Wound healing activity of Solanum xanthocarpum Schrad. & Wendl. fruits

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1 Indian Journal of Natural Products and Resources Vol. 1 (4), December 2010, pp Wound healing activity of Solanum xanthocarpum Schrad. & Wendl. fruits Neeraj Kumar 1*, Dhan Prakash 2 and Pankaj Kumar 3 * 1 Department of Pharmacy, Sri Ram Murti Smarak College of Engineering and Technology, Bareilly , Uttar Pradesh, India 2 Amity Institute of Herbal Research & Studies, Amity University, Sector-125, Gautam Budh Nagar , Uttar Pradesh 3 Faculty of Pharmacy, Moradabad Educational Trust Group of Institutions, Moradabad , Uttar Pradesh Received 9 February 2009; Accepted 9 November 2010 Solanum xanthocarpum Schrad. & Wendl. (Family-Solanaceae), commonly known as Yellow-Berried Nightshade, fruits are used traditionally in the folklore for treatment of various kinds of wounds. The present study was undertaken to verify the effects of its fruits on experimentally induced excision and incision wound models in Sprague Dowlay rats. Aloe vera Linn. was used as standard wound healing agent. The methanolic fruit extract was formulated at 10% w/w in white petroleum jelly. In excision wound model, compared to the control, per cent wound healing was significantly higher (51.2%, 36% and 30% at 4 th, 8 th and 12 th day of wound creation) in S. xanthocarpum treated group. In incision wound model, tensile strength of the healing tissue after treatment was also found to be significantly higher (37.5%) as compared to the control. The results suggested that methanolic fruit extract of S. xanthocarpum possess significant wound healing potential. Keywords: Solanum xanthocarpum, Solanaceae, Yellow-Berried Nightshade, Wound healing, Wound contraction, Incision wound, Excision wound, Tensile strength. IPC code; Int. cl. 8 A61K 36/00 Introduction Wound is a breach in the normal tissue continuum resulting in a variety of cellular and molecular sequelae 1. Wound healing is body's natural process of regenerating dermal and epidermal tissues 2. The healing cascade is activated when platelets come in contact with exposed collagen leading to platelet aggregation and release of clotting factors resulting into contact with exposed collagen. It leads to platelet aggregation and release of clotting factors resulting in deposition of a fibrin clot at the site of injury 3. Natural products are still the primary and most preferable health care system in some parts of the world and described by traditional herbal medicine practitioners 4-6. Wound healing is an important area of clinical medicine explained in most of the Ayurvedic texts since about 5000 BC under the heading Vranaropaka 4. The wound as a medical problem was discussed by Maharishi Agnibesha in Agnibesha samhita (latter known as Charak Samhita ) as verna and Maharshi Sushruta in Sushruta samhita also elaborated on this subject 7. *Correspondent author neerajsitm@yahoo.com Phone: (Mob.); Fax: S. xanthocarpum is a very prickly perennial herb with local names Kantkari, Kateli and Yellow-Berried Nightshade and found in Southeast Asia, Malaya and tropical Australia. It is very commonly found throughout India in plains from seashore to hills up to 1000 m of height. In literature it is some times dealt with as a synonym of Solanum surattense Burm.f. It is used in Dhasamoolaristha and in combination with other expectorants, demulcents and aromatics, astringent and heart disease. Fumigation with the vapour of the burning seeds of this plant was in high repute to cure toothache and when smoked like tobacco, the natives believed that smoke killed the insects, which they assumed caused the pain. In Bengal the plant was used as a diuretic in dropsy and root paste was utilized by the Mukundara tribals of Rajasthan for the treatment of hernia 8. Steroidal alkaloid solasodine, present in fruits and the glycol-alkaloid content of fruits collected from plants growing in Jammu & Kashmir were reported to be 3.5% (total alkaloids: 1.1%) and plant samples collected from Kolkata contained solasodine of %. Seeds yield 19.3% of greenish yellow, semi-drying oil with a characteristic odour. The unsaponifiable matters of fruits contain two sterols, one of which is carpesterol.

