普洱茶对低血糖症的影响

Wan-hongDu,§Sheng-MingPeng,†,#Zhong-huaLiu,‡LingShi,‡Li-FengTan,†andXiao-QingZou*,††KeyLaboratoryofEnvironmentallyFriendlyChemistryandApplicationsofMinistryofEducation,CollegeofChemistry,XiangtanUniversity,Xiangtan411105,Hunan,People’sRepublicofChina‡NationalResearchCenterofEngineeringandTechnologyforUtilizationofFunctionalIngredientsfromBotanicals,HunanAgricultureUniversity,Changsha410128,Hunan,People’sRepublicofChina§TheFourthDepartmentofInternalMedicine,163HospitalofPLA,Changsha410003,Hunan,People’sRepublicofChinaABSTRACT:Thewaterextractionandcompositionofpu-erhtea,aswellasthehypoglycemiceffectofthewaterextractofpu-erhtea(WEPT)invivoandinvitro,arereportedtoinvestigateitshypoglycemiceffectondiabetes.High-performanceliquidchromatographyandcolorimetricmethodsareusedtoanalyzetheteacatechins,caffeine,polyphenols,aminoacids,andpolysaccharidesoftheWEPT.TheeffectoftheWEPTonglucoseuptakebyculturedHepG2cellsandtheinhibitioneffectofratintestinalsucrase,maltase,andporcinepancreaticamylasearedeterminedinvitro.Then,thebloodglucoseandinsulinlevelsofintragastricallydbadministeredWEPTonfastingandoralglucosetolerancetest(OGTT)usingtype2diabeticdb/db(BKS.Cg-m+/+Lepr/J)micearedeterminedinvivo.TheresultsshowedthattheWEPTdose-dependentlyandsignificantlyincreasedglucoseuptakebyHepG2cellsandinhibitedratintestinalsucrase,maltase,andporcinepancreaticamylaseactivity.TheWEPTintragastricallygivenfor4weekssuppressedtheincreaseinbloodinsulinandglucoselevelsofdb/dbmicefastedovernight.InOGTT,theWEPTimprovedimpairedglucosetoleranceandamelioratedretardedinsulinresponseat60and120minindb/dbmice.TheseresultssuggestthattheWEPThasbeneficialeffectsonglucosehomeostasisintype2diabetesandinamendmentofinsulinresistance.KEYWORDS:waterextractofpu-erhtea,hypoglycemiceffect,glucoseuptake,α-glucosidase,insulinresistance,db/dbmice■INTRODUCTION

carbohydrateswillleadtoasharpriseofPPG.FrequentType2diabetesmellitus(T2DM)isagroupofmetabolicconsumptionofsuchfoodsorhighglycemicindexfoodsisdisorderscharacterizedbyhyperglycemia,resultingfromsuggestedtoplayacausativeroleindevelopmentoftype2resistancetoinsulinactionand/orinadequateinsulinsecretion.diabetes.7Introductionofα-amylaseandα-glucosidaseChronichyperglycemiaisstronglyassociatedwiththeincreasedinhibitorintothediethasbeenproposedtobeeffectiveinriskofkidney,eye,andnervecomplications(microvascularretardingcarbohydratedigestion,resultinginreducedPPG.8complications),aswellasincreasedriskofstroke,heartdisease,Identifyingeffectiveα-amylaseandα-glucosidaseinhibitorfromandamputations(macrovascularcomplications),whicharenaturalsourcescanbebeneficialinthepreventionandresponsibleforthemajorityofmorbidityandmortalityintreatmentofdiabetesmellitus.

patientswithT2DM.

Tea(Camelliasinensis)isoneofthemostpopularbeveragesTheoverallloweringofglucoseisofpivotalimportanceinconsumedworldwide.Generally,teacanbebroadlyclassifiedthetreatmentofdiabetes,withprovenbeneficialeffectsonaccordingtoproductionmethodasunfermentedtea(greenmicro-andmacrovascularoutcomes.1Furthermore,evidencetea),semi-fermentedtea(oolongtea),fullyfermentedteasuggeststhat“glucosevariability”maybeanindependentrisk(blacktea),andpost-fermentedtea(pu-erhtea).Pu-erhteaisafactorforcardiovascularcomplicationsindiabetes.2Thekindofpost-fermentedteaproducedmainlyintheYunnantherapeuticchallengeliesintheneedforintensiveglycaemicprovinceofChinaandwidelyconsumedbytheChinese.Thecontrolandmaintenanceofglycaemiawithinastrictnormalkeyprocessofpu-erhteapreparationissecondaryfermenta-narrowrange.Somestudieshavesuggestedthatanelevatedtion,inwhichmicroorganismsplayaveryimportantroleinpostprandialglucose(PPG)levelisacriticalcomponentofproducingthetaste,color,fragrance,andfunctionalcompo-diabetes-relatedvascularcomplicationsbecausePPGisanents.Duringthefermentationprocess,catechinsareoxidizedcontributortobothacuteglucosefluctuationsandchronicandthenpolymerizedtoformbisflavanol,theaflavins(TFs),sustainedhyperglycemia.3TheGermanDiabetesInterventionthearubigins(TRs),andotherhigh-molecularcomponents.StudyshowedthatcontrollingPPGhasagreatereffectonSomestudieshaveshownthatpu-erhteaanditscomponentscardiovasculardiseaseandall-causemortalitythancontrollinghavemanybiologicalandbiochemicaleffects,suchasfastingplasmaglucose(FPG)levels.4Studieshavealsoshownpreventionofcancerandcardiovasculardiseases.9,10Italso

