New xanthone glycosides from comastoma pedunculatum

Article abstract of DOI:10.1055/s-0032-1315079

Five new xanthone glycosides, comastomasides AE (15), were isolated from aqueous ethanol extracts of the aerial portions of Comastoma pedunculatum. The structures of these compounds were elucidated by spectroscopic analysis methods. Compounds 15 were evaluated for their hepatoprotective activity and cytotoxicity against four human cancer cell lines by in vitro assays. Among them, compounds 3 and 5 exhibited potent hepatoprotective activity. However, none of the compounds displayed cytotoxic activity.

Full text of DOI:10.1055/s-0032-1315079

Letters 1591
tory, anti-HIV, and antioxidant activities [3]. This report de-
scribes the isolation and characterization of five new xanthone
New Xanthone Glycosides from
Comastoma pedunculatum
Yongqi Qiao, Yi Yuan, Baosong Cui, Ying Zhang, Hui Chen,
Shuai Li, Yan Li
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, PR China
glycosides from the aerial parts of Comastoma pedunculatum
(l Fig. 1). Spectroscopic methods were used to elucidate the
"
structures of these new compounds. Compounds 1–5 were eval-
uated for hepatoprotective activity against D-galactosamine-in-
duced toxicity and cytotoxicity in four human cancer cell lines.
Compound 1 was obtained as a yellow amorphous powder, and
its molecular formula was determined to be C36H38O18 by HR‑E-
SI‑MS. The UV spectrum for compound 1 displayed absorption
maxima at 257, 273, 297, and 327 nm, indicating the presence of
a xanthone skeleton [4]. IR absorption bands at 3457, 1650, 1606,
1564, and 1484 cm−1 indicated the presence of a hydroxyl group,
conjugated carbonyl, aromatic ring, and C‑C double bond, respec-
Abstract
!
Five new xanthone glycosides, comastomasides A–E (1–5), were
isolated from aqueous ethanol extracts of the aerial portions of
Comastoma pedunculatum. The structures of these compounds
were elucidated by spectroscopic analysis methods. Compounds
1–5 were evaluated for their hepatoprotective activity and cyto-
toxicity against four human cancer cell lines by in vitro assays.
Among them, compounds 3 and 5 exhibited potent hepatopro-
tective activity. However, none of the compounds displayed cyto-
toxic activity.
1
"
tively. The H‑NMR of compound 1 (l Table 1) showed a signal at
δ
H 12.44 that corresponded to the proton of the phenolic hydrox-
yl, indicating that the phenolic proton was hydrogen bonded to
the carbonyl group. In addition, there were two ortho-coupled ar-
omatic proton signals at δH 7.37 and 6.64 and two meta-coupled
aromatic proton signals at δH 6.77 and 6.65; these data were used
to assign the tetrasubstituted xanthone moiety as an aglycone.
Another group of aromatic proton signals corresponded to ABX-
coupled aromatic protons at δH 7.14, 6.63, and 6.91. The pair of
coupled olefinic protons at δH 7.33 and 6.28 with a coupling con-
stant of J = 15.0 Hz confirmed that the configuration of the C‑C
double bond was E. These data were identical to the proton sig-
nals of the caffeic acid moiety. In addition, there were two
anomeric proton signals which corresponded to sugars protons.
The proton signal at δH 5.01 corresponded to the β-linkage of
the sugar moiety in D-glucopyranose, and the other signal at δH
4.44 confirmed the presence of the β-linkage of the sugar moiety
in D-xylopyranose. There were also 11 other sugar proton signals
between 3.04 and 3.96 ppm. These data suggest that 1 is a xan-
thone glycoside. There were also three oxygenated methyl
groups at δH 3.94, 3.88, and 3.74 present in compound 1. Further-
Key words
Comastoma pedunculatum · Gentianaceae · xanthone glyco-
sides · hepatoprotective activity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
The herbs of Comastoma pedunculatum (Royle ex D. Don) Holub
(Gentianaceae) have been used to treat hepatitis, liver fibrosis,
and cholecystitis in traditional Tibetan medicine. These herbs
have hepatoprotective, anti-inflammatory, antioxidant, and nor-
malizing gallbladder properties. Phytochemical investigations of
the Comastoma genus have uncovered a number of structurally
diverse metabolites, such as xanthones, flavonoids, and oleanolic
acid from C. pulmonarium [1] and C. pedunculatum [2]. Xan-
thones are the characteristic chemical constituents of the Comas-
toma genus. These compounds exhibit a wide range of biological
properties including anticancer, antimicrobial, anti-inflamma-
more, the 13C‑NMR (l Table 2) and DEPT spectra of compound 1
"
displayed thirty-six carbon signals corresponding to thirteen sp2-
hybridized quaternary carbons (two carbonyl, eight oxygenated,
and three quaternary carbons), eighteen methine carbons (nine
sp2-hybridized and nine oxygenated sp3-hybridized carbons),
two sp3-hybridized oxygenated methylene carbons, and three
methoxy carbons. On acid hydrolysis of compound 1, the sugar
Fig. 1 Structures of compounds 1–5.
