Three new sulfated triterpenoids from the roots of Gypsophila pacifica

Article abstract of DOI:10.1080/10286020.2011.575067

Three new sulfated triterpenoids (1-3), along with one known compound (4), were isolated from the roots of Gypsophila pacifica Kom. The structures of the new compounds were established as 3β-O-sulfate gypsogenin 28-O-β-d-glucopyranosyl ester (1), 3β-O-sulfate gypsogenin (2), and 3β-O-sulfate quillaic acid (3) on the basis of 1D, 2D NMR, and HR-ESI-MS methods.

Full text of DOI:10.1080/10286020.2011.575067

Journal of Asian Natural Products Research
Vol. 13, No. 6, June 2011, 529–533
Three new sulfated triterpenoids from the roots of Gypsophila pacifica
Jian-Guang Luo, Wei Nie and Ling-Yi Kong*
Department of Natural Medicinal Chemistry, China Pharmaceutical University,
Nanjing 210009, China
(Received 11 January 2011; final version received 22 March 2011)
Three new sulfated triterpenoids (1–3), along with one known compound (4), were
isolated from the roots of Gypsophila pacifica Kom. The structures of the new
compounds were established as 3b-O-sulfate gypsogenin 28-O-b-D-glucopyranosyl
ester (1), 3b-O-sulfate gypsogenin (2), and 3b-O-sulfate quillaic acid (3) on the basis
of 1D, 2D NMR, and HR-ESI-MS methods.
Keywords: Gypsophila pacifica; Caryophyllaceae; sulfated triterpenoids
1. Introduction
we describe the isolation and structural
elucidation of the new compounds.
The genus Gypsophila, which belongs to
the family Caryophyllaceae, comprises
more than 150 species distributed through-
out the world. Some of these species have
long been used as pharmaceutical and
ornamental plants [1]. In northern China,
18 species and a variety are reported.
Xinjiang is the center of distribution and
differentiation with the number of species
gradually decreasing eastwards [2].
2. Results and discussion
Compound 1, obtained as a white amor-
phous powder, was assigned to the
molecular formula C36H56O12S as deter-
mined from HR-ESI-MS (m/z: 711.3433
[M 2 H]2). The ESI-MS of 1 exhibited
the [M 2 H]2 ion (m/z 711), [M þ NH4]þ
ion (m/z 730), and [M þ Na]þ ion (m/z
735). The IR spectrum with absorption
bands at 1229 cm21 indicated the presence
of a sulfate group in 1 [6]. Acid hydrolysis
of 1 afforded D-glucose as component
sugar and aglycone, followed by treatment
with BaCl2, gave white precipitates, thus
demonstrating the existence of sulfate
residues [7]. The 13C NMR spectral data,
summarized in Table 1, showed 36 carbon
resonances, indicating the presence of a
sugar moiety with a triterpenoid aglycone.
The 1H NMR spectrum (Table 1) indicated
the presence of six angular CH3 groups (dH
0.80, 0.88, 0.91, 1.05, 1.21, and 1.24), a
vinyl H-atom at dH 5.39 (br s), and an
Gypsophila pacifica Kom., a small
perennial herb, is a typical representative
of the genus Gypsophila, widely distrib-
uted 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 L. var.
lanceolata Bge) to treat fever, consump-
tive disease, and infantile malnutrition
syndrome [3]. In previous chemical
studies, triterpenoid saponins were
reported from G. pacifica [4,5]. Our
continuing research for constituents from
its roots resulted in the isolation of four
sulfated triterpenoids 1–4 (Figure 1), of
which 1–3 are new compounds. Herein,
*Corresponding author. Email: cpu_lykong@126.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.575067
http://www.informaworld.com

