全 文 :Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn 141
Structural determination of two triterpenoid saponins from Gymnocladus
chinensis Baill.
Kan Wang, HongZheng Fu *
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
Abstract: Two triterpenoid saponins (compound I and II) have been isolated from Gymnocladus chinensis Baill., and compound I
was determined as a new compound. The structure of compound I was assigned as 2β,23dihydroxyacacic acid3OαL
arabinopyranosyl21O{(6S)2E2,6dimethyl6O[4O(6S)2E2,6dimethyl6OβDglucopyranosylαLarabinopyranosyl
2,7octadienoyl}28OβDxylopyranosyl(1→3)βDxylopyranosyl(1→4)αLrhamnopyranosyl(1→2)[αLrhamnopyranosyl
(1→6)]βDglucopyranoside by extensive MS and NMR studies.
Keywords: 1D/2D NMR; Triterpenoid saponins; Gymnocladus chinensis Baill.
CLC number: R284.1 Document code: A Article ID: 1003–1057(2009)2–141–05
Received date: 20090318.
Foundation item: National Natural Science Foundation of China (Grant
No. 30772639).
* Corresponding author. Tel.: 861082805212; fax: 861082802724;
email: drhzfu@yahoo.com.cn
1. Introduction
Gymnocladus chinensis Baill. is a rich source of
saponins. Since Takao Konoshima isolated several
saponins from its dry fruits in 1980s, over 10 triter
penoid saponins were reported [1–5] . Furthermore, its
diverse biological activities, such as antitumor and
antiHIDS activities [6] , prompted us to investigate its
chemical constituents. The constituents of nBuOH
extract were isolated by the bioassaydirected fractiona
tion, and we carried out purification of the nBuOH
extract and structural identification of two saponins.
The structures were established by MS, 1D and 2D
NMR experiments, including DQFCOSY, HMQC,
HMBC, TOCSY and NOESY. Compound I is sepa
rated for the first time from Gymnocladus chinensis
Baill..
2. Materials and methods
2.1. General experimental procedures
A Lab Alliance HPLC system consisted of two
Series III pumps and a Model 201 detector was
used. TOFMS spectra were obtained on SHIMA
DZU MALDITOF AXIMA CFR plus, and GCMS
analysis was carried out with FINNIGAN TRACE
instrument.
2.2. Plant materials
The dry fruits of Gymnocladus chinensis Baill.
were purchased from Sichuan Province, where is the
major cultivation area. They were identified by Yu Qiu,
Senior Pharmacist at Chengdu Laimei Pharmaceutical
Co, Ltd. A voucher specimen was deposited at State
Key Laboratory of Natural and Biomimetic Drugs,
Peking University.
2.3. Extraction and isolation
The airdried and powdered fruits (7.5 kg) were
extracted consecutively with 95% EtOH and 50%
EtOH. The EtOH extract was concentrated under
vacuum and the residue was suspended in water, and
extracted successively with EtOAc and nBuOH.
The nBuOH soluble portion was concentrated under
reduced pressure, and the viscous concentrate was
passed through a Diaion HP20 column and succes
sively eluted with 20% MeOH, 50% MeOH, 70%
MeOH, MeOH, and EtOAc. The 70% MeOH eluate
portion was chromatographed on silica gel eluted with
a stepwise gradient mixture of CHCl3–MeOH–H2O
(30:10:1, 10:5:1, 6:4:1) and finally with MeOH, giving
seven fractions. Fraction D was subjected to column
chromatography on ODS silica gel eluted with
MeOH–H2O (7:3), to yield a crude saponin fraction.
Further purification of the fraction by combination
of preparative and semi preparative reverse phase
HPLC successively yielded compound I (12.3 mg)
and compound II (15.2 mg).
K. Wang et al. / Journal of Chinese Pharmaceutical Sciences 18 (2009) 141–145 142
2.4. Acid hydrolysis and determination of the
absolute configuration of the monosaccharide
units
Each solution of Saponin I and II (0.5 mg) in 1 M
HCl (dioxane–H2O, 1:1, 200 mL) was heated at 80 °C
for 10 h under an Ar atmosphere. After dioxane was
removed, each solution was extracted with EtOAc to
obtain the aglycone. The aqueous layer was neutralized
by passing through an ionexchange resin (Amberlite
MB3, Organo, Tokyo, Japan) column and concentrated
under reduced pressure to dryness, to afford a residue
of the sugar fraction. The residue was dissolved in
pyridine (0.1 mL), to which 0.08 M Lcysteine methyl
ester hydrochloride in pyridine (0.15 mL) was added.
