JouRNAl．Qr FuNCTIONAL F00DS

22（2016）490−503

Availableoniineatwww．sciencedirect．com

SciehceDiredELSEVIER jou…同omepage：WWW・eisevier・cOmIIocate仰An竜一dbesitye鯖ectsofAsianday鳳owel
CorrImeIi調ComrrIurIIS，lnmicewimhighfatdiet−inducedobesityandin3T3−Llcells
ShioHNagaia，乱すW加ねia，MakioShib帥Ob，KoF可imo青年・＊aLaboratOリザBiod垂nse釧dReg血tio時Oso加Uniuers申すPh nmocc諦coISci飢CcS．4・20・lNosqhar0．
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Osa融569・1094．Io qn

A R TI C L E i N F O

A B S T R A C T

Artic厄histo7y‥

Asiandaynower，Comme臨ocommunistea（CCr）．decreasedmeb（Nlyweightgainandreduced

Receivedll December2015

themassofviscehlandsubcutaneousadiposedssueinthehigh−tatdiet（HFD〉−tedmice・

ReceivedinrevisedfomGFebruary

ThesemmglucoSeandtTiacylglycemllevelswerel°WeredinCCTadministeredHFD−fed

2016

mice．Moreover，CCTSllghtlyimprovedtheirinsulinsensitivi TbeluddatethemolecularAccepted8Febma町2016

mechanismofanti−adipogenice純膜ofC．communis．wefocusedonthe触vonoidsthathave

Availableonnne

Vahousphysiol0両alhnctions．Them如OrnⅣonOids徹）mC．00mmuniswerepuf並edbyHPLC．
andtheirstructmesdetenninedbyNMRandmassspeetrometry．GlucoluteolinOuteolin−
7−0−glucoside），aaavonoidofC．co771mtlrlis，reducedthein億acellulartriacylglycerollevelin

KヴWords：

Asiandaynower

3T3−LIceus．Thetranscriptionlevelofglucosetransporter4（GLUT4）andglucoseuptake

CommeIinocommunis

WeredecreaSedbyglucolute°lin．TheseresultsindicatethatCCrreducedthebodyweight

And一〇besity

gainandslightlyimpr°VedtheinsulinsensitivityinHFD・fedmice．Gluc01uteolinre−

Tea

PreSSedtheaccumulationofintracellularHpidsbysuppressingGLUT4−mediatedglueose

GLUT4

uptakeintoadipocytes．

①2016とIsevierLtd．Allrightsreserved・

Gluc°luteounincreaseintheadipoSetissuemassandisassodatedwithme
1．

InnoducGon

developmentofnumerouspathologicaldi5°rders（Attie＆Scherer，2009；Comiereta1．，2008；Pi−Sunyer，2009）．Adipocytes
Obesityisaglobalhealthproblemanditsprevalenceisrapidly playchticalrolesinmaintainingenergybalance・Theseceus
increasing（Finucaneeta1．，2011）．0besityiscausedbyan storelipidsasanenergysoureeandreleasefattyacidsin

CorltSPOndingatlthor．LaboratoryofBi°defenseandReguladon，OsakaUniversityofPhamaceudcalSciences．4−20−1Nasahara，

mkat−

SuH．0saka569−1094，Japan．ml．：鴫1726001215；ぬX：鴫1726的1215・E・mailaddress：fujimohegly．oups．acjp（K・Fujim°坤Abb†etJiation：C／馳P．CCAAr／enhancer−bindingprotein；PmR，PeroXisomeproliftratoractivatedreceptor；GLUT，glucosetransporter；

CCT．C．00mm…応tea；WA：I．whiteadiposedssue；Hm，high−fatdiet；LrD．1°W−fatdiet；qPCR．quantitadvePCRTBP，珊HA−bindingprotein；

N曲A．n°nでStehaedfattyadd；lTT．insulintoleramCeteStiMS，maSSSPeCtTOmetry；UHPLC，ultm−HPLC；DMEM．Dulbecco●smodi丘edEadesmedium；2−NBDG，2−deoxy−2−【（7−nitr0−2．1，3−benzoxadiazo1−4−yl）amin〇十D専ucose；VW附，VisceralWAT；SWAT．subeutaneousWAlr；aP2．
fattyacidbhdingpr°tein4；LPL．up°PrOteinhpase；ACC．acetyl−CoAcarboxylase；mS．fattyacidsynthase；SCD，StearOyl−CoAdesatumse；
JrrGL．adip∝ytethかeride叫ase；HSL，h°mOneSenSidvelipase；MGL，monO由yceridehpase；TNrqtumourneCrcSisEaetorα；H・−6．inteneukin−
6；MCP−1，monoCyteChemcattractantprotein−l

http：〟dx．doi．orglO．1016／う．jff．2016．02．012
1756−4646102016とlsevierLtd．AllrightsneseⅣed．

JOURNAL Or FuNCTlONAL F00DS

22（20I6）490−503491

responsetovahousnutritionalsignalsand／orenergyinsu綿−More°Ver，glucoluteoHnOuteoun−7−0−glucoside），Whichisan

eiency（Spiegelman＆fuer．2001）・Adipocytesareals°known

abundantnavonoidinC．communis．repressedtheaccumula・

asend°Crinecellsthatsecreteanumberofadipocytokines

donofhtraceuularhpidsByrepressingGLUT4・mediatedducose（Antuna−Puente，Feve，Fellahi，＆Bastard，2008；Atde＆Scherer，

uptakeintomouseadipocytic3T3−LIcells・Thus．CCTde−2009；Ga職C．Oakh址，＆Steinberg，2010；Rasouli＆Kem，2008）・

CreaSedthenuthent一mediatedbodyweightgainseeninHFD・Adipogenesisiscontrolledviaacomplexprocessinduding

tedmiceandcouldbeuse請Eerthedevelepmentofanti一Changesinhomonesensidvityandgeneexpression・Anumber〇besityandanti−diabeticfoodsandbeverage・

ofpnoteins，includingtIanSCriptionEactorsinvolvedinthenegu−

1ationofadipogenesis，havebeenidentiAed（Lefterova＆LaZar，2009；Rosen．2005；Rosen，Walkey，Puigserver，8ISpiegelman，

2000）．Amongthem，CCANr／enhancer・bindingpr°teins〈C／EBPs）andperoxisomeprol rator・aCtivatedreceptors（PPARs）

2．

Materialsandmethods

2．1．

坤0mかれずC．commu血stea（CCD

arekeyadipogenesis・regulatingtransChpdoniactors（Lefterova＆Lazar，2009；Rosen，2005；R°Seneta1．，2000）．Thesetranscl由一
donねetorsregulatetheexpressionofanumberofadip°geneSis・
relatedgenest°COntroladipogenesis（adipoqtedifferendation）・

