Asian Pacific Journal of Tropical Medicine (2011)921-924 921

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Document heading doi: Healing promoting potentials of roots of benghalensis L. in albino rats Krishna Murti1*, Upendra Kumar2, Mayank Panchal3

1Singhania University, Jhunjhunu, Rajasthan, 2College of Pharmacy, Azamgarh, Uttar Pradesh, India 3Vidyabharti Trust College of Pharmacy, Umrakh, , India

ARTICLE INFO ABSTRACT

Article history: Objective: Ficus To screen the wound healing activity of aqueous and ethanolic extract of roots of Received 1 June 2011 benghalensis Methods: ie . Both the extracts were evaluated for wound healing by three models . Received in revised form 15 August 2011 Accepted 15 October 2011 incision, excision and dead space wound. In incision and dead space the extracts were applied 10 Available online 20 November 2011 daily topically till the th post wounding days while in excision model it was till the complete epithelialization process. Standard group were administered Povidone iodine ointment topically daily. The breaking strength, percentage of wound contraction, period of epithelialization, dry Keywords: Results: granulation weight and hydroxyproline content were observed. The result of the present Ficus benghalensis study showed that both extracts were able to increase the breaking strength (incision model), Incision decrease period of epithelialization, increase percentage wound contraction (excision model), Excision increaseP hydroxyprolineConclusions: content (Dead space wound model) significantly compared with control Dead space group ( <0.05). Based on result we conclude that aqueous extract is more effective Hydroxyproline estimation than ethanolic extract. However, it is needed more research to be carried out especially on Phytochemical analysis toxicity studies of ethanolic extract. Povidone iodine

