Two new glycosides from the whole plant of Anemone rivularis var. flore-minore

Article abstract of DOI:10.1016/j.phytol.2012.07.003

Phytochemical investigation on the whole plant of Anemone rivularis var. flore-minore led to the isolation of a new labdane-type diterpene glycoside (1) and a new trihydroxyfuranoid lignanoid glycoside (2), together with three known triterpene and triterpenoid glycosides (3-5). The structures of the two new compounds were elucidated as β-d-glucopyranosyl (13S)-13-hydroxy-7-oxo- labda-8,14-diene-18-oate (1) and (7S,7′R,8R,8′S)-7′-butoxy-7, 9′-epoxy-4,4′,9-trihydroxy-3,3′-dimethoxylignane 9-O-β-d-glucopyranoside (2), on the basis of extensive spectral analysis and chemical evidence. Compound 1 is characterized by a glucose (Glc) esterified C-18 carboxyl group, which is a rarely encountered labdane-type diterpene glycoside in nature. The two new compounds (1 and 2) reported here are the first examples of diterpene glycoside and lignanoid glycoside found in the genus Anemone, and the known triterpene and triterpenoid glycosides (3-5) are identified for the first time from the title plant.

Full text of DOI:10.1016/j.phytol.2012.07.003

Phytochemistry Letters 5 (2012) 668–672
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Two new glycosides from the whole plant of Anemone rivularis var. flore-minore
a,c,1
a,b,1
a,
b
a
a
Yu Ding
, Xiang-Rong Tian
, Hai-Feng Tang *, Jun-Tao Feng , Wei Zhang , Wen-Li Hai ,
a
a
Xiao-Yang Wang , Yi Wang
a
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, PR China
b
Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Research & Development Center of Biorational Pesticide, Northwest A & F University,
Yangling 712100, PR China
c
Department of Pharmacy, Dalian Sanatorium of Shenyang Military Region, Dalian 116013, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Phytochemical investigation on the whole plant of Anemone rivularis var. flore-minore led to the isolation
of a new labdane-type diterpene glycoside (1) and a new trihydroxyfuranoid lignanoid glycoside (2),
together with three known triterpene and triterpenoid glycosides (3–5). The structures of the two new
Received 9 May 2012
Received in revised form 25 June 2012
Accepted 3 July 2012
compounds were elucidated as
b-D-glucopyranosyl (13S)-13-hydroxy-7-oxo-labda-8,14-diene-18-oate
Available online 17 July 2012
0 0 0 0 0 0
(
1) and (7S,7 R,8R,8 S)-7 -butoxy-7,9 -epoxy-4,4 ,9-trihydroxy-3,3 -dimethoxylignane 9-O-b-D-gluco-
pyranoside (2), on the basis of extensive spectral analysis and chemical evidence. Compound 1 is
characterized by a glucose (Glc) esterified C-18 carboxyl group, which is a rarely encountered labdane-
type diterpene glycoside in nature. The two new compounds (1 and 2) reported here are the first
examples of diterpene glycoside and lignanoid glycoside found in the genus Anemone, and the known
triterpene and triterpenoid glycosides (3–5) are identified for the first time from the title plant.
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Anemone rivularis var. flore-minore
Diterpene glycoside
Lignanoid glycoside
Triterpenoid glycoside
1
. Introduction
The genus Anemone (Ranunculaceae) consists of about 150
2011a,b). As part of our ongoing search for new bioactive natural
products from the plant, a new diterpene glycoside (1), a new
lignanoid glycoside (2), along with three known triterpene and
triterpenoid glycosides (3–5) were isolated. To the best of our
knowledge, the present study is the first example of the occurrence
of diterpene glycoside and lignanoid glycoside in genus Anemone,
and all of the known compounds were identified for the first time
from this species. Herein, we report the isolation and structural
elucidation of the two new glycosides and three known
compounds from the whole plant of A. rivularis var. flore-minore
(Fig. 1).
species with a near global distribution, of which about 50 species
were found in the northwest and southwest of China (Wang, 1980).
