大鼠灌胃蒙药复方阿拉坦-5后诃子酚性成分的药代动力学研究.pdf 7页

'中国科技论文在线http://www.paper.edu.cnPharmacokineticsofTerminaliaPhenolicsinRatsafterOralAdministrationofaMongolianCompoundMedicine#5Alatan-5**WuDandan,AzzayaJukov,MaChaomei(SchoolofLifeSciences,InnerMongoliaUniversity,Huhhot010021)Abstract:ThispaperreportsthepharmacokineticpropertiesofTerminaliaphenolicsafteradministrationofAlatan-5whichisacompoundMongolianmedicinecontainingthefruitofT.chebula10incombinationwithfourothermedicines.Gallicacid,ellagicacid,casuarinin,andchebulicacidweredetectedandquantifiedintheratplasma.GallicacidshowedthelargestAUCvalueamongthesefourconstituentswhenAlatan-5wasadministrated,anditsAUCvaluewaslargerthanwhenT.chebulafruitwasusedalone.WhiletheAUCvaluesoftheotherconstituentsweresmallerwhenAlatan-5wasusedthanwhenT.chebulafruitwasadministratedalone.Thesedataindicatedthatgallicacidandother15TerminaliaphenolicscouldplayimportantrolesinthepharmacologiceffectsofAlatan-5andthatothermedicinesinthiscompoundMongolianmedicinecouldgreatlyalterthepharmacokineticbehaviorsofTerminaliaphenolics.Keywords:TraditionalMongolianmedicines;Alatan-5;Terminaliaphenolics;Pharmacokinetics200IntroductionAlatan-5isacompoundMongolianmedicinecomposedofthefruitofTerminaliachebulaRets.(400g),thefruitofPunicagranatumL.(100g),theprocessedseedofMomordicacochinchinensis(Lour.)Spreng.(40g),thefecesofTrogopterusxanthipes(110g),andthefecesofSusscrofaL.(310g).ThepowderofthiscompoundMongolianmedicineisclinicallyusedasa[1].25digestionaidandforliverdiseasesThisresearchwascarriedouttoinvestigatethepharmacokineticbehaviorstheTerminaliaphenolicsafteroraladministrationofAlatan-5torats.1Experimental1.1ChemicalsandreagentsHPLCgrademethanolwasfromFisherScientific(FairLawn,NJ,USA).Formicacidwas30fromAlfaAesar,UK.GallicacidandellagicacidwerefromSigma-Aldrich(China).Casuarinin[2]andchebulicacidwereisolated,purifiedandidentifiedasdescribedinourpublishedpaper.The[3]4β-(L-acetylcysteinyl)-epicatechinpreparedinourpreviousworkwasusedasaninternalstandard(I.S.).ThepurityofthesestandardcompoundsandI.S.were>98%,andtheirstructuresareshowninFigure1.Foundations:ThisworkwassupportedbySpecializedResearchFundfortheDoctoralProgramofHigherEducation(20131501110008).Briefauthorintroduction:WuDandangotherbachelordegreeinInnerMongoliaUniversityin2014andherMasterdegreeinthesameUniversityin2017.SheisworkingontheresearchofNaturalProductsandFunctionalFoodsCorrespondanceauthor:MaChaomeigotbachelordegreeinBeijingMedicalCollege,ChinaandPhDinToyamaMedicalandPharmaceuticalUniversity,Japan.Shehasbeendoingresearchonnaturalproductsandfunctionalfoods.E-mail:cmma@imu.edu.cn-1- 中国科技论文在线http://www.paper.edu.cn35Fig.1StructuresofTerminaliaPhenolicsandAnInternalStandard(I.S.)forPharmacokineticInvestigation1.2UHPLCandLC–MSconditionsAnAgilent1290infinityUPLC-MSsystemwitha6430tripleQuadmassspectrometerfromAgilent(AgilentTechnologiesSingapore(International)Pte.Ltd.,Singapore)wasusedinthis40study.ChromatographycolumnwasanAgilentZORBAXRRHDEclipsePlusC18column(50mm×2.1mm;particlesize:1.8μm).Water(containing0.1%formicacid)andmethanolwereusedassolventAandB,respectivelyinthemobilephaseandprogrammedas:5–50%(B)in0–3.5min,50–100%(B)in3.5–4.5min,and100%(B