The mixture was kept at 60 °C for 1.5 h. After reaction
mixture was dried in vacuo, the residue was trimethyl
silylated with 1trimethylsilylimidazole (0.1 mL) for 2 h.
Each mixture was partitioned between nhexane and
H2O (0.3 mL), and the hexane extract was analyzed
by GCMS under the following conditions: capillary
column, DB1MS (30 m×0.25 mm×0.25 µm), column
temperature, 260 °C; injection temperature, 90 °C;
carrier gas, He. In the acid hydrolyzate of Saponin I
and II, Lrhamnose, Dglucose, Larabinose, and D
xylose were confirmed by comparison with the reten
tion times of the Lrhamnose, Dglucose, Larabinose,
and Dxylose derivatives prepared in a similar way,
which showed retention times of 18.41, 19.62, 17.64
and 18.40 min, respectively [7] .
3. Results and discussion
Compound I was obtained as a white powder and
showed positive LibermannBurchard and Molish
reactions. Compound I gave the quasimolecular ion
at m/z 2034.9 [M+Na + ] and 2050.9 [M+K + ] in the
TOFMS spectrum, corresponding to the molecular
formula C95H150O45.
The 1 H NMR spectrum showed signals for six
methyl groups at δ 1.21, 1.62, 1.17, 1.76, 1.00 and
1.24 (each 3H, s), and a characteristic olefinic proton
at 5.64 (brs). The 13 C NMR spectrum revealed a pair
of characteristic olefinic carbons at δ 143.3, 123.6
and a carbonyl carbon at 174.7. The assignments of
other proton and carbon signals of the aglycone
were accomplished by analysis of the DQFCOSY,
NOESY, HSQC, HMBC and DEPT experiments.
The 1 H and 13 C NMR values were in full agreement
with those reported in the literature for acacia acid [1–5]
(Fig. 2, Table 1).
In addition, the 1 H NMR spectrum revealed signals
for four methyl groups at δ 1.89, 1.53, 1.86, 1.50
(each 3H, s), and the 13 C NMR spectrum showed
four pairs of characteristic olefinic carbons at δ 143.4
and 128.6, 143.9 and 115.0, 142.2 and 128.0, 143.6
and 111.6 , as well as two carbonyl carbons at 167.9,
168.1. All the results indicated that there were two
monoterpene moieties, by the comparison of NMR
data with those reported in literature, and the assign
ments of other proton and carbon signals were also
accomplished by means of 2D NMR (Fig. 2, Table 1).
The 1 H and 13 C NMR spectra showed eight sugar
anomeric protons at δ 5.95 (1H, d, J 8.0 Hz, glu1H),
4.79 (1H, d, J 7.2 Hz, ara1H), 5.67 (1H, brs, rha1H),
5.28 (1H, brs, rha 1H), 5. 77 (1H, brs, rha1H), 5.36
(1H, d, J 6.6 Hz, xyl1H), 5.56 (1H, d, J 7.8 Hz,
xyl1H), 5.15 (1H, d, J 5.4 Hz, xyl1H). On the basis
of the combined analysis of HMQC, the carbons
were revealed at δ 95.3, 100.0, 101.71, 101.73, 104.0,
104.3, 105.1 and 105.9. Starting from the anomeric
protons of each sugar unit, all the hydrogens within
each spin system were assigned using DQFCOSY
with the aid of TOCSY and NOESY experiments,
while the carbons were assigned by HMQC and further
confirmed by HMBC experiments. The monosaccha
rides were identified as two glucopyranoses, two
xylopyranoses, three rhamnopyranoses and one
arabinopyranose (Table 1).
These data also indicated αconfiguration at the
anomeric position of the rhamnose and arabinose, β
configuration at the anomeric position of the glucose
and xylose. The absolute configuration of the sugar
residues obtained by acidic hydrolysis was determined
as Lrhamnose, Dglucose, Larabinose, and Dxylose
through GCMS analysis and comparison with authentic
sugar samples.
