Pharmacological Investigation on the Wound Healing Effects of ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701ointment (Glaxosmithkline), urethane (SD finechemical,Mumbai), Surgical thread (no. 000) and acurved needle (no. 9), Tensiometer.2.2 Experimental animals: The study wasconducted on Albino rats (Wistar) of 200-250 gand maintained under standard conditions (roomtemperature 24- 27oC and humidity 60-65%) with12 h light and dark cycle. The food in the form ofdry pellets (Amrut Lab., Pune) and water wereavailable ad libitum . The animal experiments wereapproved by the ethics committee of the institute.2.3 Excision wound model:-The animals were divided into three major groupsviz. control, standard and test with six animals ineach group. The control group was treated withsimple ointment base B.P. The standard group wastreated with Fluticasone (Glaxosmithkline makecontaining 0.005% (w/w)) ointment. The testgroups were treated with ointments withconcentrations of drug viz. 2% (w/w) incorporatedin simple ointment base. The rats were anesthetizedby administering urethane (0.5 ml/kg b. w. i.p.). Afull thickness of the excision wound of circulararea (approx. 500 mm 2 ) and 2 mm depth was madeon the shaved back of the rats 30 min later theadministration of urethane injection. The woundingday was considered as day 0 (Fig. 1). The woundswere treated with topical application of theointments as described above till the wounds werecompletely healed. The wounds were monitoredand the area of wound was measured on 4, 8, 12,16 post-wounding days and the mean % woundclosure is reported in Table 1. The period ofepithelization was calculated as the number of daysrequired for falling of the dead tissue remnantswithout any residual raw wound (Nayak et al.,2007). % of wound contraction (Muthusamy et al.,2008):% of wound contraction =wound area on day 0 − wound area on day n------------------------------------------- × 100wound area on day 0where n = number of days 4th, 8th, 12th, and 16thday.2.4 Incision wound model:The animals were divided into three major groupsviz. control, standard and test with six animals ineach group. The control group was treated withsimple ointment base B.P. The standard group wastreated with Fluticasone (Glaxosmithkline makecontaining 0.005% (w/w)) ointment. The testgroups were treated with ointments withconcentrations of drug viz. 2% (w/w) incorporatedin simple ointment base. The rats were anesthetizedby administering urethane (0.5 ml/kg b. w. i.p.).Excision wounds of about 6 cm in length and 2mmin depth were made with sterile scalpel on theshaved back of the rats 30 min later theadministration of urethane injection. The partedskin was kept together and stitched with black silkat 0.5cm intervals (Fig. 3). Surgical thread (no.000) and a curved needle (no. 9) were used forstitching. The continuous thread on both woundedges were tightened for good closure of thewounds. The wounds of animals in the differentgroups were treated with topical application of theOintments as described above, for the period of 10days. The wounding day was considered as day 0.When wounds were cured thoroughly, the sutureswere removed on the 8th post-wounding day (Fig.2) and the tensile strength of the skin that is theweight in grams required to break open thewound/skin was measured by tensiometer on the10th day reported in Table 3 (Nath et al., 2006).3. Statistical analysis : Statistical comparison wasperformed using one way analysis of variance(ANOVA) and for multiple comparisons versuscontrol group was done by Dunnett’s test. P value< 0.05 were considered statistically significant.4. Result :Excision wound model :Control rats showed atime dependent increase in % wound contractionfrom 14.53 ± 0.98 to 30.33 ± 1.87 from day 4 th today 8 th and 46.67 ± 1.98 to 60.5 ± 2.09 from day12 th to day 16 th .While complete wound closure andepithelization was observed on 16 th day of woundinduction compared with day 0 which was taken as0% (figure-1). The mean epithelization period was30.16 days.Fluticasone (standard drug) rats showed a timedependent increase in % wound contraction from26.2 ± 0.74 to 44.17 ± 1.58 from day 4 th to day 8 thand 63.67 ± 2.08 to 92 ± 2.62 from day 12 th to day16 th .While complete wound closure andepithelization was observed on 16 th day of woundinduction compared with day 0 which was taken as0%. The mean epithelization period was 22.67days.GA (Test drug) rats showed a time dependentincrease in % wound contraction from 17.33 ± 0.67to 34.17 ± 1.27 from day 4 th to day 8 th and 55.33 ±1.29 to 80.17 ± 2.12 from day 12 th to day16 th .While complete wound closure andepithelization was observed on 16 th day of woundinduction compared with day 0 which was taken as0%. The mean epithelization period was 25.76days.Incision wound model : Control rats showed meanwound breaking strength and epithelization time ofVol. 2 (3) Jul – Sep 2011 www.ijrpbsonline.com 1067

