黄连解毒汤1

Comparativepharmacokineticsofbaicalinafteroraladministration

ofpurebaicalin,Radixscutellariaeextractand

Huang-Lian-Jie-Du-Tangtorats

TongLu,JueSong,FangHuang,YuanxiongDeng,LinXie,

GuangjiWang,XiaodongLiu∗

CenterofDrugMetabolismandPharmacokinetic,ChinaPharmaceuticalUniversity,Nanjing,210009JiangsuProviencePRChina

Received4March2006;receivedinrevisedform19May2006;accepted22September2006

Availableonline13October2006

Abstract

Huang-Lian-Jie-Du-Tang(HLJDT)isanimportant“heat-clearing”multiherbremedyoftraditionalChinesemedicine,andRadixscutellariae(ScutellariabaicalensisGeorgi,Labiatae)isakeyingredientherbinit.BaicalinandwogonosidearetwomaineffectiveingredientsenrichedinRadixscutellariae.Inthepresentstudy,pharmacokineticdifferencesofbaicalinfollowingoraladministrationofpurebaicalin,Radixscutellariaeextract,baicalinco-administratedwithextractoftheotherthreeherbsofHLJDTandHLJDTwereinvestigatedinmaleS.D.ratswithapproximatelythesamedoseof200mg/kgbaicalin.ThepharmacokineticcomparisonofwogonosidewasconductedonlyinRadixscutellariaeextractandHLJDT.PlasmaconcentrationsofbaicalinandwogonosideweredeterminedusingHPLCmethod.UnpairedStudent’st-testwasusedforstatisticalcomparison.Theresultsindicatedthatbaicalinandwogonosidedemonstratedbimodalphenomenonintheplasmaprofile.SomeingredientsintheotherthreeherbsofHLJDT,notinRadixscutellariaeitself,hadpharmacokineticinteractionwithbaicalinandwogonosideandhencedecreasedtheirsystematicexposurelevel(p<0.01).Theabsorptionsiteofbaicalinwaspreliminaryevaluatedinratusinginsituabsorptioninstomachanddifferentintestinalsegmentsandresultsrevealedtheexistenceofdouble-siteabsorptionofbaicalin.Thefirstabsorptionsitewasinupperintestinal,probablyviadirectlyabsorptionofbaicalin;whilethesecondabsorptionsitewasincolonintheformofaglygon.©2006ElsevierIrelandLtd.Allrightsreserved.

Keywords:Huang-Lian-Jie-Du-Tang;Baicalin;Wogonoside;Pharmacokineticdifferences;Bimodalphenomenon;Double-siteabsorption

1.Introduction

Huang-Lian-Jie-Du-Tang(HLJDT,orOren-gedoku-toinJapanese)isanimportantmultiherbremedyintraditionalChi-nesemedicine(TCM)andwasfirstdescribedbyWangTao(intheChineseTangDynasty)inhistreatise“WaiTaiMiYao”.HLJDThasattractedagreatdealofattentionforitshealingeffectsongastrointestinaldisorders,inflammatorydisease,anditsbeneficialeffectstopreventthedevelopmentofliverdis-ease,hypertensionandcerebrovasculardisease(Ji,2003).ThetraditionalremedyisanaqueousextractofRhizomacoptidis(CoptischinensisFranch,Ranunculaceae),Radixscutellariae(ScutellariabaicalensisGeorgi,Labiatae),Cortexphellodendri

∗

Correspondingauthor.Tel.:+862583271006;fax:+862585306750.E-mailaddress:xdliu@cpu.edu.cn(X.Liu).

(PhellodendronamurenseRupr,Rutaceae)andFructusgarde-niae(GardeniajasminoidesEllis,Rubiaceae)withtheratioof3:2:2:3andalltheherbsareofficiallylistedintheChinesephar-macopoeia(PharmacopoeiaofPRChina,2005).

