A novel adduct of ECG fused to piceid and four new dimeric stilbene glycosides from: Polygonum cuspidatum

Article abstract of DOI:10.1039/c6ra11135a

Polyflavanostilbene B (1), an unusual adduct of epicatechin-3-O-gallate fused to piceid through a carbon-carbon bond, four new dimeric stilbene glycosides (2-5), three new stilbene glucosides (6-8), one new flavan glucoside (9), and six known compounds were isolated from the rhizome of Polygonum cuspidatum. The structures of these compounds were elucidated using spectroscopic data, including electronic circular dichroism (ECD) and Rh₂(OCOCF₃)₄-induced CD spectra. All of the compounds were screened for their inhibitory activity against α-glucosidase using acarbose as a positive control (IC₅₀ = 385 μM), and strong inhibitory activity against α-glucosidase was observed for compound 8 (IC₅₀ = 3.04 μM).

Full text of DOI:10.1039/c6ra11135a

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PAPER
A novel adduct of ECG fused to piceid and four new
dimeric stilbene glycosides from Polygonum
cuspidatum†
Cite this: RSC Adv., 2016, 6, 60741
Ya-nan Yang,‡ Fu-shuang Li,‡ Fu Liu, Zi-ming Feng, Jian-shuang Jiang
and Pei-cheng Zhang*
Polyflavanostilbene B (1), an unusual adduct of epicatechin-3-O-gallate fused to piceid through a carbon–
carbon bond, four new dimeric stilbene glycosides (2–5), three new stilbene glucosides (6–8), one new
flavan glucoside (9), and six known compounds were isolated from the rhizome of Polygonum
cuspidatum. The structures of these compounds were elucidated using spectroscopic data, including
Received 29th April 2016
Accepted 16th June 2016
electronic circular dichroism (ECD) and Rh
2 3 4
(OCOCF ) -induced CD spectra. All of the compounds were
screened for their inhibitory activity against a-glucosidase using acarbose as a positive control (IC50
¼
DOI: 10.1039/c6ra11135a
3
85 mM), and strong inhibitory activity against a-glucosidase was observed for compound 8 (IC50 ¼ 3.04
www.rsc.org/advances
mM).
gallate fused to piceid through a carbon–carbon bond (1), four
new dimeric stilbene glycosides (2–5), three new stilbene
Introduction
The genus Polygonum comprises more than 300 species around glucosides (6–8), and one new avan glucoside (9), as well as six
the world and approximately 120 species distributed in China. known compounds (10–15) (Fig. 1). The structures of these
The rhizome of Polygonum cuspidatum, named HuZhang in compounds were using spectroscopic data (1D and 2D NMR,
China, is a traditional Chinese medicine (TCM) that has been UV, IR, ORD, HRESIMS, ECD, and Rh (OCOCF ) -induced CD)
2 3 4
documented in the Chinese Pharmacopoeia (1977–2015 and comparison to literature data. Additionally, the inhibitory
editions), and it is conventionally used for the treatment of activities of compounds 1–15 against a-glucosidase were
1,2
diabetes, inammation, hyperlipemia, and infection. Modern examined. Compound 8 exhibited strong inhibitory activity
pharmacological studies on P. cuspidatum have revealed against a-glucosidase with an IC50 value of 3.04 mM.
3
4
signicant biological activity, including antioxidant, antiviral,
antibacterial, antitumour, and neuroprotective effects.
5
6
7
Results and discussion
8
9
10
Various anthroquinones,
stilbenes,
avonoids,
and
11
phenols, have been isolated from this plant. In particular, Compound 1 was isolated as a white powder. Its molecular
stilbenes (resveratrol and piceid) and anthraquinones are formula was determined as C H O from the HRESIMS ion at
4
2
40 19
ꢀ
considered to be the major constituents of P. cuspidatum and m/z 847.2094 [M ꢀ H] (calcd for C H O : 847.2091), indi-
4
2
39 19
are generally regarded as the index for quality assessment of cating 23 degrees of unsaturation. IR absorptions revealed the
1
2
ꢀ1
ꢀ1
this herb.
presence of hydroxyl (3372 cm ) and carbonyl (1613 cm
Previously, we investigated the bioactive components of the groups, in addition to aromatic rings (1513 and 1450 cm ).
)
ꢀ
1
1
0
0
rhizome of P. cuspidatum. This study revealed a novel adduct
The H NMR spectrum (Table 1) of 1 displayed a set of AA BB
of epicatechin-3-O-gallate and piceid known as polyava- system aromatic protons at dH 7.04 (2H, d, J ¼ 8.5 Hz) and 6.53
1
3
nostilbene A, two novel naphthalene-fused piceid glycosides, (2H, d, J ¼ 8.5 Hz), three meta-coupled aromatic protons at d
H
14
and a series of naphthalene glucosides. In the current study, 6.18 (1H, s), 6.15 (1H, s), and 6.12 (1H, s), three ABX system
we report the isolation of an unusual adduct of epicatechin-3-O- aromatic protons at d 6.96 (1H, dd, J ¼ 8.5, 1.5 Hz), 6.90 (1H, d,
H
J ¼ 1.5 Hz), and 6.68 (1H, d, J ¼ 8.5 Hz), a singlet aromatic
proton at d
a galloyl moiety at d
H
6.00 (1H, s), and two characteristic signals of
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, People's Republic of China. E-mail: pczhang@imm.
ac.cn
H
6.88 (2H, s). In addition, four methine
4.45 (1H, d, J ¼ 10.5 Hz), 5.39 (1H, dd, J ¼ 10.5, 4.0
protons at d
H
Hz), 5.18 (1H, br s), and 5.36 (1H, br s) and two methylene
protons at dH 2.76 (1H, dd, J ¼ 17.5, 4.5 Hz) and 3.03 (1H, d, J ¼
†
Electronic supplementary information (ESI) available: 1D NMR, 2D NMR HRMS,
IR, ECD and Rh (OCOCF induced CD spectra. See DOI: 10.1039/c6ra11135a
These authors contributed equally.