thatoralantidiabeticagentsthattargetPPGslowandevenreversetheprogressionofcarotidintima-mediathickening.5Theseresults,whichhavealsobeenconfirmedbyotherstudies,Received:June5,2012

suggestthatPPGisanindependentRevised:September7,20126riskfactorforvascularAccepted:September9,2012complicationsindiabetes.Ingestionofhighlyavailable

Published:

September10,2012

©2012AmericanChemicalSociety

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JournalofAgriculturalandFoodChemistryArticlehasantiobesity,11,12antibacterial,13antioxidative,14,15antiviral,16andhypolipidemic17,18effects.

polysaccharide,Theanthronecolorimetricmethod22wasusedtoAneffectivemeanstocontrolthebiologicalnetworkthattometerunderliesdiseasesisneededtotreatcomplexdiseases,suchastestedatawavelengthandtheabsorbanceof620nm.Standardwasrecordedsolutionsusingofaglucosespectropho-assayteadiabetesorcancer.However,monotherapyactingonasingleAssayintheofsameSucrase,mannerMaltase,toobtainandaAmylasecalibrationActivity.curve.

were23targetisoftenlimitedinitstherapeuticeffectbecauseoftheandinherentrobustnessandredundanceMatsuomaltaseSucrasebodyandactivityIzumori.was23Brieassayedfly,fouraccordingmaleWistartotheratsmethodof180describedby19ofbiologicalnetworkstoexternalperturbationsandchanges.Multi-targettherapeuticsisoftenmoreeffectivecomparedtomonotherapiesforcomplexcervicalweightquicklydislocation,werefastedand12ahbeforeuse.Theratswere−killed240gbyofdiseases,suchasdiabetes.Ingeneral,herbalmedicinesarecomplexmixturesofdifferentactivecompoundsthatoftenactmicroscopeTheexcisedmucosalandsegment(40−45cm)ofjejunumwassurfacerinsedwaswithice-coldsalinesolution(0.9%NaCl).inasynergisticfashionandexerttheirfullbeneficialeffectsashomogenizedslide.totalextracts.Inthisstudy,thewaterextractionand(0.9%compositionofpu-erhteaarepresented.TheglucoseuptakethenNaCl)andtogetherAllofcentrifugedwiththecollectedremovedbygentlyscrapingwithaamucosafromthefourratswereat9-fold4°Cvolumefor30minofcoldat12saline000rpm,solutionandpromotioneffectisexploredinvitrotodiscusstheinfluenceofactivitythethewaterextractofpu-erhtea(WEPT)onthehepaticglucosesubstratesofsupernatantmaltaseandwassucrase.usedasaMaltosecrudeenzymeandsucrosesolutionweretoassaythemetabolismthatmainlyaffectsFPG.Theinhibitioneffectsofwasofmaltaseandsucrase,respectively.ThereleasedusedasDsucrase,maltase,andamylaseareperformedtostudythediagnosticdeterminedkitscolorimetricallyusingtheglucoseoxidasemethods-glucosepotencyoftheWEPTinretardingcarbohydratedigestionandasThereducingPPGinvitro.Todeterminetherelationshipbetweenαsubstrates.porcine(NanjingThepancreaticJianchengprocedureamylasefordeterminingwasBioengineeringdeterminedtheenzymaticusingInstitute).

solubleactivitystarchFPG,PPG,andinsulinvalueswiththeWEPTtreatment,anenzymatic-amylasewasalignedwiththeprotocoloftheSigma-Aldrichoforalglucosetolerancetest(OGTT)wasperformedonobeseconcentrationassayofα-amylase(EC3.2.1.1).The50%inhibitionanddiabeticdb/db(BKS.Cg-m+/+Leprdb/J)miceinvivo.the(IC50)wasobtainedfromthelinearregressioncurveofTheexperimentsareexpectedtobevaluableinfurtherGlucoseconcentrationsUptakeandAssay.inhibitory24understandingthepotentialusesofpu-erhteafortheandHepG2rates.

cellsdevelopmentculturecultured■

oftherapeuticandpreventiveagentsfordiabetes.

