Isolation and characterization of new minor triterpenoid saponins from the buds of Lonicera macranthoides

Article abstract of DOI:10.1016/j.carres.2013.01.019

Three new minor compounds, 18-oleanene (1), lupine (2), and 12-oleanene (3) type triterpenoid saponins, along with four major known triterpenoid saponins were isolated from the buds of Lonicera macranthoides. Their structures were elucidated by spectrosco

Full text of DOI:10.1016/j.carres.2013.01.019

Carbohydrate Research 370 (2013) 76–81
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Isolation and characterization of new minor triterpenoid saponins
from the buds of Lonicera macranthoides
a
b
a
c
a,
⇑
Jiang Liu , Jing Zhang , Feng Wang , Yuan-feng Zou , Xing-fu Chen
a
Division of Pharmaceutical Botany, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, PR China
College of Horticulture, Sichuan Agricultural University, Ya’an 625014, PR China
School of Pharmacy, University of Oslo, PO Box 1068, Blindern, 0316 Oslo, Norway
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 October 2012
Received in revised form 21 January 2013
Accepted 24 January 2013
Available online 4 February 2013
Three new minor compounds, 18-oleanene (1), lupine (2), and 12-oleanene (3) type triterpenoid
saponins, along with four major known triterpenoid saponins were isolated from the buds of Lonicera
macranthoides. Their structures were elucidated by spectroscopic and chemical analyses. 18-oleanene
and lupine-type triterpenoid saponins were first isolated from this plant. The isolated compounds were
evaluated for their cytotoxic activities against HSC-2 cells.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Lonicera macranthoides
Caprifoliaceae
Triterpenoid saponin
1
Lonicera is one of the most important genera in the caprifolia-
ceae family of honeysuckle plants. Four species, including Lonicera
macranthoides, L. hypoglauca, L. confusa, and L. fulvotomentosa, were
recorded in the Chinese Pharmacopoeia (2010 edition);1 these
species were commonly known as Lonicerae Flos (Shanyinhua in
Chinese). Lonicerae Flos has long been used in China and Southeast
Asia to treat acute fever, headache, pharyngodynia, respiratory
infection, pyocutaneous disease, and epidemic disease.2 Many
studies concerning the chemical constituents of L. macranthoides
have been conducted, leading to the isolation of a series of
secondary metabolites, including phenolic acids, iridoids, and
of the D NMR spectra of 1 and a comparison between 1 and 4 indi-
cated that a linear tetra-glycoside and a linear bi-glycoside at-
tached to C-3 and C-28 of the aglycon, respectively, were
identical to those of 4. However, 1 differed from 4 in terms of
the locus of an olefinic group in the aglycon moiety, since the ole-
C C
finic carbon was observed at d 134.0 and d
138.4 in the 13C NMR
spectrum of 1. In the HMBC spectrum of 1, the olefinic proton at d
5.16 showed a long-range correlation with the quaternary carbon
at d 33.0 assignable to C-20, with which the methyl protons due
to Me-29 at d 1.02 and Me-30 at d 1.00 in turn exhibited long-range
correlations. These spectral data provided evidence for the pres-
–5
6
–11
18(19)
2
saponins.
In this article, we present the isolation and structure
ence of a double bond at
D
. Analysis of D NMR data (HMQC,
elucidation of these new minor triterpenoid saponins based on
extensive spectroscopic and chemical studies.
The methanol extract of the dried buds of L. macranthoides was
successively chromatographed on macroporousresin, ODS, Sepha-
dex LH-20 CC, and finally purified by preparative HPLC to afford
three new triterpenoid saponins (1–3) and four known congeners
DQF, and HMBC), the aglycon part of 1 was revealed to be a triter-
pene of 3,23-dihydroxyolean-18-en-28-oic acid. The monosaccha-
ride residues in the glycosidic part of 1 were assigned by HPLC
analyses with authentic sugars after acid hydrolysis of 1 and opti-
cal rotation measurement for each isolated residue, indicating that
the sugar part was composed of
(Ara), and -glucose (Glc). In addition, the b-anomeric configura-
tion of the glucose unit was determined by the
constant (7.6–8.3 Hz), and arabinose and rhamnose were deter-
mined as the -configurations by the pseudo triplet and broad sin-
L-rhamnose (Rha), L-arabinose
(Fig. 1). Compounds 1, 2, and
3
were determined to be
D
3
C
65
H
106
O
32 on the basis of a quasi-molecular ion at m/z 1421.65
JH1, H2 coupling
+
[
M+Na] by HR-ESI-MS. The IR spectrum showed strong absorption
ꢀ1
bands at 3388 and 1727 cm , typical for the hydroxyl and car-
bonyl groups, respectively.