Anemone is well known as a rich source of glycosides, especially
triterpenoid glycosides that showed a range of biological and
pharmacological activities including antitumor, antiperoxidation,
antibacterial, insect deterrence, etc. (Wang et al., 2011; Chen et al.,
2
009; Sun et al., 2008; Wang et al., 1998). Anemone rivularis var.
flore-minore Maxim. is a perennial herbaceous plant distributed
mainly in the north and south slopes of Tsinling Mountains,
Shaanxi province of China. The entire plants and rhizome of this
species have been used as folk medicines for treatment of hepatitis,
muscle and joint pain, stranguria, emission, edema, etc. (Wang,
2. Results and discussion
Compound 1 was obtained as a hygroscopic amorphous
1
980). Our previous chemical investigations on this species have
powder. The positive ion mode HR-ESI-MS showed a pseudomo-
+
led to the isolation of a new structurally interesting gypsogenin
type triterpenoid saponin, as well as several known oleanolic acid
and hederagenin type triterpenoid saponins, which are meaningful
for the chemotaxonomy of A. rivularis var. flore-minore (Ding et al.,
lecular ion peak at m/z 519.2573 [M+Na] (calcd. for C26
H
40
O
9
Na,
5
19.2570), which enabled the determination of the molecular
1
40 9
formula as C26H O with a degree of unsaturation as seven. The H
NMR spectrum showed four tertiary methyl proton signals at
.00 (3H, s, H -17), 1.50 (3H, s, H -16), 1.25 (3H, s, H -19) and 1.24
3H, s, H -20), one vinyl group signals at 5.20 (1H, dd, J = 10.7,
-15), 5.59 (1H, dd, J = 17.2, 1.9 Hz, H -15), and 6.14
d
H
2
3
3
3
(
3
d
H
1.9 Hz, H
a
b
d
H
*
Corresponding author at: Department of Pharmacy, Xijing Hospital, Fourth
(
1H, dd, J = 17.2, 10.7 Hz, H-14), and one Glc anomeric proton
Military Medical University, 127 Changle West Rd., Xi’an 710032, PR China. Tel.: +86
0
13
signal at
d
H
6.29 (1H, d, J = 8.1 Hz, Glc H-1 ). The C NMR and DEPT
2
1
9 84775471; fax: +86 29 84775471.
E-mail address: tanghaifeng71@163.com (H.-F. Tang).
spectra gave 26 carbon signals including a tetrasubstituted double
bond 130.3 (C-8) and 166.5 (C-9), a conjugated carbonyl 198.8
1
Both authors contributed equally.
d
C
d
C
874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2012.07.003

Y. Ding et al. / Phytochemistry Letters 5 (2012) 668–672
669
Fig. 1. Structures of compounds 1–5 from Anemone rivularis var. flore-minore.
(
1
C-7), a carboxyl or ester function
46.4 (C-14) and 111.9 (C-15), a quaternary carbon
linked with a hydroxyl group, and a sugar unit ( 95.8, 74.2, 78.9,
1.1, 79.4, 62.2). These H and C NMR signals were assigned with
d
C
175.9 (C-18), a vinyl group
d
C
the optical rotation of the methanolysis product of 1, methyl 13(S)-
2
3
d
C
72.5 (C-13)
13-hydroxy-7-oxo-labda-8,14-diene-18-oate
([a
]
D
+24.08),
d
C
which was close to those of the synthetic labdane-type diterpene
1
13
22
7
13-epi-isomanool ([
a
]
D
+27.58), while its 13(R) isomer isomanool
+73.58) showed a huge difference of the optical rotation
(Bigley et al., 1960). Based on the above analysis, compound 1 was
undoubtedly deduced as -glucopyranosyl (13S)-13-hydroxy-7-
1
1
22
the aid of H– H COSY, HSQC and HMBC spectra as shown in Table
, and they suggested that compound 1 was a labdane-type
([a
]
D
1
13
diterpene glycoside. The key C NMR data of 1 were similar to
those of 13-hydroxy-7-oxo-labda-8,14-diene (Wu and Lin, 1997),
and the difference was a Glc esterified C-18 carboxyl group as
b-D
oxo-labda-8,14-diene-18-oate (1).
