全 文 ：天然产物研究与开发 NatProdResDev2008, 20:839-841
文章编号:1001-6880(2008)05-0839-03
　
　
　ReceivedJune11, 2007;AcceptedAugust8, 2007
　FoundationItem:ThisworkwassupportedbyNaturalSciencesFundof
HuaihaiInstituteofTechnology.
＊CorrespondingauthorTel:86-518-5895427;E-mail:glei77@sohu.com
金叶子中抑制膜结合型 PGE2 合酶 -1表达活性物质的分离与鉴定
郭　雷 1＊ ,龚邦强 2
1淮海工学院海洋学院 , 连云港 222005;2华东理工大学生物工程学院 , 上海 200237
摘　要:在从天然产物筛选抗炎物质的过程中 , 应用有机溶媒萃取 、浓缩及层析分离纯化后 , 从金叶子中得到三
个化合物:槲皮素(1)、番石榴苷(2)、栎素(3)。化合物 2为首次从此植物中分离鉴定 , 化合物 1显示了强烈的
抑制 mPGES-1基因表达的活性 , 其 IC50值为 35.40 μg/mL。
关键词:金叶子;膜结合型 PGE2合酶-1;类风湿性关节炎;槲皮素;番石榴苷;栎素
中图分类号:R284.2;Q946.91 文献标识码:A
IsolationandIdentificationofmPGES-1
ExpressionInhibitorfromCraibiodendronyunnanense
GUOLei1＊ , GONGBang-qiang2
1MarineCollege, HuaihaiInstituteofTechnology, Lianyungang222005 , China;
2BioengineeringCollege, EastChinaUniversityofScienceandTechnology, Shanghai200237 , China
Abstract:Inthecourseofsearchingforanti-inflammatoryagentsfromnaturalproducts, threeknowncompoundswerei-
solatedfromtheethanolextractoftheleavesofCraibiodendronyunnanenseW.W.Smithandidentifiedasquercetin
(1), quercetin3-O-α-L-arabinopyranoside(guaijaverin, 2), quercetin3-O-α-L-rhamnopyranoside(quercitrin, 3).Com-
pound2wasisolatedforthefirsttimefromthisplant.Amongthesecompounds, compound1 showedsignificantbioactivi-
tyagainstmPGES-1 expressionwithIC
50
valueof35.40 μg/mL.
Keywords:Craibiodendronyunnanense;mPGES-1;rheumatoidarthritis;Quercetin;Guaijaverin;Quercitrin
Introduction
Rheumatoidarthritis(RA)isanautoimmunedisease
thatcauseschronicinflammationinsynovialtissuesand
jointsresultinginjointdestructionandseverepain.
ProstaglandinE2(PGE2)playsanimportantroleinthe
physiopathologyofRA, actingasamediatorofinflam-
mationandpain.Thus, regulationoftheproductionor
actionofPGE2 hasimportantmeaninginthetreatment
ofRA.