Direct evidence for the sugar sequence and their
linkage sites at aglycone C3 was derived from the
HMBC experiment, which showed unequivocal corre
lation between resonances at δ 5.77 (1H, brs, rhaC H1)
and 82.6 (aglycone C3). Similarly, the sequence of
the sugar unit at aglycone C28 was indicated by the
crosspeaks between δ 5.95 (1H, d, J 8.0 Hz, gluA H1)
and 174.7 (aglycone C28), δ 5.67 (1H, brs, rhaA H1)
and 77.4 (gluA C2), δ 5.28 (1H, brs, rhaB H1) and
66.9 (gluA C6), δ 5.15 (1H, d, J 7.6 Hz, xylA H1)
and 82.6 (rhaA C4), δ 5.36 (1H, d, J 6.6 Hz, xylB H1)
and 77.5 (xylA C3). The sequence of the sugar unit at
C21 was indicated by the crosspeaks between δ 5.56
(1H, d, J 7.8 Hz, gluB H1) and 78.6 (monoterpene
(araO) C6), 4.79 (1H, d, J 7.2 Hz, araA H1) and
79.9 (monoterpene (21O) C6), 6.24 (dd, J 9.0, 7.0 Hz,
aglycone H21) and 167.9 (monoterpene (21O) C1)
(Fig. 1, Table 1) .
K. Wang et al. / Journal of Chinese Pharmaceutical Sciences 18 (2009) 141–145 143
On the basis of these evidences, the compound was
identified as 2β,23dihydroxyacacic acid3OαL
arabinopyranosyl21O{(6S)2E2,6dimethyl6O
[4O(6S)2E2,6dimethyl6OβDglucopyranosyl
αLarabinopyranosyl2,7octadienoyl}28OβD
xylopyranosyl(1→3)βDxylopyranosyl (1→4)αL
rhamnopyranosyl(1→2)[αLrhamnopyranosyl(1→6)]
βDglucopyranoside. This is the first time that the
13 C NMR data and complete assignments for 1 H NMR
of compound I were provided.
Compound II was obtained as a white powder and
showed positive LibermannBurchard and Molish
reactions. Compound II gave the quasimolecular ion
at m/z 1951.8 [M+Na + ] and 1967.8 [M+K + ] in the
TOFMS spectrum, corresponding to the molecular
formula C90H144O44.
The NMR spectrum of compound II showed a
close resemblance to that of compound I, except for
the oligosaccharide chain at aglycone C21. 1 H NMR
spectrum revealed signals for two methyl groups at
δ1.88, 1.49 (each 3H,s), and the 13 C NMR spectrum
showed two pairs of characteristic olefinic carbons
at δ 142.2 and 128.6, 143.8 and 115.2, as well as a
carbonyl carbons at 167.8. All the results indicated
that there were monoterpene moieties (Fig. 2, Table 2).
The signals of two methyl groups at δ 0.81 (t, J
7.2 Hz) and 1.17 (d, J 7.2 Hz) in the 1 H NMR spectrum
indicated the existence of a 2methylbutyroyl group,
and the absolute configuration of the 2methylbutyroyl
group was determined after the assignments of other
proton and carbon signals by analysis of 2D NMR
spectra (Fig. 2, Table 2).
13 C 1 H 13 C 1 H
Aglycone 28O GluA
1 44.6 2.38 (dd, J 9.6,7.0 Hz), 1 95.3 5.95 (1H, d, J 8.0 Hz)
1.30 (br d, J 13.5 Hz) 2 77.4 4.21
2 70.5 4.10 3 78.0 4.05
3 82.6 4.12 4 70.9 3.94
4 42.2 — 5 76.7 4.22
5 47.4 1.91 6 66.8 3.49, 4.20
6 19.0 1.68, 2.10 6O GluB
7 33.2 1.71, 1.76 1 104.3 5.56 (1H, d, J 7.8 Hz)
8 40.3 — 2 75.3 4.01
9 47.7 1.44 3 78.0 3.92
10 37.2 — 4 71.7 4.04
11 24.1 2.28, 2.14 5 78.1 3.9
12 123.6 5.64 6 62.6 4.15, 3.45
13 143.3 — 1→2 RhaA
14 42.2 — 1 101.7 5.28 (1H, brs)
15 36.0 2.27, 1.70 2 70.1 4.40
16 73.6 5.16 3 82.6 4.42
