全 文 ：PhenolicglucosidesfromtheleavesofPierisjaponica
YAOGuang-min, WANGYu-bo, WANGLi-quan, QINGuo-wei＊
(ShanghaiInstituteofMateriaMedica, ShanghaiInstitutesforBiologicalSciences,
ChineseAcademyofSciences, Shanghai201203 , China)
Abstract:TheaimofthestudyistoinvestigatechemicalconstituentsoftheleavesofPierisjaponica.
Theisolationandpurificationoftheconstituentswereperformedbyvariouschromatographyandspectral
analysis.Threenewphenolicglucosides, erythro-syringoylglycerol4-O-β-D-glucoside(1), 1-(2-β-D-
glucopyranoxyl-4-methoxyl-6-hydroxyphenyl)-3-hydroxyl-1-propanone (3), erythro-1-(4-hydroxyl-3-
methoxyphenyl)-2-[ 4-(3-β-D-glucopyranoxypropyl)-2, 6-dimethoxyphenoxy] -1, 3-propanediol(4), along
withfiveknownphenolicglucosides, syringoylglycerol8-O-β-D-glucoside(2), magnoleninC(5),
syringaresinolmono-β-D-glucoside(6), 3-(4-hydroxyl-3-methyphenyl)-1-propanol-1-O-β-D-glucoside
(7)and3, 5-dimethoxyl-4-hydroxybenzylalcohol4-O-β-D-glucoside(8)wereisolatedandidentifiedfrom
theplantleaves.Compounds1and2 inhibitedsignificantly(P<0.01)theproliferationofmurineTand
Bcelsatconcentrationof1×10-6 mol· L-1 , invitro.
Keywords:Pierisjaponica;Ericaceae;phenolicglucoside;immuno-suppressiveactivity
CLCnumber:R284.1　　　Documentcode:A　　　ArticleID:0513-4870(2008)03-0284-07
Received2007-11-08.
ProjectsupportedbyNationalNaturalScienceFoundationofChina
(30170104).
＊Corespondingauthor　Tel:86-21-50805853,
Fax:86-21-50807088,
E-mail:gwqin@mail.shcnc.ac.cn
马醉木叶的酚性化学成分
姚广民 , 王玉波 , 汪礼权 , 秦国伟＊
(中国科学院 上海生命科学院 上海药物研究所 , 上海 201203)
摘要:为了研究马醉木(Pierisjaponica)叶的化学成分 , 采用各种色谱方法对其乙醇提取物进行分离 ,所得成分
用各种波谱特别是一维和二维 NMR进行结构测定 , 最后分离鉴定出 8种酚性成分 (1 ～ 8), 其中 erythro-
syringoylglycerol4-O-β-D-glucoside(1)、 1-(2-β-D-glucopyranoxyl-4-methoxyl-6-hydroxyphenyl)-3-hydroxyl-1-propanone
(3)、 erythro-1-(4-hydroxyl-3-methoxyphenyl)-2-[ 4-(3-β-D-glucopyranoxypropyl)-2, 6-dimethoxyphenoxy] -1, 3-propanediol
(4)为 3种新化合物 , 其余 5种已知化合物 syringoylglycerol8-O-β-D-glucoside(2)、 magnoleninC(5)、 syringaresinol
mono-β-D-glucoside(6)、 3-(4-hydroxyl-3-methyphenyl)-1-propanol-1-O-β-D-glucoside(7)和 3, 5-dimethoxyl-4-
hydroxybenzylalcohol4-O-β-D-glucoside(8)为该植物中首次报道 ,化合物 1和 2在体外试验中显著抑制 T和 B细胞
的增殖。
关键词:马醉木;杜鹃花科;酚性葡糖苷;免疫抑制活性
　　Pierisjaponica(Wal)D.Don(Ericaceae)isan ornamentalshrub, whichcanbeeasilycultivatedasa
floweringpot-plant.Theleaves, stems, androotsof
theplantareusedasChinesefolkmedicinetocure
vomitinganddiarheacausedbysunstrokeandasan
insecticideandalotionfortreatmentofringwormand
scabies[ 1] .Previousphytochemicalworkontheplant
·284· 药学学报 ActaPharmaceuticaSinica2008, 43(3):284-290
DOI :10.16438/j.0513-4870.2008.03.001
resulted in isolation of dihydrochalcones[ 2] ,
diterpenoids[ 3] , steroidsandtriterpenoids[ 4] .Inour
ongoingsearchforbiologicalyactivesubstancesfrom
Chinesemedicinalplants, weinvestigatedtheleavesof
P.japonicaandisolatedthreenewphenolicglucosides
1, 3 and4 , alongwithfiveknownphenolicglucosides
(2 , 5-8), whichwerefirstreportedfromtheplant.
