Effects of Bilwadi Churna (Bc) on Experimentally Induced Wound ...

International Journal <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701____________________________________________Research Paper<strong>Effects</strong> <strong>of</strong> <strong>Bilwadi</strong> <strong>Churna</strong> (<strong>Bc</strong>) on Experimentally InducedWound Healing in RatsDevesh D, Gosavi* and Ranjana KaleDepartment <strong>of</strong> Pharmacology, MGIMS, Sewagram Wardha, Maharashtra, India.INTRODUCTION“Wound” can happen to any one by any means atany age, human body has got the ability to “heal”the insult inflicted to it. Healing is thephysiological process, but gets disturbed in fewpathological conditions like Diabetes and in case <strong>of</strong>chronic ulcers this process <strong>of</strong> healing gets delayed.“<strong>Bilwadi</strong> <strong>Churna</strong>”(BC) is the mixture with provenhypoglycemic activity.prepared by mixing following ingredients in equalparts. Initially each ingredient was prepared in apowder form individually and then mixed togetherin equal parts to measured as their weights.The ingredients are:1) Andrographis paniculata (Kalmegh) All parts,2) Emblica <strong>of</strong>ficinalis (Amalaki) fruits,3) Trigonella foenum-graecum (Methi) fruits,4) Syzigium cumini (Jambu) seeds,5) Sesamum indicum (Teel) seeds,6) Aegle marmelos (Bilwa) fruits, and7) Azadirachta indica (Neem) leaves.(Names in the parenthesis are the ayurvedicSanskrit names <strong>of</strong> the herbs)The preparation <strong>of</strong> this formulation involves simpleprocedures since it is in the plain powder form andall the ingredients are easily available in rural area.Non healing wounds are one <strong>of</strong> the most commoncomplications seen in diabetes patients. Thereforethis study has been carried out to know the effect <strong>of</strong>“<strong>Bilwadi</strong> <strong>Churna</strong>” on wound healing that can beeffectively helpful in non healing diabetic ulcers.This powder was used by a untrained traditionalhealers in an area surrounding our institute. As themost <strong>of</strong> the ingredient have hypoglycemic actionwhich is already mentioned in various publication,we have decided to concentrate on treating thecomplication <strong>of</strong> diabetes, i.e. diabetic ulcers.MATERIALS AND METHODSPreparation <strong>of</strong> powderThe contents <strong>of</strong> the preparation includedAndrographis paniculata , Aegles marmelos ,Embilica <strong>of</strong>ficinalis ,Trigonella foenum graecum,Syzigium cumini , Sesamum indicum, andAzadirachta indica.They were taken in equal quantity and mixed indistilled water. The freshly prepared suspensionwas used for this purpose orally.Calculation <strong>of</strong> DoseThe dose was calculated by extrapolating thehuman dose on the rats.PlaceThe study was conducted at MGIMS, Sewagram.TimeDaily at 10 a.m.Ethics committee approvalThe study was conducted after seeking the approvalby Institutional Ethics Committee.AnimalsThirty male albino rats, weighing between 100-200gm, aged 6-8wks were purchased from NationalInstitute <strong>of</strong> Nutrition, Hyderabad. The rats were feda standard diet and water given food and water adlibitum in our animal house facility and maintainedunder standard laboratory conditions. Rats weremaintained on 12 hourly light and dark cycle.Animal identification was done by cage numberallotted to them.The rats were divided into three groups <strong>of</strong> 10animals each.First group acted as a test group which received1.25gm/kg <strong>of</strong> BC orally.Second group also acted as a test group whichreceived 2.25gm/kg <strong>of</strong> BC per oral route.Third group acted as control and received 0.2ml<strong>of</strong> distilled water orally.Procedure <strong>of</strong> incised woundAnimals were anaesthetized using Inj.Ketaminegiven intraperitoneally. An excision wound wasinflicted by cutting away approximately 500mm 2full thickness <strong>of</strong> shaved skin <strong>of</strong> a predeterminedarea on the anterior-dorsal side <strong>of</strong> each rat.Thewound was observed and cleaned with salinesoaked cotton swabs twice daily.The raw area <strong>of</strong> the wound was traced on butterpaper after inflicting the wound [day 0] and then on4 th ,8 th , 12 th and 16 th day respectively. The tracedarea on butter paper was retraced on a graph paperand the area was measured in terms <strong>of</strong> mm 2 .Thedegree <strong>of</strong> wound healing was calculated aspercentage closure <strong>of</strong> wound area as compared tothe original area using formula:Vol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 20