Radixscutellariae(Huang-QininChinese),akeyingredientherbinHLJDT(Minister),hasbeenwidelyusedinTCMforthetreatmentofinflammation,fever,hepatitisandallergicdis-easesandhypertension,etc.(Wangetal.,1998).Flavonoidsarethemainactiveingredients(Fig.1).Amongthem,baicalinhasbeenusedasaphytochemicalmarkerforthequalitycontrolofRadixscutellariaeinChinesepharmacopoeia,andwogonosideisanothermainflavonoid(Qietal.,1998;Wuetal.,2005).Phar-macologicalstudieshaveindicatedthatbaicalinandwogonosidesharemanybeneficialactivitieswithRadixscutellariaewithrespecttoanti-inflammatary,anti-allergic,anti-oxidantandhep-atoprotectiveproperties(Chouetal.,2003;Jangetal.,2003;Kimetal.,2005;Lim,2003).

0378-8741/$–seefrontmatter©2006ElsevierIrelandLtd.Allrightsreserved.doi:10.1016/j.jep.2006.09.036

T.Luetal./JournalofEthnopharmacology110(2007)412–418413

Fig.1.ChemicalstructuresoffourRadixscutellariaeactiveflavonoids.

PharmacokineticstudiesonthemainflavonidsofRadixscutellariaehavebeenfullycarriedout.Itisgenerallyassumedthatbaicalinispoorlyabsorbedfromthegastrointestinaltractinitsnativeformandmustbehydrolyzedbymicrofloraenzymes(bacterial␤-glucuronidase)inguttoitsaglycone,baicaleininhumanandrat(Yimetal.,2004;Zuoetal.,2002).AsubstantialportionoftheaglyconeissubsequentlyconjugatedtobalcalininthegutmucosalcellbyUDP-glucuronosyltransferaseandapproximatelyhalfoftheconjugateisexcretedbackintothegutlumen,mainlythroughMRP2.Theunconjugatedbaicaleinandabsorbedbaicalinarethenextensivelymetabolizedinliverandexcretedviabile(Abeetal.,1990;Akaoetal.,2000,2004).AfteroraladministrationofbaicalinorRadixscutellariaeextract,theglucuronides/sulfatesofbaicaleinarepredominantinthebodyfluid(plasmaandurine)inratandhuman.Themainmetabolitesarebaicalein6-O-glucuronide-7-O-sulfateandbaicalein6,7-di-O-glucuronide(Fengetal.,2005;Laietal.,2003a;Mutoetal.,1998;Xingetal.,2005a).Similarwithbaicalin,wogonosideisfirstlymetabolizedbymicrofloraenzymesandsubsequentlycirculatedandexcretedmainlyinconjugatedformfollowingintakeofRadixscutellariaeextractinratandhuman(Chenetal.,2002;Laietal.,2003a;Zuoetal.,2002).Accordingly,itcouldbededucedthatbaicalin,wogonosideandotherphaseIIconjugatesmayberesponsibleforthein-vivoeffectsofRadixscutellariae.

Giventhemulti-ingredientcharacterofherbalmedicines,thelikelihoodofherb–druginteractionsistheoreticallyhigherthandrug–druginteractions.Inrecentyears,multiplecasereportsofherb–druginteractionshavebeenpublished(Huetal.,2005;Izzo,2005).Herb–herbinteractionhasalsobeenfoundinthedecoctingprocessofRhizomacoptidisandRadixscutellariae,RadixetRhizomaRhei(RheumpalmatumL.,Polygonaceae)orRadixglycyrrhizae(GlycyrrhizauralensisFisch,Legumi-nosae)(Cheng,2005).TheaimofthisstudyistoexplorewhethertherearesomeherbalingredientsinHLJDTaffect-ingthepharmacokineticbehaviorofbaicalinandwogonosideinRadixScutellaria(herb–herbinteraction).Duetolackofstandardsofotherconjugatedmetabolites,weselectedbaicalintocomparethepharmacokineticdifferencesafteroraladmin-istrationofpurebaicalin,Radixscutellariaeextract,baicalinco-administratedwithextractoftheotherthreeherbsofHLJDT(co-administratedmixture)andHLJDTtorats.Thepharmacoki-neticcomparisonofwogonosidewasconductedonlyinRadixscutellariaeextractandHLJDT.Tofurtherclarifytheabsorptionmechanismofbaicalininrats,theinsituabsorptionmethodwasalsoused.

