Xanthone glycoside constituents of Swertia kouitchensis with α-glucosidase inhibitory activity

Article abstract of DOI:10.1021/np400082g

Ten new xanthone glycosides, kouitchensides A-J (1-10), and 11 known analogues were isolated from an n-butanol fraction of Swertia kouitchensis. The structures of these glycosides were determined on the basis of extensive spectroscopic data interpretation and comparison with data reported in the literature. In an in vitro test, compounds 2, 4, 5, 6, 11, 12, and 13 (IC ₅₀ values in the range 126 to 451 μM) displayed more potent inhibitory effects against α-glucosidase activity than the positive control, acarbose (IC₅₀ value of 627 μM).

Full text of DOI:10.1021/np400082g

Article
pubs.acs.org/jnp
Xanthone Glycoside Constituents of Swertia kouitchensis with
α‑Glucosidase Inhibitory Activity
Luo-Sheng Wan, Qiu-Xia Min, Yong-Long Wang, Yao-Dong Yue, and Jia-Chun Chen*
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Pharmaceutical Science Department of Tongji
Medical School, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China
S
* Supporting Information
ABSTRACT: Ten new xanthone glycosides, kouitchensides
A−J (1−10), and 11 known analogues were isolated from an
n-butanol fraction of Swertia kouitchensis. The structures of
these glycosides were determined on the basis of extensive
spectroscopic data interpretation and comparison with data
reported in the literature. In an in vitro test, compounds 2, 4,
5, 6, 11, 12, and 13 (IC50 values in the range 126 to 451 μM)
displayed more potent inhibitory effects against α-glucosidase
activity than the positive control, acarbose (IC50 value of 627 μM).
Swertia kouitchensis Franch. (Gentianaceae), widely distributed in
mainland China, has been used as a treatment for hepatitis and
diabetes.1 Previous work has also found that this plant can
control postprandial hyperglycemia by inhibiting α-glucosidase
activity.2 In order to find new inhibitors from S. kouitchensis,
α-glucosidase was used as a target to guide the chemical investi-
gation of this plant. It was found that an n-butanol-soluble
fraction of S. kouitchensis extract showed inhibitory activity against
α-glucosidase (Table S1, Supporting Information). Following
purification of this fraction, 10 new xanthone glycosides,
kouitchensides A−J (1−10), and 11 known analogues were
found. Xanthones and their derivatives have been studied
extensively for their inhibitory effects against α-glucosidase.3−6
Herein, the isolation and identification of compounds 1−10 and
the inhibitory activities of all compounds isolated against
α-glucosidase are reported.
spectra of these compounds showed characteristic carbonyl and
hydroxy groups in the ranges 1618−1650 and 3200−3435 cm−1,
respectively, and the UV absorption bands were in the ranges
226−269 and 312−379 nm, typical of a xanthone chromophore.7
Kouitchenside A (1) gave a molecular formula of C27H32O16
by HRESIMS (m/z 611.1648 [M − H]−). After acid hydrolysis, 1
gave D-xylose and D-glucose (see Experimental Section).8 The 1H
NMR and 13C NMR data (Table 1) of 1 were similar to those
reported for 1,7-dihydroxy-3,4,8-trimethoxyxanthone,9 except for
signals associated with two additional sugar residues. Moreover,
when compared to 1,7-dihydroxy-3,4,8-trimethoxyxanthone, the
carbonyl carbon C-9 in 1 displayed an upfield shift of about 5
ppm in the 13C NMR spectrum, along with the absence of a signal
1
for a chelated hydroxy group in the H NMR spectrum, sug-
gesting that the sugar unit replaced the hydroxy group at C-1.10
The assumption was confirmed by an HMBC correlation of C-1
(δC 154.3) with the anomeric proton (δH 4.90) of the glucose
unit. Further, the anomeric proton (δH 4.18) of the xylose moiety
was long-range coupled with C′-6 (δC 69.0) of Glc. The relative
configurations of the Glc and Xyl residues were both deduced to
be β, based on the coupling constants and comparison of the
NMR spectra with those of known compounds.11 Thus, 1 was
defined structurally as 1-O-[β-D-xylopyranosyl-(1→6)-β-D-
glucopyranosyl]-7-hydroxy-3,4,8-trimethoxyxanthone.
