New triterpenoid saponins from the roots of Gypsophila perfoliata Linn.

Article abstract of DOI:10.1016/j.carres.2011.07.027

Nine new triterpenoid saponins (1-9) have been isolated from the roots of Gypsophila perfoliata Linn. Their structures were established on the basis of extensive NMR (¹H, ¹³C, HSQC and HMBC) and ESIMS studies.

Full text of DOI:10.1016/j.carres.2011.07.027

Carbohydrate Research 346 (2011) 2206–2212
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
New triterpenoid saponins from the roots of Gypsophila perfoliata Linn.
⇑
Qing Chen, Jian-Guang Luo, Ling-Yi Kong
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 March 2011
Received in revised form 28 July 2011
Accepted 31 July 2011
Available online 5 August 2011
Nine new triterpenoid saponins (1–9) have been isolated from the roots of Gypsophila perfoliata Linn.
Their structures were established on the basis of extensive NMR ( H, C, HSQC and HMBC) and ESIMS
studies.
1
13
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Gypsophila perfoliata Linn.
Caryophyllaceae
Triterpenoid saponins
ꢀ
1
. Introduction
exhibited a quasi-molecular ion peak at m/z 1873.7716 [MꢀH]
(
129
calcd for C84H O46: 1873.7760), which was consistent with the
The genus Gypsophila, belonging to the family Caryophyllaceae,
molecular formula of C84
HCl afforded the aglycone and monosaccharides. The monosaccha-
rides were identified as -rhamnose, -fucose, -galactose, -xylose,
-quinovose, and -glucuronic acid (1:1:1:4:1:1) by GC–MS analy-
128
H O46. Acid hydrolysis of 1 with 2 M
comprises about 150 species widely distributed in Asia and Europe.
Of these, more than 18 species grow in mainland China, especially
L
D
D
D
1
in Xinjiang province. Some species have applications in either tra-
D
D
ditional Chinese medicines (TCMs) or folklore herbs to treat fever,
consumptive disease, and infantile malnutrition syndrome. Triter-
sis. The aglycone was identified as quillaic acid by co-TLC compar-
ison with a standard sample, which was also confirmed on the
2
1
13
penoid saponins with biological activities have been reported in
our previous phytochemical investigations from this genus.3 Gyp-
sophila perfoliata Linn., a perennial herbaceous plant, that is
approximately 70 cm tall, grows in forest grasslands, wet river-
sides, saline alkaline soils, steppe sands at 500–1000 m above sea
basis of the H and C NMR spectroscopy (Table 1) by comparing
–5
3
13
to the reported data. The downfield C NMR chemical shift at d
C
1
3
83.7 and the upfield C NMR chemical shift at d
C
175.9 suggested
that 1 was a 3, 28-O-bidesmoside. Compound 1 showed nine ano-
meric proton signals at d 6.17 (s), 5.90 (1H, d, J = 8.5 Hz), 5.50 (1H,
d, J = 7.0 Hz), 5.28 (1H, d, J = 7.5 Hz), 5.11 (1H, d, J = 7.2 Hz), 5.06
(1H, d, J = 8.0 Hz), 5.03 (1H, d, J = 8.5 Hz), 4.99 (1H, d, J = 7.5 Hz)
and 4.85 (1H, d, J = 7.0 Hz) displayed the correlations with anomer-
1
level. To date, there is only one report on the chemical identifica-
6
tion of the essential oil of this plant. In our continuing search for
bioactive triterpenoid saponin constituents, nine new triterpenoid
saponins were isolated from the roots of G. perfoliata.
ic carbon signals at d
C
101.4, 94.4, 104.1, 105.0, 105.4, 106.3, 105.7,
1
13
1
06.8, and 103.7 in HSQC spectrum, respectively. The H and
C
NMR data (Tables 1 and 2) of the monosaccharide residues were
assigned starting from the readily identifiable anomeric protons
by means of the TOCSY, HSQC, and HMBC spectra obtained for this
2
. Results and discussion
The concentrated n-BuOH-soluble fraction of the ethanol ex-
compound. The b-anomeric configurations for the
acid, -fucose, -quinovose, -galactose and -xylose units, the
-anomeric configurations for -arabinose were determined by
their large JH1,H2 coupling constants of 7–8 Hz, and the -anomer-
C
ic configuration of -rhamnose was judged by its C-5 (d 67.8). The
D-glucuronic
tract of G. perfoliata roots was purified by precipitation with aceto-
acetate. The resulting crude saponin mixture was further subjected
to multiple chromatographic steps over MCI gel and reversed-
phase C18 and finally purified by prep-HPLC, yielding nine saponins
D
D
D
D
a
L
3
a
3
L
(1–9, Fig. 1). Among them, saponins 4/5, 6/7 and 8/9 were obtained
sequence of the sugar residues were unambiguously defined by the
HMBC experiment. Cross peaks due to long-range correlations
as three inseparable mixtures of their (trans)- and (cis)-acylated
derivatives.
Compound 1 was obtained as an amorphous powder. Its high-
resolution ESI mass spectrum (HR-ESIMS, negative-ion mode)
between C-3 (d
C-2 (d 78.6) of GlcA and H-1 of Gal (d
and H-1 of Xyl (d 5.28) indicated that the trisaccharide moiety of 1
attached to C-3 was established to be b- -galactopyranosyl-(1?2)-
b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosyl. Similarly, the
C
83.7) of the aglycone and H-1 of GlcA (d
H
4.85),
C
H
5.50), C-3 (d 86.0) of GlcA
C
H
D
⇑
Corresponding author. Tel./fax: +86 25 8327 1405.
E-mail address: cpu_lykong@126.com (L.-Y. Kong).