Thus．fcrthepreventionofobesityanddevelopmentofanti一〇besityfo°dsandbeverage，elucidationoftheregulatory
mechanismofobesity（adipogenesis）isimp°rtant・C．e°mmunistea（CCT〉1eaveswereobtainedfr°mAobana

Shokkenn（Osaka．Japan）andJAKUSATSU〈Shiga，Japan）・The
tealeaves（1g）wereextractedinS00mLofhotwaterat80oC
forlSmin．Theresultanth°tWater−eXtlaetSofC．communiSWere

designatedasCCTc°eledtor°0mtemperature，andusedin
experimentswithin2days・

Natunalc°mPOundstromplantsandvegetableSWithanti一〇besitye縦etshavebeeninvesdgated・Flavonoids，Whichare

Well・knownnaturalpolyphenoliccompounds●arefoundin

2．2．

A証malsmゆfmits，Vegetables，andbeverages〈DiCar1°．Mascolo，122°，＆

Capasso，1999）．Thec°mpoundshavevariousbiolo由Calactivi−

tiessuchasand−eanCer（Adhami，Syed，Khan，＆Mukhtar，2012；

Nishiumietal．．2011），antioxidant（Pandey＆Rizvi．2009）．anti−

innammat°Ty（Ratheeeta1．，2009）．andanti・obesity（Meydani＆Hasan，2010；Wangeta1．，2014）e旋cts・Commenna00mmunis，knownastheAsiandaynower，iswidely

disthbutedthroughouttheworld・C・COmmtLnishasbeentra−

ditionauyusedinChinesemedicne（Bingetal・，2009；Shibano，Kakutani，Tbniguchi．Yasuda，＆Baba，2008；Shihetal・．2002；
Ybun，Park，＆Ch°．2004；Zhangetal・，2013），forithasavariety
ofbiologicalactivities．Suchas anti−innammatory．anti・
bacterialand・hypergIycaemic．mdandoxidante熊請S・Moreover，
C．commtmiscontainsanumberoEchemicalconstituents，SuChasnavonoids．a膿aloids．polysaccharides．terpenes，andsterols・

TWoofthesec°nStituents，1・deoxynqiirimydn（DNJ）and（2R．
3R．4R，SR）・2，5・bis但ydroxymethyl〉−3，4・dihydroxypyn°lidineMice（C57BUeJ，5−Week一〇ldmales）werepurchasedfl●°mJapan

SLC〈ShiZuoka，Japan）andmaintainedina12・hlight／12−hdakph。toperi。dinahumidity・andtempemture一ccntrolledrocm（55％at240C）．Themiceweredividedinto4groupslN＝8−10

miceforeachHFDgroupandN＝4mieeforeachl°W血tdiet〈LfD〉group1°fSimiIaraveragebodyweights・Theywerepr°−

VidedCCTorwater．respective巾todhnkandeachgr。uPWaS
fedeithertheLfD〈rR・2，4．8％ねt；FunabashiFam，Chiba．Japan）

ortheHFD（35％fat；ResearehDiet．NewBmnswick，Nl，USA〉・
These4groupsweredesignatedasCC肌FD，CCT／HFD．water／
LfD．andwater／HFDgroups，reSPeCtively間はterorCCrandbod

WereaVailableod！ibitum・Bodyweightwasmeasuredweekly

fbr8weeks．TheanimalstudywasapprovedbytheAnimal
CommitteeofOsakaUniversityofPhamaceudcalSciences，andanimalswerehandledinaccordancewithlhepnnciplesand
guidelinesestabushedbythec°mmittee・PMDP），inhibittheacdvity°fαTglueosidase（Shibanoetal・．2008；

Ybuneta1．．2004）．theenzymethatdigestsstarchesandou−

2．3．（nant殖otio動けmRNAteuel切quqM証a帥ePCR

gosaccharidesinlhesmauintesdnes・

血PCR）

InstLnnaCdvatesglucosetransportintocellsviagluc°Se

tnnsporters（GLUTs），aCdvationofwhichistherate−umiting

mtalRNAwasextnctedbytheuseofThPureIs°ladonReagent

Stepduringglucoseuptate（Muretta＆Mastick，2009〉・GLUTA（RocheDiagnostics．Mannheim，Gemany）accordingtothein・

isdominantlyexpressedinadiposedSSueSandmusele，Where

StmCtionsprescribedbythemanufacturer・WhenRNAwas

ltplaysacentralroleinglucosehomeostasisintheirdssues

preparedfromtissues．theyweredisruptedinTmreIsola・（Huang＆Czech，2007）．GLUT4istranslocatedt°theplasmationReagentbyuSingaBeadbeater・typehom°geniseruTOlmembraneをomthecytosolwheninsulinbindstotheinsulin（TArmC．Saitama，Japan）．Afterremovaloftheceudebrisandrecept°T．Mostoftheglucoseincorporatedint°Cellsisused

lipidsbycentrmgati°n，thesupematantwasthenudHsedt°

tosynthesiSe却mHoweve事eXCeSSinc°rP°rad°n°fducoseint°

PrePa鵬RNA・

CeuSincreaSeStheirintraceuularlipidlevels，Whichcanin−

Firs章一StrandcDNAswereslmthesisedwithtotalRNA〈lu9，creasetheprWalenceofdiabetesand°besityThus．theconnol

randomhexamer（mkara・Bio，KyotoJapan），andRevermAce°fglucoseuptakeintocellsisimp°rtantもrreguladonofthe

ReversenanscriptaSefrbyob°．csaka．lapan）at420Cfor60min

intracellularlipidlevel．

afterdenaturadonat720Cbr3min，andthiswaSfollowedby

mlhepresentstudyiWeinvestigatedtheand・Obesitye徹的ts

heat−inactivadonofthererversetranSCriptaseat990Cfor5min・

OfC．commurIistea（CCT）inhigh・ぬtdiet（HFD〉一缶dmice．and

ThecDNASWerehlrtherutiIisedforquantitativePCR（qPCR）

fbundthatCCI

malySiS．

decreasedthebodyweightgainbyreducing

thevisceraJandsut鷺utaneouSWhiteadiposedssueMMmmass

nanscriptionlevelsofthedeSiredgenesweremeasu皿edby

andslightlyimprovedtheinsulinsensidvityinHFD−fedmice・

using a LightCycler System（Roche Diagnostics）with

492

JOURNAL OF FuNCTlONAL F00DS

22（2016）490−503

THUNDERBIRDSYBRqPCRMix（Tbyobo）oraStepOnePlus

ObtainedwithanMS700Vmassspectr°meterOEOL，System（ThemoFisherScientihc，Waltham．MA，USA）with

