New triterpenoid saponins from the aerial parts of Comastoma pedunculatum

Article abstract of DOI:10.1080/10286020.2014.984699

Three new saikosaponin analogs, comastomasaponins I-K (1-3), were isolated from the aerial portions of Comastomapedunculatum. The structures of these compounds were elucidated on the basis of spectroscopic data analysis, and their nitric oxide production inhibitory activity was evaluated invitro.

Full text of DOI:10.1080/10286020.2014.984699

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New triterpenoid saponins
from the aerial parts of
Comastomapedunculatum
Yi Yuana, Yong-Qi Qiaoa, Bao-Song Cuia, Li Tangb, Shao-Peng Yuana,
Qi Houa & Shuai Lia
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing
100050, China
b College of Life and Environmental Science, Central University
for Nationalities, Beijing 100081, China
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Published online: 01 Dec 2014.
To cite this article: Yi Yuan, Yong-Qi Qiao, Bao-Song Cui, Li Tang, Shao-Peng Yuan, Qi Hou & Shuai
Li (2014): New triterpenoid saponins from the aerial parts of Comastomapedunculatum, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2014.984699
To link to this article: http://dx.doi.org/10.1080/10286020.2014.984699
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Journal of Asian Natural Products Research, 2014
http://dx.doi.org/10.1080/10286020.2014.984699
New triterpenoid saponins from the aerial parts of Comastoma
pedunculatum
Yi Yuana, Yong-Qi Qiaoa, Bao-Song Cuia, Li Tangb, Shao-Peng Yuana, Qi Houa
and Shuai Lia*
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China; bCollege of Life and Environmental Science, Central University for Nationalities,
Beijing 100081, China
(Received 19 September 2014; final version received 2 November 2014)
Three new saikosaponin analogs, comastomasaponins I–K (1–3), were isolated from
the aerial portions of Comastoma pedunculatum. The structures of these compounds
were elucidated on the basis of spectroscopic data analysis, and their nitric oxide
production inhibitory activity was evaluated in vitro.
Keywords: Gentianaceae; Comastoma pedunculatum; saikosaponin analogs; structure
elucidation; NO production inhibition
1. Introduction
2. Results and discussion
Comastoma pedunculatum (Royle ex
D. Don) Holub, belonging to the genus
Comastoma of Gentianaceae, which is
named “Sangdou” in Tibet, has been used
to treat hepatitis, liver fibrosis, and
cholecystitis in traditional Tibetan medi-
cine [1]. Previous phytochemical studies
on this genus have demonstrated the
presence of xanthones, flavonoids and
triterpenoids, steroids, lignins, coumarins,
and long-chain alkanes [2–5]. In the
course of our investigations on the
constituents of C. pedunculatum, we have
reported the isolation and characterization
of xanthones [6,7], flavone glycosides [8],
and triterpenoid saponins [8,9]. In this
paper, we report the isolation and struc-
tural elucidation of a new oleanane-
triterpene and two new ursane-triterpene
saponins (Figure 1). Furthermore, the
nitric oxide (NO) production inhibitory
activity in vitro of the compounds is
reported herein.
Comastomasaponin I (1) has the molecular
formula C48H78O18, as determined by
HR-ESI-MS analysis (m/z 965.5094
[M þ Na]þ). The proton and carbon
NMR spectra displayed features charac-
teristic of triterpenoid saponins. Here, 30
of the 48 carbons were assigned to the
triterpenoid skeleton and 18 to a trisac-
charide moiety. In the 1H NMR spectrum,
singlets corresponding to six tertiary
methyl groups at dH 0.85, 0.86, 0.90,
0.96, 0.96, and 1.07 (Table 1) correlated
with the signals in the 13C NMR spectrum
for six sp3 carbons at dC 13.1, 18.8, 17.0,
21.9, 32.2, and 24.8 (Table 2) in the HSQC
spectrum. Two carbinol protons (dH 4.27
and dH 4.50) and the correlated carbon
signals (dC 81.6 and 76.5), and two
hydroxymethyl groups (dH 3.69, 4.37 and
4.21, 4.39) and the correlated carbon
signals at dC 64.0 (double carbons), were
observed in the HSQC spectrum. The UV
spectrum included absorption maxima at
*Corresponding author. Email: lishuai@imm.ac.cn
q 2014 Taylor & Francis

2
Y. Yuan et al.
O
CH2OH
OH
OH
RO
RO
CH2OH
3
CH2OH
3
6
6
2 R=Fuc
Glc
Glc
Glc
6
1 R=Fuc
Glc
Glc
3
3 R=Fuc
Glc
2
Glc
Figure 1. The structural formulas of compounds 1–3.