Compound 2 was obtained as a hygroscopic amorphous
substitute for the C-18 tertiary methyl. The HMBC correlations
powder. The molecular formula of 2 was deduced as C30
42 12
H O
0
from
d
H
1.04 (H -3), 1.86 (H-5), 1.25 (H
3
-19) and 6.29 (Glc H-1 ) to
according to the positive HR-ESI-MS from the pseudomolecular ion
a
+
d
C
175.9 (C-18) proved this assignment (Fig. 2).
peak at m/z 617.2570 [M+Na] (calcd. for C30
H
42
O
12Na, 617.2574).
1
The presence of Glc unit in compound 1 was established by
In the H NMR spectrum of 2, two sets of 1,3,4-trisubstituted
methanolysis followed by TLC analysis of the corresponding 1-O-
benzene ring signals at
H-5, H-2 ), 7.30 (1H, dd, J = 8.1, 1.7 Hz, H-6), 7.19 (1H, m, H-5 ) and
7.02 (1H, dd, J = 8.0, 1.8 Hz, H-6 ), two methoxy singlets at
d
H
7.40 (1H, d, J = 1.7 Hz, H-2), 7.22 (2H, m,
0
0
methyl carbohydrate derivative (Tian et al., 2009a), along with its
1
13
0
H and C NMR data in accordance with terminal Glc esterified
d
H
3.75
0
with a carboxy group (Shao et al., 1995; Xiong et al., 2001). The Glc
was determined to be D-configuration by acidic hydrolysis
followed by comparison of the GC retention times of the
corresponding trimethylsilylated hydrolysate with those of the
authentic samples prepared in the same manner (Tian et al.,
(3H, s, 3-OCH
3.26 (1H, m, H
3
) and 3.83 (3H, s, 3 -OCH
3
), one butoxy substituent at
00
00
00
d
H
a
-1 ), 3.43 (1H, m, H
b
-1 ), 1.50 (2H, m, H
2
-2 ), 1.31
0
0
00
(2H, m, H
2
-3 ) and 0.79 (3H, t, J = 7.4 Hz, H
3
-4 ), and one Glc
000
anomeric proton signal at
d
H
4.65 (1H, d, J = 7.8 Hz, Glc H-1 ) were
observed. Besides, the H NMR spectrum exhibited signals
attributable to two oxygen-bearing methene groups at 3.36
-9),
1
2
009b), and the
b-configuration for the sugar unit was deduced by
d
H
the coupling constant (J = 8.1 Hz) of the anomeric proton signal
and careful analysis of NOESY spectrum. The relative stereconfi-
gurational structure of 1 was based on the NOESY experiment
(1H, dd, J = 9.9, 3.5 Hz, H
a
-9), 4.07 (1H, dd, J = 9.7, 3.2 Hz, H
b
0
0
4.19 (1H, m, H -9 ) and 4.56 (1H, dd, J = 8.7, 4.8 Hz, H -9 ), two
a
b
oxygen-bearing methine groups at
d
H
5.18 (1H, d, J = 7.9 Hz, H-7)
0
13
(
Hoberg et al., 1999). NOE interactions from H
3
-20 to H
2
-12 and
and 4.42 (1H, m, H-7 ). In the C NMR spectrum, thirty carbon
signals can be observed. Except six hexose, four butoxy and two
methoxyl carbon signals, the other eighteen carbons could be
H
3
-19 implied that methyls at C-4 and C-10, and methene at C-13
are on the same side of the diterpene glycoside (Fig. 2). The
absolute configuration at C-13 was proposed as 13S on the basis of
1
1
ascribed to lignan skeleton. The result was confirmed by key H– H
Table 1
1
3
1
a
5 5
C NMR (125 MHz) and H NMR (500 MHz) data of compound 1 in C D N.
Position
1
d
C
mult.
d
H
mult. (J in Hz)
Position
15
d
C
mult.
