New oleanane-type triterpenoid saponins isolated from the seeds of Celosia argentea

Article abstract of DOI:10.1080/10286020.2013.879120

Three new oleanane-type triterpenoid saponins named celosins H (1), I (2), and J (3) were isolated from the seeds of Celosia argentea L. Their structures were characterized as 3-O-β-d-xylopyranosyl-(1 → 3)-β-d- glucuronopyranosyl-polygalagenin 28-O-β-d-gl

Full text of DOI:10.1080/10286020.2013.879120

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New oleanane-type triterpenoid
saponins isolated from the seeds of
Celosia argentea
a
b
a
b
Xu Pang , Hai-Xia Yan , Zhen-Fang Wang , Miao-Xuan Fan , Yang
a
b
a
a
a
Zhao , Xin-Tong Fu , Cheng-Qi Xiong , Jie Zhang , Bai-Ping Ma &
b
Hong-Zhu Guo
a
Beijing Institute of Radiation Medicine, Beijing, 100850, China
b
Beijing Institute for Drug Control, Beijing, 100035, China
Published online: 23 Jan 2014.
To cite this article: Xu Pang, Hai-Xia Yan, Zhen-Fang Wang, Miao-Xuan Fan, Yang Zhao, Xin-Tong
Fu, Cheng-Qi Xiong, Jie Zhang, Bai-Ping Ma & Hong-Zhu Guo (2014) New oleanane-type triterpenoid
saponins isolated from the seeds of Celosia argentea, Journal of Asian Natural Products Research,
16:3, 240-247, DOI: 10.1080/10286020.2013.879120
To link to this article: http://dx.doi.org/10.1080/10286020.2013.879120
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 3, 240–247, http://dx.doi.org/10.1080/10286020.2013.879120
New oleanane-type triterpenoid saponins isolated from the seeds
of Celosia argentea
a1
Xu Pang , Hai-Xia Yan , Zhen-Fang Wang , Miao-Xuan Fan , Yang Zhao ,
b1
a
b
a
b
a
a
a
Xin-Tong Fu , Cheng-Qi Xiong , Jie Zhang , Bai-Ping Ma * and Hong-Zhu Guo *
b
a
b
Beijing Institute of Radiation Medicine, Beijing 100850, China; Beijing Institute
for Drug Control, Beijing 100035, China
(Received 12 October 2013; final version received 23 December 2013)
Three new oleanane-type triterpenoid saponins named celosins H (1), I (2), and J (3)
were isolated from the seeds of Celosia argentea L. Their structures were characterized
as 3-O-b-D-xylopyranosyl-(1 ! 3)-b-D-glucuronopyranosyl-polygalagenin 28-O-b-D-
glucopyranosyl ester, 3-O-b-D-glucuronopyranosyl-medicagenic acid 28-O-b-D-
xylcopyranosyl-(1 ! 4)-a-L-rhamnopyranosyl-(1 ! 2)-b-D-fucopyranosyl ester, and
3
-O-b-D-glucuronopyranosyl-medicagenic acid 28-O-a-L-arabinopyranosyl-(1 ! 3)-
[b-D-xylcopyranosyl-(1 ! 4)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-fucopyranosyl
ester by NMR, MS, and chemical evidences, respectively. In our opinion, celosins H–J
could be used as chemical markers for the quality control of C. argentea seeds.
Keywords: Celosia argentea; triterpenoid saponins; celosins H–J; NMR
1
.
Introduction
kinds of seeds. For the purpose of
obtaining chemical markers for the quality
control of C. argentea seeds, phytochem-
ical investigation was carried out on the
extract of the C. argentea seeds, and three
new compounds named celosins H–J (1–
Celosia argentea L. (Amaranthaceae) is an
annual herbaceous plant widely distributed
in China, especially in southern China.