2
3
)
4
17.5 Hz) were observed. Finally, a doublet at d
H
4.61 (1H, d, J ¼
‡
7.5 Hz) caused by an anomeric proton and the proton signals
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Fig. 1 Chemical structures of 1–15.
0
0
between d
H
3.15 and 3.64 indicated the presence of a glycosyl congurations of C-2, C-3, C-7 , and C-8 . Cellulase hydrolysis of
0
group. Aer cellulose hydrolysis, the sugar unit of 1 was 1 resulted in 1a (the aglycone) and D-glucose. The 8 S congu-
conrmed to be the D-conguration by GC analysis of its tri- ration was supported by a positive Cotton effect at 357 nm in the
16
methylsilyl L-cysteine derivative.
Rh (OCOCF ) -induced CD spectrum of 1a (Fig. S7, ESI†). The
2 3 4
The C NMR spectrum (Table 2) of 1 revealed a total of 42 threo conguration between the two chiral centres at C-7 and C-
1
3
0
0
carbon signals, six of which were assigned to an O-glucose unit;
8
was established using the coupling constant (10.5 Hz)
the remaining 36 carbons were assigned to a C6–C2–C6 unit and between H-7 and H-8 . From the above analysis, the absolute
a avan aglycone with an additional galloyl moiety. A careful conguration of C-7 was determined to be R. In addition, the
0
0
17
0
comparison of the 1D NMR signals of 1 with the corresponding 2R,3R congurations were conrmed by the small coupling
15
data of epicatechin-3-O-gallate (ECG), suggested the presence constant of H-2/H-3 and the negative Cotton effect at 280 nm in
18
of an ECG moiety in 1, which was veried by HMBC correlations the ECD spectrum. Based on these results, the structure of 1
from H-3 to C-2, C-4, C-10, C-11, and C-17, and from H-19, H-23 was assigned as shown in Fig. 1, and the compound was
to C-17. By considering the remaining eight degrees of unsa- accorded the trivial name polyavanostilbene B.
turation and the molecular formula, the C6–C2–C6 unit was
elucidated as the 4 ,8 ,11 ,13 -tetrahydroxyl diphenylethane with a molecular formula of C40 17 on the basis of HRE-
moiety. The connectivity of these two moieties was established SIMS (m/z 819.2478 [M + Na] , calcd for C40
Compound 2 was obtained as a white amorphous powder,
0
0
0
0
44
H O
+
H
44
O
17Na:
1
0
0
by analysing the HMBC spectrum (Fig. 2). Correlations from H- 819.2471). Its H NMR spectrum exhibited two sets of AA BB
0
0
7
to C-7, C-8, and C-9 were observed, suggesting that the C-7 is system aromatic protons at d
H
7.34 (2H, d, J ¼ 8.0 Hz), 6.89
connected to the C-8 of the ECG moiety by a C–C bond. The (2H, d, J ¼ 8.0 Hz), 6.79 (2H, d, J ¼ 8.0 Hz), and 6.43 (2H, d, J ¼
0
sugar moiety was conrmed to be at C-11 position of the 8.0 Hz), three meta-coupled aromatic protons at d 6.53 (1H, s),
H
0
0
0
aglycone by the HMBC correlation of H-1 with C-11 .
Examination of the ECD and Rh (OCOCF
CD spectra of 1 allowed for determination of the absolute olenic protons at d
6.40 (1H, s), and 6.23 (1H, s), one 1,2,3,5-tetrasubstituted
-induced aromatic protons at d 6.54 (1H, s) and 6.21 (1H, s), two trans-
7.27 (1H, d, J ¼ 16.0 Hz) and 6.70 (1H, d,
2
3
)
4
H
H
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1
Table 1 H NMR data of 1–5 in DMSO-d
6
(500 MHz, d in ppm, J in Hz)
No.
1
2
3
4
5
2
3
4
5.18, br s
5.36, br s
3.03, d (17.5)
6.21, s
6.13, s
6.44, s
6.48, s
2.76, dd (17.5, 4.5)
6
7
8
9
6.00, s
6.54, s
6.70, d (16.0)
7.27, d (16.0)
6.27, s
6.59, d (16.0)
6.87, d (16.0)
6.06, s
6.60, d (12.0)
6.51, d (12.0)
6.06, s
6.58, d (11.5)
6.51, d (11.5)
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
7
8
1
1
1
1
1
1
2
3
4
5
6
0
1
2
3
4
5
6
9
7.34, d (8.0)
6.79, d (8.0)
7.35, d (8.5)
7.01, d (8.5)
6.86, d (8.5)
6.47, d (8.5)
6.87, d (8.5)
6.46, d (8.5)
6.90, d (1.5)
6.79, d (8.0)
7.34, d (8.0)
7.01, d (8.5)
7.35, d (8.5)
6.47, d (8.5)
6.86, d (8.5)
6.46, d (8.5)
6.87, d (8.5)
6.68, d (8.5)
6.96, dd (8.5, 1.5)
6.88, s
3
6.88, s
0
0
0
0
0
0
0
0
7.04, d (8.5)
6.53, d (8.5)
6.40, s
6.23, s
6.49, s
6.25, s
6.41, s
6.20, s
6.23, s
6.16, s
6.53, d (8.5)
7.04, d (8.5)
4.45, d (10.5)
5.39, dd (10.5, 4.0)
6.15, s
6.53, s
6.40, s
5.49, d (3.0)
4.47, m
7.03, d (8.5)
6.54, d (8.5)
6.27, s
6.32, s
5.57, d (10.0)
4.37, d (10.0)
6.89, d (8.0)
6.43, d (8.0)
5.61, d (10.5)
4.49, d (10.5)
6.94, d (8.0)
6.34, d (8.0)
5.66, d (10.5)
4.29, d (10.5)
7.10, d (8.0)
6.33, d (8.0)
0
0
0
0
0
0
1
2
3
4
6.18, s
6.43, d (8.0)
6.89, d (8.0)
4.85, d (7.0)
3.46, m
3.36, m
3.27, m
6.54, d (8.5)
7.03, d (8.5)
4.87, d (7.5)
3.19, m
3.32, m
3.17, m
6.34, d (8.0)
6.94, d (8.0)
4.86, d (7.5)
3.46, m
3.34, m
3.27, m
6.33, d (8.0)
7.10, d (8.0)
5.02, d (7.0)
3.50, m
3.33, m
3.23, m
6.12, s
0
0
0
0
0
0
0
0
0
0
0
0
4.61, d (7.5)
3.15, m
3.21, m
3.25, m
3.21, m
3.55, m
3.21, m
3.52, m
3.42, m
3.46, m
3.19, m
3.54, m
3.34, m
3.51, m
3.64, m
3.75, d (11.0)
4.66, d (7.0)
3.16, m
3.36, m
3.20, m
3.66, m
4.57, d (6.0)
3.15, m
3.33, m
3.31, m
3.73, m
4.63, d (7.5)
3.14, m
3.20, m
3.18, m
3.73, m
4.57, d (7.5)
3.13, m
3.19, m
3.19, m
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
3.22, m
3.54, m
3.19, m
3.46, m
3.43, m
3.50, m
3.19, m
3.51, m
3.65, d (11.5)
3.66, m
3.61, d (11.5)
3.64, m
1
7
J ¼ 16.0 Hz), two aliphatic protons at d
H
5.57 (1H, d, J ¼ 10.0 stilbene glycoside 1, which was previously isolated from P.
