Four new triterpenoid glycosides from the seed residue of Hippophae rhamnoides subsp. sinensis

Article abstract of DOI:10.1080/10286020.2013.879383

Four new triterpenoid saponins (1-4) were isolated from the seed residue of Hippophae rhamnoides subsp. sinensis, named 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-13-ene-19-one-28-oic acid 28-O-β-d-glucopyranosyl ester (1), 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-13-ene-19-one-30-hydroxyolean-28-oic acid 28-O-β-d-glucopyranosyl ester (2), 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranosyl-13-ene-19-one-28-oic acid 28-O-β-d- glucopyranosyl ester (3), and 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranosyl-13-ene-19-one-30-hydroxyolean-28-oic acid 28-O-β-d-glucopyranosyl ester (4), and their structures were elucidated on the basis of spectroscopic and chemical methods.

Full text of DOI:10.1080/10286020.2013.879383

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Four new triterpenoid glycosides
from the seed residue of Hippophae
rhamnoides subsp. sinensis
a
a
a
a
a
Chao Chen , Wen Gao , Liang Cheng , Yan Shao & De-Yun Kong
a
State Key Laboratory of New Drug & Pharmaceutical Process,
Shanghai Institute of Pharmaceutical Industry, Shanghai, 200040,
China
Published online: 23 Jan 2014.
To cite this article: Chao Chen, Wen Gao, Liang Cheng, Yan Shao & De-Yun Kong (2014) Four new
triterpenoid glycosides from the seed residue of Hippophae rhamnoides subsp. sinensis, Journal of
Asian Natural Products Research, 16:3, 231-239, DOI: 10.1080/10286020.2013.879383
To link to this article: http://dx.doi.org/10.1080/10286020.2013.879383
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 3, 231–239, http://dx.doi.org/10.1080/10286020.2013.879383
Four new triterpenoid glycosides from the seed residue of Hippophae
rhamnoides subsp. sinensis
Chao Chen, Wen Gao, Liang Cheng, Yan Shao and De-Yun Kong*
State Key Laboratory of New Drug & Pharmaceutical Process, Shanghai Institute of
Pharmaceutical Industry, Shanghai 200040, China
(
Received 8 October 2013; final version received 24 December 2013)
Four new triterpenoid saponins (1–4) were isolated from the seed residue of
Hippophae rhamnoides subsp. sinensis, named 3-O-[b-D-glucopyranosyl(1 ! 2)-b-D-
glucopyranosyl-(1 ! 3)]-[a-L-rhamnopyranosyl-(1 ! 2)]-a-L-arabinopyranosyl-13-
ene-19-one-28-oic acid 28-O-b-D-glucopyranosyl ester (1), 3-O-[b-D-glucopyranosyl
(
1 ! 2)-b-D-glucopyranosyl-(1 ! 3)]-[a-L-rhamnopyranosyl-(1 ! 2)]-a-L-arabinopyr-
anosyl-13-ene-19-one-30-hydroxyolean-28-oic acid 28-O-b-D-glucopyranosyl ester (2),
-O-[b-D-glucopyranosyl(1 ! 2)-b-D-glucopyranosyl-(1 ! 3)]-[a-L-rhamnopyranosyl-
3
(
1 ! 2)]-b-D-glucopyranosyl-13-ene-19-one-28-oic acid 28-O-b-D-glucopyranosyl
ester (3), and 3-O-[b-D-glucopyranosyl(1 ! 2)-b-D-glucopyranosyl-(1 ! 3)]-[a-L-
rhamnopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-13-ene-19-one-30-hydroxyolean-28-
oic acid 28-O-b-D-glucopyranosyl ester (4), and their structures were elucidated on the
basis of spectroscopic and chemical methods.
Keywords: Hippophae rhamnoides subsp. sinensis; hippophosides A–D; triterpenoid
saponins
1
.