242, 250, and 260 nm, indicating the
presence of a conjugated heteroannular
diene. This assignment was further sup-
ported by the four olefinic carbon signals
observed at dC 125.8, 127.0, 136.4, and
133.3 and their correlations with two broad
doublet-like vinyl signals at dH 5.70 and
6.53 in the HSQC spectrum. On the basis
of the detailed analysis of the NMR data
and comparison with the literature [10,11],
it was concluded that the triterpenoid
skeleton of 1 is 3b,16b,23,28-tetrahy-
droxy-olean-11,13-diene. HMBC corre-
lation analysis (Figure 2) further
supported this conclusion. The three
anomeric proton signals at dH 4.99, 5.26,
and 5.08 and the three corresponding
carbons at dC 106.1, 106.2, and 105.5
indicated a trisaccharide moiety. The
saccharides of 1 were determined to be
glucose and fucose by acid hydrolysis and
TLC analysis. The absolute configurations
of the sugars were determined by high-
performance liquid chromatography
(HPLC) analysis to be D [9]. The three
sets of glucopyranosyl and fucopyranosyl
signals (Tables 1 and 2) were assigned by
detailed analysis of the NMR data (1H,
13C, COSY, HSQC, and HMBC) and
comparison with the literature [10].
The anomeric configurations of the
glucopyranosyl and fucopyranosyl moi-
eties were defined as b based on their large
3JH1,H2 coupling constants of 7.5–8.0 Hz.
The linkage between the fucopyranosyl
and glucopyranosyls was established by
analysis of the HMBC correlations
between dH 4.99 (H-10 of Fuc) and dC
81.6 (C-3 of the aglycone), dH 5.26 (H-100
of Glc) and dC 85.0 (C-30 of Fuc), and dH
5.08 (H-1000 of Glc) and dC 70.2 (C-600 of
Glc). The trisaccharide was elucidated as a
[b-D-glucopyranosyl-(1 ! 6)]-b-D-gluco-
pyranosyl-(1 ! 3)-b-D-fucopyranosyl
moiety. Therefore, 1 was determined to be
3b,16b,23,28-tetrahydroxy-olean-11,13-
diene-3-O-[b-D-glucopyranosyl-(1 ! 6)]-
b-D-glucopyranosyl-(1 ! 3)-b-D-fuco-
pyranoside.
The molecular formula of comastoma-
saponin J (2) is C48H78O18, identical to
that of comastomasaponin I (1), as
determined by HR-ESI-MS analysis (m/z
965.5092 [M þ Na]þ). Detailed analysis
of the NMR spectra (Tables 1 and 2)
revealed that the trisaccharide signals of 2
and 1 were nearly identical. The aglycone
signals, however, displayed significant
differences. In the triterpenoid skeleton,
the four tertiary methyl singlets at dH 0.90,
0.96, 1.20, and 1.37 and the two secondary
methyl singlets at dH 0.88 (br s) and 1.04

Journal of Asian Natural Products Research
3
Table 1. 1H NMR spectroscopic data of compounds 1–3 (500 MHz in pyridine-d5).
