depurative, diuretic, emetic, hemostatic, lactogogue, narcotic, purgative, rubefacient, styptic, vermifuge, and vulnerary 5, 6 . Latex is applied topically to bee and wasp stings 7 and to dress sores and ulcers and inflamed tongues 8 . Duke and Wain 6 list it for homicide, piscicide, and raticide as well. Colombians drink the leaf decoction for venereal disease 9 . The latex was strongly inhibitory to watermelon mosaic virus 10 . Jatropha tanjorensis 11 Shrubs, 3-4 m high; stem long, stout, dichotomously branched; branches puberulous when young, glabrous when mature. excised at the dorsal interscapular region. Rat wounds were left undressed to the open environment. This model was used to monitor wound contraction and epithelization time. The simple ointment B.P., reference standard drug (0.2% w/w nitrofurazone ointment), stigmasterol ointment (0.2% w/w), hexane, chloroform and methanol extract ointments of 3%, 4% and 5% w/w in simple ointment base (where 3g, 4g and 5 g of the extracts was incorporated in 100 g of simple ointment separately) were applied everyday till the wound was completely healed. The progressive changes in wound area were monitored planimetrically by tracing the wound margin on a graph paper every alternate day. The changes in healing of wound, i.e., the measurement of wound area on graph paper were expressed as unit (mm 2 ). Wound contraction was expressed as percentage reduction of original wound size 13, 14 .

Incision Wound Model
Twelve groups of 6 animals in each group were taken and anaesthetized under light ether anesthesia. The incision wound 13,14 with one full thickness paravertebral incision of 6 cm length was made including the cutaneous muscles of the depilated back of each rat. Full aseptic measures and no local or systemic antimicrobials were not used throughout the experiment. After incision, the parted skin was kept together and stitched with sutures, 1 cm apart. The continuous threads on both wound edges were tightened for good adaptation of wound and it was left undressed. The ointment of all the extracts 3%, 4% and 5% w/w, stigmasterol ointment (0.2% w/w), standard drug (nitrofurazone ointment) and simple ointment B.P. were applied to the wound twice daily, until complete recovery to the respective groups of animals 15, 16 .

Measurement of Healing
On the 9 th day after wounding, the sutures were removed and tensile strength was measured on 10 th day. For measuring the tensile strength, the rats were again anesthetized and each rat was placed on a stack of towels at the middle of the board. The amount of the towels could be adjusted in such a way so that the wound was at the same level as the tips of the arms. The clamps were then carefully clamped on the skin of the opposite edges of the wound. The longer pieces of the fishing line were placed on the pulley and finally to the polyethylene bottle. The position of board was adjusted to rate water from a large reservoir, until the wound began to open. The amount of water in the polyethylene bottle was weighed and equated as the tensile strength of the wound. The tensile strength induced by the extracts, stigmasterol ointment (0.2% w/w), and the nitrofurazone ointment-treated wounds was compared with Control.

DISCUSSION
The term "wound" is defined as a disruption of normal anatomical structure and more importantly function. Therefore, "healing" is the complex and dynamic process that results in the restoration of anatomical continuity and function 17 . Acute wounds normally heal in a very orderly and efficient manner characterized by four distinct but overlapping phases such as hemostasis, inflammation, proliferation and remodeling 18 . These steps are orchestrated in a controlled manner by a variety of bioactive molecules like growth factors, cytokines, their receptors and matrix molecules 19

Excision Wound Model
Nath and Dutta 21 isolated the protease namely curcain from the latex of J. curcas and reported its effective wound healing property by excision wound model in mice. Ointments of 0.5% and 1.0% (w/w) curcain were compared with 0.2% (w/w) nitrofurazone ointment and 0.15% (w/w) propamidine isethionate cream. Curcain ointments healed the wound completely within 10 days of treatment whereas the nitrofurazone ointment and propamidine isethionate cream took 15 and 20 days respectively to show the same results. Esimone et al., 22 tested ointments at 0.5 g/10 g, 1 g/10 g and 1.5 g/10 g of the methanol leaf extract in J. curcas, standard gentamycin and blank ointment on the excision wound in rats and their respective wound area on 21 day was 1.33±0.07, 0.2±0.03, 0.00±0.00, 0.00±0.00, and 3.90±0. 28 Figure 1). Treated excision wounds showed an increased rate of wound contraction, leading to faster healing as confirmed by the increased wound healing area compared to control.

