New triterpenoid saponins from the roots of Gypsophila pacifica Kom.

Article abstract of DOI:10.1016/j.carres.2009.08.015

Six new triterpenoid saponins (1-6) have been isolated from the roots of Gypsophila pacifica Kom. Their structures were established on the basis of extensive NMR (¹H, ¹³C, TOCSY, HSQC, and HMBC) and ESIMS studies.

Full text of DOI:10.1016/j.carres.2009.08.015

Carbohydrate Research 345 (2010) 68–73
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
New triterpenoid saponins from the roots of Gypsophila pacifica Kom.
*
Wei Nie, Jian-Guang Luo, Ling-Yi Kong
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2009
Received in revised form 7 August 2009
Accepted 13 August 2009
Available online 19 August 2009
Six new triterpenoid saponins (1–6) have been isolated from the roots of Gypsophila pacifica Kom. Their
structures were established on the basis of extensive NMR (1H, 13C, TOCSY, HSQC, and HMBC) and ESIMS
studies.
Ó 2009 Published by Elsevier Ltd.
Keywords:
Gypsophila pacifica Kom.
Caryophyllaceae
Triterpenoid saponins
1. Introduction
Compound 1 was obtained as a white, amorphous powder. It
was assigned the molecular formula C74H116O39 from its nega-
The genus Gypsophila, a genus of the family Caryophyllaceae,
comprises more than 150 species distributed throughout the
world. Some of these species have long been used as pharmaceuti-
cal and ornamental plants.1 Gypsophila pacifica Kom. is a small
perennial herb widely distributed in the northeast regions of
China. Its roots have been used as a substitute for the traditional
Chinese medicine Yin-Chai-Hu (roots of Stellaria dichotoma var.
Lanceolata Bge) to treat fever, consumptive disease, and infantile
malnutrition syndrome.2 A series of triterpenoid saponins have
been reported from this plant during the 1960s.3–5 Some triterpe-
noid saponins have been reported in our previous saponins inves-
tigation from this genus.6 In our continuing search for triterpenoid
saponins constituents, six new oleanane-type triterpenoid sapo-
nins were isolated from the roots of G. pacifica. The structural elu-
cidation of these triterpenoid saponins was based on chemical
methods and spectroscopic techniques.
tive-ion (HRESIMS m/z 1627.7022 [MꢀH]ꢀ). On acid hydrolysis
with 2 M HCl, 1 afforded the sugars and aglycone. The sugars were
identified as
D-glucuronic acid,
D-galactose, D-xylose, D-fucose,
L-rhamnose, and
L-arabinose in the ratio of 1:1:2:1:1:2 based on
GC–MS analysis of their chiral derivatives. And the aglycone was
identified as gypsogenin by co-TLC comparison with standard sam-
ple, which was also confirmed on the basis of the 1H and 13C NMR
(Table 1) referring to the reported data.7 The downfield 13C NMR
chemical shift at dC 84.6 and the upfield 13C NMR chemical shift
at dC 176.3 suggested that 1 was a bidesmosidic saponin with gly-
cosidic linkages at C-3 through an ether bond and at C-28 through
an ester bond.
The anomeric proton signals at dH 6.45 (s), 6.01 (d, J = 8.2 Hz),
5.56 (d, J = 7.7 Hz), 5.34 (d, J = 7.7 Hz), 5.17 (d, J = 7.2 Hz), 5.04 (d,
J = 7.0 Hz), 4.98 (d, J = 7.8 Hz), and 4.82 (d, J = 7.5 Hz) displayed
the correlations with anomeric carbon signals at dC 101.1, 94.5,
104.0, 104.8, 105.0, 106.8, 106.7, and 103.8 in HSQC spectrum,
respectively, which showed that 1 contained eight sugar units.
The 1H and 13C NMR data (Table 2) of the monosaccharide resi-
dues were assigned starting from the readily identifiable anomer-
ic protons by means of the TOCSY, HSQC, and HMBC spectra
obtained for this compound. The b-anomeric configurations for
2. Results and discussion
The 70% EtOH extract of the roots of G. pacifica was partitioned
by water, EtOAc, and n-BuOH. The n-BuOH and H2O soluble frac-
tions were subjected to chromatographic purification over a silica
gel column and repeated RP-C18 column, followed by prep-HPLC
purification, respectively, to finally afford six new triterpenoid sap-
onins (Chart 1).
the
and the
mined by their large JH1,H2 coupling constants at 7–8 Hz. And
D
-glucuronic acid,
D-fucose,
D
-galactose and
D-xylose units,
a
-anomeric configurations for
L-arabinose were deter-
3
the
a-anomeric configuration of L-rhamanose was judged by its
C-5 (dC 67.8).6 The sequence of the sugar residues was subse-
quently determined by HMBC experiments. The linkage of the
sugar units at C-3 of the aglycone was established from the fol-
lowing HMBCs: H-1 of galactose (dH 5.56) with C-2 of glucuronic
* Corresponding author. Tel.: +86 25 8539 1232; fax: +86 25 8530 1528.
E-mail address: cpu_lykong@126.com (L.-Y. Kong).