Hz) and 4.37 (1H, d, J ¼ 10.0 Hz), and two glucopyranosyl cuspidatum but the absolute congurations were not assigned.
0
0
anomeric protons at 4.85 (1H, d, J ¼ 7.0 Hz) and 4.66 (1H, d, J ¼ The absolute congurations of C-7 and C-8 were identied by
1
3
7
.0 Hz). The C NMR spectrum of 2 showed 40 carbon reso- the same method as that used for 1. The coupling constant
0
nances (Table 2), consisting of 12 carbon signals of two (J
7
0
,8
0
¼ 10.0 Hz) conrmed the threo conguration of C-7 and
0
glucose units and 28 skeleton carbon signals corresponding to C-8 . Hydrolysis of 2 with cellulase yielded 2a and D-glucose,
two C6–C2–C6 units. In the HMBC spectrum (Fig. 2), the which was conrmed by GC analysis of its trimethylsilyl
0
0
correlation peaks of H-8 at d
H
4.37 with C-1 (d
C
139.7), C-2 (d
C
L-cysteine derivative. The absolute conguration of C-7 in 2a
1
20.4), and C-3 (d
C
157.1) helped verify the linkage point was dened as S based on a positive Cotton effect at 350 nm in
0
0
between C-8 and C-2. Furthermore, two glucose units were the Rh (OCOCF ) -induced CD spectrum (Fig. S15, ESI†). C-8
determined to be located at C-3 and C-3 based on correlations was then assigned the R absolute conguration, in accordance
between H-1 (d 4.85) with C-3 (d 157.1) and H-1 (dH 4.66) with the 7 ,8 -threo conformation. Thus, the structure of 2 was
with C-3 (d
2
3 4
0
00
000
0
0
H
C
0
C
158.1) in the HMBC spectrum. This spectroscopic established as shown (Fig. 1), and this compound was given
data suggested that 2 had the same planar structure as dimeric the trivial name stilbenedimer A.
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13
0
0
Table 2
C NMR data of 1–9 in DMSO-d
6
(125 MHz, d in ppm)
indicated the 7 R,8 R congurations for 3a. Thus, 3 was estab-
lished as shown and was named stilbenedimer B.
No.
1
2
3
4
5
6
7
8
9
Compound 4 had the same molecular formula as 2, as
indicated by the sodiated molecular ion peak observed at m/z
1
2
3
4
5
6
7
8
9
139.7 140.0 141.0 140.6 139.4 139.4 140.1
+
76.8 120.4 117.1 121.7 122.1 105.6 105.2 107.9 81.4 819.2463 [M + Na] in the HRESIMS. A characteristic coupling
1
68.8 157.1 157.7 157.7 156.3 158.9 158.5 159.0 66.0 constant (J7,8 ¼ 12.0 Hz) between H-7 and H-8 in the H NMR
26.3 101.8 104.0 102.2 102.6 102.9 103.2 103.5 28.0
spectrum (Table 1) suggested the presence of a cis-stilbene
153.7 156.1 157.0 156.7 155.9 158.4 158.4 158.8 155.7
95.6 106.9 105.0 109.6 109.5 106.6 107.5 105.8 94.9
154.2 124.7 126.4 129.0 128.3 125.3 125.2 125.6 156.8
moiety in 4 instead of the trans-stilbene moiety observed in 2.
The presence of the aglycone (4a) was determined by the
0
108.6 130.7 130.0 130.2 129.6 128.5 128.6 128.5 96.6 hydrolysis of 4 with cellulose. The threo conguration of C-7
0
0
154.4 128.2 131.0 127.9 127.3 128.1 128.0 129.1 155.2 and C-8 was veried by the coupling constant between H-7 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
2
3
4
5
6
7
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
97.9 127.8 127.6 130.9 130.6 128.1 128.0 128.6 101.2
130.2 115.8 116.6 115.3 114.7 115.6 115.6 115.9 130.2
114.3 157.4 157.0 157.1 157.5 157.4 157.4 157.7 115.2
144.5 115.8 116.6 115.3 114.7 115.6 115.6 115.9 145.0
0
H-8 (J
0
0
¼ 10.5 Hz). This result, combined with the positive
7
,8
Cotton effect at 358 nm in the Rh
2
(OCOCF
3
)
4
0
-induced CD
0
spectrum of 4a (Fig. S31, ESI†), indicated the 7 S,8 R congu-
144.9 127.8 127.6 130.9 130.6 128.1 128.0 128.6 144.9 ration of 4. Consequently, 4 was elucidated as an isomer of 2
115.2
117.5
165.5
119.8
108.9
145.6
138.7
145.6
114.9 and named stilbenedimer C.