[
D
D
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.07.027

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
2207
Xyl'''
O HO
Xyl''
1
R = OH R = HO
O
1
2
HO
O
O
C
R1
OH
OH
GlcA
Xyl
HOOC
O HO
2
3
R = H R =H
1 2
HO
HO
O
OH Ara
O
O
O
CHO
O
Qui
O
Fuc
O
OH
R = H R2=
OH OH
O
1
HO
O
O
O
O
OH
O
O
HO
HO
OH
OH
O
Rha
O
Gal
Xyl'
O
HO
R O
O
2
OH
HO
OH
O
4
5
R1= S1 R2=H R3=H
C
GlcA
R OOC
R1= S2
R1= S1
R2=H R3=H
3
O
OR
1
OHO
HO
HO
O
Me
O
Fuc
O
O
CHO
OH OH
O
6
R2=H R3=CH3
R2=H R3=CH3
Xyl OH
HO
R2O
O
O
Rha
Xyl'
R = S
7
8
1
2
Me
O
OH
O
Gal
HO
HO
O
O
S1
R3=CH3
R1=H
R1=H
R2=
OH
OH
HOH C
2
O
HO
R2= S2 R3=CH3
9
HO
Glc OH
O
S1=
S2=
H CO
O
H CO
3
3
Figure 1. The structures of compounds 1–9.
Table 1
1
3C NMR data (d) for the aglycone moieties of compounds 1–9 (125 MHz, C
anosyl-(1?4)-
syl-(1?4)]-b-
between H-1 of xylose (d
a
-
L
-rhamnopyranosyl-(1?2)-[b-
D
-quinovopyrano-
5 5
D N)
D
-fucopyranosyl with the long-range correlations
1
2
3
4/5
38.1
6/7
38.1
8/9
38.1
000
00
H
4.99) with C-3 of xylose (d
C
84.3),
0
0
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38.2
25.2
83.7
55.2
48.4
20.6
32.0
40.3
49.2
36.2
23.7
38.1
25.3
84.7
55.0
48.7
20.7
32.5
40.1
47.8
36.2
23.2
122.4
144.0
42.2
28.4
23.7
47.0
41.9
46.3
30.7
33.9
32.2
210.2
11.1
15.7
17.3
25.9
176.4
33.0
23.7
38.1
25.2
83.7
55.1
48.6
20.7
32.6
40.2
47.8
36.2
23.6
122.5
144.0
42.3
28.6
23.2
47.1
42.0
46.3
30.7
33.9
32.3
210.4
11.0
15.7
17.4
25.9
176.5
33.1
23.6
H-1 of xylose (d
5.11) with C-4 of rhamnose (d
with C-2 of fucose (d 73.1), H-1 of quinovose (d
fucose (d 83.6), and H-1 of fucose (d 5.90) with C-28 of the agly-
cone (d 175.9) in the HMBC spectrum. Besides the above informa-
5.06) with C-3 of xylose (d
86.6), H-1 of xylose
H
C
25.2
82.4
55.1
49.6
20.6
32.6
40.2
47.8
36.2
22.8
122.5
144.1
42.0
28.2
23.7
46.4
42.2
47.1
30.8
33.9
32.5
210.3
11.1
15.7
17.5
26.0
176.5
33.1
23.8
25.2
84.6
55.0
48.9
20.7
32.5
40.2
47.8
36.2
23.6
122.5
144.0
42.0
28.1
23.9
46.4
42.1
47.1
30.8
33.9
32.5
210.2
11.2
15.7
17.5
26.0
176.5
33.1
23.7
25.2
84.6
55.1
48.8
20.6
32.6
40.2
47.8
36.3
23.8
122.5
144.1
41.9
28.2
23.8
46.4
42.2
47.1
30.8
33.9
32.5
210.3
11.2
15.8
17.5
26.0
176.5
33.1
23.7
(d
H
C
83.7), H-1 of rhamnose (d
H
6.17)
5.03) with C-4 of
C
H
C
H
C
1
tion of the sugars and aglycone, there were other signals in H and
13
C NMR spectra of 1, suggesting the presence of two acetyl
H C H C
groups: d 1.94 (3H, s), d 170.3, 20.7 and d 2.02 (3H, s), d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
170.1, 20.5. The location of two acetyl groups at Qui-3 and Qui-4
was determined by the HMBC correlations between H-3 (d 5.58)
and H-4 (d 5.02) of quinovose with the carbonyl carbons of the
two acetyl groups (d 170.3 and 170.1), respectively. On the basis
of the above results, the structure of 1 was established as 3-O-b-
122.1
144.5
41.5
36.2
74.5
47.4
42.1
46.9
30.7
35.9
32.8
209.8
10.9
15.8
17.4
27.1
175.9
33.2
24.5
D
-galactopyranosyl-(1?2)-[b-
onopyranosyl quillaic acid 28-O-b-
xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)-a-L
D
-xylopyranosyl-(1?3)]-b-
-xylopyranosyl-(1?3)-b-
-rhamnopyr-
-quinovopyranosyl-(1?4)]-b-D-
D-glucur-
D
D-
D
anosyl-(1?2)-[3,4-di-O-acetyl-b-
fucopyranoside.