a
glet of their anomeric protons. In the HMBC experiment (Fig. 2),
All signals observed in the upfield (d
and 1.04) and midfield (d 5.16) regions of the H NMR spectrum
Tables 1 and 2) suggested 1 to be a triterpene glycoside. Analysis
H
0.71, 0.84, 0.97, 1.00, 1.02,
the downfield-shifted carbon at C-3 of the aglycon showed long-
range correlation with the proton at d 4.52 (H-1) of the Ara resi-
H
due, of which C-2 was in turn connected to the H-1 of Rha via an
ether linkage from the correlations between the anomeric proton
1
H
(
at d
HMBC correlation between the H-1 (d
82.9) of Rha, as well as that between H-1 (d
H
5.22 (H-1) of Rha and C-2 (d
C
76.7) of Ara unit. An additional
4.56) of Glu I and C-3 (d
4.43) of Glu II and
⇑
Corresponding author. Tel.: +86 28 86290971; fax: +86 28 86290870.
E-mail address: chenxf.64@sohu.com (X. Chen).
H
C
H
0
008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.019

J. Liu et al. / Carbohydrate Research 370 (2013) 76–81
77
3
0
29
2
9
30
21
2
9
30
21
2
0
1
9
20
19
2
1
19
18
13 17
H
20
12
1
8
22
12
12
26
1
8
22
22
1
0
1
O
1
3
17
16
11
11
2
5
13
O
O
2
6
25
17
16
25
1
4
14
28
26
1
4
14
28
1
4
28
1
4
2
3
9
9
2
3
9
16
1
8
15
OR2
2
3
OR2
OR2
1
0
8
15
10
8
15
27
27
27
7
5
R O
7
7
1
5
5
6
R1O
R1O
6
6
24
23
2
4
23
24
23
HO
1
2
3-7
HO
HO
Figure 1. The Structures of compounds 1–7.
Table 1 (continued)
Table 1
3 3
NMR data (CD OD-d
, d in ppm)a for aglycon moieties of compounds 1–3
No.
1
2
3
No.
1
2
3
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
29
1.02 (s)
30.9
29.4
4.59 (br s) 110.3 0.90 (s)
.72 (br s)
1.69 (s)
33.5
4
1
2
0.93
.72
1.76
.89
40.1
0.93
1.70
1.76
1.86
3.59
39.9
0.93
1.72
1.76
1.89
3.61
39.8
30
1.00 (s)
19.6
0.93(s)
24.1
1
26.8
26.8
26.4
Assignments were assigned on the basis of DQF, HMQC, HMBC, and ROESY spectra.
1
a
Signals are designated as follows: s, singlet; br s, broad singlet; d, doublet; dd,
doublet of doublets; overlapped signals were reported without designating
multiplicity.
3
4
5
6
3.61
82.4
44.1
48.6
18.7
82.4
44.1
48.4
18.8
82.3
44.0
48.6
18.8
1.21
1.36
1.16
1.31
1.42
1.43
1.21
1.36
1.45
1.43
1.45
C
C-4 (d 80.2) of Glu I completed the linkage of the tetra-saccharide
chain at C-3 of the aglycon part. Followed by the same analysis, the
28-O-bi-glycoside structure of 1 was characterized on the basis of
7
8
9
1
1
1.43
1.35
1.37
35.2
41.9
52.6
37.9
22.2
34.9
42.0
52.0
37.8
22.1
34.9
40.7
48.2
37.6
24.1
1.32
1.35
0
1
the HMBC cross-peaks between H-1 (d
H
5.50) of Glc III and C-28 (d
77.0) of the aglycon part, as well as those between H-1 (d 4.34)
69.7) of Glc III. The unequivocal sequence of
C
1.28
1.62
1.25
1.65
2.32
1.03
1.83
5.24 (br s)
1
H
1
.62
1.25
.57
of Glc IV and C-6 (d
C
1
2
27.2
26.9
123.8
1
saccharide chains was also supported by ROESY peaks between
the anomeric protons and the proton in the adjacent residue. Thus,
the structure of 1 was determined as 3-O-b-
1?4)-b- -glucopyranosyl-(1?3)- -rhamnopyranosyl-(1?2)-
-arabinopyranosyl-23-hydroxyolean-18-en-28-oic acid O-b- -glu-
copyranosyl-(1?6)-b- -glucopyranosyl ester. Compared with
common bisdesmosidic saponins with saccharide chains at C-3
and C-28 of the aglycon, the aglycon of 1 possessed a rare olean-
8-en moiety. This was the first reported occurrence of an olean-
18-en-type saponin in the family caprifoliaceae.