The molecular formula of kouitchenside B (2) was determined
as C28H34O16 by HRESIMS (m/z 649.1749 [M + Na]+). Its 1H
and 13C NMR spectra (Table 1) were similar to those of
compound 1, except that an additional signal for a methoxy
group at δH 3.86 replaced the hydroxy group signal at δH 9.49
(OH-7) in 1, indicating that this additional methoxy group
RESULTS AND DISCUSSION
The n-butanol fraction of S. kouitchensis was subjected to column
chromatography to yield 10 new compounds (1−10). The IR
■
Received: January 27, 2013
Published: June 27, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Table 1. 1H and 13C NMR Data of 1−3 and 3a in DMSO-d6 (δ in ppm, J in Hz in parentheses)
1
2
3
3a
position
δH
6.94, s
δC
δH
6.92, s
δC
δH
7.07, s
δC
δH
δC
1
154.3
98.0
154.2
97.8
150.5
99.4
12.83, s, OH
6.49, s
154.8
94.3
2
3
156.8
130.9
149.8
148.6
112.9
123.2
146.9
145.2
117.0
107.7
175.2
56.3
156.9
130.7
149.3
149.8
112.6
119.8
149.1
147.4
117.1
107.6
175.0
56.4
151.8
129.3
145.0
148.8
112.8
123.3
146.6
145.2
116.8
107.9
175.8
56.0
155.1
125.2
143.7
149.6
113.3
124.4
146.8
145.3
114.6
102.7
181.0
56.3
4
9.11, s, OH
8.72, s, OH
4a
4b
5
7.22, d (9.1)
7.31, d (9.1)
9.49, s, OH
7.35, d (9.3)
7.54, d (9.3)
7.20, d (9.1)
7.32, d (9.1)
9.40, s, OH
7.23, d (9.1)
7.38, d (9.1)
9.55, s, OH
6
7
8
8a
8b
9
3-OCH3
4-OCH3
7-OCH3
8-OCH3
Glc-1′
2′
3′
4′
5′
6′
3.93, s
3.81, s
3.93, s
3.90, s
3.90, s
3.81, s
60.9
3.82, s
60.9
3.86, s
56.6
3.79, s
60.8
103.4
73.4
75.8
69.6
76.1
69.0
3.81, s
61.0
3.80,s
60.8
104.7
73.6
76.1
70.2
77.7
61.1
61.0
4.90, d (7.6)
3.40, m
3.32, m
3.25, m
3.63, m
4.03, m
3.62, m
4.17, d (7.6)
2.92, m
3.08, m
3.16, m
3.66, m
3.01, m
4.92, d (7.6)
3.41, m
3.34, m
3.24, m
3.63, m
4.03, m
3.62, m
4.16, d (7.6)
3.92, m
3.09, m
3.17, m
3.67, m
3.00, m
103.1
73.3
4.73, d (5.0)
3.40, m
3.31, m
3.16, m
3.39, m
3.77, m
3.50, m
a
75.9
69.6
76.1
69.0
Xyl-1″
2″
3″
4″
5″
104.2
73.4
76.7
70.0
65.7
104.2
73.4
76.8
70.0
65.7
a
a
Overlapped signals.
should be located at C-7 of the aglycone. This deduction was
further supported by the HSQC and HMBC spectra, which
showed the additional methoxy group (δC 56.6/δH 3.86) to have
a long-range correlation with C-7 (δC 149.1). Thus, 2 was
assigned as 1-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyrano-
syl)-3,4,7,8-tetramethoxyxanthone.
Accordingly, the structure of 3a was deduced as 1,4,7-trihydroxy-
3,8-dimethoxyxanthone.
Kouitchenside D (4) was assigned the molecular formula
C27H32O15 on the basis of its HRESIMS data (m/z 619.1640
[M + Na]+). The NMR data (Table 2) of 4 were similar to those
of 3,5,8-trimethoxyxanthone-1-O-glucopyranoside,12 except for a
group of signals from an additional xylose residue. In the HMBC
spectrum, a cross-peak between the anomeric proton (δH 4.16)
of Xyl and C′-6 (δC 68.7) of Glc indicated these two sugar
residues to be connected in a 1→6 manner. The relative
configuration of the Xyl residue was deduced as β from the
coupling constant (J = 7.5 Hz). According to the data obtained, 4
was elucidated as 1-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyr-
anosyl]-3,5,8-trimethoxyxanthone.
Kouitchenside E (5) gave a molecular formula of C28H34O16
by HRESIMS (m/z 627.1932 [M + H]+). Its 13C NMR data
(Table 2) were similar to those of 4 with the exception of a signal
for an additional methoxy group, downfield shifts of C-5, C-7,
and C-8a (2.7, 42.8, and 4.2 ppm, respectively), and upfield shifts
of C-4b, C-6, and C-8 (6.9, 12.3, and 13.0 ppm, respectively).