2 NEERAJ KUMAR et al: WOUND HEALING ACTIVITY OF SOLANUM XANTHOCARPUM FRUITS 471 Stem, flowers and fruits are bitter and carminative, employed as anthelmintic, in indigestion, cough, asthma and pains in chest, being used in the form of a decoction and also prescribed for relief in burning sensation in the feet accompanied by vesicular watery eruptions 9. It has high concentration of solasodine, a starting material for the manufacture of cortisone and sex hormones and scientifically reported as antifungal, antispermatogenic, antiandrogenic, antinociceptive and hypoglycemic The present study was undertaken to verify the effects of its fruits on experimentally induced excision and incision wound models in Sprague Dowlay rats and its efficacy was compared with Aloe vera Linn. which is considered to be a good wound healing agent. Materials and Methods Collection and identification of the plant The fruits of S. xanthocarpum used in present studies were obtained from the local market of Bareilly, Uttar Pradesh and were identified on the basis of its physical and morphological characteristics and the voucher specimen (Srmscet/Pharma/09/087) has been deposited in the department of Pharmacognosy, SRMS College of Engineering and Technology, Bareilly. The fruits were crushed to small pieces using mortar and pestle and powdered in an electric grinder. Experimental animals Healthy inbreed Sprague-Dowlay rats of either sex weighing g were procured from Indian Veterinary Research Institute, Bareilly. The experimental protocol was approved by Institutional Animal Ethical Committee for the purpose of control and supervision of experiments on animals (715/02/c/CPCSEA). The rats were anaesthetized prior to and during inflection of the experimental wounds. The surgical interventions were carried out under sterile conditions using Ketamine anaesthesia (80 mg/kg body weight). Animals were closely observed for any infection and those which show signs of infection were separated and excluded from the study. After inflection of wounds, 6 animals each were randomly distributed into 3 groups (n=6) according to their similarity in weight. Group1=control-treated with white petroleum jelly only, Group2 = experimentaltreated with 10% (w/w) S. xanthocarpum extract in white petroleum jelly and Gruop3 = standard- 10% (w/w) Aloe vera cream. Chemicals Aloe vera Cream (Glenmark Pharm. Ltd., Himachal Pradesh, composition: Aloe extract 10% w/w and Vitamin E, 0.5% w/w), Ketamine HCl (Themis Medicare Ltd., Mumbai), White Petroleum Jelly [Central Drug House (P) Ltd., New Delhi]. Extraction of S. xanthocarpum fruits The coarsely powdered fruits of S. xanthocarpum were extracted with methanol using hot Soxhlet method. The extract was concentrated by using vacuum rotary evaporator (Buchi type) at 45 o C and finally dried in vacuum desiccators (yield is 4.6%) then 2.5 g of dried extract was mixed with 25 g of white petroleum jelly base (10% w/w). Acute dermal toxicity The acute dermal toxicity was performed according to the OECD Guidelines (OECD 410), a limit test of 2000 mg/kg did not show any sign of lethality or moribund state and the gross behaviour of the animals was normal and no signs of dermal toxicity were observed. Creation of excision wound and wound area measurement The animals were anaesthetized. An impression was made on the dorsal thoracic region 1 cm away from ear. The particular skin area was shaved one day prior to the experiment. The skin of impressed area was excised to the full thickness to obtain a wound area 14 of about 500 mm 2. Wound area was measured by tracing the wound on a millimeter scale graph paper. The percentage of wound contraction was calculated of original wound size (500 mm 2 ) for each animal on 4, 8 and 12 th days of post wounding for final analysis of results 15 (Plate 1). Creation of incision wound and tensile strength measurement A 5 cm long abdominal incision was made in shaved area of anaesthetized rat and closed with interrupted sutures (Mersilk, Ethicon Aurangabad) at a distance of 1 cm. These were housed individually in different cages 16. On 12 th day of post wounding, the animals were sacrificed by cervical dislocation and wound areas from each animal were removed carefully. Wound stripes of equal size (width) were cut and both ends of each strip were fixed with a pair of steel clip, one clip was allowed hanging on a stand and other clip with a polyethylene bag. It was then gradually filled with water till the wound strip was broken at the site of wound. The amount of water required to break the wound was noted and expressed as tensile strength of wound in grams 17 (Plate 2).

3 472 INDIAN J NAT PROD RESOUR, DECEMBER 2010 Plate 1 Excision wound healing: A. Excision wound on first day; B. Excision wound on 4 th day; C. Excision wound on 8 th day; D. Excision wound on 12 th day Plate 2 Incision wound healing: A. Incision wound on first day; B. Incision wound on 4 th day; C. Incision wound 8 th day; D. Incision wound on 12 th day

4 NEERAJ KUMAR et al: WOUND HEALING ACTIVITY OF SOLANUM XANTHOCARPUM FRUITS 473 Results and Discussion The methanolic extract of fruits of S. xanthocarpum (10% w/w) ointment did not show any signs of lethality or moribund state and gross behavior of the animals was normal without any signs of dermal toxicity, when performed a limit test of 2000 mg/kg (OECD guidelines 410). The methanolic extract of fruits (10% w/w) ointment had produced significant (P<0.01) increase in mean % wound contraction (104, 58 and 43%) after 4 th, 8 th and 12 th day of treatment. Similarly a significant (P<0.01) increase in tensile strength (60%) on 12 th day was observed, as compared with control (Plate 1 & 2). The wound contraction and tensile strength of experimental ointment under test were in close proximity with control Aloe vera 10% cream (Fig. 1 & 2). Wound healing is the process of repair that follows injury to the skin and other soft tissues. After injury, an inflammatory response occurs and the cells below the dermis begin to increase collagen production and latter, the epithelial tissue is regenerated 4. Wound contraction is a process that occurs through out the healing process, commencing in the fibroblastic stage whereby the area of the wound undergoes shrinkage 18. The present study was undertaken to evaluate whether fruit extract of this plant could promote wound healing in experimentally produced wounds in Fig. 1 Per cent wound contraction in excision wound model Fig. 2 Tensile strength (g) in incision wound model