JapaLn）．SpectrumdataofthenavonoidswereshowninSupple一

PoIPerSYBRGreenPCRMasterMix（Them°FisherScientihc〉

mentarymaterials・

andgene・SpeCi鉦primersets（TableSl〉・ThemRNAlevelsof

tbkyo，

Thepurityofeachofthese4navonoidcompoundswas

thedesiredgenesweren°maHsedtothelevel°ftheTATA・

dledkedbyultra−HPLCq間PLC，Nexera；ShimadzuKyoto，Japan〉

bindingproteinけBP）．

uslngadiode・amydetector・ACosmosi12・5C18・MS・IIcolumn

2．4．

u址SedforUHPLCanalysiswitham°bilephasell％（V／V）acedc（100×2．Ommi．d．；2．5叫n；NacalaiTesque．Kyoto，Japan〉was
Semmbiochemicolmorke†S

acidiandacetonitmeataradoof85：15（V／vrThenowratewas

SeruminsulinlevelsweremeasuredbyuslngaMouseInsulin

O．4mL／min，Columnoventemperaturewas400C，andthede−

EHSA綱すぐ恥部；Shibaya缶GunmaJapanrSemmtriacy固ycerol，

tecti°nWaVelengthwasat400ー200nm．

non・eStermedfattyacid（NEm），tOtalcholesterol，low−density

Hpoprotein（LDL），andhigh−density坤°PrOtein（HDL）levelswere

2．8．

C（州cuImredetemhnedbyusingtheLtypeTGMtest，NEm−C，Choles−
teroIM．L・Iy uDL・C，andL・TyPeHDL−CKits．respeくねvely（Wako
PureChemical，Osaka，Japan）acc°rdingtothemanufactur−ersinstructions．

Mouseadipocytic3T3−Llcells（HumanScienceResearchRe−
SOurCeSBank．0saka，Japan）wereculturedinDulbeccos

mod漁edねgle●smedium〈DMEM；Sigma，St・Louis．MO，USA〉
C°ntaininglO％（V／V〉fcetalcalfsemm〈Sigma〉andandbi°tics

2．5．Insl相調tolemnCefest（I7n（NacalaittiSque），andmaintainedinahumidi魚edatm0−
SPhere°f5％C02at370C・Adipocytedifrerendationof3T3−LI

Micewereねstedfor16hphortothemInsulin（0・75mU／g
ofbodyweight；HUMULIN⑰；郎Lillylhdianapolis．lN．USA）was

CeuS川mSinidatedbyineubadonfbr2daysinDMHMwithinlⅢn（10uyml；Sigma），1両Idexamethasone（Sigma），andO・SmM

intraperitonealJyinjeeted．BloodwascoueetedfT°matailvein

3−isobuty1−1−methykanthine（NacalaiTesque）・Onday2．theandglucosewasimmediatelymeasuredbyusingaMEDISAFE

mediumwasreplacedwithDMEMcontaininginsuhn（10uy

MINIBl°odGlucoseMonitoringSystemr rlumo．Tbkyo，Japan）・

ml）al°neandthenchangedevery2days・OilRedOstaining

Thebl°OdglucoselevelsweremeasuredatO，15．30．60．90andoftheintracellularlipidswascarriedoutasdeschbedprevi−

120minafterinjecti°nofinsulin．

ously〈Fujimori，Yano．8tUeno，2012）・

2．6．

gmdonandisolqdon〆COnS五億6m喰￠2．9．

C側圧o継idりaSSqy

C．commulnis

Theaerialparts（400g）°fC・COmmunisweredriedandre−

nuxed3血neswith70％〈V／v）ethanol（3L）fbr3h・Thesoludon
WaSthenconcentratedunderreducedpressuret°glVean
extTaCt（75・59，Whichwassubsequentlychromatographedon
aDiaionHP−20colum（MitsubishiChemical，lbkyoJapan）．ARer

thecolumnhasbeenwashedwithwater．theadsorbedma・
terialwaselutedwithmethan°l（2L）．Theelutedfhcdonwas

CelltoxicitywasassessedasdescribedpreviouSly（F山imori8［

Shibano．2013）．Inbhef，3T3−LIcellswereseededin96・Wen
Platesandallowedtoattachovemightat37。C・Thecellswere
incubatedf°r6daysinDMEMc°ntainingvariousconcentra−

tions（0−50小4）ofeachofthepu舶ednavon°ids‥orientin，
isooriendn．is°malmnedn3・〇・m血oside，andduc°luteoun．Cen

toxidtywasmeasuredwithcellCountReagentSF（Nacalai
TeSque〉accotdingtotheinstrucdonsindieatedbymanufactu記事

nemconcentratedinuaettot°giveanavonoid血cti°n（51・79・

ThisfractionwaschromatographedonasiIicagel（400g）
column（5．3i．d．×56cm）withadich1°romethane・methanoI

2．10．Measurem飢tQfintmcel皿αrfridCylgbCt汀Ollelle1

mobilephase，andallflactionsweresubsequentlysutljected

toHPLCseparation（DevelosilODS・510i・d・×250mm．Nomum
Chemical，Aichi，Japan〉orNucleosilC−18ABlOi・d・×250mm（Macherey−Nagel，Dtiren，Gemany）withelud°nWithl％（V／V）
aceticacid：aCetOnitrile＝88：12or85：15〈V／V）．

3T3−LIcellswereallowedt°difrerentiateintoadipocytesfor

edaysinDMEMwithorwithouteaehofthepuriaednavo−
n°ids（50ltM）・IntTaCenularthacylglycerolleveBweremeasured

byusingaWAKOLabAssaymglycerideKit叩批oPureChemi・cal）．ProteinconcentrationsweredeteminedwithPierceBCA

2．7．

S畔dmIid釧章的（南on〆conSdmcntsげPTOteinAssayReagentqhemoFisherSdenti的．withBSAused

C．commu轟sbNMRa融mQSS坤cdrome巾（M9astheStandard．

9°ur naVonoid compounds．i．e．．orientin，iso°rientin，

2．11．Gtycrd職l餌SeaS紬yisorhammetin3−0−mtinoside，andglueoluteolin，Wereidenti・

hedbycomparisonoftheirspectra1匹−NMR．鯵C−NMR．andMS〉

3T3−LIceuswereallowedtodifferentiateintoadipoeytesfcr

datawitmherep°rtedvalues（Kato＆M°rita，1990；Park，Kim，

6daysinDMEMinmepresenceorabsenceofgluc°luteolin・＆Kim，2000；Sakakibara，DiSeo．Nakatani∵rimmemann．＆Onday5，themediumwaschangedtophenolred五eeDMEM

Mabry，1976）．lH−and13C−NMRspectrawererecorded°na（Sigma）Containinginsulinwithorwithoutducoluteolin（50画・VNMRS−400speCtIOmeter（Agilent龍chn°1°gies；SantaClara，

Onday6，theculturemediumwascollectedandusedt°

GA．USA）．operatingat400MHzforprotonandl00MHzformeasuretheglycerolreleasedintothemediumbyusingFree