1
1
1
3
4
5
2.28(d,12.4) 42.3
48.8
39.4
43.6
30.8
145.0
42.6
28.9
D-glucopyranosyl-
1.18
.70
1.60
.02
30.5
1.19
1.71
1.42
2.63
0.92
1.28
1.65
1.87
(
L
D
a-
L
a-
1
1
6
34.1
32.9
24.6
D
2
D
1
1
7
8
50.5
138.3
57.9
50.6
48.1
43.0
1.7
2.85
(
dd,3.5,13.1)
1.17
.25
1
1
9
5.16 (br s)
1.33
134.0
3.38
48.3
47.3
1
1
2
2
0
1
33.0
34.5
151.8
31.5
31.6
33.4
The H NMR spectrum (Tables 1 and 2) of 2 clearly showed the
1.46
2.2
1.48
2.16
3.32
3.52
0.59 (s)
0.87 (s)
0.95 (s)
1.00 (s)
1.25
1.55
1.27
1.54
3.33
3.51
0.70 (s)
0.97 (s)
0.79 (s)
1.16 (s)
presence of five tertiary methyl groups at d
H
0.59, 0.87, 0.95, 1.00,
4.59 and 4.72. In addi-
tion, the C NMR spectrum of 2 contained a hydroxymethyl car-
bon at d 82.4 (C-3), two olefinic carbons at d 151.8 (C-20) and
10.3 (C-29), and a carboxylic carbon at d 176.3 (C-28). The H
1
.44
1.45
.22
3.33
.51
and 1.69 and one exo-methylene group at d
H
2
2
2
3
34.6
64.6
37.6
64.5
33.2
64.5
1
3
2
C
C
3
1
1
C
2
2
2
2
2
4
5
6
7
8
0.71 (s)
0.97 (s)
1.04 (s)
0.84 (s)
13.6
17.9
16.8
15.6
13.5
17.3
16.7
15.1
176.3
13.8
16.6
17.9
26.4
178.1
13
and C NMR spectra indicated that 2 was a 23-hydroxybetulinic
acid derivative by comparing its spectroscopic data with previ-
ously published data.
gested the presence of six sugar residues, which was the same as 1.
Following the same analysis approach as used for 1, the glycoside
1
2–14
1
The D NMR spectroscopic data also sug-
177.00

7
8
J. Liu et al. / Carbohydrate Research 370 (2013) 76–81
Table 2
3 3
NMR data (CD OD-d
, d in ppm)a for sugar moieties of compounds 1–3
No.