These observations suggested that this additional methoxy group
is located at C-7, and this was confirmed by an HMBC cor-
relation between the additional methoxy group (δH 3.89) and
C-7 (δC 148.5). Thus, 5 could be proposed as 1-O-[β-D-
xylopyranosyl-(1→6)-β-D-glucopyranosyl]-3,5,7,8-tetramethox-
yxanthone.
Kouitchenside C (3) gave a molecular formula of C21H22O12,
on the basis of its HRESIMS data (m/z 489.0991 [M + Na]+).
On acid hydrolysis, 3 gave D-glucose. Comparison of the
spectroscopic data (Table 1) of 3 with those of 1 showed that
they were superimposable in terms of the xanthone core, while
the methoxy group (δH 3.81) at C-4 in 1 was replaced by a
hydroxy group (δH 9.11) in 3, and also signals associated with a
Xyl residue were absent in 3. In the HMBC spectrum, the
anomeric proton of the Glc was correlated with C-1 (δC 150.5).
From this information and from the coupling constant (J = 5.0
Hz) of the Glc anomeric proton, the structure of 3 was elucidated
as 1-O-(β-D-glucopyranosyl)-4,7-dihydroxy-3,8-dimethoxyxanthone.
The xanthone aglycone (3a) of 3 was also found to be a new
compound. The molecular formula of kouitchensone (3a), the
acid hydrolysis product of 3, was deduced as C15H12O7 by
HRESIMS (m/z 303.0511 [M − H]−). Its NMR spectra (Table 1)
were similar to the xanthone core of 3, except that 3a exhibited a
chelated hydroxy group at δH 12.83 (1H, s, OH-1) and the signal
at C-9 was shifted downfield from δC 175.8 (in 3) to 181.0 (in 3a),
suggesting the presence of a chelated hydroxy group at C-1.10
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Table 2. 1H and 13C NMR Data of 4−6 in DMSO-d6 (δ in ppm, J in Hz in parentheses)
4
5
6
position
δH
δC
δH
δC
δH
δC
b
1
158.8
100.5
164.2
95.0
158.9
100.2
164.3
94.8
11.20, s, OH
157.0
a
2
6.75, brs
6.76, d (2.3)
6.73, d (2.3)
6.73, s
98.4
158.2
129.1
148.9
144.9
139.9
121.8
109.3
153.0
107.5
102.7
184.3
3
a
4
6.75, brs
4a
157.2
145.8
141.3
116.5
105.7
152.5
113.6
108.0
174.3
56.2
157.6
138.9
144.0
104.2
148.5
139.5
117.8
107.5
174.4
56.1
4b
5
6
7.34, d (9.1)
6.84, d (9.1)
7.23, s
7.50, d (9.0)
6.75, d (9.0)
7
b
8
11.62, s, OH
8a
8b
9
a
a
3-OCH3
4-OCH3
5-OCH3
7-OCH3
8-OCH3
Glc-1′
2′
3′
4′
5′
6′
3.87,
3.87,
s
s
3.89,
s
s
3.85, s
3.91, s
61.1
57.2
a
56.4
3.96, s
56.5
56.9
60.9
102.4
73.4
75.8
69.5
75.9
68.6
a
3.89,
3.80, s
56.1
102.7
73.4
75.9
69.5
76.0
68.7
3.74, s
4.89, d (7.2)
3.37, m
4.92, d (7.6)
3.41, m
5.10, d (7.0)
3.34, m
100.1
73.1
76.4
69.5
75.7
68.5
a
a
3.30, m
3.32, m
3.33, m
3.24, m
3.25, m
3.24, m
3.60, m
3.61, m
3.65, m
3.94, m
3.96, m
3.92, m
3.61, m
3.63, m
3.63, m
Xyl-1″
2″
3″
4″
5″
4.16, d (7.5)
2.95, m
104.2
73.4
76.6
69.7
65.7
4.18, d (7.5)
2.97, m
104.1
73.4
76.6
69.7
65.7
4.16, d (7.5)
2.98, m
104.1
73.3
76.5
69.4
65.6
a
a
3.07, m
3.09, m
3.08, m
3.20, m
3.23, m
3.30, m
3.66, m
3.68, m
3.69, m
2.97, m
3.00, m
2.97, m
a
b
Overlapped signals. Signals may be interchanged.
The molecular formula of kouitchenside F (6) was determined
found to be 7.5 Hz. Therefore, 7 was determined to be 1-O-[β-D-
glucopyranosyl-(1→6)-β-D-glucopyranosyl]-3,5-dimethoxyxanthone.