2.Materialsandmethods2.1.Materials

Baicalin,baicalein,andwogoninstandardswerepurchasedfromtheNationalInstitutefortheControlofPharmaceu-ticalandBiologicalProducts(Beijing,China).Wogonoside(>95%)waskindlyprovidedbyProfessorMei-LanLiu(Insti-tuteofChineseMaterialMedica,ChinaAcademyofTradi-tionalChineseMedicine).Baicalin(>95%)wassuppliedbymedicinalchemistryofChinaPharmaceuticalUniversity.Ace-tonitrilewasofchromatographicgrade(FisherCompany,Inc.,USA);alltheotherreagentswereofanalyticalgrade.Milli-Qwater(Millipore,Bedford,MA)wasusedthroughoutthestudy.

2.2.PreparationofRadixscutellariaeextractandHLJDTRhizomacoptidis,Radixscutellariae,CortexphellodendriandFructusgardeniaeweremixedintheratioof3:2:2:3andthetotalweightwas1kg.Themixturewasdecoctedtwicebyrefluxingwithwater(1:10andthen1:5w/v)for1h,andthesolutionobtainedwasconcentratedtogiveanextractof219.5g.Radixscutellariae200gwastreatedasaboveandgaveanextractof53.3g.Thedriedpowderwasstoredat4◦Cbeforeuse.AllcomposeddecoctionpiecesinHLJDTwerepurchasedfromKai-XinHerbalShop(Nanjing,China)andauthenticatedbyDoc.Li-NaChen(Departmentofpharmacognosy,ChinaPharmaceu-ticalUniversity,Nanjing,China).ThevoucherspecimensweredepositedinDepartmentofpharmacognosy,ChinaPharmaceu-ticalUniversity.

2.3.ContentofbaicalinandwogonosideinRadixscutellariaeextractandHLJDT

Tocalculatetheadministereddose,thecontentsofbaicalinandwogonosideinHLJDTandRadixscutellariaeextractwerequantitativelyanalyzed.Theextractpowderwasultra-sonicatedwith70%ethanolfor1h;thesuspensionwasthendiluted100times.Aftercentrifugingat15,000rpmfor10min,thesuper-natantwasanalyzedusingamodifiedHPLCmethod(Laietal.,2001).Thecontentsofbaicalinandwogonosideweredeter-minedtobe4.42and1.08%inHLJDTand17.63and4.82%inRadixscutellariaeextract,respectively.2.4.Animals

TheanimalstudieswereapprovedbytheAnimalEthicsCommitteeofChinaPharmaceuticalUniversity.MaleSprague–Dawleyrats,weighing250–280g,weresuppliedbySino-BritishSippr/BKLabAnimalLtd.(Shanghai,China).Theratsweremaintainedinanair-conditionedanimalquarteratatemperatureof22±2◦Candarelativehumidityof50±10%.Waterandfood(laboratoryrodentchow,Nanjing,China)wereallowedadlibitum.Theanimalswereacclimatizedtothefacil-itiesforfivedays,andthenfastedwithfreeaccesstowaterfor12hpriortoeachexperiment.

414T.Luetal./JournalofEthnopharmacology110(2007)412–418

2.5.Invivostudy

Ratsweredividedintofourgroupsrandomly(n=5)andadministratedanoraldoseof200mg/kgbaicalin(intheformofpurecompoundorco-administratedmixture),1.07g/kgRadixscutellariaeextract(188mgbaicalinand47mgwogonoside/kg)or4.38g/kgHLJDT(193mgbaicalin,and43mgwogono-side/kg),allofwhichweresuspendedin0.5%carboxymethylcellulosesodiumsalt(CMC-Na)aqueoussolutionjustbeforeeachexperiment.Bloodsamples(200␮l)werecollectedinheparinizedeppendorftubeviatheoculichorioideaeveinbeforedosingandsubsequentlyat0.083,0.167,0.333,0.5,1,2,3,4,6,8,10,12,16and24hfollowingoraladministration.Aftercentrifugingat5000rpmfor10min,theplasmasampleswereobtainedandfrozenat−20◦Cuntilanalysis.2.6.Insituabsorptionstudy

