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Antiurolithic activity of Origanum vulgare is mediated through multiple pathways
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Permanent Link: http://ufdc.ufl.edu/AA00008937/00001
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Title: Antiurolithic activity of Origanum vulgare is mediated through multiple pathways
Series Title: BMC Complementary and Alternative Medicine
Physical Description: Archival
Language: English
Creator: Khan, Aslam
Bashir, Samra
Khan, Saeed R.
Gilani, Anwar H.
Publisher: BioMed Central
Publication Date: 2011
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Abstract: Background: Origanum vulgare Linn has traditionally been used in the treatment of urolithiasis. Therefore, we investigated the crude extract of Origanum vulgare for possible antiurolithic effect, to rationalize its medicinal use. Methods: The crude aqueous-methanolic extract of Origanum vulgare (Ov.Cr) was studied using the in vitro and in vivo methods. In the in vitro experiments, supersaturated solution of calcium and oxalate, kidney epithelial cell lines (MDCK) and urinary bladder of rabbits were used, whereas, in the in vivo studies, rat model of urolithiasis was used for the study of preventive and curative effect. Results: In the in vitro experiments, Ov.Cr exhibited a concentration-dependent (0.25-4 mg/ml) inhibitory effect on the slope of nucleation and aggregation and also decreased the number of calcium oxalate monohydrate crystals (COM) produced in calcium oxalate metastable solutions. It also showed concentration-dependent antioxidant effect against DPPH free radical and lipid peroxidation induced in rat kidney tissue homogenate. Ov.Cr reduced the cell toxicity using MTT assay and LDH release in renal epithelial cells (MDCK) exposed to oxalate (0.5 mM) and COM (66 μg/cm2) crystals. Ov.Cr relaxed high K+ (80 mM) induced contraction in rabbit urinary bladder strips, and shifted the calcium concentration-response curves (CRCs) towards right with suppression of the maximum response similar to that of verapamil, a standard calcium channel blocker. In male Wistar rats receiving lithogenic treatment comprising of 0.75% ethylene glycol in drinking water given for 3 weeks along with ammonium chloride (NH4Cl) for the first 5 days, Ov.Cr treatment (10-30 mg/kg) prevented as well as reversed toxic changes including loss of body weight, polyurea, crystalluria, oxaluria, raised serum urea and creatinine levels and crystal deposition in kidneys compared to their respective controls. Conclusion: These data indicating the antiurolithic activity in Ov.Cr, possibly mediated through inhibition of CaOx crystallization, antioxidant, renal epithelial cell protective and antispasmodic activities, rationalizes its medicinal use in urolithiasis.
General Note: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials
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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution.
Resource Identifier: doi - 10.1186-1472-6882-11-96
System ID: AA00008937:00001

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RESEARCHARTICLE OpenAccessAntiurolithicactivityof Origanumvulgare is mediatedthroughmultiplepathwaysAslamKhan1,2,3 ,SamraBashir2,SaeedRKhan3andAnwarHGilani2,4*AbstractBackground: Origanumvulgare Linnhastraditionallybeenusedinthetreatmentofurolithiasis.Therefore,we investigatedthecrudeextractof Origanumvulgare forpossibleantiurolithiceffect,torationalizeitsmedicinaluse. Methods: Thecrudeaqueous-methanolicextractof Origanumvulgare (Ov.Cr)wasstudiedusingthe invitro and in vivo methods.Inthe invitro experiments,supersaturatedsolutionofcalciumandoxalate,kidneyepithelialcelllines (MDCK)andurinarybladderofrabbitswereused,whereas,inthe invivo studies,ratmodelofurolithiasiswasused forthestudyofpreventiveandcurativeeffect. Results: Inthe invitro experiments,Ov.Crexhibitedaconcentration-dependent(0.25-4mg/ml)inhibitoryeffecton theslopeofnucleationandaggregationandalsodecreasedthenumberofcalciumoxalatemonohydratecrystals (COM)producedincalciumoxalatemetastablesolutions.Italsoshowedconcentration-dependentantioxidant effectagainstDPPHfreeradicalandlipidperoxidationinducedinratkidneytissuehomogenate.Ov.Crreducedthe celltoxicityusingMTTassayandLDHreleaseinrenalepithelialcells(MDCK)exposedtooxalate(0.5mM)and COM(66 g/cm2)crystals.Ov.CrrelaxedhighK+(80mM)inducedcontractioninrabbiturinarybladderstrips,and shiftedthecalciumconcentration-responsecurves(CRCs)towardsrightwithsuppressionofthemaximum responsesimilartothatofverapamil,astandardcalciumchannelblocker.InmaleWistarratsreceivinglithogenic treatmentcomprisingof0.75%ethyleneglycolindrinkingwatergivenfor3weeksalongwithammoniumchloride (NH4Cl)forthefirst5days,Ov.Crtreatment(10-30mg/kg)preventedaswellasreversedtoxicchangesincluding lossofbodyweight,polyurea,crystalluria,oxaluria,raisedserumureaandcreatininelevelsandcrystaldepositionin kidneyscomparedtotheirrespectivecontrols. Conclusion: ThesedataindicatingtheantiurolithicactivityinOv.Cr,possiblymediatedthroughinhibitionofCaOx crystallization,antioxidant,renalepithelialcellprotectiveandantispasmodicactivities,rationalizesitsmedicinaluse inurolithiasis.BackgroundUrolithiasis,theformationofurinarystones,isoneofthe