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596

1592 Letters
Table 1 1H NMR spectra of compounds 1–5 (DMSO-d6).
No.
1
2
3
4
5
1
2
6.82, d (9.0)
6.84, d (9.0)
6.71, d (9.0)
3
7.37, d, (9.0)
7.35, d (8.0)
7.37, d (9.0)
7.41, d (9.0)
7.12, d (9.0)
4
6.64, d, (9.0)
6.63, d (8.0)
4a
4b
5
6.77, br s
6.65, br s
6.76, br s
6.67, br s
6.31, br s
6.45, br s
6.66, br s
6.62, br s
6.20, br s
6.46, br s
6
7
8
8a
8b
9
glc-1
glc-2
glc-3
glc-4
glc-5
glc-6
5.01, d, (7.5)
5.00, d (7.0)
4.87, d (7.0)
5.02, d (8.0)
3.34, overlapping
3.30, overlapping
3.10, m
4.81, d (7.0)
3.34, overlapping
3.30, overlapping
3.10, dd, (9.0, 9.0)
3.58, m
3.32, overlapping
3.32, overlapping
3.12, dd (9.0, 9.0)
3.57, m
3.36, overlapping
3.30, overlapping
3.16, m
3.35, overlapping
3.30, overlapping
3.16, m
3.48, m
3.54, m
3.44, m
3.96, br d (13.0)
3.60, m
3.96, br d (12.0)
3.59, br s
3.93, br d (12.0)
3.62, dd (12.0, 6.0)
4.47, d (8.0)
3.96, br d (12.0)
3.60, m
3.94, br d (12.0)
3.64, dd (12.0, 6.0)
4.51, d (8.0)
xyl-1
xyl-2
xyl-3
xyl-4
xyl-5
4.44, d (8.0)
4.44, d (8.0)
4.43, d (8.0)
4.62, dd (9.0, 8.0)
3.30, overlapping
3.40, br s
4.62, dd (9.0, 8.0)
3.30, overlapping
3.40, br s
4.63, dd (9.0, 8.0)
3.32, overlapping
3.39, overlapping
3.76, br s
4.63, dd (9.0, 8.0)
3.34, overlapping
3.40, overlapping
3.70, overlapping
3.07, dd (10.5, 10.5)
4.63, dd (9.0, 8.0)
3.35, overlapping
3.40, overlapping
3.76, overlapping
3.09, dd (10.5, 10.0)
3.76, overlapping
3.04, dd (11.0, 11.0)
3.76, overlapping
3.04, m
3.05, dd (11.0, 11.0)
1′
2′
7.14, br s
6.85, br s
7.21, br s
7.15, br s
7.24, br s
3′
4′
5′
6.63, d (8.0)
6.91, d (8.0)
7.33, d (15.0)
6.28, d (15.0)
6.70, d (8.0)
6.64, d, (8.5)
6.92, br d (8.5)
7.34, d, (16.0)
6.28, d (16.0)
6.72, d (8.0)
6′
6.85, br s
6.99, br d (8.0)
7.45, d (16.0)
6.39, d (16.0)
7.00, br d (8.0)
7.48, d (16.0)
6.44, d (16.0)
7′
7.37, d (15.0)
6.34, d (15.0)
8′
9′
OCH3
3.94, s, (6-OCH3)
3.88, s, (2-OCH3)
3.74, s, (3′-OCH3)
12.44, s, (1-OH)
3.94, s, (6-OCH3)
3.86, s, (2-OCH3)
3.74, s, (3′,5′-OCH3)
12.43, s, (1-OH)
3.80, s, (2-OCH3)
3.92, s, (6-OCH3)
3.81, s, (4-OCH3)
3.74, s, (3′-OCH3)
13.25, s, (1-OH)
9.44, s, (4′-OH)
3.78, s, (3′-OCH3)
3.76, s, (3′-OCH3)
OH
13.42, s, (1-OH)
13.33, s, (1-OH)
9.49, s, (4′-OH)
9.04, s, (4′-OH)
moiety was determined to be composed of glucose and xylose by
TLC [5]. NOESY experiments were used to further determine the
substitution of the xanthone moiety and position of the three