1
2
3
dH
dC
dH
dC
dH
dC
C-3: Ara
Ara
Xyl
1
2
3
4
5
4.52 (pt, 2.8)
3.68
3.69
3.75
3.51 (m)
104.9
76.7
74.1
69.8
65.7
1
2
3
4
5
4.48 (pt, 2.8)
3.68
3.69
104.9
76.7
74.1
69.8
65.7
1
2
3
4
5
4.56 (d, 7.6)
3.60
3.63
3.42
3.28
3.82 (m)
Rha-(1?2Xyl)
5.32 (br s)
4.10
3.83
3.56
105.7
78.6
75.1
71.6
66.7
3.75
3.51 (m)
3.84 (m)
Rha-(1?2Ara)
5.18 (br s)
4.07
3.83
3.56
3.92
1.23 (d,6.2)
Glu I-(1?3Rha)
4.53 (d,8.2)
3.25
3.55
3.68
3.38
3.91 (m)
3.70 (m)
3.84 (m)
Rha-(1?2Ara)
1
2
3
4
5
6
5.22 (br s)
4.07
3.83
3.56
3.92
101.6
71.6
82.9
72.6
70.0
18.1
1
2
3
4
5
6
101.6
71.6
82.9
72.6
70.0
18.1
1
2
3
4
5
6
101.6
71.8
83.0
72.5
70.1
18.1
4.25
1.28 (d,6.2)
1.24 (d,6.2)
Glu I-(1?3Rha)
4.56 (d,7.6)
3.25
3.55
3.57
3.38
3.88 (m)
3.66 (m)
Glu I-(1?3Rha)
1
2
3
4
5
6
4.56 (d,7.6)
3.25
3.55
3.68
3.38
3.91 (m)
105.6
75.1
76.1
80.2
78.1
61.6
1
2
3
4
5
6
105.6
75.1
76.1
80.2
78.1
61.6
1
2
3
4
5
6
105.6
75.1
76.1
80.2
78.2
61.6
3.70 (m)
Glu II-(1?4Glu I)
Glu II-(1?4Glu I)
4.40 (d,8.2)
3.27
3.56
3.50
Glu II-(1?4Glu I)
4.43 (d,7.6)
3.27
3.56
3.50
1
2
3
4
5
6
4.43 (d,8.3)
3.27
3.56
3.50
3.47
3.68 (m)
104.6
74.9
76.4
71.1
78.4
62.4
1
2
3
4
5
6
104.6
74.9
76.4
71.1
78.4
62.4
1
2
3
4
5
6
104.6
74.9
76.4
71.0
78.6
62.4
3.47
3.47
3.68 (m)
3.86 (m)
Glc III
3.684 (m)
3.86 (m)
Glc III
3.86 (m)
C-28: Glc III
1
2
3
4
5
6
5.50 (d,8.2)
3.40
3.37
3.49
3.44
95.8
75.2
78.0
70.9
77.8
69.7
1
2
3
4
5
6
5.46 (d,8.2)
3.40
3.37
3.49
3.44
2.80 (m)
4.13 (dd,1.4,11.6)
Glc IV-(1?6Glc III)
4.33 (d,7.6)
3.36
3.50
3.32
3.47
3.37 (m)
3.85 (m)
95.2
75.2
78.0
70.9
77.8
69.7
1
2
3
4
5
6
5.37 (d,7.6)
3.40
3.37
3.49
3.44
2.80 (m)
4.11 (dd,1.4,11.6)
Glc IV-(1?6Glc III)
4.36 (d,7.6)
3.36
3.50
3.32
3.47
3.60 (m)
3.90 (m)
95.8
75.1
78.0
71.0
77.8
69.5
2.80 (m)
4.16 (dd,2.1,11.7)
Glc IV-(1?6Glc III)
1
2
3
4
5
6
4.34 (d,8.3)
3.36
3.50
3.32
3.47
3.37 (m)
105.0
74.0
77.9
71.4
78.1
62.8
1
2
3
4
5
6
104.7
74.0
77.9
71.4
78.1
62.8
1
2
3
4
5
6
104.7
73.9
77.9
71.4
78.1
62.8
3.85 (m)
Assignments were assigned on the basis of DQF, HMQC, HMBC, and ROESY spectra.
a
Signals are designated as follows: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; pt, pseudotriplet; m, multiplet; overlapped signals were reported
without designating multiplicity.
structure of 2 was characterized on the basis of the HMQC, HMBC,
and ROESY results. Taking into consideration that -arabinose,
-rhamnose, and -glucose were identified in the hydrolysate of
the corresponding glycosidic fraction, the structure of 2 was
assigned as a newly isolated lupine glycoside, 3-O-b- -glucopyran-
osyl-(1?4)-b- -glucopyranosyl-(1?3)- -rhamnopyranosyl-(1?2)-
-arabinopyranosyl]-23-hydroxyolean lup-20(29)-en-28-oic acid
O-b- -glucopyranosyl-(1?6)-b- -glucopyranosyl ester.