Carbon，WithtetramethylsilaneasanintemalStandard・Fast

GlyceroIAssayReagent（CaymanChemical．AnnArtNlr，MI，USA）

atombombardment−maSSSPeCtrome叫（mB−MS）spectIaWere

acc°rdingtothemethodprescribedbymemanufacturer・JouRNAL OF FuNCTlONAL F00DS

493

22（20I6）490−503

CCTeffecdvelysuppressedHFD−inducedbodyweightgainby

2．12．2−Deoxyglucoseuptakeassay

reducingtheWATmassinthemice．
3T3−LIcellswere allowedtodifferentiatefor6daysinto

adipocytesin96−Wellplatesin DMEM with or without

3．2．
glucoluteolin．Themediumwasremoved．andthecellswere

WaShed3timeswithPBSO，followedbyincubationfor30min
at370CinPBS（一〉containing150ug／m12−deoxy−2−I（7−nitr0−
2，1，3−benzoxadiaz01−4−yl）amino十D一glucose（2−NBDG；Cayman

Chemical〉Eor30min．Thebu儲erwasthendisCarded，andthe
Cellswerewashed3timeswithPBS（−），afterwhichthenuo−
rescencewasobseⅣedinPBS〈−）underanuoresCentmicroscope

訪ね〆CCronSemmbiochemicdpommetersin

HFD・induced°besemice（cc〝HFDgroup）

ThesemmtnacylglycerQllevelintheCCT／HFDgr°uPWaSloweredascomparedwitmhatinthewater／HFDgmup（Tablel）・
However，tnaCylglycer°llevelsremainedunchangedbetween

theCCT／LFDandwater／LFDgroups〈Tablel）・Tbtalch01es−
terQl，HDl．−，andLDL−ChelesterCllevelsintheCCT／HFDgroup（CKX−41FL．01ympus，

lbkyo，Japan）．Flu°reSCenCeWaSmea−

SuredbyusinganEnspire2300Mu血nodePlateReader（Perkin−

Elmer，Waltham，MA，USA）．The2−NBDGuptakelevelwas
expressedasthedegreeofelevadonc°mparedwithhatofhe

Werealmostthesameasthoseofthewater／HF．Dgroup匹blel）・
Thus，CCTdecTeaSedthesemmdacy固ycerellevelsirLtheHFD一缶dmice．

Vehicle−treatedcells．

3．3．

TlWOgrOupSWereCOmparedbyuslngStudent
WayanalysIS°fvahanceandTukey

gxpreSSionすきdipogeneSis−relatedg飢eSinu附rd

CCTadmirIjsteredHHDTjedmice（CCl2．13．5mdsdcalanQlysis

st・teSt・One−

spos証°CteStWereuSed

toc°mParemOrethan2groupswithcomparablevariances・性iFDgnotJP）

WはmeasuredthemRNAlevels°fadipogenic，lipogenic，and

lipolyticgenesintheWATofeachHFD・Eedgroup・ThemRNAlevelsoftheadipogenicPPARYLC／EBPα，Eattyadd−bindingprotein

4（aP2），CD36，1ipoproteinlipase（LPL），andGLUT4geneswere

p＜0．05wasconsideredsignihcant・Slgni魚CantlydecreasedintheWATandsWAToEtheCCT／
HFDgroup，aSCOmparedwitheach。fthoseinthewatel／HFD

group〈Fig．2A）．Incontrast，thetranscripdonlevelsoftheacetyl−

3．

Results

C°Acarboxylase（ACC），fattyaddsynthase（mS），andstearoyL

3．1．

DecreaSeinbodytpeightgaintlyadministratjorIQf

wereeitherunaltered orratherincreasedin thevWnrI

CoAdesaturase（SCD〉genes，Whichareinvolvedinhpogenesis，
andSWAToftheCCT／HFDgr°uP，aSCOmParedwitheachofthose

CCTtoHFD−inducedobesemicelCCT／HFDgrotIP）

inthewater／HFDgroup（Fig．2B）・Theseresultsindicatethat

C．communis（Fig．lA）hasavahetyofbioactivepropertiesasa

theexpressionofadipogenicgeneswassuppressedbyCCTin

Chinesemedicine（Bingetal・，2009；ShibanoetaL2008；ShihtheW附rofHFD−tedmice，SuggeSdngitsabilitytosuppressadi−

eta1．，2002；Youneta1．，2004；Zhangetal・，2013）・Tbinvesti−

pogenesisinadiposetissues・

gate the effects°fC．communis on obesityln mice，We

Then．WemeasuredthetranscTiptionleveloftheLipolytic

administeredCCT〈Fig．lB〉tomiceoneithertheLFDorHFD・genesincludingadipocytetTiglycerideHpase（ATCL），h°mOne−

After8weeks，thebodyweightgalnintheCCt／HFDgr°upWaS

SenSitivelipase（HSL），andmon°glycerideupase（MGL〉inthe

Sign漁cantlyreducedasc°mparedwiththatinthewater／

WLrI

HFDgroup，Whoselevelsweresimilart°th。SeOftheCCT／

oftheATGL，HSL．andMGLgenesintheCCTJHFDgroupwas

oftheCCT／HFDandwateIlHFDgroups・ThemRNAlevel

LFDandwater／LFDgroups（Fig・lCandD）・Inaddition，there

Slgni魚cantlyreduced，aSCOmparedwitheach°fthewater／

WaSnOSigrl血cantdi維renceinfQQdintakebetweenthegroups

HFDgroup（Fig・2C）・InC°ntraSt，theserumNEmlevelwasthe（Fig．1g），Tbinvesdgatechangesinfatweight，Weis01atedsub−

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Obesityisthoughttobeastateofchroniclowrgradeinnam−

Next，Weelucidatedthemolecularmechanismoftheanti−

mation．When adipocytes are expanded and／orenlarged，adip。genice錐ectsofC・COmmunisbyuslnganinUitrocell・

in珊tlationofmacTOphageintoadiposetissuesisincreased・

culturesystem・We血cuSed。nthenavonoidsinC・Communis

andmesecredonofpro・innammat°fyCytokinessuchastumour

beeausetheyareabundandycontainedinC・COnmTLmisandhave

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that of chemokines such as monocyte chem°attraCtant

Panchal，2015；Nijveldteta1．．2001；Wangetal・，2014〉・So，We

protein−1（MCP・1〉言Selevated・TheseEactorsareasSodated

puriRedthemfromC・COmmunis・TheaehalpartsofC・COmmu・

Withthepathogenesis。finstninresistanceandtype2diabe−

nisweでeextnctedinmethanol，andthenavonoidconstituentstes（Osbom＆01efsky，2012；Suganami，mnaka．＆Ogawa．