International Journal <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Percentage closure=1- area on day <strong>of</strong> measurement x 100Area on day[0]An average percentage wound closure area <strong>of</strong> thewound was determined and “SD” values werecalculated. The statistical analysis was performedby using student’s unpaired ‘t’ test.Histopathological studies. For histopathologicalstudies the samples were taken from the margin <strong>of</strong>the healing wound on the 7 th , 14 th , 21- st day andsent for histopathological studies. The parametersstudied were epithelization fibroblast formationinflammation and neovascularization. Onmicroscopic examination the observations weregiven numbers from 1 to 5. The number 5 impliesmaximum similarity with the normal tissuesurrounding the wound. Number 1 denotes the leastsimilarity. Less number <strong>of</strong> days were chosen toavoid interference with the healing process.RESULTS<strong>Bilwadi</strong> <strong>Churna</strong> was found to have the woundhealing property.Histopathological studies showed significantneovascularization 4 th day onwards andinflammatory response up to 7 th (??) day in the testwounds (Table 1). Epithelization was faster in thetest wound as compared to the control wound.The study showed no quantitative differences infibroblast levels in any <strong>of</strong> the days tested.The wound contractions were measured aspercentage reduction in the wound area every 4days. The progressive decrease in the wound areawere monitored periodically by tracing the woundmargin on a tracing paper an the area assessedusing a graph paper. Percentage reduction in thewound area was significantly high in the testwound (Table 2).DISCUSSIONBC contains many ingredients which are havingknown hypoglycemic action. Apart from this someingredients like Embilica <strong>of</strong>ficinalis, Azadirachtaindica, Aegle marmelos , Sesamum indicum andSyzigium cumini, have individually been reportedto have wound healing properties.In this study ,we found that wound healing timewas significantly reduced when rats received BC .Also, there was no secondary infection in woundsin BC treated groups.Mechanism by which it acts and exact contribution<strong>of</strong> the individual ingredients towards this propertycan not be commented upon.In the ancient system <strong>of</strong> ayurveda, when an excess<strong>of</strong> one taste causes a disease ,the opposite taste isintroduced in the diet with herbs and food to reducethe imbalance. Diabetes is a disease <strong>of</strong> excesssweetness so bitter herbs and food must be used tocounteract the imbalance .Neem being very bitterwas <strong>of</strong>ten used to treat diabetes in ayurveda.Modern clinical studies reveal that Neem has beeneffective in treating and preventing diabetes 1.A variety <strong>of</strong> biological activity <strong>of</strong> Azadirachtaindica (Neem) has been reported in literature.Literature revealed antifertility 2 , hypoglycaemic 3 ,wound healing 4 and analgesic 5 activity <strong>of</strong>Azadirachta indica 6 .Studies have been conducted which provides firmevidence to support that topical application <strong>of</strong>emblica represents a feasible and productiveapproach to support dermal wound healing. Duringwound healing, the wound site is rich in oxidants,such as hydrogen peroxide, mostly contributed byneutrophils and macrophages. Ascorbic acid andtannins <strong>of</strong> low molecular weight, namelyemblicanin A (2,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-2-keto-glucono-δ-lactone)and emblicanin B (2,3,4,6-bis-(S)-hexahydroxydiphenoyl-2-keto-glucono-δ-lactone)present in Emblica <strong>of</strong>ficinalis (emblica), have beenshown to exhibit a very strong antioxidant action 7 .Syzygium cumini (L.) Skeels (synonym Eugeniajambolana), popularly known as "jambolão",belonging to Myrtaceae family is one <strong>of</strong> the mostcommonly medicinal plants used to treat diabetesmellitus