)in4.5–7minataflowrateof0.4ml/min.ElectrosprayionizationinMRMmodewasusedfordetectioninthefollowingparameters:drygas45(N2)12l/min,drygastemperature350ºC,nebulizinggas(N2)pressure60psi,massrange100–1000m/z.TheMRMdetectionparameterswereoptimizedaslistedinTable1.Tab.1TheOptimizedMSParametersforTerminaliaPhenolicsandAnInternalStandardabcanalyteMWPrecursorProductsF(V)CE(eV)Retentionioniontime(min)gallicacid17016912590100.95ellagicacid302301284150303.86casuarinin93693593521011.82chebulicacid356355337100100.53dI.S.45145045011002.37abcd50Note:molecularweight;fragmentorvoltage;collisionenergy;I.S.:4β-(L-acetylcysteinyl)-epicatechin.1.3PreparationofthemethanolextractofAlatan-5ThepowderofAlatan-5(100g)wasextractedwithmethanol(800ml)underrefluxfor2h.Theextractwasfilteredandthefiltratewasevaporatedundervacuumtodrynesstoobtain28gofAlatan-5extract.551.4Animals,drugadministrationandbloodsamplingFemaleWisterrats(180±20g)fromtheLaboratoryAnimalCenterofInnerMongoliaUniversitywereusedinthisresearch.ProceduresoftheanimalexperimentswereperformedaccordingtotheregulationsoftheCommitteeonUseofAnimalSubjectsinResearchofInner-2- 中国科技论文在线http://www.paper.edu.cnMongoliaUniversity.Ratswereacclimatedin12-hlight/darkcyclefor1weekwithfoodand60waterprovidedadlibitumpriortotheexperiments.Afteracclimation,theratsweredeprivedoffoodbutallowedaccesstowateradlibitumfor12handthendividedintogroupsof3ratseach.T.chebulafruitpowderwassuspendedindistilledwaterandgavagedtoratsatthedosageof5.56g/kgbodyweight.ThedosageofamethanolextractofAlatan-5was5.54g/kgbodyweight(equivalentto16.21g/kgofAlatan-5and6.76g/kgofT.chebulafruit).Bloodsamples(each0.365ml)werecollectedfromthefossaorbitalisveniplexinheparinizedcentrifugetubesat15min,30min,1h,2h,4h,6h,8h,10h,12h,24hoursoftimeintervalsandwereimmediatelycentrifugedat7000rpm(3287g)for2minatroomtemperature,and0.1mloftheplasmawascollectedandstoredat−20ºCuntilLC-MSanalysis.1.5PreparationofsamplesolutionforUPLC-MSanalysis70Differentkindsandratiosofsolventsweretestedfortheefficacyinprecipitatingbloodproteinsanda0.4mlofamixtureofMeOH-EtOH(1:1,v/v)to0.1mlratplasmawasfoundtobegood.Themixturewasvortexedfor30sfollowedbycentrifugationat6000rpm(2415g)for10minatroomtemperature.ThesupernatantwasappliedtoaWAT036810Sep-PakPlustC18cartridge(WatersCorporation,Milford,MassachusettsUSA)andthecartridgewaselutedwith175mlMeOHfortwice.ThecombinedMeOHsolutionwasconcentratedwithavacuumconcentratortodryness.Theresiduewasdissolvedin0.1mlofmethanolcontaining1μg/mlofI.S.Thesolutionwasfilteredwithamicrofilter(0.22μm)andthefiltrate(2μl)wasanalyzedbyUPLC-MSwiththeoptimizedparameters.1.6Calibrationcurves80Thestocksolutionofstandardswasdilutedwithmethanoltoaseriesconcentrationsofstandardsolutionscontainingsameconcentrationofinternalstandard,and100μlofwhichwasmixedwith900μlblankplasmatoobtainsolutionsforcalibrationcurvesatconcentrationsof12.5to0.0488μg/ml.Concentrationswithsignal-to-noiseratiosof3:1and10:1weredefinedasthelimitsofdetection(LOD)andquantification(LOQ),respectively.851.7MethodvalidationPrecisionandintra-dayaccuracywereobtainedbyrepeatedanalysisofthestandardsolutionfor6timesinoneday.Theinter-dayaccuracywasobtainedbyanalysisofthestandardsolutionat3differentdays.TherecoveryofTerminaliapolyphenolswasevaluatedbycomparingthetheoreticconcentrationsandmeasuredconcentrationsofsolutionsspikedwithknown90concentrationsoftheanalytes.1.8DeterminationofthepharmacokineticbehaviorofTerminaliapolyphenolsinratplasmaafteroraladministrationofAlatan-5AUC,Tmax,Cmax,t1/2,CLandotherpharmacokineticparameterswereobtainedusingthepharmacokineticsoftware,DAS3.0withnon-compartmentalmodel(Theclinicresearchcenter,95ShanghaiUniversityofTraditionalChineseMedicine,China).2Results2.1CalibrationcurvesandvalidationresultsofthequantificationmethodAnUPLC–MSmethodinMRMmodewasusedforthequantificationoftheTerminaliapolyphenolsinplasma.ThemethodwasvalidatedaccordingtotheguidanceofUSFoodandDrug[4]100Administration.The4β-(L-acetylcysteinyl)-epicatechinwaschosenasaninternalstandarddue-3- 中国科技论文在线http://www.paper.edu.cntoitsstructuralsimilaritywiththeanalytes.Underthiscondition,fourTerminaliapolyphenols,casuarinin,chebulicacid,ellagicacid,andgallicacidwerewelldetectedandquantified.LinearregressionswereplottedusingthepeakarearatiosofTerminaliapolyphenolstointernalstandardv.s.concentrationsofTerminaliapolyphenolsinarangeof4.17-0.0057μg/ml,exceptfor105casuarininthatrangedat12.5-0.017μg/ml.Theintra-dayaccuracywasfoundtorangefrom4.6to2.8%.Theinter-dayaccuracyvariedbetween7.9to3.5%.Therecoveryratesofthesecompoundsrangedfrom81.3to103.9%(Table2).Tab.2ValidationoftheMethodforDeterminationofTerminaliaPhenolicsinPlasma2AnalyteRetentionLODLOQCalibrationequationRRecoverytime(min)(μg/mL)(μg/mL)(%)gallicacid0.960.00190.0057Y=4.749x-0.0830.99995.7ellagicacid3.830.00190.0057Y=0.874x+0.0640.98994.4casuarinin1.800.00570.017Y=0.795x-0.0470.99281.3chebulicacid0.550.00190.0057Y=2.857x-0.1900.996103.92.2PharmacokineticbehaviorofTerminaliapolypenolsinratplasmaafteroral110administrationofAlatan-5orT.chebulafruitIntheclinicpracticeofMongolianMedicine,thepowderofAlatan-5isadministrateddirectly,so,wetriedtogivethepowderofAlatan-5torats.However,thepowderofAlatan-5couldnotbegavagedtoratsduetoitsstickinessandlargeamount.So,weusedmethanoltoextractAlatan-5,asmethanolhasextractingabilityformostconstituents.Thepharmacokinetic115behavioroftheTerminaliaPhenolicsinratsafteroraladministrationofthemethanolextractofAlatan-5wasinvestigatedusingthevalidatedUPLC-MSmethod.Theplasmaconcentration–timecurvesofgallicacid(1),ellagicacid(2),casuarinin(3),chebulicacid(4)arepresentedinFigure2.Thepharmacokineticparametersobtainedfromanon-compartmentalpharmacokineticanalysisarepresentedinTable3andcomparedwiththoseafteroraladministrationofthefruitpowderofT.120chebula.Fig.2ThePlasmaConcentration–timeCurvesofGallicacid(1),Ellagicacid(2),Casuarinin(3),andChebulicAcid(4)inRatsafterOralAdministrationofAlatan-5MethanolExtract-4- 