Xanthone glycosides from swertia bimaculata with α -glucosidase inhibitory activity

Article abstract of DOI:10.1055/s-0034-1368299

Seven new xanthone glycosides (1-7) were isolated from the n-butanol extract of Swertia bimaculata, together with six known compounds (8-13). Their structures were elucidated on the basis of extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, a

Full text of DOI:10.1055/s-0034-1368299

502 Original Papers
Xanthone Glycosides from Swertia bimaculata
with α-Glucosidase Inhibitory Activity
Authors
Yao-Dong Yue, Yu-Tang Zhang, Zhao-Xia Liu, Qiu-Xia Min, Luo-Sheng Wan, Yong-Long Wang, Zuo-Qi Xiao,
Jia-Chun Chen
Affiliation
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical Sciences,
Huazhong University of Science and Technology, Wuhan, Peopleʼs Republic of China
Key words
Abstract
hibitory activities in vitro, and compounds 3, 4,
and 7 exhibited significant activities to inhibit α-
glucosidase. Meanwhile the effects of different
substitutions on the α-glucosidase inhibitory ac-
tivity of xanthone glycosides from S. bimaculata
are also discussed.
"
l Swertia bimaculata
!
"
l Gentianaceae
Seven new xanthone glycosides (1–7) were iso-
lated from the n-butanol extract of Swertia bima-
culata, together with six known compounds (8–
13). Their structures were elucidated on the basis
of extensive spectroscopic analyses (1D- and
2D‑NMR, HRESIMS, UV, and IR) and comparison
with data reported in the literature. All the com-
pounds were evaluated for their α-glucosidase in-
"
l xanthone glycosides
"
l α‑glucosidase inhibition
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
"
Introduction
tion of S. bimaculata. As shown in l Fig. 1, here
!
we report the isolation of seven new xanthone
glycosides (1–7), along with six known ones (8–
13). The α-glucosidase inhibitory activity and the
structure-activity relationships of all isolated
compounds were also evaluated.
The medicinal plants from the genus Swertia
(Gentianaceae) are used as traditional Asian med-
icines for the treatment of various digestive dis-
eases [1–3]. For example, Swertia mileensis,
known as “Qing-Ye-Dan”, has been documented
in the Chinese Pharmacopoeia (1977–2010 edi-
tions) to cure hepatitis clinically [1]. Previous
work has also found that Swertia. chirayita [4],
Swertia japonica [5], Swertia punicea [6], Swertia
kouitchensis [7], Swertia macrosperma [8], and
Swertia bimaculata [9] can alleviate the hypergly-
cemic status in diabetic animals. Phytochemical
investigations have indicated that the main con-
stituents from this genus are xanthones [10], tri-
terpenoids [11], secoiridoid glucosides [12], and
flavonoids.
S. bimaculata (Siebold & Zuccarini), a well-known
ethnomedicine to cure hepatitis and dyspepsia
[13], was recorded as Zhang Yacai in Jiuhuang
Bencao by the Chinese Ming Dynasty 600 years
ago. According to our previous work, the n-buta-
nol fraction of the ethanol extract from the whole
plant of S. bimaculata showed an antidiabetic ef-
fect in vitro and in vivo [9]. However, the active
constituents from the n-butanol fraction of S. bi-
maculata remained unclear. Therefore, a system-
atic study was initiated to investigate the chemi-
cal constituents isolated from the n-butanol frac-
Results and Discussion
!
The n-butanol extract of the whole herbs of S. bi-
maculata was chromatographed over silica gel,
Sephadex LH-20, Toyopearl HW-40C, and ODS,
and further purified by semipreparative HPLC to
yield seven new xanthone glycosides (1–7), to-
received
revised
October 27, 2013
January 1, 2014
accepted
February 25, 2014
Bibliography
DOI http://dx.doi.org/
10.1055/s-0034-1368299
Published online April 7, 2014
Planta Med 2014; 80: 502–508
© Georg Thieme Verlag KG
Stuttgart · New York ·
"
gether with six known ones (8–13) (l Fig. 1).
Compound 1 was isolated as a yellow amorphous
powder. Its molecular formula was deduced to be
C28H34O17 on the basis of HRESIMS (m/z 641.1748
[M – H]−, calcd. for C28H33O17, 641.1723). After ac-
id hydrolysis, compound 1 gave D-xylose and D-
glucose (see Acid hydrolysis and sugar analysis).