WerePu融edasdescribedintheMaterialsandMethods・

2012；1ぬm，Covington．Ravussin，Redman，＆Pennington．

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2012）．

glucoluteonn．whicharethemajornavonoidsinC・Commun屯Whenwemeasuredthetranscriptlonlevelofthesemac−

WeneisolatedbypreparadveHPLCIandthencheckedbyUHPLC

rophagemarkergenesinmeVWAToftheCCrmDandwater／

rorpurity．whichwassh°Wntobemorethanatleast99％

HFDgroups〈Fig・3），theresultsshowedthatthemRNAleve1（Fig．SA）．Finally．weobtainedeachofthepu舶ednavon°ids°ftheTN職，IL−6，andMCP・lgeneswasreducedinthevWiAT

h・omC．Communis（4009：orientin（178mg，0，045％）．isoohen血°ftheCCT／HFDgroup．ascomparedwithlhatofthesegenes（22mg，0．0055％），isorhamnedn−3・〇・rudn°Side（64mg．0・016％），inlhewater／HFDgmup．althoughtheirlevelsinmeLIDgroups

andgluc°1uteolin（461mg．0・12％）・Furtherm°re．WeC°n−

WerenotChanged．TheseresultsrevealthatCCTsuppressed

hmedtheirchemicalstntCtureSbaSedonspectlmlanalysesぐH・

theinnammadoninthevⅧTintheHFD−fedmice．

NMR，13C−NMR，tWO・dimensionalNMR．andMS；Fig．5A五mset〉・
Next，WeeXaminedthesenavonoidsforpossiblecytotox−

icitytowards3T3−LIceus．Thecellswereculturedfor6dayS
3．5．

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Tbinvestigatethee鵬ctofCcToninsulinsensidvity，Wecon−

thepu噺ednavonoidsusedinthisstudy（（．ig・SB）・

ductedtheITTin LFD−orHrD・fed micewithorwithout
administration ofCCTThe serum gluc°Selevelin the

3．7．

CCTyHFDgr。uPWaSSigniacantlyreducedasc。mparedwith

lipidaC関れ融donin紺3−Llc鵬訪ねsQf如Vonoidspt．一郎edfJomc・Communison

thatinthewatetJHFDgroup（Fig．4A）．whereassemminstLun
levelsbetweenthesegroupSWereSimHar，ald1°ughtheirlevels

InhIrthering。urStudylWeinvestigatedthee脆ctsof。hendn・

intheHFD−fedgToupSWereSughtlyhigherthanthoseinthe

isoorientin膏Orhamnedn3・0−rlutinoside．andgluceluteolinon

LFD・fedgroups（Fig．48）．InsldinsensidvityintheCCT／HFDgrouplipidacCumulationin3T3−LIcellS・Thecenswereallowedto

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di縦rentiateintoadipocytesfor6daysinthepresenceor

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absence ofeach purined navonoid．and thereafter the

CCtJLmandwatern．FDgroups（Fig．4C）・Thetlefbre，CCrslightly

intracellularlipidswerestainedwithOilRedO・Thenumber

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髄g．4−Im（A）SemmglucoselevelineadlgTouP．MicewenefastedoYemight，afterlyhichtheirsemwaspmpared．Dataamsho馴れasdlemeanS±S．B．Ql＝4−10）
aneshomasmeans士S．言．（N＝4−10）

pく0．01，asindicatedbythebracketB．￠）SemminsuIinlevelmeadlgrouP．Data

JI＜0．01，aeindicatedbythebmcketB．（qlTrineadlgrouP．Bleedglucose

concentntlontmsmeasuredathdicatedtimepointB．Dataa隠8hovmasthemeans土S．B．Ql＝4−5）．

COmparedwiththatofthoseintheundifferentiatedones

Thus，WeneXtinvestlgatedthemolecularmechanismofsup−（Fig．6A）．whereasthenumberofintracellularlipiddr°Pletswas

pressi°nOfadip°eytediEferentiati°nbygluccluteelin・

Clearlydecreasedwhenoriendn，is°rhamnetin3−0−rudnoside

Orgluc°lute°un．butn°tisoohentin．werepresent（Fig・6A〉・
Then，WemeaSuredthelevelsofintraceuulartnacylglycer°l

3．8．

ChongeinexprcssionIeuelげqdipog飢eSis−rclated

geneSbyglucoluteo嗣in3T3−LlceIIs

inthenav°nOid・treatedcells．3T3・LIcellswereallowedt°dif−

ferendateint°adipocytesfor6daysinDMEMwith°rWithout

Tbdeteminethenatureofthismechanism．Weinvestigatedeachofthenav°n°ids．Theintl．aCellulartnacylglycerollevelin

Changesintheexpressionleveloftheadipogenicgenesin

thedifferendatedcellswasclearlyenhancedascomparedwith

glucoluteolin−treated3T3−LIcells．ThemRNAlevelofthethatiJl＝heundifterentiatedcells（Fig．6B）・Whenwecausedthe

adipogenicPPARγ，C／EBPα，aP2，andLPLgeneswassigni魚−Cellstodifferendateintoadipocytesinthepresenceofeach

Cantlyelevatedduringadipocytedifterendadonintheabsence

ofthenav°n°ids，theintracellulartrlaCylglycerollevelin

ofthenav°noid，butreducedinitspresence（Fig．7A）．

°hendn一〇rglucoluteolinlJeatedcellswasdecreasedascom−

Next，WeinvestigatedthemRNAlevelofthelipogenicgenes

paredwiththatinthedifferentiatedcells（Fig．68）．Incontrast，inlheglucoluteolinlreated3T3−LIcells．Whenthecellswere

isoorientinandisorhamedn3−0−rudnosidedidn°taffectthe

al1°Wedtodiffenentiateintoadipocytesfor6daysinDMmIWith

acCumulationofintracellulartriacylglycerol（Fig．6B）．These

orwithoutglueoluteolin，thetranscnptlOnlevelofthelipo−

resultsindicatethatglucoluteoLinhasthemostsuppressive

genicgenessuchasACC，mS，andSCDwasenhancedduring

effectontheaccumulad°nofintracellulaJ虻pidsin3T3−LIceus．

adipocytedifferentiationof3T3−LIcellsintheabsenceof）ouRNAL OFFuNCTIONAL P00DS

22（20I6）490−503

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notthatoftheHSLandMGLgenes，WaSdecreasedascom−

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paredwiththatinthevehicle−treateddifferentiatedcells（Fig・7C）．Inaddidon，melevelofglycerolreleasednt）mthecells

WaSnOtChangedwhenthecellswereanowedtodifferend−
ateintoadipocytesinthemediumwithducoluteolin（Fig・7D）・
Theseresultsrevealthatglucoluteelinsuppressedadipogen−esis，butnotlipogenesisorlipolysis，inadipocytes．

