Phenolic glycosides from Ficus tikoua and their cytotoxic activities

Article abstract of DOI:10.1016/j.carres.2013.09.008

Four new phenolic glycosides, named 2-ethylene-3,5,6-trimethyl-4-phenol-1- O-β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside (1), 3-methoxy-4-O-β-d-apiofuranosyl-(1→2)-β-d- glucopyranosylpropiophenone (2), 3-hydroxy-1-(4-O-β-d-glucopyranosyl-3- methoxyphenyl)

Full text of DOI:10.1016/j.carres.2013.09.008

Carbohydrate Research 382 (2013) 19–24
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Phenolic glycosides from Ficus tikoua and their cytotoxic activities
a
a
a
a
a
a
Zhi-Yong Jiang , Shi-Yuan Li , Wen-Juan Li , Jun-Ming Guo , Kai Tian , Qiu-Fen Hu ,
a,b,
⇑
Xiang-Zhong Huang
a
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry and Biotechnology, Yunnan University
of Nationalities, Jingming South Road, Chenggong New District, Kunming, Yunnan 650500, PR China
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,
b
Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 August 2013
Received in revised form 21 September
Four new phenolic glycosides, named 2-ethylene-3,5,6-trimethyl-4-phenol-1-O-b-D-xylopyranosyl-(1?6)-
b-
-hydroxy-1-(4-O-b-
methyl-2-butenyl)benzoic acid-O-b-
tikoua, together with six known compounds: 3,4,5-trimethoxyphenol-1-O-b-
glucopyranoside (5), 3,4,5-trimethoxyphenol-1-O-b- -glucopyranoside (6), 3-methoxy-4-O-b-
syl-(1?6)-b- -glucopyranosylpropiophenone (7), baihuaqianhuoside (8), 3,5-dimethoxy-4-hydroxybenzoic
acid-O-b- -glucopyranoside (9) and 2-methoxy-4-allylphenyl-1-O-b- -apiofuranosyl-(1?6)-b- -glucopy-
D
-glucopyranoside (1), 3-methoxy-4-O-b-
D
-apiofuranosyl-(1?2)-b-
D
-glucopyranosylpropiophenone (2),
0
3
D-glucopyranosyl-3-methoxyphenyl)propan-1-one (3) and 4-hydroxy-3,5-bis(3 -
2
013
D
-glucopyranoside (4), were isolated from the ethanol extract of Ficus
-apiofuranosyl-(1?6)-b-
-apiofurano-
Accepted 21 September 2013
Available online 1 October 2013
D
D-
D
D
D
Keywords:
Phenolic glycosides
Ficus tikoua
D
D
D
ranoside (10). The structures of the four new compounds were elucidated by chemical methods and MS
and IR, as well as 1D and 2D NMR analyses. The cytotoxicities of the 10 compounds against HeLa, K562,
HL60 and HepG2 cell lines were assessed.
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Ficus tikoua Bur., belonging to the Moraceae family, is a Miao
ethnomedicine in China and has long been used for the treatment
of injuries, rheumatism, diarrhoea and cough caused by lung heat.
A recent pharmacological investigation of this plant showed that
Ficus tikoua possessed anti-tumour, anti-diabetes and anti-
sylpropiophenone (7),5 baihuaqianhuoside (8),6–9 3,5-dime-
thoxy-4-hydroxybenzoic acid O-b-
-glucopyranoside (9)10 and
2-methoxy-4-allylphenyl-1-O-b- -apiofuranosyl-(1?6)-b- -gluco-
pyranoside (10). The cytotoxicity of the 10 isolates was tested by
the MTT method with HeLa, K562, HL60 and HepG2 cell lines.
Herein, we describe the isolation, structural elucidation and cyto-
toxic activity of the 10 isolates.
