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Metabolomics analysis of Tripterygium wilfordii formulation based on theory of detoxicity compatibility

基于“异类相制”的雷公藤复方配伍减毒代谢组学研究



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MetabolomicsanalysisofTripterygiumwilfordiformulationbasedon
theoryofdetoxicitycompatibility
XIETong1,2,ZHOUXueping2,LINLili1,2,XUJianya1,2,SHENCunsi1,2,FENGZhe2,ZHOULingling3,SHANJinjun1,2
(1JiangsuKeyLaboratoryofPediatricRespiratoryDisease,InstituteofPediatrics,NanjingUniversityof
ChineseMedicine,Nanjing210023,China;
2TheFirstColegeofClinicalMedicine,NanjingUniversityofChineseMedicine,Nanjing210023,China;
3SchoolofPharmacy,NanjingUniversityofChineseMedicine,Nanjing210023,China)
[Abstract] TripterygiumwilfordiHook.f.inducedhepatotoxicitywasthemainlimitationforitsusageinclinicQingluoTongbifor
mulationshowedobviousatenuationforhepatotoxicityinclinicandfundamentalresearchinvivoToexplorethepotentialmechanismof
theatenuation,weconductedastudyontheplasmametabolomicprofilesofT.wilfordiandQingluoTongbiformulationinratsbya
sensitivegaschromatographymassspectrometry(GCMS/MS)methodInplasmasamples,atotalof72compoundswereanalyzedby
EIsourceMS,andweresuccessfulyidentifiedbymatchingNISTdatabaseThesemiquantificationresultswerethencalculatedby
OPLSDAmodelwithSIMCAP130softwareThethreegroupswereclearlydistinguishedinOPLSDAscoreplotInaddition,the
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observationvaluesofQingluoTongbiformulationshowedtheobvioustrendtowardsthecontrollevels,suggestingthedetoxicityefectof
theformulationVariationmetaboliteswerefurtheranalyzedbyVIPandOneWayANOVAs,andtheresultsshowedasignificantin
creaseincompoundsofglycogenicaminoacids,suchasalanine,proline,serineandglutamineaftertheadministrationofT.wilfordi,
indicatedthatthetissueproteinsweredecomposedandaminoacidswereleakageintobloodQingluoTongbiformulationcouldreverse
theaminoacidsintonormallevelOnthecontrary,thelevelsofglucose,lacticacidandhydroxybutyratedecrease,andtheformula
tioncanrelievethedisorderinthelevelsoflacticacid,suggestingtheregulationoftheenergymetabolismAdditionaly,thelevelof
branchedchainaminoacidwasdecreased,suggestedthetoxicitywasinduced,buttheformulationcannotincreaseitintothenormal
levelsNevertheless,altheaboveresultssuggestedthattheclassicalQingluoTongbiformulationdisplayedtheliverprotectionefectby
adjustingtheaminoacidlevelsandregulatingtheenergymetabolismQingluoTongbiformulationwasdevelopedbasedontraditional
Chinesemedicinetheory“detoxicitycompatibility”,andcontainedPanaxnotoginseng(Burk)FHChentonourishbloodandab
sorbclotsModernpharmacologysuggestedthatitsliverprotectionefectwascorelatedwiththepromotionofproteinsynthesisAnoth
erimportantherbisRehmanniaglutinosaLibosch,whichcanregulatetheenergymetabolismBothwereconsistentwiththemetabolo
micresultsinthisstudy,whichexplainedthepotentialmechanismof“detoxicitycompatibility”theoryTherefore,thecurentlydevel
opedmetabolomicapproachandtheobtainedresultswouldbehighlyusefulforthecomprehensivetoxicitystudiesforotherherbalmedi
cinesandvariouscomplexdeoxicityformulations
[Keywords] detoxicitycompatibility;Tripterygiumwilfordi;metabonomics;hepatotoxicity;QingluoTongbiformulation
doi:10.4268/cjcmm20160625
  
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teomic,andmetabolomicdataintegrationstrategies[J]Biomark
Insights,2015,10(suppl4):1
[7] ReilyMD,TymiakAAMetabolomicsinthepharmaceuticalin
dustry[J]DrugDiscovTodayTechnol,2015,13:25
[8] XiongYH,XuY,LangL,etalGaschromatographymass
spectrometrybasedprofilingofserumfatyacidsinacetamino
pheninducedliverinjuredrats[J]JApplToxicol,2014,34
(2):149
[9] GonzalezE,LiempdS,CondeVancelsJ,etalSerumUPLC
MS/MSmetabolicprofilinginanexperimentalmodelforacute
liverinjuryrevealspotentialbiomarkersforhepatotoxicity[J]
Metabolomics,2012,8(6):997
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