530
J.-G. Luo et al.
presence of a sulfate group in the
molecule, the position of the sulfate
group was assigned to C-3 on the basis
of the downfield chemical shifts of H-3
(Dd þ 1.10) and C-3 (Dd þ 7.3), which
are consistent with the presence of a
sulfate group [7–9]. Thus, the structure of
this new compound was established as 3b-
O-sulfate gypsogenin 28-O-b-D-glucopyr-
anosyl ester.
Compound 2 was isolated as a white
amorphous powder. The negative-ion HR-
ESI-MS of 2 exhibited a pseudo-molecular
ion at m/z 549.2905 [M 2 H]2 (calcd for
C30H45O7S, 549.2891), consistent with a
molecular formula of C30H46O7S, 162
1
mass units less than 1. The H and 13C
NMR spectra of 2 (Table 1) resembled to
those of 1, except for the absence of
glucosyl signals and downfield shifts of C-
28 from dC 176.3 to 182.0, which revealed
that C-28 of 2 was non-substituted.
Combining the evidence of HSQC and
HMBC experiments, the structure of 2 was
elucidated to be 3b-O-sulfate gypsogenin.
Compound 3 had a molecular formula
of C30H46O7S as established by HR-ESI-
MS (m/z: 565.2819 [M 2 H]2). The IR
spectrum showed the presence of OH
(3435 cm21), CO (1704 cm21), and ZSO3
(1231 cm21) functions. These suggested
that 3 also possessed a sulfate group in the
molecule. The 1H and 13C NMR spectra of
compound 3 were similar to those
of compound 2, except for the presence
of 16-OH in 3. This was evidenced by
carbon shifts at dC 36.6 (C-15) and dC 75.3
(C-16) of 3 with respect to the correspond-
ing values of dC 28.8 and 24.1 in 2. The
appearance of H-16 as a broad singlet at dH
5.31 indicated that 16-OH was a-oriented.
The above detailed NMR (Table 1)
analysis identified the aglycone as quillaic
acid [10], which was further confirmed by
the HSQC and HMBC experiments.
Concerning the sulfate group, the down-
field shifts of the H-3 (Dd þ 1.1) and C-3
(Dd þ 9.6) signals of 3, compared with a
non-substituted moiety, indicated that the
Figure 1. Structures of 1–4.
aldehyde proton at dH 9.80 (s). Moreover,
an anomeric proton signal at dH 6.30 (d,
8.2) and an oxygenated methylene proton
signal at dH 5.14 (1H, dd, 4.0, 12.5)
showed the b-configuration of sugar
moiety and 3-OH, respectively. After
assignments of the NMR (Table 1) data
on the basis of HSQC and HMBC
experiments (Figure 2), the aglycone in 1
was determined as gypsogenin [8], and the
carbon signals appeared at dC 95.6, 73.9,
78.2, 70.8, 78.7, and 62.0 were assigned to
C-10 –C-60 of D-glucose. When comparing
the data of 1 (Table 1) with those of
gypsogenin, the resonances for C-3 and C-
28 were shifted from dC 71.9 to 79.2, and
from dC 178.4 to 176.3, respectively,
which revealed that the OH group at C-3
and COOH group at C-28 was substituted.
The HMBC correlations of the proton at
dH 6.30 (H-1 of glucose) with the carbon at
dC 176.3 (C-28 of aglycone) confirmed
that the sugar residue was attached to C-28
of aglycone (Figure 2). Since HR-ESI-MS,
IR, and acid hydrolysis of 1 suggested the