Introduction
was elucidated as C H O by the high-
59 94 27
resolution electrospray ionization mass
spectrometry (HR-ESI-MS) data at m/z
Hippophae rhamnoides L. (Sea buckthorn),
a plant of family Elaeagnaceae, has been
used for application as food and medicine
in eastern countries, especially in tra-
ditional Mongolian and Tibetan medicine
in China [1]. Previous investigations
demonstrated that the seed extracts have
sorts of biological effects, such as antiox-
idant [2], antilipemic [3], and antineoplastic
þ
1
257.5886 [M þ Na] . The acid hydroly-
sis, derivatization, and GC analysis gave
D-glucose, L-rhamnose, and L-arabinose
1
in 1. The H NMR spectrum (Table 1)
revealed signals due to seven tertiary
methyl groups (d 0.60, 0.94, 0.96, 0.97,
1
.09, 1.13, 1.13) and five anomeric protons
1
3
(d 4.68, 5.04, 5.14, 6.14, 6.16). The
NMR spectrum (Table 2) showed signals
C
[4]. As a part of our continuing search for
bioactive components from the seed residue
of H. rhamnoides subsp. sinensis, we
present in this report the isolation and
structure elucidation of four new oleanane-
type saponins (1–4) (Figure 1).
of seven tertiary methyl carbons at d 16.6,
17.0, 17.4, 20.9, 24.4, 25.3, and 28.2;
a pair of olefinic carbons at d 132.8 (C,
C-18) and 152.0 (C, C-13); five anomeric
carbons at d 96.4, 101.0, 102.8, 104.8, and
106.2; six non-hydrogenated carbons at d
37.3, 39.5, 43.2, 45.4, 45.5, and 53.0; and
2
.
Results and discussion
Compound 1 was obtained as a white
amorphous powder. Its molecular formula
two carbonyl carbons at d 174.8 and 208.1.
The spectroscopic features suggested that
*Corresponding author. Email: deyunk@aliyun.com
q 2014 Taylor & Francis

2
32
C. Chen et al.
R2
3
0
OH
2
9
HO
6''''
5''''
O
20
4''''
4''
O
5''
2''
O
O
2
1
O
O
19
HO
HO
6'''''
5'''''
1
2
1
8
22
2''''
3''
5'''
1''
HO
4
3''''
1''''
6'''
25
11
13
17
S1=
'''''
2
6
14
O
O
HO
1
6
1
4
9
Me
O
1'''
2'''
OH
2
3
10
8
7
15
28
HO
1'''''
2
'''''
HO
3
'''''
OH
5
27
4'''
HO
3'''
6
HO
6'
R O
1
O
'
O
5
HO
HO
4'
24
23
2'
1'
3'
OH
HO
6''
5
HO
6''''
5''''
4
''
''
4''''
O
O
O
O
HO
1
2
3
4
R =S , R =CH
3
HO
HO
6'''''
5'''''
O
1
1
2
2''
1''
S2=
1''''
3''
5'''
3'''' 2''''
HO
R =S , R =CH OH
1
1
2
2
3
4
'''''
O
6
'''
HO
HO
R =S , R =CH
1'''
2'''
1
2
2
Me
O
1'''''
2'''''
3'''''
OH
HO
R =S , R =CH OH
3'''
1
2
2
2
4'''
HO
OH
Figure 1. Structures of compounds 1–4.
1
was an oleanane-type saponin with five
(d, J ¼ 6.0 Hz)] and a carbinolic proton
0
00
sugars. The HMBC plot showed long-
range correlations of the carbonyl carbon
at d 208.1 (C-19) with 3H-30 (d 0.97) and
H-5 (d 4.66) was clearly revealed by
corresponding cross-peaks observed in
1
1
the 2D H– H COSY spectrum of 1 (see
Supplementary Material), in agreement
with the presence of a rhamnose moiety.
All monosaccharides were in the pyranose
form, and the anomeric configurations
were a- for arabinose and rhamnose and
b- for glucoses, determined by comparing
the d values with the compounds in the
literature [6–8]. In the HMBC spectrum,
the following correlations were observed:
3
H-29 (d 1.13), and the olefinic carbons at
d 132.8 (C, C-18) and 152.0 (C, C-13)
shifted to downfield, which established
that a CvC bond existed between C-13
and C-18, and the carbonyl carbon was at
C-19. The carbon chemical shifts, regard-
ing C-29 and C-30 of 1, were almost
identical with the compounds 1 and 3 in
the literature [5]. In addition, the long-
range correlations of the C-3 (d 88.2)
0
00
00
0000
00000
C-1 (d 101.0) with H-2 (d 4.58), C-1
00
00
with the anomeric proton H-1 (d 4.68),
00
(d 102.8) with H-3 (d 4.14), and C-1
0
0
0000
C-1 (d 104.8) with H-5 (d 3.60), and
C-28 (d 174.8) with the anomeric proton
(d 106.2) with H-2 (d 3.88) (Figure 2).