The H NMR spectrum (Tables 1 and 2) of 3 showed the exis-
tence of six methyl groups of aglycon by signals at d 0.70, 0.79,
.90, 0.93, 0.97, and 1.16. The double-bond signals at d 123.8
singlet to doublet (d
downshifted from d 104.9 to d
-rhamnose, -glucose, and -xylose (Xyl) as the carbohydrate moi-
eties, which indicated that the Ara was replaced by a Xyl in 3. From
the result of the HMBC experiment (Fig. 2), the downfield-shifted
carbon at C-3 of the aglycon showed long-range correlation with
H
4.47, d, J = 7.6 Hz), and C-1 in Ara
L
C
C
105.7. Acid hydrolysis of 3 yielded
L
D
L
D
D
D
D
a-L
a
-
L
the proton at d
in turn connected to the H-1 of Rha via an ether linkage from the
correlations between the anomeric proton at d 5.32 (H-1) of Rha
and C-2 (d 78.6) of Xyl unit. Followed by the same analysis, other
H
4.47 (H-1) of the Xyl residue, of which C-4 was
D
D
1
H
H
C
0
C
parts of the glycoside structure of 3 were characterized on the
1
3
2
and 145.0 in the C NMR spectrum indicated that aglycon was
basis of the D NMR data. Cumulatively, on the basis of above
1
2
type, and the 1D NMR spectrum of the aglycon
an oleanane
D
evidences, the structure of 3 was established as 3-O-b-
anosyl-(1?4)-b- -glucopyranosyl-(1?3)- -rhamnopyranosyl-
(1?2)-b- -xylopyranosyl]-23-hydroxyolean hederagenin O-b-
glucopyranosyl-(1?6)-b- -glucopyranosyl ester.
The other four known compounds 4–7 were identified as Macr-
D-glucopyr-
1
moiety of 1 was the same as 4. The D NMR spectroscopic data sug-
gested the presence of six sugar residues. Comparison of the chem-
ical shifts and coupling values indicated that most of the anomeric
signals of 3 were similar to 4, except the Ara and Rha parts (Fig. 3).
Specifically, the proton peak of H-1 in Ara was changed from broad
D
a-L
D
D-
D
1
0
3
15
anthoidin B (4), Macranthoidin A (5), Dipsacoside B (6), and

J. Liu et al. / Carbohydrate Research 370 (2013) 76–81
79
O
OH Ara
O
O
HO
O
O
OH
OH
O
OH
HO
O
O
O
O
HO
Glc-III
HO
HO
O
O
OH
Rha
HO
Glc-IIOH
OH
O
HO
OH
Glc-I
OH
HO
Glc-IV
1
HO
OH
O
O
OH Ara
O
O
O
OH
Glc-III
HO
O
OH
OH
HO
O
O
HO
HO
O
O
HO
OH
HO
HO
O
O
O
RhaOH
OH
Glc-IV
HO
Glc-IIOH
Glc-I
OH
HO
OH
2
O
O
Xyl
HO
HO
HO
O
O
O
OH
OH
O
OH
Glc-III
O
O
O
O
O
HO
HO
HO
O
OH
Rha
HO
Glc-IIOH
HO
OH
Glc-I
OH
O
OH
Glc-IV
HO
3
HO
OH
Figure 2. Selected key HMBC correlations for compounds 1–3.
Leiyemudanoside A (7)16 by comparing the NMR data with those
reported in the literature.
The isolated compounds were evaluated for their cytotoxic
activities against HSC-2 cells. Only compound 2, a lupine-type tri-
terpenoid saponin, showed weak cytotoxic activity against HSC-2
chromatograph; Shimadzu RID-10A refractive index detector; Pre-
parative column: YMC-Pack ODS-A (250 ꢁ 20 mm i.d., S–5
lm,
12 nm).
1.2. Plant material
cells with the IC50 values of 68.6
were less active in the HSC-2 cell line (the IC50 >100
l
M. However, other compounds
M).
l
The buds of Lonicera macranthoides were collected in 2011 from
Suining, Sichuan province, China; the plant was identified by
Professor Xingfu Chen (Sichuan Agricultural University, China). A
voucher specimen of this plant was deposited in the research lab-
oratory of College of Agronomy, Sichuan agricultural University.