oldestknowndiseases.Archaeologicalfindingsgiveprofoundevidencethathumanshavesufferedfromkidney andbladderstonesforcenturies,evenexaminationsof Egyptianmummieshaverevealedkidneyandbladder stones[1]. Itisthethirdmostcommonproblemoftheurinary tractwithanestimatedlifetimeriskof2-5%inAsia, 8-15%inEuropeandAmericaandaround20%inthe MiddleEast.Itisassociatedwithhighrateofrecurrence, whichisaround10-23%peryear,50%in5-10yearsand 75%in20years.Onceafflicted,thesubsequentrelapse rateisincreasedandtherecurrenceintervalisshortened [2-5].Moreover,itsannualincidencesareincreasingand alsotheageofonsetisdecreasing,perhapsduetochange inlifestyles,dietandclimate[6]. Improvementintherapyofkidneystoneswithmodern techniqueslikeextracorporealshockwavelithotripsy (ESWL),ureteroscopy(URS),andpercutaneousnephrolithotomy(PNL),hasrevolutionizedtheurologicalpractice buttherecurrenceinkidneystoneisnotaltered.ESWLis lesseffectiveincalciumoxalatemonohydrate(COM)and cystinestonesthancalciumoxalatedihydrate(COD)and uricacidstones[7].Inadditiontothehighcastandthe *Correspondence:anwar.gilani@aku.edu Contributedequally2NaturalProductResearchDivision,DepartmentofBiologicalandBiomedical Sciences,AgaKhanUniversityMedicalCollege,Karachi-74800,Pakistan FulllistofauthorinformationisavailableattheendofthearticleKhan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 2011Khanetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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issuesofrecurrencewiththesemeasurestherearemultiplesideeffects,whichincluderenaldamage,ESWL inducedhypertension,renalimpairment,severhaematuria, steinstrasse(multiplesmallstoneblockingureter), pancreatitis,infectionandpersistentresidualfragmentsas potentialnidusfornewstoneformation.Whilethesecomplicationscanleadtolargeperfusionofthecollectingsystem,extravasationsofirrigatingfluid,urosepsis,ureteral injuryanddelayedbleedingmayalsooccur[8,9].Pharmacologicalagentsincludealimitedchoicelikecitrateand thiazidediuretics,whichhavelimitedefficacyinaddition totheirlesstolerability[10,11].Ontheotherhand,there isgrowinginterestofpublicinherbalmedicine,particularlyinthetreatmentofurolithiasispartlybecauseoflimitedchoiceinthepharmacotherapy.Moreover,herbal remediesareknowntocontainmultipleconstituents, actingthroughmultiplepathwaysneededinurolithiasis, forexample,antispasmodic,diuretic,painrelieving [12,13].Inthisstudy,weevaluated Origanumvulgare L foritsantiurolithicactivity,inanattempttorationalizeits folkloricuseinurolithiasisandtoseeifitexhibitscalcium oxalatecrystallizationinhibitory,antioxidant,renalcell protective,antispasmodicanddiureticactivities,whichare likelytocontributeinitsantiurolithiceffect. Origanum vulgare Linn(family,Lamiaceae)isdistributedthroughout Asia,EuropeandNorthAmerica[14]andiscommonly knownasWildMarjoramandWinterSweetandlocally inPakistanasMirzanjosh,Sathra[15,16].Itiswidelyused inthetraditionalmedicineaslithotriptic,diureticand antispasmodicalongwithothermedicinaluses,suchas stimulant,expectorant,antibacterial,anticancer,antiinflammatory,antioxidantandlaxative[15-17].MethodsChemicalsandreagentsAllchemicalusedwereofanalyticalgrade.Listand sourcesofchemicalusedinthestudyareprovidedinthe additionalfile1.AnimalsExperimentswereperformedincompliancewiththerulingsoftheInstituteofLaboratoryAnimalResources, CommissiononLifeSciences,NationalResearchCouncil [18]andapprovedbytheEthicalCommitteeforResearch onAnimals(ECRA)oftheAgaKhanUniversity,Karachi, Pakistan. Wistarrats(180-220g)ofeithersexusedforthisstudy weresourcedlocallyandhousedattheanimalhouseof theAgaKhanUniversity,keptinplasticcages(4734 18cm3)withsawdust(renewedafterevery48hrs),under acontrolledtemperatureof23-25Cand12hrslight-dark cycle.Animalshadaccesstofoodandwaterad-libitum throughoutthestudy.How ever,foodwaswithdrawn 24hrsbeforeandduring6hrsofdiureticstudy,andwhile collecting24hrsurinesamples.PlantmaterialandextractionTheplantof Origanumvulgare wascollectedfromthe NorthernAreasofPakistan,identifiedbytaxonomistProf. Jhandarshah,Vicechance llorShaheedBenazirBhutto University,KhyberPakhtun khwa,Pakistanandvoucher specimen(OV-PL-02-08-72)wassubmittedtotheherbariumoftheDepartmentofBiologicalandBiomedical Sciences,theAgaKhanUniversity,Karachi.Theaerial partoftheplantmaterialwascleanedofadulterantsand keptsoakedforthreedays intheaqueous-methanol (30:70)withoccasionalshaking,atroomtemperature.The filtrationwascarriedoutu singamuslinclothandthen throughWhatmanqualitativ egrade1filterpaper.This procedurewasrepeatedtwiceandthenallthefiltrates obtainedwerecombinedandconcentratedonarotary evaporator(RE-111,Buchi,Flawil,Switzerland)accompaniedwithB-700recirculationchillerandawaterbath model461at40Ctoathickpastymasscalledascrude extract(Ov.Cr),yieldingapproximately12%[19,20].PreliminaryphytochemicalanalysisThecrudeextractof Origanumvulgare ,wasscreenforthe presenceofdifferentphytochemicalgroupssuchasalkaloids,saponins,coumarins,sterols,terpenes,tanninsand flavonoidsbyusingmethodsfollowedpreviousstudies [21,22].Invitro experiments InvitrocrystallizationstudiesKineticstudy Theeffectofthetestmaterialonkinetics ofcalciumoxalate(CaOx)crystallizationwasdetermined bythetimecoursemeasurementofturbiditychanges duetothecrystalnucleationandaggregationaftermixingmetastablesolutionsofcalcium(Ca++)andoxalate (Ox).StocksolutionsofCaCl2(8.5mM)andNa2C2O4(1.5mM),containing200mMNaCland10mMsodium acetatewereadjustedtopH 5.7[23].Anaggregometer (Chrono-LogCorporation,USA)devisedforplatelet aggregationstudiesbasedonthemeasurementofoptical densityat620nmwasusedtoinvestigatetheeventof CaOxcrystallization[24].T heslopesofnucleation(SN) andaggregationphases(SA)werecalculatedusinglinear regressionanalysis.Usingtheslopes,thepercentageinhibitionwascalculatedas[(1 -Sm/Sc)100],whereSmis slopeinthepresenceofmodifier;K.CitorOv.Cr,andSc isslopeofthecontrolexperiment. Incubationstudy Todeterminetheeffectofincubation onCaOxcrystalformation,stocksolutionsofCaCl2and Na2C2O4similartothoseinthekineticstudywereused. CaCl2solutions,containingdifferentconcentrationsoftheKhan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page2of16