wasplates.inAfterDMEM.thecellsHepG2reachedcells80werewasplatedobtainedinto96-wellfromATCCtissueWEPTreplacedMATERIALSANDMETHODS

positive(0.01,0.03,byDMEM−90%confluence,themediumand0.1containingg/L),variousconcentrationsoftheReagents.Caffeine(CAF),(−)-epigallocatechin3-gallateglucosidasereference(EGCG),(−)-epigallocatechin(EGC),(±)-catechin(DL-C),(−)-ep-hpositivecompound),orrosiglitazoneacarbose(10(10μmol/L,μmol/L,usedusedasaasαa-icatechinatofgallate(GCG),(EC),(α−-amylase)-epicatechin(EC3.2.1.1),3-gallateand(ECG),acarbose(−)-gallocatechinwerepurchased3-and100treatment,themediumreferencewascompound)removedaddedandalltocellsthewerewells.incubatedAfter24NBDG.thenμMwashed2-NBDG5timesinphosphate-buwithadditionalfferedPBSsalineto(PBS)removefor15minfromwavelengthFluorescence2-deoxyglucoseSigma-Aldrich.(2-NBDG)2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-wasobtainedfromMolecularProbes.DTX880of485nmandintheanemissioncellswaswavelengthmeasuredofat535anexcitationexcess2-HepG2MousemultifunctionnmusingaExperiments.counterAnimal(Beckmanhusbandry,Co.,care,Ltd.).

andexperimentalgrownincellshigh-glucose[fromAmericanDulbeccoType’smodiCulturefiedCollectionEagle’smedium(ATCC)](DMEM)wereprocedures(purchasedLaboratorywereconductedincompliancewith(obtainedfromfromSijiqingHyclone)BiologicalcontainingEngineering10%fetalMaterials,bovineserumHangzhou,(FBS)guidelines,China).CommitteeasAnimalapprovedCareby”theNationalInstitutionalInstitutesthe“PrinciplesofAnimalofHealthCare(NIH)CentermutationSampling.ofAllNanjinganimalsTheUniversity.

werepurchasedfromModelAnimalResearchpu-erhteasampleusedwasYunnanpu-erh2008totalof48(BKS.Cg-m(IACUC).obeseand+/+MicediabeticLeprhomozygousdbdb/db/J)werefor(BKS.Cg-musedthefordiabetes+/+invivospontaneousandUseLeprdbstudies.AprovidedbyBio-ResourcesInnovativeDevelopmentOfficeofthemicePeoplediabeticwereusedforinvivostudies,and/J)maleconducted’sGovernmentwereandensuretosampleestablishofYunnanProvince.Preliminaryassayswerehomogeneitytheappropriateandrepresentativeness.amountofsamplesEachforanalysisofthedarkhousedmiceat(C57BLKS/J)22±wereusedasatotalcontrols.of8age-matchedAlloftheanimalsnon-teaweekscycleof12h.Food2°Candand55water±5%wererelativegivenhumidity,withalight/boiledsamplesdistilled(100waterg)was(1andcut0.2intoLofsmalleachpiecesbulk;and45andsoaked35mintwiceeachinoftime).daybodyofweightage,differentdosesoftheWEPT(100,200,adlibitum.and400Frommg/kg8wereconcentratedAftertheleavesandwerefreeze-dried.separatedthroughThefreeze-driedfiltration,wholeextractsextractswereacarbosefor28days.perRosiglitazoneday)wereadministered(5mg/kgofbybodyintragastricweightperroutday)onceandathencontrols.(10mg/kgofbodyweightperday)wereusedasexperiments.

mixed,ground,andpassedthrougha40-meshsieveforlatercontrols.

Age-matchednon-diabeticmice(C57BLKS/J)werepositiveusedasBloodCollection.ThefastingbloodwascollectedfromratsafteraofHigh-PerformanceTeaCatechinsandLiquidCaffeine.ChromatographyHPLC(Shimadzu,(HPLC)Japan)Analysiswas12performedtoidentifycatechinsandcaffeine.Thechromatographicthehovernightfast.PlacetheratinaplexiglasstubeandconditionsInmm,5μm);weremobileasfollows:phaseShim-packcomposedODSofmobileC18columnphase(4.6A,double-×150heldthesidebloodoftheglucosetailoverdetermination,theveinwithgentlywashpuncturegauzesoakedwith75%ethanol.distilledintoperpendicularrate,1.1water;mL/min;mobileandphasequantiB,ficationDMF/MeOH/HACofcatechinsand(40:2:1.5);caffeineatfl278owbloodabloodnm.

ofcollectionglucose(90°angle)tothetailandcollecttheveintheusingbloodadirectlyneedleneedlestrip.parallelInthewithbloodtheinsulintailintodetermination,theveinuntilinsertaflashasaccharidesMeasurementsofExtracts.ofPolyphenols,ThecontentofAminotheextractsAcids,oftheandtotalPoly-teathebloodisseenanduseaevacuatedbloodcollectiontubetocollectOGTT.blood.