5 474 INDIAN J NAT PROD RESOUR, DECEMBER 2010 Table 1 Effect of topical application of 10% w/w methanolic extract of fruits of S. xanthocarpum on wound healing in rats Groups % wound contraction in excision wound model Tensile strength (g) in 4 th day 8 th day 12 th day incision wound model Control (white petroleum jelly) 21.08± ± ± ±6.35 S. xanthocarpum (10% w/w ointment) 43.02±1.24* 59.3±1.19* 88.01±2.02* ±5.12* Aloe vera (10% w/w cream) 44.23±1.28* 58.63±2.07* 87.56±1.23* ±7.19* All values are mean ± SD, *P<0.01 rats. The results substantiated its use in folklore medicine for the treatment of wounds. The extract applied topically increased the tensile strength (37.5%) and wound contraction (30%) in different models after 12 days as compared to control and was in close proximity to standard cream (Table 1). It might be either due to enhanced contractile property of myofibroblasts or increased number of myofibroblast recruited in the wound area. In excision wound model it enhanced the process of epithilialization significantly (P<0.01). It appears that S. xanthocarpum was able to promote epithilialization either by facilitating the proliferation of epithelial cells or by increasing the viability of epithelial cell. The faster wound contraction rate from the S. xanthocarpum treatment may be due to antioxidant, antimicrobial, and antiseptic properties of the extract and by stimulation of interleukin-8, an inflammatory α-chemokine that affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. It may increase the gap junctional intracellular communication in cultured fibroblasts and induce a more rapid maturation of granulation tissue 19. In the incision wound model, increase (37.5%) in tensile strength of treated wounds may be due to the increase in collagen formation per unit area and stabilization of the fibers 20. Tensile strength depends upon the Van der Waals force interaction among the hydrogen ion bonds of the triple helix collagen, leading to twisting of the collagen fibers. The more twisting of fibers that occurs, the greater the tensile strength and hence the better healing of wounds 21. Conclusion The methanolic extract of fruits of S. xanthocarpum applied topically, effectively increased (30%) the contraction of open wound, tensile strength (37.5%) after 12 days and significantly (P<0.01) enhanced wound healing process as compared to control. The extract did not show any dermal toxicity up to 2000 mg/kg, on topical application. Further, present investigation substantiated scientifically the use of this plant in Ayurveda for wound healing. References 1 Pieree GF and Mustoer TA, Pharmacologic enhancement of wound healing, Annu Rev Med, 1995, 46, Patil MB, Jalalpure SS and Ali Ashraf, Preliminary phytochemical investigation and wound healing activity of leaves of Argemone mexicana Linn., Indian Drugs, 2001, 38(6), Nayak BS, Investigation of wound healing activity of Lantana camara Linn. in Sprague Dowlay rats using a burn wound model, Int J Appl Res Nat Prod, 2008, 1(1), Biswas TK and Mukherjee B, Wound healing potential of Pterocarpus santalinus Linn; A Pharmacological Evaluation, Lower Extremity Wounds, 2004, 3(3), Falanga V, The chronic wound: Impaired wound healing and solution in the context of wound bed preparation, blood cells mole, Blood Cells Mol Dis, 2004, 32(1), Clark RA, Fibrin and wound healing, Ann N Y Acad Sci, 2001, 936, Singh A and Singh DK, Molluscicidal activity of Lawsonia inermis and binary tertiary combinations with other plant derived molluscicides, Indian Journal Exp Biol, 2001, 39, Ozgen U, Coskun M, Kazaz C and Secen H, Naphthoquinones from the roots of Onosma argentatum, Truk J Chem, 2004, 28, Nayak BS and Pinto PML, Catharanthus roseus flower extract has wound healing activity in Spraque Dowley Rats, BMC Comp Med, 2006, 6(41). 10 Ehrich HP and Hunk TK, Effect of cortisone and anabolic steroids on tensile strength of healing wound, Ann Surg, 1969, 170(2), Kumar N and Gupta AK, Wound-healing activity of Onosma hispidum (Ratanjot) in Normal and Diabetic Rats, J Herb Spices Med Plants, 2009, 15(4), Mother herbs and agro products, Solanum xanthocarpum, accessed on dated , url- com/solanum-xanthocarpum.html 13 Singh OM, Subharani K, Singh NI, Devi NB and Nevidita L, Isolation of steroidal glycosides from Solanum xanthocarpum and studies on their antifungal activities, Nat Prod Res, 2007, 21(7), Biswas TK and Mukherjee B, Plant medicines of Indian origin for wound healing activity: A review, Lower Extremity Wounds, 2003, 2(1), Shanbhag TV, Sharma C, Adiga S, Bairy LK, Shenoy S and Shenoy G, Wound healing activity of alcoholic extract of Kaempferia galanga in wistar rats, Ind J Physiol Pharmacol, 2006, 50(4),

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