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Ov.Crorpotassiumcitrate,werealiquoted(0.5ml)tothe flat-bottomedtubesina24wellplate(IwakiMicroplate withlid,ScitechDiv.,AsahiTechnoGlass,Japan).Toeach ofthesetubessodiumoxalate(Na2C2O4)solution(0.5ml) wasadded[25].Theplateswerethenincubatedinashakingwaterbathatthe90oscillations/minatatemperature of37Cfor45minutes.Abundanceandthemorphology ofthecrystalsineachtubewerethenobservedunder invertedmicroscope(NikonCorporation,Tokyo,Japan).DeterminationofantioxidantactivityAntioxidantpotentialofthetestmaterialswasestimated invitro byfreeradicalscavengingandlipidperoxidation inhibitoryactivities.FreeradicalscavengingactivityForfreeradicalscavengingactivity,a0.1mMsolutionof 2,2-Diphenyl-1-picrylhydrazyl(DPPH)radicalinmethanolwaspreparedand1mlofthissolutionwasaddedto 3mlofthetestmaterialatdifferentconcentrationspreparedinmethanol[26].Solutionswereincubatedfor 30minatroomtemperatureandthenabsorbancewas measuredat517nm.DecreasingoftheDPPHsolution absorbanceindicatesanincreaseoftheDPPHradicalscavengingactivity.LipidperoxidationinhibitoryactivityToassesslipidperoxidationi nhibitoryactivity,thekidneysisolatedfromWistarratwerehomogenizedwith electrichomogenizer(Zero-Max),inicecoldPBS (50mmol/l,pH7.4)asdescribedearlier[27].Celllines(MDCK)ExperimentMDCKcellsweremaintainedassub-confluentmonolayersat37Cin5%CO2.Thecultureweregrownin 75cm2Falcontissuecultureflasksina1:1DMEM nutrientmixtureandF-12medium(DMEM/F-12)containing10%FBS,2%streptomycin/penicillin,pH7.4. Themediumwaschangedevery3to4days.XTTassayforcellviabilityMediawasaliquotedtodesignatedwells,containingconfluentMDCKcells,ofa96wellplate.TheXTTCellViabilityAssayKitwasusedtodeterminethecellviability. Cellswereincubatedwithandwithout(Control)plant extractsfor24hrs.Then50 Loftheworkingactivation solution,preparedbymixingofonebottleofXTTsolutionandoneoftheactivationreagentPMSsuppliedwith kit,wasaddedtoallthesamplesandcontrol,andincubatedfor2-4hrs.Opticaldensityabsorbencywasreadat 450-490nmonaBio-Rad3550microplatereader(BioRad,Hercules,CA).LactateDehydrogenase(LDH)ReleaseMediawasaliquatedtodesignatedwellsofa96well plate.TheCytoTox96Non-RadioactiveCytotoxicity assaykitwasusedtodetermineLDHpercentrelease. Substrate(suppliedwithkit)wasaddedtoallsamples, positivecontrol(MDCKcells lysedwithlysissolutionsuppliedwithkit),andblanks(acclimazationmedia).The platewasincubatedatroomtemperaturefor30minutes inthedark.Stopsolution(suppliedwithkit)wasadded toallsamples,positivecontrol,andblanks.Opticaldensityabsorbencywasreadat490nmonaBio-Rad3550 microplatereader(Bio-Rad,Hercules,CA).AntispasmodicactivityAntispasmodicactivityoftheplantextractwasevaluated againstcarbachol(CCh)andhighK+(80mM)-induced contractionsinstripsofrabbiturinarybladder[28].The wholeurinarybladderofrabbitwasdissectedout,and dividedinto3-4verticalstrips.Eachpreparationwas mountedina10-20mltissuebathcontainingKreb sHenseleitsolution,maintainedat37Candcontinuously aeratedwithcarbogen.Atensionof1gwasappliedto eachtissuethroughouttheexperiment.Isometric responseswererecordedonaGrassModelPolygraph and/orPowerlab4/24dataacquisitionsystemattached tocomputerrunningChart5.3software(ADInstruments,Sydney,Australia). Thetissueswereallowedto equilibrateforaperiodofabout1hrbeforetheaddition ofanydrug,duringwhichthetissuewaswashedwith freshbathingfluidatanintervalofevery15-20minutes. Followingtheequilibrium,preparationswerestabilized,byrepeatedlytreatingwith1 MCCh,untilconstantresponseswererecorded.Thenthespasmolytic activityoftheplantextract wasdeterminedbyadding theplantextractsonCChorhighK+-inducedsustained contractioninrabbitbladder,inacumulativefashionto obtainconcentration-dependentinhibitoryresponse [29].IC50values(concentrationcausing50%inhibition) werecalculatedfromthesecurvesandcalculatedasa measureofspasmolyticpotencyoftherelaxantdrugs.CalciumchannelblockingactivityTheconcentration-responsecurves(CRCs)ofCa++were constructedintheabsenceandpresenceofincreasing concentrationsoftestdrugtoconfirmtheCa++antagonist actionofthetestsubstance.ThetissuewasallowedtostabilizeinnormalKreb s-Henseleitsolution,whichwasthen replacedwithCa++-freeKreb s-HenseleitsolutioncontainingEGTA(0.1mM)for30mininordertoremoveCa++fromthetissues.ThissolutionwasfurtherreplacedwithK+-richandCa++-freeKreb s-Henseleitsolution.Following anincubationperiodof30min,CRCsofCa++wereconstructedintheabsenceandthepresenceofdifferentconcentrationsofthetestmaterials[30].Isometricresponses wererecordedonaGrassMo del7Polygraphand/or Powerlab4/24dataacquisitionsystemattachedtocomputerrunningChart5.3software(ADInstruments, Sydney,Australia).Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page3of16