The UV spectrum of compound 1 in MeOH dis-
played the characteristic absorption at λmax 247,
278, and 345 nm for the xanthones. The IR spec-
trum of compound 1 suggested the presence of
hydroxyl groups (3417 cm−1), hydrogen-bonded
ketone (1649 cm−1), and the aromatic rings
(1606, 1581, and 1486 cm−1) [14]. The 1H‑NMR
spectrum displayed signals for four methoxy
ISSN 0032‑0943
Correspondence
Prof. Dr. Jia-Chun Chen
Tongji School of Pharmaceutical
Sciences
Huazhong University of Science
and Technology
Hangkong Road 13
Wuhan 430030
Peopleʼs Republic of China
Phone: + 862783692793
Fax: + 862783692793
homespringchen@126.com
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

Original Papers 503
Fig. 1 Chemical structures of compounds 1–13.
groups (δH 4.07, 3.99, 3.90, and 3.84) and two ortho-coupled pro-
tons (δH 7.44 and 6.70, J = 8.4 Hz) (l Table 1). The characteristic
Compound 2 was isolated as a yellow amorphous powder. Its mo-
lecular formula was established as C28H34O17 on the basis of HRE-
SIMS (m/z 641.1710 [M – H]−, calcd. for C28H33O17, 641.1723).
After acid hydrolysis, compound 2 gave D-xylose and D-glucose.
The xanthone aglycone (2a) of 2 (Fig. 37S) was the same as 1a by
comparison with the 1H‑NMR spectrum (Fig. 36S). This indicated
that the glucose residue of compound 2 should be located at C-1
or C-8. In the HMBC spectrum, the glucosyl group was proposed
at C-8 by the correlations from H-1′ (δH 4.86, J = 7.6 Hz) and H-6
(δH 7.49, J = 8.4 Hz) to C-8 (δc 150.3), and the correlation of H-1″
(δH 4.21) with C-6′ (δc 68.3) suggested that the xylose moiety was
attached to C-6′ of the glucosyl group. The relative configurations
of two glucose residues were also deduced to be β by the coupling
constants (J) of 7.6 and 7.4 Hz. Thus, the structure of compound 2
was defined as 8-O-[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyra-
nosyl]-1-hydroxy-2,3,4,5-tetramethoxyxanthone.
"
peak at δH 12.26 permitted the assignment of a hydroxyl group
at C-1 or C-8. Two ortho-coupled protons at H-6 (δH 7.44,
J = 8.4 Hz) and H-7 (δH 6.70, J = 8.4 Hz) were confirmed unambig-
uously through the HMBC experiment. In the 1H‑NMR spectrum
(Fig. 36S) of 1a (the xanthone aglycone of compound 1), two pro-
tons at δH 11.28 and 11.76 permitted the assignment of two hy-
droxyl groups at C-1 and C-8 [15]. This indicated that the glucose
residue of compound 1 was located at C-1 or C-8. The glucosyl
group was proposed at C-1 by an HMBC correlation from H-1′
(δH 5.02, J = 7.6 Hz) to C-1 (δc 144.3). In the HMBC spectrum, the
correlation of H-1″ (δH 3.91) with C-6′ (δc 68.2) suggested that
the xylose moiety was attached to C-6′ of the glucosyl group.
The relative configurations of two glucose residues were deduced
to be β by the coupling constants (J) of 7.6 Hz and 7.4 Hz [16]. The
positions of the four methoxys were confirmed by HMBC and
HSQC spectra. In the HMBC spectrum of 1, δH 3.84, 4.07, 3.99,
and 3.90 correlated with C-4 (δc 137.0), C-3 (δc 152.9), C-2 (δc
Compound 3 was isolated as a yellow amorphous powder, and its
molecular formula was deduced to be C27H32O16 from HRESIMS
at m/z 647.1432 [M + Cl]− (calcd. for C27H32O16Cl, 647.1384). After
acid hydrolysis, compound 3 also gave D-xylose and D-glucose.
"
142.4), and C-5 (δc 139.5), respectively (l Fig. 2). Thus, the struc-
1
"
ture of compound 1 was defined as 1-O-[β-D-xylopyranosyl-
(1 → 6)-β-D-glucopyranosyl]-8-hydroxy-2,3,4,5-tetramethoxyx-
anthone.
Its H‑NMR spectra data (l Table 1) were similar to those of com-
pound 1, except for a methoxy group (δH 3.99) that was substi-
tuted by a singlet aromatic proton (δH 6.90). The proton at δH
6.90 was assigned to H-2 due to the long-range coupling correla-
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

504 Original Papers
Table 1 NMR data of compounds 1–4 (in DMSO-d6, δ in ppm and J in Hz).