methoxy groups. The NOESY correlations between the following
defined the linkage position of three methoxy groups and the
1,2,6,8-tetrasubstituted xanthone moiety: 6-OCH3 and H-5, H-7;
2-OCH3 and H-3, 1-OH; and 3′-OCH3 and H-2′. To determine the
linkage position of the xanthone, caffeic acid, D-glucose, and D-
xylose moieties, 2D NMR experiments were performed. The pro-
losyl (4.62) moiety to C-9′ (165.5), which is the α,β-unsaturated
conjugated ester carbonyl of the caffeic acid moiety, confirmed
that the caffeic acid moiety was linked to the C-2 position of the
xylosyl moiety. The position of H-2 on the xylosyl (4.62) moiety
1
was further supported by H-1H COSY correlations between H-1
(4.44) and H-2 (4.62) on this moiety. Ultimately, compound 1 was
deduced to be 1-hydroxy-2,6-dimethoxy-8-O-[2-(4′-hydroxy-3′-
methoxy-E-cinnamyl)-β-D-xylopyranosyl-(1–6)-β-D-glucopy-
ranosyl]-xanthone and named comastomaside A.
"
tons and protonated carbons of compound 1 (l Tables 1 and 2)
The molecular formula of compound 2 was determined to be
"
were unambiguously assigned by heteronuclear single quantum
coherence (HSQC). The heteronuclear multiple bond correlation
(HMBC) spectrum helped to determine the structural moieties
C
37H40O19 by HR‑ESI‑MS. The NMR spectrum of 2 (l Tables 1
and 2) showed similarities to that of compound 1 with the excep-
tion of the signals corresponding to the aromatic ring of the caf-
feic acid moiety. One of the phenolic hydroxyl groups on the caf-
feic acid moiety in compound 1 was replaced by signals corre-
sponding to a methoxy group in compound 2. The NOESY corre-
lations between 3′- and 5′-OCH3 and H-2′, H-6′ defined the loca-
tion of the methoxy group and further confirmed that the addi-
tional methoxy group was linked to C-5′ of the caffeic acid moiety
in compound 2. Other correlations were the same as those of
"
and connectivity of compound 1. HMBC correlations (l Fig. 2)
from the anomeric proton signal of glucose (5.01) to C-8 (159.2)
indicated that the glycosyl moiety was located at the C-8 posi-
tion. The correlations from the anomeric proton signals of the xy-
losyl (4.44) to the C-6 position of the glycosyl (68.9) moiety indi-
cated that the xylosyl moiety was located at the C-6 position of
the glycosyl moiety. In addition, correlations from H-2 on the xy-
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596

Letters 1593
Table 2 13C NMR spectra of com-
pounds 1–5 (DMSO-d6).
No.