in Brazil 8 . Different parts <strong>of</strong> this plant,such as seeds, bark, fruit, and leaves have beenused in traditional medicine as a remedyfor diabetes mellitus in many countries 9,10 .The results obtained in this study suggest apotential application <strong>of</strong> Syzygium cumini fortreatment <strong>of</strong> skin wounds and further investigationsshould be conducted in order to explore thisapplication. Other medicinal plants containingphenolic compounds, including tannins, as majorconstituents are used topically for care and repair<strong>of</strong> skin wounds 11,12 .Preliminary studies indicate that a decoction <strong>of</strong> thedry leaves <strong>of</strong> the jamun exhibits hypoglycaemicproperty.Mahapatra et al did some preliminarystudies on the glycemic effect <strong>of</strong> jamun seeds andfound that they produce hypoglycaemic effect.Thefruit,bark and seeds have been found to possessantidiabetic properties.The seed extracts also havea significant effect on key carbohydrate metabolicenzymes such as hexokinase and glucose-6-phosphatase in diabetic rats.The hypoglycemicproperty is closely related with its antioxidantproperty 13 .Trigonella foenum-graecum L (Fenugreek) is alegume, rich in soluble dietary fiber and protein 14-16.Fenugreek seeds and its extract have exhibitedhypoglycemic and hypocholesterolemic activity inanimal and human models 17-22 . Inclusion <strong>of</strong>fenugreek in the daily diet in amounts <strong>of</strong> 25-100gm can serve as an effective therapy in the clinicalmanagement <strong>of</strong> diabetes 23. Its hypoglycemicactivity is ascribed to the presence <strong>of</strong> solubleVol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 21

International Journal <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701dietary fiber 19 saponin fraction, and 4-hydroxyisoleucine, a free amino acid 20 . Thehypoglycemic activity <strong>of</strong> fenugreek seed is also dueto glucose-dependent insulin secretion frompancreatic beta cells and soluble dietary fiber(50%) that can slow the rate <strong>of</strong> glucoseabsorption 16 .The poor acceptability <strong>of</strong> fenugreek seeds in thediet, due to its bitter taste, poses a problem 24 .Aegle Marmelos is also one <strong>of</strong> the most commonlyused plants in diabetes.Various parts <strong>of</strong> the tree,including the fruit, possess medicinal properties. Inpharmacological trials, both the fruitand root showed antiamoebic and hypoglycaemicactivities 25,26 .The aqueous extract <strong>of</strong> leaf possessesa hypoglycaemic effect 27 .The aqueous extracts <strong>of</strong> the stem and root bark areused to treat malaria, fever, jaundice, andskin diseases 28,29 .Andrographis paniculata (Burm. F.) Nees is atraditional medicinal plant belonging to the family<strong>of</strong> Acanthaceae. This annual herb is indigenous tosoutheast Asia, China and India. Andrographispaniculata (AP) has a broad range <strong>of</strong>pharmacological effects and high therapeutic value.It is claimed to possess antihepatotoxic,antibacterial, antimalarial, antihepatitis,antitrombogenic, anti-inflammatory, antipyreticproperties and has been used for the treatment <strong>of</strong>snake bites. In Malaysia, this plant has reputableuse in treating diabetes and hypertension, (30) andits aqueous extract was effective in reducing theblood glucose level <strong>of</strong> diabetic animal models 30-32 .The seeds <strong>of</strong> S. indicum L (Pedaliaceae) are usedtraditionally in the folklore for the treatment <strong>of</strong>various kinds <strong>of</strong> wounds. The present study wasundertaken to verify the effect <strong>of</strong> S. indicum seedsand its oil on experimentally induced excisionwound, incision wound, burn wound and deadspace wound models in rats 33 .The effect <strong>of</strong> diabetes on wound healing is a verycomplex issue that continues to leave manyunanswered questions. There continues to be anempiric fact that diabetic patients are at increasedrisk for the development <strong>of</strong> infection and impairedwound healing. The precise underlying defect(s)continue to be a focus <strong>of</strong> research; however, it isgenerally agreed that improved glycemic controlplays a significant role in lowering the risk <strong>of</strong>wound healing complications. Diabetic patientsneed to pay special attention to their diabetes carein general as well as to the critical illness, wound,or ulcer that has brought them to our care.Insulin is an anabolic hormone which promotesprotein synthesis and utilisation <strong>of</strong> glucose.Diabetes affects the metabolism <strong>of</strong> carbohydrates,proteins and fats, which play an important role incellular activities, proliferation, and migration andwoundhealing(Cooper,1990).Proteins are the structural units <strong>of</strong> healing wounds.Collagen and proteoglycans are the importantproteins in the context <strong>of</strong> wound healing (Deodharand Rana, 1997). A lack <strong>of</strong> insulin in diabeticwounds results in more protein degradationuncoupled from protein synthesis (Marchesini et al,1982). Collagen formation is reduced and theexisting collagen lacks tensile strength (Gottrupand Andreassen, 1981). This also adversely affectsfibroblast and polymorphonuclear (PMN) cellfunctions (Sawant, 1993).Insulin is integral to the metabolism <strong>of</strong>carbohydrates. The majority <strong>of</strong> cells--with notableexceptions being red blood cells and lens tissuecells--are dependent on insulin for the intracellularshift <strong>of</strong> glucose, which is the major source <strong>of</strong>energy for cellular functions. Fibroblasts and PMNcells require glucose to carry out their vitalfunctions in wound healing.The formulation is found to be effective in earlyhealing <strong>of</strong> wounds, which again the rural people arevery prone to because <strong>of</strong> the nature <strong>of</strong> their work,can be prove to be helpful tool in village doctorskit.So, it becomes all the more necessary forundertaking vigorous research on alternative andaffordable medicines and specially those that canbe prepared using locally available resources, thiseffort is an attempt in that direction.Table 1: Table showing the effects <strong>of</strong> BC on histologicalParameters <strong>of</strong> wound healingControl BC1 BC2 CoEpithelization 1.25 ± 0.50 3.50± 1.38 4.15± 0.76 4.25± 0.5Inflammation 4.00± 0.00 3.5± 0.55 3.95± 0.45 2.5± 0.00Fibroblast 4.00± 0.00 3.63± 0.41 3.53± 0.40 3.00± 0.00Neovascularization 3.5± 0.58 3.5± 0.55 3.72± 0.50 2.00± 0.00Vol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 22

International Journal <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701REFERENCES1. http://www.planetayurveda.com/neem_capsules.htm.2. Sharma PL. Fitoterapia.1987;58:239.3. Prakash AO. Indian Drugs. 1991;28:183.4. Kumar A, Indian J Veterinary. 1994;70(1):42.5. Lal J. Fitoterapia. 1990;61:75.6. . Kamlesh J, Wadher CL, Lakhotia and MJ.Umekar Formulation and Evaluation <strong>of</strong> Cream<strong>of</strong> Azadirachta indica leaves extracts on SkinRenewal rate International Journal <strong>of</strong> ChemTech Research. 2009;1(1):88-95.7. Sumitra Miriyala, ManikandanPanchatcharam, GayathriVinayaSubramani, Mahendran Panchatcharamand Suguna Lonchin. Emblica<strong>of</strong>ficinalis exerts wound healing action throughup-regulation <strong>of</strong> collagen and extracellularsignal-regulated kinases (ERK1/2) WoundRepair and Regeneration. 2009;17(1):99-107.8. Teixeira CC. Rava CA, Silva PM, Melchior R,Argenta R, Anselmi F, Almeida CRC andFuchs FD.Absence <strong>of</strong> antihyperglycemic effect<strong>of</strong> jambolan in experimental and clinicalmodels. J Etnopharmacol. 2000;71:343–347.9. Rahman AU and Zaman K. Medicinal plantswith hypoglycemic activity. J.Ethnopharmacol. 1989;26:1–55.10. Teixeira CC, Pinto LP, Kessler FHP, KnijnikL, Pinto CP, Gastaldo GJ and Fuchs FD. Theeffect <strong>of</strong> Syzygium cumini (L.) Skeels onpost–prandial blood glucose levels in non–diabetic rats and rats with streptozotocin–induced diabetes mellitus. J. Ethnopharmacol.1997;56:209–213.11. Dweck AC. Herbal medicine for the skin.Their chemistry and effects on skin andmucous membranes. Personal Care Mag.2002;3:19–21.12. Antimicrobial activity <strong>of</strong> Syzygiumcumini(Myrtaceae) leaves extract. BrazilianJournal <strong>of</strong> Microbiology. 