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701390 ± 22.673 g and 25.67 days respectively, whileGA showed mean wound breaking strength andepithelization time of 474 ± 21.986 g and 17.14days respectively. Again wound healing property ofGA was comparable with fluticasone (Table-3).5. Discussions and conclusion:Wound healing process consists of different phasessuch as granulation, collagenation, collagenmaturation and scar maturation which areconcurrent but independent to each other. Hence inthis study two different models were used to assessthe effect of herbal ointment on various phases.The result showed that Glycyrrhizic Acid,Ammonium Salt (GA) ointment possesses adefinite pro-healing action. This was demonstratedby a significant increase in the rate of woundclosure and by enhanced epithelialization.Significant increase in tensile strength, andcollagen levels were observed, which was furthersupported by histopathological studies and gain ingranuloma breaking strength. This indicatedimproved collagen maturation by increased crosslinkingwhile an increase in dry granuloma weightindicated higher protein content.Literature concluded that GA documented to haveantidiabetic (Kalaiarasi 2009), potent antioxidantand free radical scavenging effect (Beskina,2006).Active oxygens are effective as cytotoxic and todamage cells by inactivating and modifyingcellular components. The low concentration ofoxygen free radicals stimulate fibroblastproliferation, also when free radical scavengers areadded to cultured fibroblasts in absence of freeradical generating system, they inhibited thymidineincorporation, indicating the growth promotingaction of free radicals (Shukla and Patnaik, 1998).Ascorbic acid has been shown to be involved incollagen gene expression. It was also found insome burn injury cases ointment containingsuperoxide dismutase (SOD), which stimulateswound healing. It was observed that asiaticosideenhanced induction of antioxidant levels at aninitial stage of healing, which may be importantcontributory factor in the healing property (Shuklaet al., 1999). The antioxidant enzymes (superoxidedismutase and catalase) are known to quench thesuperoxide radical and thus prevent the damage ofcells caused by free radicals (Shirwaikaret al.,2003). So scavenging effect might be one of themost important components of wound healing.Also plant reported to have antioxidant activity(Beskina ,2006) may be responsible to supportwound healing.Thus the enhanced wound healing may be due tothe free radical scavenging action of the GA as wellas enhanced antioxidant enzyme level in granulomatissues. The GA were found to be powerfulantioxidant to protect thermally injured miceoxidative damage (Utsunomiya, 1999).In conclusion, the results of study showed that theGA ointment effectively stimulates woundcontraction; increase tensile strength of incisionand excision as compared to control group. Thesefinding could justify the inclusion of this drug inthe management of wound healing.Acknowledgements:The authors would like to acknowledge R.D.’scollege of pharmacy, Bhor, Pune, India, forproviding necessary facilities to carry out the study.Table 1:Effect of topical application of ointments containing GA on wound contraction of excision woundGroups 4 th day 8 th day 12 th day 16 th day Epithelialization time(days)Wound Control 14.53±0.98 30.33±1.87 46.67±1.98 60.5±2.09 30.16Standard -Fluticasone(0.005% w/w)20.2±0.74** 44.17±1.58** 63.67±2.08** 92±2.62** 22.67**Test - GA (2%w/w)17.33±0.67* 34.17±1.27* 55.33±1.24*80.17±2.12**25.76*Values are expressed as mean ± S.E.M., n=6, Fluticasone (0.005% w/w), GA (2% w/w), *p

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 2: Effect of topical application of ointments containing GA on Haematoxylin and eosin stained wound sections were scored.Groups C ED PMN MNC N FP NV EP UWound Control +++ +++ ++ ++ + + + - ++Standard - Fluticasone(0.005% w/w)- + + + - ++ ++ ++ -Test - GA (2% w/w) ++ + + - - + ++ ++ +Haematoxylin and eosin stained sections were scored as mild (+), moderate (++) and severe(+++) for epidermal and/or dermal re-modeling. Epithelialization and ulceration were scored aspresent (+) and absent (−). C, congestion; Ed, edema; PMN, polymorphonuclear cells; MNC,mononuclear cells; N, necrosis; FP, fibroblast proliferation; NV, neovascularization; Ep,epithelialization; U, ulceration.Table 3: Effect of topical application of ointments containing GA on epithilization time and tensile strength of Incisionwound.Groups Epithelialization time (Day) Tensile strength (g)Wound Control 25.67 390±22.673Standard - Fluticasone (0.005% w/w) 14.89** 565±27.564**Test - GA (2% w/w) 17.14* 474±21.986*Values are expressed as mean ± S.E.M., n=6, Fluticasone (0.005% w/w), GA (2% w/w), *p

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Figure1: Excision wound image of rat at day 0.Figure 2 :Before Incision wound Shaved image of rat at day 0.Figure3: Incision wound image of rat at day 0.Vol. 2 (3) Jul – Sep 2011 www.ijrpbsonline.com 1070

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Histopathological study:ControlStandardTestCollagen fibre ,blood vesels formationsVol. 2 (3) Jul – Sep 2011 www.ijrpbsonline.com 1071

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