Ratsweredividedintothreegroupsrandomly(n=5).Theywereanesthetizedwithdiethylether,fastenedandexposedtoaheatinglamptomaintaintheirnormalbodytemperature.Theabdominalcavitywasopenedbyamidlineincisionof3–4cm.Groupone,acannulawasinsertedintostomachandthepyloruswasligatedwithsurgicalsilkforstomach-limitedadministra-tion.Grouptwo,acannulawasinsertedintoduodenumforabsorptionbytheentireintestine(IDadministration).Groupthree,theproximalileumwaslocatedandligatedfollowedbyintraduodenaladministrationforabsorptiononlybyintraduo-denalandjejunum(IDadministrationwithileumligation).Aftersurgery,theintestinalsegmentwasplacedbackintotheabdominalcavity.Theincisionwasthensuturedandcoveredbysaline-wettedcottontokeeptheintestinalsegmentmoist.Thebloodsamples(200␮l)werecollectedinheparinizedeppendorftubeviajugularveinat0.083,0.167,0.333,0.5,1and2hforstomach-limitedadministration,0.083,0.167,0.333,0.5,1,2,3,4,6,8,10,12,16and24hforIDadministrationand0.083,0.167,0.333,0.5,1,2,3,4,5hforIDadministrationwithileumligationafteradministrationof200mgbaicalin/kgtorats.Eachbloodsamplewithdrawnwasreplacedbyanequalvolumeofheparinized0.9%saline(20IUheparin/ml)2.7.Assayprocedure

Theassayofbaicalinandwogonosideinratplasmawasdevelopedandvalidatedinourlaboratory(Luetal.,2005).The

plasmasampleswerepretreatedbyproteinprecipitationandsep-aratedbyHPLConaShimadzuShim-packVP-ODScolumn(150mm×4.6mm,5␮m)usingamobilephasecomposedofA(0.05%phosphoricacidand5mMmonosodiumphosphate)andB(acetonitrile)withlinergradientelution.TheLCbinarygradi-entprogramconsistedofaninitial25.5%Bfor16min,followedbya3.5minlinergradientsegmentofincreasingBfrom25.5to45%,andthenlinerdecreasedto25.5%Bin2minandthecolumnwasre-equilibratedat22.5%Bfor4.5min.Separationwasperformedat40◦Cwithaflowrateof1.5ml/minandthetotaltimeofasinglerunwas26min.Thedetectionwavelengthwassetat276nm.TheLLOQs(Lowerlimitofquantification)oftheassaywere0.156␮g/mlforbaicalinand0.107␮g/mlforwogonside,respectively.Goodlinearitywasobtainedfrom0.156to10.0␮g/mlforbaicalin,andfrom0.107to7.0␮g/mlforwogonoside.2.8.Dataanalysis

Theareaundertheplasmaconcentration-timecurve(AUC(0–τ))wasestimatedbynon-compartmentalmethodsusingthebioavailabilityprogrampackage(BAPP,version2.0,CenterofDMPK,ChinaPharmaceuticalUniversity).Terminatehalf-life(T1/2)wasdefinedasT1/2=0.693/keandthekewasdeter-minedbyunweightlinearregressionoflogarithmicalplasmaconcentrationversustimeforthelast4–5datapointsusingMicrosoftExcel.CmaxandTmaxvalueswereobtaineddirectlyfromtheobservedconcentrationversustimedata.AnunpairedStudent’sttestwasusedforcomparison.Allresultswereexpressedasarithmeticmean±standarddeviation(S.D.).3.Results

3.1.PharmacokineticofbaicalinandwogonosideinratsafteroraladministrationofRadixscutellariaeextractandHLJDT

PlasmaconcentrationsofbaicalinandwogonosideweredeterminedafteroraladministrationofRadixscutellariaeextracttoratsandtheplasmaprofiledemonstratedbimodalphenomenon(Fig.2).Thefirstpeakoccurredatabout20minandthesecondat8h.Theconcentrationofwogonosidewaslowerthanthatofbaicalin,whichmightbecauseofthelowercontentintheextract.ComparedwithRadixscutellariaeextract,

Fig.2.Comparativeplasmaconcentration–timeprofileofbaicalinandwogonosideafteroraladministrationofRadixscutellariaeextract(188mg/kgbaicalinand47mg/kgwogonoside)(᭹)andHLJDT(193mg/kgbaicalinand43mg/kgwogonoside)(󰀄)torats(n=5).(A)Baicalin;(B)wogonoside(**p<0.01vs.HLJDT).

T.Luetal./JournalofEthnopharmacology110(2007)412–418

415

g58k1152/....g1203m)3h±±±±4(d22249/n13911....aT4455nilacia)bLg/kh/ggk*m(**3e*10s21429o00001d....(/0000)T␶–±±±±D0(JLC387176972121HAU....0000dna)edi)slomn/*oh***gg4636o98w␮12....(4110)gτk–±±±±0/(g5mC7942493....774AU123531dnani)lalcm***i/ag*365b␮4200....g(0000k2/g,±±±±xma4678m161788C....42101(tcart)xleme/ag803i␮1843....r(1100al1le,±±±±txua5950cm3025sC....5511xidaRfo513)1906nh....o(1111it2a±±±±r,txsa0000inm0550....iT9876mdalaro4199r)1412eh....t(0000fa1±±±±,exda3738ism3635....oT0000nogowdttccnaaarrtt.nxxTieeDlaeeJcaaLiamiirrHbraa.ollllsffoeevnttuu1sorcc0iesTsT.ttc0eoxxiDiDbimodalphenomenonofbaicalinandwogonsideinratplasmaalsoexist,buttheconcentrationofthesecondpeak(Cmax,2)andthevaluesofAUC(0–τ)/doseweresignificantlydecreased(p<0.01)afteroraladministrationofHLJDT(Table1,Fig.2).ResultsindicatedtheexistenceofcertainingredientsinHLJDTthataffectthepharmacokineticbehaviorofbaicalinandwogonoside.

3.2.Pharmacokineticcomparisonofbaicalininratsafteroraladministrationofpurebaicalinandco-administratedmixture

Previousstudyreportedthatbaicaleincouldbeabsorbedeas-ilyinthesmallintestine,andtheTmaxwas0.5hafteroralintakeofbaicaleintorats(Laietal.,2003b).Thecontentsofaglyconsweredeterminedtobe1.01%baicaleinand0.45%wogonininRadixscutellariaeextractinthisstudy.Initially,wethoughtthatthefirstpeakwasresultfromtherapidabsorptionofbaicaleinandwogonin.InordertodeterminewhethertheabsorptionofbaicalincouldcontributetothefirstpeakornotandwhethertheingredientsaffectingthepharmacokineticbehaviorofbaicalinandwogonosideoriginatedfromRadixscutellariaeitselfortheotherthreeherbsinHLJDT,pharmacokineticcomparisonofbaicalinbetweenpurebaicalinandco-administratedmixturewasconductedinrats.Theplasmaconcentrationsofbaicalinweredeterminedandpharmacokineticparameterswereesti-mated(Table2).ResultswerealsocomparedwiththoseacquiredfromratsgivenRadixscutellariaeextract,containingapproxi-matelythesamedoseofbaicalin(Fig.3).

AsshowninFig.3andTable2,theplasmaprofilestilldemon-stratedbimodalphenomenonafterintakeofpurebaicalin,whichconfirmedtheabilityofrapidabsorptionofbaicalinresultingintheappearanceofthefirstpeak.SimilartotheratsgivenHLJDT,lowerplasmaconcentrationandreducedAUC(0–τ)ofbaicalinwerefoundinratgivenco-administratedmixturecom-paredwithpurebaicalin(p<0.01).Onthecontrary,however,theplasmaprofileandpharmacokineticparametersofbaicalininRadixscutellariaeextractwerealmostequalwiththoseinpurebaicalin.Theseresultsfurthersuggestedthattheingre-dientsinteractingwithbaicalinandwogonosideexistedintheotherthreeherbsinHLJDT,notinRadixscutellariae.