Proton
1
2
3
Aglycone
H-1
1.11 (d, 8.5)
1.84a
0.98a
1.82 (br d, 12.5)
2.03a
0.98a
1.80 (br d, 12.0)
2.03a
H-2
2.08a
2.32 (br d, 10.0)
4.27 (dd, 10.0, 4.0)
1.71 (br d, 9.5)
1.32a
2.29 (br d, 10.0)
4.22a
1.63 (brd, 11.0)
1.38a
1.73 (br d, 12.5)
1.26 (br d, 10.5)
1.55 (br d, 10.5)
2.07a
2.29 (br d, 10.0)
4.20 (br d, 10.0)
1.61 (br d, 11.5)
1.38a
H-3
H-5
H-6
1.80a
1.72 (br d, 12.5)
1.24 (br d, 10.5)
1.53a
H-7
1.33a
1.43 (d, 9.5)
2.10a
H-9
2.06a
H-11
H-12
H-15
5.70 (d, 10.5)
6.53 (br d, 10.5)
1.58 (br d, 12.5)
2.21 (t, 12.5)
4.50 (t, 9.5)
5.86 (d, 10.5)
5.75 (dd, 10.5, 1.5)
1.72 (dd, 12.5, 5.0)
2.05a
4.52 (br d, 9.0)
1.47 (br d, 6.5)
0.88a
5.86 (d, 10.5)
5.75 (br d, 10.5)
1.72 (dd, 13.0, 5.0)
2.04a
H-16
H-18
H-19
4.50a
1.46 (br d, 11.0)
0.87a
1.84 (d, 15.0)
2.49 (d, 15.0)
H-20
H-21
1.82a
1.46a
1.80a
1.45a
1.32a
1.72a
1.49a
1.54a
H-22
H-23
1.61 (br d, 13.5)
2.95 (br d, 13.5)
3.69 (d, 10.5)
4.37a
1.13 (br d, 13.0)
2.62 (br d, 13.0)
3.69 (d, 10.5)
4.34a
1.24 (br d, 13.0)
2.63 (br d, 13.0)
3.68 (d, 10.5)
4.32a
H-24
H-25
H-26
H-27
H-28
0.90 s
0.96 s
0.90 s
0.96 s
1.37 s
1.20 s
1.00 s
0.95 s
1.38 s
1.19 s
0.85 s
1.07 s
4.21a
3.25 (d, 7.0)
4.34a
1.04, d (5.5)
0.88 br s
3.24 (d, 6.0)
4.35a
1.04, d (5.0)
0.88 br s
4.39a
0.96 s
H-29
H-30
Fucosyl
H-1
H-2
H-3
0.86 s
4.99 (d, 8.0)
4.53 (dd, 9.0, 8.0)
4.15 (dd, 9.0, 2.5)
4.36a
3.87–3.89 m
1.52 (d, 5.5)
4.96 (d, 8.0)
4.48a
4.93 (d, 8.5)
4.50a
4.13a
3.86a
H-4
H-5
4.33a
4.22a
3.86–3.88 m
1.51 (d, 6.5)
3.76 (q, 6.0)
1.47 (d, 6.0)
H-6
Glucosyl (at C-3 of Fuc)
H-1
H-2
H-3
H-4
H-5
H-6
5.26 (d, 8.0)
5.24 (d, 7.5)
3.97 (dd, 9.0, 7.5)
4.18 (t, 9.0)
4.04 (t, 9.0)
4.10 m
5.03 (d, 8.0)
3.90 (dd, 8.5, 8.0)
4.21a
4.02 (dd, 9.0, 8.0)
4.22 (t, 9.0)
4.06 (t, 9.0)
3.74a
4.22a
4.12a
4.25 (br d, 10.5)
4.84 (br d, 10.5)
4.23a
4.17a
4.81 (br d, 10.5)
4.76a
(Continued)

4
Y. Yuan et al.
Table 1 – continued
Proton
1
2
3
Glucosyl (at C-2 of Glu)
H-1
H-2
H-3
H-4
H-5
H-6
5.10 (d, 7.5)
4.05a
4.11a
4.16a
3.80a
4.33a
4.48 (br d, 13.5)
Glucosyl (at C-6 of Glu)
H-1
H-2
H-3
H-4
H-5
H-6
5.08 (d, 7.5)
4.05 (dd, 9.0, 7.5)
4.22a
5.06 (d, 7.5)
4.02 (t, 7.5)
4.19a
5.01 (d, 7.5)
4.01 (dd, 9.0, 7.5)
4.19a
4.26a
4.24a
4.23a
3.87–3.89 m
4.26a
4.51 (dd, 12.0, 1.5)
3.86–3.88 m
4.31a
4.51 (dd, 12.0, 1.5)
3.87a
4.30 (br d, 11.0)
4.41 (br d, 11.0)
a Overlapped with other signals, assignments confirmed by 2D COSY, HSQC, and HMBC experiments.
(d, J ¼ 5.5 Hz) (Table 1) were correlated
with the six sp3 carbon signals at dC 13.0,
18.7 (triplex carbons), 19.6, and 19.9
(Table 2), which were correlated in the
HSQC spectrum, respectively. Two doub-
let-like vinyl signals at dH 5.75 and 5.86
were correlated with two sp2 carbons at dC
129.4 and 133.2 in the HSQC spectrum.