Journal of Asian Natural Products Research
531
Table 1. 1H (500 MHz) and 13C NMR(125 MHz) spectral data for 1–3a.
1b
2c
3c
No.
dC
dH
dC
dH
dC
dH
1
2
3
4
5
6
7
8
38.6 1.44 (m), 0.94 (m)
28.4 2.28 (m)
79.2 5.14 (dd, 4.0, 12.5)
39.1
28.8
80.4
55.9
48.6
21.4
33.8
40.9
48.2
36.9
24.0
123.4
145.3
43.0
28.8
24.1
47.6
42.8
47.2
31.6
34.9
33.2
206.9
9.8
1.41 (m), 0.90 (m)
2.23 (m)
4.84 (dd, 4.0, 12.0)
–
1.49 (m)
1.53 (m), 1.24 (m)
1.62 (m)
–
1.70 (m)
–
2.01 (m), 1.86 (m)
5.35 (br s)
–
39.2 0.84 (m), 1.34 (m)
30.3 2.08 (m)
80.2 4.85 (dd, 4.2, 12.0)
55.9 –
47.6 1.41 (m)
55.1
47.6 1.51 (m)
–
20.4 1.58 (m), 1.34 (m)
33.0 1.68 (m)
21.3 1.38 (m), 1.14 (m)
33.4 1.60 (m)
39.9
47.1 1.76 (m)
35.7
–
41.0
48.2 1.66 (m)
36.9
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Glc
10
–
–
23.2 2.07 (m), 1.95 (m)
122.3 5.39 (br s)
24.9 2.07 (m), 1.95 (m)
123.1 5.31 (br s)
144.0
42.0
–
–
145.2
41.0
–
–
–
28.0 2.29 (m), 1.68 (m)
23.8 2.07 (m), 0.90 (m)
2.19 (m), 1.62 (m)
2.02 (m), 0.84 (m)
–
3.21 (m)
1.72 (m), 1.19 (m)
–
1.28 (m), 1.18 (m)
1.96 (m), 1.69 (m)
9.31 (s)
1.20 (s)
0.82 (s)
1.01 (s)
1.15 (s)
–
0.94 (s)
0.91 (s)
36.6 2.09 (m), 1.48 (m)
75.3 5.31 (br s)
46.8
41.5 3.30 (m)
–
47.2
42.8 3.00 (m)
–
46.0 1.79 (m), 1.25 (m)
30.6
47.6 1.25 (m), 1.69 (m)
30.7
–
–
33.8 1.32 (m), 1.14 (m)
32.3 2.04 (m), 1.75 (m)
205.4 9.80 (s)
9.7 1.24 (s)
15.9 0.80 (s)
36.2 1.14 (m), 1.22 (m)
31.4 1.75 (m), 2.04 (m)
206.8 9.32 (s)
9.9 1.41 (s)
16.2 0.81 (s)
16.1
17.7
26.4
182.0
33.5
24.0
17.1 1.05 (s)
26.0 1.21 (s)
17.8 1.02 (s)
27.3 1.71 (s)
176.3
–
176.0
–
33.0 0.91 (s)
23.8 0.88 (s)
33.5 0.87 (s)
24.4 0.97 (s)
95.6 6.30 (d, 8.2)
73.9 4.20 (m)
78.2 4.27 (m)
70.8 4.35 (m)
78.7 4.03 (m)
62.0 4.41 (m), 4.48 (m)
20
30
40
50
60
Notes: aAssignments were made on the basis of HSQC and HMBC experiments.
b Measured in C5D5N.
c Measured in CD3OD.
sulfate group was in position C-3 of the
aglycone. Accordingly, the structure of
new compound was established as 3b-O-
sulfate quillaic acid.
The appearance of sulfated or sulfur-
containing triterpenoids is quite rare. From
the species of the Caryophyllaceae family,
only eight compounds of this type are
reported [6,7,12–14]. It is noteworthy to
mention that four sulfated triterpenoids
were simultaneously isolated from the
roots of G. pacifica in our present study.
Compound 4 was identified as 3b-O-
sulfate oleanolic acid by direct comparison
of their spectral analysis with the literature
[11].