D
D
D
1
1
1
bacterial activity. Only a few phytochemical studies on the stems
and leaves of F. tikoua are documented,1 and there has been no
chemical investigation on the rhizomes to the best of our
knowledge. In our previous cytotoxic experiments on natural
medicines, 90% ethanol extract of the rhizome of F. tikoua was
found to possess significant cytotoxic activity. To elucidate the
anti-tumour bio-active constituents from this ethnomedicine, the
rhizome of F. tikoua was phytochemically investigated to afford
four new phenolic glycosides, 2-ethylene-3,5,6-trimethyl-4-phenol-
,2
Compound 1 was obtained as a white amorphous powder. The
positive ESIMS gave a quasi-molecular ion peak at m/z 495
+
([M+Na] ), in agreement with the molecular formula C22
H
32
O11 re-
vealed by the HRESIMS. The IR suggested the existence of hydroxyl
ꢀ
1
ꢀ1
(3447 cm ), olefin (1646 cm ) and aromatic groups (1601, 1505
ꢀ
1
and 1457 cm ) in compound 1. After the hydrolysis of 1 with 10%
HCl in MeOH, -glucose and -xylose were identified by a compar-
ison with the authentic sugar samples on TLC (developed by
n-BuOH/AcOH/H O 4:1:5, upper layer; and PhOH/H O 4:1). In the
H NMR spectrum, three olefinic proton signals due to a mono-
substituted double bond at d 6.94 (1H, dd, J = 11.6, 18.4 Hz, H-7),
5.48 (1H, dd, J = 2.4, 11.6 Hz, H-8a) and 5.32 (1H, dd, J = 2.4,
18.4 Hz, H-8b) were observed, as well as three singlet methyl sig-
nals at d 2.23 (3H, s), 2.20, (3H, s) and 2.16 (3H, s). The anomeric
proton signals at d 4.68 (1H, d, J = 7.6 Hz, H-1 ) and 4.12 (1H, d,
J = 7.6 Hz, H-1 ) suggested the presence of two b-linked sugar
moieties. The C NMR spectrum displayed 22 carbon signals: three
methyls at d 14.3, 14.2 and 13.2; one mono-substituted double
bond at d 134.7 (C-7, d) and 120.2 (C-8, t); a fully substituted
D
D
1
4
3
1
-O-b-
D
-xylopyranosyl-(1?6)-b-
D-glucopyranoside (1), 3-methoxy-
-glucopyranosylpropiophenone (2),
-O-b-
D
-apiofuranosyl-(1?6)-b-
D
2
2
1
-hydroxy-1-(4-O-b-
-one (3) and 4-hydroxy-3,5-bis(3 -methyl-2-butenyl)benzoic acid
D
-glucopyranosyl-3-methoxyphenyl)propan-
0
O-b-D
-glucopyranoside (4) (Fig. 1), as well as six known compounds,
-apiofuranosyl-(1?6)-b- -glucopy-
ranoside (5), 3,4,5-trimethoxyphenol-1-O-b- -glucopyranoside
-apiofuranosyl-(1?6)-b- -glucopyrano-
3
,4,5-trimethoxyphenol-1-O-b-
D
D
3
D
4
0
(6),
3-methoxy-4-O-b-
D
D
0
0
1
3
⇑
Corresponding author. Tel.: +86 871 65913013; fax: +86 871 65910017.
E-mail addresses: jiangzy2010@163.com (Z.-Y. Jiang), xiangzhongh@126.com
(
X.-Z. Huang).
0
008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.09.008

2
0
Z.-Y. Jiang et al. / Carbohydrate Research 382 (2013) 19–24
aromatic ring at d 150.4 (C-4, s), 147.0 (C-1, s), 130.8 (C-2, s), 129.6
C-5, s), 125.3 (C-6, s) and 122.4 (C-3, s); one b- -glucopyranosyl
moiety at d 104.8 (C-1 , d), 75.9 (C-2 , d), 77.8 (C-3 , d), 71.6 (C-4 ,
baihuaqianhuoside (8)6–9 showed a high similarity, with the excep-
tion that there was one more oxygenated methylene and one less
methyl in compound 3. Acid hydrolysis of 3 resulted in D-glucose,
which was determined by a TLC comparison with the authentic
(
D
0
0
0
0
0
0
12,13
d), 77.7 (C-5 , d) and 69.5 (C-6 , t);
and one b-
D
-xylopyranosyl
00
00
00
group at d 104.6 (C-1 , d), 74.7 (C-2 , d), 77.3 (C-3 , d), 71.1
sample and GC analysis. Considering that compound 3 had 16 mass
0
0
00
12,14
6–9
(
C-4 , d) and 66.5 (C-5 , t).
-glucose and -xylose were verified by GC analysis of their
trimethylsilyl -cysteine derivatives. The long-range HMBC correla-
tions (Fig. 2) of H-7 (d 6.94) with d 147.0 (C-1), 130.8 (C-2) and
22.4 (C-3); H-9 (d 2.23) with 130.8 (C-2), 122.4 (C-3)
The absolute configurations of the
units more than baihuaqianhuoside (8),
it could be concluded
D
D
that compound 3 was a derivative of baihuaqianhuoside (8), with
a hydroxyl added at C-9. This was further verified by the HMBC
correlation (Fig. 2) between d 3.95 (2H, t, J = 6.0 Hz, H-9) and C-7
L
1
1
1
d
(d 199.7) and the H– H COSY cross peak (Fig. 2) between d 3.95
(2H, t, J = 6.0 Hz, H-9) and d 3.20 (2H, t, J = 6.0 Hz, H-8). The other
and 150.4 (C-4); H-10 (d 2.16) with d 150.4 (C-4), 129.6 (C-5),
and 125.3 (C-6); and H-11 (d 2.20) with d 147.0 (C-1), 129.6 (C-
1
1
HSQC, HMBC and H– H COSY correlations (Fig. 2) were used for
the other proton and carbon assignments. Thus, the structure of
5
) and 125.3 (C-6) suggested that the mono-substituted double
bond is located at C-2, and the three methyls are attached to C-3,
compound 3 was elucidated to be 3-hydroxy-1-(4-O-b-
anosyl-3-methoxyphenyl)propan-1-one (3) (Fig. 1).