Other additional heteronuclear long-range
interactions involving proton and carbon
atoms are described in the Supplementary
Material. On the basis of this evidence,
the structure of compound 1, named
hippophoside A, was elucidated to be 3-O-
[b-D-glucopyranosyl(1 ! 2)-b-D-gluco-
pyranosyl-(1 ! 3)]-[a-L-rhamnopyrano-
syl-(1 ! 2)]-a-L-arabinopyranosyl-13-
ene-19-one-28-oic acid 28-O-b-D-gluco-
pyranosyl ester (1).
0
H-1 (d 6.14) (Figure 2) confirmed that the
five sugars were present in two saccharide
chains, one with arabinose attached to C-3
and the other to C-28. The unambiguous
identification of the individual spin sys-
tems associated with the five monosac-
charides and the assignments of their
NMR resonances were established by the
combined use of 1D and 2D NMR techni-
ques (see Supplementary Material). For
example, the spin–spin coupling of
Compound 2 was isolated as a white
amorphous powder. Its molecular formula
000
a secondary methyl group 3H-6 [d 1.56

Journal of Asian Natural Products Research
233
d

2
34
C. Chen et al.

Journal of Asian Natural Products Research
235
1
3
Table 2 – continued
Table 2. C NMR spectral data of 1–4 (1 at
00 MHz, 2–4 at 150 MHz, in pyridine-d , d ,
1
J in Hz).
5
C
Position
1
2
3
4
0
00
3
-O-Glc
Position
1
2
3
4
0
000
000
000
000
000
000
1
2
3
4
5
6
102.8
84.5
78.2
71.0
78.4
62.0
102.9
84.4
78.1
70.9
78.4
61.9
100.6
84.8
77.9
70.6
78.3
61.8
100.6
84.8
77.9
70.6
78.3
61.8
0
0
0
0
0
1
2
3
4
5
6
7
8
9
39.4
26.7
88.2
39.5
56.2
18.4
34.8
45.4
51.2
37.3
28.2
36.0
152.0
43.2
27.3
22.9
53.0
132.8
208.1
45.5
34.2
31.8
28.2
17.0
16.6
17.4
20.9
174.8
25.3
24.4
39.3
26.7
88.2
39.5
56.1
18.3
34.8
45.5
51.2
37.2
28.2
31.1
152.1
43.2
27.3
22.9
53.0
133.6
207.9
52.1
31.9
34.0
28.1
17.0
16.6
17.5
21.0
175.1
21.1
66.6
39.4
26.8
88.4
39.5
56.0
18.2
34.8
45.4
51.0
37.2
28.2
35.9
152.1
43.1
27.2
22.9
53.0
132.8
208.1
45.5
34.2
31.8
28.0
17.0
16.6
17.4
21.0
174.8
25.3
24.3
39.4
26.8
88.4
39.5
56.1
18.3
34.8
45.5
51.1
37.2
28.2
31.1
152.1
43.2
27.3
22.9
53.0
133.6
207.9
52.1
31.9
34.0
28.1
17.0
16.6
17.5
21.0
175.1
21.0
66.6
0000
2
-O-Glc
0
0
0
0
0
0
0000
0000
0000
0000
0000
0000
1
2
3
4
5
6
106.2
76.1
78.3
70.6
78.7
62.5
106.2
76.0
78.3
70.6
78.7
62.4
106.7
76.0
78.0
70.4
78.7
62.0
106.8
76.1
78.0
70.4
78.7
62.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
was elucidated as C H O by the HR-
59 94 28
ESI-MS data at m/z 1273.5833
þ
[
M þ Na] . The acid hydrolysis, derivati-
zation, and GC analysis suggested the
presence of D-glucose, L-rhamnose, and
L-arabinose in 2 as 1. Comparing the 1D
and 2D NMR spectroscopic data of 2 with
those of 1, both were almost super-
imposable, except for some differences
of the signals of aglycone. There were only
six tertiary methyl carbons at d 16.6, 17.0,
17.5, 21.0, 21.1, and 28.1 in 2, but one
more methylene carbon at d 66.6 (C-30).