118.7
Compound 5, a white amorphous powder, had the same
molecular formula (C H O Na, m/z 819.2468) as 4. The UV,
4
0
44 17
IR, and NMR spectroscopy data of 5 indicated that it was an
optical isomer of 4. Cellulase hydrolysis of 5 resulted in 5a and
D-glucose. In the Rh
a negative Cotton effect at 356 nm indicated the 7 R congu-
ration in 5a (Fig. S39, ESI†). Meanwhile, the 8 S conguration
was conrmed by the large coupling constant between H-7
2
(OCOCF
3
)
4
-induced CD spectrum,
0
3
108.9
0
0
133.7 147.8 146.7 148.5 148.0 100.8 100.6 99.4 97.9
129.6 106.8 105.9 107.2 106.8 73.2 75.0 74.0 73.5
0
0
0
0
0
0
0
0
0
0
114.5 158.1 158.4 158.6 157.7 76.7 77.4 74.4 74.2 and H-8 (J
7
0
,8
0
¼ 10.5 Hz). Based on the aforementioned
154.8 102.0 102.0 102.1 101.5 69.7 69.9 70.8 69.7 information, 5 was identied as shown (Fig. 1) and named
114.5 157.4 157.7 158.0 157.5 75.0 77.0 75.4 77.0
stilbenedimer D.
129.6 108.6 107.4 109.5 107.9 66.3 60.5 66.1 60.4
Compound 6 was obtained as a white amorphous powder
49.6 74.0 74.5 74.4 74.0
and had a molecular formula of C26
protonated molecular ion peak at m/z 553.1920 [M + H] (calcd
for C26 13: 553.1916) in the HRESIMS. The UV, IR, and NMR
32
H O13, as evidenced by the
74.7 52.7 50.5 54.0 54.4
148.4 132.9 132.6 133.1 132.5
106.6 129.9 129.2 131.1 130.6
157.7 114.5 114.6 114.7 114.3
101.3 154.9 155.2 155.4 154.8
157.3 114.5 114.6 114.7 114.3
108.1 129.9 129.2 131.1 130.6
+
0
0
0
0
0
0
1
2
3
4
33
H O
18
spectroscopic data of 6 were closely related to those of piceid,
a known compound that was isolated as the major component
from the rhizome of P. cuspidatum. Comparison of the NMR
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
00
00
00
100.5 101.5 100.4 101.2 100.6 98.5 97.6 119.8 119.6 spectra with those of piceid demonstrated these compounds
73.3 74.4 73.3 74.0 73.3 72.1 72.1 109.2 108.9 differed only by the presence of an additional glucose moiety in
76.7 77.0 77.1 77.0 77.1 73.2 73.2 146.0 145.5
69.2 69.6 69.8 69.6 69.8 70.2 70.1 139.0 138.5
76.7 76.6 76.9 76.6 77.0 72.6 72.2 146.0 145.5
60.3 60.6 60.5 60.3 60.8 60.8 60.7 109.2 108.9
1
6
. The H NMR spectrum displayed two anomeric protons at d
H
4
.78 (1H, d, J ¼ 7.5 Hz) and d 4.69 (1H, d, J ¼ 3.0 Hz), which
H
signied the presence of two glucose units with one b-linkage
1
3
165.4 165.0 and one a-linkage. In the C NMR spectrum, the resonance for
0
100.6 101.1 100.4 100.3
73.2 73.3 73.1 73.1
77.2 76.7 76.8 76.6
69.4 69.5 69.3 69.3
76.9 76.7 77.1 76.8
60.5 60.8 60.6 60.3
C-6 (d
66.3) of 6 was shied signicantly downeld compared
C
0
to piceid. This suggested that the a-glucose was attached to C-6
of the b-glucose, which was further supported by the HMBC
correlation between H-1 and C-6 . Aer acid hydrolysis, the
glucose unit was conrmed to be D-conguration by GC anal-
ysis of its trimethylsilyl L-cysteine derivative. Using the data
above, the structure of 3 was elucidated and determined to be
0
0
0
0
piceid-6 -O-a-D-glucopyranoside.
The molecular formula of 3 was determined to be the same
Compound
7 exhibited the same molecular formula
44
as 2 (C40H O17), based on the positive HRESIMS ion observed
(
6
C H O ) as that of 6. Comparison of the NMR data for 7 and
2
6
32 13
+
at m/z 819.2464 [M + Na] . Careful analysis of the UV, IR, and
NMR spectroscopic data revealed that 3 had the same planar
structure as 2. The coupling constant (3.0 Hz) of the two
showed that the two compounds differed by the location of the
0
0
a-D-glucose unit. The key HMBC correlation from H-1 (d 5.19)
to C-2 (d
C-2 . All of the aforementioned data indicated that the structure
of 7 was piceid-2 -O-a-D-glucopyranoside.
H
0
C
75.0) conrmed the location of the a-D-glucose unit at
0
0
0
aliphatic protons H-7 (d 5.49) and H-8 (d 4.47) suggested that
H
H
0
they were in the erythro conformation. Cellulase hydrolysis of 3
resulted in 3a and D-glucose. In the Rh (OCOCF -induced CD
experiment (Fig. S23, ESI†), a negative Cotton effect at 361 nm
2
3 4
)
Compound 8 was obtained as a white amorphous powder.
26
The molecular formula of 8 was C27H O15S, as conrmed by
60744 | RSC Adv., 2016, 6, 60741–60748
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Fig. 2 Key HMBC correlations of 1, 2, and 9.