H
d mult. (J in Hz)
36.4 t
a
b
a
a
b
1.28 m
1.97 m
1.47 m,
111.9 t
a 5.20 dd (10.7, 1.9)
b 5.59 dd (17.2, 1.9)
1.50 s
2
3
19.6 t
37.6 t
b
2.25 m
16
17
28.4 q
11.8 q
1.04 td (13.3, 3.9)
2.42 m
2.00 s
4
5
6
44.0 s
51.2 d
37.2 t
18
19
20
175.9 s
27.8 q
16.6 q
1.86 m
1.25 s
1.24 s
a
b
2.99 dd (17.0, 3.0)
3.37 dd (16.9, 14.8)
0
7
8
9
198.8 s
130.3 s
166.5 s
42.1 s
Glc-1
95.8 d
74.2 d
78.9 d
71.1 d
79.4 d
62.2 t
6.29 d (8.1)
0
2
4.15 dd (8.9, 8.4)
4.24 brt (8.9)
4.31 d (9.5)
0
3
0
1
1
1
1
1
0
1
2
3
4
4
0
24.7 t
a 2.35 m, b 2.56 m
a 1.80 m, b 1.83 m
5
3.98 m
0
41.5 t
6
a 4.34 dd (11.8, 4.6)
b 4.41 dd (11.8, 2.3)
72.5 s
146.4 d
6.14 dd (17.2, 10.7)
a
1
1
Assignments aided by the DEPT, H– H COSY, HSQC, HMBC, TOCSY, and NOESY experiments.

6
70
Y. Ding et al. / Phytochemistry Letters 5 (2012) 668–672
0
chemical shifts of H-8, H
2
2
-9 and H
2
-9 in 2 were similar to those of
0
a, indicating that 2 has the same configuration to 2a (7 R). In
addition, the coupling constant of H-7 (8.9 Hz) in 2 revealed an
antiperiplanar orientation of H-7 and H-8 . In the NOESY spectrum
Fig. 3), H-8 showed NOE correlations with H-2, H-6 and H-7 ,
whereas H-8 showed NOE correlations with H-7, H
. However, no NOE correlations between H-8 and H-2 , H-6 were
observed. These evidences further verified the 7 R configuration of
. Consequently, the structure of 2 was determined to be
7S,7 R,8R,8 S)-7 -butoxy-7,9 -epoxy-4,4 ,9-trihydroxy-3,3 -
dimethoxylignane 9-O- -glucopyranoside.
The known compounds 3–5 were determined to be 3-O-
arabinopyranosyl-hederagenin (3) (Kizu and Tomimori, 1982), 3-
-glucopyranosyl-(1!2)-
O- -arabinopyranosyl-oleanolic ac-
id (4) (Liao et al., 2001) and hederagenin (5) (Kizu and Tomimori,
982), respectively, by comparing their spectroscopic data and
0
0
0
0
(
0
0
2
-9, H-2 and H-
0
0 0
6
0
2
(
0
0
0
0
0
0
b
-D
a-L-
Fig. 2. Key HMBC and NOESY correlations of compound 1.
b
-
D
a-L
1
1
3
COSY, HSQC and HMBC correlations as shown in Fig. 3. The
C
chemical evidences with those published in the literature
(Supplementary material).
NMR data of
2
were similar to those of compound 2a
0
0
0
0
0
0
[
2a = (7S,7 R,8R,8 S)-7,9 -epoxy-4,4 ,7 ,9-tetrahydroxy-3,3 -
In conclusion, the present study reports a new labdane-type
diterpene glycoside (1) and a new trihydroxyfuranoid lignan
glycoside (2), together with three known triterpene and triterpe-
noid glycosides (3–5) from the whole plant of A. rivularis var. flore-
minore. To the best of our knowledge, compound 1 possess a new
aglycone (13S)-13-hydroxy-7-oxo-labda-8,14-diene-18-oic acid,
which is a rarely encountered labdane-type diterpene in nature.
The two new compounds isolated from this species are first
examples of diterpene glycoside and lignanoid glycoside found in
the genus Anemone. Besides, the known triterpene and triterpenoid
glycosides are identified for the first time from A. rivularis var. flore-
minore.
dimethoxylignane 9-O-b-D-glucopyranoside] which was previous-
ly isolated from the leaves of Osmanthus fragrans var. aurantiacus
Machida et al., 2010), except for significant upfield shift of C-7 (
0
(
8
d
C
0
4.6, +7.1 ppm) and downfield shift of C-8 (
d
C
48.7, ꢀ7.1 ppm).