17 51.63 — 4 73.8 4.28
18 40.8 3.47 5 69.5 4.20
19 47.8 1.45, 2.96 6 18.8 1.59 (3H, d, J 6.5 Hz)
2.70 (t, J 10.0 Hz) 1→6 RhaB
20 35.4 — 1 101.7 5.14 (1H, brs)
21 76.7 6.24 2 71.8 4.24
22 36.4 2.21, 2.566 3 72.0 4.18
23 66.3 3.76, 4.23 4 73.6 3.94
24 14.9 1.24 s 5 77.6 4.26
25 17.7 1.68 s 6 18.9 1.48 (3H, d, J 5.7 Hz)
26 17.6 1.21 s 3O RhaC
27 27.3 1.78 s 1 104.0 5.77 (1H, brs)
28 174.7 — 2 71.8 4.70
29 29.2 1.01 s 3 72.3 4.58
30 19.2 1.17 s 4 73.6 4.29
Monoterpene moiety (21O) 5 69.2 4.44
1 167.9 — 6 17.5 1.69 (3H, d, J 6.4 Hz)
2 128.6 — 1→4 XylA
3 143.4 7.12 (t, J 10.0 Hz) 1 105.9 5.15 (1H, d, J 7.6 Hz)
4 23.9 2.17, 2.40 2 75.2 3.99
5 40.8 1.44, 1.53 3 77.5 4.12
6 79.9 — 4 69.0 4.06
7 143.9 6.23 5 67.0 4.15, 3.43
8 115 5.37, 5.20 1→3 XylB
9 12.7 1.89 s 1 105.1 5.36 (1H, d, J 6.6 Hz)
10 23.7 1.53 s 2 74.7 3.90
Monoterpene moiety (araO) 3 88.0 4.06
1 168.1 — 4 69.3 4.12
2 128.0 — 5 67.1 4.15, 3.43
3 142.2 6.93 (t, J 10.0 Hz) 6OAraA
4 23.7 2.26, 2.20 1 100.0 4.79 (1H, d, J 7.2 Hz)
5 40.6 1.80, 1.69 2 72.3 4.40
6 78.6 — 3 73.6 4.16
7 143.6 6.06 4 69.3 4.07
8 111.6 5.15, 5.50 5 66.3 4.19, 3.43
9 12.5 1.86 s
10 23.6 1.50 s
Table 1. 13 C NMR and 1 H NMR date of compound I (pyridined5)
Figure 1. Compound I isolated from Gymnocladus chinensis Baill..
CH 2 OH
C
HO
O
O
OH
O
O
O
OH
OH
O
C
O
O
O
OH OH
O
CH 3 O
OH
O
OH
O
O
OH
OH
O
O
OH
OH
OH
O
OH OH
OH
CH 3
O
OH OH
OH
CH 3
C
O
O
O
OH
OH
HO
OH
H
H
H
H
H
H
H
H
H
H
GluA
GluB
RhaA
RhaB
RhaC
AraA
XylA
XylB
Figure 2.HMBC and DQFCOSY correlations of aglycone, monoterpene
and 2methylbutyroyl group of compounds I and II.
CH 2 OH
C HO
OR 2
OH
O
OR 3
C
O
H
C
CH 3
CH 2 CH 3 3
5
1 2 4
C
O
O
1
2
4
6
9
10
3
7
8
5
HMBC DQF—COSY
CH 2 OH
C
HO
O
O
OH
O
O
O
OH
OH
O
C
O
O
O
OH OH
O
CH 3
O
OH
O
OH
O
O
OH
OH
HO
OH
O
OH
O
O
O
OH
OH
OH
O
OH OH
OH
CH 3
O
OH OH
OH
CH 3
O
H
H
H
H
H
H
H
H
H
144 K. Wang et al. / Journal of Chinese Pharmaceutical Sciences 18 (2009) 141–145
The sugar sequence and their linkage sites at C21
was indicated by the crosspeaks between δ 5.27 (1H,
d, J 7.6 Hz, gluB H1) and 81.3 (monoterpene (araO)
C6), 4.77 (1H, d, J 7.3 Hz, araA H1) and 79.9
(monoterpene (21O) C6), 167.8 (monoterpene (21O)
C1) and 6.22 (dd, J 10.0,5.5 Hz, aglycon H21),
4.21(1H, araA H4) and 176.7 (2methylbutyroyl C1).
By comparing with the values for the corresponding
signals of 1D and 2D NMR spectra reported in the
literature, it appeared that compound II was a known
saponin as 2β,23dihydroxyacacic acid3OαL
arabinopyranosyl21O{(6S)2E2,6dimethyl6O
[3OβDglucopyranosyl(4O(2methylbutyroyl))
αLarabinopyranosyl]2,7octadienoyl}28OβD
xylopyranosyl(1→3)βDxylopyranosyl(1→4)αL
rhamnopyranosyl(1→2)[αLrhamnopyranosyl(1→6)]
βDglucopyranoside.
Acknowledgements
This project was supported by the National Nature
Science Foundation of China (Grant No. 30772639).
References
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Chem. Pharm. Bull. 1984, 32, 2617.
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Chem. Pharm. Bull. 1984, 32, 4833–4841.