1
2
3
4
5
1
153.6
139.2
119.7
108.7
143.9
158.6
94.9
153.6
139.2
119.6
108.7
143.9
158.7
95.0
150.4
104.5
120.0
142.3
147.9
159.1
97.7
150.3
104.7
120.5
142.4
147.9
158.9
94.8
148.9
104.5
120.0
139.7
147.8
158.9
97.0
2
3
4
4a
4b
5
6
165.8
99.1
165.8
99.1
168.3
101.6
159.9
102.6
108.3
180.4
102.0
73.3
165.8
98.5
165.4
101.6
159.5
103.8
108.2
180.4
101.2
73.1
7
8
159.2
105.3
108.9
180.5
100.7
73.1
159.2
105.3
108.9
180.5
100.9
73.1
159.3
104.6
108.5
181.1
100.6
73.1
8a
8b
9
glc-1
glc-2
glc-3
glc-4
glc-5
glc-6
xyl-1
xyl-2
xyl-3
xyl-4
xyl-5
Cinnamyl
1′
76.3
76.3
76.0
76.3
75.8
70.0
70.0
69.4
70.0
69.1
75.6
75.6
76.0
75.6
75.6
68.9
69.0
68.0
69.0
67.7
101.7
73.3
101.8
73.3
101.6
73.5
101.7
73.3
101.6
73.1
74.5
74.6
74.4
74.5
74.0
69.8
69.8
69.8
69.8
69.4
65.8
65.8
65.8
65.8
65.5
125.5
110.7
147.9
149.3
115.3
122.9
144.7
114.5
165.5
124.4
105.9
148.0
138.3
148.0
105.9
145.1
114.9
165.6
125.8
111.0
148.2
149.3
115.5
123.0
144.9
114.9
165.7
125.6
110.8
148.2
149.3
115.4
123.0
144.7
114.6
165.5
125.5
110.6
147.6
146.9
115.1
122.8
144.6
114.6
165.4
2′
3′
4′
5′
6′
7′
8′
9′
OCH3
2-
56.7
56.6
4-
55.7
56.6
55.6
56.2
56.6
6-
56.3
55.6
56.3
56.0
56.0
3′-
5′-
55.3
compound 1. HMBC (l Fig. 3) and 1H-1H COSY correlations
confirmed that the linkage positions of the glycosyl, xylosyl, and
caffeic acid moieties were identical to those in compound 1. Thus,
the structure of compound 2 was deduced to be 1-hydroxy-2,
6-dimethoxy-8-O-[2-(4′-hydroxy-3′,5′-dimethoxy-E-cinnamyl)-
ring moiety of the xanthone nucleus, one of the phenolic hydrox-
yl groups on the aromatic ring in compound 3 was replaced by a
signal corresponding to a methoxy group in compound 4. The
NOESY correlations between 6-OCH3 and H-5, H-7 determined
the location of the additional methoxy group and further con-
firmed that the methoxy group was linked to C-6 of the xanthone
nucleus. Thus, the structure of compound 4 was deduced to be 1-
hydroxy-4,6-dimethoxy-8-O-[2-(4′-hydroxy-3′-methoxy-E-cin-
namyl)-β-D-xylopyranosyl-(1–6)-β-D-glucopyranosyl]-xanthone
and named comastomaside D.
"
β-D-xylopyranosyl-(1–6)-β-D-glucopyranosyl]-xanthone
named comastomaside B.
The molecular formula of compound 3 was determined to be
35H36O18 by HR‑ESI‑MS. The NMR spectrum of compound 3
and
C
"
(l Tables 1 and 2) was similar to that of compound 1 with the ex-
ception of the substitution of the xanthone nucleus and a missing
methoxy group (l Fig. 4). NOESY correlations between the fol-
The molecular formula of compound 5 was determined to be
"
"
C
34H34O18 by HR‑ESI‑MS. Its NMR spectrum (l Tables 1 and 2)
lowing determined the locations of the two methoxy groups and
the 1,4,6,8-tetrasubstituted xanthone moiety in compound 3: 4-
OCH3 and H-3; and 3′-OCH3 and H-2′. Thus, the structure of com-
pound 3 was deduced to be 1,6-dihydroxy-4-methoxy-8-O-[2-
(4′-hydroxy-3′-methoxy-E-cinnamyl)-β-D-xylopyranosyl-(1–6)-
β-D-glucopyranosyl]-xanthone and named comastomaside C.
The molecular formula of compound 4 was determined to be
showed similarities to that of compound 3 with the exception of
the signals corresponding to the aromatic ring of the xanthone
nucleus. The two phenolic hydroxyl groups on the aromatic ring
in compound 3 were replaced by signals corresponding to two
methoxy groups in compound 5. The enhanced NOE between 3′-
OCH3 and H-2′ confirmed that the methoxy group was connected
at the C-3′ position of the cinnamyl moiety. In addition, hydroxyl
groups were confirmed to be connected to C-4 and C-5 of the
xanthone nucleus. Thus, the structure of compound 5 was de-
C36H38O18 by HR‑ESI‑MS. The NMR spectrum of compound 4
"
(l Tables 1 and 2) showed similarities to that of compound 3.
With the exception of the signals corresponding to the aromatic
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596

1594 Letters
Fig. 2 Main HMBC (H→C) correlations of com-
pound 1.