Hence, it was deduced that one of the
methyl carbons in 1 changed to methylene
carbon with a hydroxyl, and this was
confirmed by the methyl carbons at d 24.4
(C-30) and d 25.3 (C-29) in 1 substituted
by the signals at d 21.1 (C-29) and d 66.6
(C-30) in 2. The position of methylene is
further confirmed by HMBC correlations
from C-30 (d 66.6) to 3H-29 (d 1.47), C-20
(d 52.1) with 3H-29 (d 1.47), and C-19
(d 207.9) with 3H-29 (d 1.47). Moreover,
the NOESY correlations of 3H-27 (d 1.18)
with 3H-29 (d 1.47) suggested that the
orientation of C29 was a. Thus, 2 was
determined to be 3-O-[b-D-glucopyranosyl
6
7
8
9
0
8-O-Glc
0
1
2
3
4
5
6
96.4
74.0
78.7
71.3
79.3
62.5
Ara
104.8
74.4
82.2
69.1
65.4
96.4
74.0
78.8
71.2
79.4
62.4
Ara
104.9
74.4
82.4
69.3
65.6
96.4
74.0
78.8
71.2
79.4
62.4
Glc
104.9
78.1
87.8
68.8
77.1
62.6
96.4
74.1
78.8
71.2
79.4
62.4
Glc
104.9
78.1
87.8
68.8
77.1
62.6
0
0
0
0
0
3
-O
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
0
0
2
-O-Rha
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
101.0
72.3
72.5
74.0
69.8
18.2
101.0
72.3
72.5
73.9
69.8
18.2
101.2
72.3
72.4
73.8
69.8
18.2
101.2
72.3
72.4
73.8
69.8
18.3
(
1 ! 2)-b-D-glucopyranosyl-(1 ! 3)]-[a-
L-rhamnopyranosyl-(1 ! 2)]-a-L-arabino-
pyranosyl-13-ene-19-one-30-hydroxyolean-

2
36
C. Chen et al.
OH
3
H C
CH
3
CH
3
O
O
O
O
OH
CH
3
HO
HO
HO
H
OH
H
CH
3
H
O
O
HO
HO
H
O
O
O
O
HO
HO
HO
O
O
O
HO
HO
HO
O
O
O
O
O
HO
O H
HO
H
O H
HO
HO
HO
H
HO
HO
O
Me
OH HO
O
OH
H
O
OH
H
Me
OH HO
O
HO
OH
HO
1
OH
2
OH
3
H C
CH
3
CH
3
O
O
O
O
HO
O
CH
3
HO
O
CH
3
HO
HO
HO
H
HO
HO
HO
H
H
O
H
HO
O
O
HO
O
O
O
HO
O
O
HO
HO
HO
OO
OH
O
O
O
H
HO
HO
HO
HO
HO
H
O H
HO
HO
HO
H
O
O
OH
H
H
Me
OH HO
O
Me
HO
O
OH
HO
HO OH
OH
3
4
HMBC
NOESY
Figure 2. Selected HMBC and NOESY correlations for compounds 1–4.
0000
inner glucose, and C-2 (d 84.8) of
2
8-oic acid 28-O-b-D-glucopyranosyl ester
0
0000
(2) and was named hippophoside B.
the inner glucose with H-1
(d 5.30)
Compound 3 was obtained as a white
amorphous powder. Its molecular formula
was elucidated as C H O by the HR-
of the glucose. Hence, the structure
of 3-O-[b-D-glucopyranosyl(1 ! 2)-b-D-
glucopyranosyl-(1 ! 3)]-[a-L-rhamno-
pyranosyl-(1 ! 2)]-b-D-glucopyranosyl-
13-ene-19-one-28-oic acid 28-O-b-D-glu-
copyranosyl ester (3) was deduced for 3,
named hippophoside C.
6
0 96 28
ESI-MS data at m/z 1287.5979
13
þ
1
[
M þ Na] . The H and C NMR signals
of 3 were assigned after DEPT, HSQC,
and HMBC experiments. A comparison of
1
13
the H and C NMR signals of 3 with
those of 1 clearly revealed that the
aglycone part of 3 was identical to that of
Compound 4 was obtained as a white
amorphous powder. Its molecular formula
was elucidated as C H O by the
0 96 29
6
1
, and 3 was suggested to be 13-ene-19-
HR-ESI-MS data at m/z 1303.5944
þ
one-28-oic acid with five monosaccharide
units, but one saccharide chain with four
monosaccharides at C-3 was different
from 1. Acid hydrolysis suggested that
the monosaccharides of this compound
were L-rhamnose and D-glucose. The
sequence of the glycan parts and glycoside
sites were also proven by the following
HMBC correlations: C-3 (d 88.4) with
[M þ Na] . Acid hydrolysis of 4 gave the
same results as those of 3. Compound 4
had the same two sugar chains as 3 by
1
13
comparison of the H and C NMR
spectroscopic data of 4 with those of the
moieties of 3. Moreover, the aglycone part
of 4 was identical to that of 2 by comparison
1
13
oftheir H and CNMR spectroscopic data.