D／isoorienth D讐書誌3D／9−ucoiuteoiin

3．9．

Glt．coluteoii証nducedSumreSSionqfGLUm−

mediatedglucoseup億kein373−Llcc帖

Next．weinvestlgatedtheexpression°ftheGLUT481ucose
transporteringlucoluteolin巾eatedadip°SeCells，aSitisdomi−

nandyexpressedinadipoeytesandmuscle（Muretta＆Masdck，B2009）．lntheabsenceofthisnav°n°id，theexpressionlevel°f

theGLUT4genewasenhancedduringadipogenesis，aSCOm−
paredwiththatintheundifterentiatedcells．whereasinits︵u壇OJdの三宝︶一〇﹂8主旦ゝoQtL

PreSenCethislevelwasreduced（Fig．8A）・

青Finally，WemeaSuredglucoseuptakeintothegluccluteonn−

treatedcellsbytheuseof2−NBDG．anuorescentglucose
analogue・When3T3−Lleellswereanowedtodi儲erentiateinto

adipocytesfbr6daysinthepresence°fgluc01uteom，thelevel°f2−NBDGuptakewassigni鉦anuydecreased，aSComPared
Withthatbythevehicle巾eateddifEerendatedcens（Fig・8Band
C）・TheseresultsindiCatethatgluc°luteolinsuppressedGLUT4−

mediatedgluc°SeuPtakeintoadipoeytes．

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Discussion−

iSOrhamnetin3−0−rutinOSide

AdipocytedifEerentiationandfataccumulationareass°Ci−

gIucoiute01h
DatedwitmheoccurTenCeanddevel°PmentOfobesityObesity
isariskfactorforanumberofdiseasessuchascardiovascu−

rig．6−Supp腿ssionofaccumulationofinnacenna重職pids

tlyC．commmisnavonoide．い）oilRedoBtainingofde（undifferentiatedceug：U）wemallowedtodi細Brentiateinto

adipo電ytes（difleenGatedcellB：DIfOredaysinmedium

Onlyin patients with exCeSSive°besity（BMl≧27−30〉

3−0−mtinoeideorglucoluteoⅢl（50LLM）．Bar＝50Lun．
The

CeIls山ndi餓けendatedcells：U；ttIhite00lumn）vemcausedtod讃tpenGate（di鯖e腿ndatedceus：D）intoadipocytesfcre
dayeiJ＝heabsenceゆeycolumn）orpnesenCeofodentin，（501〇・mtinosideorglucoluteotin

M；bla臓00lum間）．DataaかePnesentedasmemeans士S．D．

mentandprevendonofobesityarecftticalinlheclinicalheld・

Although，tOday，thereareseveralanti・Obesitydrugsthatde・

CreaSefoodintakebyreducingappetite，theymaybeutilisedtIUimorwilhouto轟entin，isoo轟en血．isematnneGn

isooriendn，ieomamnetin3

Scherer，2009；Comieretal，2008）．Hyperplasdccl絶Sityiscaused
tJyanincreaseinlhenumberorsizeofadipocytesorboth．TTeat・

intTaceIIuaIIuPidsin373・Llceus．mecellB￠）lntracemlartriacylgtycercllevelh373−Llcells

1ardiseases，hypertension，anddiabetesmellitus（Attie＆

pく0．01，33indicatedbymebmcketB．

（Cunningham＆Wivi°tt，2014；）enseneta1．，2014）．Moreover．
SOmeefthesedrugshavethesideefrectsonpsychiatricand／
OrCardiovascularsystems（Cunningham＆Wiviott，2014；
Dietrich＆Horvath，2012）．Therefore，thedevelopmentofnovelanti−obesitydmgswithoutside−e旋etsisstronglyneededclini−

Cally∴Tbs°lvethisproblem．elueidationofthemolecularmechanism°fregulatingobesityisabsolutelyneeded．

S°medietarysupplementsandnaturalproductshavea
Variety°fthempeudee絶ctsonmarydisorderswimoutcausing
gluc°lute°lin（Fig．7B）．Theexpressi°nlevelofthemSgenewas

Sideetrectsor．ifso，thosecausmgveIyHttleinconvenience，

reducedwhentheceuswereanowedtodiffetlendateiJl＝hepres−althoughtheygenerallyhaveweakertherapeutieeftectsthan

enceofglucoluteolin（Fig．7B）．However，themRNAlevelofthe

thoseofthephamaceudcalmedicines．坤idemiol0両alstudies

ACCandSCDgenesinthediEferentiatedcellSWaSnotChanged

havedem°nStratedthatsomedietaryplantc°nStituentsare

eveninthepreSenCeofglucolute°un（rig．7B）．

associatedwithareducedriskofdeveloplngmetabolicdis−

Then．Weinvestigatedthetranscriptionlevelofthelipo一

eases（Meydani＆Hasan，2010；WangetaL，2014）．rlavonoids

明iclHiGL，HSL，andMGLgenes．Theexpressi°nlevelofallof

areweu−knownnaturalpolyphenoliccompoundsfbundinveg−

thesegeneswasincreasedwhenthecellsweremadetodif−

etables，fmits，andbeveragesandhavevariousbi°10gicalrerentiatefor6daysintheabsenceofthenavonoid（Fig．7C）．

acdvities（DiCarloeta1．，1999；Nishiumieta1．．2011；Pandey

H°WeVer，initspresence，themRNAleveloftheATGLgene，but＆Rizvi，2009；Ratheeet al・，2009）．In Uitrostudies have

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髄g．7−Supp郵esSionofe浸P調Bsionofadipogenic，HPogenic，md叫olyticgeneBinglucoluteoun・treated3T3・LlceIls・（AIE義Pnessionlevelofmeadipogenfcgenesinglucolutt的鵬n・tJeated373−LIceu〇・ceIIs山ndi鉦8entiatedcdls：U；tPhite

00IIJmnS）veかeCausedtodiffIlendate（diHerendatedceus：DIintoadipocytesfbredaysintheabseneeerりcelumns）or

pmaenceOfBIucduteoml（50山；black00lumnS）・Dataaかememe種ns±S．D・），＜0・01，asmdicatedbythebmckeB・￠）Suppressionofdne寄anscripGenleveIoftheIipogenicgeneBinduceluteelhtmated373−LlceuB・Undi鯖erenGatedcellBO；tqhite00ltlmnS）ureかeCausedtodiff mndateO）intoadipocytesfbredaysinDMHMWimoutolqColum的OrVim
glucoluteoun（50中嶋blackcolumTIS）．セく〇・01，asindicatedbymebmcketB．（qReduc丘onid‖hemRNAIevelofmelipelytic
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Veredi髄腿n68tedandBIycemlreleasewasaBdescribedinmeMateriaIsandMemods．N．D．：nOtdetected．