D-glucopyr-
0
C-5 and C-6. Simultaneously, HMBC correlations (Fig. 2) of H-1
00
0
(
d 4.68) with d 147.0 (C-1) and H-1 (d 4.12) with d 69.5 (C-6 ) were
Compound 4 was isolated as a white amorphous powder, with
the molecular formula C23 , revealed by the HRESIMS peak
observed, demonstrating that the b-
D
-xylopyranose was located at
32 8
H O
0
+
the C-6 of the inner glucopyranose, and the inner glucopyranose
was linked at C-1 in the aromatic ring. The carbon signal in the
low field at d 150.4 (C-4, s) suggested the existence of the hydroxyl
at C-4. These deductions were supported by the HRESIMS and
at m/z 459.1986 ([M+Na] ). A comparison of the NMR data with
0
18
those of 4-methoxy-3,5-bis(3 -methyl-2-butenyl)benzoic acid
0
suggested that compound 4 had a similar 4-hydroxy-3,5-bis(3 -
methyl-2-butenyl)benzoic acid nucleus. However, compound 4
1
1
H– H COSY, ROESY and HMBC (Fig. 2) correlations. Consequently,
the structure of compound 1 was assigned as 2-ethylene-3,5,6-tri-
contained one more b-
bis(3 -methyl-2-butenyl)benzoic acid, as shown by the TLC and
D
-glucopyranose unit than 4-methoxy-3,5-
0
18
methyl-4-phenol-1-O-b-
side (1) (Fig. 1).
D
-xylopyranosyl-(1?6)-b-D-glucopyrano-
GC analysis of the alkaline methanolysis product of compound 4.
1
0,13,19,20
According to the previous literatures,
signal should appear at the relatively higher field (with a chemical
shifts smaller than 96.0 ppm) when a -glupyranose was linked at
the anomeric carbon
Compound 2 was obtained as a white amorphous powder and
had the molecular formula C21 12, deduced by the HRESI peak
12Na , calcd 497.1634). The IR spectrum
showed absorption bands at 3444, 1679, 1595, 1511 and
H
30
O
D
+
at m/z 497.1643 (C21
H
30
O
a carboxyl function. In our experiments, the anomeric carbon sig-
0
nal at d 95.9 (s, C-1 ) at a relatively higher field indicated that the
ꢀ1
1
461 cm , corresponding to hydroxyl, carbonyl and aromatic ring
groups, respectively. The hydrolysis of 2 with 10% HCl in MeOH lib-
erated -glucose and -apiose, identified by a comparison with the
authentic sugar samples on TLC (n-BuOH/AcOH/H O 4:1:5, upper
layer; PhOH/H O 4:1). The absolute configurations of the -glucose
and -apiose were also confirmed by GC analysis. The H NMR
b-
D
-glucopyranose might be linked at the carboxyl group. This idea
0
was substantiated by the HMBC correlation between H-1 (d 5.69
1H, d, J = 7.8 Hz) and C-7 (167.2, s). The other 2D NMR correlations
(Fig. 2) further confirmed the above deduction. Lastly, the structure
D
D
2
0
2
D
1
of compound 4 was characterised as 4-hydroxy-3,5-bis(3 -methyl-
D
2-butenyl)benzoic acid O-b-
D-glucopyranoside (4) (Fig. 1).
spectrum showed three aromatic proton signals at d 7.63 (1H, dd,
J = 1.6, 8.8 Hz), 7.56 (1H, d, J = 1.6 Hz) and 7.18 (1H, d, J = 8.8 Hz);
one methoxy at d 3.89 (3H, s); one methylene at d 3.02 (2H, q,
J = 7.2 Hz); one methyl at d 1.17 (3H, t, J = 7.2 Hz); together with
The known compounds 5–10 were identified by comparing the
3
–11
NMR data with those reported in the literature.
All 10 isolates were tested for their cytotoxic activities by the
2
1
MTT method against HeLa, K562, HL60 and HepG2 cell lines. As
summarised in Table 3, compounds 1, 2, 4, 7, 8 and 10 showed
superior cytotoxicity against the HeLa cell line, with IC50 values
0
two anomeric proton signals at d 5.11 (1H, d, J = 7.6 Hz, H-1 ) and
00
15–17
5
.54 (1H, d, J = 1.2 Hz, H-1 ).