中国科技论文在线http://www.paper.edu.cn125Tab.3ComparisonofthePharmacokineticParametersofTerminaliaPhenolicsinRatafterOralAdministrationoftheFruitPowderofT.chebulaandAlatan-5MethanolExtractgallicAcidellagicAcidchebulicacidcasuarininabunitFCA5FCA5FCA5FCA5AUC(0-t)μg/L*h478.22338.128803.80.13038.17.728401.10.278AUC(0-∞)μg/L*h478.217526.928803.80.13038.17.988401.10.278MRT(0-∞)h3.0220.36.83.55.63.91.0046.8T1/2h1.6158.70.70.32.21.30.1731.1Tmaxh0.51.00.54.02.00.251.04.0Cmaxng/mL285.3813.59019.90.03810.43.408315.20.037CLL/h84.80.0036.71024.813391.929.31.11277.3abNote:ThedosageofT.chebulafruitpowder(FC)was5.56g/kg.ThedosageofAlatan-5methanolextract(A5)was5.54g/kgbodyweight(equivalentto16.21g/kgofAlatan-5and6.76g/kgofT.chebulafruit).130AsshowninFigure2andTable3,alltheseconstituents,gallicacid(1),ellagicacid(2),casuarinin(3),andchebulicacid(4),reachedtheirmaximumplasmaconcentrations(Tmax)within4h.GallicaciddisplayedthelargestAUCvaluesinadministrationofAlatan-5.Moreover,theAUCvalue(Table3)ofgallicacidinAlatan-5wasmuchlargerthanthatinthefruitofT.chebula(17526.9v.s.478.2μg/L*h),suggestingthattheinfluenceofothermedicinesinAlatan-5135ontheabsorptionofgallicacidwasremarkable.ItshouldbenotedthatthefruitsofPunica[5]granatumwhichalsoexistedinAlatan-5werefoundtocontaingallicacid(1.2μg/mg).ThoughtheamountoffruitsofP.granatuminAlatan-5ismuchlowerthanthatinT.chebulafruit(1:4),andthecontentofgallicacidinthefruitofP.granatumismuchlessthaninthefruitofT.chebula[5,6](1.2μg/mgv.s.3.5μg/mg),gallicacidinthefruitofP.granatummightcontributetosome140extenttothelargeAUCvalueofthiscompound.Ontheotherhand,administrationofAlatan-5resultedinsmallerAUCvaluesoftheotherconstituents,especiallyellagicacidandcasuarinin,thanadministrationofT.chebulafruitalone,andtheCLvalueswerelargerinAlatan-5thaninthefruitofT.chebula,suggestingthatothermedicinesimprovedtheclearanceofellagicacidandcasuarinininAlatan-5.ThedifferentpharmacokineticbehaviorofTerminaliaphenolicsbetween145administrationofT.chebulaandadministrationofAlatan-5indicatedthatothermedicinesinthiscompoundMongolianmedicine(prescription)couldgreatlyalterthepharmacologicbehaviorsofTerminaliaphenolics.[7,8][9-11]Terminaliaphenolicshavebeenreportedtohavesignificantantiviral,anti-diabetes,[12]andantibacterialactivities.Chebulicacidhasbeenreportedtohavebioactivitiesincluding[13]150protectionofhepatocytesagainstoxidativetoxicity,protectionagainsttheprogressionof[14]endothelialcelldysfunctioninducedbyadvancedglycationendproducts,etc.Ellagicacidis[15]knowntohaveliverprotectioneffects.Thedeductionfrompresentstudiesoftheirrapidabsorptionintocirculation,andthelargeAUCvaluesofsomeTerminaliaphenolics,suggestedthattheseconstituentscouldplaynotablerolesintheinvivobiologicalactivityofT.chebulafruits155andAlatan-5.3ConclusionThepharmacokineticprofilesofTerminaliaphenolics,gallicacid(1),ellagicacid(2),casuarinin(3),andchebulicacid(4),wereinvestigatedafteradministrationofacompoundMongolianmedicine-Alatan-5andthefruitofT.chebula.GallicacidshowedthelargestAUC160valueamongthesefourconstituentswhenAlatan-5wasadministrated,anditsAUCvaluewaslargerthanwhenT.chebulafruitwasused.WhiletheAUCvaluesoftheotherconstituentsweresmallerthanthosewhenT.chebulafruitwasadministratedalone.Thedifferentpharmacokinetic-5- 中国科技论文在线http://www.paper.edu.cnbehaviorsofTerminaliaphenolicsbetweenadministrationofT.chebulaandadministrationofAlata-5extractindicatedthatothermedicinesinthiscompoundMongolianmedicinecouldgreatly165alterthepharmacokineticbehaviorsofTerminaliaphenolics.GallicacidandotherTerminaliaphenolicscouldplayimportantrolesinthepharmacologiceffectsofT.chebulaandAlatan-5.References170[1]ChinesePharmacopoeiaCommittee.DrugstandardsofMinistryofpublichealthofChina(MongolianmedicineFascicule).Beijing:ChemicalIndustry.Press;1998;120-121.