Position
1
2
3
4
δH
δc
δH
δc
δH
δc
δH
δc
1
144.3
142.4
152.9
137.0
147.2
144.3
139.5
120.9
108.6
153.6
108.8
110.6
181.2
103.5
74.1
12.79, s
149.9
134.9
153.5
131.8
144.5
146.2
143.1
118.7
111.4
150.3
111.3
105.0
181.6
102.3
73.4
154.6
96.8
12.90, s
6.55, s
158.2
95.2
2
6.90, s
3
158.3
130.8
150.3
144.4
139.4
120.8
108.8
153.8
108.7
105.3
181.0
101.3
73.4
159.5
128.0
147.8
146.2
143.2
118.8
111.9
150.4
111.4
103.0
181.0
102.6
73.3
4
4a
4b
5
6
7.44, d (8.4)
7.49, d (8.4)
7.27, d (8.4)
7.42, d (8.4)
6.68, d (8.4)
12.40, s
7.47, d (8.4)
7.28, d (8.4)
7
6.70, d (8.4)
12.26, s
8
8a
8b
9
Glc-1′
2′
3′
4′
5′
6′
5.02, d (7.6)
3.44, m
3.24, m
3.13, m
2.87, m
3.86, d (9.4)
3.44, m
3.91, d (7.4)
2.82, m
3.31, m
3.17, m
3.53, m
2.72, m
3.99, s
4.86, d (7.6)
3.41, m
3.29, m
3.18, m
3.08, m
4.01, d (9.4)
3.56, m
4.21, d (7.4)
3.01, m
3.56, m
3.27, m
3.67, m
2.96, m
3.80, s
5.09, d (7.6)
3.43, m
4.84, d (7.6)
3.40, m
76.3
76.1
3.32, m
76.2
3.30, m
76.0
69.9
69.8
3.24, m
69.9
3.20, m
69.8
76.4
76.6
3.07, m
76.7
3.09, m
76.7
68.2
68.3
4.01, d (9.4)
3.59, m
69.0
4.01, d (9.4)
3.56, m
68.4
Xyl-1″
2″
103.6
73.1
76.3
69.4
65.3
103.9
73.3
76.3
69.6
65.6
4.15, d (7.4)
2.93, m
104.2
73.2
76.0
69.6
65.7
4.22, d (7.4)
3.01, m
104.0
73.4
76.3
69.6
65.6
3″
3.66, m
3.56, m
4″
3.17, m
3.18, m
5″
3.68, m
3.67, m
2.99, m
2.96, m
2-OMe
3-OMe
4-OMe
5-OMe
61.6
61.4
61.4
57.5
60.6
61.5
61.4
57.0
4.07, s
4.05, s
3.98, s
3.85, s
3.90, s
56.5
60.9
57.4
3.92, s
3.81, s
3.94, s
56.6
60.9
57.0
3.84, s
3.92, s
3.90, s
3.95, s
Fig. 2 Key HMBC (H→C) correlations of com-
pound 1.
tions with C-4 (δc 130.8), C-3 (δc 158.3), C-1 (δc 154.6), and C-8b
(δc 105.3). The glucosyl group was also proposed at C-1 by an
HMBC correlation from H-1′ (δH 5.09, J = 7.6 Hz) to C-1 (δc
154.6). Thus, the structure of compound 3 was defined as 1-O-
[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-8-hydroxy-
3,4,5-trimethoxyxanthone.
Compound 4 was isolated as a yellow amorphous powder. The
molecular formula of 4 was established as C27H32O16 by its HRE-
SIMS (m/z 647.1388 [M + Cl]−; calcd. for C27H32O16Cl, 647.1384).
After acid hydrolysis, compound 4 also gave D-xylose and D-glu-
cose. The xanthone aglycone (4a) of 4 was identified as 1,8-dihy-
droxy-3,4,5-trimethoxyxanthone by comparison with the
13C‑NMR data reported in the literature (Fig. 38S) [17]. This indi-
cated that the glucose residue of compound 4 should be located
at C-1 or C-8. The HMBC spectrum of 4 showed that H-1′ (δH
4.84, J = 7.6 Hz) and H-6 (δH 7.47, J = 8.4 Hz) had a correlation with
C-8 (δc 150.4), which indicated that the glucose residue should
be located at C-8 of the aglycone. The correlation of H-1″ (δH
4.22) with C-6′ (δc 68.4) suggested that the xylose moiety was at-
tached to C-6′ of the glucosyl group. The relative configurations of
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

Original Papers 505
Table 2 NMR data of compounds 5–7 (in DMSO-d6, δ in ppm and J in Hz).