The coupling constant of the ano-
meric proton ascribable to the glucose suggested that the glucose
less than 25 lM. Compounds 3, 5, 6, 8 and 9 showed good inhibi-
was b-linked, and the b-linkage of apiose was deduced by compar-
tory activity against K562 cells. For the HL60 cell line, compounds
4, 6 and 7 exhibited a similar potency as the positive control, and
compounds 1 and 3 showed noticeable activity against HepG2
cells, with IC50 values smaller than 20 lM. Cisplatin, an approved
agent for clinical anti-tumour treatment, was employed as a posi-
ing the J value of anomeric proton with those of methyl-
J = 4.6 Hz) and methyl-b- -apiose (J = 2.4 Hz) in the previous
report. The C NMR also displayed two sugar moieties corre-
sponding to a b- -glucopyranose with signals at d 100.2 (d), 77.3
d), 78.7 (d), 71.3 (d), 78.2 (d) and 62.4 (t);1
nose with signals at d 110.2 (d), 77.9 (d), 80.8 (s), 75.4 (t) and 66.1
a-D-apiose
(
D
1
5
13
D
2,13
(
and a b-D-apiofura-
tive control in our cytotoxicity bioassay.
In conclusion, our research described the isolation of 10 pheno-
lic chemicals from this plant for the first time. Furthermore, the
cytotoxic activities of all the isolates were assessed in vitro.
Although the cytotoxicities of compounds 1–10 were not compara-
ble to the positive control in our tested cell lines, these results re-
vealed the phenolic constituents in F. tikoua might be the main
anti-tumour bioactive components, which provide a scientific sup-
port to the ethnomedicine for the anti-tumour activity.
1
5–17
(
t);
as well as one carbonyl at d 202.2; an aromatic ring with
signals at d 152.2 (s), 150.6 (s), 132.4 (s), 123.6 (d), 115.6 (d) and
1
12.1 (d); and a methoxy at d 56.4 (q). Comparing the NMR data
6–9
with those of the known compound baihuaqianhuoside (8)
sug-
gested that compound 2 possessed the same aglycone as that of
baihuaqianhuoside (8) and contained one more apiose sugar moi-
ety. The additional b-
tached at the C-2 of the inner b-
D
-apiofuranosyl unit was assigned to be at-
0
D
-glucopyranose based on the
00
0
HMBC correlation between H-1 (d 5.54) and d 77.3 (C-2 ). The
1
1
other HMBC and H– H COSY (Fig. 2) data further confirmed this
1. Experimental
deduction. Accordingly, the structure of compound 2 was deter-
mined to be 3-methoxyl-4-O-b-
pyranosylpropiophenone (2) (Fig. 1).
Compound 3 was also isolated as a white amorphous powder
and assigned the molecular formula C16 based on the
D
-apiofuranosyl-(1?6)-b-
D
-gluco-
1.1. General experimental procedures
Optical rotations were determined on a Horiba SEPA-300
polarimeter. UV spectra were recorded on a Shimadzu UV-210A
spectrophotometer. GC experiments were conducted on an Agilent
7890A instrument. IR spectra were obtained on a Bio-Rad FTS-135
22 9
H O
+
+
HRESIMS peak at m/z 381.1169 ([M+Na] , C16
22 9
H O Na , calcd
3
81.1162). comparison of the NMR data with those of
A

Z.-Y. Jiang et al. / Carbohydrate Research 382 (2013) 19–24
21
1
0
H CO
3
2
O
7
9
O
4
O
8
7
CH
HO 6'
OH
3
1
2
OH
4
5
8
9
4
'
1'
6
'
1
O
''
O
5 CH3
CH3
OH
2'
O
5
OH
''
O
O
10
OH
4
''
O
1
1'
OH
11
5
''
OH 1''
2''
OH OH
OH
2'' OH
4''
OH
3
''
1
2
5
''a
1
0
H3CO
4
4
''a
''b
2
O
1''a
7
6
'
O
4
OH
3
2''a
OH
O
HO
1
1
HO
O
5
8
9
O
OH
OH
O
7
5
1
'
6
2''b
OH
OH
1''b
OH
OH
5
''b
3
4
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
HO
MeO
O
O
HO
O
O
O
O
O
O
OH
OH
OH
OH
O
OH
O
OH
OH
O
OH OH OH
OH
OH
OH OH
5
6
7
H3CO
O
MeO
O
O
O
HO
OCH3
O
O
OH
HO
O
O
O
O
OH
OH
OH
OCH3
OH
OH
OH
OH OH
OH
OH
OH
OH
8
9
10
Figure 1. The structures of compounds 1–10.
spectrometer. 1D and 2D NMR spectra were recorded on a Bruker
AM-400 NMR, with TMS as an internal standard. MS were recorded
on a VG Auto Spec-3000 spectrometer. Column chromatography
Resources, State Ethnic Affairs Commission & Ministry of Educa-
tion, Yunnan University of Nationalities.