0
These findings suggested that the hydroxyl group at C-7 in 2a was
replaced by a butoxy group in 2. The correction of the linkage of the
0
butoxy group to C-7 was confirmed by the HMBC correlation from
00
0
H
a
-1 to C-7 . Methanolysis of 2 yielded methyl-
which was identified on the basis of R value by TLC analysis, and
the D-configuration for Glc was confirmed by the retention time by
GC analysis with the same method of 1. The -configuration of D-
b-D-glucoside,
f
b
Glc in 2 was deduced on the coupling constant (J = 7.8 Hz) of the
anomeric proton, and the linkage of the Glc unit attached at C-9
was also confirmed by the observation of the correlation between
3
. Experimental
0
00
anomeric proton signal of Glc H-1 to C-9 in the HMBC experiment.
3.1. General
0
0
Hence, the planar structure of 2 was elucidated as 7 -butoxy-7,9 -
0
0
epoxy-4,4 ,9-trihydroxy-3,3 -dimethoxylignane
pyranoside.
9-O-
b
-D
-gluco-
The optical rotations were measured on a Perkin-Elmer 343
polarimeter. The circular dichroism (CD) spectra were determined
on a Jasco-J-715 spectropolarimeter. The ESI-MS and HR-ESI-MS
spectra were obtained on a Micromass Quattro mass spectrometer.
0
0
There are four chiral carbon atoms (C-7, C-8, C-8 and C-7 ) in 2.
The first three are in the tetrahydrofuran ring, and their absolute
configurations could be suggested by CD spectra data in
comparison with those of 2a with the similar structure. The CD
spectrum of 2 showed two positive Cotton effects [De +16.7
1
D and 2D NMR spectra experiments were measured in C
on Bruker INOVA-500 NMR spectrometer with tetramethylsilane
TMS) as an internal standard. GC were performed on
Finnigan Voyager apparatus using an l-Chirasil-Val column
0.32 mm ꢁ 25 m) with an initial temperature of 180 8C at the
rate of 5 8C/min. Separation and purification were performed by
column chromatography (CC) on silica gel H (10–40 m, Qingdao
Marine Chemical Inc., Qingdao, China), reversed-phase silica gel
Lichroprep RP-18, 40–63 m, Merck Inc., Darmstadt, Germany)
5 5
D N
(
a
(
+
210 nm), +4.6 (237 nm)] similar to those of 2a [De +15.2 (206 nm),
3.5 (235 nm)], indicating that the C-7, C-8, C-8 in 2 possess S, R,
0
(
0
2
and S configurations, respectively. It has been reported that H -9
1
signal in the H NMR spectrum was upfield by the anisotropic
m
0
0
effect between the aromatic group at C-7 and H
However, the H -9 and H-8 signals were downfield by the
anisotropic effect between the aromatic group at C-7 , H
2
-9 in 2a.
2
(
m
0
2
-9 and
and Sephadex LH-20 (Pharmacia Inc., NJ, USA). HPLC was carried
out on a Dionex P680 liquid chromatograph equipped with a UV
0
1
H-8 in the 7 S-isomer of 2a (Machida et al., 2010). The H NMR
1
1
Fig. 3. Key H– H COSY (a), HMBC (a) and NOESY (b) correlations of compound 2.

Y. Ding et al. / Phytochemistry Letters 5 (2012) 668–672
671
1
70 UV/Vis detector at 206 nm using a YMC-Pack R&D ODS-A
3.3.1. b-D-Glucopyranosyl (13S)-13-hydroxy-7-oxo-labda-8,14-
column (250 ꢁ 20 mm i.d.) for semi-preparation. TLC detection
diene-18-oate (1)
2
3
was achieved by spraying the silica gel plates (Qingdao Marine
Hygroscopic amorphous powder, [
a
]
D
ꢀ21.48 (c 0.15, MeOH);
1
13
Chemical Inc., Qingdao, China) with 20% H
heating.
2
SO
4
in EtOH followed by
5 5 5 5
H NMR (500 MHz, C D N) and C NMR (125 MHz, C D N) data,
+
+
see Table 1; Positive ESI-MS m/z 1015 [2M+Na] , 519 [M+Na] ;
ꢀ
ꢀ
Negative ESI-MS m/z 531 [M+Cl] , 495 [MꢀH] ; Positive HR-ESI-
+
3.2. Plant material
MS m/z 519.2573 [M+Na] (calcd. for C26
H
40
O
9
Na, 519.2570).
