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1 ISSN: X CODEN: IJPTFI Available Online through Research Article PHYTOCHEMICAL EVALUATION & PHARMACOLOGICAL SCREENING OF WOUND HEALING & ANTIOXIDANT ACTIVITY OF PLUMBAGO ZEYLANICA V. Asha Jyothi * & Butool Fathima Shadan Womens College of Pharmacy, Khiratabad, Hyderabad & Department of Pharmacology ashajyothivadlapudi@gmail.com Received on Accepted on Abstract: Traditional medicine in India with thousands of flora in it has not yet been explored. Many such medicines need scientific evaluation and evidence based research to be conducted on those plants for them to become medicines of great potential, one of such plants is Plumbago zeylanica. A wound is defined as a break in the epithelial integrity of skin. Wound is defined simply as the disruption of the cellular and anatomic continuity of a tissue that may be produced by physical, chemical, thermal, microbial or immunological insult to the tissue. In the present study the wound healing activity of the plumbago zeylanica is studied in the incision woung model on Sprague dwaley rats. The tensile strength of the wound was measured by the tensiometer. The results in comparison of untreated sham operated group with the standard and the test the asymptomatic significance is displayed and the significance level is p<0.05. Key words: Wound healing, Skin, Plumbago zeylanica. Introduction: A wound is defined as a break in the epithelial integrity of skin. Wound is defined simply as the disruption of the cellular and anatomic continuity of a tissue that may be produced by physical, chemical, thermal, microbial or immunological insult to the tissue (Robson et al., 2001; Raina et al., 2008).Wound healing process begins with the restoration of a damaged tissue as closely as possible to its natural state and wound contraction is the course of shrinkage in wounded area. The healing primarily depends on the repairing ability of the tissue in addition to the type and degree of damage and general health status of the tissue (Robbins and Cotran, Pathologic basis of disease, 8 th edition, 2009). IJPT Nov-2013 Vol. 5 Issue No Page 5879

2 Materials: Plant: Plumbago zeylanica plant was collected commercially from cultivator. The plant was chopped and dried in shade. It was the subjected to size reduction by mechanical grinder. The powdered material was subjected to soxhlet extraction with ethanol; the extract obtained was dried and used for preclinical studies. Chemicals: All the chemicals: Ethanol (China-Changshu Yangyuan Chemical), Ketamine (Sigma Aldrich), Xylazine (Sigma Aldrich), Atropine sulphate (Sigma Aldrich), Neosporin (Glaxo Smith Kline), Clobeta GM cream- Laborate (Clobetasol propionate, Gentamicin, Miconazole nitrate & Zinc oxide with borax cream) used for the study were of analytical grade. Animals: Twenty four (24) albino Male Sprague Dawley rats weighing between gms were used for the study. They were kept in polypropylene cages and allowed to acclimatize to the environment for two weeks before the commencement of the experiment. The animals were fed with standard animal pellet grower mash and allowed access to water ad libitum. Methodology: Incision Wound Model: Adult male Wistar or Sprague-Dawley strain weighing g were used. They were divided into four groups of six each. After fasting overnight, rats were anaesthesized with Ketamine (70mg/kg, IP) or Xylazine (5mg/kg, IP) and Atropine sulphate (0.2 ml, SC). Under anaesthesia the dorsal skin was shaved using electronic clipper. A 2cm long incision wound was made through the skin and cutaneous muscles in the dorso-lumbar region with the help of sharp scalpel (Ehrlich and Hunt, 1969). After the incision was made, the parted skin was kept together and stitched with black silk at 0.5 cm intervals; surgical threads (No.2) and a curved needle (No.11) were used for stitching. The wounds were left undressed (Lee K.H., 1968). Extracts were topically applied to the wound once a day. The animals were placed in individual cages for recovery with free access to food and water. The experimental room was maintained between 25 C and 28 C with natural light and dark cycles. When wounds were cured thoroughly the sutures were removed on the day before the tensile strength was tested i.e. on 8 th day and formulations were continued to be applied regularly. For the measurement of wound tensile strength, the rats were anaesthetised on days 9 th after surgery. Regular observation of IJPT Nov-2013 Vol. 5 Issue No Page 5880