Inthepastseveralyears, aninducibleformof, mem-
braneassociatedprostaglandinE2 synthase1 (mPGES-
1)hasbeenidentifiedandshowntobeinduciblein
variousmodelsofpainandinflammationincludingRA,
whereitappearstobelinkedwithcyclooxygenase2
(COX-2)andpromotingdelayedPGE2 synthesis[ 1] .In
theratmodelofadjuvant-inducedarthritis(AIA), a
diseasemodelofhumanRA, asignificantinductionof
mPGES-1isdemonstratedintheafectedpawandthe
increaseinmPGES-1levelcoincideswithlocalincrea-
sesinCOX-2 expressionandPGE2 production[ 2] .In
colagen-inducedarthritis(CIA), anothermodelofhu-
manRA, theimpactofmPGES-1 deletionwasevalua-
ted.Incontrasttothewild-typemice, mPGES-1-defi-
cient(mPGES-/-)micedisplayamarkedreductionin
inflammatoryresponsesandareprotectedfrom his-
topathologicaldeterioration(hyperplasia, boneandcar-
tilageerosion)[ 3] .Furthermore, inRApatients, strong
expressionofmPGES-1 isdetectedinsynovialtissues
[ 4] .TheseresultsprovidedefinitiveevidencethatmPG-
ES-1representsapotentialtargettodevelopnovelanti-
inflammatoryandanalgesicagents[ 5] .Inourresearch
work, wefirstlytransfectedA549 celswiththerecom-
binantDNAincludingmPGES-1 genepromoterandlu-
ciferasereportgene, constructedacel-basedscreening
modelM-1 [ 6] .Bythiscel-basedassayM-1, we
screenedourextractbankforinhibitorsofmPGES-1
expresionfrom naturalproductsandfoundthatan
EtOAcfractionfromethanolextractoftheleavesof
CraibiodendronyunnanenseW.W.Smithshowedstrong
inhibitorybioactivityagainstmPGES-1 expression.U-
singthemPGES-1 expressionbioassayasaguide, the
chromatographyofthefractionafordthreeknowncom-
pounds:quercetin(1), quercetin3-O-α-L-arabinopyr-
anoside(guaijaverin, 2), quercetin3-O-α-L-rhamnopy-
ranoside(quercitrin, 3), and2 (guaijaverin)hasnot
beenreportedpreviouslyfromthisplant(Fig.1).
MaterialsandMethods
Generalexperimentalprocedures
MeltingpointsweredeterminedusinganXT-4Aappa-
ratusandareuncorected.UVspectraweremeasured
withUV240 spectrophotometer.IRspectraweremeas-
uredwithBio-RadRTS-135 apparatus.NMRspectra
wererecordedwithBruckerAM-400NMRspectrometer
usingTMSasinternalstandard.MSwereobtainedona
MAT711 massspectrometer.Silicagel(QingdaoHaiy-
angChemicalGroupCo., China)andSephadexLH-20
(Amersham, Sweden)wereusedforcolumnchroma-
tography.
Plantmaterial
TheleavesofCraibiodendronyunnanenseW.W.Smith
werecolectedfromDali, YunnanofChina, inSeptem-
ber2004.Avoucherspecimen(G04010)wasdeposi-
tedattheherbariumofourlaboratory, EastChinaUni-
versityofScienceandTechnology, Shanghai, China.
Biologicalactivityevaluation
ThebiologicalactivitywasdeterminedagainstM-1 cel
lineaccordingtothemethodgivenbyGuoLetal[ 6] .
Briefly, 200 μLofM-1 celsat1×105 cels/mLwas
addedintoeachwelofwhite48-welplateovernight.
Themediawereremovedthenextdayand200 μL
freshmediawasaddedintoeachwel.Andthen, 2 μL
ofthecompound(quercetin, quercitrin, orguaijaverin)
dissolvedinDMSOwasaddedintoeachwel, andeach
concentrationofthecompoundhas3 repeats, 2 forlu-
ciferaseassayand1for3-(4, 5-dimethyl-2-thiazodyl)-
2, 5-diphenyltetrazoliumBromide(MTT)assay, while
2 μLofDMSOwasaddedintoeachwelascontroland
theplatewasincubatedovernight.Afterincubation, the
supernatantineachwelwasremoved, and100μLCel
CultureLysisReagentwasaddedintoeachweland
waspipetedenough.Finaly, 20 μLofcelextractswas
addedinto100 μLluciferasesubstrate.Luciferaseac-
tivitywasmeasuredbyusingTD2020 Luminometer.
Theluminometerreadingwascorectedbytheabsor-
bance(A)valueat570 nmandtheinhibitionratio
wasdefinedas(DMSOreading-compoundsread-
ing)/DMSOreading ×100%.Duringtheisolation
process, theactivityofalfractionswasmonitoredusing
thecel-basedassayM-1.
Extractionandisolation
ThedriedleavesofCraibiodendronyunnanenseW.W.
Smith(1.0kg)wereextractedwith80% EtOH(8 L
×3, 1 heach)underreflux.Theethanolextractwas
concentratedunderreducedpressuretoafordacrude
extract(21 g).