Inthepresentpaper, wedescribetheisolationand
structuralelucidationof1, 3 and4 aswelasinvitro
immuno-suppressiveactivitiesof1 and2.
Resultsanddiscussion
Compound1　Colorlessneedles, hasamolecular
formulaofC17H26O11 determinedbyHRESIMSat
m/z:429.137 7 [ M+Na] + , calculated429.137 3.
Hydrolysisof1 withβ-glucosidaseafordedglucoseas
detectedbyco-TLC, suggestingthat1 isaglucoside.
TheIRspectrumindicatedthepresenceofhydroxyl
(3 408 cm-1)andaromaticrings(1 595cm-1).The
1HNMRand13CNMRspectra(Table1)of1 were
verysimilartothoseofsyringoylglycerol8-O-β-D-
glucoside2, aknowncompoundisolatedfromthesame
plant, exceptforthechemicalshiftsofC-3, C-4, C-5,
andC-8.Inthe13CNMR spectra, theC-8 of1
resonatedatahigherfield(δ75.6)thanthatof
compound2(δ87.5), whiletheoxygenatedaromatic
carbonsof1 shifteddownfieldatδ139.5 (C-4)and
152.2 (C-3 andC-5)comparedwiththoseof2.
Thus, 1 shouldbeanisomerof2 withdiferenceat
thepositionofthesugarlinkage.IntheHMBC
spectrumof1, acrosspeakbetweenanomericproton
H-1′(δ4.84)ofglucoseandC-4 (δ139.5)was
observed, indicatingthatC-4 wasglucosylated.The
β-configuration ofthe glucopyranosy moiety was
deducedfromcouplingconstant(J=7.2 Hz)ofthe
anomericprotonH-1′andrelativestereochemistryof
C-7 andC-8 wasexpectedtobeofanerythreo-form
fromthecouplingconstantJ7, 8 =5.3 HzofH-7[ 10, 11] .
Thus, compound1 wasdeterminedaserythreo-1-
(4-β-D-glucopyranoxyl-3, 5-dimethoxyphenyl)-1, 2, 3-
propanetriol, i.e.erythro-syringoylglycerol4-O-β-D-
glucoside.
Compound3　Colorlessneedles, hadamolecular
formulaofC16H22O10 determinedbyHRESIMSat
m/z:397.111 0 [ M+Na] + , calculated397.111 1.
Hydrolysisof3 withβ-glucosidaseafordedglucoseas
detectedbyco-TLC, suggestingthat3wasaglucoside.
TheIRspectrumof3 showedthepresenceofhydroxyl
(3 419 cm-1), conjugatedketone(1 620cm-1)and
aromaticring(1 593, 1 514 cm-1).The1HNMR
spectra(Table1)showedsignalsforamethoxygroup
(δ3.79, s), twometaaromaticprotons(δ:6.15
and 6.28, J=2.2 Hz), onechelated phenolic
hydroxyl(δ13.41), a-CH2CH2O- moietyanda
glucoseunit.Inthe13CNMR andDEPTspectra
(Table1), sixteencarbonsignalswereassignedtobe
acarbonylgroup(δ204.5), sixaromaticcarbon
singnals, one methoxylgroup (δ55.5), one
oxygenatedmethylene(δ56.9), one methylene
(δ47.1)andaglucoseunit(δ:100.5, 77.3,
76.6, 73.1, 69.7 , and 60.6).Furtherstudies
showedthatthe1HNMRand13CNMRdataof3were
similartothoseofasebotin(2′, 4, 6′-trihydroxy-4′-
methoxydihydrochalcone 2′-O-β-D-glucopyranoside),
aknowncompoundisolatedfrom sameplant[ 2, 12] ,
exceptforabsenceofap-hydroxyphenylmoietyand
presenceofahydroxylatpropaneterminal.Inthe
HMBCspectrumof3, theC-4 (δ165.2)corelated
with methoxy group protons (δ3.79 ), H-3
(δ6.28)andH-5(δ6.15), suggestingthemethoxy
groupatC-4 , whichwasfurthersupportedbythe
NOESYcorelationsofthemethoxygroupprotonswith
H-3 andH-5.Furthermore, theHMBCcorelationsof
C-2 (δ160.1)withH-3 andH-1′ofglucoserevealed
thatthe glucose unitwas connected atC-2.