Compound 2 was obtained as a white, amorphous powder. It
was assigned the molecular formula C74 37 from its nega-
D
H
114
O
ꢀ
tive-ion (HR-ESIMS m/z 1593.6966 [MꢀH] ). Upon acid hydrolysis
with 2 M HCl 2 afforded the monosaccharides and aglycone. The
monosaccharides were identified as
-xylose, -fucose, -rhamnose, and
D
-glucuronic acid,
D-galactose,
D
D
L
D-quinovose based on GC–MS
analysis of their chiral derivatives. The aglycone was identified as
gypsogenin by co-TLC comparison with standard sample, which
was also confirmed on the basis of H and C NMR spectroscopy
1
13
hexasaccharide moiety of 1 attached to C-28 was established to be
b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?3)-b- -xylopyr-
5
13
D
D
D
(Table 1) by comparison to the reported data. The downfield
C

2
208
Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
Table 2
1
H and 13C NMR data for sugar moieties of compounds 1–3 (C
5
D
5
N)
1
2
3
3-O-
13C
1H
13C
1H
13C
1H
GlcA
1
2
3
4
103.7
78.6
86.0
71.4
76.5
4.85 (7.0)
4.29
4.22
3.91
3.95
104.3
78.4
86.1
71.6
76.6
4.82 (7.5)
4.26
4.20
4.72
3.93
105.5
78.3
86.7
71.6
76.5
5.17 (7.0)
4.25
4.03
4.58
3.99
5
6
*
*
*
Nd
Nd
Nd
Gal
1
2
3
4
104.1
73.6
75.4
70.2
76.4
61.6
5.50 (7.0)
4.39
4.46
3.65
4.17
104.0
73.6
75.4
70.7
76.5
61.6
5.46 (7.8)
4.42
4.09
4.15
4.17
104.1
73.6
75.5
70.8
76.5
61.5
5.51 (7.2)
4.43
3.97
4.11
3.91
5
6
4.36, 4.48
4.38/4.46
4.38, 4.49
Xyl
1
2
3
4
105.0
75.2
78.7
70.8
67.3
5.28(7.5)
4.12
4.33
4.10
4.22, 4.23
105.1
75.2
78.4
69.6
67.7
5.19 (7.5)
3.91
4.28
4.20
3.71, 4.22
105.1
75.2
78.6
69.6
67.1
5.21 (7.7)
4.11
4.09
4.23
3.77, 4.29
5
28-O-
Fuc
1
2
3
4
94.4
73.9
76.4
83.6
71.4
17.0
5.90 (8.5)
4.48
4.15
3.97
3.92
94.3
74.7
76.5
83.7
71.3
17.0
5.90 (8.0)
4.48
4.17
4.02
3.89
94.3
74.7
76.6
83.7
71.3
17.0
5.94 (7.9)
4.08
4.18
3.99
3.92
5
6
1.49 (6.0)
1.48 (6.3)
1.48 (6.3)
Qui
1
2
3
4
105.7
73.1
76.2
74.3
70.2
17.7
5.03(8.5)
3.97
5.58
5.02
3.66
105.7
73.1
76.1
74.3
70.2
17.6
5.01(7.8)
3.98
5.56
5.02
3.67
105.8
73.1
76.2
74.4
70.2
17.7
5.05 (8.1)
4.03
5.61
5.06
3.70
5
6
1.22 (6.0)
1.22 (6.2)
1.24 (6.1)
Rha
1
2
3
4
101.4
71.7
72.3
83.7
68.3
18.5
6.17
4.67
4.57
4.29
4.35
1.53(5.5)
101.5
71.3
72.4
85.3
68.3
18.5
6.20
4.38
4.56
4.24
4.37
1.64 (6.2)
101.5
70.1
72.4
85.4
68.1
18.5
6.23
4.56
4.56
4.26
4.36
1.61 (6.1)
5
6
Xyl
0
1
2
3
4
5
Xyl
1
2
3
4
5
Xyl
1
105.4
75.3
86.6
68.9
66.9
5.11 (7.2)
3.93
3.98
3.97
3.36, 4.12
107.6
75.4
76.5
70.7
67.3
4.95 (7.4)
4.09
4.17
4.15
3.47, 4.17
107.1
74.5
86.2
68.8
67.7
4.96 (7.2)
4.52
4.20
4.08
3.73, 4.26
0
0
106.3
73.0
84.3
69.6
67.2
5.06 (8.0)
4.46
4.00
4.22
3.66, 3.70
000
106.8
76.5
78.6
71.0
66.4
4.99 (7.5)
3.97
4.10
4.41
3.74, 4.54
2
3
4
5
Ara
1
2
4
5
105.0
71.4
72.9
69.3
66.9
170.4
20.7
5.28
4.73
4.42
4.31
6
3.45, 4.20
Qui-3-OAc
Qui-4-OAc
170.3
170.3
20.7
170.0
20.6
2
0.7
170.1
0.5
1.94
2.02
1.95
2.03
1.95
2.03
170.1
20.6
2
*
nd = not detected.

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
2209
NMR chemical shift at d
shift at d 176.4 suggested that 2 was a bidesmosidic saponin with
glycosidic linkages at C-3 through an ether bond and at C-28
C
84.7 and the upfield 13C NMR chemical
compound. The b-anomeric configurations for the
acid, -fucose, -galactose, -quinovose and
determined by their large 3JH1,H2 coupling constants of 7–8 Hz.