Aftera12hovernightfastonday28,db/dbmicewerepolyphenolstotalpolyphenolwascontentdeterminedwasusingdeterminedtheFolinat760−Dennisnmbymethod.20comparisonThetogivenastandardwaterdiTotalaminocurveacidspreparedwereusingestimatedtannicspectrophotometricallyacidasastandardreference.bythesolutionintragastrically;fferentdosesof0.5thehWEPT,rosiglitazone,acarbose,ordistilledninhydrinassayatawavelengthof570nm.21samples120,andwerewas180collectedintragastricallylater,minaftersequentiallyadministeredglucose(2g/kgofbodyweight)glucoseadministration.

fromthetotailtheveinanimals.beforeandBlood60,10127

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JournalofAgriculturalandFoodChemistryArticleMeasurementofBloodGlucoseandInsulin.BloodglucoseafromTable2.ContentsofRegularIngredientsintheWEPTglucometerthetailveinwasmeasuredusinganAccu-Chekcomfortconcentration(Rocheenzyme-linkedintheDiagnostics,serumwasmeasuredBasel,Switzerland).bycommerciallyTheavailableinsulingrouppolyphenols

(%)amino(%)acidcatechin(%)polysaccharide

tea

(%)ProductsWEPT

30.79±0.43

1.06±0.01

1.20±0.02

1.21±0.01

blooda28.

glucoseCompany,immunosorbantconcentrationsLtd.).Duringassays(ELISAs;AmericanLaboratorywerethemeasuredexperimentalondaysperiod,0,7,14,the21,fastingandThepercentagerepresentsper100gofWEPT.EachvaluerepresentsStatisticalAnalysis.Datawereshownasthemean±standardthemean±SD(n=3).

deviationby(SD).Differencesbetweenacarbose(10μmol/L)significantlypromotedtheglucoseandone-wayanalysisofvariation(ANOVA),individualfollowedgroupsbywereDunettanalyzed’stestuptakeofHepG2cellscomparedtothevehiclecontrol0.5%of■

<0.05leastwassigniconsideredficantdifferencesignifi(LSD)cant.

test.AdifferencewithapvalueH2O(p<0.05).

RESULTS

EffectoftheWEPTontheBodyWeightofDiabeticdb/dbMice.Thebodyweightsofthegroupofdb/dbmiceCatechinsandCaffeineContents.Therepresentativewerehigherthanthoseinthecontrolgroup(p<0.01),asHPLCpatternsoftheWEPTareillustratedinFigure1.

showninTable4.Thebodyweightsofthedb/dbmicegiven200and400mg/kgofWEPTwerelowerthanthatofthetype2diabeticdb/dbmice(p<0.05or0.01)onday28.Forthepositivecontrol,thebodyweightsofdb/dbmicegivenacarboseandrosiglitazonewerelowerthanthoseofthemodeldb/dbmice(p<0.01)onday28.

EffectoftheWEPTontheFastingBloodGlucoseofDiabeticdb/dbMice.Thefastingbloodglucoselevelsofthegroupofdb/dbmicewerehigherthanthoseinthecontrolgroup(p<0.01),asshowninTable5.Thefastingbloodglucoselevelsofdb/dbmicegiven400mg/kgofWEPTwerelowerthanthoseofthetype2diabeticdb/dbmice(p<0.05orFigure0.01)ondays21and28,thesameas200mg/kgonday28(pCAF,ca1.ffHPLCeine;DL-C,chromatogram(±)-catechin;oftheWEPTEC,(−(UV)-epicatechin;signalat278EGCG,nm):<0.05).Forthepositivecontrol,thefastingbloodglucose(levelsofdb/dbmicegivenacarboseandrosiglitazonewere(−−)-epigallocatechin)-epicatechingallate.

gallate;GCG,gallocatechin-gallate;andECG,lowerthanthoseofthemodeldb/dbmice(p<0.05or0.01)fromday14today28.

EffectoftheWEPTontheFastingBloodInsulinLevelCaffeineandsixmajorcatechinforms,namely,(−)-epigallo-ofDiabeticdb/dbMice.Thefastingbloodinsulinlevelsofcatechin(EGC),(±)-catechin(DL-C),(−)-epicatechin(EC),thegroupofdb/dbmicewerehigherthanthoseinthecontrol(−)-epigallocatechingallate(EGCG),gallocatechin-gallategroup(p<0.01),asshowninFigure3.Thefastingblood(GCG),and(−)-epicatechingallate(ECG),weredetectedinsulinlevelsofdb/dbmicegiven400mg/kgofWEPTwere(Table1),butEGCwasnotdetectedintheWEPT.

lowerthanthoseoftype2diabeticdb/dbmice(p<0.05)onContentsofRegularIngredientsintheWEPT.Activeday28.Forthepositivecontrol,thefastingbloodinsulinlevelsingredients,includingteapolyphenols,freeaminoacids,ofdb/dbmicegivenacarbosewerenotaffectedsignificantly,catechins,andpolysaccharides,wereanalyzedandareshownwhereasthoseoftherosiglitazonegroupwerelowerthanthoseinTable2.

ofthemodeldb/dbmicegroup(p<0.01)onday28.