0008-6215/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.carres.2009.08.015

W. Nie et al. / Carbohydrate Research 345 (2010) 68–73
69
O
C
O
C
O
GlcA
Fuc
HO
HOOC
O
O
R1O
HO
R2O
Glc I
O
O
CHO
Xyl
O
O
R2O
COOH
O
HO
HO
HO
OR1
Rha
O
O
HO
Glc III
OH
O
O
O
OH
HO
R3O
HO
HO
HO
O
HO
OH
Glc II
OH
OH
R1
R2
β -D-Gl c (IV)
β -D-Gl c α -D-Gal
R1
β -D-Gal
H
R2
β - D-Xyl
β -D-Gal
R3
3
4
H
1
2
α -L-Ara'-(1 4)- α - L-Ara
β -D- Xyl
O
OH
R1
R3O
O
O
HO
O
O
Fuc
R2O
CHO
O
HO
OH
OH
O
Gal
HO
O
Rha
R4O
O
OH
R5O
OH
R1
COOH
CH2OH
R2
β - D-Xyl
α -L-Ara
R3
R4
β -D-Glc
α -L-Ara- (1 3)-β -D-Xyl
R5
5
6
α -L- Ara
β -D-Gl c'
α -L-Rha'
β -D-Gl c
Chart 1. Structures of triterpenoid saponins 1–6.
Table 1
13C NMR (150 MHz, C5D5N) data for aglycone moieties of compounds 1–6
with C-3 of xylose (dC 86.3), H-1 of xylose (dH 4.98) with C-4 of
rhamnose (dC 85.4), H-1 of rhamnose (dH 6.45) with C-2 of fucose
(dC 73.7), and H-1 of fucose (dH 6.01) with C-28 of the aglycone
(dC 176.3) (Fig. 1). On the basis of the data obtained, the structure
Carbon
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
37.9
25.0
84.6
54.9
48.3
20.4
32.4
39.9
47.6
36.0
23.0
37.8
25.0
84.5
54.9
47.5
20.5
32.1
39.9
47.0
36.0
23.0
38.7
23.7
75.2
53.3
52.0
23.2
32.8
40.1
48.2
36.6
23.7
38.5
23.6
84.8
53.2
51.9
22.8
32.6
40.0
48.0
36.5
23.1
38.3
25.5
84.6
55.3
49.0
20.7
30.9
40.5
49.4
36.4
23.1
37.9
24.9
84.6
55.1
48.1
20.5
31.6
40.1
49.1
36.0
23.5
of 1 was established as 3-O-b-
xylopyranosyl-(1?3)]-b- -glucuronopyranosyl gypsogenin 28-O-
-arabinopyranosyl-(1?4)- arabinopyranosyl-(1?3)-b-
-rhamnopyranosyl-(1?2)-b- -fucopyr-
D-galactopyranosyl-(1?2)-[b-D-
D
a-
L
a
-
L
D-
xylopyranosyl-(1?4)-
a
-
L
D
anosyl ester.
Compound 2, a white amorphous powder, exhibited in HRE-
SIMS (m/z 1387.6192 [M+Na]+), which was consistent with the
molecular formula of C64H100O31. Acid hydrolysis afforded gypsog-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
enin and the monosaccharide components were identified as
D-
122.2
143.9
42.0
28.3
23.5
46.8
41.8
46.1
30.5
33.5
32.1
210.1
10.8
15.5
17.2
25.7
176.3
32.9
23.5
122.5
143.9
42.0
28.3
23.4
46.8
41.8
46.1
30.5
33.7
32.3
210.2
11.0
15.6
17.2
25.7
176.4
32.9
23.4
122.7
144.1
42.0
28.2
21.2
47.0
41.6
46.0
30.7
33.8
32.4
181.1
12.7
16.0
17.3
26.1
176.1
33.1
23.6
122.6
144.0
41.9
28.1
21.2
46.8
41.5
45.9
29.9
33.7
32.2
181.6
12.6
15.9
17.2
26.0
176.1
32.9
23.1
121.8
144.4
41.8
36.1
74.3
47.7
42.3
47.1
30.1
32.3
31.8
210.4
11.3
14.5
17.7
27.4
176.3
33.3
23.9
122.3
144.4
41.3
36.1
74.2
47.2
41.9
46.7
30.6
34.2
32.6
209.6
10.7
14.1
17.2
26.9
175.8
33.0
24.3
glucuronic acid, -galactose, -fucose, -rhamnose, and -xylose
D
D
L
D
(1:1:1:1:2) by GC–MS. Similar to 1, the aglycone of 2 was also
determined to be gypsogenin with sugar linkages at positions C-3
and C-28 on the basis of the 1H-, 13C NMR (Table 1). The NMR data
(Table 3) assignments of sugar moieties were accomplished by a
combination of TOCSY, HSQC, and HMBC experiments. The exact
linkage for the sugar units of 2 was established using the HMBC
spectrum. From the long-range correlations H-1 (dH 5.43) of xylose0
and C-4 (dC 87.3) of xylose, H-1 (dH 4.97) of xylose and C-4 (dC 86.7)
of rhamnose, H-1 (dH 6.38) of rhamnose and C-2 (dC 73.6) of fucose,
and H-1 (dH 5.97) of fucose and C-28 (dC 176.4) of the aglycone, it
was deduced that oligosaccharide at C-28 was consistent with the
data in the literature8 as well. The sequence of the sugar chain at C-
3 of the aglycone was also established by a combination of H-1 (dH
5.13) of galactose and C-3 (dC 87.7) of glucuronic acid, and H-1 (dH
4.65) of glucuronic acid and C-3 (dC 84.6) of the aglycone in HMBC
spectrum. From the above evidences, the structure of 2 was eluci-
dated as 3-O-b-
gypsogenin
D
-galactopyranosyl-(1?3)-b-
28-O-b- -xylopyranosyl-(1?3)-b-
(1?4)-a-L-rhamnopyranosyl-(1?2)-b-D-fucopyranosyl ester.