The HMBC cross-peaks of C-28 (d 175.1)
0
0
00
00
0
00
H-1 (d 4.78) of the inner glucose, C-2
withH-1 (d6.31)andC-3(d88.4)withH-1
0
(
(
d 78.1) of the inner glucose with H-1
d 6.47) of the rhamnose, C-3 (d 87.8) of
(d 4.78) indicated that the sugar chains
ether-linked was attached to C-3 and C-
28, respectively. Besides, the NOESY
00
0000
the inner glucose with H-1 (d 5.15) of the

Journal of Asian Natural Products Research
237
correlations of 3H-27 (d 1.18) with 3H-29
d 1.47) suggested that the orientation
Co., Ltd, Yantai, China) was used for thin
layer chromatography (TLC). Fractions
were monitored by TLC on silica-gel
plates sprayed with 10% H SO in EtOH,
(
of C29 was a. Thus, 4 was determined to
be 3-O-[b-D-glucopyranosyl(1 ! 2)-b-D-
glucopyranosyl-(1 ! 3)]-[a-L-rhamnopyr-
anosyl-(1 ! 2)]-b-D-glucopyranosyl-13-
ene-19-one-30-hydroxyolean-28-oic acid
2
4
followed by heating.
3.2 Plant materials
28-O-b-D-glucopyranosyl ester (4), named
hippophoside D.
The seeds of H. rhamnoides subsp. sinensis
were collected at Datong County of
Qinghai Province, China, in July 2008 and
identified by Dr Min-Sheng Cao of Qinghai
General Health Bio-Science Co., Ltd.
A voucher specimen (SIPITCM-080715)
has been deposited at the Shanghai Institute
of Pharmaceutical Industry.
3
.
Experimental
3
.1 General experimental procedures
Optical rotations were determined with a
Perkin-Elmer-341 polarimeter (San Jose,
CA, USA). UV–vis spectra were recorded
using a Techcomp 8500 spectrometer
3.3 Extraction and isolation
(
Shanghai, China). IR spectra were
recorded in KBr pellets on a Nicolet-
NEXUS-670-FTIR spectrophotometer
The seed residues of H. rhamnoides
subsp. sinensis, which had been extracted
through supercritical fluid extraction, were
obtained from Qinghai General Health Bio-
Science Co., Ltd (Xining, Qinghai, China).
The air-dried seed residues of H. rham-
noides subsp. sinensis (12 kg) were extracted
twice with 70% EtOH (120 l each) for 1 h.
The extracts were combined and concen-
trated under reduced pressure to give a
residue (900 g), which was suspended in
30% EtOH, and then passed through a D101
macroporous resin column eluted with
(Madison, WI, USA). NMR spectra were
obtained with a Varian INOVA-400/600
1
instrument (Palo Alto, CA, USA). The H
1
3
and
relative to solvent signals at dH/C
.49/39.5 (DMSO-d ) relative to tetra-
C NMR chemical shifts were
2
6
methylsilane. MS was measured on a
Waters Q-Tof micro YA019 mass spec-
trometer (Leederville, WA, USA). Pre-
parative HPLC was performed on a
Shimadzu HPLC system consisting of
two LC-8A pumps and an SPD-M10A
detector. For preparative purposes, a
Shimadzu PRC-ODS (15 mm, i.d. 20 mm
EtOH–H O (30:70, 72 l and 70:30, 72 l
2
(v/v)) to yield two fractions (Frs A and B).
Fr. B (47g) was chromatographed on a
silica-gel column (10 £ 60 cm) eluted with
CHCl –MeOH–H O (9:1:0.1, 8:2:0.2,
£ 250 mm; Kyoto, Japan) was used.