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Invivo experiments DeterminationofdiureticactivityThediureticactivityofthetestmaterialwasstudiedon Wistarratsofeithersex(180-220g)asdescribedpreviously[31].Animalsweredividedwithmatchedbody weightandsexintogroupsof6animalseach.Normaland positivecontrolgroupsweregivenbygavagessaline (20ml/kg)andstandarddiuret icdrug:hydrochlorothiazide(HCT),10mg/kgofbodyweight,respectively.The restofthegroupsweregivendifferentdosesofthetest materialdissolvedinsaline.Subsequently,theanimals wereplacedindividuallyinmetabolicanddiureticcages. Theurinewascollectedingraduatedcylindersfor6hrsat 2hrsintervals.Totalurineexcretedoutwascollectedand thevolumewasdetermined.StudyonanimalmodelofurolithiasisAntiurolithicactivityofthetestmaterialwasdetermined usinganimalmodelofCaOxurolithiasis[32,33].Male Wistarrats(weighing180-220g)weredividedwith matchedbodyweightsintogroupsof6-8animalseach, whichwerethenrandomlyselectedtoreceivevarious treatmentsforpreventiveandcurativestudy. Preventivestudymodel Tostudythepreventiveeffectof thetestmaterial,ratsservingasnormalcontrol,received intra-peritoneal(i.p.)injectionsofnormalsaline(2.5ml/ kg),oncein24hrs.Whereas,untreatedgroupreceivedi.p. injectionofnormalsaline(2.5ml/kg)alongwithrenal CaOxcrystaldepositsinducing(lithogenic)treatmentfor 21days.TherenalCaOxdepositsinductionwasachieved bygivingwatercontaining0.75%(w/v)ethyleneglycol (EG)and1%(w/v)ammoniumchloride(AC)for5days, followingthisthewatersupplywasswitchedto0.75%EG alone[32,33].Treatedgroupsreceivedi.p.injectionofthe extractdissolvedinsalineoncein24hrsandsimultaneouslyreceivedcrystaldepositsinducingtreatmentsimilartotheuntreatedgroup.Basedonthemedicinaluse andliteratureavailableon Origanumvulgare ,wheredoses of20-60mg/kghavebeenusedinrats[34,35],weused logdoses10and30mg/kgforitsdiureticandantiurolithic effect,optimizedinourpilotstudy.Animalweightand activitywereregularlymonito redtoassesstheiroverall healthandthoselookinglethargicorwhohavelostexcessiveweightwereexcludedfromthestudy.Waterintake wasdeterminedand24hrsurinesampleswerecollected immediatelybeforetheonsetandattheendoftotal21 daysoftreatment,forwhichanimalswerehousedindividuallyinmetabolicanddiureticcages.The3hrsmorning urineforcrystalluriastudywasalsocollectedbeforecollecting24-hrsurineattheendof21daysoftreatments. Thenumberofthecrystals/mm3wascountedunderlight microscopeusinghaemocytometer. Curativestudymodel Inthestudyforthecurative effect,CaOxdepositswereinducedinthekidneysof ratsinbothuntreatedandtreatedgroupswithEG (0.75%)andAC(1%)treatmentfor21days,following theplanasgivenwiththestudyonthepreventiveeffect. Thereafter,lithogenictreatmentwaswithdrawnand treatmentwithvehicleandthetestmaterialwasrespectivelystartedtotheuntreatedandthetreatedgroupsfor anotherperiodof14days[33].Normalcontrol,received notreatmentforthefirst21daysstudyperiod,but receivedi.p.injectionsofnormalsaline(2.5ml/kg),duringthenext14days.Animalweightsandtheiractivity wereregularlymonitored,whereas,24hrsurinesamples werecollectedimmediatelybeforeandafterthecrystals depositsinduction(lithogenic)treatmentandattheend oftotaltreatmentperiod(35days).3-hrsurinesample wascollectedafterboth21and35days. FollowingvolumeandpHdetermination,24hrsurine sampleswerestoredat-20Cuntilanalyzed.Bloodwas collectedthroughcardiacpuncturefromanimalsunder etheranaesthesiaforserumseparationinordertoassess serumcreatinineandbloodureanitrogen(BUN).Animals weresacrificedandthekidneyswerefixedin10%neutral bufferedformalin,processed,embeddedinparaffinwax, sectionedat5 mandstainedwithHaematoxylinand Eosin(H&E)andbyPizzolato smethod,forcalcium oxalatecrystals[36],formicroscopicexamination.CalciumoxalatecrystaldepositioninkidneyCrystaldistributionwithinthekidneyswasdetermined byusingthesemiquantitaivescoringbythemethods usedbyVanachayangkuletal.,[37].Brieflythecrystal depositsinstainedsectionswithvisibleinafieldof10 magnificationwerecountedandseveritygradeswere assignedas0=<1crystals,1= 10,2= 30,3= 50, 4= 75and5=>75crystals.Mostofthecrystalswere locatedintheoutermodularlyandcorticalregionofthe kidney.BiochemicalanalysisofurineandserumUrinesamplesforOx,Ca++andMg++,citrate,anduric acid(UA)andserumsamplesforcreatinineandBUN contentsweredeterminedby usingcommerciallyavailablekits(Companynamesforkitsaregivenintheadditionalfile),whileurinaryinorganicphosphateand proteinweredeterminedbyusingthemolybdenumblue reaction[38]andLowrys[39]methodrespectively.DataAnalysisThedataexpressedaremeanstandarderrorofmean (SEM)andthemedianeffectiveconcentration(EC50value)with95%confidenceintervals(CI).Allstatistical comparisonsbetweenthegroupsaremadebymeansof t-test(comparisonbetweentwogroups)orOneWay AnalysisofVariance(ANOVA)withposthocDunnett sKhan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page4of16