D
-glucuronopyranosyl
acid (dC 78.4), H-1 of xylose (dH 5.34) with C-3 of glucuronic acid
(dC 86.0), and H-1 of glucuronic acid (dH 4.88) with C-3 of the
aglycone (dC 84.6). Similarly, the sugar chain at C-28 was also
established from the following HMBCs: H-1 of arabinose0 (dH
5.04) with C-4 of arabinose (dC 78.1), H-1 of arabinose (dH 5.17)
D
D
-xylopyranosyl-
The HRESIMS of compound 3 showed a quasimolecular ion peak
at m/z 1157.5367 [M+Na]+, compatible with the molecular formula

70
W. Nie et al. / Carbohydrate Research 345 (2010) 68–73
Table 2
13C and 1H NMR data for sugar moieties of 1, 5, and 6 (C5D5N)
Position
1
5
6
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
3-O-Sugars
3-O-Sugars
3-O-Sugars
GlcA
GlcA
Glc
1
2
3
4
1
2
3
4
5
6
103.8
78.4
86.0
71.5
77.1
171.7
4.82 (d, 7.5)
4.33
4.21
4.45
4.64
1
2
3
4
5
6
103.7
78.2
86.2
71.2
77.2
172.4
4.93 (d, 7.5)
4.37
4.28
4.42
4.54
103.4
78.1
85.9
71.3
75.6
62.5
5.45 (d, 7.8)
4.31
4.14
4.02
3.97
5
6
4.46,4.30
Gal
1
2
3
4
Gal
1
2
3
4
Gal
1
2
3
4
104.0
73.7
75.2
70.0
76.3
61.4
5.56 (d, 7.7)
4.47
4.14
4.56
3.98
104.2
73.5
75.4
70.4
76.4
61.3
5.56(d, 7.6)
4.49
4.19
4.58
4.07
103.9
73.5
75.2
70.8
76.2
61.7
5.58 (d, 7.6)
4.46
4.23
4.60
3.97
5
6
5
6
5
6
4.52,4.40
4.45
4.53,4.40
Xyl
1
2
3
4
Xyl
1
2
3
4
Ara
1
2
3
4
104.8
75.1
78.3
70.7
67.0
5.34 (d, 7.7)
3.96
4.13
4.13
4.23,3.70
105.3
75.2
78.5
70.7
67.2
5.34 (d, 7.7)
3.98
4.13
4.06
4.17,3.68
105.1
72.3
74.8
68.3
67.8
5.21 (d, 7.2)
4.44
4.15
4.24
4.26,3.45
5
5
5
28-O-Sugars
28-O-Sugars
28-O-Sugars
Fuc
1
2
3
4
Fuc
1
2
3
4
Fuc
1
2
3
4
94.5
73.7
76.4
72.9
72.3
16.6
6.01 (d, 8.2)
4.67
4.17
3.96
3.89
95.3
73.8
76.6
82.1
72.4
16.9
6.03 (d, 8.2)
4.62
4.16
3.97
3.93
94.7
73.7
75.9
82.3
71.4
15.6
6.08 (d, 8.2)
4.69
4.17
3.96
3.91
5
6
5
6
5
6
1.48 (d, 6.0)
1.71 (d, 6.2)
1.71 (d, 6.3)
Rha
1
2
3
4
Rha
1
2
3
4
Rha
1
2
3
4
101.1
71.5
72.1
85.4
67.8
18.2
6.45 (s)
4.82
4.64
4.30
4.42
102.0
71.2
82.7
78.1
68.7
18.1
6.07 (s)
4.72
4.68
4.34
4.45
101.3
71.7
83.0
78.4
68.7
18.8
6.32 (s)
4.82
4.52
4.36
4.47
5
6
5
6
5
6
1.65 (d,4.5)
1.76 (d, 6.1)
1.76 (d, 6.2)
Xyl
1
2
3
4
Glc
1
2
3
4
Xyl
1
2
3
4
106.7
75.3
86.3
68.6
67.0
4.98 (d, 7.8)
3.97
4.03
4.08
4.23,3.44
105.1
75.3
78.6
71.4
78.2
62.7
5.49 (d, 7.7)
4.06
4.15
4.66
3.94
106.2
75.4
86.4
69.1
66.8
5.14 (d, 7.8)
3.92
3.95
4.05
4.14,3.70
5
5
6
5
4.50,4.30
Ara
1
2
3
4
Glc0
1
2
3
4
Glc
1
2
3
4
105.0
73.2
74.0
78.1
66.3
5.17 (d, 7.2)
4.43
4.23
4.27
4.47,3.79
105.2
75.5
78.6
71.5
78.3
62.5
5.34 (d, 7.8)
4.06
4.16
4.02
3.93
104.7
75.1
78.4
71.7
78.2
62.5
5.32 (d, 7.8)
3.90
4.10
3.98
3.90
5
5
6
5
6
4.51,4.24
4.46,4.30
Ara0
1