Subsequently, thechelatedphenolichydroxylgroup
shouldbeatachedtoC-6.The β-glucopyranosyl
linkagewasdeduced from the coupling constant
(J=7.2Hz)oftheanomericproton.Onthebasisof
theaboveevidence, compound3 wasdeterminedas1-
(2-β-D-glucopyranoxyl-4-methoxyl-6-hydroxyphenyl)-
3-hydroxyl-1-propanone.
Compound4　wasobtainedasanamorphous
powder.Aquasi-molecularionpeakatm/z593.219 8
[ M+Na] + inHRESIMSsuggestedamolecularformula
ofC27H38O1 3 , indicatingninedegreesofunsaturation.
ItgaveapositiveresponsetoalcoholicFeCl3 regent,
andafordedglucosewhenhydrolyzedwithβ-gluco-
sidase, suggestingthatcompound4 isaglucosidewith
phenolichydroxylgroup.The 1HNMR spectrum
(Table2)showedsignalsforasetofABX-type
aromaticprotonsatδ:6.98(d, J=1.3 Hz), 6.56
(dd, J=8.2, 1.3 Hz)and6.24(d, J=8.2 Hz),
twoequivalentmetaaromaticprotonsatδ6.56, three
methoxygroupsatδ:3.80 (6H)and3.82 (3H),
aliphaticprotonsfora“-CH2 -CH2 -CH2O-” moiety, a
1, 2, 3-trioxygenatedpropylmoiety, andaglucose
unit.The13CNMR andDEPTspectra(Table2)
·285·YAOGuang-min, etal:PhenolicglucosidesfromtheleavesofPierisjaponica
Table1　1HNMRand13CNMRdataof1, 3 (inDMSO-d6)and13CNMRdataof2 (inCD3OD).JinHz
No.　　 1δH δC
2
δC
3
δH δC
1 133.3(s) 132.8(s) 106.2(s)
2 6.64(s) 105.0(d) 105.4(d) 160.1(s)
3 152.2(s) 149.3(s) 6.28(d, J=2.2) 93.3(d)
4 139.5(s) 136.3(s) 165.2(s)
5 152.2(s) 149.3(s) 6.15(d, J=2.2) 95.1(d)
6 6.64(s) 105.0(d) 105.4(d) 165.1(s)
7 4.49(d, J=5.3) 72.7(d) 75.1(d) 204.5(s)
8 3.48(m) 75.6(d) 87.5(d) 3.22(t, J=6.1) 47.1(t)
3.29(t, J=6.1)
9 3.21(dd, J=5.7, 10.6) 62.8(t) 63.3(t) 3.72(t, J=6.1) 56.9(t)
3.37(dd, J=5.5, 10.6)
1′ 4.84(d, J=7.2) 102.9(d) 105.3(d) 5.0(d, J=7.2) 100.5(d)
2′ 3.17(m) 74.3(d) 75.6(d) 3.31(m) 73.1(d)
3′ 3.00(m) 77.2(d) 78.1(d) 3.15(m) 77.3(d)
4′ 3.12(m) 70.0(d) 70.0(d) 3.28(m) 69.7(d)
5′ 3.16(m) 76.6(d) 77.9(d) 3.31(m) 76.6(d)
6′ 3.40(dd, J=5.7, 11.7) 61.0(t) 62.6(t) 3.45(dd, J=5.7, 11.5) 60.6(t)
3.58(dd, J=5.2, 11.7) 3.75(dd, J=5.5, 11.5)
3-OCH3 3.73(s) 56.4(q) 56.9(q)
4-OCH3 3.79(s) 55.5(q)
5-OCH3 3.73(s) 56.4(q) 56.9(q)
6-OH 13.41(brs)
Table2　1HNMRand13CNMRdataofcompound4andnymphaeoside[ 13] (inCD3OD).JinHz
Position　　　 4δH δC