Compound 2 was thus determined to be an
ursane-triterpene. In the NMR spectrum,
signals characteristic of a hydroxymethyl
group at position 23 were observed at dH
3.69, 4.34, and dC 64.0, along with
oxygenated sp3 carbons C-3 and C-16
(dH 4.22 and 4.52; dC 81.6 and 65.1).
Signals characteristic of an epoxide bridge
between the quaternary carbon C-13 (dC
84.0) and the secondary carbon C-28 (dH
3.25 and 4.34, dC 72.8) were observed.
Detailed analysis of the NMR spectra and
comparison with the literature led to the
assignment of the triterpenoid skeleton of
2 as 3b,16b,23-trihydroxy-13,28-epox-
yurs-11-ene [12,13]. This conclusion was
further supported by HMBC correlation
analysis (Figure 3). Therefore, 2 was
determined to be 3b,16b,23-trihydroxy-
13,28-epoxyurs-11-ene-3-O-[b-D-gluco-
pyranosyl-(1 ! 6)]-b-D-glucopyranosyl-
(1 ! 3)-b-D-fucopyranoside.
Comastomasaponin K (3) has the
molecular formula C54H88O23, as
determined by HR-ESI-MS analysis (m/z
1127.5618 [M þ Na]þ). The proton and
carbon NMR spectra displayed features
characteristic of triterpenoid saponins.
Detailed analysis of the NMR spectra
(Tables 1 and 2) revealed that the aglycone
signals of 3 and 2 were nearly identical.
Acid hydrolysis of 3 released glucose and
fucose, as confirmed by TLC analysis. The
linkage between the fucopyranosyl and
glucopyranosyl units was established by
analysis of the HMBC correlations between
dH 4.93 (H-10 of Fuc) and dC 81.6 (C-3 of the
aglycone), dH 5.03 (H-100 of Glc) and dC
86.3 (C-30 of Fuc), dH 5.10 (H-1000 of Glc)
and dC 85.5 (C-200 of Glc), and dH 5.01 (H-
10000 of Glc) and dC 69.5 (C-600 of Glc)
(Figure 4). The tetrasaccharide chain was
elucidated as
a b-D-glucopyranosyl-
(1 ! 6)-[b-D-glucopyranosyl-(1 ! 2)]-b-
D-glucopyranosyl-(1 ! 3)-b-D-fucopyra-
nosyl group. Therefore, 3 was determined to
be 3b,16b,23-trihydroxy-13,28-epoxyurs-
11-ene-3-O-b-D-glucopyranosyl-(1 ! 6)-
[b-D-glucopyranosyl-(1 ! 2)]-b-D-gluco-
pyranosyl-(1 ! 3)-b-D-fucopyranoside.
Compounds 1–3 were also investigated
for their inhibitory effect on the production

Journal of Asian Natural Products Research
5
Table 2. 13C NMR spectroscopic data of
compounds 1–3 (125 MHz in pyridine-d5).
Table 2 – continued
Carbon
1
2
3
Carbon
1
2
3
Glucosyl (1 ! 6)
Aglycone
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-1
C-2
C-3
C-4
C-5
C-6
105.5
105.5
75.5
78.4
71.7
78.4
62.6
105.0
74.9
78.0
71.1
78.0
61.7
38.4
26.0
81.6
43.6
47.3
18.2
32.3
40.4
54.5
36.5
127.0
125.8
136.4
44.2
34.8
76.5
44.4
133.3
38.3
32.6
35.1
29.9
64.0
13.1
18.8
17.0
21.9
64.0
32.2
24.8
38.6
26.0
81.6
43.7
47.3
17.6
31.7
42.3
52.9
36.2
129.4
133.2
84.0
46.1
36.2
65.1
48.0
62.2
41.0
38.4
31.3
30.1
64.0
13.0
18.7
19.9
18.7
72.8
18.7
19.6
38.2
25.7
81.6
43.3
46.9
17.2
31.3
41.9
52.5
35.8
129.1
132.8
83.7
45.8
35.8
64.7
47.6
61.8
40.7
38.0
30.9
29.7
63.7
12.5
18.2
19.6
18.3
72.4
18.3
19.2
75.5
78.4
71.7
78.4
62.6
C-9
of NO in lipopolysaccharide (LPS)-stimu-
lated macrophages in mice. The positive
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
Fucosyl
C-1
control,
dexamethasone
(DEX)
(1 £ 1026 M), exhibited an inhibitory rate
(IR) of 86.5%. However, compound 1
inhibited NO production of LPS-stimulated
macrophages with an IR value of 82.3%
(Table 3). Actually, compound 1 did not
exhibit NO production; however, the effect
was considered due to the inhibition of the
mice macrophage proliferation.