3 healing patterns are to be done from day 1-10 and parameters to measure wound healing are tensile strength, percentage wound contraction and period of epithelialisation. Measurement of Tensile strength: Two stands were arranged facing opposite to each other. Thread was attached to both the stands. Stand L was kept stationery. Artery forceps was attached to stand L. Stand R was allowed to move and artery forceps was attached to it with the help of thread. On the other side of the thread a cup was hanged in which weights were placed accordingly. The arrangement was setup as shown in Fig Incision Wound Model Adult male Sprague-Dawley strain rats weighing gm. They are divided into four groups of six each. After fasting overnight. Rats are anaesthesized with ketamine (70mg/kg, IP) or xylazine(5mg/kg, IM) and atropine (0.2 ml, SC). Under anesthesia the dorsal skin is shaved using electronic clipper. A 2cm long incision wound is made through the skin and cutaneous muscles in the dorso-lumbar region with the help of sharp scalpel. After the incision was made, the parted skin was kept together and stitched with black silk at 0.5 cm intervals; surgical threads (No.2) and a curved needle (No.11) were used for stitching. The wounds are treated according to specific group. Then rats are treated with formulations beginning on the day of surgery (day 0) till day 10. during this time the healing pattern of the wounds is noticed. The groups were divided into: Fig-1: Incision wound model for the wound healing activity. IJPT Nov-2013 Vol. 5 Issue No Page 5881

4 a. Group 1: Blank the animals with no wound and no vehicle. Fig-2: Healthy animal b. Group 2: Control group the animals with incision wound and liquid paraffin. Fig-3: Untreated animal c. Group 3: Standard group the animals with incision wound and standard drug (Neosporin in Clobeta GM cream). Fig-4: Treated animal d. Group 4: Test group animals with incision wound and ethanolic extract of Plumbago zeylanica. IJPT Nov-2013 Vol. 5 Issue No Page 5882

5 Fig 5: Extract treated animal The animals are carefully observed from day 0. They are placed in individual cages for recovery and should have free access to food and water. The experimental room is maintained between 25 C and 28 C with natural light and dark cycles. When wound were cured thoroughly the sutures were removed on the day before the tensile strength is tested i.e. on 8 th day and formulations were continued to be applied regularly. For the measurement of wound tensile strength, the rats are anesthetised on days 9 after surgery. Regular observation of healing patterns was done from day 1-10 and parameters to measure wound healing are tensile strength, percentage wound contraction and period of epithelialisation. Measurement of Tensile Strength: Both the artery forceps were clipped to the either sides of the incision wound. Weights were added in the cup until wound dehiscence takes place. These weights determine the wound breaking strength. Fig 6: Dehiscence of wound during the tensile strength measurement using tension meter. IJPT Nov-2013 Vol. 5 Issue No Page 5883

6 Results: Yield Calculation Calculate the % dry weight as follows: % yield of the extract was found to be 96.4% Phytochemical Evaluation: % h = The preliminary phytochemical investigation of extract of Plumbago zeylanica shows presence of Carbohydrate, Tannin, Alkaloid, Saponins and Flavonoids in ethanolic extract. The presence of Carbohydrates was confirmed by Molisch s test, Fehling s test & Barfoed s test.while, Alkaloids by Mayer s test, Wagner s test, & Dragendroff s test.the presence of Flavonoid was confirmed by Shinoda test & Lead acetate test. While of Glycoside by Borntrager s test; Phenolics & Tannins by Bromine water test; Saponins by Foam test (reported by Khandelwal). As, ethanolic extract shows presence of most of these compounds, its extract were selected for these study. Table-1: Phytochemical Investigation of Extract of Plumbago zeylanica. S.NO. NAME OF COMPOUNDS ETHANOLIC EXTRACT OF PLUMBAGO ZEYLANICA 1. Alkaloids Carbohydrates Glycosides + 4. Saponins + 5. Fat and Oils + 6. Flavonoids Tannins Steroids ++ + = slightly present; ++ = moderately present; +++ =highly present; - = absent IJPT Nov-2013 Vol. 5 Issue No Page 5884