Sucrase,Maltase,andAmylaseInhibitionEffectsofEffectoftheWEPTontheOGTTofDiabeticdb/dbtheWEPT.Inthisstudy,α-glucosidaseinhibitionassaysfortheMice.EffectoftheBloodGlucose.Theincreasedpost-OGTTWEPTwereconductedusingratsmallintestinalsucraseandbloodglucosevaluesofthegroupofdb/dbmicewerehighermaltaseinvitro.TheWEPTshowedinhibitoryeffectsonratthanthoseinthecontrolgroup(p<0.01),asshowninTable6.intestinalsucrase,maltase,andporcinepancreaticamylase,asTheincreasedbloodglucosevaluesofdb/dbmicegiven400showninTable3,butlesspotentcomparedtoacarbose(ICmg/kgofWEPTwerelowerthanthoseofthetype2diabetic4.35±0.59,6.63±0.70,and.19±6.77μmol/Lforsucrase,50=db/dbmicegroup(p<0.05)at1and3hpost-OGTT.Forthemaltase,andamylase,respectively).

positivecontrol,theincreasedbloodglucosevaluesofdb/dbGlucoseUptakePromotionEffectoftheWEPT.ThemicegivenacarboseandrosiglitazonewerelowerthanthoseofglucoseuptakeofHepG2cellstreatedwiththeWEPTandthemodeldb/dbmice(p<0.05or0.01)at1,2,and3hpost-positivereferencerosiglitazoneandacarbosewasalsoOGTT.

determinedtoevaluatethehypoglycemiceffectsoftheEffectoftheBloodInsulin.Thepost-OGTTbloodinsulinWEPT(Figure2).TheresultsshowthattheWEPT(0.03levelofdb/dbmicedidnotincreasesignificantlycomparedand0.1mg/mL)androsiglitazone(10μmol/L)butnot

withcontrol,whereasthebloodinsulinlevelofdb/dbmice

Table1.ContentsofFiveMajorCatechinFormsandCaffeineDeterminationintheWEPTbyHPLCaCAF(%)DL-C(%)EC(%)EGCG(%)GCG(%)ECG(%)8.51±0.15

0.57±0.01

0.23±0.00

0.02±0.00

0.06±0.00

0.15±0.00

aThepercentagerepresentsper100gofWEPT.Eachvaluerepresentsthemean±SD(n=3).CAF,caffeine;DL-C,(±)-catechin;EC,(−)-epicatechin;EGCG,(−)-epigallocatechingallate;GCG,gallocatechin-gallate;andECG,(−)-epicatechingallate.

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JournalofAgriculturalandFoodChemistryArticleTable3.InVitroInhibitoryaEffectsofVariousConcentrationsoftheWEPTonRatIntestinalSucrase,Maltase,andPorcinePancreaticAmylaseconcentration(g/L)

sucrasemaltaseamylaseinhibitionratio

0.10.01±0.000.01±0.000.01±0.000.30.09±0.010.12±0.030.07±0.0210.27±0.070.29±0.040.21±0.0330.46±0.020.49±0.030.36±0.0310

0.84±0.020.85±0.010.73±0.03IC50(g/L)

3.47

±0.36

3.14

±0.38

5.13

±0.61

aconcentrationsEachvaluerepresentsandinhibitorythemeanrates.

±SD(n=3).IC50isthe50%inhibitionconcentrationobtainedfromthelinearregressioncurveoftheriskfactors,suchasobesity,hypertension,andhighglycaemicvariabilitywithlittletonoriskforhypoglycemia.25Intensiveeffortshavebeenexertedtosearchforeffectiveandsafeα-glucosidaseinhibitorsinnaturalmaterialstodevelopphysiologicalandfunctionalfoodforthepreventionandcureofdiabetes.Kohetal.investigatedtheabilityofgreen,oolong,andblackteasininhibitinghumansalivaryα-amylaseandmammalianα-glucosidaseandfoundthattheinhibitoryprofilesofteasarecorrelatedtotheirmajorpolyphenolcontent,TFs,andcatechins.7Lietal.indicatedthattwocomponents,EGCGandECG,arepotentα-glucosidase(EC3.2.1.20,fromSaccharomycescerevisiae)inhibitorsfoundinpu-erhtea,withIC50lower26thannitrophenyl-αthatofacarbose,acommercialα-glucosidaseinhibitor.αFigurereaction-glucosidaseα-Glucosidase-fromtheS.cerevisiaeassayusesp-Disdirectlyinto-glucopyranosideproportionalp-nitrophenyl(PNPG)to(PNP).thattheenzymeTheishydrolyzedactivity.rateofbyThistherosiglitazone2.Eff(10ectsμmol/L),ofvariousandconcentrationsacarbose(10μmol/L)oftheWEPTontheglucose(g/L),uptakemethodisasatisfactoryandefficientscreeningtest,butthe0.05%versusofHepG2thecontrol.

cells(valuesarethemean±SD;n=12).(∗)pdb/dbmice,asshowninTable7.