ꢀ
the negative HRESIMS ion observed at m/z 621.0929 [M ꢀ H]
Compound 9 was isolated as a white amorphous powder, and
(
25 28
calcd for C27H O15S: 621.0920), and likely contains a sulfate the molecular formula C28H O15 was determined by the positive
group. A detailed analysis of the spectroscopic data revealed HRESIMS ion (m/z 627.1316 [M + Na] ). The IR spectrum revealed
that 8 was a piceid derivative with a galloyl moiety. The HMBC absorption bands attributed to hydroxy groups (3363 cm ),
correlation observed between H-2 (dH 4.92) and C-7 (d 165.4), carbonyl groups (1698 cm ), and aromatic rings (1610, 1528,
4.92) indicated that 1504, and 1453 cm ). The H NMR spectrum (Table 3) of 9
the galloyl moiety was linked at C-2 of glucose. The unusual revealed a set of ABX system aromatic protons at d
+
ꢀ1
0
00
ꢀ1
C
0
ꢀ1
1
together with the downeld shi of H-2 (d
H
0
H
6.67 (1H, d, J ¼
0
0
downeld shi of the H-6 (d
H
4.16, 3.81), C-6 (d
C
66.1) was an 1.5 Hz), 6.63 (1H, d, J ¼ 8.0 Hz) and 6.52 (1H, dd, J ¼ 8.0, 1.5 Hz),
0
important indicator that the sulfate group was attached at C-6
two meta-coupled aromatic protons at d
H
6.11 (1H, d, J ¼ 1.5 Hz)
of glucose. Aer acid hydrolysis, the sugar unit was conrmed and 5.85 (1H, d, J ¼ 1.5 Hz), and two characteristic signals of
to be D-glucose by GC analysis of its trimethylsilyl L-cysteine a galloyl moiety at d 6.94 (2H, s) in the downeld region. Addi-
H
derivative. On the basis of these data, the structure of 8 was tionally, two oxygenated methylene protons at dH 4.33 (1H, d, J ¼
0
0
determined to be piceid-2 -galloyl-6 -sulfate.
7.5 Hz) and 3.59 (1H, m), two methylene protons at dH 2.45 (1H,
1
Table 3 H NMR data of 6–9 in DMSO-d
6
(500 MHz, d in ppm, J in Hz)
No.
6
7
8
9
2
3
4
6.63, s
6.40, s
6.75, s
6.33, s
6.57, s
6.17, s
4.33, d (7.5)
3.59, m
2.26, dd (16.5, 8.5)
2.45, overlap
6
7
8
6.59, s
6.56, s
6.59, s
6.11, d (1.5)
6.87, d (16.5)
6.99, d (16.5)
7.41, d (8.5)
6.74, d (8.5)
6.84, d (16.5)
7.02, d (16.5)
7.39, d (8.5)
6.74, d (8.5)
6.83, d (16.5)
6.93, d (16.5)
7.42, d (8.5)
6.71, d (8.5)
5.85, d (1.5)
1
1
1
1
1
1
1
1
2
3
4
5
6
0
1
2
3
4
5
6.67, d (1.5)
6.74, d (8.5)
7.41, d (8.5)
6.74, d (8.5)
7.39, d (8.5)
6.71, d (8.5)
7.42, d (8.5)
6.63, d (8.0)
6.52, dd (8.0, 1.5)
5.11, d (8.0)
4.96, dd (8.0, 8.0)
3.58, m
3.34, m
3.43, m
3.56, m
3.72, d (11.0)
6
0
4.78, d (7.5)
3.44, m
3.27, m
3.27, m
3.52, m
3.62, m
5.00, d (8.0)
3.42, m
3.50, m
3.19, m
3.35, m
3.40, m
3.53, m
5.19, d (3.5)
3.90, m
3.44, m
3.35, m
3.19, m
3.53, m
3.72, m
5.01, d (8.0)
4.92, dd (8.0, 8.0)
3.57, m
3.26, m
3.70, m
0
0
0
0
0
3.81, m
4.16, d (10.0)
3.75, m
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4.69, d (3.0)
3.21, m
3.22, m
3.09, m
3.08, m
3.45, m
6.97, s
6.97, s
6.94, s
6.94, s
3.57, m
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overlap), 2.26 (1H, dd, J ¼ 16.5, 8.5 Hz), and one anomeric proton identied by L. Ma (Institute of Materia Medica, Peking Union
1
3
at d 5.11 (1H, d, J ¼ 8.0 Hz) were also observed. The C NMR Medical College and Chinese Academy of Medical Sciences,
H
spectrum of 9 showed 28 carbon resonances (Table 2) corre- Beijing 100050, P. R. China). A voucher specimen (ID number:
sponding to the glucose unit, galloyl moiety, and 3,5,7,3 ,4 -pen- ID-S-2593) was deposited at the Institute of Materia Medica,
tahydroxyavane. The HMBC correlation (Fig. 2) between H-2 (d
165.0) indicated that the galloyl moiety was Sciences, Beijing 100050, P. R. China.
attached to C-2 of the glucose. Furthermore, the linkage point of
0
0
0
H
Peking Union Medical College and Chinese Academy of Medical
00
4
.96) and C-7 (d
C
0
the glucose was identied by the correlation between the anomeric
0
Extraction and isolation
proton H-1 and C-5 in the HMBC experiment. Acid hydrolysis of 9
0
0
gave the 3,5,7,3 ,4 -pentahydroxyavane (9a), gallic acid, and Powdered P. cuspidatum (20 kg) was extracted with 80% EtOH
glucose. Using the same method as described in 8, the D-cong- under reux (3 ꢁ 2 h). The EtOH extract was concentrated under
uration of glucose was conrmed. The coupling constant of H-2 (J reduced pressure to give a residue (4.2 kg), which was sus-
¼
7.5 Hz) indicated that H-2/H-3 was in a trans conguration. This pended in H O (4 L) and partitioned with, consecutively,
2
observation, along with the negative Cotton effect at 281 nm in the petroleum ether (PE) (3 ꢁ 4 L), EtOAc (3 ꢁ 4 L), and n-BuOH (3
ECD spectrum of 9a (Fig. S64, ESI†), permits assignment of the ꢁ 4 L). Aer evaporation of the n-BuOH solvent under reduced
19
absolute congurations of C-2 and C-3 as 2R,3S. Thus, 9 was pressure, the extract (1375 g) was suspended in H
dened as (+)-catechin-5-O-b-D-(2 -O-galloyl)-glucopyranoside.