2
3
4
Ara
1
2
3
4
Ara
1
2
3
4
5
Rha0
1
2
3
106.8
72.9
74.3
69.4
66.7
5.04 (d, 7.0)
4.47
4.12
4.23
4.23,3.70
102.9
73.8
74.7
70.0
67.2
5.46 (d, 7.0)
4.52
4.24
4.35
4.26,3.44
106.6
71.8
74.8
69.8
67.8
5.02 (d, 7.8)
4.44
4.15
4.24
4.26,3.45
5
5
103.4
73.9
72.6
73.4
72.6
16.9
4.90 (s)
4.88
4.72
4.57
4.53
4
5
6
1.69 (d, 6.3)
C54H86O25. On acid hydrolysis, 3 afforded gypsogenic acid as the
aglycone8 (Table 1), and
-glucose as component sugars by GC–
MS. The long-range correlations between dH 1.54 (H-24) and dC
181.1 indicated that dC 181.1 belonged to C-23 of the aglycone,
so dC 176.1 belonged to C-28. The chemical shifts of dC 176.1
(C-28) revealed that the sugar chain was attached to C-28 of the
aglycone. The sequences of the tetrasaccharide unit and sugar–
aglycone linkage were determined from the HMBC spectrum of 3,
which afforded key 1H–13C long-range correlations between H-1
of Glc I (dH 6.18) and C-28 (dC 176.1) of the aglycone, H-1 (dH
D

W. Nie et al. / Carbohydrate Research 345 (2010) 68–73
71
ogenic acid 28-O-b-
D
-glucopyranosyl-(1?6)-b-
D
-glucopyranosyl-
(1?6)-[b-D-glucopyranosyl-(1?3)]-b-D-glucopyranosyl ester.
O
Compound 4 was obtained as an amorphous powder. The molec-
ular formula of 4 was determined to be C60H96O30 from data of the
HRESIMS (m/z 1319.5914 [M+Na]+). 1H and 13C NMR spectra indi-
cated that compound 4 had the same aglycone to 3. The chemical
shifts of dC 84.8 (C-3) and dC 176.1 (C-28) revealed that compound
4 was a bidesmosidic glycoside. Among the five sugar units in the
C
GlcA
O
Xyl
HOOC
O
HO
O
HO
HO
O
CHO
OH
H
H
O
O
O
HO
HO
Fuc
OH
OH
molecule, four were identified as D-glucose, and one as D-galactose
O
Gal
O
HO
by GC–MS after acid hydrolysis of 4. The b anomeric configurations
Rha
H
OH
3
Xyl
H
Ara'
O
for the glucose units were determined from their JH1,H2 coupling
Ara
O
H
O
O
HO
O
constants (7–8 Hz), and the galactose unit was determined to be of
HO
HO
O
O
OH
HO
3
-configuration based on the J H1,H2 (3.5 Hz) values observed.6
OH
a
a
HO
OH
H
H
OH
The HMBC spectrum of 4 showed correlations between H-1 (dH
5.42) of Gal-1 and C-6 (dC 68.2) of Glc III-6, between H-1 (dH 5.03)
of Glc III-1 and C-6 (dC 68.5) of Glc I-6, and between H-1 (dH 5.24)
of Glc II-1 and C-3 (dC 88.2) of Glc-6, which suggested the linkage
of C-1 of Glc I to C-28 of the aglycone, C-1 of Glc II to C-3 of Glc I, C-
1 of Glc III to C-6 of Glc I, and C-1 of Gal to C-6 of Glc III, respectively.
The sequence of the sugar chain at C-3 of the aglycone was also
established by a combination of dH 5.22 (H-1 of Glc) and dC 84.8
H
Figure 1. Key HMBCs of 1.