Table1416T.Luetal./JournalofEthnopharmacology110(2007)412–418

Table2

Pharmacokineticdifferencesofbaicalinafteroraladministrationofpurebaicalin(200mg/kg),co-administratedmixture(200mg/kgofbaicalin)andRadixscutellariaeextract(188mg/kgbaicalin)toratsAdministrationmethodPurebaicalin

Co-administratedmixtureRadixscutellariaeextract

Tmax,1(h)0.27±0.150.19±0.060.33±0.14

Tmax,2(h)8.25±0.717.00±2.149.00±1.15

Cmax,1(␮g/ml)5.92±1.914.78±1.015.35±1.1

Cmax,2(␮g/ml)4.91±0.57**2.50±0.674.14±0.4

AUC(0–τ)(␮gh/ml)52.39±4.23**29.72±4.8451.27±4.14

T1/2,(h)3.71±0.734.96±1.614.32±1.1

Mean±S.D.,n=5;**p<0.01vs.co-administratedmixture.

Fig.4.Theplasmaconcentration-timeprofileofbaicalinafteroraladminis-tration(᭹),IDadministrationwithentireintestinalabsorption(󰀄)andIDadministrationwithileumligation(󰀇)purebaicalin(200mg/kg)torats(n=5).

3.3.Pharmacokineticofbaicailininratsusinginsituabsorption

Toourknowledge,itwasthefirsttimereportingtherapidlyabsorption(approximately20min)ofbaicalinafteroraldosing.Inthisstudy,weusedtheinsituabsorptionmethod(stom-achanddifferentintestinalsegments)tofurtherinvestigatetheabsorptionsiteofbaicalin.Afterstomach-limitedadmin-istration,onlytraceofbaicalincouldbedetected.TheplasmaprofilesdemonstratedbimodalphenomenonwhenbaicalinwasIDadministrated,butthetimetoreachCmax,2(Tmax,2)wassignificantlydelayed(12±0.0hversus8.25±0.71h)andCmax,2wasalsodecreased(3.95±0.74␮g/mlversus4.91±0.57␮g/ml)comparedwithoraladministration(Fig.4),whichmightbecauseoftheloweredintestinalabsorptionrateunderthenon-physiologicalconditionofIDadministratedrats.ItwasinterestingthattheCmax,1inIDadministrationgroupwashigherthanthatinoraladministrationgroup(8.42±1.23␮g/mlversus5.92±1.91␮g/ml),themechanisminvolvedhereinwasunknown.Intheileum-ligatedgroup,thefirstpeakstillappeared(Fig.4),andvalueofCmax,1(9.52±1.11␮g/ml)wassimilartothat(8.42±1.23␮g/ml)inIDadministrationgroup.Basedontheresultsofinsituabsorption,wecouldmakeaqualitativeconclusionthatbaicalinhasdouble-siteabsorptionkinetic,thefirstsitewasinupperintestinalandthesecondsitewasincolon.4.Discussion

Bimodalphenomenonofbaicalinandwogonosideintheplasmaprofilewasrevealedinthepresentstudyanddouble-siteabsorptionwasfurtherconfirmedusingbaicalinasamodelcompound.Thefirstpeakoccurredatabout20minafteroral