Fig. 3 Main HMBC (H→C) correlations of com-
pound 2.
Fig. 4 Main HMBC (H→C) correlations of com-
pound 3.
duced to be 1,4,6-trihydroxy-8-O-[2-(4′-hydroxy-3′-methoxy-E-
cinnamyl)-β-D-xylopyranosyl-(1–6)-β-D-glucopyranosyl]-xan-
thone and named comastomaside E.
Compounds 1–5 were evaluated for hepatoprotective activity
against D-galactosamine-induced toxicity in WBF344 cells. At a
concentration of 10−5 M, compounds 3 and 5 showed potent
"
hepatoprotective activities (l Table 3), whereas the other com-
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596

Letters 1595
1.5 × 60 cm] eluted with CHCl3-MeOH (1:1), to obtain a yellow
Table 3 Hepatoprotective activity of compounds 1–5 against D-galactos-
solid (257 mg) which was purified by preparative HPLC [All-
sphere C18 column, (250 × 10 mm, 5 µm); 2.5 L of MeOH‑H2O
(35:65); 2.0 mL/min] to obtain compounds 3 (10 mg) and 5
(9 mg).
amine-induced toxicity in WB-F344 cellsa.
Compound
Cell survival rate
(% of normal)
Inhibition
(% of control)
Normal
100.23 0.52
24.14 0.16
37.12 0.22**
29.33 0.26
29.19 0.26
35.12 0.33*
29.06 0.19
33.20 0.20*
Isolates: Comastomaside A (1): yellow amorphous solid (94.3%
purity), m.p. 284–285°C, [α]2D0 − 144.2 (c 0.10, DMSO). UV
(DMSO) λmax(log ε): 257 (4.41), 273 (4.28), 297 (4.21), 324
Control
Bicyclolb
17.10
6.58
1
2
3
4
5
"
(4.38) nm; NMR data, see l Tables 1 and 2; ESI‑MS (pos): 781
6.58
[M
36H38O18Na, 781.1950).
+ +
Na]+; HR‑ESI‑MS: 781.1967 [M Na]+ (calcd. for
14.47
6.58
C
Comastomaside B (2): yellow amorphous solid (98.2% purity),
m.p. 270–272°C, [α]D20 − 33.1 (c 0.10, DMSO). UV (DMSO) λmax
11.84
a Results are expressed as means SD (n = 3; for normal and control, n = 6); * p < 0.05,
** p < 0.01. Compounds were tested at 1 × 10−5 M; b Positive control substance bicy-
"
(log ε): 257 (4.29), 269 (4.30), 318 (4.26) nm; NMR data, see l Ta-
bles 1 and 2; ESI‑MS (pos): 811 [M + Na]+, HR‑ESI‑MS: 811.2067
[M + Na]+ (calcd. for C37H40O19Na, 811.2056).
clol
Comastomaside C (3): yellow amorphous solid (99.9% purity),
m.p. 260–262°C, [α]D20 − 54.6 (c 0.10, DMSO). UV (DMSO) λmax
(log ε): 257 (4.38), 268 (4.30), 299 (4.24), 324 (4.39) nm; NMR da-
ta, see l Tables 1 and 2; ESI‑MS (pos): 767 [M + Na] , HR‑ESI‑MS:
767.1789 [M + Na]+ (calcd. for C35H36O18Na, 767.1794).
pounds tested exhibited no significant activity at a concentration
of 10−5 M. Compounds 1–5 were also evaluated for cytotoxicity
against four human cancer cell lines (HCT-8, Bel-7402, BGC-823,
and A2780) and determined to be inactive (IC50 > 10 µM).
+
"
Comastomaside D (4): yellow amorphous solid (98.2% purity),
m.p. 277–279°C, [α]D20 − 55.5 (c 0.10, DMSO). UV (DMSO) λmax
(log ε): 257 (4.46), 272 (4.40), 295 (4.18), 356 (4.49) nm; NMR da-
Materials and Methods
!
+
"
ta, see l Tables 1 and 2; ESI‑MS (pos): 781 [M + Na] , HR‑ESI‑MS:
781.1936 [M + Na]+ (calcd. for C36H38O18Na, 781.1950).