5.24) of Glc II and C-3 (dC 88.4) of Glc I, H-1 (dH 4.98) of Glc III and
C-6 (dC 69.0) of Glc I, H-1 (dH 5.07) of Glc IV and C-6 (dC 68.9) of Glc
III, respectively. Accordingly, compound 3 was elucidated as gyps-
Table 3
13C and 1H NMR data for sugar moieties of 2–4 (C5D5N)
Position
2
3
4
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
3-O-Sugars
28-O-Sugars
3-O-Sugars
GlcA
Glc I
Glc
1
2
3
4
1
2
3
4
5
6
104.6
79.4
87.7
73.8
79.2
172.3
4.65 (d, 7.5)
4.26
4.17
4.51
4.43
1
2
3
4
5
6
95.0
72.6
88.4
68.8
78.2
69.0
6.18 (d, 7.8)
4.09
4.19
4.28
3.88
105.2
75.1
78.3
71.9
78.2
62.7
5.22 (d, 7.8)
4.01
4.19
4.14
3.91
5
6
4.57,4.32
4.51,4.41
28-O-Sugars
Gal
1
2
3
4
Glc II
Glc I
106.0
74.8
75.7
71.5
77.9
63.0
5.13 (d, 7.5)
4.43
4.14
4.54
3.92
1
2
3
4
5
6
105.7
75.4
78.3
71.6
78.6
62.8
5.24 (d, 7.6)
3.97
4.14
4.19
3.94
1
2
3
4
5
6
94.9
72.1
88.2
68.5
77.1
68.5
6.19 (d, 8.0)
4.09
4.24
4.25
4.03
5
6
4.39
4.44,4.24
4.55,4.24
28-O-Sugars
Fuc
1
2
3
4
Glc III
Glc II
95.9
73.6
77.5
74.4
73.6
18.1
5.97 (d, 8.2)
4.64
4.13
3.94
3.87
1
2
3
4
5
6
105.3
75.2
78.4
71.5
77.6
68.9
4.98 (d, 7.8)
3.96
4.18
4.00
3.84
1
2
3
4
5
6
105.7
75.5
78.3
71.9
78.2
62.4
5.24 (d, 7.8)
4.02
4.18
4.13
3.98
5
6
1.47 (d, 6.2)
4.55,4.28
4.52,4.39
Rha
1
2
3
4
Glc IV
Glc III
102.5
73.0
73.7
86.7
69.4
19.7
6.38 (s)
4.79
4.64
4.26
4.42
1
2
3
4
5
6
105.3
75.6
78.4
71.6
78.4
62.7
5.07 (d, 7.8)
3.99
4.14
4.17
3.90
1
2
3
4
5
6
105.0
75.0
78.2
71.4
75.9
68.2
5.03 (d, 7.8)
3.99
4.12
3.92
4.09
5
6
1.66 (d, 6.3)
4.44,4.24
4.46,4.34
Xyl
1
2
3
4
Gal
1
2
3
4
108.8
76.5
87.3
71.0
68.4
4.97 (d, 7.6)
3.99
4.03
4.13
4.17,3.46
100.5
70.5
71.4
70.9
72.6
62.5
5.42 (d, 3.5)
4.69
4.58
4.62
4.583
5
5
6
4.45,4.32
Xyl0
1
2
3
4
105.1
76.2
79.8
70.4
68.6
5.43 (d, 7.2)
3.96
4.14
4.04
4.27,3.74
5

72
W. Nie et al. / Carbohydrate Research 345 (2010) 68–73
(C-3) of the aglycone. On the basis of the above results, compound 4
is 3-O-b- -glucopyranosyl gypsogenic acid 28-O- -galactopyran-
osyl-(1?6)-b- -glucopyranosyl-(1?6)-[b- -glucopyranosyl-(1?3)]-
b- -glucopyranosyl ester.
Compound 5 had a molecular composition C76H120O42 as deter-
spectrometer. 1D and 2D NMR spectra were recorded at 300 K on
Bruker, ACF-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz),
with TMS as internal standard. Mass spectra were obtained on a
MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESIMS)
and a Micro Q-TOF MS (HRESIMS), respectively. TLC was performed
on precoated silica gel G (Qingdao Haiyang Chemical Co. Ltd) and
detection was achieved by 15% H2SO4–EtOH for saponins, and ani-
line–phthalate reagents for sugars. Sephadex LH-20 (Pharmacia)
D
a-D
D
D
D
mined from HRESIMS analysis (quasimolecular ions at m/z
1727.7213 [M+Na]+). Acid hydrolysis of 5 yielded quillaic acid,
and
D
-glucuronic acid,
D-galactose, D-xylose, D-fucose, L-rhamnose,
L-arabinose, and
D-glucose (1:1:1:1:1:1:2) as component sugars.
and RP-C18 (40–63 lm, Fuji) were used for column chromatogra-
Detailed NMR analysis established the aglycone to be quillaic acid9
(Table 1). The 1H and 13C NMR data showed eight anomeric proton
signals at dH 6.07 (s), 6.03 (d, J = 8.2 Hz), 5.56(d, J = 7.6 Hz), 5.49 (d,
J = 7.7 Hz), 5.46 (d, J = 7.0 Hz), 5.34 (d, J = 7.7 Hz), 5.34 (d,
J = 7.8 Hz), and 4.93 (d, J = 7.5 Hz) and the corresponding anomeric
carbon signals at dC 95.3, 102.0, 104.2, 105.1, 102.9, 105.3, 105.2,
and 103.7. The 1H and 13C NMR chemical shift assignments were
accomplished by a combination of HSQC, TOCSY, and HMBC
experiments. It was evident that 5 had the same trisaccharides
linked to C-3 of the aglycone as in 1, as the expected sequence
correlations were observed. The linkage of the remaining five sugars
at C-28 was determined from the following HMBCs. The long-range
correlations observed in the HMBC spectrum between the 1H NMR
resonances at dH 5.46 (H-Ara-1) and the 13C NMR resonances at dC
82.1 (C-Fuc-4), between dH 6.07 (H-Rha-1) and dC 73.8 (C-Fuc-2),
between dH 5.33 (H-Glc-1) and dC 82.7 (C-Rha-3), between dH 5.49
(H-Glc0-1) and dC 78.1 (C-Rha-4), and between dH 6.03 (H-Fuc-1)
and dC 176.3 (C-28), respectively, showed that the pentosaccharide
phy. Preparative HPLC was carried out using Agilent 1100 Series
with Shim-park RP-C18 column (200 ꢁ 20 mm i.d.) and 1100 Series
Multiple Wavelength detector.