(
CC) was performed using silica gel (SiO
Meigao Chemical Company, Qingdao, China), Sephadex LH-20
20–150 m; Pharmacia Fine Chemical Co. Ltd, Sweden), MCI gel
CHP-20P (70–150 m, Mitsubishi Chemical Corporation, Tokyo,
Japan), and Lichroprep Rp-18 gel (40–63 m, Merck, Germany).
The authentic sugars including -glucose, -xylose and -apiose
2
; 200–300 mesh; Qingdao
1.3. Extraction and isolation
(
l
The dried and powdered rhizomes of Ficus tikoua (11 kg) were
extracted with 90% EtOH (80 L) under reflux three times. The ex-
tract was concentrated in vacuo and partitioned between water,
petroleum ether, EtOAc and n-BuOH to provide petroleum ether
(320 g), EtOAc (280 g) and n-BuOH (368 g) fractions. The n-BuOH
fraction was subjected to silica gel column chromatography (CC)
l
l
D
D
D
were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO,
USA).
with a gradient elution of CHCl
70:30:3, 65:35:5 (v/v/v)) to yield seven fractions (Frs. 1–7). Fr. 2
15 g) was subjected to silica gel CC (200 g) and eluted with CHCl
MeOH/H O (90:10:1) to afford five fractions (Frs. 2a–2e). Fr. 2c
(3.2 g) was subjected to MCI gel CC (80 g, MeOH/H O 75:25) to give
three fractions. The third fraction was further purified by Rp-18 CC
(MeOH/H 75:25) to yield compounds (130 mg) and
(270 mg). Fr. 2d (2.1 g) was also subjected to MCI gel CC (80 g,
3 2
/MeOH/H O (90:10:0, 80:20:2,
1
.2. Plant material
(
3
/
The rhizomes of Ficus tikoua were collected in Chuxiong,
2
Yunnan province, PR China, in September 2010, and identified by
Professor Deding Tao from the Kunming Institute of Botany, Chi-
nese Academy of Sciences. A voucher specimen (2010-09-06) was
deposited in the Key Laboratory of Chemistry in Ethnic Medicinal
2
2
O
3
9

2
2
Z.-Y. Jiang et al. / Carbohydrate Research 382 (2013) 19–24
+
ESIMS (pos.): m/z 495 [M+Na] ; HRESIMS (pos.): m/z 495.1851
H CO
+
+
3
[
M+Na] (calcd for C22
H
32
O11Na , 495.1842).
O
O
O
HO
1
.4.2. Compound 2
CH3
26:4
D
White amorphous powder; ½
a
ꢁ
ꢀ80.5 (c 0.225, MeOH); UV:
OH
OH
OH
MeOH
max
ꢀ1
½
k
(log
e
) 275 (2.87); IR (KBr) cm : 3444, 2978, 1679, 1595,
1
13
O
1511, 1461, 1267, 1130, 1069; H and C NMR data, see Tables 1
O
CH3
O
+
O
O
and 2; ESIMS (pos.): m/z 497 [M+Na] ; HRESIMS: m/z 497.1643
[M+Na] (calcd for C21
O
CH3
+
+
OH
OH
OH
OH
H
30
O
12Na , 497.1634).
OH
OH
OH
OH OH
1.4.3. Compound 3
2
D
5:0
White amorphous powder; ½
a
ꢁ
ꢀ62.5 (c 0.110, MeOH); UV:
1
2
MeOH
max
ꢀ1
½
k
1
(log
e
) 278 (2.90); IR (KBr) cm : 3445, 2975, 1684, 1592,
1
13
514, 1463, 1274, 1108, 1067; H and C NMR data, see Tables 1
+
and 2; ESIMS (pos.): m/z 381 [M+Na] ; HRESIMS: m/z 381.1169
H3CO
O
+
+
[
M+Na] (calcd for C16
H
22
O
9
Na , 381.1162).
O
O
OH
HO
HO
OH
O
O
1.4.4. Compound 4
O
24:8
D
OH
OH
White amorphous powder; ½
a
ꢁ
ꢀ28.9 (c 0.160, MeOH); UV:
OH
OH
MeOH
max
ꢀ1
½k
(log
e
) 266 (3.05); IR (KBr) cm : 3446, 2978, 1650, 1598,
1
13
OH
1450, 1267, 1122, 1070, 995, 863; H and C NMR data, see Tables
OH
+
1
and 2; ESIMS (pos.): m/z 459 [M+Na] ; HRESIMS: m/z 459.1986
3
Figure 2. Key HMBC (?) and 1H– H COSY (
4
+
+
[
M+Na] (calcd C23
32 8
H O Na , 459.1994).
