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In Vivo Topical Wound Healing Activity of Punica Granatum Peel Extract on Rats

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In Vivo Topical Wound Healing Activity of Punica Granatum Peel Extract on Rats Original Article In Vivo Topical Wound Healing Activity of Punica Granatum Peel Extract on Rats NITIN NEMA*1, SAURABH ARJARIYA 1, SM BAIRAGI 2, MEGHA JHA 3, MD KHARYA 4 1Sagar Institute of Pharmaceutical Sciences Sagar (M.P.) India 2MES College of Pharmacy Sonai (M.H.) India 3Pinnacle Biomedical research Institute, Bhopal (M.P.) 4Department of Pharmaceutical Sciences, Hari Singh Gour Central University Sagar (M.P.) . ABSTRACT Punica granatum is a well-known fruit for its antioxidant, hepatoprotective, anticancer, antibacterial potential. The methanolic extract of Punica granatum , in the form of an ointment with two different concentrations (10% and 15% w/w ointment of extract in simple ointment base) was evaluated for wound healing potential in an excision wound model in rats. The results were comparable to standard drug Nitrofurazone ointment. It was observed that the wound contracting abilities of 10% and 15% extract ointments (97.8%, 98.4%) were significantly (P < 0.05) greater than that of the control. The wound closure time was less and the percentage of wound contraction was much more with the 15%w/w extract ointment treated group. On 18th day 100% contraction was observed Address for which was almost similar to that of the nitrofurazone ointment group. Correspondence 10%w/w extract ointment group of animals showed significant wound contraction from the 18th day onwards and achieved 100% Sagar Institute of with the wound closure time of 20th days. Both concentrations of the Pharmaceutical methanolic extract of Punica granatum ointment showed significant Sciences Sagar (M.P.) responses when compared with the control group. Thus methanolic extract of Punica granatum proves to be a potential wound healing E-mail: agent. nitin.nema85@ gmail.com Keywords : Nitrofurazone, Punica granatum, antioxidant, ointment, hepatoprotective. INTRODUCTION The pomegranate ( Punica granatum consumed fresh and more recently in L. ) fruit has been used for centuries in beverage form as juice 1. Pomegranate juice ancient cultures for its medicinal purposes. possesses potent antioxidant activity that For a long time, pomegranate fruit is widely results in marked protection of nitric oxide American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org NEMA et al___________________________________________________________________ (NO) against oxidative destruction, thereby healing effect of Pomegranate (Punica resulting in augmentation of the biological granatum) peel extract in Wistar rats. actions of NO 2. PFE (pomegranate fruit extract) was found to contain anthocyanins MATERIALS AND METHODS (such as delphinidin, cyanidin, and pelargonidin) and several hydrolyzable Plant material tannins (such as punicalin, pedunculagin, Fresh peel of Punica granatum were punicalagin, gallagic, and ellagic acid esters collected in area free of pesticides and other of glucose), which account for 92% of the contaminants from the local vendor of district antioxidant activity of the whole fruit 3. Sagar, Madhya Pradesh and shed dried. The Several compounds have been isolated from collected peels were crushed. Punica granatum such as tannins, punicalagin, ellagic acid, hydroquinone Preparation of Extract pyridinium, delphinidin, cyanidin, and The peel powder (100 g) was pelargonidin. extracted by stirring using a magnetic stirrer Natural products are a source of with 600 ml of methanolic at 30 °C for 4 h. synthetic and traditional herbal medicine The extract was filtered through Whatmann and are still the primary health care system 4. filter paper no. 41 for removal of peel An important aspect of the use of traditional particles. The residue was re-extracted with medicinal remedies and plants in the 500 mL of MeOH and filtered. The extracts treatment of burns and wounds is the were pooled and concentrated under vacuum potential to improve healing and the same at 40°C using a Speedvac system (SC110A, time to reduce the financial burden 5. Wound Savant, USA). healing occurs in three stages: inflammation, proliferation, and remodeling. The Animals proliferative phase is characterized by Wistar rats (150-200g) were used in angiogenesis, collagen deposition, this study. They were given water ad libitum granulation tissue formation, and fed with commercial food pellets. The epithelialization and wound contraction 6. In study protocol was approved by institutional angiogenesis, new blood vessels grow from animal ethics committee, Pinnacle endothelial cells. In fibroplasias and Biomedical Reasearch Institute, Bhopal, granulation tissue formation, fibroblasts Madhya Pradesh India. Following overnight grow and form a new, provisional starving, animals