And the -anomeric configuration of -rhamnose was judged by
its C-5 resonance (d 68.3). The sequence of the sugar residues
D
-glucuronic
C
D
D
D
D-xylose units were
through an ester bond. The anomeric proton signals at d 6.20 (s),
a
L
4
5
.90 (1H, d, J = 8.0 Hz), 5.46 (1H, d, J = 7.8 Hz), 5.19 (1H, d,
J = 7.5 Hz), 5.01 (1H, d, J = 7.8 Hz), 4.95 (1H, d, J = 7.4 Hz) and 4.82
1H, d, J = 7.5 Hz) gave correlations with anomeric carbon signals
C
was subsequently determined by HMBC experiments. The linkage
of the sugar units at C-3 of the aglycone was established from
(
at d 101.5, 94.3, 104.0, 105.1, 105.7, 107.6 and 104.3 in HSQC spec-
trum, respectively, which showed that 2 contained seven sugar
units. The H and C NMR data (Table 2) of the monosaccaride res-
idues were assigned starting from the readily identifiable anomeric
protons by means of the HSQC, and HMBC spectra obtained for this
the following HMBC correlations: H-1 of galactose (d
C-2 of glucuronic acid (d 78.4), H-1 of xylose (d 5.19) with C-3
of glucuronic acid (d 86.1), and H-1 of glucuronic acid (d 4.82)
with C-3 of the aglycone (d 84.7). Thus, the sugar chain attached
to C-3 was determined to be 3-O-b-
H
5.46) with
C
H
1
13
C
H
C
D
-galactopyranosyl-(1?2)-[b-
Table 3
1
5 5
H NMR data for sugar moieties of compounds 4–9 (500 MHz, C D N)
4
5
6
7
8
9
3-O-
GlcA
1
2
4.79 (7.2)
4.21
4.79 (7.2)
4.21
4.79 (7.6)
4.23
4.79 (7.6)
4.23
4.85 (7.3)
4.28
4.85 (7.3)
4.28
3
4.16
4.16
4.19
4.19
4.19
4.19
4
4.27
4.27
4.21
4.21
4.18
4.18
5
3.96
3.96
4.37
4.37
3.99
3.99
6
OCH
Gal
1
3
3.71
3.71
3.73
3.73
5.46 (7.7)
4.41
5.46 (7.7)
4.41
5.44 (7.8)
4.41
5.44 (7.8)
4.41
5.51(7.6)
4.45
5.51 (7.6)
4.45
2
3
4.08
4.08
3.90
3.90
4.45
4.45
4
4.51
4.51
4.51
4.51
4.55
4.55
5
3.96
3.96
3.92
3.92
3.93
3.93
6
4.42, 4.51
4.42, 4.51
4.38, 4.46
4.38, 4.46
4.41, 4.47
4.41, 4.47
Xyl
1
2
5.17 (7.8)
3.93
5.17 (7.8)
3.93
5.15 (9.2)
3.90
5.15 (9.2)
3.90
5.25(7.8)
3.95
5.25 (7.8)
3.95
3
4.06
4.06
4.06
4.06
4.08
4.08
4
4.21
4.21
4.05
4.05
4.26
4.26
5
3.35, 4.15
3.35, 4.15
3.35, 4.16
3.35, 4.16
3.38, 4.18
3.38, 4.18
28-O-
Fuc
1
2
6.06 (8.0)
4.03
6.00 (8.0)
4.03
6.07 (8.0)
4.04
6.02 (8.0)
4.04
6.10 (7.9)
4.13
6.05 (7.9)
4.13
3
4.40
4.40
4.43
4.43
5.69
5.69
4
5.68
5.68
5.67
5.67
4.40
4.40
5
4.21
4.21
4.48
4.48
4.08
4.08
6
1.24
1.24
1.28
1.28
1.29
1.29
Rha
1
2
3
4
6.03, s
5.17
4.82
4.50
4.47
1.73
5.96, s
5.17
4.82
4.50
4.47
1.73
6.03, s
5.17
4.81
4.50
4.19
1.76
5.93, s
5.17
4.81
4.50
4.19
1.76
6.06, s
5.17
4.82
4.51
4.51
1.74
5.98, s
5.17
4.82
4.51
4.51
1.74
5
6
Glc
1
2
5.31 (7.6)
4.40
5.31 (7.6)
4.40
5.30 (7.8)
3.90
5.30 (7.8)
3.90
5.31 (7.9)
3.95
5.31 (7.9)
3.95
3
3.96
3.96
4.37
4.37
4.38
4.38
4
4.04
4.04
4.03
4.03
4.07
4.07
5
3.86
3.86
4.09
4.09
3.84
3.84
6
4.15, 4.51
4.15, 4.51
4.14, 4.43
4.14, 4.43
4.18, 4.45
4.18, 4.45
Xyl
1
2
5.38 (7.0)
4.08
5.38 (7.0)
4.08
5.39 (6.5)
4.09
5.39 (6.5)
4.09
5.39 (7.9)
4.09
5.39 (7.9)
4.09
3
4.06
4.06
4.54
4.54
4.08
4.08
4
4.14
4.14
4.14
4.14
3.83
3.83
5
3.35, 4.15
3.35, 4.15
3.70, 4.21
3.70, 4.21
3.62, 4.22
3.62, 4.22
p-Methoxycinnamoyl
0
0
0
0
0
0
1
2
3
00
00
,6
,5
7.40 (8.5)
6.94 (8.5)
6.54 (16)
7.87 (16)
7.94 (8.5)
6.92(8.5)
6.00 (13)
6.85 (13)
7.41 (8.8)
6.92 (8.9)
6.53 (16)
7.87 (16)
7.94 (8.9)
6.92 (9.0)
6.00 (12.9)
6.85 (12.9)
7.41 (8.8)
6.92 (8.9)
6.54(15.9)
7.88(15.9)
7.95 (8.8)
6.90 (8.9)
6.01(12.9)
6.86(12.9)
a
b
CO
OMe
3.62
3.61
3.62
3.61
3.68
3.63

2
210
Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
D
-xylopyranosyl-(1?3)]-b-
sugar chain at C-28 was also established to be b-
1?3)- -rhamnopyranosyl-(1?2)-[3,4-di-O-acetyl-b-
pyranosyl-(1?4)]-b- -fucopyranosyl from the following HMBC
correlations: H-1 of xylose (d 4.95) with C-4 of rhamnose (d
6.20) with C-2 of fucose (d 74.7), H-1
83.7), and H-1 of fu-
176.4). There were two
D
-glucuronopyranosyl. Similarly, the
-xylopyranosyl-
-quinovo-
with the carbonyl carbons of the two acetyl groups (d 170.4, d
70.1), respectively. On the basis of the data obtained, the struc-
ture of 2 was established as 3-O-b- -galactopyranosyl-(1?2)-
b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosyl gypsogenin
b- -xylopyranosyl-(1?4)- -rhamnopyranosyl-(1?2)-[3,4-di-O-
acetyl-b- -quinovopyranosyl-(1?4)]-b- -fucopyranoside.
