International Journal of Innovative Pharmaceutical Sciences and Research www.ijipsr.com A STUDY OF THE WOUND HEALING PROPERTIES OF SUDANESE Aerva javanica. IN DIABETIC RATS 1 Nada Mohammed Osman Abbas, 2 Yahia Mohamed El Imam, 3 Ghada M Ahmed 1,2 Faculty of Pharmacy, Khartoum College of Medical Sciences, Khartoum- 10995, SUDAN 3 Faculty of Science and Technology, Omdurman Islamic University, SUDAN Abstract The wound healing activity of 70% ethanolic extract of the whole air dried plant of Aerva javanica (Burm.f.) Juss. ex J.A. Schultes, was studied on streptozotocin-induced diabetic Wistar rats using an excision wound model. The healing activity was made by assessing the ability of the extract to heal the wounds once compared to the standard which in this case was tetracycline ointment (1%) and the control which received only petroleum jelly. The wounds were measured on days 0, 4, 8, 12 and 14, were on day 14 there was found a complete closure of the wounds of the animals receiving the extract. The animals treated with the extract exhibited 100% wound closure by the 14 th day unlike the standard and the control which have both shown an incomplete wound closure on that day owing to an 88 % and approximately 61 % closure on day 14 respectively. Upon comparison epithelialization was faster in the animals that were treated with the extract rather than both the animals those treated with the standard and those treated with the control. Aerva javanica showed that it promotes significant wound healing in diabetic rats this could be promising for further evaluation of this activity in humans is suggested. Keywords: Aerva javanica, diabetic, streptozotocin, excision wounds, wound healing. Corresponding Author Dr. Nada Mohammed Osman Abbas Faculty of Pharmacy Khartoum College of Medical Sciences, Khartoum-10995 Khartoum, SUDAN Email: nadamoabbas@gmail.com Mobile: +249918040731 Available online: www.ijipsr.com March Issue 111
INTRODUCTION Aerva javanica (family: Amaranthaceae) also known as kapok bush or Um shaar in regions in Sudan [1]. This plant is widely spread in the drier parts of tropical and sub-tropical Africa and Asia, and is commonly found at roadsides, stony rough valley's and hill slopes [2]. Aerva javanica mainly contains terpenes, phenolic compounds, flavonoids, saponins and tannins [3,4]. Traditionally Aerva javanica is used in wound healing, also used externally to remove swelling, and to relieve inflammation [2,5]. In one study, the plant has shown significant antimicrobial activity against Klebsiella penumoniae, Staphylococcus epidermidis, Pseudomonas aeruginosa and Staphylococcus aureus [4]. Diabetic wound complications are challenging and hindering and present a difficult encounter in the clinical practice. The pathogenesis of delayed wound healing with diabetic wounds is still not entirely comprehended, while there are evidences from a number of studies that involved human and animal models showed that there are rather a few irregularities in the different phases of wound healing process [6]. Therefore from its traditional use in wound healing to its antimicrobial activity this study was carried out to evaluate the wound healing ability in diabetic rats. METHOD AND MATERIAL SAMPLE COLLECTION AND PREPARATION: The plant was collected locally from Khartoum State in Sudan on October 2014 and was then identified in the herbarium of the Medicinal and Aromatic Plants Research Institute (MAPRI). The plant was immediately air dried in the shade and then pulverized by a mechanical grinder. About 100 gm of the ground plant were transferred to be extracted in a Soxhlet extractor. The first step was defatting using 300 ml of petroleum ether (60-80 o C), then extraction was done using 300 ml 70% ethanol for 12 hours. After Soxhlet extraction was complete the extract was concentrated using a rotary evaporator after which the residual extract was oven dried at 35-40 o C. The greasy extract was then used for the experiment. EXPERIMENTAL ANIMALS Healthy male and female Wistar rats weighing from 90-120 gm were used. The animals were fed on normal food and water ad libitum, the temperature was maintained at 24-28 o C, a relative humidity (30% - 70%) and a 12/12 hour light/dark cycle. The animals were fasted for 16 hours but had free access to the water. Available online: www.ijipsr.com March Issue 112
GROUPING OF THE ANIMALS After being weighed and their initial blood glucose level measured, the animals were divided into 3 groups each group containing 5 animals each. Group 1 contained rats that received the extract; Group II were the control receiving on the Vaseline and Group III which received the standard treatment using tetracycline ointment. INDUCTION OF DIABETES The streptozotocin, citric acid and sodium citrate were all purchased from Sigma-Aldrich. The animals were divided into 3 groups, all groups were weighed then fasting blood sugar was noted before the induction of diabetes. Diabetes mellitus was induced by the administration of 60 mg/kg of streptozotocin in freshly prepared 0.1M citrate buffer (ph 4.5) given intraperitoneal. Three days later, the fasting blood sugar was tested to confirm the diabetic status of the animals. The blood glucose was estimated using Accu-Chek Active glucometer (Roche), and the blood was collected from the coccygeal veins. EXCISION WOUND The animals were inflicted with excision wounds according to Morton and Malone [7] method. All the animals were anesthetized with slight vapor inhalation of diethyl ether then the back of the rats was shaved. The area of the wound was outlined with methylene blue using a circular stainless steel stencil. Then a full