The molecular formula of kouitchenside H (8) was
determined to be C27H32O16 by HRESIMS (m/z 611.1620
[M − H]−). The NMR spectra (Table 3) of 8 were very similar to
those of 7, except for an additional chelated hydroxy group signal
at δH 12.49 and a downfield shift of the C-9 (6.0 ppm) signal,
indicating that a hydroxy group is located at C-8.10 Upfield shifts
were observed in C-5, C-7, and C-8a (8.7, 15.2, and 14.1 ppm,
respectively), with a downfield shift for C-8 (37.1 ppm). Thus, 8
was defined as 1-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyr-
anosyl]-8-hydroxy-3,5-dimethoxyxanthone.
as C26H30O16 by HRESIMS (m/z 597.1483 [M − H]−). After
acid hydrolysis, 6 gave D-glucose and D-xylose. The NMR signals
(Table 2) of 6 were similar to those of corymbiferin 3-O-β-D-
glucopyranoside,13 except for additional signals for a xylose
residue and the downfield shift of C′-6 of the glucose unit (δC
from 61.8 to 68.5), suggesting that the Xyl residue is located at
C′-6 of Glc. This inference was supported by the correlation
between the anomeric proton (δH 4.16) of Xyl and C′-6 (δC
68.5) of Glc in the HMBC spectrum. From this information, in
addition to the coupling constant (J = 7.6 Hz) of the Xyl
anomeric proton, 6 was assigned as 3-O-[β-D-xylopyranosyl-(1→6)-
β-D-glucopyranosyl]-1,8-dihydroxy-4,5-dimethoxyxanthone.
The molecular formula of kouitchenside I (9) was established
as C26H30O15 by HRESIMS (m/z 605.1483 [M + Na]+). On acid
hydrolysis, 9 gave D-glucose and L-rhamnose. The NMR spectra
(Table 3) of 9 were similar to those of 7-O-β-D-glucopyranosyl-
1,8-dihydroxy-3-methoxyxanthone,15 except for a group of
signals from an additional rhamnose residue and the downfield
shift of C′-2 of a glucose residue (δC from 73.2 to 76.1). This
suggested that the Rha residue is located at C′-2 of Glc. The
assumption was supported by the HMBC spectrum, in which the
Rha anomeric proton (δH 5.21) showed a correlation with C′-2
(δC 76.1) of the Glc unit. The relative configuration of the Rha
Kouitchenside G (7) was obtained as a pale yellow powder. Its
molecular formula was established as C27H32O15 by HRESIMS
(m/z 619.1632 [M + Na]+). The NMR data (Table 3) of 7 were
closely comparable to those of 1-O-(β-D-xylopyranosyl-(1→6)-
β-D-glucopyranosyl)-3,5-dimethoxyxanthone,14 except that the
signals for a xylose residue were replaced by signals associated
with a glucose residue. Its HMBC spectrum displayed long-range
correlations between the anomeric proton (δH 5.00) of Glc-1 and
C-1 (δC 159.2) of the aglycone and between the anomeric proton
(δH 4.21) of Glc-2 and C′-6 (δC 68.9) of Glc-1. These indicated
that the Glc-2 residue is linked to the Glc-1 residue by a (1→6)
linkage. The coupling constant of the Glc-2 anomeric proton was
residue was deduced to be α by comparison with literature 13
C
NMR spectroscopic data.16 Accordingly, the structure of 9 was
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Table 3. 1H and 13C NMR Data of 7−10 in DMSO-d6 (δ in ppm, J in Hz in parentheses)
7
8
9
10
position
δH
δC
δH
δC
δH
δC
δH
δC
a
1
159.2
100.8
164.8
95.6
159.1
99.5
11.78, s, OH
6.38, brs
161.9
97.4
13.26, s, OH
6.36, brs
162.9
97.0
2
6.86, brs
6.84, brs
6.83, brs
6.82, brs
3
165.7
94.9
167.2
92.9
166.2
92.0
4
6.60, brs
6.57, brs
4a
158.4
144.6
147.8
115.7
123.9
116.5
123.0
106.8
174.5
56.3
158.6
144.0
139.1
119.8
108.7
153.6
108.9
105.3
180.5
56.3
157.6
150.0
105.5
125.1
139.7
150.0
107.4
101.8
184.1
56.3
156.5
151.6
113.3
126.4
146.1
145.5
115.0
103.4
180.5
56.1
4b