Incision wound model
Tensile strength for control was 432±13.30 g (Table 2; Figure 2). The increased tensile strength was 591±14.60 g, 584±10.90 g, 535±10.10 g and 526±10.00 g for stigmasterol, methanol, chloroform and hexane extracts (5% w/w) respectively. While the standard ointment-treated group showed 597±14.80 g. At 4% and 3% w/w, the extracts showed decreased tensile strength such as 581±10.40 g and 578±10.20 g to methanol extract, 532±9.70 g and 528±9.50 g to chloroform extract and 521±9.80 g and 519±9.60 g to hexane extract. It is well-accepted that several local growth factors help in the wound healing process. It is possible that the test extracts may have a growth factorlike activity or have the ability to stimulate the expression of growth factors like the basic fibroblast growth factor (bFGF). The bFGF has the broadest range of target cells such as endothelial cells, fibroblasts, myoblasts, etc 23 . Wound contraction is mediated by specialized myofibroblasts found in the granulated tissue 24 . The increase in tensile strength of treated wounds may be due to increase in collagen concentration and stabilization of the fibers 13, 25 .

Excision and Incision Wound Models
Shetty et al., 26 recorded very effective property to accelerate wound healing process to the crude bark extract of J. curcas in Wistar albino rats. Odoh et al., 27 reported the wound healing property of methanol leaf extract in J. curcas by incision and excision wound models in rats. The wound healing effect at 200 mg/kg was compared to that of standard, cicatrin, and showed significant healing potential (P<0.05) in dosedependent manner for the increase in wound contraction rate and skin breaking strength and decrease in epithelization period. they are now proved to have adverse effects in the human body. In view of these developments, so much of attention has been paid recently to the extracts of biologically active compounds isolated from plant species used in herbal medicinal system 31 . Pro-inflammatory cytokines were implicated to stimulate the synthesis of platelet activating factors by the recruited monocytes which in turn induce several angiogenic factors and chemokines 32 . Moon et al., 33 reported the pronounced improvement of type-I collagen material invasion by β-sitosterol which acts as an angiogenic factor in wound healing. Angiogenesis is the growth of new vascular capillary channels from pre-existing vessels and is of fundamental importance in a number of physiological processes such as embryonic development, reproduction, wound healing and bone repair 34 .

Figure 2: Effect of various solvent extracts of leaves and stigmasterol ointment on incision wounds
When wounding occurs it is accompanied within quite a short time by pain, reddening and edema of the surrounding tissue. These are all classical symptoms of inflammation and are caused by the release of the eicosanoids, prostaglandins and leutkotrienes and of ROS. The release of other factors such as the cytokines is also important which is caused by neutrophils aggregating at the wound site and producing proteolytic enzymes and ROS as antimicrobial defenses and as aids to the debridement of dead tissue. As the test extracts of J. tanjorensis exhibited potent anti-inflammatory and antioxidant activities these properties could have contributed to wound healing in part. Apart from the role of antioxidants in removing products of inflammation, they are also beneficial in wound healing in some other means. Antioxidants counter the excess proteases and ROS often formed by neutrophils accumulation in the wound area and protect protease inhibitors from oxidative damage. Fibroblasts and other cells may be killed by excess ROS and skin lipids will be made less flexible. So, antioxidant substances will reduce the possibility of occurrence of these adverse events. Because of these factors, overall antioxidant effects appear to be important in the successful treatment of wounds 35 . In the present study, the test extracts were shown to be strongly antioxidant due to the presence of flavonoids and polyphenols. Open wounds are particularly prone to infection, especially by bacteria and superficial mycotic agents and also provide an entry point for systemic infections. Infected wounds heal less rapidly and also often result in the formation of unpleasant exudates and toxins that will be produced with concomitant killing of regenerating cells 35 . Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa are the most common wound pathogens with≥10 3 CFU/g tissues which were classified as infection 36 . The presence of saponins, flavonoids and other phenolics in the test extracts and their potent polyvalent activity due to their antiinflammatory, antioxidant and antimicrobial properties could have contributed to the wound healing because of their detergent ability to remove grease, dirt and bacteria from tissue and act as antimicrobials 337,38 . Methanol extract exhibited better activity than of all. Further, the wound healing property of stigmasterol isolated from methanol extract supports the present findings totally.

CONCLUSION Excision Wound Model
The time for wound closure to methanol extract ointment (5% w/w) and stigmasterol (0.2% w/w) is similar to that of nitrofurazone ointment (0.2% w/w) 16±2 days and 18±2 days to chloroform and hexane extract in a dose-dependent manner. Incision wound model Activity is in the order of stigmasterol, methanol, chloroform and hexane extracts (5% w/w) respectively.

Excision and Incision Wound Models
Test extracts exhibit potent anti-inflammatory and antioxidant activities due to the presence of flavonoids and polyphenols which could have contributed wound healing in part; and Activity against Candida albicans indicates healing capacity of superficial skin infection.