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test. p valuelessthan0.05isregardedassignificant. CRCswereanalyzedbynon-linearregressionusing GraphPadPrism(GraphPadSoftware,SanDiego,CA, USA).ResultsPhytochemicalScreeningsThephytochemicaltestshowedthepresenceofsaponins,alkaloids,coumarins,sterol,terpenes,flavonoids andtanninsinOv.Cr.Invitro Experiment EffectoninvitrocrystallizationKineticstudy EffectofOv.CronvariousphasesofCaOx crystallizationasdeterminedbytimecoursemeasurement ofturbidityunderstandardconditions(4.25mMCa++and 0.75mMOx)isgiveninFigure1,whichshowstypicaltracing(Figure1Aand1B)oftheexperimentinthepresence ofOv.Crandpotassiumcitrate.Inthecontrolcurve,the initialriseinturbidity;the nucleationphase ,onattaining itsmaximumafterabout15015sec,followedbyaslow Figure1 CalciumOxalatecrystallizationstudy .Effectof Origanumvulgare (Ov.Cr)andPotassiumCitrate(k-Cit)oncalciumoxalate crystallization.(A)and(B)arethetypicaltracingofthecontrolandinthepresenceofOv.Crandpotassiumcitrate.Panel(C)isconcentrationresponsecurvesofOv.CrandpotassiumcitrateonSAoftheturbiditycurves,while(D)showsthe%inhibitionontheSN.Symbolsshownare meanS.E.M.(n=3).SNandSArepresentslopeofnucleationandslopofaggregationrespectively. Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page5of16

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decrease;the aggregationphase .Ov.CrinhibitedtheSAwithamedianinhibitoryconcentrationof0.47mg/ml (0.31to0.69;95%CI),similartopotassiumcitratewhich causesinhibitionwithIC50valueof0.42mM(0.31to0.58; 95%CI)asshowninFigure1C.Ov.Crcaused10.73, 162.3,201.7and352.8%inhibitionofSNatthe concentrationof0.5,1,2and4mg/ml,whilethecitrate caused201.7,312.31,424.0and654.2%inhibitionataconcentrationof0.5,1,2and4mM,respectively (Figure1D). Incubationstudy Intheincubationstudy,mixingthe metastablesolutionsofCa++andOxresultedintheformationofCaOxcrystals,predominatelyofthedumbbell shapedcalciumoxalatemonohydrate(COM).Ov.Cr causedaconcentration-dependent(1-4mg/ml)decrease intheCaOxcrystalformation(Figure2A&2B)andalso decreasedthenumberandsizeofCOMcrystals;similarly citratealsodecreasedthenumberandsizeofcrystals formed(Figure2C&2D).AntioxidantactivityFreeradicalscavengingactivity Ov.CrcausedinhibitionofDPPHfreeradicalwithIC50valueof6.28 g/ml (5.41-7.27;95%CI),whilethecontroldrugbutylated hydroxytoluene(BHT)inhibitedDPPHwithIC50value of3.41 g/ml(3.16-3.68;95%CI)asshowninthe Figure3A. Lipidperoxidationinhibitoryactivity Ov.Crinhibited the invitro lipidperoxidationinducedinratkidney Figure2 CalciumOxalateincubationstudy .Representativephotographs,underinvertedmicroscope(200x),ofCaOxcrystalsdevelopedinthe metastablesolutionsintheabsence(AandC)andinthepresenceofcrudeextractof Origanumvulgare (Ov.Cr)2mg/ml(B)and2mMK-Citrate (D). Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page6of16

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homogenateby69.34.7%and84.15.0%(Figure4B), whileBHTcaused30.82.6and71.63.8%inhibition oflipidperoxidationat50and150 g/ml,respectively (Figure3B).EffectonKidneyEpithelialCellLines(MDCK)Effectoncellviability Ov.Crhadnotoxiceffecton MDCKcellsupto0.1mg/ml.H owever,itsignificantly ( p <0.01)reducedthecellviabilityathigherconcentrations(Figure4A). EffectOv.CronMDCKcellsafterexposuretoOxalate andCOMcrystals Thecellviabilitywasdecreased( p < 0.001)afterexposureto0.5mMOxor66 g/cm2of COM.However,aftertheco-exposureofOv.Cr,thecell viabilitywasmildlyincreasedat0.03mg/mlwhilesignificantly( p <0.01)increasedascomparedtoOxorCOM ataconcentrationof0.1mg/ml(Figure4Band4C). EffectofOv.Croncellmembranedamage LDHrelease wassignificantlyincreased( p <0.001)afterexposureto 0.5mMOxor66 g/cm2COMvs.untreatedcontrol. However,co-exposureofOv.Cr(0.1and0.3mg/ml)significantlydecreasetheLDHrelease(Figure4Dand4E).EffectonUrinarybladderOv.Crcausedconcentration-dependentinhibitionof bothCCh(1 M)andhighK+(80mM)-inducedcontractionsinrabbiturinarybladderpreparations(Figure 5A)withanEC50valuesof0.061(0.04-0.08)and0.068 mg/ml(0.05-0.08)respectivel y.Similarly,verapamilwas foundtobemorepotentagainsttheK+thantheCChinducedcontraction.ItrelaxedbothCChandK+-inducedcontractions(Figure5C)withIC50valuesof 0.08 M(0.07-0.10)and0.04 M(0.03-0.5)respectively. Ov.Cr(0.03-0.1mg/m1)causedrightwardshiftofthe Ca++CRCsaccompaniedbysuppressionofthe maximumcontractileeffect,similartothatcausedby verapamil(0.01-0.03 M),asshowninFigure5Band 5D.Invivo experiments DiureticeffectOv.Crincreasedsignificantly( p <0.05)theurineoutput inWistarratsatthedoseof10mg/kg,whiletherewas noaffectseenatlower(3mg/kg)orhigherdose (30mg/kg).HCT(10mg/kg)wasusedasreference drug,whichsignificantly( p <0.01)increasedtheurine output(Figure6).EffectonanimalmodelofUrolithiasisPreventiveeffect Inpreventivestudy,alltheparameters, likebodyweights,24hrswa terintake,urinevolume, urinarypHandcomposition,recordedbeforethetreatmentwerenotsignificantlydifferentamongthegroups. Theparametersrecordedatday0andattheendof3 weeksoftreatmentperiodarelistedintheadditional file2. Inthe3-hrsmorningurinesampleofrats,significantly moreandbiggerCaOxcrystalsmostlyofCODwere observedinlithogenicgroupascomparedtothesaline group.Whereas,Ov.Crsignificantlyreducedurinarycrystalcountaswellasdecreasedcrystalsize(Figure7).Atthe endofthetreatmentasignificant( p <0.01vs.Normal) lossinbodyweightswascausedbytheEGandACconsumptioninthelithogenicgroupascomparedtothenormalsalinegroup.Theco-administrationofOv.Cr(10-30 mg/ml)preventedthelossinbodyweightsofrats( p < 0.01vs.lithogenicgroup).24hrsurinevolumeandwater intakewerehigher( p <0.01)inthelithogenicgroupcomparedtothatofnormalsalineanimals.UrinepHwasalso Figure3 Antioxidantactivity .Concentration-responsecurvesofthefreeradicalscavengingactivityofthebutylatedhydroxytoluene(BHT)and Ov.Cr,whilebar-chart(B)representinglipidperoxidationinhibitoryactivityoftwodifferentconcentrationsofOv.CrandBHT.Inhibitionis measureas%oftherespectivecontrolexperiments.ThevaluesshownaremeanSEM(n=3). Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page7of16