501

JouRNAL OF FuNCTJONAL FooDS∴22（2016）490−503

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treated373・LIceus．celIs恒ndil撤汀entiatedceDB：U；tA，hitecclumn）we鴫differentiated（di鯖erentiatedceIIs：D）ihtOadipocytes

fbredaysinDMEMtN向hout（greycelumn）orl高thglucoluteoHn60山き；black00lumn）・ThetmnsdipGonlevelofdAeGLUT4

genewase粗minedbyqPcRDataanePresentedasmemeane士S．D．

p＜0．01，88indicatedbythebmcl【etS・￠）Supp聯sion

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NBDG，anuoneScentglucose

analogue．Bar＝100Lm．（c）MeasurementOf2・NBDGIevel．373・LlceusWeredi鉦腿ntiatedintoadipoqtesbredaysas
descTibedin血elegendofFig・88・2−NBDcuptakeintomeceuevasmeasurednuommetricauy・medataaneかePnesentedas

mevaluemIativetomatoftheundi縦腿ntiatedceus．Dataa腿p鳩sentedaememeans±S．D．

pく0．01，asindicaledbyme

bmcketdem°nStratedthatnavon°idssuppresstheprogressionofadi−

macrophagemarkergenesinthevW椎rofHFD壷dmicewas

pogenesis〈Andersen，Rayalam，Della−Fera，＆Baile，2010；

repressedbytheadministration°fCCTtotheHrD・tedmice

Nishiumieta1．，2011；Pandey＆Rizvi，2009；Wangetal・，2014〉・（Fig・3〉・S°menaVOn。idssuchasapigenin，luteelin，hsedn，quer−

Forexample，quercetinandapigeninrepressadipocytedi絶r−

Cetin，and catechins（tea−polyphenQls）can modulate

endationbyactivadngAMP−aCdvatedproteiT日出nase（Ahn，Lee，

innammation（Funakoshi・Tag°，Nakamura，Tago，Mashino，＆

事由m，Park，＆Ha，2008；Ono＆Fujimori，2011；Zhangetal・，2013）・

Kasahara，2011；Gonzalez−Gallego，Garcia−Mediavilla，Avieularinsuppressestheaccumulationofintracellularlipids

Sanchez−Campos，＆Tunon，2010；Siriwardhanaetal・，2013；

byinhibiangqEBPα−aCdvatedGLUT4−mediatedglucoseuptake

Wangeta1．，2014）．Therefore．wethoughtthatotheI

inadipocytes（FLJimori8・Shibano，2013〉・Fisednlowerstheac−nisconsdtuentsmiかtahi°C°ntributet°theseeEtects・Thereもre．

CumulationofintracellularlipidsviareductionofGLUT4−

WeS。ughttohndnovelC・COmmuniseonstituentswithanti・C・COmmu−mediatedgluc°SeuPtakebyinhibitingmTORsignalling

adiFOgenice脆cts，andfocluSedonthenavonoidsofC・COmmunis・

OITatanabe，Hisatake，＆Fujimoh，2015〉・

FinauyiWefbundthatglucoluteolin，amajornavonoidinC・COm・

Asmentionedearlier．DNJandDMDPasc°nStituentsof

mtLrtjs（Shibanoetal．．2008），hadananti−adipogeniceftectin

C．commtlnis，haveanti−diabetice絶ctsbyinhibitingtheacdv−

3T3−LIcells・Thesuppressionmechanismofadipogenesisby

ityofα−glucosidase（Shibanoetal・，2008；Ybunetal・，2004），

gluc。Iuteolinhasneverbeendehned，aLthoughanumberof

Whichdigestsstarchesandoligosaccharidesinthesmallin−navon°idssuchasquercetinhaveanumberofbeneAcialeffects

testines．Inlinewiththathnding．weshowedthatCCT−

SuChasinhibitionofapoptosisandadipogenesis（Meydani＆administeredmiceontheHFDhadlowersemmglucoselevels

Hasan．2010；Wangeta1．．2014）．Theadiposetissuemassmay（Fig・4A）・WealsofoundthatCCrdecreasedthegaininbody

bereducedduetoadecreaseinthenumberofadiposecells

WeightbyreducingthetatofmiceontheHFD（Fig・lCandD）．

thr°ughtheirapoptosisortoinhibition。fadipogenesis・

aswellasslightlyimprovedtheirinsulinsensidvity（Fig・4C〉・

However，glucoluteonnhasnoapparenttoxicertel費in3T3−LI

Mor00Ver，theexpressi°nlevelofsome蘭ammadon−aSS∝iated

Cells（Fig．58），indicatingthatglucoluteolindecreasedthe

502

JOURNAL OF FuNCTIONAL F

00DS

22（2016）490−503

accumulation°fintracellulartriacylglycerol（Fig・eAandB）

throughrepressionofGLUT4−mediatedrepressionofglucose

Appendix：Supplementarymaterial

uptakebythecells（Fig・8A一℃）・
Glucosetransportintocellsistherate−limitingstepof

glucoseuptake．anditisinvelvedintheregulationofglucose

Supplementarydatatothisarticlecanbefoundonlineat
doi：10．1016相性2016．02．012・

hom∞StaSis（Muretta＆Masdく丸2009）．Glucoseuptakeisdghdy
regulatedbyinsulin，andGLUT4isresp°nSibleEbrglueose

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Phyt°medicine：Jnternatio関りournalqfPhytotheTaPyand