1. Introduction [6] healingFicus benghalensisactivity . Linn (Family-) is commonly A wound may be defined as a break in the epithelial knownFicus benghalensis as or vata or vada tree in Ayurveda. integrity of the skin or may also be defined as a loss or is a remarkable tree from India that breaking of cellular and anatomic or functional continuity sends down its branches and great number of shoots, of living tissue[1]. Wound healing studies are mainly aimed which take root and become new trunks. This tree is to detect various means and factor influencing healing considered to be sacred in many places in India[7]. process, so they could be either used or avoid in clinical Traditionally all parts of the are astringent, acrid, practice to favorably alter the healing process[2]. Wound sweet, refrigerant, anodyne, vulnerary, depurative, healing process involves several steps such as coagulation, anti-inflammatory, ophthalmic, styptic, antiarthritic, inflammation, formation of granulation tissue, matrix diaphoretic, antidiarrhoeal, antiemetic and tonic[8]. It is formation, remodeling of connective tissue, collagenization used in Ayurveda for the treatment of diarrhea, dysentery and aquisation of wound strength. During the formation of and piles, teeth disorders, rheumatism, skin disorders like new tissue, endothelial cells proliferate and form new blood sores and to boost immune system, as a hypoglycemic[9]. vessels. There are several reports stating that the extracts of Bark contains tannins, wax, esters and glucoside, 20- several are used for wound healing properties[3-5]. tetratriaconthene-2-one, 6-heptatriacontene-10-one, Some of plants possessing prohealing activity have been pentatriacontan-5-one, beta sitostirol-alpha-D-glucoseviz Tridexscientifically procumbens, analyzed. Trigonella The wound foenumgraecum, healing potential Leucas of and meso-inositol. Two flavonoid compounds, . 5, lavandulaefolia Aloe vera 7-dimethylether of leucopelargonidin 3-0-alpha-L- and have shown promising rhamnoside and 5,3-dimethyl ether of leucocyanidin 3-0- alpha-D galactosyl cellobioside were present in the bark Ficus benghalensis[9,10] *Corresponding author: Krishna Murti, Research Scholar, Singhania University, of . PharmacologicalFicus benghalensis evaluation Jhunjhunu, Rajasthan, India. has shown the various extract of has Tel: +91-9328832853 shown anthalmintic[9], analgesic[10], anti-inflammatory[10], E-mail: [email protected] Krishna Murti et al./Asian Pacific Journal of Tropical Medicine (2011)921-924 922 Ficus benghalensis antioxidants[11], antidiabetic[12], Immunomodulatory[13] The ethanolic & aqueous extract of was [14] p.o. and antimicrobial activity in experimentalFicus benghalensis animals. The administered orally ( ) to all group of animals. ethnomedicinal use of the roots of in 2.6. Incision wound model wound healing has not been systematically investigated so far. Therefore the present study was designed to determine the healing activity of roots in the wound model in rats. The rats werei.p. anesthetized by administering ketamine (0.5 mL/kg bw. ). Incision wounds of about 6 cm in length 2. Materials & methods and 2 mm in depth were made with sterile scalpel on the shaved back of the rats 30 min later the administration 2.1. Materials of ketamine injection. The parted skin was kept together and stitched with black silk at 0.5 cm intervals. Surgical Ficus benghalensis thread (No. 000) and a curved needle (No. 9) were used for Roots of L. growing in natural habitant stitching. The continuous thread on both wound edges were in Modasa, Gujarat, India, was collected in October, 2010 tightened for good closure of the wounds. The wounds of and identified by Associate Professor Dr. MS Jangid, animals in the different groups were treated with drug by Department of Botany, Modasa, Hemchandra Gujarat oral administration as described above, for the period of 10 University by carrying out macroscopic and microscopic days. The wounding day was considered as day 0. When evaluation and has been submitted in the institute for future wounds were cured thoroughly, the sutures were removed reference purpose. on the 8th post-wounding day and the tensile strength of the 2.2. Preparation of the root extract skin that is the weight in grams required to break open the wound/skin was measured by tensiometer on the 10th day [19,20] Ficus reported . benghalensis Dried and coarsely 500 g powdered roots of 2.7. Excision wound model was extracted with 90% (v/v) ethanol in soxhlet apparatus for 36 h and aqueous extract was prepared by using maceration technique of extraction. After filtration, the A circular piece of full thickness [approximately 500 (mm. filtrate was concentrated on water ℃bath using petridish. The sup.2)] was removed from a predetermined dorsal area[21]. temperature was maintained at 55 . The powdered extract The wound were traced on 1-(mm.sup.2) graph paper was dried and weighed. on the day of wounding and subsequently on alternate 2.3. The preliminary phytochemical analysis days until healing was complete. Changes in the wound area were calculated, giving an indication of the rate of wound contraction. The number of days required for The preliminary phytochemical studies were performed falling of the eschar without any residual raw wound was for testing different chemical groups present in ethanolic & determined as the period of epithelization. [15-18] aqueous extract . 2.8. Dead space wound model 2.4. High performance Thin Layer Chromatography (HPTLC) profile The dead space wounds were created by making a small transverse incision in the lumber region on either side of Chromatography was performed on 3 cm 伊10 cm HPTLC vertebral column in each animal. Two polypropylene tubes plates coated with 0.25 mm layer of silica gel 60 F254 (2.5 cm 伊 0.5 cm) were inserted subcutaneously one on (Merck, Germany). Before using, the℃ plates were washed either side of vertebral column and pushed cephal head for with methanol and activated at 110 for 5 min. Samples 3-4 cm for the final implantation to harvest the granulation were applied as 4 mm wide bands and 6Ⅳ mm apart by using a tissue. The animals were treated with the extracts from 0 Camag (Muttenz, switzerland) Linomat sample applicator day to 9th post-wounding day considering wounding day as equipped with 100 毺L syringer. A constant application rate zero. Granulation tissue formed on the implanted tubes was of 5 毺L/s was used. carefully dissected out on the 10th post-wounding day and 2.5. Animals the tensile strength was measured by continuous constant water flow technique[20]. Mean value gives the breaking strength℃ for a given group. The tissue was dried in oven at Wistar albino rats of either sex weighing between 180 and 60 for 24 hours and the dry weight was noted. The acid 200 g were obtained from Jai Foundation Research, Vapi hydrolysate of the dry tissue was used for the estimation of (Gujarat). The study was approved by the Institutional Ethics the hydroxyproline content in the tissue[22]. Committee for animal experimentation Vidyabharti Trust 2.9. Hydroxyproline estimation College of Pharmacy, Umrakh, Gujarat (VBT/IAEC/10/12/30) and all the procedures on animals were carried out as per CPCSEA guidelines, India. These animals were used for the Hydroxyproline is an uncommon amino acid present in the wound healing activity studies. The animals were stabilized collagen fibres of granulation tissues. Its estimation helps for 1 week. They were maintained in standard conditions clinically to understand progress rate at which the healing at room temperature, (60依5)% relative humidity and 12 h process is going on in the connective tissue of the wound. light dark cycle.ad libitum They had been given standard pellet diet For the determination of hydroxyproline content, the wound℃ and water throughout the course of the study. tissues were excised and dried in a hot air oven at 60-70 Krishna Murti et al./Asian Pacific Journal of Tropical Medicine (2011)921-924 923 ℃ FOR 6NHCl 130 constant weight and were hydrolysed in at extract ointmentsP were significantly greater than that of for 4 hour in sealed glass tubes. The hydrolysate was the control ( <0.05). The aqueous extract ointment treated neutralized to pH 7.0 and was subjected to Chloramine-T group showed significant wound healing from the fourth day oxidation for 20 min. The reaction was terminated by onwards, which was comparable to that of the standard drug, addition of 0.4 M perchloric acid and colour℃ was developed povidone iodine ointment. The wound closure time was with the help of Ehrlich reagent at 60 . The absorbance lesser, as well as the percentage of wound contraction was was measured at 557 nm using a spectrophotometer much higher in the aqueous extract ointment treated group (Shimadzu 1700, Japan). The amount of hydroxyproline in (13.33 days for 100% contraction which was almost better the samples was calculated using a standard curve prepared than that of the povidone iodine treated group). Ethanolic with pure l-hydroxyproline[23]. extract ointment treated group showed significant wound 2.10. Statistical Analysis contraction from the fourth day onwards and achieved 100% with the wound closure time of 14.17 days.