[2]AjalaOS,JukovA,MaCM.HepatitisCvirusinhibitoryhydrolysabletanninsfromthefruitsofTerminaliachebula.Fitoterapia,2014,99:117-123.[3]MengHC,Ma,CM.Flavan-3-ol-cysteineandacetylcysteineconjugatesfromediblereagentsandthestemsof175Cynomoriumsongaricumaspotentantioxidants.FoodChem,2013,141:2691-2696.[4]USFoodandDrugAdministration,CenterforDrugEvaluationandResearch,Rockville,MD,GuidanceforIndustry:BioanalyticalMethodValidation,200.[5]WangCX,YueLL,XuHY,Uneer,HuangY,ZhangHP,GaoJ,MaCM.SimultaneousQuantificationof7ComponentsinDifferentPartsofPunicagranatumFruitsUsingUltra-HighPerformanceLiquid180Chromatography-TripleQuadrupoleMassSpectrometry(UPLC-QQQMS).FoodScience,2016,37(4):139-143.[6]JukovA,AjalaOS,GaoJ,MaCM.QuantificationofSixBioactiveConstituentsintheThreePartsofTerminaliaFruitbyLiquidChromatography-quadropoleMassSpectrometry.JournalofInnerMongoliaUniversity(NaturalScienceEdition),2016,47(1):90-95.[7]LinLT,ChenTY,LinSC,ChunCY,LinTC,WangGH,AndersonR,LinCC,RichardsonCD.185Broad-spectrumantiviralactivityofchebulagicacidandpunicalaginagainstvirusesthatuseglycosaminoglycansforentry.BMCMicrobiol,2013,13:187-202.[8]ChengH,LinC,LinT.Antiherpessimplexvirustype2activityofcasuarininfromthebarkofTerminaliaarjunaLinn.AntiviralRes,2002,55:447-455.[9]RenYL,HimmeldirkK,ChenXZ.Synthesisandstructure-activityrelationshipstudyofantidiabetic190penta-O-galloyl-D-glucopyranoseanditsanalogues.JMedChem,2006,49(9):2829-2837.[10]YangMH,VasquezY,AliZ,KhanIA,KhanSI.ConstituentsfromTerminaliaspeciesincreasePPARαandPPARγlevelsandstimulateglucoseuptakewithoutenhancingadipocytedifferentiation.JEthnopharmacol,2013,149:490-498.[11]SinghI,SinghPK,BhansaliS,ShafiqN,MalhotraS,PandhiP,PalA.Singh.Effectsofthreedifferentdoses195ofafruitextractofTerminaliachebulaonmetaboliccomponentsofmetabolicsyndrome,inaratmodel.PhytotherRes,2010,24:107-112.[12]ZhongX,ShiY,ChenJ,XuJ,WangL,BeierRC,HouX,LiuF.PolyphenolextractsfromPunicagranatumandTerminaliachebulaareanti-inflammatoryandincreasethesurvivalrateofchickenschallengedwithEscherichiacoli.BiolPharmBull,2014;37:1575-1582.200[13]LeeHS,JungSH,YunBS,LeeKW.IsolationofchebulicacidfromTerminaliachebulaRetz.anditsantioxidanteVectinisolatedrathepatocytes.ArchToxicol,2007,81:211-218.[14]LeeHS,KooYC,SuhHJ,KimKY,LeeKW.PreventiveeffectsofchebulicacidisolatedfromTerminaliachebulaonadvancedglycationendproduct-inducedendothelialcelldysfunction.JEthnopharmacol,2010,131:567-574.205[15]GuL,DengWS,LiuY,JiangCH,SunLC,SunXF,XuQ,ZhouH.Ellagicacidprotectslipopolysaccharide/D-galactosamine-inducedacutehepaticinjuryinmice.IntImmunopharmacol,2014,22:341–345.210-6- 中国科技论文在线http://www.paper.edu.cn大鼠灌胃蒙药复方阿拉坦-5后诃子酚性成分的药代动力学研究武丹，AzzayaJukov，马超美(SchoolofLifeSciences,InnerMongoliaUniversity,Huhhot010021)215摘要：阿拉坦-5是由诃子等5种药材组成的蒙药复方。本文研究大鼠灌胃阿拉坦-5后诃子酚性成分的药代动力学性质。喂药后，在大鼠血清中检测到了没食子酸、鞣花酸、木麻黄鞣宁和诃子裂酸。其中，灌胃阿拉坦-5后没食子酸的AUC曲线下面积最大，并且其面积大于灌胃诃子单药材时，而其他3种成分的AUC曲线下面积在灌胃阿拉坦-5时要小于灌胃诃子单药材时。这些数据提示，没食子酸及其他诃子酚性成分在阿拉坦-5的药效中发挥着重要220作用，并且阿拉坦-5复方中的其他药材可明显改变这些成分的药代动力学性质。关键词：传统蒙药；阿拉坦-5；诃子酚性成分；药代动力学中图分类号：R917-7-'