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Figure4 EffectonMDCKcells .EffectsofvariousconcentrationsofOv.CronMDCKcellsurvivalinacclimatizationmedia(A).BandCshowthe protectiveeffectofOv.Crafterexposureto0.5mMoxalateor66mg/cm2COMrespectively.While(D)and(E)showsthepercentincreasein LDHreleaseagainstcontrolbyMDCKcellsexposedOx.(0.5mM)andCOM(66 g/cm2)for24hrs.DatashownaremeanSEMoftwoseparate experimentswith3independentreplicates.* p <0.05,** p <0.05and*** p <0.001 Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page8of16

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reduced,thoughnottoasignificantextent.Aco-treatmentwithOv.Crsignificantlyreduced( p <0.05)polyurea andwaterintakecomparedtolithogenicgroup.Similarly, oxalateexcretionwassignificantlyincreased( p <0.01)in lithogenicanimals,whereasCa++excretionwasdecreased ( p <0.05).Urinecontentsofcitrate,phosphate,UA,Mg2 +,Na+andK+didnotaltertoasignificantlevel.CoadministrationofOv.Cr(10-30mg/kg)tolithogenicgroup significantly( p <0.05)decreasedoxalateexcretion, whereasurinaryexcretionofcitrateandCa2+wassignificantlyincreased( p <0.05).EGtreatmentcausedimpairmentofrenalfunctionsoftheuntreatedrats(lithogenic group)asevidentfromtotalproteinlossandraisedBUN andserumcreatinine( p <0.05),whichwerepreventedin theanimalstreatedwithOv.Cr.(pleaseseeadditionalfile 2). Kidneysexcisedfromlithogenicgroupwereenlarged. Histologicalpreparations ofkidneysofnormalsaline Figure5 Antispasmodiceffect .Concentration-responsecurvesofcrudeextractofOv.Cr(A)andverapamil(B)onK+(80mM)andCCh(1 M)inducedcontractionsandtheconcentration-responsecurvesofCa++constructedintheabsenceandpresenceofincreasingconcentrationsof Ov.Cr(C)andverapamil(D)inisolatedrabbiturinarybladder.ThesymbolsrepresentmeanSEM(n=4-6). Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page9of16

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groupdidnotshowanycrystallinedeposits,whereas,a highscoreofcrystallinedepositswasobservedinall regionsofkidneysinthelithogenicgroup.However,Ov. CrtreatmentsignificantlyloweredtheCaOxcrystal deposits( p <0.5)ascomparedtountreated(lithopgenic)group(Figure8and9A). Curativeeffect Inthecurativestudy,alltheparameters wererecordedbeforestartofthetreatment(day0), after3weeksofthecrystalsdepositsinduction,and thentwoweeksafterthewi thdrawalofthecrystals depositiontreatment(0.75%EG+1%ACindrinking water).Theparametersrecordedforthecurativestudy aregivenintheAdditionalfile3. Thegroups,whichconsumedthelithogenictreatment forthefirst21days,developedthelithogenicparameters ascomparedtothenormalsalinegrouplikeinthepreventivestudy.Thiswassuggestedbyanetlossinthe bodyweights,asignificantincreasein24hrswater intake,urinevolume,oxalate,uricacid,totalprotein, anddecreasedurinarypHandCa++contents,impaired renalfunctionsuggestedbyincreasedserumcreatinine, BUNandtotalurinaryproteinloss(Additionalfile3). Post-inductiontreatmentwithOv.Cr(10and30mg/kg) reversedthelossinbodyweights,impairedurinaryand serumfunctions,crystalluriaanddepositionofcrystals inthekidneymorequicklythanthecontrolgroup (Additionalfile3). AftertwoweeksofOv.Crtreatmentinthecurative study,renalCaOxcrystaldepositswerefoundin4out of6rats.However,theyweresignificantlyless( p <0.5) thantheuntreatedgroup(Figure9BandFigure10).DiscussionInviewofthemedicinalusesof Origanumvulgare in urolithiasis,weevaluateditscrudeextractforthepossibleantiurolithiceffectalon gwithantioxidant,antispasmodicanddiureticactivitiesusingthe invitro assays and invivo ratmodels. Inthisstudy,theplantextractinhibitedtheCaOx crystalnucleationandaggr egationinaconcentrationdependentmanner,similartocitrate,awell-knowninhibitorofCaOxcrystallizationandclinicallyusedforthe managementofurolithiasis[40].Similarly,intheincubationstudy,Ov.Crcausedadecreaseincrystalcount andtransformedCOMtoCODcrystallikethatof citrateandMg2+[41].COMcrystalsareconsideredto bemoreharmfulthanCODbecauseoftheirtendency toattachwiththemembranetoformaggregates[42] andaremorelikelytoattach withthekidneyepithelial cellsthanCaOxdehydrate,resultingintheformationof kidneystones[42,43].Calcif icationisamultifactorial Figure6 Diureticeffect .EffectoftheOv.Crand hydrochlorothiazide(HCT)onurinevolumecollectedin6hrs (valuesshownaremeansSEM,n=6-8).* p <0.05,** p <0.01 Figure7 ImagesofCrystalluria .Imagesofcalciumoxalatecrystalsin3hrsmorningurinecollectedfromNormalcontrol(A),Lithogenic Control(B)andtreatedwithcrudeextractof Origanumvulgare (Ov.Cr)(C),underlightmicroscopeat400magnification. Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page10of16

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phenomenon[44],arisingasaresultofacascadeof eventsinitiatedbysupersaturation,includingcrystal nucleation,growth,aggregationandretention[45]. Althoughsupersaturationisnottheonlycriticalstep involvedintheformationofkidneystonesasseveral studieshaveidentifiedmanyinhibitorsofcalciumoxalateandcalciumphosphatec rystallizationincluding ionicormacromolecules[46] ,yetitistheprerequisite Figure8 MicroscopicimagesofKidneysectionsinpreventivestudy .RepresentativemicroscopicimagesoftheHandEstainofthekidney sectionsfromnormal(A),Lithogenicgroup(B)andTreated(C)withOv.Cr.A1,B1andC1showthepolarizedimagesofthesections. Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page11of16