2
O (2 L) and
0
subjected to column chromatography over macroporous resin,
Based on MS data, NMR data and comparison with litera- eluting successively with H O, 30% EtOH, 50% EtOH, 70%
2
tures, known compounds (Fig. 1) were identied as 7-O-(b-D- EtOH, and 95% EtOH (30 L each). The 30% EtOH was removed
2
2
2
0
1
2
glucopyranosyloxy)-5-hydroxy-1(3H)-isobenzofuranone (10),
under reduced pressure and the fraction (86.4 g) was subjected
2,4,6-trihydroxyacetophenone-3-C-b-D-glucopyranoside (11),
to chromatography over Sephadex LH-20 with H O–MeOH in
2
2
5
,4,6-trihydroxy-acetophenone-4-O-b-D-glucopyranoside (12),
gradient as the mobile phase to yield 25 fractions (Fr. A–Fr. Y)
20
23
,7-dihydroxyphthalide (13), altechromone A (14), and 7- on the basis of HPLC-DAD analysis.
24
demethylsiderin (15).
Fr. B was subjected to reversed-phase preparative HPLC, using
MeOH–H O (24 : 76) as the mobile phase, to give 11 (25 mg) and
2 (22 mg). Compound 10 (12 mg) was obtained from Fr. C by
2
1
Experimental
recrystallization. Fr. D was puried by preparative RP-HPLC using
General experimental procedures
MeOH–H O (28 : 72) as the mobile phase to yield 14 (53 mg). Fr. E
2
2
was puried by preparative RP-HPLC using MeOH–H O (31 : 69)
as the mobile phase to yield 13 (13 mg). Fr. I was subjected to
The optical rotations were measured on a Jasco P-2000 polar-
imeter (JASCO, Easton, MD, U.S.A.). ECD spectra were recorded
with a JASCO J-815 spectrometer. IR spectra were recorded on
Sephadex LH-20 column and eluted with MeOH–H O (from
2
20 : 80 to 60 : 40) to afford 20 fractions (Fr. I-1–I-20). Fr. I-13 was
a Nicolet 5700 spectrometer (Thermo Scientic, Waltham, MA,
1
further puried using preparative RP-HPLC with MeOH–H O
2
U.S.A.) using an FT-IR microscope transmission method.
H
1
3
(50 : 50) as the mobile phase to yield 15 (8 mg). Fr. N was subjected
NMR (500 MHz), C NMR (125 MHz), and 2D NMR spectra were
run on Bruker 500 MHz NMR spectrometer (Bruker-Biospin,
Billerica, MA, U.S.A.) using TMS as internal standard and the
values are given in ppm. High-resolution electrospray ionization
mass spectrometry (HRESIMS) was performed on an Agilent
to Sephadex LH-20 column and eluted with MeOH–H
0 : 80 to 60 : 40) to afford 20 fractions (Fr. N-1–N-20). Fr. N-9 was
further puried using preparative RP-HPLC with MeOH–H
33 : 67) as the mobile phase to yield 6 (11 mg), 7 (10 mg), 3 (10
mg), and 5 (6 mg). Fr. N-10 was further puried using preparative
RP-HPLC with MeOH–H O (40 : 60) as the mobile phase to yield 2
2
O (from
2
2
O
(
1100 series LC/MSD ion trap mass spectrometer (Agilent Tech-
2
nologies, Waldbronn, Germany). HPLC-DAD analysis was per-
formed using an Agilent 1200 series system with an Apollo C18
column (250 mm ꢁ 4.6 mm, 5 mm; Grace Davison). GC analysis
was performed using an Agilent 7890A series system with
a capillary column, HP-5 (30 m ꢁ 0.25 mm, with a 0.25 mm lm,
Dikma). Preparative HPLC was carried out on a Shimadzu LC-
(13 mg). Fr. O was subjected to Sephadex LH-20 column and
2
eluted with MeOH–H O (from 20 : 80 to 60 : 40) to afford 20
fractions (Fr. O-1–O-20). Fr. O-18 was further puried using
preparative RP-HPLC with MeOH–H O (34 : 66) as the mobile
2
phase to yield 4 (10 mg). Fr. Q was subjected to Sephadex LH-20
column and eluted with MeOH–H O (from 20 : 80 to 60 : 40) to
2
6AD instrument with an SPD-20A detector (Shimadzu Corp.,
afford 15 fractions (Fr. Q-1–Q-15). Fr. Q-12 was further puried
Tokyo, Japan), using a YMC-Pack ODS-A column (250 mm ꢁ 20
mm, 5 mm; YMC Corp., Kyoto, Japan). Column chromatography
was performed with macroporous resin (Diaion HP-20, Mitsu-
bishi Chemical Corp., Tokyo, Japan), Rp-18 (50 mm, YMC Corp.),
Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden).
using preparative RP-HPLC with MeOH–H
mobile phase to yield 9 (11 mg). Fr. R was subjected to Sephadex
LH-20 column and eluted with MeOH–H O (from 20 : 80 to
0 : 40) to afford 20 fractions (Fr. R-1–R-20). Fr. R-11 was further
puried using preparative RP-HPLC with MeOH–H O (33 : 67) as
2
O (19 : 81) as the
2
6
2
the mobile phase to yield 8 (10 mg). Fr. U was subjected to
Plant material
Sephadex LH-20 column and eluted with MeOH–H O (from
2
The rhizome of P. cuspidatum was purchased from Beijing Pu 20 : 80 to 60 : 40) to afford 30 fractions (Fr. U-1–U-30). Fr. U-25 was
Sheng Lin Pharmaceutical Co., Ltd. in Beijing, People's further puried using preparative RP-HPLC with MeOH–H
Republic of China, in Feb 2010. The plant material was (38 : 62) as the mobile phase to yield 1 (24 mg).