5
6.94, d (9.0)
7.58, d (9.0)
7.33, d (9.3)
7.65, d (9.3)
6
7.42, d (7.8)
7.33, t (7.9)
7.63, d (7.8)
7.41, d (9.3)
6.68, d (9.3)
12.49, s, OH
7
a
8
11.47, s, OH
8a
8b
9
3-OCH3
3.90, s
3.93, s
3.88, s
3.87, s
5-OCH3
3.95, s
56.2
3.88, s
56.7
Glc-1′
2′
3′
4′
5′
5.00, d (7.6)
3.43, m
3.33, m
3.25, m
3.71, m
4.01, m
3.66, m
102.3
73.5
5.12, d (7.6)
3.44, m
3.34, m
3.25, m
3.74, m
3.99, m
3.64, m
100.7
73.3
5.09, d (7.7)
3.56, m
3.30, m
3.21, m
3.45, m
3.65, m
3.45, m
98.8
76.1
76.9
69.8
77.6
60.6
5.10, d (7.6)
3.45, m
3.22, m
3.67, m
3.08, m
3.55, m
3.46, m
103.4
74.4
76.6
69.7
77.4
60.5
76.0
76.3
69.8
69.7
75.8
75.6
6′
68.9
68.8
Glc
4.21, d (7.6)
Glc
4.19, d (7.1)
Rha
Rha
1″
2″
3″
4″
5″
6″
103.7
73.6
76.8
70.1
77.0
61.1
103.6
73.5
76.7
70.1
76.9
61.0
5.21, brs
3.44, m
100.2
70.5
70.6
72.0
68.3
18.0
5.27, brs
3.21, m
101.3
69.5
70.4
71.8
70.2
17.9
2.98, m
3.13, m
3.05, m
3.06, m
3.66, m
3.40, m
2.97, m
3.13, m
3.05, m
3.06, m
3.66, m
3.41, m
3.71, m
3.70, m
3.18, m
3.31, m
3.95, m
3.99, m
1.10, d (6.1)
1.18, d (6.1)
a
Signals may be interchanged.
elucidated as 7-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyr-
anosyl]-1,8-dihydroxy-3-methoxyxanthone.
norswertianolin (18),17 neolancerin (19),22 7-O-[α-L-rhamno-
pyranosyl-(1→2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methox-
yxanthone (20),23 and 7-O-[β-D-xylopyranosyl-(1→6)-β-D-
glucopyranosyl]-1,8-dihydroxy-3-methoxyxanthone (21).24
All isolated compounds were evaluated for α-glucosidase
inhibitory effects. The IC50 values, defined as the compound
concentration that inhibits α-glucosidase activity by 50%, are
summarized in Table 4. Among them, compounds 2, 4, 5, 6, 11,
Kouitchenside J (10) gave a molecular formula of C26H30O15
from its HRESIMS data (m/z 605.1483 [M + Na]+) and again
afforded gave D-glucose and L-rhamnose on acid hydrolysis. The
spectroscopic data (Table 3) of 10 were similar to those of 7-O-β-
D-glucopyranosyl-1,8-dihydroxy-3-methoxyxanthone,15 except
for additional rhamnose residue signals and the absence of a
signal for a chelated hydroxy group. In addition, an upfield shift of
C-9 (3.4 ppm) signal was observed, suggesting that C-8 or C-1 is
also substituted.10 The downfield shifts of C-5, C-7, and C-8a
(7.5, 6.1, and 7.7 ppm, respectively) and the upfield shift of C-8
(4.3 ppm) indicated that C-8 is substituted. Moreover, in the
HMBC spectrum, the Glc anomeric proton (δH 5.10) was
correlated with C-8 (δC 145.5), while the Rha anomeric proton
(δH 5.27) was correlated with C-7 (δC 146.1). Thus, together
with the comparison of the two sugar signals with those of 9,
compound 10 could be proposed as 7-O-(α-L-rhamnopyrano-
syl)-8-O-(β-D-glucopyranosyl)-1-hydroxy-3-methoxyxanthone.
The known compounds isolated were identified by compar-
ison of their NMR data with literature data as swertianolin
(11),17 mangiferin (12),181-O-[β-D-xylopyranosyl-(1→6)-β-D-
glucopyranosyl]-8-hydroxy-3,7-dimethoxyxanthone (13),15 1-O-
[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-3,5-dimethox-
yxanthone (14),14 1-O-primeverosyl-3,7,8-trimethoxyxanthone
(15),19 1-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-8-
hydroxy-3,7-dimethoxyxanthone (16),20 triptexanthoside D (17),21
Table 4. Inhibitory Effects of Compounds 1−13 and Acarbose
a
against α-Glucosidase
compound
IC50 (μM)
compound
IC50 (μM)
1
2
3
4
5
6
7
1503 119
383 18
701 51
360 39
371 22
184 23
956 35
8
693 47
>1717
9
10
714 55
126 23
296 52
451 41
627 28
11
12
13
acarbose
a
Each value represents the mean SD (n = 3).