1
1
. Experimental
.1. General methods
1
.3. Extraction and isolation
The following instruments were used to obtain physical data:
JEOL spectrometer (600 MHz for 1H NMR, 150 MHz for C NMR)
and ESI-MS spectra that recorded with Agilent 6510 Q-TOF LC/
MS apparatus. The following instrumental conditions were used
for column chromatography: Diaion HP-20, Chromatorex ODS
DM1020T, and Sephadex LH-20. Shimadzu LC-6AD liquid
13
The buds of L. macranthoides (2.0 kg) were extracted three times
with MeOH under reflux for 3 h. Evaporation of the solvent under
reduced pressure provided the MeOH extract (202.1 g). MeOH ex-
tract was passed through Diaion HP-20 column chromatography

8
0
J. Liu et al. / Carbohydrate Research 370 (2013) 76–81
Figure 3. Comparison of anomeric NMR Spectroscopic data (A: 1H NMR, B: 13C NMR) between compounds 3 and 4.
and twice sequentially eluted with H
O-eluted fraction (80.5 g) and MeOH-eluted fraction (120.5 g).
MeOH-eluted fraction (120.5 g) was chromatographed on an
ODS column using gradient of MeOH/H (20:80?40:
0?60:40?80:20)?MeOH to yield 15 fractions (Fraction 1–15).
2
O and MeOH to give
2
using [MeOH/H O (65:35)] as the solvent to afford compounds 1
(3.0 mg), 2 (4.5 mg), 3 (2.6 mg), 4 (98.7 mg), 5 (11.5 mg), 6
(12.2 mg), and 7 (2.3 mg).
H
2
a
2
O
6
1.4. Determination of absolute configurations of sugars
Part of fraction 13 (2.0 g) was purified over Sephadex LH-20,
eluting with MeOH to yield 3 fractions (Fraction 13A–13C). Then
fraction 13A was separated by reversed-phase preparative HPLC
Solutions of compound 1–3 (1.0 mg) in 5% H
(1:1, v/v, 1.0 ml) were heated under reflux for 1 h (80 °C). After
2 4
SO –1, 4-dioxane

J. Liu et al. / Carbohydrate Research 370 (2013) 76–81
81
cooling, the reaction mixture was neutralized with Amberlite IRA-
and the cell growth was evaluated using an MTT reduction assay
procedure.
ꢀ
17
4
00 (OH form) and the resin was removed by filtration. On re-
moval of the solvent from the filtrate under reduced pressure,
the residue was subjected to HPLC analysis under the following
Acknowledgment
conditions: HPLC column, Kaseisorb LC NH
i.d. ꢁ 250 mm; detection, optical rotation [Shodex OR-2; Mobile
phase, CH CN–H O (17:3, v/v); flow rate 1.0 mL/min]. Identifica-
tion of sugar present in the aqueous layer was carried out by com-
parison of their retention time with those of authentic samples; t
were 6.3 min ( -rhamnose, negative optical rotation), 6.7 min
-xylose, positive optical rotation), 7.1 min ( -arabinose, positive
-glucose, positive optical rota-
2
-60-5, 4.6 mm
This study was funded by a Grant from Sichuan Provincial Crop
breeding research project Application in the 12th Five-Year Period
(No. 2011NZ0098-12-01).
3
2
R
L
Supplementary data
(D
L
optical rotation), and 11.6 min (
tion), respectively.
D
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
0
1.019.
1
1
.5. Identification
References
.5.1. Compound 1
Amorphous powder, ½
2
D
5
a
ꢂ
–21.0° (c = 0.1, MeOH). High-resolu-
1. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic
of China; People’s Medical Publishing House: Beijing, 2010; Vol. 1, pp 28–29.
+
tion positive-ion ESI-MS: Calcd for
65 106
C H O32Na (M+Na)
2
.
Chen, M.; Wu, W. W.; Shen, G. Q.; Luo, S. Q.; Li, H. T. Yaoxue Xuebao 1994, 29,
17–620.
3. Mao, Q.; Cao, D.; Jia, X. S. Yao Xue Xue Bao 1993, 28, 273–281.
ꢀ1
1
1
421.6532. IR (film, cm ): 3388(OH), 2934(CH), 1727 (C@O).
H
6
1
3
NMR (600 MHz, CD
3
OD-d
3
), see Tables
1
and 2;
C
NMR
4
5
.