3. Experimental
3.1 General experimental procedures
Optical rotation was measured in MeOH
solution in a JASCO P2000 digital
polarimeter with a sodium lamp (589 nm)
(JASCO Corp., Tokyo, Japan). The UV
spectra were obtained using JASCO V-650
spectrophotometer (JASCO Corp.). IR
spectra were acquired on a Nicolet 5700
spectrophotometer (Thermo Electron
Scientific Instrument Crop., Madison, WI,
USA). NMR spectra were recorded on a
Varian Unity Inova 500 FT-NMR spec-
trometer (Varian, Inc., Palo Alto, CA,
USA). Chemical shifts are reported in parts
per million (d), and coupling constants (J)
are expressed in hertz, using residual
C5D5N (dH 7.22 and dC 123.87) as an
internal standard. Mass spectra were
obtained on Mass Agilent 1100 Series
LC-MSD-Trap-SL (ESI-MS) and 6210
ESI-TOF (HR-ESI-MS) spectrometers
(Agilent Technologies, Santa Clara, CA,
USA). Silica gel 60 (Qingdao Ocean
106.1
71.5
85.0
72.1
71.1
17.3
106.1
71.5
85.0
72.1
71.1
17.3
104.9
70.7
86.3
71.2
70.7
17.0
C-2
C-3
C-4
C-5
C-6
Glucosyl (1 ! 3)
C-1
C-2
C-3
C-4
C-5
C-6
106.2
106.3
75.3
78.4
71.7
77.3
70.2
103.9
85.5
77.5
70.7
77.3
69.5
75.3
78.4
71.7
77.3
70.2
Glucosyl (1 ! 2)
C-1
C-2
C-3
C-4
C-5
C-6
106.8
76.1
76.8
70.2
78.7
62.2

6
Y. Yuan et al.
30
29
12
26
11
25
28
CH2OH
16
OH
OH
O
3
27
O
HO
HO
O
O
O
1'
OH
O
CH2OH
24 23
1''
OH
HO
1'''
HO
OH
HO
Figure 2. The key HMBC correlations of compound 1.
29
28
30
O
12
26
11
25
16
OH
3
27
OH
O
O
HO
HO
HO
O
1'''
HO
CH2OH
23
1'
OH
O
O
O
1''
OH
Figure 3. The key HMBC correlations of compound 2.
24
OH
HO
O
OH
OH
O
O
HO
HO
HO
1''''
O
CH2OH
1'
OH
O
O
O
1''
HO
OH
HO
O
HO
HO
O
HO
1'''
OH
Figure 4. The key HMBC correlations of compound 3.

Journal of Asian Natural Products Research
7
Table 3. Inhibitory rate values of NO
production for the compounds in LPS-stimu-
lated macrophages.
from Gonghe County (3000–4000 m
elevation), Qinghai Province, China, in
August 2006 and identified by Prof.
Pengcheng Lin from Qinghai University
for Nationalities. A voucher specimen
(HMH200608A) was deposited in the
College of Life and Environmental
Science, Central University for National-
ities of China, Beijing.
MTT cell
Concentration NO growth IR
Compound
(M)
IR(%)
(%)
LPS
DEX
1
2
3
/
/
1 £ 1026
1 £ 1025
1 £ 1025
1 £ 1025
86.5
82.3
20.0
2.5
8.6
62.0
4.9
1.3
3.3 Extraction and isolation
The air-dried aerial portions of C.