3.2. Plant material
The roots of G. pacifica were collected from Xifeng region, Liaon-
ing Province, China, in October 2005. The botanical origin of mate-
rial was identified by Professor Minjian Qin, Department of
Medicinal Plants, China Pharmaceutical University, and the vou-
cher specimens (No. 051020) were deposited at the Department
of Natural Medicinal Chemistry, China Pharmaceutical University,
Nanjing, China.
3.3. Extraction and isolation
The roots of G. pacifica (8.9 kg) were ground into powders, and
then extracted with 70% aqueous ethanol (v/v) three times (10 L,
2 h each) under reflux. After evaporation, the residue was sus-
pended in water and partitioned by EtOAc, n-BuOH, and water.
The n-BuOH-soluble portion (268 g) was fractionated by MCI gel,
which was eluted with MeOH/H2O (0:10, 3:7, 1:1, 7:3, and 10:0)
to give five fractions (fractions 1–5); fractions 3 (MeOH/H2O, 1:1)
and 4 (MeOH/H2O, 7:3) were further subjected to repeated RP-
C18 column with MeOH/H2O (4:6?9:1) and then the eluents of
fraction 3.4 (MeOH/H2O, 7:3) were further separated by HPLC
(MeCN–0.05% TFA in H2O, 32:68, UV detection at 210 nm), to yield
pure 1 (5.6 mg, tR = 14.5 min), and 2 (13 mg, tR = 26.2 min), respec-
tively. The eluents of fraction 4.4 (MeOH/H2O, 7:3) were subjected
to a silica gel column (200–300 mesh), which was eluted with
CHCl3/MeOH/H2O (7:3:0.5) to give 3 (18 mg) and 4 (4.2 mg). The
part of H2O-soluble portion (50 g) was fractionated by MCI gel,
which was eluted with MeOH/H2O (0:10, 3:7, and 10:0) to give
three fractions (fractions 1–3). Fraction 2 (3:7 MeOH/H2O) was
subjected to a silica gel column (100–200 mesh), which was eluted
with CHCl3/MeOH (1:1) and 100% MeOH to give fraction 2.1 and
fraction 2.2. Fraction 2.2 (100% MeOH) was further separated by
HPLC (MeCN–0.05% TFA in H2O, 26: 74, UV detection at 210 nm)
to yield pure 5 (3.7 mg, tR = 18.0 min) and 6 (3.2 mg, tR = 21.2 min),
respectively.
residue O-b-
-rhamnopyranosyl-(1?2)-[
-fucopyranosyl was linked to the quillaic acid unit at C-28. On the
D-glucopyranosyl-(1?3)-[b-
D-glucopyranosyl-(1?4)]-
a-
L
a-L
-arabinopyranosyl-(1?4)]-b-
D
basis of all the foregoing evidences, 5 was elucidated as 3-O-b-
galactopyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b-
onopyranosyl quillaic acid 28-O-b- -glucopyranosyl-(1?3)-[b-
glucopyranosyl-(1?4)]- -rhamnopyranosyl-(1?2)-[
pyranosyl-(1?4)]-b- -fucopyranosyl ester.
D
-
D
D
-glucur-
D
D
-
a-L
a-L-arabino-
D
Compound 6 as amorphous powder possessed the molecular for-
mula C81H130O44, as determined by HRESIMS in the positive-ion
mode (HRESIMS m/z 925.3957 [M+HCOOHꢀ2H]2ꢀ). Acid hydrolysis
of 6 afforded
and
D-glucose, D-galactose, D-xylose, D-fucose, L-rhamnose,
-arabinose in the ratio of 2:1:1:1:2:2 by GC–MS. The downfield
L
chemical shifts of C-3 and upfield chemical shifts of C-28 of the agly-
cone and HMBCs revealed that 6 were bidesmosidic glycosides. The
positions of the sugar residues were unambiguously defined by the
HMBC experiment. Across peaks due to long-range correlations
between C-3 (dC 84.6) of the aglycone and H-1 of Glc (dH 5.45), C-2
(dC 78.1) of Glc and H-1 of Gal (dH 5.58), and C-3 (dC 85.9) of Glc
and H-1 of Ara (dH 5.21) indicated that the trisaccharide moiety of
6 attached to C-3 was established to be b-
(1?2)-[ -arabinopyranosyl-(1?3)]-b- -glucopyranosyl. Similarly,
the hexasaccharide moiety of 6 attached to C-28 was established
to be -arabinopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)-[b-
glucopyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)-[ -rhamno-
pyranosyl-(1?4)]-b- -fucopyranosyl. Therefore, the structure 3-O-
b- -galactopyranosyl-(1?2)-[ -arabinopyranosyl-(1?3)]-b-
glucopyranosyl quillaic acid 28-O- -arabinopyranosyl-(1?3)-b-
-xylopyranosyl-(1?4)-[b- -glucopyranosyl-(1?3)]- -rhamno-
pyranosyl-(1?2)-[ -rhamnopyranosyl-(1?4)]-b- -fucopyranosyl
D-galactopyranosyl-
a-L
D
a-
L
D
D
-
3.3.1. Compound 1
a-L
a-L
White powder, ½a D25
ꢂ
+6.5 (c 0.14; C5H5N); IR (KBr)
mmax: 3429,
D
2932, 1740, 1656, 1067 cmꢀ1 1H NMR (500 MHz, C5D5N) dH 0.74,
.