The seeds of C. argentea (Semen Celo-
siae) are used as a traditional Chinese
medicine, named ‘Qing Xiang Zi’ in
Chinese, for removing liver-heat, improv-
ing eyesight, clearing wind-heat, and
lowering the blood pressure [1]. The
pharmacological investigation on the
extract of C. argentea seeds showed that
it had hepatoprotective effect and anti-
tumor activity [2,3]. To our knowledge, in
many places of China, the seeds of
Amaranthus tricolors, which also belong
to Amaranthaceae family, are always
misused as the C. argentea seeds or are
incorporated unlawfully as adulterants into
C. argentea seeds due to their almost
identical physical features. By RP-HPLC
analyses of their constituents, we found the
obvious difference between these two
3
) were isolated as the main ingredients in
these seeds, and their structures were
characterized by NMR, MS, and chemical
evidences (Figure 1). This paper mainly
presented the isolation and structure
elucidation of these new compounds.
2. Results and discussion
Compound 1 was obtained as a white
amorphous powder. Its molecular formula
C H O was determined by HR-ESI-
4
7 72 20
2
MS ion [M 2 H] at m/z 955.4583.
1
By combined analyses of the H NMR,
1
3
C NMR, and HSQC spectra, three
anomeric proton signals at d 4.99 (1H, d,
0
J ¼ 7.8 Hz, H-1 ), 5.31 (1H, d, J ¼ 7.8 Hz,
0
0
000
H-1 ), 6.31 (1H, d, J ¼ 7.8 Hz, H-1 ) and
*Corresponding authors. Email: mabaiping@sina.com; guohz@bidc.org.cn
q 2014 Taylor & Francis

Journal of Asian Natural Products Research
241
3
0
29
21
2
0
1
9
1
OH
HO
O
1
2
22
8
1
3
1''
1
1
18
15
2
O
2
5
26
7
O
1
4
16
HO
1
4
9
O
1'''
2
3
10
8
H C
3
O
O
5
7
27
O
O
HOOC
O
6
1'''
HO
HO
1
'
HO
HO
HO
1'''' HO
O
HO
O
OH
OH
OH
O
24
CHO
OH
23
S1
HO
HO
1''
OH
1
OH
O
1
''
HO
O
O
1'''
H C
O
OH
3
O
O
HO
HO
HO
1
'''''
OR
O
OH
HOOC
HO
HO
O
OH
HO
O
O
1
'
1''''
OH
COOH
OH
2
3
R=S1
R=S2
S2
Figure 1. The structures of compounds 1–3.
three corresponding anomeric carbon
0
(C-1, þ 6.1 ppm) and 69.9 (C-2, þ
44.9 ppm) deduced the aglycone of 1 and
had a hydroxyl group at C-2. In ROESY
spectrum, the correlation between H-24 at
d 1.73 and H-25 at d 1.48 (H-25) revealed
the b-configuration of C -Me. H-3 at d
00
signals at d 105.1 (C-1 ), 106.2 (C-1 ),
000
9
5.8 (C-1 ) were observed, which
revealed 1 contained three sugar units.
Acid hydrolysis of 1 followed by GC
analysis of its derivative enabled further
identification of the existence of D-
glucuronic acid, D-xylose, and D-glucose.
The b-configurations of D-glucuronic acid,
D-xylose, and D-glucose were determined
by the J1,2 values of 7.8 Hz, 7.8 Hz, and
2
4
4.15 had correlation with H-2 at d 4.74 and
did not show correlation with H-24 at d
1.73, suggesting the b-configurations of
C -OH and C -OH. By combined analyses
2
3
1
13
1
1
of the H NMR, C NMR, HSQC, H– H
COSY, HMBC, and ROESY spectra, the
aglycone structure of 1 was identified as
polygalagenin [6], and all their proton and
carbon signals were assigned (Table 1).