12, and 13 (with IC50 values of 383, 360, 371, 184, 126, 296, and
451 μM, respectively) displayed more potent inhibitory effects
than the positive control, acarbose (IC50 = 627 μM), with the
latter comparable to a reported value.25 Interestingly, on further
analysis, it was observed that the presence of an O-glc(6−1)-xyl
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Kouitchenside A (1): yellowish gum; [α]25D −69.0 (0.1, MeOH); UV
(MeOH) λmax (log ε) 255 (4.21), 310 (3.75), 370 (3.35) nm; IR (KBr)
νmax 3369, 2932, 1619, 1589, 1311, 1064, 827 cm−1; 1H and 13C NMR
data, see Table 1; HRESIMS m/z [M − H]− 611.1648 (calcd for
C27H31O16, 611.1618).
residue resulted in relatively more effective inhibitors than other
diglycoside units. A hydroxy group located at C-1 or C-8
(compounds 6 and 11) enhanced the inhibitory effects of the
compounds, while a diglycoside residue located at C-7
(compounds 9, 17, 20, and 21) produced steric hindrance and
lowered the inhibitory activity.
Kouitchenside B (2): pale yellow, amorphous powder; [α]25D −43.9
(0.07, MeOH); UV (MeOH) λmax (log ε) 230 (4.20), 255 (4.20), 310
(3.79), 360 (3.48) nm; IR (KBr) νmax 3401, 2926, 1660, 1601, 1481,
1
1419, 1122, 1070, 797 cm−1; H and 13C NMR data, see Table 1;
EXPERIMENTAL SECTION
■
HRESIMS m/z [M + Na]+ 649.1749 (calcd for C28H34O16Na,
649.1739).
General Experimental Procedures. Optical rotations were
measured on an AA10R digital polarimeter. UV spectra were run on a
Cary-50 UV−vis spectrophotometer. IR spectra were recorded on an
Avater-360 FT-IR spectrophotometer with KBr pellets. 1D- and 2D-
NMR spectra were recorded on a Bruker AV-400 spectrometer (400
Kouitchenside C (3): yellow, amorphous powder; [α]25D −52.1 (0.09,
MeOH); UV (MeOH) λmax (log ε) 240 (4.15), 265 (4.26), 320 (3.74)
nm; IR (KBr) νmax 3372, 2918, 1656, 1627, 1484, 1419, 1100, 823 cm−1;
1H and 13C NMR data, see Table 1; HRESIMS m/z [M + Na]+ 489.0991
(calcd for C21H22O12Na, 489.1003).
1
MHz for H and 100 MHz for 13C, respectively). Chemical shifts are
expressed in δ (ppm) and are referenced to the solvent peaks at δH 2.50
and δC 39.5 for DMSO-d6, respectively, and coupling constants are in
Hz. HRESIMS were measured by an Agilent 6520 Q-TOF LC-MS mass
spectrometer for compounds 2−7, 9, and 10 and a Bruker micrOTOF-
Q MS spectrometer for compounds 1, 3a, and 8. Preparative HPLC was
performed on a Hitachi Spectra Series HPLC system equipped with an
L-2130 pump and a UV L-2400 detector in a YMC-ODS column
(10 mm × 250 mm, 5 μm; flow rate at 2.0 mL/min; wavelength
detection at 254 nm). GC analysis was carried out on an Agilent 7820A
GC system using an HP-5 (30 m × 0.32 mm × 0.25 μm) column;
detection FID; carrier gas N2; injection temperature 250 °C, detection
temperature 250 °C, column temperature 180 °C.
Hydrolysis of Kouitchenside C (3) (ref 8). Compound 3 (10 mg)
was refluxed with 2 N CF3COOH in aqueous MeOH (25 mL) for 3 h at
60 °C. The reaction mixture was then evaporated to dryness with
MeOH until neutral and diluted with H2O (10 mL). After being
extracted with EtOAc (3 × 10 mL), the EtOAc layer was concentrated to
afford 3a.