Nymphaeoside
δH δC
1 134.3(s) 139.9
2 6.98(d, J=1.3) 112.0(d) 7.11(brs) 112.4
3 149.3(s) 151.6
4 147.4(s) 146.0
5 6.24(d, J=8.2) 116.3(d) 7.08(d, J=8.0) 119.2
6 6.78(dd, J=1.3, 8.2) 121.1(d) 6.93(d, J=8.0) 120.6
7 4.91(d, J=4.9) 74.5(d) 4.99(d, J=5.2) 73.7
8 4.17(m) 88.0(d) 4.22(m) 87.2
9 3.55, 3.86(overlap) 62.0(t) 3.57(m) 61.4
1′ 140.4(s) 138.0
2′, 6′ 6.56(s) 108.4(d) 5.57(s) 106.8
3′, 5′ 154.8(s) 154.3(s)
4′ 135.4(s) 134.6
7′ 2.69(t, J=6.3) 33.9(t) 2.65(t, J=8.0) 33.4
8′ 1.91(m) 33.1(t) 1.82(m) 35.8
9′ 3.54, 3.91(overlap) 70.3(t) 3.57(m) 62.1
3-OCH3 3.82(s) 56.9(q) 3.85(s) 56.4
3′, 5′-OCH3 3.80(s) 57.2(q) 3.83(s) 56.6
1″ 4.25(d, J=7.8) 105.0(d) 5.38(brs) 101.4
2″ 3.19(m) 75.7(d) 4.12(brs) 72.2
3″ 3.25(m) 78.4(d) 3.95(m) 72.0
4″ 3.26(m) 72.2(d) 3.48(t, J=9.5) 73.8
5″ 3.34(m) 78.7(d) 3.91(m) 70.7
6″ 3.66, 3.84(overlap) 63.3(t) 3.16(3H, d, J=5.9) 18.8
·286· 药学学报 ActaPharmaceuticaSinica2008, 43(3):284-290
displayedsignalsforthreemethoxygroups, four
methylene(twooxygenated), sevenmethinegroups
(twooxygenatedandfivearomaticrings), seven
quaternarycarbons(fiveoxygenated)andaglucose
moiety.Above1HNMRand13CNMRdatawerevery
similartothoseofnymphaeosideA, i.e. 1-[ 3-
methoxyl-4-α-L-rhamnopyranosyloxyl-phenyl] -2-[ 4-(3-
hydroxypropyl)-2, 6-dimethoxyphenyl] -1, 3-
propanediol[ 13] , exceptforthediferenceofthesugar
anditslinkageposition.Comparedwith13CNMRdata
ofnymphaeosideA, thesignalofC-9′in4 wasfound
toshiftdownfieldforδ8.2 , indicatingthattheglucose
moietyshouldbeconnectedtoC-9′.TheHMBC
correlationbetweentheanomericprotonH-1″(δ
4.25)oftheglucoseandC-9′(δ70.3)further
confirmedthisdeduction.Alargercouplingconstant
(J=7.8 Hz)oftheanomericprotonindicatedaβ-
formoftheglucosepyranosymoietyandasmaler
couplingconstant(J=4.9 Hz)ofH-7 suggestedan
erythreo-formoftherelativestereochemistryofC-7 and
C-8[ 10, 11] .TheHMQCandHMBCanalysesalowedfor
thecompleteasignmentsofthe1HNMRand13CNMR
signalsof4.Therefore, itsstructurewasdeterminedas
erythro-1-(4-hydroxyl-3-methoxyphenyl)-2-[ 4-(3-β-D-
glucopyranoxypropyl)-2 , 6-dimethoxy-phenoxy] -1, 3-
propanediol.ThekeyHMBCcorrelationsof4 were
showninFigure2.