3.4. Dead space wound model The mean value依SEM was calculated for each parameter. Results were statistically analyzed by one-way-analysis’ P of- variance (ANOVA) followed by posthoc dunnet s test. <0.05 In the dead space wound study, there was a significant was considered as significant. increase in granuloma breaking strength in both extract treated groups when compared to control (Table 2). There 3. Results was significant increase in Ficushydroxyproline benghalensis content in both extract treated groups of . Standard 3.1. Phytochemical Analysis drug povidone iodineP also showed significant increase as compared to control( <0.05).

Result of preliminaryFicus benghalensis phytochemical screening showed 4. Discussion that the roots of L. contained saponins, tannins, alkaloids and flavanoids while other constituents like amino acids, carbohydrate were absent. Further the Wound healing process consists of different phases such presence of these chemical constituents was confirmed by as granulation, collagenization, collagen maturation and HPTLC analysis. scar maturation which are concurrent but independent to 3.2. Incision wound model each other. Therefore, in this study three different models were used to establishFicus the healingbenghalens potentialsis of aqueous and Ethanolic extracts of Ficusin various benghalensis phases. The breaking strength of the incision wounds was increased From the result, it is evident that in extract treated groups Ficussignificantly, benghalensis from 305.20 依15.15 in possesses a definite potential healing action. The breaking control to 502.30依2.26 Ficusin benghalensis aqueous extract strength of the incision wounds was increased in aqueous 455 80 2 82 andP . 依 . in ethanol extract & ethanolic extract treated groups. Deposition of newly ( <0.05). The results were also comparable to standard drug synthesized collagens at the wound site increases the povidone iodine (429.70依3.21). collagen concentration per unit area and hence the tissue [24] 3.3. Excision wound model tensile strength . In excision wound healing modelFicus the benghalensisaqueous & ethanolic extract of the root of the plant showed Table 1 showed that the wound contracting ability of two significant increase in percentage closure by enhanced

Table 1 Percentage wound contraction and period of epithelialization in excision wound model(Mean依SE). Percentage of wound contraction (%) Period of epithelialization Groups Day 2 Day 4 Day 8 Day 12 Day 16 (Days) Control 3.50依0.87 13.33依0.24 44.50依0.23 71.60依0.54 79.66依0.32 19.67依0.61 Povidone iodine 10.50依1.23 43.83依1.54 73.80依1.67 84.50依1.23 92.80依0.23 16.00依0.26* Ficus benghalensis aqueous extract 14.60依0.12 66.50依0.21 85.33依0.65 98.15依0.13 100.00依0.00 13.33依0.42* Ficus benghalensis ethanol extract 13.66依0.12 54.50依2.21 74.16依1.26 90.10依2.32 94.83依1.32 14.17依0.48* P * : <0.05 compared with control group. Table 2 Granulation tissue breaking strength, dry granulation tissue weight and hydroxyproline content on 10th day old of dead space model(Mean依SE). Groups Granulation breaking strength Dry granulation weight (mg/g) Hydroxyproline content (mg/mL) Control 261.20依3.23 56.33依4.43 33.00依1.12 Standard Povidone iodine 351.80依2.54* 80.67依1.50* 40.80依0.25* Ficus benghalensis aqueous extract 515.80依5.04* 97.33依0.56* 56.49依0.57* Ficus benghalensis ethanol extract 479.00依2.98* 86.83依1.35* 45.08依0.45* P * : <0.05 compared with control group. Krishna Murti et al./Asian Pacific Journal of Tropical Medicine (2011)921-924 924 Ficus benghalensis J Basic the bark of in plants of different age. epithelialization. This enhanced epithelialization may be Clini Pharm 1 Ficus benghalensis 2010; (2): 107-113. due to the effect of extracts on enhanced Indian medicinal plants: A compendium of 500 species collagen synthesis. Similarly, in dead space model there [8] Sala ⅢAV. . was a significant increase in granuloma tissue breaking Vol- . Madras: Orient Longman Ltd; 1997, p. 20-26. 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