Analytical GC was carried out on an
3
2
Agilent 6890N system (H flame ioniz-
2
7:3:0.5, and 6:4:1 (v/v)) and MeOH, each
15 l, to give Frs B1–B11. Fr. B9 (3.23 g)
was loaded on silica-gel CC, eluted with
CHCl –MeOH–H O (8:1:0.1, 6:1:0.1, and
ation detector; Minneapolis, MN, USA)
and a capillary column (30 m £ 0.32 mm
£ 0.25 mm; Abel AB-5, Appleton, WI,
USA). Column chromatography (CC) was
performed on silica gel (100–200 or 200–
3
2
4:1:0.1 (v/v)) and MeOH, each 0.5 l, to give
Frs B9-1–B9-4. Fr. B9-3 (1.02 g) was
subjected to Sephadex LH-20 column eluted
3
00 mesh; Shanghai Sanpont Co., Ltd,
Shanghai, China), D101 macroporous
resin (Cangzhou Bon Adsorber Technol-
ogy Co., Ltd, Cangzhou, China), and
Sephadex LH-20 (GE Healthcare Bio-
Sciences AB, Uppsala, Sweden). Silica gel
HSGF254 (Yantai Jiangyou Guijiao Kaifa
with MeOH and pre-HPLC (MeOH–H O,
2
60:40 (v/v); 7 ml/min; UV detection
254 nm), and compounds 1 (35 mg, tR:
25.461 min) and 3 (25 mg, t : 15.869 min)
R
were obtained, respectively. Fr. B9-4
(1.36 g) was separated by a Sephadex

2
38
C. Chen et al.
2
1
LH-20 column eluted with MeOH and Pre-
HPLC (MeOH–H O 52:48 (v/v); 7 ml/min;
(KBr) nmax cm : 3423, 2924, 1736, 1654,
1458, 1384, 1075, 912, 569; ESI-MS m/z:
2
þ
UV detection 254 nm) to yield compounds 2
(14 mg, t : 31.911 min) and 4 (25 mg, t :
1287
[M 2 162 2 H] . HR-ESI-MS (positive)
[M þ Na]
and
1101
2
R
R
þ
23.881 min).
m/z: 1287.5979 [M þ Na] (calcd for
C H O Na, 1287.5986).
0 96 28
6
3
.3.1 3-O-[b-D-Glucopyranosyl(1 ! 2)-
b-D-glucopyranosyl-(1 ! 3)]-[a-L-
3
.3.4 3-O-[b-D-Glucopyranosyl(1 ! 2)-
rhamnopyranosyl-(1 ! 2)]-a-L-
b-D-glucopyranosyl-(1 ! 3)]-[a-L-
rhamnopyranosyl-(1 ! 2)]-b-D-
glucopyranosyl-13-ene-19-one-30-
hydroxyolean-28-oic acid 28-O-b-D-
glucopyranosly ester (4)
arabinopyranosyl-13-ene-19-one-28-oic
acid 28-O-b-D-glucopyranosly ester (1)
25
White amorphous powder; ½aꢀ –83.2
D
(
c 0.45, MeOH). UV (MeOH) l
log 1) nm: 254 (3.31); IR (KBr) n
max
(
cm : 3423, 2927, 1736, 1638, 1458,
max
25
D
White amorphous powder; ½aꢀ –77.5
2
1
(
c 0.45, MeOH). UV (MeOH) l
log 1) nm: 256 (3.27); IR (KBr) n
1 13
384, 1075, 1028, 912, 589; for H and C
max
1
NMR spectral data, see Tables 1 and 2;
(
cm : 3421, 2926, 1735, 1655, 1458,
max
2
1
þ
ESI-MS m/z: 1257 [M þ Na] and 1071
1
384, 1262, 1075, 911, 570; ESI-MS m/z:
þ
2
M 2 162 2 H] . HR-ESI-MS (positive)
[
2
303 [M þ Na] and 1279 [M 2 H] .
1
HR-ESI-MS (positive) m/z: 1303.5944
þ
m/z: 1257.5886 [M þ Na] (calcd for
C H O Na, 1257.5880).
5
9
94 27
þ
[
M þ Na] (calcd for C H O Na,
6
0
96 29
1
303.5935).
3
.3.2 3-O-[b-D-Glucopyranosyl(1 ! 2)-
b-D-glucopyranosyl-(1 ! 3)]-[a-L-
rhamnopyranosyl-(1 ! 2)]-a-L-
arabinopyranosyl-13-ene-19-one-30-
hydroxyolean-28-oic acid 28-O-b-D-
glucopyranosly ester (2)
3.4 Acid hydrolysis of 1–4
Compounds 1–4 (10 mg each) were
refluxed in 1 mol/l HCl (1 ml) for 3 h.