pedunculatum (5.0 kg) were extracted
three times, each for 1 h, with 70% EtOH
under reflux. The combined extracts were
concentrated in vacuo, suspended in H2O,
and partitioned sequentially with pet-
roleum ether, EtOAc, and n-BuOH (satu-
rated with H2O). The n-BuOH extract
(230 g) was subjected to CC on AB-8
macroporous resin using EtOH:H2O
(20:80, 40:60, 60:40). The EtOH:H2O
(60:40) fraction (25.7 g) was chromato-
graphed over reversed-phase silica gel and
eluted in a step gradient manner with
MeOH:H2O (20:80 to 100:0) to afford 12
subfractions. Subfraction 4 (3.63 g) was
fractionated over silica gel with CHCl3:
MeOH (6:1 to 0:1) to yield a mixture
(864 mg) that was further separated by RP-
18 HPLC in MeOH:H2O (60:40) to isolate
2 (81.0 min, 2.0 ml/min, 7 mg). Subfrac-
tion 5-2 (211 mg) were purified by RP-18
HPLC in MeOH:H2O (70:30) to give 3
(34.3 min, 2.0 ml/min, 12 mg). Subfraction
8 (4.92 g) was submitted to RP-18 CC,
eluted with MeOH:H2O (40:60 to 70:30),
and further separated by RP-18 HPLC in
MeOH:H2O (65:35) to isolate 1 (98.9 min,
2.0 ml/min, 206 mg).
Chemical Co., Ltd, Qingdao, China, 200–
400 mesh), RP-18 (YMC Co., Ltd, Kyoto,
Japan, 40–60 mm), and Sephadex LH-20
(Amersham Pharmacia Biotech, Uppsala,
Sweden) were used for column chroma-
tography (CC). Precoated silica gel plates
(Yantai Chemical Industry Research Insi-
titute, Yantai, China, GF254) and precoated
RP-18F254 plates (Merck KGaA, Darm-
stadt, Germany) were used for analytical
thin-layer chromatography. HPLC was
performed using a Lab Alliance Prep-100
pump equipped with a Lab Alliance
Model-201 UV–vis detector at 210 nm
(Scientific Systems, Inc., Woburn, MA
USA) and a semipreparative reversed-
phase column (Allsphere ODS-2 5 mm,
250 mm £ 10 mm, Grace Alltech, Deer-
field, IL, USA). Analytical HPLC was
performed using SHIMADZU LC-20AT
HPLC Pump with a SPD-M20A detector at
250 nm (SHIMADZU, Corp., Tokyo,
Japan) and a COSMOSIL 5C18-AR-II
HPLC column (4.6 ID £ 150 mm) (Nacalai
Tesque, Inc., Kyoto, Japan). D-Glucose, L-
glucose, D-fucose, and L-fucose were from
Alfa Aesar A Johnson Matthey Company
(Tianjin, China), and L-cysteine methyl
ester hydrochloride and o-toly isothiocya-
nate were from J&K Scientific Ltd
(Beijing, China).
3.3.1 Comastomasaponin I (1)
White amorphous powder (MeOH–H2O);
20
½aꢀD 29.9 (c ¼ 0.032, MeOH); UV
3.2 Plant material
(MeOH) lmax (log 1) 201 (3.86), 242
(4.07), 250 (4.11), 260 (3.91) nm; IR
(microscope) nmax: 3355, 2924, 2855,
The aerial portions of C. pedunculatum
(Rogle ex D. Dou) Holub were collected

8
Y. Yuan et al.
1655, 1598, 1454, 1417, 1384, 1365, 1314,
1269, 1166, 1118, 1071 cm21; 1H and 13
reported procedure [9]. Compound 1
(2 mg) was hydrolyzed by 1 M HCl
(1 ml) at 1008C for 2 h. The reaction
mixture was dried under vacuum. After the
addition of water, the acidic solution was
evaporated to again remove HCl. After
drying under vacuum, the residue was
dissolved in pyridine (0.4 ml) containing
L-cysteine methyl ester hydrochloride
(2 mg) and heated at 608C for 2 h. o-Toly
lisothiocyanate (2 ml) was then added and
the mixture was heated at 608C for 2 h.
Each reaction mixture was directly ana-
lyzed by reversed-phase HPLC using a
SHIMADZU LC-20AT HPLC pump with
a SPD-M20A detector at 250 nm. A COS-
MOSIL packed 5C18-AR-II 4.6 ID £
150 mm HPLC column was used [temp,
358C; flow, 0.8 ml/min; eluate, CH3CN–
H2O (25:75) containing 50 mM H3PO4].