7
aglycone C NMR data of 1 with those of
.8 Hz, respectively [4,5]. Comparison of
1
3
3
-O-b-D-galactopyranosyl-(1 ! 2)-[b-D-
xylopyranosyl-(1 ! 3)]-b-D-glucurono-
pyranosyl gypsogenin 28-O-a-L-arabino-
pyranosyl-(1 ! 4)-a-L-arabinopyranosyl-
1
13
The H and C NMR data of each sugar
unit were assigned starting from the
identifiable anomeric protons by means
(
1 ! 3)-b-D-xylopyranosyl-(1 ! 4)-a-L-
1
1
rhamnopyranosyl-(1 ! 2)-b-D-fucopyra-
nosyl ester, isolated from Gypsophila
pacifica Kom. [5], showed that the
aglycone structure of 1 was almost
identical to gypsogenin, the aglycone of
the reference compound, except the dis-
tinguished difference of C-1 to C-3 in A
ring portion. The carbon signal at d 44.0
of the H– H COSY, HSQC–TOCSY,
HSQC, and HMBC spectra (Table 2). The
glycosylation positions and sugar
sequence of 1 were confirmed due to the
following long-range HMBC correlations
0
of H-1 of GlcA at d 4.99 with C-3 at d
0
0
0
83.3, H-1 of Xyl at d 5.31 with C-3 of
00
0
GlcA at d 85.9, and H-1 of Glc at d 6.31

2
42
X. Pang et al.
1 13
Table 1. H and C NMR spectral data for aglycone moieties of 1–3.
1
2
3
No
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
H
d (J in Hz)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
44.0
69.9
83.3
54.8
48.4
20.4
32.5
40.3
48.4
36.3
23.9
122.8
144.2
42.4
2.18 m, 1.16 m
4.74 m
4.15 m
–
1.47 m
1.46 m
1.76 m, 1.24 m
–
1.67 m
–
2.04 m
5.39 t (3.0)
44.5 2.27 d (13.2), 1.16 m 44.5 2.28 d (12.6), 1.28 m
70.6
86.3
53.0
52.6
21.3
33.1
40.5
48.7
36.9
24.0
122.8
144.0
42.3
4.82 m
4.75 d (3.6)
–
3.00 m, 2.07 m
1.78 m
1.69 m, 0.90 m
–
1.75 m
–
1.98 m, 0.98 m
70.6
86.2
53.0
52.7
21.3
33.3
40.5
48.7
40.0
24.0
122.8
144.0
42.3
28.1
23.5
47.0
4.85 br s
4.74 d (3.0)
–
3.00 m
1.86 m, 1.82 m
1.66 m, 0.91 m
–
1.77 m
–
2.13 m, 2.00 m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
5.38 br s
–
5.38 br s
–
–
–
–
–
28.2 2.30 dt (3.0, 13.2),1.10 m 28.3
2.08 m, 1.39 m
2.09 m, 1.89 m
–
2.32 m, 1.39 m
1.92 m, 0.97 m
–
23.4
47.0
42.4
46.2
30.8
34.0
32.5
207.0
11.8
16.8
17.6
26.2
176.4
33.1
23.7
1.94 m, 1.00 m
23.4
47.0
–
3.16 dd (9.6, 4.2)
1.74 m, 1.22 m
–
42.1 3.08 dd (10.8, 3.0)
42.2 3.11 dd (13.3, 3.0)
46.2
30.8
34.0
32.4
180.7
14.2
17.0
17.4
26.1
176.6
33.1
23.8
1.72 m, 1.18 m
46.3
30.8
33.9
32.5
180.7
14.3
17.0
17.5
26.2
176.7
33.2
23.9
1.73 m, 1.18 m
–
1.26 m, 1.16 m
2.05 m, 1.68 m
–
–
1.28 m
2.10 m, 1.76 m
–
1.08 m
1.82 m, 1.47 m
9.73
1.73 s
1.99 s
1.55 s
1.12 s
1.23 s
–
0.82 s
0.87 s
2.02 s
1.61 s
1.18 s
1.25 s
–
1.48 s
1.09 s
1.25 s
–
0.89 s
0.86 s
0.82 s
0.90 s
0
000
with C-28 at d 176.4. Consequently, the
structure of 1 was elucidated as 3-O-b-D-
xylopyranosyl-(1 ! 3)-b-D-glucurono-
pyranosyl-polygalagenin 28-O-b-D-gluco-
pyranosyl ester, named celosin H.