Position
5
6
7
δH
δc
δH
δc
δH
δc
1
154.4
101.3
158.0
130.1
151.0
144.2
139.4
120.6
108.6
153.9
108.7
104.4
180.7
102.0
73.4
154.4
101.0
157.6
130.0
151.1
144.2
139.4
120.6
108.6
153.9
108.8
104.6
180.7
101.9
73.7
154.7
102.3
159.8
130.5
151.0
144.2
139.3
120.4
108.5
153.9
108.6
103.6
180.5
101.9
73.1
2
6.82, s
6.85, s
6.75, s
3
4
4a
4b
5
6
7.40, d (8.4)
6.66, d (8.4)
12.51, s
7.41, d (8.4)
6.67, d (8.4)
12.49, s
7.40, d (8.4)
6.66, d (8.4)
12.54, s
7
8
8a
8b
9
Glc-1′
2′
3′
4′
5′
6′
4.88, d (7.6)
3.39, m
4.91, d (7.6)
3.44, m
3.31, m
3.25, m
3.59, m
4.03, m
3.66, m
4.97, d (7.6)
3.43, m
3.48, m
3.50, m
3.54, m
3.73, m
3.67, m
3.30, m
76.1 a
69.6
76.1
74.5
3.30, m
69.4
79.5
3.10, t (8)
3.99, m
76.3
76.0
75.3
68.0
68.2
60.0
3.63, m
Glc-2/Xyl
1″
2″
3″
4″
5″
4.21, d (7.4)
3.01, m
3.53, m
3.27, m
3.69, m
2.99, m
104.0
73.3
4.25, d (7.4)
2.97, m
103.4
73.3
76.5
70.1
76.9
4.33, d (7.4)
3.03, m
103.1
73.3
76.5
70.0
76.8
76.0 a
69.3
3.17, m
3.19, m
3.05, m
3.08, m
65.6
3.07, m
3.21, m
6″
3.66, m
3.46, m
3.89, s
3.86, s
61.0
3.73, m
3.37, m
3.90, s
3.85, s
61.0
4-OMe
5-OMe
3.90, s
3.86, s
60.7
57.3
60.8
57.3
60.7
57.3
a: Assignments may be interchanged
two glucose residues were also deduced to be β by the coupling
constants (J) of 7.6 Hz and 7.4 Hz. Thus, the structure of com-
pound 4 was defined as 8-O-[β-D-xylopyranosyl-(1 → 6)-β-D-
glucopyranosyl]-1-hydroxy-3,4,5-trimethoxyxanthone.
of compound 5, except that the signals for a xylose residue were
replaced by signals associated with a glucose residue. Its HMBC
spectrum displayed the correlation between the proton at the
anomeric center of Glc-2 (δH 4.25) and C-6′ (δc 68.2) of Glc-1. This
suggested that the Glc-2 residue was linked to the Glc-1 residue
by a (1 → 6) linkage [10]. The coupling constant of the proton at
the anomeric center of Glc-2 was found to be 7.4 Hz. Therefore,
the structure of compound 6 was defined as 1-O-[β-D-gluco-
pyranosyl-(1 → 6)-β-D-glucopyranosyl]-3,8-dihydroxy-4,5-di-
methoxyxanthone.
Compound 5 was obtained as a yellow amorphous powder. The
HRESIMS spectrum of 5 exhibited an [M – H]− ion at m/z
597.1446 (calcd. for C26H29O16, 597.1461), corresponding to the
molecular formula C26H30O16. After acid hydrolysis, compound 5
1
"
gave D-xylose and D-glucose. The H‑NMR (l Table 2) data of 5
was similar to those of 1-O-β-D-glucopyranosy-3,8-dihydroxy-
4,5-dimethoxyxanthone (9), except for one additional proton at
δH 4.21 (J = 7.4 Hz) and a group of glycosyl protons between δH
3.69 and δH 2.99. The 13C‑NMR spectrum displayed five carbon
signals (δc 104.0, 73.3, 76.0, 69.3, and 65.6) attributed to a xylose
moiety, and C-6′ of the glucosyl group shifted from δc 60.6 to
68.0. In the HMBC spectrum, the correlation of H-1″ (δH 4.21)
with C-6′ (δc 68.0) suggested that the xylose moiety was attached
to C-6′ of the glucosyl group. The relative configurations of two
glucose residues were deduced to be β by the coupling constants
(J) of 7.6 Hz and 7.4 Hz. Thus, the structure of compound 5 was
defined as 1-O-[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyrano-
syl]-3,8-dihydroxy-4,5-dimethoxyxanthone.