0
0
0
0
0
0
The whole plants of A. rivularis var. flore-minore were collected
3.3.2. (7S,7 R,8R,8 S)-7 -Butoxy-7,9 -epoxy-4,4 ,9-trihydroxy-3,3 -
in the north of Tsinling Mountain, Shaanxi Province of China in
September 2009, and identified by Professor Ji-Tao Wang from
Shaanxi University of Chinese Medicine. A voucher specimen (No.:
dimethoxylignane 9-O-b-D-glucopyranoside (2)
2
3
Hygroscopic amorphous powder [
a
]
D
ꢀ17.48 (c 0.10, MeOH);
CD (c 5.6 ꢁ 10ꢀ M, MeOH): +16.7 (210 nm), +4.6 (237 nm);
5
1
H
1
3
2
0090901) was deposited in the Department of Pharmacy, Xijing
NMR (500 MHz, C
5
D
5
N) and C NMR (125 MHz, C
5
D
5
N) data, see
+
+
Hospital, Fourth Military Medical University.
Table 2; Positive ESI-MS m/z 633 [M+K] , 617 [M+Na] ; Negative
ꢀ
ꢀ
ESI-MS m/z 629 [M+Cl] , 593 [MꢀH] , 498, 362; Positive HR-ESI-
+
3.3. Extraction and isolation
42
MS m/z 617.2570 [M+Na] (calcd. for C30H O12Na, 617.2574).
The air-dried whole plants of A. rivularis var. flore-minore
3.4. Methanolysis of glycosides 1 and 2
(
(
7.7 kg) were crushed and then extracted with 70% EtOH
3 ꢁ 61 L) under reflux at room temperature. The organic solvent
Each glycoside (4 mg) was dissolved in 5% HCl–MeOH (4 mL)
and refluxed for 12 h (80 8C). The reaction mixture was evaporated
under reduced pressure to remove residual HCl. The resulting
was concentrated to afford a crude extract (1.8 kg). The extract
2
was suspended in H O and then partitioned successively with
petroleum ether (3 ꢁ 0.4 L) and n-BuOH (4 ꢁ 0.4 L). The n-BuOH
2 3
residue was partitioned between H O and CHCl3. The CHCl layers
extract (150 g) was separated into 10 fractions (Fr. 1–10) on a
of glycosides 1 and 2 were concentrated in vacuo to yield methyl
(13S)-13-hydroxy-7-oxo-labda-8,14-diene-18-oate with the opti-
silica gel column using a step gradient elution of CHCl
O (100:1:0–7:3:0.5) according to their TLC profiles. Fr. 3 (4.5 g)
was eluted with CHCl –MeOH (1:1) on Sephadex LH-20 to remove
pigments, and then subjected to CC over silica gel eluting with
CHCl –MeOH–H O gradient (from 15:1:0 to 8:1:0.1) to give 3
subfractions (Fr. 3.1–Fr. 3.3). Fr. 3.2 (470 mg) was subjected to CC
over reversed-phase silica gel eluting with MeOH–H O (70:30),
and then further purified by semi-preparative HPLC using MeOH–
O (50:50) as the mobile phase at a flow rate of 7.0 mL/min to
afford compounds 1 (10.6 mg) and 2 (14.2 mg). Fr. 2 (2.3 g) was
subjected to CC over silica gel eluting with CHCl –MeOH–H
gradient (from 20:1:0 to 8:1:0.1) to give 4 subfractions (Fr. 2.1–Fr.
.4). Fr. 2.2 was subjected to CC over Sephadex LH-20 eluting with
CHCl –MeOH (1:1) to remove pigments, and then further purified
by semi-preparative HPLC using MeOH–H O (66:34) as the mobile
phase at a flow rate of 7.0 mL/min to afford compounds 3
36.0 mg) and 4 (7.5 mg). Fr. 4 (1.2 g) was subjected to CC over
silica gel eluting with CHCl –MeOH–H O (9:1:0.1), and further
purified by semi-preparative HPLC with MeOH–H O (85:15) as
the mobile phase at a flow rate of 7.0 mL/min to afford compound
(14.0 mg).