532
J.-G. Luo et al.
3.3 Extraction and isolation
The roots of G. pacifica (8.9 kg) were
ground into powders and then extracted
with 70% aqueous EtOH (v/v) three times
(10 l, 2 h each) under reflux. After
evaporation, the residue was suspended
in water and partitioned by EtOAc,
n-BuOH, and water. The EtOAc portion
(60 g) was fractionated by silica gel
column (100–200 mesh), which was
eluted with CHCl3/MeOH with gradually
increasing polarity to give five fractions
(fractions 1–5); fraction 3 (500 mg,
CHCl3/MeOH, 10:1, v/v) was further
subjected to repeated silica gel column
with CHCl3/MeOH gradually to yield pure
2 (105 mg), 3 (7 mg), and 4 (4 mg),
respectively. The n-BuOH-soluble portion
(268 g) was fractionated by MCI gel,
which was eluted with MeOH/H2O
(0:100, 30:70, 50:50, 70:30, and 100:0,
v/v) to give five fractions (fractions 1–5);
fraction 5 (180 mg, MeOH/H2O, 100:0,
v/v) was further purified by repeated silica
gel column (CHCl3/MeOH, 3:1, v/v) to
afford pure 1 (28 mg).
Figure 2. Key HMBC correlations for
compound 1.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured with a
JASCO P-1020 polarimeter. IR (KBr
disks) spectra were recorded by Bruker
Tensor 27 spectrometer. 1D and 2D NMR
spectra were recorded at ACF-500 NMR
instrument (1H: 500 MHz, 13C: 125 MHz),
with TMS as an internal standard. Mass
spectra were obtained on an MS Agilent
1100 Series LC/MSD Trap mass spec-
trometer (ESI-MS) and a Micro Q-TOF
MS (HR-ESI-MS), respectively. TLC was
performed on a precoated silica gel G
(Qingdao Haiyang Chemical Co. Ltd,
Qingdao, China), and detection was
achieved by 15% H2SO4 –EtOH for
triterpenoids. Silica gel H (Qingdao
Haiyang Chemical Co. Ltd) and Sephadex
LH-20 (Pharmacia, Amersham Bios-
ciences, Uppsala, Sweden) were used for
column chromatography.
3.3.1 3b-O-Sulfate gypsogenin 28-O-b-
D-glucopyranosyl ester (1)
A white amorphous powder (MeOH);
[a]2D5 þ 5.95 (c 0.01; MeOH); IR (KBr)
n
max: 3418, 2948, 1735, 1639, 1229,
1073 cm21. H NMR (500 MHz, C5D5N)
and 13C NMR (125 MHz, C5D5N) spectral
data, see Table 1. ESI-MS m/z: 711
[M 2 H]2, 730 [M þ NH4]þ, 735
[M þ Na]þ; HR-ESI-MS m/z: 711.3433
[M 2 H]2 (calcd for C36H55O12S,
711.3420).
1
3.2 Plant material
The roots of G. pacifica were collected
from Xifeng region, Liaoning Province,
China, in October 2005. The botanical
origin of material was identified by Prof.
Minjian Qin, Research Department of
Traditional Chinese Medicinal Resources,
China Pharmaceutical University, and the
voucher specimens (No. 051020) have
been deposited at the Department of
Natural Medicinal Chemistry, China Phar-
maceutical University, Nanjing, China.
3.3.2 3b-O-Sulfate gypsogenin (2)
A white amorphous powder (MeOH);
[a]2D5 þ 42.03 (c 0.08; MeOH); IR (KBr)
n
max: 3442, 2948, 1690, 1638, 1244,
1078 cm21. H NMR (500 MHz, CD3OD)
and 13C NMR (125 MHz, CD3OD) spec-
tral data, see Table 1. ESI-MS m/z: 549
1

Journal of Asian Natural Products Research
533
[M 2 H]2, 568 [M þ NH4]þ, 573
Acknowledgements
[M þ Na]þ; HR-ESI-MS m/z: 549.2905
[M 2 H]2 (calcd for C30H45O7S,
549.2891).
This research work was financially supported by
theNationalNaturalScienceFoundationofChina
forOutstandingYoungScientists(No.30525032)
and the National Key Scientific and Technologi-
cal Special Projects (2009ZX09502-011).
3.3.3 3b-O-Sulfate quillaic acid (3)
References
A white amorphous powder (MeOH);
[a]2D5 þ 10.81 (c 0.09; MeOH); IR (KBr)
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