Table3　Efectofcompounds1 and2 onmurine
lymphocyteproliferationinducedbyconcanavalinA
(ConA)(5mg· mL-1)orlipopolysaccharide(LPS)
(10mg·mL-1)
Group Concentration/mol· L-1
[ 3H] TdRincorporation×10-3 /min-1
ConA-inducedT
celproliferation
LPS-inducedB
celproliferation
Negativecontrol 2.294±0.400 2.985±0.386
Positivecontrol 39.102±4.298 3.6064±0.700
(ConAorLPS)
　　 1 1×10-7 37.014±2.413 32.791±2.846
1×10-6 27.793±4.170＊＊＊ 24.410±3.909＊＊＊
1×10-5 38.922±7.592 35.504±1.695
Negativecontrol 6.272±0.181 4.772±0.684
Positivecontrol 80.704±2.692 74.008±0.798
(ConAorLPS)
　　 2 1×10-7 68.539±3.023 20.798±1.508
1×10-6 56.600±4.379＊＊ 22.049±1.976＊＊
1×10-5 8.096±3.255 28.245±2.019
Resultsarerepresentedasmean±S.D.basedonthree
independentexperiments.n=3;＊＊P<0.01, ＊＊＊P<0.001 vs
controlgroup
Fiveknownphenoliccompoundswereisolatedand
identifiedbycomparingtheirspectroscopicdata(MS,
1HNMRand 13CNMR)withliteraturevaluesas
syringoylglycerol8-O-β-D-glucoside(2)[ 5] , magnolenin
C(5)[ 6] , syringaresinolmono-β-D-glucoside(6)[ 7] ,
3-(4-hydroxyl-3-methylphenyl)-1-propanol1-O-β-D-
glucoside(7)[ 8] and3, 5-dimethoxyl-4-hydroxybenzyl
alcohol4-O-β-D-glucoside(8)[ 9] , whichalwerefirst
reportedfromtheplant.The13CNMRdataof5 was
alsodeterminedforthefirsttime.Compounds1 and2
inhibitedsignificantly(P<0.01)theproliferationof
murineTandBcelsattheconcentrationof1 ×10-6
mol· L-1 invitro, asshowninTable3.TheMTT
assaydemonstrated thatinhibitingproliferation of
thesecompoundswasnotinvolvedwiththegeneral
cytotoxity.
Figure1　Thestructuresofcompounds1-4
Figure2　KeyHMBC(H※C)corelationsof
compound4
Experimental
MeltingpointsweredeterminedonadigitalSGW
X-4 apparatusandareuncorected.Opticalrotations
were measured on a Perkin ElmerModel341
polarimeter.UVspectrawererecordedonaShimadiu
·287·YAOGuang-min, etal:PhenolicglucosidesfromtheleavesofPierisjaponica
UV-2550 UV-visiblespectrophotometer.TheFT-IR
spectrawererecordedonanIR-514 spectrophotometer
withKBrpelets.1HNMRand13CNMRspectrawere
recordedonaBrukerAM-400 spectrometerwithTMS
astheinternalstandard. 2D NMR spectrawere
recorded on a Bruker DRX-500 spectrometer.
HR-ESIMSandESIMSwerecariedoutonaJEOL
SX-102, usingPEG-400 ascalibrationmatrix.
Plantmaterial　TheleavesofPierisjaponica
(Wal)D.DonwerecolectedintheHuangshan
mountainofAnhuiprovince, China, inJune2003 and
identifiedbyProfessorBing-yangDingofSchoolofLife
and Environmental Sciences, Wenzhou Normal
Colege.Avoucherspecimen(No.20030715)is
depositedintheherbarium ofShanghaiInstituteof
MateriaMedica, ChineseAcademyofSciences.
Extractionandisolation　 Theair-driedand
powderedleavesofP.japonica(10kg)wereextracted
with95% EtOH.TheEtOHextractwasevaporated
underreducedpressuretogivearesidue(2 kg)that
wasthensuspendedindistiledwaterandpartitioned
successivelywithpetroleumether, CHCl3 , EtOAcandn-BuOH.Then-BuOHextractwaschromatographedon
asilica-gelcolumn, usingmixturesofCHCl3 and
MeOHaseluent, togivefractionsA-G.FractionFwas
chromatographedonareversed-phaseC18 columnwith
MeOH-H2O(0 -80%)assolventtogivefractions
1-4, fractions1, 2 and4 werethenpurifiedby
SephadexLH-20 togivecompounds1 (100 mg), 2
(8mg), and3 (12 mg), respectively.FractionE
waschromatographedonareversed-phaseC18 column
withMeOH-H2O(30% -80%)assolventtogive
threefractions, eachfractionwaspurifiedbySephadex
LH-20 togivecompounds4 (14 mg), 7 (15 mg),
and8 (25mg).FractionDwaschromatographedona
reversed-phaseC18 column and then purified by
SephadexLH-20 togivecompound 5 (12 mg).