1
) correlations of compounds 1–4.
1
.5. Sugar identification
MeOH/H
O 70:30) and Sephadex LH-20 (eluted with MeOH) to afford
compounds 1 (65 mg), 6 (201 mg) and 10 (70 mg). Fr. 3 (20.6 g)
was purified by silica gel CC eluting with CHCl /MeOH/H
90:10:1?80:20:2) to provide five fractions (Frs. 3a–3e). Fr. 3b
3.7 g) was decoloured by MCI gel CC and further purified by suc-
cessive Rp-18 CC to furnish compounds 4 (87 mg), 2 (118 mg)
and 8 (360 mg). Fr. 3c (2.5 g) was separated by MCI gel CC, fol-
lowed by Rp-18 and Sephadex LH-20 CC (eluted with MeOH) to
yield compounds 7 (245 mg) and 5 (160 mg).
2
O 70:30) and subsequently purified on Rp-18 (MeOH/
H
2
1.5.1. Acidic hydrolysis of compounds 1–3
Each solution of compounds 1–3 (each 5 mg) in a mixture of
MeOH (1.0 mL) and 10% HCl (1.0 mL) was stirred at reflux for 4 h.
3
2
O
(
(
The hydrolysate was allowed to cool, diluted twofold with H O,
2
and extracted with EtOAc (3 ꢂ 2 mL). The aqueous layer was neu-
tralised with 2 M ammonium hydroxide and concentrated in vacuo
to give a residue in which
cose and -apiose (from 2), and
by comparison with authentic sugar samples (n-BuOH/AcOH/H O
D
-glucose and
D-xylose (from 1), D-glu-
-glucose (from 3) were identified
D
D
2
4
2
:1:5, upper layer; PhOH/H O, 4:1) on TLC (sprayed with aniline
1
1
.4. Identification
phthalate reagent, followed by heating).
.4.1. Compound 1
1.5.2. Alkaline methanolysis of compound 4
2
D
5:8
White amorphous powder; ½
aꢁ
ꢀ58.8 (c 0.171, MeOH); UV:
Compound 4 (5 mg) was treated with methanolic 3% NaOMe at
room temperature and stirred for 4 h. After being extracted with
EtOAc (3 ꢂ 2 mL), the aqueous layer was neutralised and concen-
MeOH
max
ꢀ1
k
(log
e) 247 (3.03), 292 (2.91); IR (KBr) cm : 3447, 2976,
1
13
1
646, 1601, 1505, 1457, 1263, 1127, 1060, 993, 864; H and
C
ꢀ
NMR data, see Tables 1 and 2; ESIMS (neg.): m/z 471 [MꢀH] .
trated in vacuo to afford a residue, where
D
-glucose was identified
Table 1
The 1H (400 MHz) and 13C NMR (100 MHz) data for the aglycone moieties of compounds 1–4 in CD
3
OD
No.
1
2
3
4
d
H
d
C
(mult.)
d
H
d
C
(mult.)
d
H
d
C
(mult.)
d
H
C
d (mult.)
1
2
3
4
5
6
7
8
—
—
—
—
—
—
147.0 (s)
130.8 (s)
122.4 (s)
150.4 (s)
129.6 (s)
125.3 (s)
134.7 (d)
120.2 (t)
—
132.4 (s)
112.1 (d)
150.6 (s)
152.2 (s)
115.6 (d)
123.6 (d)
202.2 (s)
32.3 (t)
—
133.0 (s)
112.4 (d)
150.7 (s)
152.4 (s)
116.2 (d)
124.0 (d)
199.7 (s)
41.8 (t)
—
121.6 (s)
130.5 (d)
129.5 (s)
159.0 (s)
129.5 (s)
130.5 (d)
167.2 (s)
—
7.56 (1H, d, 1.6)
—
—
7.18 (1H, d, 8.8)
7.63 (1H, dd, 1.6, 8.8)
—
7.60 (1H, d, 2.0)
—
—
7.24 (1H, d, 8.8)
7.66 (1H, dd, 2.0, 8.8)
—
7.68 (1H, s)
—
—
—
7.68 (1H, s)
—
—
6.94 (1H, dd,11.6,18.4)
5.48 (1H, dd, 2.4, 11.6)
3.02 (2H, q, 7.2)
3.20 (2H, t, 6.0)
5.32 (1H, dd, 2.4, 18.4)
9
1
1
2.23 (3H, s)
2.16 (3H, s)
2.20 (3H, s)
—
—
—
—
—
—
14.3 (q)
13.2 (q)
14.2 (q)
—
—
—
—
—
—
1.17 (3H, t, 7.2)
3.89 (3H, s)
8.9 (q)
56.4 (q)
3.95 (2H, t, 6.0)
3.92 (3H, s)
58.7 (t)
56.7 (q)
—
—
—
—
—
—
0
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Isopentenyl
0
0
0
0
0
0
0
0
0
0
00
00
00
00
1
2
3
4
5
a, 1 b
a, 2 b
a, 3 b
a, 4 b
3.32 (4H, d, 4.8)
5.30 (2H, m)
—
1.75 (6H, s)
1.72 (6H, s)
29.4 (t)
123.0 (d)
134.1 (s)
26.6 (q)
17.9 (q)
00
a, 5 b
d in ppm, J in Hz.