and 107.7 (C-1 ) were observed by the
use of HSQC spectrum, which revealed
that 2 contained four sugar units. Acid
hydrolysis of 2 followed by GC analysis of
its derivative identified the sugar units as
D-glucuronic acid, D-fucose, L-rhamnose,
and D-xylose. The b-configurations of D-
glucuronic acid, D-fucose, and D-xylose
were determined by the J1,2 values of
7.8 Hz, 8.4 Hz, and 7.2 Hz, respectively,
and the a-configuration of L-rhamnose
was determined by the carbon signals at d
Compound 2 was obtained as a white
amorphous powder with a molecular
formula C H O determined by HR-
5
3 82 24
2
ESI-MS ion [M 2 H] at m/z 1101.5189.
1
The H NMR spectrum showed four
anomeric proton signals at d 5.24 (1H, d,
0
J ¼ 7.8 Hz, H-1 ), 6.00 (1H, d, J ¼ 8.4 Hz,
0
0
000
000
H-1 ), 6.40 (1H, br s, H-1 ), and 5.02 (1H,
d, J ¼ 7.2 Hz, H-1 ), and four corre-
68.3 (C-5 ) [4,5]. Compound 2 had the
0000
almost same aglycone structure as 3-O-b-
D-glucopyranosyl gypsogenic acid 28-O-
b-D-galactopyranosyl-(1 ! 6)-b-D-gluco-
sponding anomeric carbon signals at d
0 00 000
05.9 (C-1 ), 94.8 (C-1 ), 101.4 (C-1 ),
1

Journal of Asian Natural Products Research
243
d
d
d
d
d
d

2
44
X. Pang et al.
0
00
00000
pyranosyl-(1 ! 6)-[b-D-glucopyranosyl-
1 ! 3)]-b-D-glucopyranosyl ester [5]
except the difference at C-1 and C-3 in A
1 ), and 105.1 (C-1 ) were observed by
means of HSQC spectrum, which revealed
that 3 contained five sugar units. Acid
hydrolysis of 3 followed by GC analysis of
its derivative identified the sugar units as
D-glucuronic acid, D-fucose, L-rhamnose,
D-xylose, and L-arabinose. The b-con-
figurations of D-glucuronic acid, D-fucose,
D-xylose and the a-configuration of
L-arabinose were determined by the J1,2
values of 7.8 Hz, 8.4 Hz, 7.8 Hz, and
7.8 Hz, respectively. And the a-configur-
ation of L-rhamnose was identified by the
(
1
3
ring by comparing their C NMR data.
Likewise, the higher carbon signal at d 44.5
(
C-1, þ 6.0 ppm)
and
70.6
(C-
2
, þ 46.0ppm) deduced the aglycone of 2
which also had a hydroxyl group at C-2.
The correlation between H-24 at d 1.99 and
H-25 at d 1.55 in ROESY spectrum
revealed the b-configuration of C -Me.