Compound 3 (Fig. 1) was obtained as a white, amorphous pow-
D
1
(
a
-
L
D
D
D
[
D
D
H
C
D
a-L
8
5.3), H-1 of rhamnose (d
H
C
D
D
of quinovose (d
cose (d
H
5.01) with C-4 of fucose (d
C
5.90) with C-28 of the aglycone (d
C
H
der. It was assigned the molecular formula C79
H
O
41 from its neg-
122
ꢀ
additional acetyl signals visible in the NMR spectra. Attachment of
these groups to C-3 and C-4 of quinovose was confirmed by HMBC
correlations between H-3 (d 5.56) and H-4 (d 5.02) of quinovose
ative-ion (HR-ESIMS m/z 1725.7371 [MꢀH] ). The overall structure
assignment was accomplished using the same protocol as in 1 and
2. Acid hydrolysis afforded gypsogenin and L-arabinose, L-rham-
Table 4
1
3C NMR data for sugar moieties of compounds 4–9 (125 MHz, C
D N)
5 5
4
5
6
7
8
9
3-O-
GlcA
1
2
3
4
5
6
OCH
Gal
1
103.7
78.4
85.6
71.2
76.0
103.7
78.4
85.6
71.2
76.0
103.9
78.5
85.7
70.9
76.6
169.8
52.1
103.9
78.5
85.7
70.9
76.6
169.8
52.1
103.9
78.4
85.7
71.0
76.6
169.8
52.2
103.9
78.4
85.7
71.0
76.6
169.8
52.2
169.8
169.8
3
104.0
73.6
75.8
70.2
76.0
61.7
104.0
73.6
75.8
70.2
76.0
61.7
104.2
73.7
76.0
70.2
75.7
61.7
104.2
73.7
76.0
70.2
75.7
61.7
104.2
73.6
76.0
70.3
75.9
61.7
104.2
73.6
76.0
70.3
75.9
61.7
2
3
4
5
6
Xyl
1
2
3
4
105.0
75.3
79.4
70.8
67.1
105.0
75.3
79.4
70.8
67.1
105.1
75.3
79.4
70.7
67.1
105.1
75.3
79.4
70.7
67.1
105.0
75.2
79.4
70.8
67.1
105.0
75.2
79.4
70.8
67.1
5
28-O-
Fuc
1
2
3
4
5
6
94.8
74.7
72.8
74.5
69.6
16.7
94.8
74.7
72.8
74.5
69.6
16.7
94.8
74.7
72.9
74.5
69.1
16.7
94.8
74.7
72.9
74.5
69.1
16.7
94.8
75.4
74.5
73.1
70.6
16.6
94.8
75.4
74.5
73.1
70.6
16.6
Rha
1
2
3
4
5
6
102.3
71.2
82.4
78.9
69.0
19.0
102.3
71.2
82.4
78.9
69.0
19.0
102.3
71.1
82.3
78.8
69.6
19.0
102.3
71.1
82.3
78.8
69.6
19.0
102.3
71.2
82.4
78.9
69.0
19.0
102.3
71.2
82.4
78.9
69.0
19.0
Glc
1
2
3
4
5
6
105.3
73.1
76.4
71.7
78.2
62.6
105.3
73.1
76.4
71.7
78.2
62.6
105.3
75.3
76.4
71.8
78.3
62.7
105.3
75.3
76.4
71.8
78.3
62.7
105.3
75.2
76.4
71.8
78.3
62.7
105.3
75.2
76.4
71.8
78.3
62.7
Xyl
1
2
3
4
105.0
75.3
78.6
71.2
67.7
105.0
75.3
78.6
71.2
67.7
105.2
75.4
76.2
71.2
67.7
105.2
75.4
76.2
71.2
67.7
105.3
75.3
78.6
71.8
67.3
105.3
75.3
78.6
71.8
67.3
5
p-Methoxycinnamoyl
0
0
0
0
0
0
0
0
1
2
3
4
127.5
130.3
114.7
161.9
116.3
145.1
167.6
55.3
127.9
133.1
113.9
161.0
117.2
143.9
166.6
55.2
127.5
130.3
114.7
161.9
116.3
145.1
167.6
55.3
127.9
133.1
113.9
161.0
117.3
143.9
166.6
55.2
127.5
130.3
114.7
161.9
116.3
145.2
167.6
55.3
127.9
133.1
113.9
161.0
117.2
143.9
166.7
55.2
00
00
,6
,5
a
b
CO
OMe

Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
2211
nose,
D
-fucose,
D
-galactose,
D
-xylose,
D
-quinovose and
D
-glucuronic
C
C
81
H
H
119
O
38, 1699.7384), consistent with the molecular formula of
1
13
acid. The linkage positions were determined by the HMBC experi-
ment. It was evident that 3 had the same trisaccharides linked to
C-3 of the aglycone as in 1 and 2 (Table 1), as the expected sequence
correlations were observed. The linkage of the remaining five sug-
ars at C-28 was determined from the following HMBC correlations
81
120
O38. The H and C NMR data of 6/7 (Tables 1, 3 and 4) as-
signed from HSQC and HMBC experiments were similar to those of
4/5, except for the appearance of one additional methyl group. The
methyl group was located at GlcA-C-6 by the HMBC correlation of
d
H
3.71 (OCH
observations, the structures of 6/7 were determined as 3-O-b-
galactopyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-6-O-methyl-
b- -glucuronopyranosyl gypsogenin 28-b- -xylopyranosyl-(1?4)-
[b- -glucopyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)-4-
-fucopyranoside (6) and its
3 C
) and d 169.8 (GlcA-C-6). On the basis of the above
between H-1 of arabinose (d
of xylose (d 4.96) with C-4 of rhamnose (d
6.23) with C-2 of fucose (d 73.1), H-1 of quinovose (d
with C-4 of fucose (d 83.7), and H-1 of fucose (d 5.94) with C-28
of the aglycone (d 176.5). Attachment of the two acetyl groups to
H
5.28) with C-3 of xylose (d
85.4), H-1 of rhamnose
5.05)
C
86.2), H-1
D-
H
C
D
(d
H
C
H
D
D
C
H
D
a-L
D
C
O-trans-p-methoxycinnamoyl-b-
C-3 and C-4 of quinovose was determined from HMBC correlations
between H-3 (d 5.61) and H-4 (d 5.06) of quinovose with the car-
bonyl carbons of the two acetyl groups (d 170.4 and 170.1), respec-
tively. On the basis of all the foregoing evidence, compound 3 was
cis-isomer (7).