Z.-Y. Jiang et al. / Carbohydrate Research 382 (2013) 19–24
23
Table 2
The 1H (400 MHz) and 13C NMR (100 MHz) data for the sugar moieties of compounds 1–4 in CD
3
OD
No.
1
2
3
4
d
H
d
C
(mult.)
d
H
d
C
(mult.)
d
H
d
C
(mult.)
d
H
C
d (mult.)
glc-
0
1
4.68 (1H, d, 7.6)
3.48 (1H, m)
3.39–3.44 (1H,
overlapped)
3.39–3.44 (1H,
overlapped)
104.8 (d) 5.11 (1H, d, 7.6)
100.2 (d) 5.08 (1H, d, 7.6)
101.9 (d) 5.69 (1H, d, 7.8)
95.9 (d)
0
2
75.9 (d)
77.8 (d)
3.76 (1H, overlapped)
3.64 (1H, m)
77.3 (d)
78.7 (d)
3.53 (1H, m)
3.48 (1H, m)
74.7 (d)
77.9 (d)
3.48 (1H, overlapped) 74.1 (d)
3.48 (1H, overlapped) 78.2 (d)
0
3
0
4
71.6 (d)
3.41–3.43 (1H, m)
71.3 (d)
3.41–3.44 (1H, m)
3.42 (1H, m)
3.87–3.90 (1H, dd, 1.6, 12.0) 62.4 (t)
3.69 (1H, dd, 5.2, 12.0)
71.2 (d)
78.4 (d)
3.43 (1H, overlapped) 71.0 (d)
3.43 (1H, overlapped) 78.8 (d)
0
5
3.23 (1H, m)
77.7 (d)
69.5 (t)
3.47 (1H, m)
3.87–3.91
78.2 (d)
62.4 (t)
0
6
3.92 (1H,dd, 5.2, 11.6)
.67 (1H, dd, 1.6, 11.6)
3.85 (1H, br d, 12.4)
3.63(1H, dd, 2.0, 12.4)
62.3 (t)
3
(1H,dd,2.0,12.0)
3
.69 (1H, dd, 5.6, 12.0)
xyl- or api-
0
0
0
0
1
2
4.12 (1H, d, 6.8)
3.06–3.10 (1H,
overlapped)
104.6
74.7
5.54 (1H, d, 1.2)
3.96 (1H, br s)
110.2 (d)
77.9 (d)
—
—
—
—
—
—
—
—
0
0
0
0
0
0
3
4
5
3.12–3.16 (1H,
overlapped)
3.43–3.45 (1H, m)
77.3
71.1
66.5
—
80.8 (s)
75.4 (t)
66.1 (t)
—
—
—
—
—
—
—
—
—
—
—
—
4.17 (1H, d, 9.6)
3
.76 (1H, overlapped)
3.76 (1H, dd, 5.2, 11.6)
3.53 (2H, br s)
3.03 (1H, m)
d in ppm, J in Hz.
Table 3
The cytotoxic activities of compounds 1–10
Samples
IC50 (lM)
Hela
K562
HL60
HepG2
9
1
2
3
4
5
6
7
8
9
1
0% Ethanol extract (
lg/mL)
17.5 ± 3.1
22.3 ± 5.2
21.5 ± 6.7
73.5 ± 8.9
16.0 ± 3.2
79.2 ± 9.7
68.0 ± 6.6
19.6 ± 5.5
23.7 ± 6.2
49.9 ± 10.5
22.4 ± 5.8
6.9
28.1 ± 5.8
76.9 ± 10.1
102.5 ± 11.3
16.3 ± 3.8
89.9 ± 9.7
18.5 ± 7.3
20.6 ± 4.4
76.3 ± 8.1
24.8 ± 6.4
20.6 ± 5.2
75.4± 6.6
12.0
29.0 ± 6.1
45.5 ± 8.8
72.8 ± 9.3
28.1 ± 7.4
23.2 ± 4.7
20.3 ± 6.2
15.1 ± 5.4
23.6 ± 4.8
41.4 ± 7.3
89.8 ± 9.1
47.9 ± 6.7
10.1
18.7 ± 4.1
15.1 ± 6.3
118.3 ± 11.2
16.9 ± 4.9
132.4 ± 10.8
121.2 ± 9.2
105.1 ± 10.6
71.1 ± 9.4
50.1 ± 8.0
44.3 ± 5.9
77.0 ± 8.1
8.5
0
a
Cisplatin
a
Positive control.
by TLC comparison with the authentic sugar sample (n-BuOH/
AcOH/H O 4:1:5, upper layer; PhOH/H O, 4:1).
t
from 1, D-glucose and D-apiose (R 13.6 min) from 2, and D-glucose
from 3 and 4 were detected.