2
4
H-3 at d 4.75 had correlation with H-2 at d
4
2
.82 and did not show a correlation with H-
4 at d 1.99, suggesting the b-configur-
0
00
carbon signal at d 68.5 (C-5 ) [4,5]. The
molecular weight of 3 was 132 Da less
13
than that of 2. Comparison of C NMR
ations of C -OH and C -OH. By combined
2
3
1
use of the H NMR, C NMR, HSQC,
13
1
1
H– H COSY, HMBC, and ROESY
spectral data of 3 with those of 2 revealed
that they had the same aglycone, medica-
genic acid, and their structural difference
could be deduced to be only in one more
terminal arabinose. By combined use of
spectra, the aglycone structure of 2 was
elucidated as medicagenic acid [7], and all
the proton and carbon signals were assigned
1
13
(Table 1). Subsequently, the H and
NMR data of the sugar moieties were also
C
1
13
1
1
the H NMR, C NMR, HSQC, H– H
COSY, TOSCY, HMBC, and ROESY
spectra, the structures of aglycone and
sugar units were identified, and the proton
and carbon signals were fully assigned
(Tables 1 and 2). The HMBC spectrum
1
assigned by means of the H– H COSY,
1
TOCSY, HSQC, and HMBC spectra
0
(Table 2). The HMBC correlations of H-1
of GlcA at d 5.24 with C-3 at d 86.3, H-1 of
0
00
0
Fuc at d 6.00 with C-28 at d 176.6, H-1 of
Rha at d 6.40 with C-2 of Fuc at 74.1, and
0
0
0
showed the cross peaks between H-1 of
00
0
000
000
H-1 of Xyl at d 5.02 with C-4 of Rha at d
5.5 revealed the glycosylation positions
GlcA at d 5.26 with C-3 at d 86.2, and H-1
of Fuc at d 6.03 with C-28 at d 176.7, H-1
000
8
00
of Rha at d 6.32 with C-2 of Fuc at d 74.3,
and sugar sequence of 2. According to the
above mentioned information, the structure
of 2 was determined as 3-O-b-D-glucur-
onopyranosyl-medicagenic acid 28-O-b-D-
xylcopyranosyl-(1 ! 4)-a-L-rhamnopyra-
nosyl-(1 ! 2)-b-D-fucopyranosyl ester,
named celosin I.
0
000
000
H-1 of Ara at d 5.20 with C-3 of Rha of
0
0000
000
82.4, and H-1 of Xyl at d 5.35 with C-4
of Rha at d 78.9, which revealed the
glycosylation positions and sugar
sequence of 3. Finally, the structure of 3
was elucidated as 3-O-b-D-glucuronopyr-
anosyl-medicagenic acid 28-O-a-L-arabi-
nopyranosyl-(1 ! 3)-[b-D-xylcopyrano-
syl-(1 ! 4)]-a-L-rhamnopyranosyl-
(1 ! 2)-b-D-fucopyranosyl ester, named
celosin J.
Compound 3 was obtained as a white
amorphous powder with a molecular
formula C H O determined by HR-
5
8 90 28
2
ESI-MS ion [M 2 H] at m/z 1233.5614.
1
The H NMR spectrum showed five
anomeric proton signals at d 5.26 (1H, d,
0
J ¼ 7.8 Hz, H-1 ), 6.03 (1H, d, J ¼ 8.4 Hz,
3
.
Experimental
0
0
000
H-1 ), 6.32 (1H, brs, H-1 ), 5.20 (1H, d,
J ¼ 7.8 Hz, H-1 ), and 5.35 (1H, d,
0
000
3.1 General experimental procedures
0
0000
J ¼ 7.8 Hz, H-1 ), and five correspond-
Optical rotations were measured with
Perkin-Elmer 343 Polarimeter (Perkin-
Elmer, Waltham, MA, USA). The
ing anomeric carbon signals at d 105.9 (C-
1
0
00 000
), 94.8 (C-1 ), 101.4 (C-1 ) 106.3 (C-

Journal of Asian Natural Products Research
245
measurements of IR were carried out using
Bruker Vertex 70 (Bruker Corporation,
Karlsruhe, Germany). Gas chromato-
graphic analyses were carried out using
an Agilent 6890N Network GC system
equipped with a FID detector and a HP-5
capillary column (30 m £ 0.25 mm £
Chinese Medicine, Beijing Institute for
Drug Control.
3.3 Extraction and isolation
The air-dried C. argentea seeds (5 kg) were
crushed and extracted with 50% aqueous
EtOH at 1208C for three times, which was
carried out for 1 h each time. The combined
extract was filtered, concentrated under
reduced pressure. The extract was then
subjected to macroporous resin SP825
column chromatography with a gradient
0
.25 mm) (Agilent Technologies, Santa
Clara, CA, USA). The HR-ESI-MS and
ESI-MS were recorded on Synapt Q/TOF
MS (Waters Corporation, Milford, MA,
USA). The NMR spectra were recorded
1
with Varian SYS-600 (600 MHz for H
1
3
mixture of EtOH–H
2
O
(0:100 !