Repeatedsilica-gelcolumnchromatographonfraction
Cafordedcompound6(85mg).
Identification
Erythreo-syringoylglycerol4-O-β -D-glucoside,
1　Colorlessneedles, mp174 -176 ℃ (MeOH).
[ α] 20D -16°(c0.11 , MeOH);UVλMeOHmax nm(logε):
207(4.22), 270(3.25);IRνKBrmaxcm-1:3 408(br,
OH), 2 922, 1 595(aromaticrings), 1 466, 1 423,
1 333, 1 236, 1 126, 1 074, 611;1HNMR(400
MHz, CD3OD)and13CNMR(100 MHz, CD3OD)
data, seeTable 1;HR-ESI-MSm/z:429.137 7
[ M+Na] +(calcd.forC17H26O11Na+, 429.137 3).
Syringoylglycerol8-O-β -D-glucoside2　Color-
lessneedles, mp204 -206 ℃ (MeOH);[ α] 29D
+7.9°(c0.63, MeOH);UVλMeOHmax nm(logε):211
(4.11), 230(3.72), 270(3.05);IRνKBrmax cm-1:
3 350(br, OH), 2 900, 1 615, 1 523(aromatic
rings), 1 466, 1 353, 1 236, 1 126, 1 078;1HNMR
(400 MHz, CD3OD)δ:3.33-3.23(4H, m, H-2′,
3′, 4′, 5′), 3.40(1H, dd, J=5, 12.0 Hz, H-9a),
3.56(1H, dd, J=4, 12.0 Hz, H-9b), 3.64(1H, dd,
J=5.6 , 12.0 Hz, H-6′), 3.82(1H, m, H-8), 3.84
(1H, dd, J=5.4, 12.0 Hz, H-6′a), 3.85(3H, s, 3, 5-
OCH3), 4.35(1H, d, J=7.2 Hz, H-1′), 4.68(1H,
d, J=6.4 Hz, H-7), 6.70(2H, s, H-2, 6), 13CNMR
(100 MHz, CD3OD)data, seeTable1.
1-(2-β-D-Glucopyranoxyl-4-methoxyl-6-hydroxy-
phenyl)-3-hydroxyl-1-propanone3　Colorlessneedles,
mp182 -184 ℃;[ α] 20D -73°(c0.03, MeOH);
UVλMeOHmax nm(logε):211(4.07), 221(4.07), 285
(4.02);IRνKBrmaxcm-1:3 419(OH), 1 620(C=O),
1 514 (phenyl), 1 431, 1 387, 1 283, 1 165 ,
1 097, 1 072;1HNMR(400 MHz, DMSO-d6)and
13CNMR(100 MHz, DMSO-d6)data, seeTable1.
HR-ESI-MSm/z:397.111 0 [ M+Na] +(calcd.for
C16H22O10Na, 397.111 1).
Erythro-1-(4-hydroxyl-3-methoxyphenyl)-2-
[ 4-(3-β-D-glucopyranoxypropyl)-2, 6-dimethoxy-
phenoxy] -1, 3-propanediol4　Amorphouspowder,
[ α] 20D -15°(c0.001, MeOH);UVλMeOHmax nm(logε):
222(4.98), 286(3.35);1HNMR (400 MHz,
CD3OD)and13CNMR(100 MHz, CD3OD)data,
seeTable2.HRESI-MSm/z:593.219 8[ M+Na] +
(calcd.forC27H38O13Na, 593.221 0).