Compound 7 was isolated as a yellow amorphous powder. Its mo-
lecular formula was deduced to be C27H32O17 on the basis of HRE-
SIMS (m/z 627.1602 [M – H]−; calcd. for C27H31O17, 627.1567).
"
The NMR (l Table 2) data of 7 were also similar to those of com-
pound 9, except for a group of signals from an additional glucose
residue and the downfield shift of C-4′ of Glc-1 (δc from 69.4 to
79.5). This suggested that the Glc-2 residue was located at C-4′
of Glc-1 [18]. This assumption was supported by the HMBC spec-
trum, in which the proton at the anomeric center of Glc-2 (δH
4.33) showed a correlation with C-4′ of Glc-1 (δc 79.5). The rela-
tive configurations of two glucose residues were deduced to be β
by the coupling constants of the protons at the anomeric center.
Accordingly, the structure of compound 7 was defined as 1-O-[β-
D-xylopyranosyl-(1 → 4)-β-D-glucopyranosyl]-3,8-dihydroxy-
4,5-dimethoxyxanthone.
Compound 6 was also isolated as a yellow amorphous powder. Its
molecular formula was assigned as C27H32O17 on the basis of
HRESIMS (m/z 627.1567 [M
627.1567). The NMR (l Table 2) data of 6 were similar to those
– ,
H]−; calcd. for C27H31O17
"
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

506 Original Papers
Compound
IC50 µM
Compound
IC50 µM
> 1000
578 39
> 1000
Table 3 Inhibitory effects of
compounds 1–13 and acarbose
against α-glucosidasea.
1
2
3
4
5
6
7
442 47
8
> 1000
9
142 17
136 14
417 32
478 45
258 19
10
11
389 23
765 54
679 58
426 45
12
13
acarbose
a Each value represents the mean SD (n = 3)
Comparisons of NMR data reported in the literature led to the
identification of the known compounds as 3-O-β-D-glucopyra-
nosyl-1-hydroxy-4,5-dimethoxyxanthone (8) [19], 1-O-β-D-glu-
copyranosyl-3,8-dihydroxy-4,5-dimethoxyxanthone (9) [20,21],
3-O-β-D-glucopyranosyl-1,8-dihydroxy-5-methoxyxanthone
(10) [22], swertianolin (11) [23,24], 7-O-[α-L-rhamnopyranosyl-
(1 → 2)-β-D-xylopyranoyl-1,8-dihydroxy-3-methoxyxanthone
(12) [25], and norswertianolin (13) [26].
Agilent 7820A GC system using an HP-5 (30 m × 0.32 mm ×
0.25 µm) column; detection FID; carrier gas N2; injection temper-
ature 250°C, detection temperature 250°C, and column temper-
ature 180°C. Preparative HPLC methanol and chemicals of analyt-
ical grade, such as methanol, dichloromethane, and ethyl acetate,
were purchased from Sinopharm Chemical Reagent Co., Ltd.
Plant materials
All isolated compounds were evaluated for α-glucosidase inhib-
itory activities. The IC50 values, defined as the compound concen-
tration that inhibits α-glucosidase activity by 50%, are summar-
The whole plant of S. bimaculata was collected in Enshi, Hubei
Province, Peopleʼs Republic of China, in October 2010. The plant
material was identified by Jia-chun Chen, Tongji School of Phar-
maceutical Sciences, Huazhong University of Science and Tech-
nology. A voucher specimen (No. 20101010) has been deposited
at the Department of Pharmacognosy of the same University.
"
ized in l Table 3. Among them, compounds 3, 4, and 7 exhibited
more potent α-glucosidase inhibitory activities (IC50s of 142 µM,
136 µM, and 258 µM, respectively) than the positive control acar-
bose (IC50 426 µM). It was observed that the methoxy group lo-
cated at C-2 (compounds 1 and 2) produced steric hindrance
and lowered the inhibitory activity. The presence of an O‑glc-
(4–1)-glc residue (compound 7) resulted in relatively more effec-
tive inhibitory activity than other diglycoside units (compounds
5 and 6). But the reason why the glycoside units located at C-1
(compound 1) exhibited more potent inhibitory activity than
the units located at C-8 (compound 2) is still unclear. Thus, fur-
ther experiments will be performed to evaluate the effects of dif-
ferent substitutions on the α-glucosidase inhibitory activity of
xanthone glycosides.