2
O
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Structure characterization
reaction was monitored by HPLC-DAD analysis. Aer cooling,
the reaction mixture was extracted with EtOAc (3 ꢁ 5 mL). The
aqueous layer was evaporated under vacuum to furnish a crude
sugar fraction. Hydrolysis of 2–5 was performed according to
the procedure described for 1.
2
5
Compound (1). White amorphous powder; [a]D ꢀ149.1 (c
0
.05, MeOH); UV (MeOH) lmax (log 3) 280 (0.26) nm; ECD
(
3
(
MeOH) lmax (D3) 285 (ꢀ3.41), 222 (ꢀ5.38) nm; IR (KBr) nmax
:
ꢀ
1
ꢀ
372, 1613, 1513, 1450 cm ; HRESIMS m/z 847.2094 [M ꢀ H]
1 13
calcd for C42
40
H O19, 847.2091); H and C NMR data, see
Tables 1 and 2.
Acid hydrolysis of 6–9
2
5
Compound (2). White amorphous powder; [a]D ꢀ32.3 (c
Compound 6 (2 mg) was dissolved in 0.5 M HCl–H O (2 mL) and
2
0
.05, MeOH); UV (MeOH) lmax (log 3) 283 (0.21), 316 (0.14) nm;
reuxed for 2 h. Aer cooling, the reaction mixture was
extracted with EtOAc (3 ꢁ 5 mL). The aqueous layer was evap-
orated under vacuum to furnish a crude sugar fraction.
Hydrolysis of 7–9 was performed according to the procedure
described for 6.
ECD (MeOH) lmax (D3) 276 (+6.54), 234 (ꢀ27.99) nm; IR (KBr)
ꢀ
1
n
max: 3328, 1603, 1514, 1453 cm ; HRESIMS m/z 819.2478 [M +
+
1
13
Na] (calcd for C40
see Tables 1 and 2.
Compound (3). White amorphous powder; [a]
.05, MeOH); UV (MeOH) lmax (log 3) 283 (0.21), 323 (0.12) nm;
ECD (MeOH) lmax (D3) 298 (ꢀ3.16), 238 (ꢀ3.33) nm; IR (KBr)
44
H O17Na, 819.2471); H and C NMR data,
2
5
D
ꢀ122.4 (c
0
Gas chromatographic (GC) analysis of glucose
ꢀ
1
n
: 3365, 1604, 1511, 1453 cm ; HRESIMS m/z 819.2464 [M +
Na] (calcd for C H O Na, 819.2471); H and C NMR data,
max
+
1
13
The crude sugar fraction was dissolved in anhydrous pyridine (1
mL), to which 2 mg of L-cysteine methyl ester hydrochloride was
4
0
44 17
see Tables 1 and 2.
Compound (4). White amorphous powder; [a]
.06, MeOH); UV (MeOH) lmax (log 3) 284 (0.21), 320 (0.13) nm;
ECD (MeOH) lmax (D3) 280 (ꢀ2.93), 214 (ꢀ19.37) nm; IR (KBr)
ꢂ
2
5
added. The mixture was stirred at 60 C for 2 h. Aer evapora-
D
ꢀ109.3 (c
tion under reduced pressure, 0.2 mL of N-trimethylsilylimida-
0
ꢂ
zole was added, and the mixture was kept at 60 C for another 2
ꢀ
1
h. The reaction mixture was partitioned between n-hexane and
n
max: 3328, 1574, 1514, 1416 cm ; HRESIMS m/z 819.2463 [M +
+
1
13
H O (2 mL each), and the n-hexane extract was analysed by GC
2
Na] (calcd for C H O Na, 819.2471); H and C NMR data,
4
0
44 17
under the following conditions: capillary column, HP-5 (30 m ꢁ
see Tables 1 and 2.
Compound (5). White amorphous powder; [a]D 170.7 (c 0.05,
MeOH); UV (MeOH) lmax (log 3) 84 (0.21), 321 (0.13) nm; ECD
2
5
0.25 mm, with a 0.25 mm lm, Dikma); detection, FID; detector
ꢂ
ꢂ
temperature, 280 C; injection temperature, 250 C; initial
ꢂ
ꢂ
ꢀ1
temperature 160 C, then raised to 280 at 5 C min , nal
(
(
MeOH) lmax (D3) 279 (+0.31), 248 (ꢀ1.34), 230 (+6.32) nm; IR
ꢀ1
temperature maintained for 10 min; carrier, N gas. D-Glucose
2
KBr) nmax: 3340, 1604, 1513, 1452 cm ; HRESIMS m/z 819.2468
+
1
13
was conrmed by comparison of the retention time of its
derivative with the authentic sugar derivatized in a similar way,
which exhibited a retention time of 19.03 min.
[M + Na] (calcd for C40
44
H O17Na, 819.2471); H and C NMR
data, see Tables 1 and 2.
2
5
Compound (6). White amorphous powder; [a]D ꢀ18.9 (c
.06, MeOH); UV (MeOH) lmax (log 3) 307 (0.43), 321 (0.42) nm;
0
ꢀ
1
IR (KBr) nmax: 3348, 1592, 1513, 1445 cm ; HRESIMS m/z
Inhibitory activity of a-glucosidase
+
1
13
5
53.1920 [M + H] (calcd for C26
NMR data, see Tables 1 and 3.