MagnoleninC5　Colorlesscrystals, mp133 -
134 ℃;UVλMeOHmax nm(logε):310(4.15);IRνKBrmax
cm-1:3 400(OH), 1 675(C=O), 1 610, 1 585,
1 500, 1 460(phenyl), 1 110;ESIMSm/z:619.2
[ M+Na] +;1HNMR(400MHz, DMSO-d6)δ:3.03
(1H, m, H-5″), 3.08(1H, m, H-8′), 3.24 -3.10
(3H, m, H-2″, 3″, 4″), 3.40(1H, dd, J=5.8, 11.6
Hz, H-6″a), 3.58(1H, dd, J=4.0 , 11.6 Hz, H-6″b),
3.67(1H, dd, J=3.3, 12.0 Hz, H-9′b), 3.74(1H,
dd, J=6.9, 12.0 Hz, H-9′b), 3.75(6H, s, 3, 5-
OCH3), 3.84(6H, s, 3′, 5′-OCH3), 4.08(1H, dd,
J=3.4 , 9.0 Hz, H-9a), 4.10(1H, d, J=9.0 Hz,
H-9b), 4.22(1H, m, H-8), 4.54(1H, d, J=7.9Hz,
H-7′), 5.14(1H, d, J=7.1Hz, H-1″), 6.61(2H, s,
H-2′, 6′), 7.33(2H, s, H-2, 6), 8.29(1H, brs,
4-OH);13CNMR(100 MHz, DMSO-d6)δ:131.7
(C-1), 106.8(C-2, 6), 147.8(C-3, 5), 134.9
·288· 药学学报 ActaPharmaceuticaSinica2008, 43(3):284-290
(C-4), 198.3(C-7), 53.5(C-8), 69.7(C-9),
131.3(C-1′), 103.9(C-2′, 6′), 152.4(C-3′, 5′),
138.8(C-4′), 83.1(C-7′), 48.9(C-8′), 60.0(C-9′),
101.9(C-1″), 74.1(C-2″), 76.6(C-3″), 69.8(C-4″),
77.4(C-5″), 60.7(C-6″), 56.5(3′, 5′-OCH3), 55.9
(3, 5-OCH3).
Syringaresinolmono-β-D-glucoside6　Amor-
phoussolid, [ α] 28D -10°(c1.4, MeOH);UVλMeOHmax
nm(logε):270 (302).IRνKBrmaxcm-1:3 350(OH),
1 590, 1 510, 1 460(phenyl);ESI-MSm/z:603.2
[ M+Na] +;1HNMR(400 MHz, CD3OD)δ:3.12
(2H, m, H-8, 8′), 3.18(1H, m, H-5″), 3.49-3.38
(3H, m, H-2″, 3″, 4″), 3.65(1H, dd, J=5.3, 12.0
Hz, H-6″a), 3.77(1H, dd, J=2.2, 12.0Hz, H-6″b),
3.85(6H, s, 3, 5-OCH3), 3.85(6H, s, 3′, 5′-OCH3),
3.90(1H, dd, J=3.2, 9.0 Hz, H-9a), 3.90(1H, dd,
J=2.0, 9.0 Hz, H-9′a), 4.25(1H, dd, J=5.3, 9.0
Hz, H-9′b), 4.26(1H, dd, J=5.0, 9.0 Hz, H-9b),
4.71(1H, d, J=4.4 Hz, H-7′), 4.76(1H, d, J=4.0
Hz, H-7), 4.85(1H, d, J=7.0 Hz, H-1″), 6.65
(2H, s, H-2′, 6′), 6.71(2H, s, H-2, 6);13CNMR
(100 MHz, CD3OD)δ:136.1(C-1), 105.3(C-2,
6), 154.9(C-3, 5), 140.0(C-4), 87.6(C-7), 56.2
(C-8), 73.4(C-9), 133.6(C-1′), 105.0(C-2′,
6′), 149.8(C-3′, 5′), 136.7(C-4′), 88.1(C-7′),
56.0(C-8′), 73.4(C-9′), 105.8(C-1″), 76.2(C-
2″), 78.3(C-3″), 71.8(C-4″), 78.8(C-5″), 63.1
(C-6″), 57.6(3, 5-OCH3), 57.3(3′, 5′-OCH3).
3-(4-Hydroxyl-3-methylphenyl)-1-propanol
1-O-β-D-glucoside7　 Amorphouspowder[ α] 20D
-21.6°(c0.5, MeOH);ESI-MSm/z:367.1[ M+
Na] +;1HNMR(400 MHz, CD3OD)δ:1.81(2H,
dt, J=6.5, 7.6 Hz, H-8), 2.63(2H, t, J=7.6 Hz,
H-7), 3.55(2H, t, J=6.5 Hz, H-9), 3.68(1H, dd,
J=12.5, 4.4 Hz, GlcH-6b), 3.84(3H, s, OCH3),
3.86(1H, dd, J=12.5, 5.0 Hz, GlcH-6a), 4.84
(1H, d, J=7.2Hz, GlcH-1), 6.74(1H, dd, J=1.8,
8.2Hz), 6.86(1H, d, J=1.8 Hz, H-2), 7.07(1H,
d, J=8.2Hz, H-5), 7.90(1H, brs, 4-OH).