dose.Akaoetal.(2000)reportedthatbaicalincouldtransformintobaicaleinbyratgastriccontentswith74%conversion,butdataofstomach-limitedadministrationinthisstudyshowedthatbaicalincouldnotbedetectedinplasma,indicatingthatbaicalinandbaicaleinwerenotabsorbedinstomach.Furtherexperimentsdemonstratedtheexistenceofthefirstpeakafterintraduodenaladministrationofbaicalininileum-ligatedrats,whichwasincontrarytothepreviousreportsandwashardtoexplainbecausetherewaslittleanaerobeintheupperintestinaltract.Summa-rizing,thefirstabsorptionsiteofbaicalinwasupperintestinalandthemechanismcouldexcludethecontributionofenterobac-teria(␤-glucuronidase)andwasprobablyduetothedirectlyabsorption.Previousstudieshadconfirmedthatmorphine-6-beta-d-glucuronideandestradiol17-beta-d-glucuronidecouldbeactivelytransportedintoblood-brainbarrierandliver,respec-tive,byglucosetransporterGLUT-1orOATP2(Bourassetetal.,2003;Shitaraetal.,2003).Therefore,itwasconceivablethatflavonoidglucuronidescouldbeactivelytransportedbyspecifictransporterintoenterocyteandhenceintosystemiccirculation.

Astwonatureoccurringflavoneglucuronides,baicalinandwogonosideweregenerallyassumedtobeabsorbedonlyintheformofaglyconincolon(high␤-glucuronidaseactivity),corre-spondingtothesecondpeakatabout8hafteroraldose(Dengetal.,2006;Laietal.,2003b;Luetal.,2005).The␤-glucuronidaseisproducedbymostEscherichiacolistrainsandotherenterobac-teria,includingsomeShigellaandSalmonellastrains,Staphy-lococcusspp.andStreptococcusspp.etc.(TrylandandFiksdal,1998).Amongthem,Streptococcusspp.andEscherichiacoliareresponsibleforthehydrolysisofbaicalin(Ishiharaetal.,2002).Itwasreportedthatbaicalinunderwententericcircula-tionandenterohepaticcirculationinratsafteroraldosing(Akaoetal.,2004;Xingetal.,2005a),whichcontributedtothesecondpeak.Thetwoimportantprocessesalsonecessitatedthe“aid”ofbacterial␤-glucuronidase.Thus,theimportanceofcolonicmicrofloraontheabsorptionandmetabolismofbaicalinandwogonosideshouldbeincreasinglyrecognized.

Someantibioticscoulddecreasethesystematicexposureofbaicalinviainhibitingintestinalbacteriaand/ordecreasingprod-uctofbacterial␤-glucuronidase.Earlierstudieshadconfirmedthepharmacokineticinteractionsofbaicalin(ormultiherbrem-edycontainingbaicalin)withantibioticsclinicallyandexperi-mentally(Ishiharaetal.,2002;Xingetal.,2005b).Forexample,afteroraladministrationofbaicalin,theabsolutebioavailabilityofbaicalin,basedontheAUCofthebaicalinparentform,was2.2±0.2%innormalrats,whileinantibiotic-treatedratsitwasestimatedat1.5±0.2%(Xingetal.,2005b).AUCofbaicalin

T.Luetal./JournalofEthnopharmacology110(2007)412–418417

ofgerm-freeratwasonly12%comparedwithnormalrat(Akaoetal.,2000).

Inthisstudy,wealsofoundthatsomeingredientsintheotherthreeherbsinHLJDT,notinRadixscutellariaeitself,hadpharmacokineticinteractionwithbaicalinandwogonoside.ThiswasdemonstratedbythesignificantlydecreasedCmax,2andAUC(0–τ)inratsgivenHLJDTorco-administratedmixture.RhizomacoptidisandCortexphellodendri,twoclassic‘heat-clearing’herbsusedinTCM,havebroad-spectrumantibacterial,antiviralandantifungaleffects(Wangetal.,1998).Berberineandpalmatinearethemainactiveingredientsandarealsousedasanaturalantibacterialagentinclinicalsetting.Resultsofrecentexperimentinourlaboratoryfoundthatberberinecoulddecreasetheplasmaconcentrationofbaicalininratwhenpurebaicalinandberberinewereco-administrated,whichwassimilartotheresultsofbaicalinco-administratedwithneomycin(datanotshown).Combiningtheexperimentaldatawithliteraturereporteddata,wecouldconcludethatthedecreasedsystemicexplorelevelofbaicalinandwogonosidemightresultfromtheinhibitionofbacterial␤-glucuronidaseactivitiesbyRhizomacoptidisandCortexphellodendriortheirmainactiveingredi-ents.