Compound (7). White amorphous powder; [a]
.05, MeOH); UV (MeOH) lmax (log 3) 306 (0.43), 321 (0.42) nm;
H
33
O
13, 553.1916); H and
C
The inhibitory activity of compounds 1–15 on a-glucosidase was
2
D
5
determined spectrophotometrically on a 96-well microplate
ꢀ17.2 (c
ꢀ
1
reader. In total, 20 mL of 0.2 U mL a-glucosidase was premixed
with 10 mL of compounds at various concentrations in 50 mL of
0
ꢀ1
IR (KBr) nmax: 3317, 1603, 1514, 1449 cm ; HRESIMS m/z
ꢂ
+
1
13
100 mM phosphate buffer (pH 7.0) at 37 C for 5 min. Then, 20
5
53.1911 [M + H] (calcd for C H O , 553.1916); H and
C
2
6
33 13
mL of 2.5 mM substrate p-nitrophenyl-a-D-glucopyranoside was
NMR data, see Tables 1 and 3.
2
5
added to the mixture to initiate the reaction. The reaction was
Compound (8). White amorphous powder; [a]D ꢀ77.5 (c
.05, MeOH); UV (MeOH) lmax (log 3) 296 (0.51), 323 (0.41) nm;
ꢂ
incubated at 37 C for 15 min and stopped by the addition of 50
0
ꢀ
1
2 3
mL of 0.4 M Na CO . a-Glucosidase activity was determined by
IR (KBr) nmax: 3297, 1701, 1603, 1514, 1450, 1218, 1047 cm
;
ꢀ
measuring the release of p-nitrophenol from p-nitrophenyl-a-D-
glucopyranoside at 400 nm. The sample contained the mixture
and the test compound, while the control consisted of the
mixture, including the solvent, but without test compound. The
sample blank contained the mixture, including the test
compound, but without a-glucosidase. Finally, the control
blank was the mixture, including the solvent, but without a-
glucosidase.
HRESIMS m/z 621.0929 [M ꢀ H] (calcd for C27
25
H O15S,
1
13
6
21.0920); H and C NMR data, see Tables 1 and 3.
2
D
5
Compound (9). White amorphous powder; [a]
ꢀ33.6 (c
0.05, MeOH); UV (MeOH) lmax (log 3) 280 (0.40) nm; ECD
(
3
MeOH) lmax (D3) 285 (ꢀ3.41), 236 (ꢀ5.38) nm; IR (KBr) nmax:
ꢀ
1
363, 1698, 1610, 1528, 1504, 1453 cm ; HRESIMS m/z
+
1
6
28
27.1316 [M + Na] (calcd for C28H O15Na, 627.1320); H and
1
3
C NMR data, see Tables 1 and 3.
The inhibition (%) of sample on a-glucosidase was calcu-
lated by the following formula:
Cellulose hydrolysis of 1–5
A solution of 1 (2 mg) in 2 mL 0.1 M HOAc–NaOAc buffer (pH
Inhibition (%) ¼ [(A(sample) ꢀ A(sample blank))/
(A(control) ꢀ A(control blank))] ꢁ 100
ꢂ
4.5) was incubated at 40 C with cellulose (2 mg) for 5 h. The
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8
H. Matsuda, H. Shimoda, T. Morikawa and M. Yoshikawa,
Bioorg. Med. Chem. Lett., 2001, 11, 1839–1842.
Conclusions
Herein, we reported the isolation and structure elucidation of
nine new compounds from the rhizome of P. cuspidatum,
9 K. Xiao, L. J. Xuan, Y. M. Xu and D. L. Bai, J. Nat. Prod., 2000,
63, 1373–1376.
including an unusual adduct of ECG fused to piceid through 10 G. S. Jayatilake, H. Jayasuriya, E. S. Lee, N. M. Koonchanok,
a carbon–carbon bond (1), four new dimeric stilbene glycosides
2–5), three new stilbene glucosides (6–8), and one new avan
R. L. Geahlen, C. L. Ashendel, J. L. McLaughlin and
C. J. Chang, J. Nat. Prod., 1993, 56, 1805–1810.
(
glucoside (9). In order to determine the absolute congurations 11 K. Xiao, L. J. Xuan, Y. M. Xu, D. L. Bai and D. X. Zhong, Chem.
for the chiral carbons, the Rh (OCOCF ) -induced CD assay was
Pharm. Bull., 2002, 50, 605–608.
combined with the characteristic coupling constant and ECD 12 Z. Y. Yang, Q. Z. Cai, N. Chen, X. M. Zhou and J. L. Hong, RSC
spectrum analysis. Further investigation of in vitro bioactivities
Adv., 2016, 6, 12193–12204.
of 1–15 revealed that compound 8 showed signicantly stronger 13 F. S. Li, Z. L. Zhan, F. Liu, Y. N. Yang, L. Li, Z. M. Feng,
inhibition activity than the positive control acarbose. This result
J. S. Jiang and P. C. Zhang, Org. Lett., 2013, 15, 674–677.
indicated that compound 8 might be useful as a glucosidase 14 F. Liu, F. S. Li, Z. M. Feng, Y. N. Yang, J. S. Jiang and
2
3 4
inhibitor agent for the treatment of type 2 diabetes.
P. C. Zhang, Phytochemistry, 2015, 110, 150–159.
15 J. M. van Der Nat, W. G. van der Sluis, L. A. Hart, H. Van Dijk,
K. T. D. de Silva and R. P. Labadie, Planta Med., 1991, 57, 65–
Acknowledgements
68.
This project was supported by the National Natural Science 16 J. Frelek, A. Klimek and P. Ruskowska, Curr. Org. Chem.,
Foundation of China (No. 81274066) and Natural Science
Foundation of Beijing Municipality (No. 7112094).
2003, 7, 1081–1104.
17 K. Xiao, L. J. Xuan, Y. M. Xu, D. L. Bai, D. X. Zhong, H. M. Wu,
Z. H. Wang and N. X. Zhang, Eur. J. Org. Chem., 2002, 3, 564–
568.
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