400mg/kgofWEPTwerelowerthanthoseofthediabetesDISCUSSION

modeldb/dbmicegroup(p<0.05)at1and3hpost-OGTT.TheseDiabetesmellitusisoneoftheprimarythreatstohumanhealthαbecauseofitsincreasingprevalence,chroniccourse,andglycemia-glucosidaseresultsprovideandmayinhibitorevidenceplayacouldthatsignifireducetheWEPTcantrolepostprandialasaneffectiveinthepreventionhyper-disablingcomplications.ControlofpostprandialhyperglycemiaandtreatmentofT2DMandpre-diabeticstates.Manystudiesisimportantinthetreatmentofdiabetes.α-Glucosidasesareindicatedthatthehealth-promotingactionsoftheteaglycosidehydrolasesfoundontheluminalsurfaceofenter-consumptioncouldbeasummationeffectofalloftheocytescontainingmaltase/glucoamylaseandsucrase/isomaltaseingredientspresentinthem,especiallythepolyphenols.Theactivity.Targetingmainlypostprandialhyperglycemia,α-digestiveenzymeinhibitorypotencyoftheWEPTcouldbeglucosidaseinhibitorsfavorablyaffectseveralcardiovascular

mainlycontributedbyitspolyphenolcontentsaccordingto

Table4.EffectoftheWEPTontheBodyWeightsofFastedDiabeticdb/dbMiceabodyweights(g)groupsday0day7day14day21day28control17.1±1.1b18.2±1.3b19.7±1.3b21.4±1.4b22.4±1.0bdb/db

27.4±1.429.2±1.331.2±1.233.1±0.835.6±0.8100mg/kgofWEPT27.4±1.429.1±1.030.9±1.532.4±1.234.1±1.1200mg/kgofWEPT27.6±1.129.6±1.031.4±1.032.3±1.232.1±1.4c400mg/kgofWEPT27.5±1.029.3±1.330.6±1.531.1±1.430.3±0.8b10mg/kgofacarbose27.6±1.029.6±1.030.8±1.231.0±1.430.8±1.7b5mg/kgofrosiglitazone

27.4

±1.4

29.3

±1.4

29.7

±1.2

29.2

±0.9

28.7

±1.6baValuesarethemean±SD;n=8.bp<0.01versusthedb/dbgroup.cp<0.05versusthedb/dbgroup.

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JournalofAgriculturalandFoodChemistryArticleTable5.EffectoftheWEPTontheFastingBloodGlucoseofDiabeticdb/dbMiceafastingbloodglucose(mol/L)groupsday0day7day14day21day28control5.20±0.43b5.25±0.45b5.26±0.38b5.27±0.48b5.23±0.43bdb/db

23.63±2.2027.42±2.4028.96±1.9530.39±2.1331.35±2.28100mg/kgofWEPT22.65±3.1926.54±3.1628.29±3.3328.46±2.7129.14±4.51200mg/kgofWEPT22.90±2.2226.37±2.0928.07±1.6827.67±1.5824.99±1.42c400mg/kgofWEPT23.11±2.5826.87±2.5327.45±2.2725.61±3.37c22.60±2.87b10mg/kgofacarbose22.60±2.5325.48±1.6822.33±2.33c20.74±2.91b19.28±2.30b5mg/kgofrosiglitazone

22.67

±3.15

25.46

±3.20

23.31

±2.34c20.62

±2.55b18.52

±3.15baValuesarethemean±SD;n=8.bp<0.01versusthedb/dbgroup.cp<0.05versusthedb/dbgroup.