Kouitchensone (3a): brownish, amorphous powder; UV (MeOH)
λmax (log ε) 235 (4.22), 270 (4.35), 335 (3.80) nm; IR (KBr) νmax 3367,
1652, 1608, 1477, 1328, 1212, 1101, 1055, 820 cm−1; 1H and 13C NMR
data, see Table 1; HRESIMS m/z [M − H]− 303.0511 (calcd for
C15H11O7, 303.0510).
Kouitchenside D (4): pale yellow, amorphous powder; [α]25D −57.3
(0.08, MeOH); UV (MeOH) λmax (log ε) 245 (4.17), 275 (3.98), 305
(3.99), 360 (3.80) nm; IR (KBr) νmax 3392, 2928, 1623, 1606, 1487,
1261, 1071, 806 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS
m/z [M + Na]+ 619.1640 (calcd for C27H32O15Na, 619.1633).
Kouitchenside E (5): pale yellow, amorphous powder; [α]25D −48.5
(0.08, MeOH); UV (MeOH) λmax (log ε) 230 (4.18), 250 (4.19), 300
(3.93), 370 (3.42) nm; IR (KBr) vmax 3400, 2922, 1624, 1596, 1297,
Plant Material. The whole plant of S. kouitchensis was collected in
Enshi, Hubei Province, People’s Republic of China, in October 2010,
and identified by one of the authors (J.-C.C.). A voucher specimen (S.k-
2010-1010) has been deposited in the University herbarium for future
reference.
Extraction and Isolation. The chopped, dried whole plants of
S. kouitchensis (15 kg) were refluxed with 120 L of 95% (v/v) EtOH−
H2O twice, two hours each time. After filtration, the filtrate was
concentrated under reduced pressure to yield a brownish residue
(3.0 kg). Part of the residue (2.5 kg) was suspended in water and
partitioned successively with petroleum ether, CH2Cl2, EtOAc, and
n-butanol, to afford five fractions.
The n-butanol fraction (890.0 g) was separated over a polyamide
resin column, using an EtOH−H2O gradient mobile phase, to give five
fractions (A−E). Fraction B (10% EtOH−H2O, 120 g) was purified by
passage over Toyopearl HW-40, eluted with CHCl3−MeOH (1:1), to
give three subfractions (Ba−Bc). Subfraction Bb was further purified
using ODS-A (MeOH−H2O gradient mobile phase) and then by
preparative HPLC (30% CH3CN−H2O) to yield compounds 1
(17 mg), 5 (21 mg), 7 (25 mg), 4 (9 mg), 2 (28 mg), 14 (13 mg),
and 15 (41 mg).
Fraction C (20% EtOH−H2O, 76 g) was subjected to separation over
ODS-A using a MeOH−H2O gradient mobile phase to give seven
subfractions (Ca−Ch). Subfraction Cb was purified by preparative
HPLC (27% CH3CN−H2O) to yield compounds 3 (18 mg) and 8 (25
mg). Subfraction Ce was separated over Sephadex LH-20 eluted with
CH2Cl2−MeOH (1:1) to yield compounds 16 (21 mg) and 17 (14 mg).
Subfraction Cf was purified by preparative HPLC (30% CH3CN−H2O)
to give compound 6 (16 mg). Subfraction Cg was purified over
Sephadex LH-20, eluted with CH2Cl2−MEOH (1:1), and then purified
by preparative HPLC (30% CH3CN−H2O) to yield compounds 9 (20
mg) and 10 (13 mg).
1
1062, 812 cm−1; H and 13C NMR data, see Table 2; HRESIMS m/z
[M + H]+ 627.1932 (calcd for C28H35O16, 627.1920).
Kouitchenside F (6): brownish, amorphous powder; [α]25 −56.8
D
(0.20, MeOH); UV (MeOH) λmax (log ε) 230 (4.11), 260 (4.25), 345
(3.82) nm; IR (KBr) νmax 3401, 2930, 1631, 1582, 1493, 1273, 1070, 817
cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [M − H]−
597.1483 (calcd for C26H29O16, 597.1461).
Kouitchenside G (7): pale yellow, amorphous powder; [α]25D −61.0
(0.07, MeOH); UV (MeOH) λmax (log ε) 245 (4.15), 299 (3.76), 340
(3.37) nm; IR (KBr) νmax 3391, 2922, 1623, 1591, 1299, 1071, 771 cm−1;
1H and 13C NMR data, see Table 3; HRESIMS m/z [M + Na]+ 619.1632
(calcd for C27H32O15Na, 619.1633).