were anesthetized with local extracellular matrix by excreting collagen anesthesia xylocaine and suitably wounded and fibronectin. Collagen, the major after shaving the area to be operated to bear component which strengthens and supports excision wound. The wounds were not extracellular tissue, contains substantial dressed and no systemic or local antimicrobial amounts of hydroxyproline, which has been agents were used. used as a biochemical marker for tissue collagen. In epithelialization, epithelial cells Excision Wounds proliferate and spread across the wound The surgical materials were sterilized surface. Wound contraction occurs as the and dorsal fur of the animals was shaved with myofibroblasts contract. Platelets release an electric clipper. The rats were anesthetized growth factors and other cytokines 7. Present with (Xylocaine®) 2% Jelly, Astra Zeneca study was conducted to evaluate the wound Pharma India ltd and anticipated area of the wound to be created was outlined on the back of the animals with methylene blue using AJPCT1[2][2013]195-200 NEMA et al___________________________________________________________________ stencil. A full thickness of the excision wound contraction was expressed as percentage of circular area of 500mm 2 and 2mm depth reduction of original wound size. was created along the markings using toothed forceps, scalpel and pointed scissors. Wound Statistical analysis contraction was monitored by measuring Results obtained from wound area, planimetrically, on alternate days threewoundmodels have been expressed as till the wounds were completely healed. mean±SEM. The data was evaluated by one way ANOVA followed by Dunnett’s t-test, P Formulation of ointment (British < 0.05 was considered as significant. pharmacopoeia, 1996) (a) Preparation of 20g simple ointment (B.P.) RESULTS & DISCUSSION base. Wool fat (1g), hard paraffin (1g), cetostearyl alcohol (1g) and white soft The least rate of wound healing was paraffin (17g) was mixed and heated gently seen in control group, which received simple with stirring then cooled. ointment. Treatment with standard group (b) 2 gm 50% methanolic extract of Punica heals the wound in a faster rate than other granatum was added separately to 20gm of group, but complete healing was obtained on base (10% ointment). day 18. The upper layer of wound was (c) 3 gm 50% methanolic extract of Punica surgically removed and subjected to granatum was added separately to 20gm of histological studies. Histological examination base (15% ointment). of the haematoxylin and eosin stained tissue of the rat wounds treated with extract and Drug administration nitrofurazone ointment have led to reduce 24 animals were divided into groups scar formation and enhanced fibroblast of four and treated as follows: proliferation, angiogenesis, keratinisation and Group 1: Simple ointment base was applied epithelialisation as compared to vehicle- once daily and served as vehicle control. treated group or control group. The Group 2: Standard drug nitrofurazone measurement of the progress of the wound ointment (0.2% w/w) was applied once daily healing induced by the standard, ointments served as positave control. and control in the excision wound model were Group 3: Punica granatum 50% methanolic shown in Table 1 . It was observed that the extract ointment (10% w/w) was applied once wound contracting abilities of 10% and 15% daily. extract ointments (97.8%, 98.4%) were Group 4: Punica granatum 50% methanolic significantly (P < 0.05) greater than that of the extract ointment (15% w/w) was applied once control. The earlier wound contraction rate of daily the methanolic extract may be due to All the above mentioned treatments stimulation of interleukin-8, an inflammatory were started from the day of wound creation a-chemokine which affects the function and and continued till 20th day of healing.The recruitment of various inflammatory cells, wound closure rate was assessed by tracing fibroblasts and keratinocytes. It may increase the wounds on day 2nd, 4th, 6th, 8th, 10th, the gap junctional intracellular 12th, 14th, 16th, 18th and 20th. Post communication in cultured fibroblasts and induces a more rapid maturation of wounding using transparency paper and a 8 permanent marker. The changes in healing of granulation tissue . wound i.e. measurement of wound on graph paper was expressed as unit (mm 2). Wound AJPCT1[2][2013]195-200 NEMA et al___________________________________________________________________ CONCLUSION enhances the biological actions of nitric oxide. Nitric Oxide 2006; 15: 93–102. Wound healing process consists of 3. Gil MI, Tomas-Barberan FA, Hess-Pierce different phases such as granulation, B, Holcroft DM, Kedar AA. Antioxidant collagenization, collagen maturation and scar activity of pomegranate juice and its maturation which are concurrent but relationship with phenolic
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