NMR and 150 MHz for C NMR) (Palo
Alto, CA, USA). TLC was carried out on
silica gel GF254 plates (Qingdao Marine
Chemical, Qingdao, China). Macroporous
resin SP825 (Mitsubishi Chemical, Tokyo,
Japan) and silica-gel H (Qingdao Marine
Chemical, Qingdao, China) were used for
column chromatography. HPLC analyses
were carried out using Agilent 1100
system equipped with an Alltech 2000ES
evaporative light scattering detector
15:85 ! 60:40 ! 95:5) to provide four
fractions: A (H O elution), B (15% EtOH
2
elution), C (60% EtOH elution), and D
(95% EtOH elution). Fr. C (20 g) was
separated over silica-gel column (8 cm
£
20cm) with CHCl –MeOH (5:1 ! 3:1)
3
to obtain four important combined frac-
tions: Fr. 60–81, Fr. 82–92, Fr. 116–124,
and Fr. 125–144. Among them, Fr. 60–81
was further separated on silica-gel column
(
8 cm £ 20cm) with CHCl –MeOH (5:1),
3
(temp: 1108C, gas: 2.4 l/min) (Alltech
Corporation, Chicago, IL, USA) and
and the tubes 28–75 was purified by semi-
preparative RP-HPLC with CH CN–
3
Techmate C18 column (4.6 mm £
0.01%TFA (Trifluoroacetic acid) in H O
2
2
50 mm, ODS, 5 mm) (Techmate Co. Ltd,
(
(
30:70, flow rate 4.5 ml/min) to give 1
256 mg, tR 31.7 min). Fr. 82–92 was
Beijing, China). Semi-preparative HPLC
separations were carried out using the
NP7000 module (Hanbon Co. Ltd, Huaian,
China) equipped with a Shodex RID 102
detector (Showa Denko Group, Tokyo,
Japan) and a Vensiul C18 column
purified by semi-preparative RP-HPLC
with CH CN–0.1%TFA in H O (33:67,
3
2
flow rate 4.5 ml/min) to obtain 2 (432mg,
tR 33.8min). Fr. 116–124 was separated on
ODS silica gel column with MeOH–H O,
2
(
8.0 mm £ 250 mm, ODS, 5 mm) (Bonna-
and then was purified by semi-preparative
RP-HPLC with CH CN–0.1%TFA in H O
Agela, Tianjin, China).
3
2
(
(
33:67, flow rate 4.5 ml/min) to give 3
46 mg, t 28.2min). An additional amount
3
.2 Plant material
R
of 3 (47 mg, t 29.7min) was obtained from
R
The seeds of C. argentea L. and A.
tricolors L. were collected from Bozhou,
Anhui Province, China in September
Fr. 125–144 by semi-preparative RP-
HPLC with CH CN–0.1%TFA in H O
3
2
(
33:67, flow rate 4.5 ml/min).
2
012. The seeds were identified by Prof.
Zhi-Guo Tao (Department of Traditional
Chinese Medicine, Beijing Institute
for Drug Control), and a voucher
specimen of C. argentea seeds (No.