Extraction and isolation
The air-dried whole plants of S. bimaculata (23 kg) were pow-
dered and extracted successively with 95% EtOH (2 × 200 L, under
reflux, each for 2 h) and 50% EtOH (2 × 200 L, under reflux, each
for 2 h) at 60°C. The obtained EtOH extracts were combined and
concentrated in vacuum to give a residue (5 kg), which was sus-
pended in H2O and partitioned with petroleum ether, CH2Cl2,
EtOAc, and n-butanol, respectively.
After removal of the solvent in vacuum, the n-butanol fraction
(900 g) was separated on an AB-8 macroporous resin column
(120 × 20 cm i.d., eluted with a mixture of H2O and EtOH from
1:0 to 0:1) to give seven fractions. Fr. B5 (200 g, eluted with
H2O:EtOH 1:1) was separated on a silica gel column (80 × 10 cm
i.d., 200–300 mesh, 3000 g; solvent system: CHCl3 -MeOH,
1:0 → 0:1, v/v; flow rate: 50 mL/min) to give 9 fractions (Fr. B5-
1→B5–9). Fr. B5-2 (2 g, eluted with CHCl3:MeOH 10:1) was sepa-
rated on Toyopearl HW-40C (130 × 4 cm i.d.; solvent system:
CHCl3-MeOH, 1:1; flow rate: 5 mL/min) and then purified by Se-
phadex LH-20 (120 × 2.5 cm i.d., eluted with MeOH) to yield com-
pound 8 (40 mg). Fr. B5-3 (20 g, eluted with CHCl3:MeOH 8:1)
was separated on a polyamide resin column (50 × 8 cm i.d., 30–
60 mesh, 1200 g, solvent system: MeOH‑H2O, 0:10 → 10:0, v/v,
flow rate: 20 mL/min) to give 5 fractions. Fr. B5-3C (200 mg,
eluted with MeOH:H2O 30:70) was then subjected to Sephadex
LH-20 (120 × 2.5 cm i.d., eluted with MeOH) to yield compound 9
(150 mg). Fr. B5-4 (30 g, eluted with CHCl3: MeOH 7:1) was sep-
arated on an ODS column (40 × 5 cm i.d., 350 g; solvent system:
MeOH‑H2O, 0:10 → 10:0, v/v) to give 9 fractions. Fr. B5-4C (2 g,
eluted with MeOH:H2O 35:65) was then subjected to Toyopearl
HW-40C (130 × 4 cm i.d.) and eluted with MeOH to give 9 frac-
tions (Fr. B5-4C1→B5-4C9). Fr. B5-4C3 (200 mg) was fractioned
by semipreparative HPLC on a YMC C18 column (MeOH–H2O–
CH3COOH, 55:45:0.1, v/v, flow rate: 1.5 mL/min) to yield com-
pounds 1 (50 mg, tR 30.2 min) and 2 (60 mg, tR 25.8 min), while
Fr. B5-4C4 (200 mg) was purified by the same way using
Materials and Methods
!
General experimental procedures
Optical rotations were measured on an AA10R digital polarime-
ter. UV spectra were run on a Carry-50 UV‑vis spectrophotome-
ter. IR spectra were recorded on an Avatar-360 FT‑IR spectropho-
tometer with KBr pellets. 1D- and 2D‑NMR spectra were re-
corded on a Bruker AV-400 spectrometer (400 MHz for 1H 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. HRESIMS were measured by an Agilent 6520
Q‑TOF LC‑MS mass spectrometer. Column chromatography was
performed with macroporous resin (Diaion AB-8; Mitsubishi
Chemical Crop.), polyamide resin (30–60 mesh, Mitsubishi
Chemical Corp.), ODS gel (50 µm, YMC), Sephadex LH-20 (GE
Healthcare), Toyopearl HW-40C (Tosoh), and silica gel (200–300
mesh; Qingdao Marine Chemical, Inc.). Semipreparative 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 1.5 mL/min; wave-
length detection at 254 nm). Fractions were monitored by TLC,
and spots were visualized by heating silica gel plates after spray-
ing with 10% H2SO4 in EtOH. GC analysis was carried out on an
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

Original Papers 507
MeOH‑H2O‑CH3COOH (55:45:0.1) as the eluent to afford com-
pounds 3 (10 mg, tR 23.4 min) and 4 (15 mg, tR 28.7 min). Fr. B5-
4C5 (150 mg) was fractioned on Sephadex LH-20 and eluted with
MeOH to yield compound 10 (30 mg). Fr. B5-4C6 (100 mg) was
subjected to semipreparative HPLC on a YMC C18 column
(MeOH–H2O–CH3COOH, 65:35:0.1, v/v, flow rate: 1.5 mL/min)
to yield compounds 11 (30 mg, tR 21.8 min) compound 12
(15 mg, tR 16.5 min).
