全 文 ：波 谱 学 杂 志第 25卷第 2期
2008年 6月　 Chinese Journal of M agnetic Resonance
Vo l.25 No.2
　 Jun.2008
Article:1000-4556(2008)02-0281-06
Structure Determination of a Triterpene Saponin Extracted
from the Barks of Pseudolarix kaempf eri by 2D NMR
YUE Zheng-gang 1 , 2 , X IAO K ai 2＊ , WU Li-jun1＊ , LI N Hong-wei 2 ,
FAN Jun-wen2 , Z HAO J ie2 , L I Y ue2 , Z HANG Li-ming 2
(1.Sch ool of Tradi ti onal Chinese M ateria Medica , Sh engyang Ph armaceut ical University , Shenyang 110016 , China;
2.Lab of T oxin & Ph armacy , Faculty of Naval Medicine , Second Milit ary Medical University ,
Shanghai 200433 , China)
Absract::A triterpene saponin w as ex tracted f rom the barks o f Pseudori x kaemp feri fo r the
fir st time , and determined to be dammar-24 (25)-ene-3β , 6α, 12β , 20 (S)-tet raol-20-O-β-D-
py ranog lucoside on the basis o f NM R analy sis.The chemical shif ts of this compound were com-
ple tely a ssigned using 2D NM R spectro scopy.
Key words:NM R , chemical shift , triterpene saponin , py ranog lucoside , Pseudorix kaemp feri
Gord
CLC number:O641　　Document code:A
Introduction
The roo t and trunk barks of Pseudorix kaemp feri Gord .(Pinacese), know n as
`Tu-Jin-Pi in t radit ional Chinese medicine , has been widely used in China for treatment
of skin diseases caused by fungal infections.Previously iso lated classes of constituents
were dite rpenes[ 1 ～ 5] and tri terpenoids[ 6 ～ 17] .As a par t of our chemical studies on the a-
queous part o f the Pseusorix kaemp f eri , we have investig ated the t ri terpene saponin in
barks of this plant collected in Jiang su Province , China.The present paper describes the
Received date:Mar.30 , 2007;Revised date:Sep.24 , 2007
Foundation i tem:This wo rk w as finan cially supported by g rants f rom the Nation al Scien tif ic Foundation of China
(30472141)and Shanghai-SK Developm ent Fund (2004005-t).
Biography:Yue Zheng-gang(1981-), Male , Shandong Rizhao , M as ter.Majored in pharmaceu tical.　＊C orrespon ding
au th or:Xiao Kai , Tel:021-25070355 , E-mai l:kaixiaocn@gmail.com , Wu Li-jun , T el:024-23986481 ,
E-mail:w uli jun 111@hotmai l.com.
i solat ion and st ructure elucidat ion of a saponin named dammar-24 (25)-ene-3β , 6α, 12β ,
20(S)-tet raol-20-O-β-D-pyranoglucoside (compound 1)by 2D NMR spect ral method .
This compound w as isolated f rom Pseudorix genus for the f irst time.
1　Experimental
Plant material:the plant w as collected in Jiang su Province of China.The specimen
w as bo tanically identi fied by vice research fellow Shen Jingui(Shanghai Insti tute of M a-
teria M edica , China).
1.1　Instruments
1
H NMR (500 MHz , MeOH-d4)and 13C NMR (125 MHz , MeOH-d4)spectra
were recorded on a Bruke r A RX-500 spect rometer wi th TM S as an internal standard and
2D NMR (500 MHz , MeOH-d4)was recorded at standard conditions.ESI-MASS spec-
t rum w as recorded on a Q-Tof-micro mass spect rometer.
1.2　Extraction and isolation
The dried ro ot bark (10 kg)of P.kaemp feri was ex tracted w ith EtOH (70 %)un-
der ref lux.After removal o f the ethano l in vacuum , the aqueous so lution was f iltered .
The fi lt rate w as then chromatog raphied sequent ially over HP-20 macroporous resin co l-
umn eluting g radient ly wi th C2H 5OH-H 2O , MCI gel column , HW-40F gel co lumn and
ODS gel co lumn elut ing w ith CH3OH-H 2O to affo rd compound 1.
2　Results and Discussion
Compound 1 was obtained as an amo rphous powder and gave po sitive reaction to the
Liebe rmann-Burchard and M olish tests fo r t riterpene saponin.The mo lecular formula
C36H6 2O9 was determined on the basis of ESI (m/z 661.4[ M +Na] +)to gether wi th 1H
and
13
C NMR spect ral data(Table 1).A quick inspection o f the 1H and 13C NM R spec-
t ra of the compound readily indicated the presence of a mono saccharide uni t through eas-
ily identif iable signals of anomeric pro ton and carbon.The 13 C and DEPT NMR spect ral
data revealed the presence o f the remaining 30 signals , which is indicative o f a t rite rpene
moiety .