whichresultsinhighglucoselevelsintheblood.ChronicameliorationofinsulinresistancewouldpreservepancreaticβcellfunctionanddelayorpreventtheonsetofT2DM.Zhangetal.showedthatEGCGcaninhibitdexamethasone-inducedinsulinresistanceandimproveinsulin-stimulatedglucoseuptakebyincreasingGLUT4translocationtotheplasmamembraneinratL6cells.28Caoetal.showedthatgreenteaextractcanincreasegeneexpressioninglucoseuptakeandtheinsulin-signalingpathwayinratsfedwithafructose-richdiet.29IntheearlieststagesofT2DM,themajordefectinvolvestheinabilityofinsulintopromoteglucoseuptakeandstorageasglycogen.Accordingly,impairedinsulinactionatthelivercausesfastinghyperglycemia.30Inthisstudy,theglucoseuptakeofHepG2cellstreatedwithvariedconcentrationsoftheWEPTwasdeterminedtoevaluatethehypoglycemiceffectsoftheWEPTinvitro.TheresultsshowedthattheWEPT(0.03and0.1μcomparedmol/L)g/L)andsignirosiglitazonetotheficantlycontrolpromoted(10μ(vehicle,glucosemol/L)0.5%HuptakebutnotofacarboseHepG2cells(102).Fromtheinvivostudy,thefastingblood2O;p<0.05;Figureglucoselevelsofdb/dbmicegiven200and400mg/kgofWEPTandthefastingbloodinsulinlevelsofdb/dbmicegiven400mg/kgofWEPTwerelowerthanthoseoftheT2DMmodeldb/dbmiceondayFigure28.Thesameresultwasobtainedforrosiglitazone(5mg/kg).db/dbmice3.Eff(valuesectofthearetheWEPTmeanon±theSD;fastingn=8).insulin(∗)plevel<0.05ofanddiabetic(∗∗)Thefastingbloodinsulinlevelsofdb/dbmicegivenacarbosep<0.01versusthedb/dbmodelgroup.

werenotaffectedsignificantly.Thedeterminantsofelevatedfastingglucoseand2hplasmaglucoselevelsinOGTTdiffer.Raisedhepaticglucoseoutputandadefectinearlyinsulinsomestudies.7,26Polyphenolsinteractwiththeenzymesecretionarecharacteristicofthefastinghyperglycemia,andprimarilyvianoncovalentinteractions,namely,hydrogen-peripheralinsulinresistanceismostcharacteristicofthelatter.bondingandπ−πinteractionswithdigestiveenzymes.7Fromourstudy,onlythe400mg/kgofWEPTsuppressedtheT2DMischaracterizedbyinsulinresistance,whichmaybeincreasedpost-loadglucoselevelsontheOGTT,althoughbothcombinedwithrelativelyreducedinsulinsecretion.Insulin200and400mg/kgofWEPTdecreasedfastingglucoselevels.enablesmuscle,fat,liver,andotherbodycellstouptakeglucoseTheameliorationofimpairedinsulinactionattheliverandthefromthebloodanduseglucoseasenergy.WhenapersonhasdefectinearlyinsulinsecretioncouldbecontributedtotheT2DM,eithernoorlowglucoseuptakeintothesecellsoccurs,

effectofthelowerdose(200mg/kg)WEPTonfastingglucose

Table6.BloodGlucoseEffectoftheWEPTontheOGTTofDiabeticdb/dbMiceaincreasedbloodglucosevaluepost-OGTT(mol/L)groupsbloodglucosepre-OGTT(mol/L)1h2h3hcontrol5.23±0.43b1.90±0.41b0.66±0.19b0.30±0.13bdb/db

31.35±2.285.82±1.713.79±1.321.96±0.94100mg/kgofWEPT29.14±4.515.11±1.323.32±1.791.75±0.82200mg/kgofWEPT24.99±1.42c3.92±1.302.51±1.221.34±0.78400mg/kgofWEPT22.60±2.87b3.12±1.06c2.21±1.040.92±0.36c10mg/kgofacarbose19.28±2.30b2.72±1.36c1.65±0.90c0.69±0.40b5mg/kgofrosiglitazone

18.52

±3.15b2.95

±1.16c1.78

±1.06c0.41

±0.36baValuesarethemean±SD;n=8.bp<0.01versusthedb/dbgroup.cp<0.05versusthedb/dbgroup.

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JournalofAgriculturalandFoodChemistryArticleTable7.InsulinEffectoftheWEPTontheOGTTofDiabeticdb/dbMiceaincreasedinsulinvaluepost-OGTT(mIU/mL)groupsinsulinpre-OGTT(mIU/mL)1h2h3hcontrol21.78±5.24b20.14±5.32b7.36.±2.011.17±0.57db/db

53.31±5.497.76±1.039.28±3.022.16±0.65100mg/kgofWEPT47.78±6.188.43±2.3310.21±3.192.70±1.12200mg/kgofWEPT48.±5.7712.15±3.10c8.79±2.241.87±0.61400mg/kgofWEPT38.56±4.88c16.61±2.76b6.21±1.041.72±0.5410mg/kgofacarbose46.78±5.659.97±1.3610.65±2.901.69±1.015mg/kgofrosiglitazone

32.48

±4.52b19.25

±5.18b6.37.

±1.06

1.41

±0.76

aValuesarethemean±SD;n=8.bp<0.01versusthedb/dbgroup.cp<0.05versusthedb/dbgroup.

levels.Thehypoglycemicmolecularmechanismofactivetype2diabetesisdueprimarilytolifestylefactorsandgenetics.compoundsintheWEPTneedsfurtherinvestigation.