Compound 8/9 had the molecular formula C81
H
120
O38, as re-
ꢀ
vealed by HR-ESIMS (m/z 1699.7386 [MꢀH] , calcd for
1
13
81 119
C H O38, 1699.7384). The H and C NMR signals of 8/9 were al-
elucidated as 3-O-b-
syl-(1?3)]-b- -glucuronopyranosyl gypsogenin
syl-(1?3)-b- -xylopyranosyl-(1?4)- -rhamnopyranosyl-(1?2)-
3,4-di-O-acetyl-b- -quinovopyranosyl-(1?4)]-b- -fucopyranoside.
Each mixture 4/5, 6/7 or 8/9 (Fig. 1) was homogeneous by HPTLC
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyrano-
most superimposable on those of 6/7 except for the fucose residue
(Tables 1 and 2). The downfield shifts observed in the HSQC spec-
trum for the Fuc-H-3/Fuc-C-3 resonances at d 5.69/d 74.5 indi-
H C
cated the primary alcoholic function of Fuc-3-OH to be acylated.
That the 4-methoxycinnamoyl group was attached to the Fuc unit
D
a-
L-arabinopyran-
D
a-L
[
D
D
but was separated into trans and cis-isomers by HPLC, but all at-
tempts to separate each saponin pair by semipreparative HPLC
were unsuccessful. This phenomenon of isomerization referred to
the effect of light on the 4-methoxycinnamoyl group in aqueous
MeOH solution. Under these conditions, the geometrical isomeric
structures of the p-methoxycinnamoyl groups showed tautomer-
like behavior. Such a phenomenon has been previously observed
at C-3 was further proved by the HMBC correlation of d
(OCH ) and d 167.6/166.7 (carbonyl group of the 4-methoxycinna-
mate). Based on this analysis, the structure of 8/9 was established
as 3-O-b- -galactopyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-6-
O-methyl-b- -glucuronopyranosyl gypsogenin 28-b- -xylopyrano-
syl-(1?4)-[b- -glucopyranosyl-(1?3)]- -rhamnopyranosyl-(1?
-fucopyranosyl (8) and its
H
5.69
3
C
D
D
D
D
D
a-L
2)-3-O-trans-p-methoxycinnamoyl-b-
D
7
8
and explained from Polygala senega, Silene jenisseensis, and Silene
cis-isomer (9).
9
fortunei.
Acid hydrolysis of compounds 4–9, obtained as white amor-
phous powders, afforded a gypsogenin,
-xylose, -rhamnose, -glucuronic acid and
cated they have same aglycone and sugar moiety.
D
-glucose,
D
-galactose,
3. Experimental
D
L
D
D
-fucose, which indi-
3.1. General experimental procedures
The high-resolution ESI mass spectrometry (HRESIMS) (nega-
tive-ion mode) of 4/5 exhibited a pseudomolecular ion peak at
Optical rotations were measured with a JASCO P-1020 polarim-
eter (cell length: 1.0 dm). IR (KBr-disks) spectra were recorded by
Brucker Tensor 27 spectrometer. Mass spectra were obtained on
a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESIMS)
and a G1969A TOF MS (HRESIMS), respectively. 1D and 2D NMR
ꢀ
m/z 1685.7216 [MꢀH] (calcd for C80
H
117
O
O
38, 1685.7228) consis-
38. The 13C NMR signals
of 4/5 (Table 4) showed six anomeric signals at d 94.8, 102.3, 103.7,
04.0, 105.0, 105.3, which correlated in the HSQC spectrum with
tent with a molecular formula of C81
H
120
1
1
H NMR spectrum (Table 3) at d 6.06/6.00, 6.03/5.96, 4.79, 5.46,
.17, 5.31, respectively. Similar to 1–3, the trisaccharide moiety
of 4/5 attached to C-3 and C-28 was established to be b- -galacto-
pyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b- -glucuronopyr-
anosyl and b- -glucopyranosyl-(1?3)-[b- -xylopyranosyl-(1?4)]-
-rhamnopyranosyl-(1?2)-b- -fucopyranosyl, respectively,
5 5
spectra were recorded in C D N at 300 K on Bruker ACF-500
1
13
5
NMR ( H: 500 MHz, C: 125 MHz) spectrometers, in which cou-
pling constants were given in Hz. Gas chromatography was done
on a Varian CP-3800 Gas Chromatograph equipped with a Saturn
2200 Mass detector. All solvents used were of analytical or chro-
matographic grade (Jiangsu Hanbang Sci. & Tech. Co. Ltd). TLC
was performed on precoated silica gel 60 F254 plates (Qingdao
Haiyang Chemical Co. Ltd), and detection was achieved by 10%
D
D
D
D
D
a-
L
D
from the HMBC spectrum. In addition to those arising from the
1
sugars and aglycone, there were other signals in H NMR spectrum,
which suggested the presence of (E/Z)-p-methoxycinnamoyl {[(E)-
MC] group: d(H) 7.87 and 6.54 [1H each, d, J = 16.0 Hz, (E)-MC–H–
H
2
SO
4
–EtOH for saponins. MCI gel (37–75
lm, Mitsubishi), and
ODS-C18 (40–63
l
m, Fuji) were used for column chromatography.