thickness excision wound of circular area of 300mm 2 and 2mm in depth was created along the markings using a sterile toothed forceps, surgical blade and pointed scissors. The entire wound was left open. The treatment was done topically at dose of 100 mgkg -1 day -1 for 14 days. The wound area was measured on day 0, 4, 8, 12 and 14, using a transparency sheet and a permanent marker. The recorded wound areas were measured on a graph paper. STATISTICAL ANALYSIS The means of wound area measurement between the groups at different time intervals was compared using one-way ANOVA. The data was analyzed using SPSS (Version 12.0, Chicago, USA) and the P value was set at <0.05. RESULTS The one way ANOVA showed significance between groups the P value 0.015. The post HOC test showed significance of the extract over the control (P < 0.012). Group III that was treated with the standard did not show significance when compared with those treated with the extract- Group Available online: www.ijipsr.com March Issue 113
I (P <0.725). On day 14 the animals treated with the extract experienced complete wound healing [Table 1 and fig 1]. Table 1: Effect of A. javanica alcoholic extract on excision wounds in terms of percentage wound closure Test Days EXTRACT Day 0 Day 4 Day 8 Day 12 Day 14 18 ±0.00 (0%) 7.7 ± 0.60 (57%) 5.6 ± 0.67 (68%) 1.2 ± 0.34 (93%) 0.00 ± 0.00 (100%) 18 ± 0.00 11.5 ±0.63 10.3 ± 0.56 9.1 ± 0.48 7.1 ± 0.40 Control (0%) (36%) (42 %) (49%) (60.5%) 18 ± 0.00 10.8 ± 0.64 6.9 ± 0.84 3.3 ± 1.33 2 ± 0.63 STANDARD (0%) (40 %) (62%) (82%) (88%) Values are mean ± SEM. Numbers in parenthesis indicate percentage wound closure. n=5 At P < 0.05 one way ANOVA showed significance between groups of 0.015 DISCUSSION Wound healing is characterized by three main stages, inflammation, proliferation, and remodeling. The proliferative phase typically demonstrates angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction. In angiogenesis, new blood vessels grow from endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new provisional extracellular matrix by excreting collagen and fibronectin. In epithelialization, epithelial cells crawl across the wound bed to cover it. Fibronectin, the major glycoprotein secreted by fibroblasts, has important functions of chemo-attraction for macrophages, fibroblasts and endothelial cells, promoting re-epithelialization and acting as a transduction agent in wound contraction. Wound contraction occurs by myofibroblasts, which Available online: www.ijipsr.com March Issue 114
establish a grip on the wound edges, bringing them in apposition [8,9]. Aerva javanica extract showed that it had wound healing properties after its topical application on streptozotocin-induced diabetic rats. The wound size reduced as early as day 4 in animals receiving the extract compared to both those in the control and those receiving the standard (tetracycline). This study shows that Aerva javanica applied topically promoted the healing in wounds that otherwise would be delayed due to diabetes as can be shown in the control group. These findings have a particular implication for the promotion of wound healing in diabetics and show the importance of further investigation and studies on this plant. The results of this study could be used for a complete evaluation of the role of the extract of A. javanica in different diabetic wound healing models, so to clarify its role in the treatment of different types of diabetic wounds. ACKNOWLEDGMENT The author is grateful for the guidance of the supervisor, Prof. Yahia Mohamed El Imam ( Professor in Khartoum College of Medical Sciences). Also thanks are extended to Dr. Ghada (Assistant Professor, Omdurman Islamic University) for her assistance throughout this study and to Mohamed Abdu (Statistician) for helping in the Statistical Analysis. Finally the author would like to thank the Faculty of Pharmacy, Omdurman Islamic University (Sudan) for providing the needed facilities for the study and the faculty of Pharmacy, Khartoum College of Medical Sciences for allowing the time to conduct the study. REFERENCES 1. El Ghazali, G.E.B., M.S. El Tohami & A.A.B. El Egami, Medicinal plants of the Sudan.Part 3 : Medicinal plants of the White Nile Provinces. Medicinal and aromatic plants institute.national council for research. 1994, Khartoum: Augustus. 116. 2. Ghazanfar, S.A., Handbook of Arabian Medicinal Plants. 1994: Taylor & Francis. 17-19. 3. Movaliya, V., Maitreyi, Z., Pharmacognostical Studies on Roots of Aerva javanica. Indian J. Pharm. Sci. Rev. Res., 2012. 16 (1): p. 34-37. 4. Srinivas, P., Reddy, S. Ram, Screening for antibacterial principle and activity of Aerva javanica (Burm. f) Juss. ex Schult. Asian Pacific Journal of Tropical Biomedicine, 2012. 2(2): p. S838-S845. 5. Muhammed Qasim Samejo, S.M., Muhammed Iqbal Bhanger, Khalid Mohammed Khan, Chemical Compositions of the Essential oil of Aerva javanica Leaves and stems. Pak. J. Anal. Environ. Chem., 2011. 13 (1): p. 48-52. Available online: www.ijipsr.com March Issue 115
6. Singhal, A.K., H. Gupta, and V.S. Bhati, Wound healing activity of Argyreia nervosa leaves extract. International Journal of Applied and Basic Medical Research, 2011. 1(1): p. 36. 7. Morton JJP, M.M., Evaluation of vulnerary activity by an open wound procedure in rats. Arch Int Pharmacodyn, 1972. 196 (1): p. 117-126. 8. Hall, J.E., Guyton and Hall Textbook of Medical Physiology. 12 ed. 2010: Elsevier Health Sciences. 9. Stadelmann, W.K., A.G. Digenis, and G.R. Tobin, Physiology and healing dynamics of chronic cutaneous wounds. The American Journal of Surgery, 1998. 176 (2): p. 26S-38S. Available online: www.ijipsr.com March Issue 116