Pu-erhteaisapopularbeverageinsouthwesternChinaandInaccordancewiththeratioofthehumanbodysurfacearea,southAsiancountries.IntraditionalChinesemedicine,pu-erhtheratdosewas100,200,and400mg/kgofWEPTperdayteaandtheadultdosewascalculatedas15,30,and60mg/kgofWEPTperday.Considerahumanwithabodyweightof60kg,“isbelievedtoinvigoratethe“spleen”andinhibitweightdampnessand”.cholesterol.Pu-erhtea11,12isahealthyChinesebeverageculturethatalsohelpsbelievesregulatethattheadultdosewas0.9,1.8,and3.6gofWEPTperday.Thepu-erhteacounteractstheunpleasanteffectsofheavyalcoholphaseItrialoforalgreenteaextract(GTE)inadultpatientsconsumption.Inthisstudy,theWEPTexhibitedα-glucosidasewithsolidtumors31andthephaseIItrialofGTEinpatientsinhibitionandglucoseuptakepromotioninHepG2cellsinwithhigh-riskoralpremalignantlesions32showedthatadoseofvitro.Fromtheinvivostudy,theWEPTreducedbothfasting1.0g/m23timesdaily(equivalentto5.2gofGTEperday)canandpostprandialhyperglycemiasignificantlyandamelioratedbetakensafelyforatleast6months.Accordingtothesestudies,insulinresistanceofT2DMdb/dbmice.RegularconsumptionoralWEPTatthedosesthatwestudiedcanbetakensafely.ofpu-erhteacancontributetothepreventionoftheprogressTheprocessingofpu-erhteaisquitedifferentfromthatofofhyperglycemiaandinsulinresistanceofT2DMwithregardblacktea,althoughbotharefermentedteas.Duringpu-erhteatotheameliorationofmetabolismdysfunction.Withtheprocessing,thefirststepissunfixationandrolling,whichcompositionofactivemoleculeswithawidevarietyofarrestsenzymaticoxidationintheleaf.Thesecondstepiswetphysiologicalandpharmacologicalfunctions,thepowerfulpilingbyprolongedbacterialandfungalfermentationinawarmeffectofpu-erhteaonmetabolismisimportanttopublichumidenvironmentundercontrolledconditions,whichhealthbecausethebenefitsthatitprovidescanbeachievedtypicallytakesfrom0.5to1year.Thebacterialandfungalwithoutdrasticchangesinthelifestyleofmodernpeople.Thus,culturesinthefermentingpilesconsistofmultiplestrainsofregularpu-erhteaconsumptionisproposedasastrategyfortheAspergillusspp.,Penicilliumspp.,yeasts,andawiderangeofothermicroflora.Duringthefermentationprocess,thepolyphenolsinthetealeavesmaybepartiallytransformedtoTFs,TRs,andotheroligomersbytheenzymesproducedbythemicrobes.IncomparisontoGTE,theWEPTcontainsless■

regulationofenergybalanceandmetabolism.

AUTHORINFORMATION

CorrespondingAuthor

polyphenolsbutalittlemorecaffeine.Studiesshowedthat*coffeeexertsasuppressiveeffectonhyperglycemiabymail:Telephone:zouxq2002@gmail.com.

+86-731-58293574.Fax:+86-731-58292251.E-improvinginsulinsensitivitypartlybecauseofcaffeine.33Andersonetal.showedthatTFsandEGCGenhanceinsulin#AuthorContributions

Sheng-MingPengistheco-firstauthor.

withthegreatestactivityinratepididymaladipocytes.34Funding

Cameronetal.foundthatthreeTFs(theaflavin3-O-gallate,WearegratefultotheprojectsupportedbytheHunantheaflavin3′-O-gallate,andtheaflavin3.3′-di-O-gallate)andProvincialNaturalScienceFoundationofChina(12JJ6081),TRsarenovelmimicsofinsulin/IGF-1actiononmammaliantheprojectsupportedbytheScientificResearchFundofFOXO1aandPEPCK.Moreover,evidencethattheeffectsonHunanProvincialEducationDepartment(10C1288),thethispathwayofEGCGdependuponitsabilitytobeconvertedNaturalScienceFoundationofXiangtanUniversityintotheselargerstructureswasprovided.35Thecatechin,(2011XZX09),theFoundationofResearchFundforthecaffeine,TF,andTRcontentsintheWEPTmaypartiallyDoctoralProgramofHigherEducationofChinacontributetoimproveinsulinresistance.Moreinvestigations(20114301120004),andtheDr.StartFundsofXiangtanshouldbeconductedtodefinethecomponentsresponsibleforUniversity(11QDZ46)forfinancialsupportforthisresearch.

theeffect.

AccordingtotheNationalHealthandNutritionExaminationNotes

Survey(NHANES),from2003to2006,31.9%ofchildrenandTheadolescentswereoverweightorobese.36Adolescentobesityhasbeenattributedtoavarietyoffactors,oneofwhichisbeverageconsumption.ProjectEAT-II(EatingAmongTeens;a5year■

authorsdeclarenocompetingfinancialinterest.

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