2007;38(2).13. Traditional medicines <strong>of</strong> modern times :antidiabetic plant :Edited By AmalaSoumyanath.119.14. Raghuram TC. Diet and diabetes mellitus. In:Bamji MB, Rao PN, Reddy V, editors.Textbook <strong>of</strong> human nutrition. Oxford and IBHpublishing Co. Pvt. Ltd: New Delhi 1996;333-45.15. Sharma RD. Effect <strong>of</strong> fenugreek seeds andleaves on blood glucose and serum insulinresponses in human subjects. Nutr Res.1986;6:1353-6416. Ali L, Azad Khan AK, Hassan Z,Mosihuzzaman M, Nahar N and Nur-e-AlamTable 2: <strong>Effects</strong> <strong>of</strong> BC on wound contracture in RatsDay 0 4 8 12 16 20Control 0 40.17 % 74.59 % 80 % 96.32 % 100 %Test 0 57.90 % 78 % 95 % 100 % 100 %M. Characterization <strong>of</strong> the hypoglycemiceffects <strong>of</strong> serum insulin responses in humansubjects. Nutr Res. 1986;6:1353-6417. Narender T, Puri A, Shweta, Khaliq T, SaxenaR and Bhatiac G. 4-Hydroxyisoleucine anunusual amino acid as antidyslipidemic andantihyperglycemic agent. Bioorg Med ChemLett. 2006;16:293-6.18. Basch E, Ulbricht C, Kuo G, Szapary P andSmith M. Therapeutic applications <strong>of</strong>fenugreek. Alter Med Rev. 2003;8:20-7.19. Pathak P, Srivastava S and Grover S.Development <strong>of</strong> food products based onmillets, legumes and fenugreek seeds and theirsuitability in the diabetic diet. Int J Food SciNutr. 2000;51:409-14.20. Gopalan C, Ramasastri BV andBalasubramanian SC. Nutritive value <strong>of</strong> Indianfood. Hyderabad: National institute <strong>of</strong>nutrition, Indian council <strong>of</strong> medical research.1998;47-91.21. Venn BJ and Mann JI. Cereal grains. Legumesand diabetes. Eur J Clin Nutr 2004;58:1443-61. Z22. Meyer KA, Kushi LH, Jacobs DR Jr, Slavin J,Sellers TA andFolsom AR. Carbohydrates,dietary fiber and incident type 2 diabetes inolder women. Am J Clin Nutr. 2000;71:921-30.23. Fiber: Nutrition Source, Harvard School <strong>of</strong>Public Health. Available from:http://www.hsph.harvard.edu/nutritionsource/fiber.html. [L.ast accessed on 2006 Oct].24. : Preethi B, Gopalpura, C Jayanthi and SonalDubey. A clinical evaluation Effect<strong>of</strong> Trigonella foenum-graecum seeds on theglycemic index <strong>of</strong> food.. IJDDC 2007;Vol27;Issue 2;41-45.25. Ponnachan PTC, Paulose CS and Panikar KR.Effect <strong>of</strong> the leaf extract <strong>of</strong> Aegle marmelos(L.) Corr. in diabetic rats. Indian J Exp Biol.1993;31:345-7.26. Kamalakkannan N and Prince PS,The effect<strong>of</strong> Aegle marmelos fruit extract instreptozotocin diabetes: a histopathologicalstudy. J Herb Pharmacother. 2009;5:87–96.27. Das AV, Padayatti PS and Paulose CS.Effect<strong>of</strong> leaf extract <strong>of</strong> Aegle marmelos (L.) onhistological and ultrastructural changes intissues <strong>of</strong> streptozotozin induced diabetic rats.Indian J Exp Biol. 1996;34:341–5.28. Nadkarni K and Nadkarni KM. Indianmaterial Medica, 3rd edn, Popular BookDepot, Bombay, India, 2005.29. Sevugan Arumugam, Subramanian Kavimani,Balamuthu Kadalmani, Abdul Bakrudeen AliVol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 23

International Journal <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Ahmed, Mohammed Abdulkadar Akbarshaand Mandali Venkateswara Rao. Antidiabeticactivity <strong>of</strong> leaf and callus extracts <strong>of</strong> Aeglemarmelos in rabbit. Science. Asia.2008;34:317-321.30. Ahmad M and Asmawi MZ. Somepharmacological effect <strong>of</strong> aqueous extract<strong>of</strong> Andrographis paniculata Nees. In: ChanKL, editor. Proceedings <strong>of</strong> the InternationalConference on the Use <strong>of</strong> Traditional Medicineand Other Natural Products in Health-Care.School <strong>of</strong> Pharmaceutical Science, University<strong>of</strong> Science Malaysia: Penang; 1993;573.31. Husen R, Pihie AH and Nallappan M.Screening for antihyperglycaemic activity inseveral local herbs <strong>of</strong> Malaysia. JEthnopharmacol. 2004;95:205-8.32. AS Syahrin, SS Amrah, KL Chan, BY Lim, NHasenan, J Hasnan and SSJ Mohsin. Effect <strong>of</strong>spray-dried ethanolic extract <strong>of</strong> Andrographispaniculata (Burm. F.) Nees on streptozotocininduceddiabetic female rats. IJDDC. 2006;Vol26( 4):163-168.33. . Kiran K and Asad MIndian. Wound healingactivity <strong>of</strong> Sesamum indicum L. seed and oil inrats. J Exp Biol. 2008;46(11):777-82...Vol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 24