BIDC20120916) was deposited in Her-
barium of Department of Traditional
3
.3.1 Compound 1
White amorphous powder, C H O ,
7 72 20
4
2
0
½aꢀ þ 28.8 (c ¼ 0.75, CH OH). IR
D
3
21
(KBr, cm ): n
3431, 2928, 1726,
max
1
1640, 1459, 1367, 1071, 571. H NMR

2
46
X. Pang et al.
1
pyridine-d , 600 MHz) and C NMR
3
(
was stirred at 608C for 1 h. After that,
hexamethyldisilazane–trimethylchlorosi-
lane (2:1) (3 ml) was added, and kept at 60 8C
for 0.5h. Finally, the supernatant was
analyzed by GC: Agilent 6890N Network
GC system; FID detector; HP-5 capillary
column (30 m £ 0.25mm £ 0.25mm); col-
umn temperature: 1808C keep 10min, 180–
2508C, programmed increase, 158C/min,
and 2508C keep 10min; carrier gas: N2
(1 ml/min); injection and detector tempera-
ture: 2708C; injection volume: 1.0 ml, split
ratio: 1/50. Consequently, the D-configur-
ations of glucose, fucose, xylose, and
glucuronic acid and the L-configurations of
rhamnose and arabinose were confirmed by
comparing the retention times with those of
standard samples, respectively. The reten-
5
(pyridine-d , 150 MHz) spectral data, see
Tables 1 and 2. HR-ESI-MS (negative): m/z
5
2
9
55.4583 [M 2 H] (calcd for C H O ,
47 71 20
9
55.4539).
3
.3.2 Compound 2
White amorphous powder, C H O ,
5
3 82 24
2
D
2
0
½
aꢀ þ 8.9 (c ¼ 0.80, CH OH). IR (KBr,
3
1
cm ): n
3426, 2932, 1716, 1455, 1385,
max
1
068, 623. For H NMR (pyridine-d5,
1
6
1
1
00 MHz) and C NMR (pyridine-d5,
3
50MHz) spectral data, see Tables 1 and 2.
HR-ESI-MS (negative): m/z 1101.5189
2
[
M 2 H]
(calcd for C H O ,
53 81 24
1101.5118).
3
.3.3 Compound 3
tion times (t , min) of monosaccharide
R
White amorphous powder, C H O ,
5
8 90 28
derivatives were as follows: D-glucose
(24.469, 25.924 min), D-fucose (22.175,
2
D
2
0
½
aꢀ þ 1.3 (c ¼ 0.80, CH OH). IR (KBr,
3
1
cm ): n
3423, 2934, 1717, 1455,
max
2
2
2
2
2
3.494 min),
2.115 min), D-glucuronic acid (25.222,
6.869 min), L-rhamnose (21.932,
D-xylose
(20.621,
1
386, 1059, 623. For H NMR (pyridine-
1
d5, 600 MHz) and C NMR (pyridine-d5,
1
3
1
2
50 MHz) spectral data, see Tables 1 and
. HR-ESI-MS (negative): m/z 1233.5614
3.325 min), and L-arabinose (20.594,
2.081min) [9].
2
[
M 2 H]
233.5540).
(calcd for C H O ,
58 89 28
1
Acknowledgments
This work was financially supported by the
special program of Hong Kong Chinese
Materia Medica Standards (SHK 2012-22)
from Department of Health, the Government
of the Hong Kong Special Administrative
Region of the People’s Republic of China.
The authors thank the technical support from
National Institute for Food and Drug Control.
3.4 Acid hydrolysis of 1–3 and GC
analysis
Compound 1 (5.0 mg) was treated with 2 N
aqueous CF COOH (5 ml) at 958C for 5 h.
3
After extraction with CH Cl for three times
2
2
(
5 ml £ 3), the aqueous layer was repeatedly
evaporated to dryness with EtOH until
neutral. After that, the glucose, xylose, and
glucuronic acid in monosaccharide mixture
were detected by TLC over silica gel
Note
1
.
These authors contributed equally to this
work.
(CHCl –MeOH–H O, 8:5:1) by compari-
3 2
son with authentic samples [8]. Likewise, the
fucose, rhamnose, xylose, and glucose acid
were detected from compound 2, and the
fucose, rhamnose, xylose, arabinose, and
glucuronic acid were detected from com-
pound 3. Furthermore, each residue of sugars
was dissolved in anhydrous pyridine (2 ml)
and was added L-cysteine methyl ester
hydrochloride (5 mg). Then, the mixture
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