C(b)/C(
C(6) and H–C(3), C(5)]; [(Z)-MC] group: d(H) 6.85 and 6.00 [1H
each, d, J = 13.0 Hz, (Z)-MC–H–C(b)/C( )]; d 7.94 and 6.92 [2H each,
d, J = 8.5 Hz, (Z)-MC–H–C(2), C(6) and H-C(3), C(5)]}. The downfield
shifts for the Fuc H-4/Fuc C-4 resonances at d 5.68/d 74.5 re-
vealed the secondary alcoholic functions Fuc-4-OH to be acylated.
An HMBC correlation was observed between d 5.68 (Fuc-4) and d
67.6/166.6 (carbonyl group of the 4-methoxycinnamoyl) proving
a
)]; d 7.40 and 6.94 [2H each, d, J = 8.5 Hz, (E)-MC–H–C(2),
Preparative HPLC was carried out using Agilent 1100 Series with
Shim-park RP-C18 column (200 ꢁ 20 mm i.d.) and 1100 Series Mul-
tiple Wavelength detector.
a
3.2. Plant material
H
C
The roots ofG. perfoliata Linn. were collected from Zhaosu County,
Xinjiang Province, People’s Republic of China, in August 2007, and
identified by Professor Rena Kasimu (College of Pharmaceutical
Sciences, Xinjiang Medical University, China). Voucher specimens
(No. 070806) were deposited at the Department of Natural Medici-
nal Chemistry, China Pharmaceutical University, Nanjing, China.
H
C
1
that the 4-methoxycinnamoyl groups was attached to the Fuc unit
at C-4. Study of the NMR spectra of 4/5 led to the establishment of
their structures as 3-O-b-
anosyl-(1?3)]-b- -glucuronopyranosyl gypsogenin 28-b-
pyranosyl-(1?4)-[b- -glucopyranosyl-(1?3)]- -rhamnopyrano-
-fucopyranosyl (4)
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyr-
D
D
-xylo-
D
a-L
syl-(1?2)-4-O-trans-p-methoxycinnamoyl-b-
and its cis-isomer (5), as new compounds.
D
3.3. Extraction and isolation
The HR-ESIMS (negative-ion mode) of compounds 6/7 exhibited
a quasimolecular ion peak at m/z 1699.7367 [MꢀH] (calcd for
The air-dried plants (1.3 kg) were extracted with 70% aqueous
ethanol (v/v) three times (10 L, 2 h each time) at reflux. After
ꢀ

2
212
Q. Chen et al. / Carbohydrate Research 346 (2011) 2206–2212
evaporation, the residue was suspended in H
with EtOAc and n-BuOH, respectively. Then the n-BuOH fraction
36 g) was purified by chromatography over a MCI gel column
eluted with 30%, 70% and 100% MeOH, respectively. The 70% MeOH
portion was fractionated by RP-C18 column (MeOH–H O, 1:9, 5:5,
:3, 8:2 and 10:0) to give five fractions (Fractions 1–5). Fraction
was further separated by HPLC (MeCN–0.05% TFA in H O,
3:57, UV detection at 210 nm), to yield 1 (11 mg), 2 (20 mg)
2
O and partitioned,
3.4. Acid hydrolysis of compounds 1–9 and determination of
absolute configuration of monosaccharides
(
Each saponin (3 mg) was heated in 2 M HCl (5 mL) at 90 °C for
2
4 h. The reaction mixture was extracted with CHCl
3
(5 mL ꢁ 3).
7
3
4
The CHCl extract was purified by chromatography on Sephadex
3
2
LH-20 (2.0 ꢁ 100 cm). Comparing the TLC with authentic samples,
the aglycone of 1 was determined as quillaic acid. Acid hydrolysis
of 2–9 was operated likewise, and the aglycone of 2–9 was deter-
mined to be gypsogenin. Each remaining aqueous layer was con-
centrated to dryness to give a residue and dissolved in pyridine
and 3 (13 mg), respectively. Fraction 4 was subjected to RP-C18 col-
umn with MeOH–H O (4:6?8:2) to give 3 fractions and then Frac-
tion 4.1 was purified by HPLC (MeCN–0.05% TFA in H O, 45:55, UV
detection at 210 nm) to yield 4/5 (9 mg). Fraction 4.2 was sub-
jected to HPLC with 48% CH CN in H O to yield 6/7 (15 mg) and
/9 (12 mg), respectively.