7 Antioxidant Activity Table-2: Standardisation and % Inhibition of Ascorbic Acid. Table-3: Measurement of anti-oxidant activity. S. No. Volu me (µl) Conce ntrati on (mg) S.NO CONC (µg) MEAN ± S.D %INHIBITION ± ± ± ± ± Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 Abs 6 Mean Std. Dev Std Err % Abs % Inhibi tion IC 50 Value of The Standard Ascorbic Acid is 1.2µg/ml. %INHIBITION %INHIBITION ± ± ± ± ± Graph 1: % Inhibition of Ascorbic acid IJPT Nov-2013 Vol. 5 Issue No Page 5885

8 HEALING PATTERNS: CONTROL STANDARD TEST DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 IJPT Nov-2013 Vol. 5 Issue No Page 5886

9 DAY 7 DAY 8 DAY 9 DAY 10 Fig 7: Healing patterns of Untreated, Standard & Test groups from Day Table 4: Measurement of Tensile Strength. S.NO UNTREATED (gm) STANDARD (gm) TEST (gm) IJPT Nov-2013 Vol. 5 Issue No Page 5887

10 TEST (gm) STANDARD (gm) UNTREATED (gm) Graph 2: Comparison of Tensile Strength of Test, Standard & Untreated groups. Table 5: SIGNIFICANT DIFFERENCES BETWEEN ALL THE GROUPS GROUPS N MEAN STANDARD DEVIATION STANDARD ERROR UNTREATED STANDARD TEST TOTAL No tearing of wound was observed above 350gms. Independent samples of distribution of tensile strength across the group using Kruskal-Wallis test was found to be and rejected the Null hypothesis. Asymptomatic significance is displayed. The significance level is Discussion: The ethanolic extracts of the roots of Plumbago zeylanica belonging to the family Plumbaginaceae, demonstrated the highest wound healing potential in experimental rats. The plant was selected based upon less amount of work done to substantiate its effects. The phytochemical results reveal the presence of tannins, alkaloids, reducing sugars and steroids in the ethanolic root extract. The constituents of the root extract, such as terpenoids and alkaloids, may play a major role in the wound healing process observed in this study. Studies have shown that flavonoids promote the wound-healing process mainly due to their astringent and antimicrobial properties which appear to be responsible for the wound healing. The wound healing IJPT Nov-2013 Vol. 5 Issue No Page 5888

11 effect of ethanolic root extracts of Plumbago zeylanica may be due to the presence of more than one active principle mentioned above. Recent studies provided strong evidence for a role of oxidative stress in the pathogenesis of non-healing ulcers. The normal physiology of wound healing depends on low levels of reactive oxygen species and oxidative stress, an overexposure to oxidative stress leads to impaired wound healing. Antioxidants are postulated to help control wound oxidative stress and thereby accelerate wound healing. The mechanism of action of the extract may be attributed to the excellent DPPH antioxidant activity which may be due to the presence of tannins, flavonoids in the extract. In the present study, wound healing potential of this plant was evaluated by creating incision wounds in rats. This method is advantageous as these wounds are less painful, heals faster with less noticeable scar. The animals are maintained in individual cages to minimize the risk of infection. The measurements of the progress of the wound healing induced by the Neosporin in Clobate GM cream, Ethanolic extract and the control group were reported. Wound healing progression was observed for a period of 10 days during which extract showed significant closure of wounds when compared with the standard drug (Neosporin in Clobetasol GM cream) and control group. It was observed that the wound contracting ability of the extract were significantly greater than that of the control, which was comparable to that of the reference standard. A better healing pattern with complete wound closure was observed in rat using test extract as shown. There were significant differences between groups in the reduction of weight. The average weight of animal in all groups increased after the treatment period. Although, hair growth was very significant in Plumbago zeylanica extract treated group since day 4 and reference standard group when compared to control. The neurological profile of all animals was observed. It was found that control group showed very strong aggressive behavior throughout the experimentation period, whereas extract treated animals showed moderate aggressiveness when compared to standard drug treated animals. In the incision wound studies, there was very excellent increase in tensile strength of the 10 day old wound due to treatment with Plumbago zeylanica extract when compared with the reference standard Neosporin in Clobate GM cream IJPT Nov-2013 Vol. 5 Issue No Page 5889