The HPLC column was washed with
MeOH after each injection. The reaction
conditions for D, L-glucose and D,
L-fucose were the same as described
above. Retention times for authentic
sugar derivatives, D-glucose (11.62 min),
L-glucose (10.61 min), and D-fucose
(15.78 min), L-fucose (17.56 min) were
used for comparison with retention times
from reaction mixtures for the saponin.
The peaks at 11.57 and 15.73 min of the
sugar derivatives from 1 coincided with
derivatives of D-glucose and D-fucose,
respectively. The absolute configuration
sugars of compounds 2 and 3 were
identified by the same method as 1.
C
NMR spectral data (Tables 1 and 2); ESI-
MS m/z 965.5 [M þ Na]þ; HR-ESI-MS
m/z 965.5094 [M þ Na]þ (calcd for
C48H78O18Na, 965.5080).
3.3.2 Comastomasaponin J (2)
White amorphous powder (MeOH–H2O);
20
½aꢀD þ 31.6 (c ¼ 0.044, MeOH); UV
(MeOH) lmax (log1) 203 (4.11) nm; IR
(microscope) nmax: 3397, 2930, 2871, 1641,
1452, 1382, 1166, 1126, 1070cm21; 1H and
13C NMR spectral data (Tables 1 and 2);
ESI-MS m/z 965.5 [M þ Na]þ; HR-ESI-
MS m/z 965.5092 [M þ Na]þ (calcd for
C48H78O18Na, 965.5080).
3.3.3 Comastomasaponin K (3)
White amorphous powder (MeOH–H2O);
20
½aꢀD þ 32.2 (c ¼ 0.045, MeOH); UV
(MeOH) lmax (log 1) 203 (3.87) nm; IR
(microscope) nmax: 3393, 2929, 2874,
1639, 1452, 1382, 1365, 1168, 1071,
1041 cm21 1H and 13C NMR spectral
;
data (Tables 1 and 2); ESI-MS m/z 1127.6
[M þ Na]þ; HR-ESI-MS m/z 1127.5618
[M þ Na]þ (calcd for C54H88O23Na,
1127.5609).
3.4 Acid hydrolysis
A solution of each compound (5 mg) in
0.20 M HCl (5 ml) was heated at 908C for
5 h. After extraction with EtOAc (3
£ 5 ml), each mixture was diluted with
H2O and extracted with EtOAc (3 £ 5 ml).
The aqueous layer was evaporated and
cryodesiccated, and the residue was
analyzed by TLC over silica gel
(CHCl3–MeOH–H2O, 6:4:1) and com-
pared with authentic samples.
3.6 Assessment of the NO inhibitory
activity of compounds 1–3
The NO inhibitory activity of compounds
1–3 was evaluated according to estab-
lished methods [14]. Adherent macro-
phages were harvested from peritoneal
cells of C57BL/6J male mice after
intraperitoneal injection of Brewer thio-
glycolate medium. Macrophages were
then seeded in 96-well plates (Costar,
Tewksbury, MA, USA) at a density of
3.5 Absolute configurations of the
sugars
The absolute configurations of glucose and
fucose were determined according to a

Journal of Asian Natural Products Research
9
1 £ 105 cells/well in RPMI-1640 sup-
Funding
plemented with 5% (v/v) newborn calf
serum (Gibco, Grand Island, NE, USA),
100 U/ml penicillin, and 100 mg/ml strep-
tomycin. The cells were cultured for 2 h
and then treated with 1 mg/ml LPS for
24 h in the presence or absence of various
concentrations of test compounds. DMSO
was used as a solvent for the compounds,
and a final concentration of DMSO of
0.1% (v/v) was present in the cell culture
medium. All incubation procedures were
performed with 5% CO2 in humidified air
at 378C. NO production was determined
by detecting the accumulation of nitrite in
the culture medium by the Griess test.
Briefly, 100 ml of the culture medium
supernatant was mixed with an equal
volume of Griess reagent (0.1% N-[1-
naphthyl] ethylenediamine and 1% sulfa-
nilamide in 5% H3PO4). Cell viability was
examined by the methylthiazolyldiphenyl
tetrazolium bromide (MTT) colorimetric
assay after 24 h of incubation with the test
compounds. All experiments were per-
formed in triplicate.
This research was financially supported by
National Science and Technology Project of
China [grant number 2012ZX09301002001003].
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Disclosure Statement
No potential conflict of interest was reported by
the author(s).