63．3436．

Phytophormocology，20．964−968・

TbbteSl．Primersusedinthisstudy

Gene

Acc．No．

PPAR†

NM Oll146

Forward

5

C／EBPO NM OO7678

5

aP2

NM O24406

CD36

NM OOl159555

GLUT4

NM OO9204−CAAGAATACCAAAGTGCGATCAAず

5

Reverse−GAGCTGGGTCTTTTCAGAATAATAAG−3−CTGGAAAGAAGGCCACCTC−3

5−AAGAGAAGGAAGCGGTCCA−355−GTTCTGGGCGTCACTCC−3

5−GGTCCCAGTCTCATTTAGCCACAGTA−3

5−GCCACGATGGAGACATAGC−3，−GCCAGACACCCCTGCTA−35−GATGTGGAACCCATAACTGGAlTCAC−3，5−GACGGACACTCCATCTGTTG−3

LPL NM OO8509

5−GCTGGTGGGAAATGATGTG−3

5−TGGACGTTGTCTAGGGGGTA−3

ACC NM133360

5−GCGTCGGGTAGATCCAGTT−3

5−CTCAGTGGGGCTTAGCTCTG−3

FAS NM O07988

5−GTTGGGGGTGTCTTCAACC−35−GAAGAGCTCTGGGGTCTGG−3

SCD NM OO9127

5，−TTCCCTCCTGCAAGCTCTAC−3

5−CAGAGCGCTGGTCATGTAGT−3

ATGL NM O01163689

5−TGACCATCTGCCTTCCAGA−3

5㌧TGTAGGTGGCGCAAGACA−3

HSL NM OlO719∴∴∴5−GCACTGTGACCTGCTTGGT−35−CTGGCACCCTCACTCCATA−3

MGL NM Ol1844−TCGGAACAAGTCGGAGGT−35−TCAGCAGCTGTATGCCAAAG−3

TNFα

5

NM O13693

MCP−l NM Ol1331

IL−6

5

NM O31168

TBP NM O13684

55−TGCCTATGTCTCAGCCTCTTC−35−GAGGCCATTTGGGAACTTCT3

一ccATCAGTCCTCAGGTATTGG−3

5−CTTCCGGACGTGAATCTTCT35㌧AGTTGACGGACCCCAAAAG−3

5−AGCTGGATGCTCTCATCAGG−3−GTGATGTGAAGTTCCCCATAAGG−3

5−CTACTGAACTGCTGGTGGGTCA−3

SuI）pIementarymatcrials

SpectrumdatafbrnavonoidsbyNMR
Orientin：FAB−MSm／2449lM＋H］＋．lH−NMR（Pyridine−d5）∂：7．87（lH，d，J＝2．0Hz，H−2），7．50（LH，dd，J二8．5，2．0Hz，H−6 ），7．27（lH，d，J＝8．5Hz，H−5 ），6．90（tH，S，Hj），6．68（lH，S，H−6），5・85（lH，d，J＝9・5Hz，
GIc−1），5．18（1H，br．dd，J＝8．5，8．5Hz，GIc−2），4．57（lH，dd，J＝12．0，2．3Hz，GIc−6），4・51（lH，dd，J＝8．5，8・5Hz，

GIc−4），4．48（lH，dd，J＝12．0，5．0Hz，GIc−6），4．45（1H，dd，J＝8．5，8．5Hz，GIc−3），4．20（lH，ddd，J＝8．5，5・0，2・3
Hz，GIc−5）．13C−NMR（Pyridine−d5）∂：182．9（C−4），164．8（C−7），164．6（C−2），162．0（C−9），157．5（C−5），151．7（C−4 ），147．8（C−3 ），122．9（C−l ），119．5（C−6 ）言16．9（C−5 ），114．6（C−2 ）言10．1（C−8），104．9（C−10），103・9（C−3），

94．6（C−6），83．0（GIc−5），80．6（GIc−3），75．6（GIc−1），72．9（GIc−2），71．9（GIc−4），62．7（GIc−6）

Isoorientin：FAB−MSm／2449［M＋H］＋．IH−NMR（CD30D）∂：7．28（lH，dd，J＝8．5，2．0Hz，H−6

d，．F2．0Hz，H−2），6．80（1H，d，J＝8．5Hz，H−5），7．27（lH，），6．45（lH，S，H−3），6．38（lH，S，H−8），4・82（lH，d，J＝7・8Hz，

GIc−1），4．13（lH，dd，J二8．5，8．5Hz，GIc−2），3．84（lH，dd，．F12．0，5．0Hz，GIc−6），3．71（lH，m，GIc−6），3．52（lH，m，GIc−3），3．50（lH，m，GIc−4），3．40（lH，m，GIc−5）．13C−NMR（CD30D）∂：182，7（C−4），163．6（C−7），164．5（C−2），
161．0（C−5），156，2（C−9），150．2（C−4 ），146．4（C−3 ），122．8（C−1 ），122・7（C−6 ）言16・3（CT5 ），114・6（C−2 ），107・3（C−6），104．7（C−10），103．0（C−3），94．0（C−8），83．0（GIc−5），79．5（GIc−3），74．0（GIc−1），71・4（GIc−2），71・9（GIc−4），
62．4（GIc−6）．

Isorhamnetin−3−0−rutinoside：FAB−MSm／乞625［M＋H］＋．lH−NMR（CD30D）∂：7．96（1H，d，．F2．1Hz，

H−2 ），7．64（lH，dd，J＝8．5，2．1Hz，H−6），6．92（lH，d，。F8．5Hz，Hj ），6．42（lH，d，J＝2．1Hz，H−8），6．21（lH，d，
J＝2．1Hz，H−6），5．25（lH，d，J＝8．5Hz，GIc−1），4．54（lH，d，J＝2．1Hz，Rham−1），3．97（lH，S，OMe），3．72（lH，dd，J＝12．0，2．5Hz，GIc−6），3．55（1H，dd，J＝12．0，5．0Hz，GIc−6），3．46（lH，m，GIc−2），3．35（GIc−3，4），3．22（1H，m），

3．20−3．78（Rham−2，3，4，5），1．10（lH，d，J＝6．5Hz，Rham−6）．13C−NMR（CD30D）∂：180．0（C−4）言66．3（C−7），
163．5（C−5），158．9（C−2），159．1（C−9），151．2（C−3 ），148．6（C−4 ），135．7（C−3），124．3（C−6 ）言23．3（C−1 ），116・5（CT5），116．4（C−2），104．9（C−10），103．0（GIc−1），102．9（Rham−1），100．4（C−6），95．3（C−8），78．5（GIc−3）77・7（GIc−5），76．4（GIc−2），74．4（Rham4），72．8（Rham−3），72．4（Rham−2），71．2（GIc−4），70．1（Rhamr5），68．8（GIc−6），57．0（OMe），18．2（Rham−6）．
GLucoluteolin：FAB−MSm／2449lM＋H］＋．IH−NMR（Pyridine−d5）∂：7．90（1H，d，J＝2．5Hz，H−2う，7．52（lH，dd，J＝8．5，2．5Hz，H−67，7．28（lH，d，J＝8．5Hz，H−5 ），6．99（lH，d，J＝2．1Hz，H−6），6．94（lH，S，H−3），6・85（lH，d，
J＝2．1Hz，H−8），5．83（lH，d，J＝7．6Hz，GIc−1），5．83（lH，d，J≒7．6Hz，GIc−1），4．58（lH，dd，J＝12．0，2．2Hz，GIc−6），4．43（lH，dd，J＝8．0，8．0Hz，GIc−3），4．42（lH，dd，J二12．0，5．3Hz，GIc−6），4．37（lH，dd，J＝8．0，8．0Hz，GIc−4），4．36（lH，dd，J＝8．0，7．6Hz，GIc−2），4．21（lH，ddd，J＝12．0，5．3，2．2Hz，GIc−5）．13C−NMR（Pyridine−d；∂：182．9（C−4），165．4（C−2），164．0（C−7），162．7（C−9）言57．9（C−5），152．0（C−4 ），147．9（C−3 ），122．8（C−1 ），119．7（C−6 ），116．9（C−5 ），114．8（C−2 ），106．6（C−10），104．2（C−6），101．9（GIc−1），100．7（C−8），95．3（C−3），79．3（GIc−3），78・6（GIc−5），74．9（GIc−2），71．2（GIc−4），62．4（GIc−6）・