12 when compared with the respective control. The above observation for tensile strength indicates the effect of Plumbago zeylanica root extract in maturation of collagen fibres. This shows wound healing potential of tested extract. Conclusion: The aim of the present study was to investigate the wound healing potential of ethanolic extract of roots of Plumbago zeylanica by using incision wound model in Sprague-dawley rats. The wound healing activity was conducted to evaluate the unexplored herbal plant material to provide a scientific evidence of their claimed pharmacological properties. The wound healing activity of the plant may be due to phytochemicals like Flavonoids, Tannins, Alkaloids, Carbohydrates and Saponins. One of these compounds may be responsible for this activity. Incision wound model is one of the simulatory modules of post operative care which replicates the true clinical conditions. This method is highly reliable, versatile and effective. The ethanolic extract has shown a greatly significant result when compared to control and standard. Wound healing properties were remarkable and highly reproducible. The tensile strength was nearly equal to healthy skin and also in comparison with the standard and control. There was a significant increase in tensile strength, and decrease in epithelization period in extract-treated group as compared to control and standard drug-treated groups. The results were significant. The study is concluded to confirm their wound healing potential and create a proof for further study. References: 1. Ann Med. Wound pathophysiology, infection and therapeutic options, 2002; 34(6): Attinger CE, Janis JE, Steinberg J, Schwartz J, Al-Attar A, Couch K. Clinical approach to wounds: débridement and wound bed preparation including the use of dressings and wound-healing adjuvants. Plast Reconstr Surg Jun; 117(7 Suppl):72S-109S. 3. Ashok Purohit, Surendra Kumar Vyas, and Keshav Bihari Vyas, Contraceptive efficacy of Plumbago zeylanica root extract (50% etoh) in male albino rats with special emphasis on testicular cell population dynamics, Anc Sci Life Jan-Mar; 27(3): Alleva R, Nasole E, Di Donato F, Borghi B, Neuzil J, Tomasetti M, alpha-lipoic acid supplementation inhibits oxidative damage, accelerating chronic wound healing in patients undergoing hyperbaric oxygen therapy, Biochem Biophys Res Commun Jul 29; 333(2): IJPT Nov-2013 Vol. 5 Issue No Page 5890

13 5. Bopaiah CP, Pradhan N. Central nervous system stimulatory action from the root extract of Plumbago zeylanica in rats. Phytother. Res., 2001, 15: Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik; Bartek, Jiri (2003). "Cell-cycle regulatory proteins in human wound healing". Archives of Oral Biology 48 (2): Dai Y, Hou LF, Chan YP, Cheng L, But PP. Inhibition of immediate allergic reactions by ethanol extract from Plumbago zeylanica stems. Biol Pharm Bull, 2004; 27(3): Dissemond J, Goos M, Wagner SN, The role of oxidative stress in the pathogenesis and therapy of chronic wounds, Hautarzt Nov; 53(11): Edwin S, Joshi SB, Jain DC.Antifertility activity of leaves of Plumbago zeylanica L. in female albino rats. Eur J Contracept Reprod Health Care, 2009; 14(3): Ehrlich, H.P. and H.K. Hunt, Effect of anabolic steroid on tensile strength of a healing wound. Ann Surg, 170: Greenhalgh, D.G. (1998). "The role of apoptosis in wound healing". The International Journal of Biochemistry & Cell Biology 30 (9): Govindarajan.R, Vijaykumar.M, Kumar.B, pushpangadan.p, Ethnopharmocological approaches to wound healing- Exploring medicinal plants of India. 13. Hiradeve S, Danao K, Kharabe V, Mendhe B.Evaluation of anticancer activity of Plumbago zeylanica Linn leaf extract. IntJ Biomed Res, 2010; 1(2):1-9. Corresponding Author: V. Asha Jyothi *, ashajyothivadlapudi@gmail.com IJPT Nov-2013 Vol. 5 Issue No Page 5891