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forthecrystalsformationintheurinarytract[47].Variouscrystalinhibitorslikepotassium-sodiumcitrateand magnesiumoxidehavebeenshowntodecreasethe saturationofCaOxandinhibitcrystalnucleation, growthandaggregationandreducedcrystallizationin urineofstoneformingpatients[48].Interferencewith crystalgrowthandaggregationthereforeseemsapossibletherapeuticstrategyforthepreventionofrecurrent stonedisease.Ov.CrinhibitsCaOxcrystalnucleation andaggregationalongwithdecreaseincountandmorphologicalchange,fromCOMtoCOD,incrystals. TheseresultsindicatethepresenceofCaOxcrystal aggregationinhibitoryconstituent(s)intheplant. Animalandcellularstudieshaverevealedthatoxalate, calciumoxalateandhydroxyapatitecrystalscauseinjury tokidneycells[49,50],causedbytheproductionof reactivespecies(ROS)whichisconsideredtobetherisk factorforthecrystallizationandcrystalsdepositionin thekidneybypromotingcrystalnucleation,aggregation, retentionandstonedevelopment[49,51].Antioxidants suchasvitaminE,catechinandseleniumhavebeen showntoprotectagainstoxidativeinjurybyoxalateand crystaldeposition[52,53].Whenstudiedforitsantioxidantactivity,Ov.CrcausedscavengingofDPPHfree radicalandinhibitedferrous-ascorbate-inducedlipid peroxidationofratkidneyhomogenatesimilartoBHT, astandardantioxidant[54],confirmingitsantioxidant activity[35,55,56]. Cytoprotectiveeffectoftheplantwasconfirmedwhen pre-treatmentofthenormalkidneyepithelial(MDCK) cellswithOv.Crsignificantlyincreasedthesurvivalrate andreducedtheLDH,amarkerofcellmembrane damage[57],releaseofMDCKcellsexposedtoOxand COMcrystals.Thisprotectiveeffectcouldbetheresult ofitsantioxidantactivity. Antispasmodicsaremorecommonlyusedinthemedicalexpulsivetherapy(MET)forthekidneystones[58]. Administrationofalpha-adrenoreceptorantagonistsor Ca++channelblockersenhancestheexpulsionrateof crystalsandreducescolicevent[59].Therefore,weevaluatedtheantispasmodiceffectofOv.Cr,andstudiedits relaxanteffectagainsthighK+(80mM)andCCh(1 M)inducedcontraction,usingrabbiturinarybladder strips.HighK+(>30mM)isknowntocausesmooth musclecontractionthoughopeningofvoltagedepended L-typecalciumchannels,thus allowinginfluxofextracellularCa++resultinginthecontractionofthesmooth muscles[20,60]andasubstancethatrelaxthehighK+inducedcontractionisconsideredtobeCCB[61]. Whereas,CChisacholinergicdrug,whichcaninduce contractioninurinarybladderthroughactivationof muscarinicreceptors,predominatelyM3subtype[62]. Ov.CrrelaxedthehighK+andCChinducedcontraction likethatofverapamil,astandardCCB[63],indicating CCBactivity,whichwasconfirmedwhenpre-treatment ofthetissuewiththeplantmaterialshiftedtheCa++CRCtotherightwithsuppressionofthemaximum response,likethatofverapamil. Inthe invivo experiment,Ov.Crcausedsignificant increaseinurineoutputatthedoseof10mg/kgsimilar tothatcausedbyHCT,astandarddiuretic,while,the administrationofnexthigherdose(30mg/kg)didnot causedanysignificantchangeinthediureticeffect, comparedtothesalinecontrol,whichcouldbedueto theco-existenceofanti-diureticcomponent(s)inOv.Cr, asplantextractmayexhibitmultipletherapeuticactivitiesprobablyonaccountofhavingamixtureof Figure9 CrystalsdepositsscoreinKidneysectionsof preventiveandcurativestudy .Calciumoxalatecrystaldeposition scoreaftertreatmentwith0.75%EG,1%NH4Cl(lithogenicgroup), Ov.Cr10and30mg/kginpreventive(A)andcurative(B)study model.Severitygradewereassignedas0=<1crystals,1= 10,2 = 30,3= 50,4= 75and5=>75crystals;dataare expressedasmeanSEM.* p <0.05,** p <0.05and*** p <0.001 vs.Normal,#p <0.05,##p <0.01and###p <0.001vs.Lithogenic group. Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page12of16

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phytochemicals.Thepresenceofsynergisticand/orsideeffectneutralizingeffectinplantsisknowntoexist[12], whichisprobablymeantbynaturenottoallowthe pharmacologicaleffectgobeyondacertainlimit,beyond whichitcouldhavebeenharmful.Diureticsincrease urinevolume,whichresultsinthereductionofsupersaturationofcrystalsformingsaltsandalsohelpinthe expulsionofalreadyformedcrystals[64,65]. Figure10 MicroscopicimagesofKidneysectionsincurativestudy .RepresentativemicroscopicimagesoftheHandEofthekidney sectionsfromnormal(A),Lithogenicgroup(B)andTreated(C)withOv.Cr.A1,B1andC1showthepolarizedimagesofthesections. Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page13of16