13 C 1 H 13 C 1 H
Aglycone 28O GluA
1 44.8 2.36 (dd, J 13.5, 6.5 Hz), 1 95.1 6.06 (1H, d, J 7.6 Hz)
1.34 (br d, J 13.5 Hz) 2 78.9 4.21
2 70.9 4.18 3 77.9 4.05
3 84.4 4.35 4 71.3 3.94
4 42.3 — 5 76.6 4.22
5 47.6 1.8 6 66.9 3.49, 4.20
6 18.3 1.86, 2.05 1→3 GluB
7 33.2 1.60, 1.74 1 106.2 5.27 (1H, d, J 7.6 Hz)
8 40.4 — 2 75.2 4.04
9 47.9 1.84 3 78 4.00
10 37.2 — 4 70.8 4.06
11 24.1 2.40, 2.20 5 78.4 4.36
12 123.7 5.65 (br, s) 6 63.1 4.48, 4.23
13 143.4 — 1→2 RhaA
14 42.3 — 1 101.6 6.25 (1H, brs)
15 35.4 2.27, 2.96 2 71.6 4.82
16 73.5 5.21 3 72.5 4.69
17 51.8 — 4 84.4 4.39
18 40.9 3.54 5 68.5 4.47
19 48.0 1.46 (dd, J 10.5, 6.0 Hz) 6 18.7 1.71 (3H, d, J 6.0 Hz)
2.94 (t, J 10.5 Hz) 1→6 RhaB
20 35.4 — 1 102.1 5.36 (1H, brs)
21 77.0 6.22 (dd, J 10.0, 5.5 Hz) 2 72.1 4.60
22 36.5 2.24, 2.75 3 72.5 4.37
23 64.2 3.64, 4.08 4 73.9 4.29
24 14.8 1.39 s 5 69.5 4.58
25 17.7 1.57 s 6 18.6 1.60 (3H, d, J 6.5 Hz)
26 17.5 1.14 s 3O RhaC
27 27.2 1.74 s 1 104.2 5.75 (1H, brs)
28 174.8 — 2 72.5 4.69
29 29.2 0.95 s 3 72.8 4.58
30 19.2 1.19 s 4 73.9 4.29
Monoterpene moiety (21O) 5 70.4 4.19
1 167.8 — 6 17.7 1.62 (3H, d, J 6.4 Hz)
2 128.6 — 1→4 XylA
3 142.2 6.94 (t, J 10.0 Hz) 1 106.5 5.16 (1H, d, J 7.6 Hz)
4 23.7 2.21, 2.26 2 75.1 4.11
5 41.2 1.72, 1.85 3 87.7 4.01
6 79.9 — 4 68.9 4.06
7 143.8 6.24 (d, J 7.5 Hz) 5 67.4 3.65, 4.29
8 115.2 5.24, 5.39 1→3 XylB
9 12.7 1.88 s 1 106.1 5.18 (1H, d, J 7.6 Hz)
10 23.6 1.49 s 2 75.2 4.02
3 78.0 4.04
1 176.7 — 4 70.8 4.11
2 5 67.4 4.29, 3.65
3 27.0 1.43, 1.75 6O AraA
4 11.5 0.81 (t, J 7.2 Hz) 1 99.9 4.77 (1H, d, J 7.3Hz)
5 16.6 1.17 (d, J 7.2 Hz) 2 71.8 4.39
3 81.3 4.29
4 69.6 4.21
5 64.3 4.09, 3.67
2Methylbutyroyl group
41.3 2.39 m
Table 2. 13 C NMR and 1 H NMR date of compound II (pyridined5)
Figure 3. Compound II isolated from Gymnocladus chinensis Baill..
K. Wang et al. / Journal of Chinese Pharmaceutical Sciences 18 (2009) 141–145 145
[3] Takao, Konoshima; Tokunosuke, Sawada; Takeatsu, Kimura.
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Prod. 2006, 69, 1577–1581.
肥皂荚Gymnocladus chinensis Baill.中两个三萜皂苷的结构鉴定
王刊, 付宏征 *
北京大学 天然药物及仿生药物国家重点实验室, 北京 100191
摘要: 从肥皂荚Gymnocladus chinensis Baill. 提取出两个三萜皂苷类化合物 (化合物I与II) , 其中化合物I为新化合物,
通过核磁方法, 鉴定其为2β,23dihydroxyacacic acid3OαLarabinopyranosyl21O{(6S)2E2,6dimethyl6O[4O(6S)2E
2,6dimethyl6OβDglucopyranosylαLarabinopyranosyl2,7octadienoyl}28OβDxylopyranosyl(1→3)βDxylopyranosyl(1→4)
αLrhamnopyranosyl(1→2)[αLrhamnopyranosyl(1→6)]βDglucopyranoside。
关键词:一维/二维核磁共振波谱技术;三萜皂苷;肥皂荚