The aerial parts of C. pedunculatum were collected in the County
of Gonghe, Qinghai province, China, in August 2006. The plant
material was identified by Prof. Lin Pengcheng. A voucher speci-
men (HMH200608A) was deposited in the College of Life and En-
vironmental Science, Minzu University of China.
Optical rotations were measured using a PE Model 343 polarim-
eter. IR spectra were recorded on a Nicolet 5700 FT‑IR spectro-
photometer. NMR spectra were collected on Varian Inova-500
and Bruker AV-500 spectrometers in DMSO, and solvent peaks
were used as references. Mass spectra were obtained on a Mass
Agilent 1100 Series LC‑MSD-Trap-SL spectrometer (ESI‑MS) and
6210 ESI‑TOF spectrometer (HR‑ESI‑MS).
The aerial parts of C. pedunculatum were dried in the shade and
chipped. The plant material (5.0 kg) was extracted with 70%
aqueous ethanol (3 × 40 L) under reflux for 1 hour. The solution
was filtered, evaporated in vacuum and successively partitioned
with petroleum ether, ethyl acetate, and n-butanol (1:1, v/v).
The n-butanol (230 g) fraction was subsequently purified on an
AB-8 porous polymer resin (10 L; column, 10 × 150 cm) and
eluted with EtOH‑H2O gradients to yield 4 fractions [HBT (20%
EtOH, 80 L), HBF (40% EtOH, 80 L), HBS (60% EtOH, 60 L), and
HBN (95%EtOH, 40 L)]. The HBF fraction (13.8 g) was applied to
CC [silica gel (mesh, 45–75 µm, 150 g); column, 3 × 120 cm] and
eluted with solvent gradients of CHCl3-MeOH (1:0 to 8:2; 10 L)
to afford fractions A–H. Fraction D (2095 mg) was further purified
by CC [ODS (mesh, 50 µm, 100 g); column, 3 × 90 cm] and eluted
with a MeOH‑H2O gradient (2: 8 to 6:4; 6.0 L) to yield subfrac-
tions D1-D5. Subfraction D2 (1236 mg) was purified by CC [Sepha-
dex LH-20 (50 g); column, 1.5 × 60 cm] and eluted with CHCl3-
MeOH (1:1) to afford a yellow solid (647 mg), which was purified
by preparative HPLC [Allsphere C18 column, (250 × 10 mm, 5 µm);
2.5 L of MeOH‑H2O (40:60); 2.0 mL/min flow rate] to obtain
compounds 1 (9 mg), 2 (7 mg), and 4 (20 mg). Fraction E
(864 mg) was further purified by CC [ODS (mesh, 50 µm; 50 g);
column, 3 × 60 cm] and eluted with a MeOH‑H2O gradient (2:8
to 6:4; 3.0 L) to obtain subfractions E1-E5. Subfraction E2
(450 mg) was purified by CC [Sephadex LH-20 (50 g); column,
Comastomaside E (5): yellow amorphous solid (93.7% purity,
HPLC); m.p. 281–284°C, [α]2D0 − 59.1 (c 0.10, MeOH). UV (MeOH)
λmax (log ε): 204 (4.65), 240 (4.57), 269 (4.44), 316 (4.49) nm;
+
"
NMR data, see l Tables 1 and 2; ESI‑MS (pos): 753 [M + Na] ,
HR‑ESI‑MS: 753.1642 [M +
Na]+ (calcd. for C34H34O18Na,
753.1637).
Acid hydrolysis TLC [5]: Compounds 1, 2, 3, 4, and 5 were dis-
solved in ethanol separately to a concentration of 0.5 mg per mL.
The solutions were applied separately at approximately 1 cm
from the bottom edge of the HPTLC silica gel plate (10 × 10 cm),
and the plate was hydrolyzed in an airtight container with hydro-
gen chloride vapor for 20 min in a 50–60°C water bath. Then, D-
glucose and D-xylose were dissolved in ethanol separately, and
the solutions were also applied separately to the base of the plate.
Glacial acetic acid (1 mL) was added to 9 mL of the lower layer
containing a mixture of chloroform-methanol-water (30:12:4),
which was used as the developing solvent. The plate was saturat-
ed for 20 min and then developed for 9 cm using an ascending
technique. The plate was subsequently soaked in a coloring re-
agent consisting of 0.93 g of aniline and 1.66 g of o-phthalic acid
in 100 mL of n-butanol, and then heated at 100°C for 15 min until
colored spots were visible. TLC analysis showed the presence of
glucose and xylose, which are the sugars present in compounds
1–5.