RhizomacoptidisandRadixscutellariaeisthemostpopularmedicinecoupleinTCM,suchasHLJDT,San-Huang-Xie-Xin-Tang(SHXXT)(Ji,2003).IthadbeenreportedthatprecipitationphenomenaoccurredindecoctingprocessofTCMcontainingthismedicinecouple.Themechanismwasthecomplexationbetweenthemainactiveingredients,flavonoidsandalkaloids,resultingthedecreasedcontentsoftheseactiveingredientssig-nificantly(Qiaoetal.,1999;YiandXu,2004).However,thedistinctprecipitatecouldnotbeobservedimmediatelyatthetimewhentwostandardsolutionsweremixed(YiandXu,2004).Lateststudyrevealedthat,underthesuccessiveprocessionofartificialgastricjuiceandartificialintestinaljuice,theprecipita-tionobtainedinthedecoctingprocessofSHXXTcouldbepartlyredissociatedandreleasetheactiveingredients(dissolvedratiowithin4h:baicalin,62.63%;berberine,79.63%)(Renetal.,2005).Therefore,afteroraladministrationofHLJDT,undissoci-atedcomplexationprobablyalsoexistedinthegutlumen,whichcoulddecreasetheexposureofbaicalinto␤-glucuronidaseandsubsequentlyreduceitsabsorption.Thephysicalandchemicalreactionbetweenflavonoidsandalkaloidsmightbeanotherrea-sonforthedecreasedsystemicexposurelevelofbaicalinandwogonoside.

Resultsofthepresentstudyindicatedthepotentialphar-macokineticinteractionbetweenflavonoidsandalkaloidsinHLJDT.Fromthispointofview,thecombinationofRhizomacoptidisandRadixscutellariaewasratherunsuitable.Ontheotherhand,however,undertheguidanceofthetheoryofTCM,Radixscutellariaecouldenhancetheefficacy(“heat-clearing”,“dampnessresolving”)anddecreasethetoxicityofRhizomaCoptidis(synergismactivity),whichwereconfirmedbytheclin-icalapplicationforthousandsofyearsandbymodernpharma-cology(CuiandZhao,2003;Lietal.,1999).ThehugenumberofactiveingredientsinTCMmakesitsuitableformulti-targetactions.DifferentfromWesternmedicine,TCMconcentratesontheoverallfunctionalstateofthepatient,whichisbecoming

atrendinmodernmedicinefortreatingcomplicateddiseases(Jiang,2005).Therefore,consideringthisaspect,themultiherbremedymightbereasonableprescriptedinspiteoftheexistenceofchemicalandpharmacokineticinteractionbetweenRhizomacoptidisandRadixscutellariae.5.Conclusions

Ourfindingssuggestedthatbaicalinandwogonosidedemon-stratedbimodalphenomenonintheplasmaprofile.Insituabsorptionexperimentrevealedtheexistenceofdouble-siteabsorptionofbaicalin.Thefirstabsorptionsitewasinupperintestinalandmightbeduetothedirectlyabsorptionofbaicalinandthesecondsitewasincolonintheformofaglygon.SomeingredientsintheotherthreeherbsinHLJDT,notinRadixscutellariaeitself,hadpharmacokineticinteractionwithbaicalinandwogonosideinthesecondabsorptionsite.Thiswasprobablycausedbytheinhibitionofbacterial␤-glucuronidaseactivi-tiesbyRhizomacoptidisandCortexphellodendriortheirmainactiveingredients.Alsothecomplexationbetweenflavonoidsandalkaloidsmightdeceasestheexposureofbaicalinto␤-glucuronidase.Themechanismisstillambiguousandfurtherprofoundresearchesarerequired.Acknowledgements

TheprojectwassupportedbytheNationalNaturalScienceFoundationofChina(No.39970862),theNational“863”Project(No.2003AA2Z347A),andProjectofNationalTraditionalChi-neseMedicineAdministrationBureau(No.02-032P32).References

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