.
Shi, J.; Chen, X.; Wan, L. Zhongguo Zhongyao Zazhi 1999, 24, 363–364.
Wang, J.; Zhao, X. Z.; Qi, Q.; Tao, L.; Zhao, Q.; Mu, R.; Gu, H. Y.; Wang, M.; Feng,
X.; Guo, Q. L. Food Chem. Toxicol. 2009, 47, 1716–1721.
(
150 MHz, CD OD-d ) see Tables 1 and 2.
3 3
1
.5.2. Compound 2
Amorphous powder, ½
tion positive-ion ESI-MS: Calcd for
6. Chen, C. Y.; Qi, L. W.; Li, H. J.; Li, P.; Yi, L.; Ma, H. L.; Tang, D. J. Sep. Sci. 2007, 30,
2
D
5
3181–3192.
a
ꢂ
ꢀ38.2° (c = 0.1, MeOH). High-resolu-
+
7.
Chen, Y.; Zhao, Y.; Wang, M.; Sun, H.; Dong, Y.; Feng, X. Chem. Nat. Compd. 2012,
7, 940–943.
65 106
C H O32Na (M+Na)
4
ꢀ1
1
1
421.6534. IR (film, cm ): 3387(OH), 2939(CH), 1732 (C@O).
H
8. Chen, Y.; Shan, Y.; Zhao, Y. Y.; Wang, Q. Z.; Wang, M.; Feng, X.; Liang, J. Y. Chin.
Chem. Lett. 2012, 23, 325–328.
1
3
NMR (600 MHz, CD
3 3
OD-d ), see Tables
1
and 2;
C
NMR
9.
Chen, Y.; Feng, X.; Wang, M.; Jia, X. D.; Zhao, Y. Y.; Dong, Y. F. Chem. Nat. Compd.
009, 45, 514–518.
(
150 MHz, CD OD-d ) see Tables 1 and 2.
3 3
2
1
1
0. Chen, M.; Luo, S.; Li, H. Chin. Chem. Lett. 1990, 1, 219–220.
1. Chai, X. Y.; Li, S. L.; Li, P. J. Chromatogr. A 2005, 1070, 43–48.
1
.5.3. Compound 3
Amorphous powder, ½
tion positive-ion ESI-MS: Calcd for
2
D
5
12. Yang, H.; Cho, Y. W.; Kim, S. H.; Kim, Y. C.; Sung, S. H. Phytochemistry 2010, 71,
892–1899.
13. Mimaki, Y.; Yokosuka, A.; Kuroda, M.; Hamanaka, M.; Sakuma, C.; Sashida, Y. J.
Nat. Prod. 2001, 64, 1226–1229.
a
ꢂ
ꢀ12.4° (c = 0.2, MeOH). High-resolu-
+
1
C
65
106
H O
32Na (M+Na)
ꢀ1
1
1
421.6522. IR (film, cm ): 3387(OH), 2939(CH), 1732 (C@O).
H
1
3
14. Ye, W.; Zhang, Q.; Hsiao, W. W.; Zhao, S.; Che, C. T. Planta Med. 2002, 68, 183–
NMR (600 MHz, CD
3 3
OD-d ), see Tables
1
and 2;
C
NMR
186.
(
150 MHz, CD OD-d ) see Tables 1 and 2.
3 3
1
1
5. Chen, J.; Xu, X. F.; Chai, X. Y.; Li, P. Zhongguo Tianran Yaowu 2006, 4, 347–351.
6. Jia, X.; Feng, X.; Dong, Y.; Zhao, X.; Wang, M.; Zhao, Y.; Sun, H. Zhongcaoyao
2
007, 38, 1452–1455.
1
.6. Bioassays for the cytotoxic activity
1
7. Ito, H.; Kobayashi, E.; Takamatsu, Y.; Li, S. H.; Hatano, T.; Sakagami, H.; Kusama,
K.; Satoh, K.; Sugita, D.; Shimura, S.; Itoh, Y.; Yoshida, T. Chem. Pharm. Bull.
2000, 48, 687–693.
HSC-2 cells were incubated in DMEM supplemented with 10%
heat-inactivated FBS for 24 h in the presence of each glycoside,