The ful l assignments o f proton and carbon signals w ere based on analysis of 1D
NMR and 2D NM R spect ra (Table 1).The 1H NMR spectrum of compound 1 show ed
eight methy l singlets at δH 0 .95 , 0.95 , 0.95 , 1.08 , 1.28 , 1 .34 , 1 .61 and 1.67 , to-
gether w ith one olefinic proton at δH 5 .10 (1H , br.s).In addi tion , the 13C NMR and
DEPT spect ra show ed eight methyl signals , tw o olefinic carbon signals at δC 126.1 (C-
24)and 132.6 (C-25).In 1H-1H COSY spect rum , one olenf inic proton at δH 5.10 (H-
24)was cor related wi th tw o methy l signals at δH 1.61(3H , s , H-26)and 1 .67 (3H , s ,
H-27)and a methane proton at δH 2.06 (2H , m , H-23), which show ed co rrelation
(1H-1H COSY)w ith δH 1.60 (1H , m , H-22a)and 1 .79 (1H , m , H-22b).In HMBC
282 波　　谱　　学　　杂　　志　　　　　　　　　第 25卷　
spect rum , the olenfinic pro ton at δH 5.10 (H-24)was co rrelated w ith δC 36.9 (C-22),
24.5 (C-23), 18 .2(C-26)and 26 .2 (C-27).Moreover , in the HMBC spect rum a me th-
y l pro ton signal at δH 1 .34 (3H , s , H-21)showed long-range co rrelation wi th the sig-
nals at δC 53.4 (C-17), 85.2(C-20)and 36.9 (C-22).Thus , the partial st ructure of the
side chain in compound 1 could be elucidated as in Chart 1.Other partial st ructures of 1
were elucidated f rom the HMBC spectrum , including a geminal dimethy l g roups (C29-
C4-C30)and three methy l g roups at tached to three quaternary carbons (C19-C10 , C18-C8 ,
C28-C14).The whole structure of the ag lycone w as deduced by 1H-1H COSY and HMBC
and the detailed interrelations be tw een 1H-1H and 1H-13C are show n in Chart 2 and Ta-
ble 1.Apparently , acco rding to it s NMR spectral characteristics , the skeleton of com-
pound 1 i s a dammar-ty pe t riterpene.The detailed analy sis of the inter relations be tw een
1H and 13C fo r the triterpene al low ed the signal assignments .The evidence above to-
gether wi th the comparison of chemical shif t s[ 18] , enabled the identification of the t ri ter-
pene ag lycone in compound 1 as dammar-24 (25)-ene-3β , 6α,12β , 20 (S)-te traol.
The anomeric pro ton (δH 4.60)and an anomeric carbon signal (δC 98.6)indicated
the presence of a monosaccharide unit in compound 1 and the 13C chemical shif ts at δC
98.6 , 75 .7 , 78.5 , 71.5 , 78.2 and 62 .8 indicated that i t is a py ranogluco side[ 19] .H-1 ,
H-2 vicinal coupling constant betw een 7 and 8 Hz show ed this sugar occurred in com-
pound 1 as the β-anome r in C1 configurat ion .The sugar linkage w as determined on the
basis o f HMBC experiment.A cro ss peak of 1H-13C long-range correlation w as observed
between the proton signal at δH 4.60 (H-1′)and the carbon signal at δC 85.2(C-20), in-
dicating that the sugar linked a t C-20 .Therefo re , compound 1 , i solated f rom Pseudola-
rix genus fo r the fir st t ime , was dete rm ined to be dammar-24 (25)-ene-3β , 6α, 12β , 20
(S)- tet raol-20-O-β-D-pyranoglucoside.