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TheantiurolithiceffectofOv.Crwasevaluatedonthe mostcommonlyappliedEG-inducedmodelforurolithiasis[66-68]andmaleWistarrats,whichdevelop changesinurineelectrolytesandCaOxsuper-saturation toagreaterextentduetotheirgreatersensitivitytoEG toxicity[69],wereused. Inthisstudy,hyperoxaluriawasinducedbyadministrationofEG(0.75%indrinkingwater)for21daysand AC(1%)wasgivenonlyforthefirst5days,asadministrationofACformorethan5daysledtoextreme weightlossandultimatelydeathoftherats[37]. Inthepreventivestudy,administrationofEGandAC resultedintheincreaseincrystalluriawithlargercrystalsduetohyperoxaluria,increaseinwaterintakeand urineoutput,whichmightbeduetotherenalimpairment[68],astherewassignificantincreaseinserum creatinine,bloodureanitrogenandtotalproteinlossin lithogenicgroupascomparedtonormal,whichhas beenrestoredbytheOv.Crtr eatment.Consistentwith somepreviousreports,crystalsdepositionbyhyperoxaluriacausedanincreaseinoxalateanddecreaseinCa2+excretioninthelithogenicgroup[70,71],whichwaspreventedbyOv.Cr.Therewashy pertrophyandextensive calciumoxalatecrystaldepositioninkidneysoflithogenicgroup.Therenaltubulesweremarkedlydilated, whichmightbeduetotheobstructionindistalrenal tubularflowbylargecrystals[68].Several invivo and in vitro studieshavedemonstratedthathyperoxaluria,a majorriskfactorforcalciumoxalatenephrolithiasis, resultsinproductionofsuperoxideandhydroxylfree radicalscausingoxidativestress,cellmembranerupture andcelldeath[53,72],andleadstoCaOxcrystaladherenceandretentioninrenaltubules[53,73].Thiscanbe speculatedthattheinhibitoryeffectofOv.Croncalcium oxalatecrystaldepositioninrenaltubulescouldhave alsobeencausedbyitsantioxidantactivity. Incurativestudy,withdrawaloflithogenictreatment after21daysevokedaspontaneousrecoveryofnephrolithicanimalsintheuntreatedgroups.However,Ov.Cr enhancedthespontaneousrecoveryinthetreatedgroup thantheuntreatedgroup,whichwasclearlyshownby thegaininbodyweight,significantdecreaseinurinary oxalate,renalcrystaldepositionandimprovementin renalfunctionscomparedtothelithogenicgroup. Phytochemicalscreeningrevealedthepresenceof saponins,alkaloids,coumari ns,sterol,terpenes,flavonoidsandtannins.Differentactivitiesobservedinthe crudeextractmightbeduetothepresenceofthese phytochemicals.Forexample,flavonoidsareknownto possessantispasmodicandCa++channelblocking [74,75],antioxidant[76]anddiuretic[77]activities. Saponinsareknowntopossessanti-crystallizationpropertybydisaggregatingthesuspensionofmucoproteins, promotersofcrystallization[9].However,the contributionofotherphytochemicalsaccountingforthe reportedactivitiescannotberuleout. Theplantshowedantiurolithicactivitybothinthe in vitro and invivo modelsinadditiontoitsantioxidant, renalepithelialcellprotective,antispasmodicanddiureticactivitiesreportedinthisstudy,allofwhichcouldbe beneficialinurolithiasis.Theplantisalsoreportedto possessanti-inflammatory[7 8]andantimicrobial[79] activities,whichcouldalsobesupplementingitsbeneficialeffect,astheinfectionandinflammationarelikely tobeassociatedwithurolithiasisprocess.Thus,theherbalremediesknowntocontainmultipleactivitiesoffer therapeuticpotentialparticularlyintheurolithiasis, wheremultipletargetsareneeded.Afewstudiesonthe effectivenessofherbalremediesinurolithiasisexists, suchas Hernairahirsute,Phylanthusniruri and Hibiscus sabdariffa ,whichshowedpromisingresultsinthemanagementofurolithiasis[8].However,mostofthesestudieswereusingthe invitro and/or invivo studieswith limitedadvancementinthepossiblemechanismsof theireffectiveness.Inthisstudy,weusedboththe in vivo and invitro modelsalongwithmultipleactivities, asmentionedabove,givingcomprehensiveinformation onthepharmacologicalbasisforitseffectivenessinurolithiasis.Theplantbeingofediblenaturewithalong historyofmedicinaluseisconsideredtoberelatively safe,however,detailedstudiesonitssafetyprofileis neededbeforerecommendingforclinicaluse.ConclusionTogetherthesedatasuggestthatthepresenceofantiurolithiceffectin Origanumvulgare againstrenalcalcium oxalatecrystaldepositsismediatedpossiblythrougha combinationofCaOxcrystalinhibitory,diuretic,antioxidant,antispasmodic,epithelialcellprotective,hypocalciuricandhypercitrauriceffectsthusactingonmultiple sites.Thisstudyrationalizesitsmedicinaluseinthe treatmentofurolithiasis.AdditionalmaterialAdditionalfile1:Chemicalsandreagents .Listofnamesandsources ofthechemicalsandreagentsusedinthestudy. Additionalfile2:TableS1 .Variousparametersfromdifferentgroupsof ratsrecordedafter21days,tostudythepreventiveeffectinanimal modelofurolithiasis Additionalfile3:TableS2 .Variousparametersfromdifferentgroupsof rats,recordedafter21and35days,tostudythecurativeeffectinanimal modelofurolithiasis Acknowledgements ThisstudywassupportedbytheHigherEducationCommission(HEC)of Pakistanas(i)indigenousM.Phill/PhDand(ii)InternationalResearchSupport InitiativeProgram(IRSIP)scholarshipsawardedtoAslamKhan.Khan etal BMCComplementaryandAlternativeMedicine 2011, 11 :96 http://www.biomedcentral.com/1472-6882/11/96 Page14of16

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Authordetails1DepartmentofPharmacology,FacultyofPharmacy,UniversityofKarachi, Karachi,Pakistan.2NaturalProductResearchDivision,Departmentof BiologicalandBiomedicalSciences,AgaKhanUniversityMedicalCollege, Karachi-74800,Pakistan.3CentrefortheStudyofLithiasis,Departmentof Pathology,CollegeofMedicine,UniversityofFlorida,USA.4Collegeof Pharmacy,KingSaudUniversity,Riyadh,SaudiArabia. Authors contributions AKcarriedoutthedraft,experimentalwork,datacollectionandevaluation, literaturesearchandmanuscriptpreparation.SBhelpedintheexperimental studydesigningandcorrectedthemanuscriptforpublication.SRKhelpedin thecellcultureexperimentsandrefinedthemanuscriptforpublication.AHG supervisedtheworkandrefinedthemanuscriptforpublication.Allauthors readandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:25June2011Accepted:17October2011 Published:17October2011 References1.LopezM,HoppeB: History,epidemiologyandregionaldiversitiesof urolithiasis. 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