Kouitchenside H (8): yellow, amorphous powder; [α]25D −44.9 (0.15,
MeOH); UV (MeOH) λmax (log ε) 240 (4.20), 280 (4.01), 310 (3.89)
nm; IR (KBr) νmax 3400, 1651, 1606, 1486, 1445, 1242, 1057, 817 cm−1;
1H and 13C NMR data, see Table 3; HRESIMS m/z [M − H]− 611.1620
(calcd for C27H31O16, 611.1618).
Kouitchenside I (9): yellow, amorphous powder; [α]25D −95.3 (0.10,
MeOH); UV (MeOH) λmax (log ε) 235 (4.19), 260 (4.23), 330 (3.95)
nm; IR (KBr) νmax 3367, 2929, 1663, 1635, 1504, 1275, 1047, 811 cm−1;
1H and 13C NMR data, see Table 3; HRESIMS m/z [M + Na]+ 605.1483
(calcd for C26H30O15Na, 605.1477).
Kouitchenside J (10): yellow, amorphous powder; [α]25 −63.3
D
(0.10, MeOH); UV (MeOH) λmax (log ε) 240 (4.18), 254 (4.21), 315
(3.95) nm; IR (KBr) νmax 3404, 2921, 1651, 1601, 1472, 1272, 1060, 821
cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z [M + Na]+
605.1483 (calcd for C26H30O15Na, 605.1477).
Acid Hydrolysis of Compounds 1−10 (ref 8). Compounds 1, 2,
and 4−10 (each 5 mg) and 3 (10 mg) were refluxed with 2 N
CF3COOH in aqueous MeOH (25 mL) for 3 h at 60 °C. The reaction
mixture was then evaporated to dryness with MeOH until neutral and
diluted with H2O (10 mL). After being extracted with EtOAc (3 × 10
mL), the aqueous layer was concentrated and compared with reference
D-glucose, D-xylose, and L-rhamnose (Sigma-Aldrich, St. Louis, MO,
Fraction D (30% EtOH−H2O, 256 g) was suspended in MeOH (0.1
g/mL) and filtered to afford a sediment and the filtrate. The sediment
(120 g) was subjected to purification by silica gel CC, using CHCl3−
MeOH (50:1−1:1) as mobile phase, to give compounds 11 (10.5 g) and
18 (16.8 g). In turn, the filtrate was subjected to passage over ODS-A
using a MeOH−H2O gradient system (0:100−100:0), to give six
subfractions (Da−Df). Compound 12 (2.3 g) was recrystallized in
MeOH from subfraction Da. Subfraction Db was purified over Sephadex
LH-20 eluted with CH2Cl2−MeOH (1:1) to give compound 19 (176
mg). Subfraction De was purified by preparative HPLC (30% CH3CN−
H2O) to give compounds 13 (23 mg), 21 (21 mg), and 20 (73 mg).
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Journal of Natural Products
Article
USA) by TLC (silica gel with CHCl3−MeOH−H2O, 6:4:1). The
residue was dissolved in pyridine (1.5 mL). Then 900 μL of HMDS−
TMCS (hexamethyldisilazane−trimethylchlorosilane, 2:1) was added,
and the mixture was stirred at 60 °C for 30 min. The supernatant was
subjected to GC analysis. Derivatives of L-rhamnose (5.64 min),
D-xylose (7.17 min), and D-glucose (13.33 min) were detected from 1−
10, separately.
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Bioassay. Inhibitory α-glucosidase (from Saccharomyces cerevisiae;
Sigma-Aldrich, St. Louis, MO, USA) activities were determined by using
p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate, according
to a reported method with minor modifications.25 Briefly, 20 μL of
enzyme solution [0.6 U/mL α-glucosidase in 0.1 M potassium
phosphate buffer (pH 6.8)] and 120 μL of the test compound in
water containing 0.5% DMSO were mixed and preincubated for 15 min
at 37 °C prior to initiation of the reaction by adding the substrate. After
preincubation, 20 μL of PNPG solution [5.0 mM PNPG in 0.1 M
potassium phosphate buffer (pH 6.8)] was added and then incubated
together at 37 °C. After incubation, 80 μL 0.2 M Na2CO3 in 0.1 M
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(21) Hideaki, O. Chem. Pharm. Bull. 1999, 47, 962−965.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Structures of known compounds (14−21), IC50 values of
extracts and known compounds (14−21), and copies of 1D-
NMR, 2D-NMR, MS, and IR spectra for compounds 1−10 and
3a. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86 27 83692793. E-mail: homespringchen@126.
com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by National Natural Science
Foundation of China [No. 30970282].
■
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