2
2
(2 mL), and then L-cysteine methyl ester hydrochloride (2 mg)
3
2
was added to the solution. The mixture was heated at 60 °C for
1 h, and chlorotrimethylsilane (0.5 mL) was added, followed by
heating at 60 °C for 30 min. Then, the solution was concentrated
to dryness and dissolved in water (1 mL ꢁ 3), followed by extrac-
tion with n-hexane (1 mL ꢁ 3). The hexane extract was subjected
to GC–MS analysis. The absolute configurations of the monosac-
8
2
D
5
Compound 1: White amorphous powder; ½
a
ꢂ
+0.9 (c 0.09,
ꢀ1
MeOH); IR (KBr) cm : 3419, 2940, 1730, 1680, 1642, 1433,
1
ꢀ
385, 1253, 1207, 1140, 1073; ESIMS m/z: 1873 [MꢀH] , HR-ESIMS
ꢀ
1
m/z: 1873.7716 [MꢀH] (calcd for C84
NMR (C
N, 500 MHz) and 13C NMR (C
given in Tables 1 and 2.
Compound 2: White amorphous powder; ½
129
H O
46, 1873.7760);
H
5
D
5
5
D
5
N, 125 MHz) data are
charides were confirmed to be
-rhamnose, -xylose, -glucuronic acid,
by comparison of the retention times of monosaccharide deriva-
tives with those of standard samples: -arabinose (13.39 min),
xylose (13.42 min), -quinovose (14.00 min),
min), -fucose(14.35 min),
(15.49 min), and
L-arabinose,
D-quinovose,
D
-fucose,
L
D
D
D-galactose, and
D-glucose
25
a
ꢂ
+5.1 (c 0.09,
D
ꢀ1
MeOH); IR (KBr) cm : 3423, 2942, 1750, 1638, 1384, 1256, 1056;
L
D-
ꢀ
ꢀ
13
ESIMS m/z: 1594 [MꢀH] , HR-ESIMS m/z: 1593.6961 [MꢀH] (calcd
D
L
-rhamnose (14.18
1
for C74
113
H O
37: 1593.6966); H NMR (C
5
D
5
N, 500 MHz) and C NMR
D
D-galactose (15.77 min),
D-glucose
(
C
5
D
5
N, 125 MHz) data are given in Tables 1 and 2.
D-glucuronic acid (17.10 min), respectively.
25
Compound 3: White amorphous powder; ½
a
ꢂ
ꢀ4.0 (c 0.05,
D
ꢀ1
MeOH); IR (KBr) cm : 3423, 2934, 1750, 1728, 1633, 1385,
1
1
ꢀ
Acknowledgments
256, 1137, 1061; ESIMS: m/z 1725 [MꢀH] , HR-ESIMS m/z:
ꢀ
121
H O
41: 1725.7388); 1H NMR
725.7371 [MꢀH] (calcd for C79
1
3
This research work was financially supported by the National
Natural Science Foundation of China (30830116, 81073009), and
a Project Funded by The Priority Academic Program Development
of Jiangsu Higher Education Institutions.
5 5 5 5
(C D N, 500 MHz) and C NMR (C D N, 125 MHz) data are given
in Tables 1 and 2.
Compound 4/5: White amorphous powder; IR (KBr) cmꢀ1: 3425,
2
926, 2854, 1679, 1633, 1385, 1256, 1205, 1141, 1076, 1046; ESIMS:
ꢀ
ꢀ
m/z 1686 [MꢀH] , HR-ESIMS m/z: 1685.7216 [MꢀH] (calcd for
1
13
C
80
117
H O
38: 1685.7228) H NMR (C
N, 125 MHz) are given in Tables 1, 3 and 4.
Compound 6/7: White amorphous powder; IR (KBr) cmꢀ1: 3423,
D
5 5
N, 500 MHz) and C NMR
References
(
5 5
C D
1
2
.
.
Lu, D. Q. Bull. Bot. Res. 1994, 4, 329–337.
The Chinese Medicine Dictionary; Shanghai People’s Publishing House: Shanghai,
2
9
931, 1679, 1632, 1605, 1513, 1444, 1386, 1257, 1161, 1077, 1048,
1977, p 2170.
ꢀ
ꢀ
13; ESIMS: m/z 1737 [M+Cl] , HR-ESIMS m/z: 1699.7367 [MꢀH]
3. Nie, W.; Luo, J. G.; Kong, L. Y. Carbohydr. Res. 2010, 345, 68–73.
4. Luo, J. G.; Ma, L.; Kong, L. Y. Bioorg. Med. Chem. 2008, 16, 2912–2920.
5. Chen, Q.; Luo, J. G.; Kong, L. Y. Chem. Pharm. Bull. 2010, 58, 412–414.
6. Schmidt, J.; Boehme, F.; Adam, G. Z. Naturforsch., C 1996, 51, 897–899.
7. Yoshikawa, M.; Murakami, T.; Ueno, T.; Kadoya, M.; Matsuda, H.; Yamahara, J.;
Murakami, N. Chem. Pharm. Bull. 1995, 43, 350–352.
1
(
calcd for C81
H
119
O
38, 1699.7384). H NMR (C
5
D
5
N, 500 MHz) and
1
3
5 5
C NMR (C D N, 125 MHz) are given in Tables 1, 3 and 4.
Compound 8/9: White amorphous powder; IR (KBr) cmꢀ1:
3
442, 2938, 1633, 1513, 1385, 1256, 1161, 1076, 1047; ESIMS:
ꢀ
ꢀ
8. Marie-Aleth, L.-D.; Bernard, H.; Cui, Z. H.; Lou, Z. C.; Hildebert, W. Phytochemistry
m/z 1737 [M+Cl] , HR-ESIMS m/z: 1699.7386 [MꢀH] (calcd for
1995, 40, 509–514.
1
13
C
81
119
H O
38, 1699.7384). H NMR (C
5 5
D N, 500 MHz) and C NMR
9.
Ghezala, G.; Tomofumi, M.; Veronique, L.; Marie-Aleth, L.-D. J. Nat. Prod. 2002,
65, 1568–1572.
5 5
(C D N, 125 MHz) are given in Tables 1, 3 and 4.