D
a
-
L
D
-
0.84, 0.86, 1.08, 1.23, 1.40 (3H each s, Me-25, 30, 29, 26, 27, 24),
3.12 (1H, br d, J = 10.8 Hz, 18-H), 4.00 (1H, m, 3-H), 5.38 (1H, br
s, 12-H), 9.95 (1H, s, 23-H); 13C NMR data of the aglycone, see Table
1. 1H and 13C NMR data of glycosidic part, see Table 2. ESIMS m/z:
1627 [MꢀH]ꢀ, HRESIMS m/z: 1627.7022 [MꢀH]ꢀ (calcd for
C74H116O39, 1627.7020).
a-L
D
D
a-L
a-L
D
ester was assigned to 6.
3. Experimental
3.1. General
3.3.2. Compound 2
White powder, ½a D25
ꢂ
ꢃ0 (c 0.20; C5H5N); IR (KBr)
mmax: 3403, 2945,
1739, 1676, 1062 cmꢀ1
.
1H NMR (500 MHz, C5D5N) dH 0.73, 0.84,
0.87, 1.01, 1.22, 1.38 (3H each, s, Me-25, 30, 29, 26, 27, 24), 3.09
(1H, m, 18-H), 3.96 (1H, m, 3-H), 5.39 (1H, br s, 12-H), 9.88 (1H, s,
23-H); 13C NMR data of the aglycone, see Table 1. 1H and 13C NMR
Optical rotations were measured with a JASCO P-1020 polarim-
eter. IR (KBr-disks) spectra were recorded by Brucker Tensor 27

W. Nie et al. / Carbohydrate Research 345 (2010) 68–73
73
data of glycosidic part, see Table 3. ESIMS m/z: 1363 [MꢀH]ꢀ, HRE-
SIMS m/z: 1387.6192 [M+Na]+ (calcd for C64H100O31Na, 1387.6141).
cone of 1 was determined as gypsogenin. Acid hydrolysis of 2–6
was operated likewise, and the aglycone of 2 was determined to
be gypsogenin, while that of 3–4 was gypsogenic acid and that of
of 5–6 was quillaic acid.
Each remaining aqueous layer was concentrated to dryness to
give a residue and was dissolved in pyridine (2 ml), and then L-cys-
3.3.3. Compound 3
White powder, ½a D25
ꢂ
+7.7 (c 0.20; C5H5N); IR(KBr)
m
max: 3410,
2931, 1738, 1649, 1064 cmꢀ1
.
1H NMR (500 MHz, C5D5N) dH 0.82,
0.83, 0.88, 1.04, 1.16, 1.54 (3H each, s, Me-30, 29, 25, 26, 27, 24),
3.16 (1H, d, J = 9.6 Hz, 18-H), 3.99 (1H, m, 3-H), 5.41 (1H, br s,
12-H); 13C NMR data of the aglycone, see Table 1. 1H and 13C
NMR data of glycosidic part, see Table 3. ESIMS m/z: 1133 [MꢀH]ꢀ,
HRESIMS m/z: 1157.5367 [M+Na]+ (calcd for C54H86O25Na,
1157.5350).
teine methyl ester hydrochloride (2 mg) was added to the solution.
The mixture was heated at 60 °C for 1 h, and trimethylchlorosilane
(0.5 ml) was added, followed by heating at 60 °C for 30 min. Then,
the solution was concentrated to dryness and taken up in water
(1 ml ꢁ 3), followed by extraction with n-hexane (1 ml ꢁ 3). The
supernatant was subjected to GC/MS analysis under the following
conditions: Varian CP-3800 Gas Chromatograph equipped with a
Saturn 2200 Mass detector (detection temperature 220 °C). Col-
3.3.4. Compound 4
White powder, ½a D25
ꢂ
+18.1 (c 0.14; C5H5N); IR(KBr)
m
max: 3427,
umn: CP-sil 5 CB capillary column (30 m, 0.25 mm i.d., 0.25 lm).
Column temperature: 150–260 °C with the rate of 8 °C/min, and
2923, 1748, 1660, 1076 cmꢀ1
.