3, 5-Dimethoxyl-4-hydroxybenzylalcohol4-O-
β -D-glucoside8　Colorlesneedles, mp175-177℃
(MeOH);[ α] 20D -20°(c2.0, MeOH);(+)-ESI-
MSm/z:369.1 [ M+Na] + , 714.9 [ 2M+Na] +;
1HNMR(400 MHz, CD3OD)δ:3.65(1H, dd, J=
12.0, 5.0 Hz, GlcH-6b), 3.76(1H, dd, J=12.0,
5.0Hz, GlcH-6a), 3.84(6H, s, OCH3 ×2), 4.54
(2H, brs, H-7), 4.84(1H, d, J=7.2 Hz, GlcH-1),
6.70(2H, brs, H-2, 6);13CNMR(100 MHz,
C5D5N)δ:134.7(C-1), 105.1(C-2, 4), 153.7(C-
3, 5), 139.6(C-4), 64.3(C-7), 105.0(Glc-1),
76.1(Glc-2), 78.4(Glc-3), 71.6(Glc-4), 78.7
(Glc-5), 62.6(Glc-6).
Enzymatichydrolysisof1, 3and4　Compound
1 (3mg)wasdissolvedinH2O(8mL)andincubated
withalmondβ-glucosidase(20 mg)for24 hat
37 ℃.Themixturewasfiltratedandthewaterphase
wasanalyzedbysilicagelHPTLC(Merck)developed
withMe2CO-2 mmol· L-1 NaOAc(17 ∶3 , v/v).
Thesugarwasdetectedbysprayingwith 0.2%
naphthoresorcinolinMe2CO-1mol·L-1 H3PO4 (5∶1,
v/v) and then heated at 105 ℃ for 5 min.
D-glucopyranose(ChemprosaHoldingAG)wasused
asstandard.Thesamemethodtoidentifythesugar
moietywasusedforcompound3 and4.
2.6 Lymphocyte proliferation test　 The
preparedspleencelsofmice(4×106)wereseeded
intoeachwelofa96-welmicroplateandvarious
concentrationsofcompounds1, 2 and5 μg·mL-1 of
concanavalinA(ConA, fromCanavaliaensiformis
TypeII, Sigma), selectivestimulionTcels, or
lipopolysaccharide (LPS, from Escherichia coli,
Sigma), selectivestimulionBcels, wereadded.The
plateswereculturedat37 ℃ with5% CO2 ina
humidifiedatmospherefor48 h.Forthelast6 h,
eachwelwaspulsedwith0.25 μCi/wel 3H-TdR
(thymidine, [ methyl-3H] , ICN Pharmaceuticals,
Inc., Irvine, CA).Thecelswereharvestedandthe
radioactivityincorporatedwascountedbyaliquid
scintilationcounter.Alcounts/minvaluesshown
werethemeanoftriplicatesampleSD.Statistical
analysiswascarriedoutbyStudentt-test.ConAorLPS
wasusedasapositivecontrol(Xiangetal.1993).
MTTasaywasperformedtoevaluatethecytotoxicityof
thecompounds.Splenocyteswereculturedina96-wel
plateat4×105 cels/ 180 μL/ welinahumidified
CO2 incubatorat37 ℃for48 hoursinthepresence
or absence ofvarious concentrations oftested
compoundse. 18 μL of5 mg·mL-1 MTT (3-
[ 4 , 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium
bromide)wasaddedtoeachwelforthefinal5 hours.
Then90 μLoflysisbufer(10% SDS, 50% N, N-
dimethylformamide, pH7.2)wasaddedtoeachwel
for6 -7 handtheOD570 valueswerereadby
microplatereader(Bio-Rad, Model550).
Acknowledgements:WethanktheNationalCenterfor
DrugScreening, Shanghai, andDepartmentofPharmacology,
ShanghaiInstituteofMateriaMedicaforassistancewiththe
bioassay.WeareindebtedtoProfessorBing-yangDingofSchool
ofLifeandEnvironmentalSciences, WenzhouNormalColege
·289·YAOGuang-min, etal:PhenolicglucosidesfromtheleavesofPierisjaponica
forcollectingtheplantmaterials.
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