Fr. B3 (80 g, eluted with H2O:EtOH 3:7) was also separated on a
silica gel column (60 × 8 cm i.d., 100–200 mesh, 1500 g; solvent
system: CHCl3–MeOH, 1:0 → 0:1, v/v; flow rate: 50 mL/min) to
give 7 fractions (Fr. B3-1→B3–7). Fr. B3–5 (6 g, eluted with
CHCl3 :MeOH 10:1) was then subjected to Toyopearl HW-40C
and eluted with MeOH to give 5 fractions (Fr. B3–5A→B3–5E). Fr.
B3–5C (200 mg) was fractioned on Sephadex LH-20 and eluted
with MeOH to yield compound 13 (10 mg). Fr. B3–5D (270 mg)
was subjected to Sephadex LH-20 using MeOH as the solvent, fol-
lowed by semipreparative HPLC on a YMC C18 column (MeOH–
H2O–CH3COOH, 55:45:0.1, v/v, flow rate: 1.5 mL/min) to give
compounds 5 (30 mg, tR 18.7 min), 6 (20 mg, tR 24.6 min), and 7
(25 mg, tR 14.8 min). The purity (≥ 95%) of all isolated compounds
was measured by HPLC analyses.
(4.31), 253 (4.34), 278 (4.04), 332 (3.92) nm; IR (KBr) νmax 3338,
2922, 1738, 1648, 1603, 1518, 1447, 1418, 1381, 1180, 1060,
13
828 cm−1
;
1H‑NMR and C‑NMR data, see l Table 2; HRESIMS:
"
m/z 627.1566 [M – H]− (calcd. for C27H31O17, 627.1567).
1-O-[β‑D-xylopyranosyl-(1 → 4)-β‑D-glucopyranosyl]-3,8-dihy-
droxy-4,5-dimethoxyxanthone (7): yellow amorphous powder;
[α]2D0 − 54.2 (0.08, MeOH); UV (MeOH) λmax (log ε) 204 (4.48),
231 (4.31), 253 (4.50), 278 (4.01), 330 (3.98) nm; IR (KBr) νmax
3390, 2924, 1739, 1648, 1602, 1523, 1418, 1339, 1253, 1064,
13
813 cm−1
;
1H‑NMR and C‑NMR data, see l Table 2; HRESIMS:
"
m/z 627.1602 [M – H]− (calcd. for C27H31O17, 627.1567).
Acid hydrolysis and sugar analysis
Compound 1 (2 mg) was dissolved in 0.1 N CF3C00H (2 mL) and
heated at 70°C for 8 h. The reaction mixture was extracted with
CH2Cl2. The aqueous layer was evaporated under vacuum and
compared with references D-glucose and D-xylose (Sigma-Al-
drich) by TLC (silica gel with CHCl3-MeOH‑H2O, 6:4:1). The res-
idue was then dissolved in pyridine (1 mL). Then 600 µL of
HMDS-TMCS (hexamethydisilazane-trimethylchlorosilane, 2:1)
was added, and the mixture was stirred at 60°C for 30 min. The
supernatant was analyzed by GC under the following conditions:
HP-5 (30 m × 0.32 mm × 0.25 µm) column; detection FID; carrier
gas N2; injection temperature 250°C, detection temperature
250°C, and column temperature 180°C. From the acid hydrolysis
of 1, D-glucose and D-xylose were confirmed by comparison of
the retention times of their derivatives with those of authentic
sugars derivatized in a similar way, which showed retention
times of 7.15 and 13.30 min, respectively [27,28]. The constituent
sugars of compounds 2–7 were identified using the same method
as for 1.
Isolates
1-O-[β‑D-xylopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-8-hydroxy-
2,3,4,5-tetramethoxyxanthone (1): yellow amorphous powder;
[α]2D0 − 72.3 (0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.38), 260
(4.32), 279 (4.13), 326 (3.78) nm; IR (KBr) νmax 3396, 2915, 1649,
1588, 1485, 1464, 1406, 1278, 1064, 814 cm−1
C‑NMR data, see l Table 1; HRESIMS: m/z 641.1748 [M – H]
(calcd. for C28H33O17, 641.1723).