Hepatoprotective activities against D-galactosamine-induced
cytotoxicity in WB-F344 cells: The hepatoprotective activities of
compounds 1-5 were determined using a 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric
assay in WB-F344 cells. Each cell suspension of 1 × 104 cells in
200 µL of Dulbeccoʼs modified Eagleʼs medium containing fetal
calf serum (3%), penicillin (100 units/mL), and streptomycin
(100 µg/mL) was placed in a 96-well microplate and precultured
for 24 h at 37°C under a 5% CO2 atmosphere. Fresh medium
(200 µL) containing the positive control bicyclol (purity > 99%,
Beijing Union Pharmaceutical Factory) or test samples were
added, and the cells were cultured for 1 h. The cultured cells were
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596

1596 Letters
exposed to 40 mM D-galactosamine for 24 h. The cytotoxic effects
Acknowledgements
!
of test samples were simultaneously measured in the absence of
D-galactosamine. The medium was replaced and contained
0.5 mg/mL MTT. After 3.5 h incubation, the medium was re-
moved, and 150 µL of DMSO was added to dissolve formazan
crystals. The optical density (OD) of the formazan solution was
measured at 492 nm using a microplate reader. Inhibition (%)
was determined using the following formula:
This work was supported by the National Science and Technology
Project of China (2011ZX09307–002–01). We are grateful to Prof.
Xiaoguang Chen for the human cancer cells screening. We thank
Mr Jianbei Li for the HRMS analyses.
Conflict of Interest
!
Inhibition (%)
=
[(ODsample
trol)] × 100
–
ODcontrol)/(ODnormal
–
ODcon-
The corresponding authors declare that this manuscript is sub-
mitted on behalf of all authors. The authors do not have any con-
flicts of interest. The copyright belongs to the publisher upon ac-
ceptance of the manuscript.
Assessment of the inhibitory activity of compounds 1–5 against
several human cancer cell lines: Human colon cancer (HCT-8),
hepatoma (Bel-7402), stomach cancer (BGC-823), ovarian cancer
(A2780), and epithelial WISH cell lines (Susan Hayflick Wistar In-
stitute) were obtained from the American Type Culture Collec-
tion (ATCC). Cells were maintained in RRMI1640 medium supple-
mented with 10% fetal bovine serum (FBS), 100 units/mL penicil-
lin, and 100 µg/mL streptomycin. Cultures were incubated at 37°
C in a humidified 5% CO2 atmosphere.
HCT-8, Bel-7402, BGC-823, A2780 cells, and human epithelial
WISH cells were seeded in 96-well microtiter plates at a concen-
tration of 1200 cells/well. After 24 h, compounds 1–5 were added
to cells. After 96 h of drug treatment, cell viability was deter-
mined by measuring the metabolic conversion of MTT (3-[4,5-di-
methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) into pur-
ple formazan crystals by active cells. MTT assay results were
monitored at 570 nm using an MK 3 Wellscan (Labsystem DRO-
GON) plate reader. All compounds were dissolved in 100% DMSO
to yield a final DMSO concentration of 0.1% in each well and
tested at five different concentrations. The concentration of each
compound was tested in three parallel wells. IC50 values were cal-
culated using Microsoft Excel software.
References
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received December 21, 2011
revised
June 20, 2012
accepted June 25, 2012
Bibliography
DOI http://dx.doi.org/10.1055/s-0032-1315079
Published online July 18, 2012
Planta Med 2012; 78: 1591–1596
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Statistical analysis: The Studentʼs t-test for unpaired observations
between control and test samples was performed to identify sig-
nificant differences, and p values less than 0.05 were considered
to be significantly different.
Correspondence
Dr. Shuai Li
Institute of Materia Medica, Department of Natural Product Chemistry
Chinese Academy of Medical Sciences and Peking Union Medical College
1 Xian Nong Tan Street
Beijing 100050
Supporting information
UV, IR, MS, 1H NMR, and 13C NMR and 2D NMR correlation spec-
tra of compounds 1–5 are available as Supporting Information.
PR China
Phone: + 861063164628
Fax: + 861063017757
lishuai@imm.ac.cn
Qiao Y et al. New Xanthone Glycosides… Planta Med 2012; 78: 1591–1596