1H NMR (500 MHz, C5D5N) dH 0.85,
0.87, 0.93, 1.04, 1.20, 1.34 (3H each, s, Me-30, 29, 25, 26, 27, 24),
3.17 (1H, m, 18-H), 4.02 (1H, m, 3-H), 5.41 (1H, m, 12-H); 13C
NMR data of the aglycone, see Table 1. 1H and 13C NMR data of gly-
cosidic part, see Table 3. ESIMS m/z: 1295 [MꢀH]ꢀ, HRESIMS m/z:
1319.5914 [M+Na]+ (calcd for C60H96O30Na, 1319.5879).
the carrier gas was He (0.8 ml/min), split ratio 1/10, injection tem-
perature: 250 °C. Injection volume: 0.5
tions of the monosaccharides were confirmed to be
-fucose, -rhamnose, -xylose, -glucuronic acid,
and -glucose by comparison of the retention times of monosaccha-
ride derivatives with those of standard samples: -arabinose
-rhamnose (12.67 min), -xylose
-galactose (14.32 min), and -glucose (14.01 min),
l
l. The absolute configura-
-arabinose,
-galactose,
L
D
L
D
D
D
D
L
3.3.5. Compound 5
(12.67 min),
(11.89 min),
respectively.
D
-fucose(12.85 min),
L
D
White powder, ½a D25
ꢂ
+5.8 (c 0.14; C5H5N); IR(KBr)
m
max: 3431,
D
D
2941, 1724, 1653, 1065 cmꢀ1
.
1H NMR (500 MHz, C5D5N) dH 0.86,
0.93, 0.97, 1.08, 1.46, 1.75 (3H each, s, Me-25, 29, 30, 26, 24, 27),
3.32 (1H, m, 18-H), 4.00 (1H, m, 3-H), 5.25 (1H, br s, 16-H), 5.37
(1H, br s, 12-H), 9.90 (1H, s, 23-H); 13C NMR data of the aglycone,
see Table 1. 1H and 13C NMR data of glycosidic part, see Table 2.
ESIMS m/z: 1703 [MꢀH]ꢀ, HRESIMS m/z: 1727.7213 [M+Na]+ (calcd
for C76H120O42Na, 1727.7146).
Acknowledgment
This research work was supported by the National Natural
Science Foundation of China for Outstanding Young Scientists
(No. 30525032).
3.3.6. Compound 6
White powder, ½a D25
ꢂ
+5.1 (c 0.24; C5H5N); IR(KBr)mmax: 3428, 2937,
Supplementary data
1729, 1662, 1069 cmꢀ1. 1H NMR (500 MHz, C5D5N) dH 0.82, 0.95, 0.98,
1.06, 1.44, 1.74 (3H each, s, Me-25, 29, 30, 26, 24, 27), 3.34 (1H, t-like,
18-H), 4.04 (1H, m, 3-H), 5.23 (1H, br s, 16-H), 5.39 (1H, br s, 12-H),
9.73 (1H, s, 23-H); 13C NMR data of the aglycone, see Table 1. 1H and
13C NMR dataofglycosidic part, see Table2. ESIMSm/z: 1805 [MꢀH]ꢀ,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.08.015.
References
HRESIMS m/z: 925.3957 [M+HCOOHꢀ2H]2ꢀ (calcd for C82H130O46
,
1. Lu, D. Q. Bull. Bot. Res. 1994, 4, 329–337. in Chinese.
2. The Chinese Medicine Dictionary, Shanghai People’s Publishing House,
Shanghai, 1977, p 2170.
925.3922).
3. Kochetkov, N. K.; Khorlin, A. J.; Ovodov, J. S. Tetrahedron Lett. 1963, 8, 477–482.
4. Kochetkov, N. K.; Khorlin, A. J. Arzneimittelforschung 1966, 16, 101–109.
5. Khorlin, A. J.; Ovodov, J. S.; Kochetkov, N. K. Zh. Obshch. Khim. 1962, 32, 782–791.
6. Luo, J. G.; Ma, L.; Kong, L. Y. Bioorg. Med. Chem. 2008, 16, 2912–2920.
7. Ghezala, G.; Tomofumi, M.; Marie-Aleth, L.-D. J. Nat. Prod. 2001, 64, 1533–1537.
8. Sibel, A.; Marie-Aleth, L.-D.; Tomofumi, M.; Erdal, B.; Serdar, G.; Ozgen, A. J. Nat.
Prod. 2007, 70, 1830–1833.
3.4. Acid hydrolysis of 1–6 and determination of absolute
configuration of monosaccharides6
Compound 1 (4 mg) was treated with 2 M HCl (4 ml) at 90 °C for
2 h. The reaction mixture was extracted with CHCl3 (3 ꢁ 5 ml). The
CHCl3 extract was purified by chromatography on Sephadex LH-20
(2.0 ꢁ 100 cm). Comparing TLC with authentic samples, the agly-
9. Fu, H.; Koike, K.; Li, W.; Nikaido, T.; Lin, W.; Guo, D. J. Nat. Prod. 2005, 68, 754–
758.