C hart 1.The piece A of com pound 1　　　　C hart 2.The st ructure and key HMBC of compound 1　　
283　第 2期　YUE Zheng-gang et al:S t ru cture Determination of a Triterpene S aponin Ex t ractedf rom the Bark s of Pseudolarix kaem p f eri by 2D NM R
Table 1　NMR data of compound 1(in MeOH-d4)
Posit ion
HM QC
δC δH
COSY HMBC
1 40.4(t) a 1.04 (1H , m) H-1b , H-2 C-2 , C-3 , C-5 , C-9 , C-10 , C-19
b 1.70 (1H , m) H-1a , H-2
2 28.0(t) 1.65 (2H , m) H-1a , H-1b , H-3 /
3 79.8(d) 3.14 (1H , m) H-2 C-2 , C-4 , C-29 , C-30
4 40.8(s)
5 62.4(d) 0.90(1H , d , J=10.6) H-6 C-1 , C-3 , C-4 , C-6 , C-7 , C-9 ,
C-10
6 69.2(d) 4.02(1H , J 1=3.5 , J 2=10.6) H-5 , H-7a , H-7b C-5 , C-7 , C-10
7 47.5(t) a 1.55 (1H , m) H-6 , H-7b C-5 , C-6 , C-8 , C-9 , C-14
b 1.64 (1H , m) H-6 , H-7a
8 42.3(s)
9 50.8(d) 1.47 (1H , dd) H-11a , H-11b C-5 , C-8 , C-10 , C-11 , C-12 ,
C-14 , C-18 , C-19
10 40.4(s)
11 31.2(t) a 1.18 (1H , m) H-9 , H-11b , H-12 C-8 , C-9 , C-10 , C-12 , C-13
b 1.86 (1H , m) H-9 , H-11a , H-12
12V 72.1(d) 3.67 (1H , m) H-11a , H-11b , H-13 C-11 , C-17 , C-28
13 49.7(d) 1.74 (1H , m) H-12 , H-17 C-12 , C-14 , C-17, C-20
14 51.6(s)
15 31.4(t) a 1.06 (1H , m) H-15b , H-16a , H-16b C-14 , C-28
b 1.60 (1H , m) H-15a , H-16a , H-16b
16 27.5(t) a 1.40 (1H , m) H-15a , H-15a , H-16b C-15 , C-17
b 1.95 (1H , m) H-15a , H-15b , H-16a
17 53.4(d) 2.28 (1H , m) H-13 , H-16a , H-16b C-12 , C-13 , C-20, C-22
18 18.0(q) 1.08 (3H , s) C-7 , C-8 , C-9 , C-14
19 18.0(q) 0.95 (3H , s) C-1 , C-9 , C-10
20 85.2(s)
21 23.1(q) 1.34 (3H , s) C-17 , C-20 , C-22
22 36.9(t) a 1.60 (1H , m) H-22b , H-23 C-17 , C-20 , C-21, C-23 , C-24
b 1.79 (1H , m) H-22a , H-23
23 24.5(t) 2.06 (2H , m) H-22a , H-22b C-22 , C-24 , C-25
24 126.1(d) 5.10(1H , brs) H-23 , H-26 , H-27 C-22 , C-23 , C-26, C-27
25 132.6(s)
26 18.2(q) 1.61 (3H , s) C-24 , C-25 , C-27
27 26.2(q) 1.67 (3H , s) C-24 , C-25 , C-26
284 波　　谱　　学　　杂　　志　　　　　　　　　第 25卷　
　　续表 1
　　Continuation of the Table 1
Posit ion
HM QC
δC δH COSY HMBC
28 17.6 (q) 0.95 (3H , s) C-8 , C-13 , C-14 , C-15
29 31.8 (q) 0.95 (3H , s) C-3 , C-4 , C-5 , C-30
30 16.4 (q) 1.28 (3H , s) C-3 , C-4 , C-5 , C-29
glcA-1′ 98.6 (d) 4.60 (1H , d , J =7.7) H-2′ C-20 , C-5′
2′ 75.7 (d) 3.08 (1H , m) H-1′, H-3′ C-1′, C-3′, C-4′
3′ 78.5 (d) 3.35 (1H , m) H-2′, H-4′ C-1′, C-2′, C-4′
4′ 71.5 (d) 3.30 (1H , m) H-3′, H-5′ C-2′, C-3′, C-5′, C-6′
5′ 78.2 (d) 3.20 (1H , m) H-4′, H-6a′, H-6b′ C-1′, C-3′, C-4′, C-6′
6′ 62.8 (t) a 3.63 (1H , m) H-5′, H-6b′ C-4′, C-5′
b 3.78 (1H , m) H-5′, H-6a′
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2D NMR对金钱松皮中的一个三萜皂苷进行结构解析
岳正刚1 , 2 , 肖　凯2＊ , 吴立军1＊ , 蔺红伟2 , 樊军文2 , 赵　杰2 , 李　玥2 , 张黎明2
(1.沈阳药科大学 中药学院 , 辽宁 沈阳 110016;2.第二军医大学海医系毒物药物研究室 , 上海 200433 )
摘要:通过核磁共振谱并结合文献对一个三萜皂苷 dammar-24 (25)-ene-3β , 6α, 12β , 20
(S)-tet rao l-20-O-β-D- py ranogluco side 进行结构解析.通过 2D NMR (1H-1H COSY 、
HMQC 、HMBC)对其进行了 NMR全归属.
关键词:NMR;全归属;2D NMR;三萜皂苷;dammar-24 (25)-ene-3β , 6α, 12β , 20
(S)-tet rao l-20-O-β-D- pyranoglucoside;金钱松
基金项目:国家自然科学基金资助项目(30472141)和上海-SK 研究与发展基金(2004005-t)资助.
＊通讯联系人:肖凯 , 电话:021-25070355 , E-mail:k aixiaocn @gmail.com , 吴立军 , 电话:024-23986481 , E-mai l:
w u lijun 111@hotmail.com .
286 波　　谱　　学　　杂　　志　　　　　　　　　第 25卷