3
–MeOH–
2
3
0
0
0
0
H
2
cal rotation of [
a]
D
+24.08, and (7S,7 R,8R,8 S)-7 -butoxy-7,9 -
0
0
3
epoxy-4,4 ,9-trihydroxy-3,3 -dimethoxylignane with the optical
2
3
rotation of [
and 2 were concentrated to afford methyl
(R = 0.63), which were subjected to co-TLC analysis with authentic
sugars, developed with n-BuOH–Pyridine–H (6:4:3) and
detected with aniline phthalate spray, while the authentic methyl
-galactosylpyranoside showed the R value of 0.54 (Tian et al.,
2009a).
a
]
D
+34.78, respectively. The H
2
O layer of glycosides 1
b-D-glucopyranosides
3
2
f
2
2
O
H
2
b
-D
f
3
2
O
3.5. Acid hydrolysis of glycosides 1 and 2
2
3
Each glycoside (1 mg) was heated with 1 mL of 2 mol/L
COOH at 120 8C for 2 h. The reaction mixture was poured into
CHCl –H O (1:1). The aqueous phase was concentrated and
2
CF
3
3
2
(
dissolved in 1-(trimethylsilyl)-imidazole and pyridine (0.1 mL).
Then, the solution was stirred at 60 8C for 5 min and dried with a
stream of N . The organic layer was analyzed by GC using an l-
2
Chirasil-Val column. In the acid hydrolysates of 1 and 2, D-Glc was
identified with the retention time of 14.56 min, while the retention
3
2
2
5
Table 2
1
3
1
a
C NMR (125 MHz) and H NMR (500 MHz) data of compound 2 in C
5 5
D N.
Position mult. mult. (J in Hz)
d
C
d
H
Position
d
C
mult.
H
d mult. (J in Hz)
0
1
2
134.3 s
111.0 d
7
84.6 d
48.7 d
71.3 t
4.42 d (8.6)
3.21 m
0
7.40 d (1.7)
8
0
9
a
b
4.19 m
3
4
5
6
7
8
9
148.7 s
147.6 s
116.3 d
119.8 d
83.5 d
51.7 d
68.8 t
4.56 dd (8.7, 4.8)
0
0
0
0
0
0
0
0
1
2
3
4
68.2 t
32.3 t
19.7 t
14.0 q
55.9 q
55.9 q
105.4 d
75.3 d
78.6 d
71.5 d
78.4 d
62.6 t
a 3.26 m, b 3.43 m
1.50 m
7.22 m
7.30 dd (8.1, 1.7)
5.18 d (7.9)
2.34 m
1.31 m
0.79 t (14.7, 7.4)
3.75 s
3-OCH
3
0
a 3.36 dd (9.9, 3.5)
b 4.07 dd (9.7, 3.2)
3 -OCH
3
3.83 s
000
Glc-1
4.65 d (7.8)
4.03 m
0
0
0
0
0
0
000
1
2
3
4
5
6
132.6 s
111.2 d
149.0 s
147.9 s
116.1 d
121.3 d
2
000
7.24 m
3
4.17 m
000
4
4.22 m
000
5
3.81 m
000
7.19 m
6
a 4.33 dd (11.7, 3.0)
b 4.44 dd (11.9, 2.7)
7.02 dd (8.0, 1.7)
a
1
1
Assignments aided by the DEPT, H– H COSY, HSQC, HMBC, TOCSY, and NOESY experiments.

6
72
Y. Ding et al. / Phytochemistry Letters 5 (2012) 668–672
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1
4.56 min (D-Glc) and 14.50 min (L-Glc).
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Acknowledgments
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glycosides from Osmanthus fragrans Lour. var. aurantiacus Makino. Helv. Chim.
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Tian, X.-R., Tang, H.-F., Li, Y.-S., Lin, H.-W., Ma, N., Zhang, W., Yao, M.-N., 2009a.
Ceramides and cerebrosides from the marine bryozoan Bugula neritina inhabit-
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This work was financially supported by grant from National
Natural Science Foundation of China (No. 30873402). The authors
are grateful to Mr. Min-Chang Wang and Mrs. Hui-Min Wang for
the measurements of NMR and MS spectra.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2012.07.003.
Tian, Y., Tang, H.-F., Qiu, F., Wang, X.-J., Chen, X.-L., Wen, A.-D., 2009b. Triterpenoid
saponins from Ardisia pusilla and their cytotoxic activity. Planta Med. 75,
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