ThecrudeextractwaspartitionedwithCHCl3 andEtO-
Acinturn.TheEtOAcfraction(7.2g)waschromato-
graphedonsilicagel(200-300mesh)elutingwiththe
CHCl3 -MeOH(1∶0 to2∶1)gradientsolventsystem.
Repeatedchromatographywiththesameeluentonlow
pressuresilicagelcolumnandSephadexLH-20 afor-
dedcompounds1(32mg), 2 (17 mg), 3 (22 mg).
ResultsandDiscussion
Asgivenabove, threeknowncompoundswereisolated
fromtheactivefractionoftheleavesofCraibiodendron
yunnanenseW.W.Smith.Theirstructureswereidenti-
fiedonthebasisoftheirphysicalandspectraldata.
Compound2 wasisolatedforthefirsttimefromthis
plant.
Fig.1　Structureofcompounds1-3
840 NatProdResDev　　　　　　　　　　　　　　　　　　　　 　Vol.20
Altheabovecompoundsobtainedfromtheleavesof
CraibiodendronyunnanenseW.W.Smithwereevalua-
tedfortheirpotentialtoinhibithumaninduciblemPG-
ES-1 expression.TheIC50 valueofEtOAcfractiona-
gainstmPGES-1 expressionwas101.64 μg/mL, The
IC50 valueofcompound1 was35.40 μg/ML.There-
sultsindicatedthatcompound1showedsignificantbio-
activityagainstmPGES-1 expressionwithIC50 valueof
35.40 μg/mL.
Quercetin(1)　Yelowpowder, mp.314-316 ℃;1H
NMR(DMSO-d6)δ:6.40(1H, d, J=2.2 Hz, H-8),
6.18(1H, d, J=1.9Hz, H-6), 7.67 (1H, d, J=2.2
Hz, H-2′), 7.53(1H, dd, J=8.5 Hz, 2.2 Hz, H-6′),
6.88(1H, d, J=8.5 Hz, H-5′);13 CNMR(DMSO-
d6 )δ:C-2 ※ C-10:146.8, 135.7, 175.8, 160.7,
98.1, 163.8, 93.3, 156.1, 103.0, C-1′※ C-6′:
121.9, 115.6, 145.0, 147.7, 115.0 , 120.0.1HNMR
and13CNMRdatawereconsistentwithliteratureval-
ues[ 7] .
Quercetin3-O-α-L-arabinopyranoside(guaijaver-
in, 2)　Yelowpowder, mp.240℃;UVλMeOHmax nm:256,
360, 207;IRυKBrmax cm-1:3478, 3376.9 and 3213.7
(OH), 1655.1 (C=O), 1612.8, 1561 and1510.1
(C6H5);FAB--MS:434 [ M-1] - , 301 [ M-1-132] -;1H
NMR(DMSO-d6)δ:6.40 (1H, d, J=2.0 Hz, H-8),
6.18(1H, d, J=2.0Hz, H-6), 7.53 (1H, d, J=2.1
Hz, H-2′), 7.65(1H, dd, J=8.5 Hz, 2.2 Hz, H-6′),
6.83(1H, d, J=8.5Hz, H-5′), 5.26(1H, d, J=5.1
Hz, H-1″), 12.63 (1H, s, 5-OH);13 CNMR(DMSO-
d6 )δ:C-2 ※ C-10:156.3, 133.6, 177.6, 161.3,
98.7, 164.2, 93.5, 156.3, 103.9, C-1′※ C-6′:
122.0, 115.8, 145.0, 148.1, 115.4, 120.9, C-1″※ C-
5″:101.4, 70.8, 71.6, 66.1, 64.3.1HNMRand13 C
NMRdatawereconsistentwithliteraturevalues[ 7, 8] .
Compound3 wasdeterminedbycomparingitsphysical
andspectradatawiththereportedvalues:quercetin3-
O-α-L-rhamnopyranoside(quercitrin, 3)[ 9] .
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841Vol.20　　　　　　GUOLei, etal:IsolationandIdentificationofmPGES-1ExpressionInhibitorfromCraibiodendronyunnanense