;
1H‑NMR and
13
−
"
8-O-[β‑D-xylopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-1-hydroxy-
2,3,4,5-tetramethoxyxanthone (2): yellow amorphous powder;
[α]2D0 − 69.2 (0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.33), 262
(4.27), 275 (4.13), 328 (3.70) nm; IR (KBr) νmax 3417, 2930, 1645,
1609, 1584, 1494, 1463 1421, 1280, 1064, 814 cm−1; 1H‑NMR and
α-Glucosidase inhibitory assay
α-Glucosidase (from Saccharomyces cerevisiae; Sigma-Aldrich)
inhibitory activities were determined by using p-nitrophenyl-α-
D-glucopyranoside (PNPG; Sigma-Aldrich) as the substrate, ac-
cording to a reported method with minor modifications [29,30].
Briefly, 20 µL of enzyme solution [0.4 U/mL α-glucosidase in
0.1 M potassium phosphate buffer (pH 6.8)] and 120 µL of the test
compound (purity ≥ 95%) in water containing 0.5% DMSO (Sig-
ma-Aldrich) were mixed and incubated for 30 min at 37°C. After
incubation, 20 µL of PNPG solution [5.0 µM PNPG in 0.1 M potas-
sium phosphate buffer (pH 6.8)] was added into each well and in-
cubated together for 30 min at 37°C. Then 80 µL 0.2 M Na2CO3 in
0.1 M potassium phosphate buffer was added to each well to stop
the reaction. The absorbance of PNP released was measured on a
UV max kinetic microplate reader (Bio Tek, Synergy 2) at 405 nm.
Blank readings (no enzyme) were substracted from each well and
the results were compared to the control (no sample). The phar-
macological inhibitor acarbose (purity ≥ 99%; Sigma-Aldrich) was
used as a positive control. Inhibition (%) was obtained by the fol-
lowing formula:
13
−
"
C‑NMR data, see l Table 1; HRESIMS: m/z 641.1710 [M – H]
(calcd. for C28H33O17, 641.1723).
1-O-[β‑D-xylopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-8-hydroxy-
3,4,5-trimethoxyxanthone (3): yellow amorphous powder; [α]D20
− 28.6 (0.04, MeOH); UV (MeOH) λmax (log ε) 215 (4.24), 251
(3.78), 335 (3.32) nm; IR (KBr) νmax 3356, 2921, 1738, 1644,
1606, 1542, 1475, 1314, 1074, 827 cm−1
data, see l Table 1; HRESIMS: m/z 647.1432 [M + Cl] (calcd. for
;
1H‑NMR and 13C‑NMR
−
"
C
27H32O16Cl, 647.1384).
8-O-[β‑D-xylopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-1-hydroxy-
3,4,5-trimethoxyxanthone (4): yellow amorphous powder; [α]D20
− 41.9 (0.06, MeOH); UV (MeOH) λmax (log ε) 213 (4.21), 252
(3.73), 335 (3.30) nm; IR (KBr) νmax 3420, 2924, 1649, 1606,
1578, 1520, 1482, 1423, 1382, 1026, 1001, 826 cm−1
and C‑NMR data, see l Table 1; HRESIMS: m/z 647.1388 [M +
Cl]− (calcd. for C27H32O16Cl, 647.1384).
;
1H‑NMR
13
"
1-O-[β‑D-xylopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-3,8-dihy-
droxy-4,5-dimethoxyxanthone (5): yellow amorphous powder;
[α]2D0 − 68.8 (0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.42), 232
(4.34), 253 (4.40), 279 (4.02), 330 (3.93) nm; IR (KBr) νmax 3397,
2924, 1649, 1606, 1581, 1515, 1486, 1423, 1249, 1197, 1060,
Inhibition (%) = [(A(control) – A(sample))/A(control)] × 100
Supporting information
1H, 13C, 2D‑NMR, and HRESIMS spectra for compounds 1–7, as
well as of 1D‑NMR spectra for compounds 1a, 2a, and 4a are
available as Supporting Information.
13
954 cm−1
;
1H‑NMR and C‑NMR data, see l Table 2; HRESIMS:
"
m/z 597.1446 [M – H]− (calcd. for C26H29O16, 597.1461).
1-O-[β‑D-glucopyranosyl-(1 → 6)-β‑D-glucopyranosyl]-3,8-dihy-
droxy-4,5-dimethoxyxanthone (6): yellow amorphous powder;
[α]2D0 − 65.9 (0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.38), 231
Yue Y-D et al. Xanthone Glycosides from… Planta Med 2014; 80: 502–508

508 Original Papers
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!
This work was supported financially by the National Natural Sci-
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Conflict of Interest
!
There is no conflict of interest among all authors.
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