The mixture was neutralized with
NaHCO3 and then partitioned with
dichloromethane. To the dried aqueous
layer, pyridine and hydroxylamine hydro-
chloride (10 mg each) were added, and the
solution was stirred at 958C for 1 h. Then,
after addition of acetic anhydride (1 ml
each), the mixture was kept overnight. The
acetylated derivatives were subjected to
GC analysis to identify the sugars.
Conditions for GC were as follows: AB-5
2
D
5
White amorphous powder; ½aꢀ – 67.8
(c 0.46, MeOH). UV (MeOH) lmax (log 1)
nm: 216 (3.22), 256 (3.26); IR (KBr) n
max
2
cm : 3422, 2925, 1735, 1655, 1458,
1
1 13
384, 1261, 1074, 912, 592; for H and C
1
NMR spectral data, see Tables 1 and 2;
þ
ESI-MS m/z: 1273 [M þ Na] and 1249
2
M 2 H] . HR-ESI-MS (positive) m/z:
[
þ
1
C H O Na, 1273.5829).
273.5833 [M þ Na]
(calcd for
5
9 94 28
(
30 m £ 0.32 mm £ 0.25 mm) column;
column temp.: from 100 to 2508C, pro-
grammed increase 108C/min and keeping
3
.3.3 3-O-[b-D-Glucopyranosyl(1 ! 2)-
b-D-glucopyranosyl-(1 ! 3)]-[a-L-
rhamnopyranosyl-(1 ! 2)]-b-D-
2508C for 5 min; injector and detector
temp.: 2508C; injection volume: 2.0 ml;
split ratio: 1:20; carrier gas: N at 1 ml/min
2
glucopyranosyl-13-ene-19-one-28-oic
acid 28-O-b-D-glucopyranosly ester (3)
[9]. Under these conditions, the standard
sugars gave peaks at t (min): 15.734 of
2
D
5
White amorphous powder; ½aꢀ –52.9
R
(
(
c 0.49, MeOH). UV (MeOH) l
log 1) nm: 217 (3.24), 253 (3.22); IR
D-glucose, 12.762 of L-rhamnose, and
12.951 of L-arabinose, respectively. Peaks
max

Journal of Asian Natural Products Research
239
at t (min): 15.724, 12.759, and 12.962 of
R
[5] S.D. Rosa, M. Mitova, N. Handjieva,
D-glucose, L-rhamnose, and L-arabinose
for 1–2, while 15.814 and 12.822 of D-
glucose and L-rhamnose for 3–4, were
observed.
S. Popov, and M. Anchev, J. Nat. Prod.
6
3, 1012 (2000).
[
6] P.T. Ky, P.T. Huong, T.K. My, P.T. Anh, P.
V. Kiem, C.V. Minh, N.X. Cuong, N.P.
Thao, N.X. Nhiem, J.H. Hyun, H.K. Kang,
and Y.H. Kim, Phytochemistry 71, 994
(2010).
References
[
1] X. Xu, Y. Gao, G. Liu, Q. Wang, and
J. Zhao, LWT—Food Sci. Technol. 41, 1223
[7] F.R. Melek, M. Toshio, S.M. Abdel-
Khalik, I.I. Mahmoud, and S.A. Mina,
Phytochemistry 64, 3089 (2004).
(
2008).
[
[
2] H.Y. Jin, Hippophae 18, 35 (2005).
3] X.Y. Yang, W.Q. Qu, Z.L. Xu, Q. Miu,
L.X. Zhu, and M. Xu, J. Chin. Mater. Med.
[
8] G.C. Gao, Z.X. Lu, S.H. Tao, S. Zhang, and
F. Wang, Carbohydr. Res. 346, 2200
(
2011).
31, 1109 (2006).
[
9] J. Zhang, X.X. Weng, W. Gao, Z.J. Zhu,
and D.Y. Kong, Chin. J. Pharm. 42, 265
(2011).
[
4] B. Sun, P. Zhang, W.J. Qu, X.L. Zhang,
X.L. Zhuang, X.Y. Zhuang, and H.J. Yang,
Chin. Med. Mater. 26, 875 (2003).