2
2
1
.5.3. Determination of absolute configuration
After being dried over P for 48 h, the above-mentioned
aqueous residue was dissolved in anhydrous pyridine (1.0 mL),
and 5 mg of -cysteine methyl ester hydrochloride was added.
1.6. Cytotoxicity assay
2 5
O
Cytotoxic activities were evaluated by the MTT (3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method21 using
HeLa, K562, HL60 and HepG2 cell lines. Briefly, the cell suspensions
L
The mixture was stirred at 60 °C for 2 h. The reaction mixture
was then concentrated in vacuo to furnish a dry residue. 0.5 mL
of N-trimethylsilylimidazole was added, and the reaction mixture
was heated at 60 °C for 1 h. Subsequently, the mixture was parti-
4
ꢀ1
(200 mL) at a density of 5 ꢂ 10 cells mL were plated in 96 well
microtitre plates and incubated for 24 h at 37 °C in a humidified
2
incubator containing 5% CO . The test compound solution (2 mL
tioned between n-hexane and H
was directly subjected to GC analysis under the following condi-
2
O (1:1; v/v). The n-hexane extract
in DMSO) at different concentrations was added to each well and
further incubated for 72 h under the same conditions. The MTT
solution (20 mL) was then added to each well and incubated for
4 h. The old medium (150 mL) containing MTT was then gently re-
placed by DMSO and pipetted to dissolve any formazan crystals
formed. The absorbance was then determined with a Spectra
Max Plus plate reader at 540 nm. Dose–response curves were gen-
erated and the IC50 values were defined as the concentration of
compound required to inhibit cell proliferation by 50%. Cisplatin,
tions: capillary column, HP-5 (30 m ꢂ 0.25 mm, with a 0.25
lm
film, Dikma); detection, FID; detector temperature, 280 °C; injec-
tion temperature, 250 °C; initial temperature 160 °C, raised to
ꢀ1
2
80 °C at 5 °C min with the final temperature being maintained
for 10 min; N gas as carrier. By comparing the retention times
) of the derivatives with those of authentic sugars prepared in
a similar way, -glucose (R 20.5 min) and -xylose (R 13.1 min)
2
(R
t
D
t
D
t

2
4
Z.-Y. Jiang et al. / Carbohydrate Research 382 (2013) 19–24
an approved agent for the treatment of many tumours, was used as
the positive control.
2. Guo, L. J.; Tan, X. Q.; Zheng, W.; Kong, F. F.; Lu, P.; Ni, D. J. Chin. Tradit. Herb.
Drugs (zhong-cao-yao) 2011, 42, 1709–1711.
3
4
.
.
Higuchi, H.; Fukui, K.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull. 1992, 40, 534–535.
Shimomura, H.; Sashida, Y.; Oohara, M.; Tenma, H. Phytochemistry 1988, 27,
644–646.
Acknowledgments
5
6
7
8
9
.
.
.
.
.
Yuan, Z.; Tezuka, Y.; Fan, W. Z.; Kadota, S.; Li, X. Chem. Pharm. Bull. 2002, 50, 73–
77.
Financial support was provided by grants from the National
Natural Science Foundation of China (NSFC Nos. 21262047,
Kong, L. Y.; Li, X.; Pei, Y. H.; Yu, R. M.; Min, Z. D.; Zhu, Y. R. Acta Pharm. Sin. 1994,
29, 276–280.
Kitajima, J.; Okamura, C.; Ishikawa, T.; Tanaka, Y. Chem. Pharm. Bull. 1998, 46,
2
1162041), the Natural science Foundation of Yunnan Province
1939–1940.
(
No. S2012FZ0227), the Program for Science and Technology Inno-
Gan, M. L.; Zhu, C. G.; Zhang, Y. L.; Zi, J. C.; Song, W. X.; Yang, Y. C.; Shi, J. G. Chin.
J. Chin. Mater. Med. (zhong-guo-zhong-yao-za-zhi) 2010, 35, 456–467.
Nagatani, Y.; Warashina, T.; Noro, T. Chem. Pharm. Bull. 2001, 49, 1388–1394.
vative Research Team in University of Yunnan Province (IRTSTYN),
the Innovation Team Project of Dai Medicine Research, Yunnan
University of Nationalities, and the Innovation Project of Graduate
1
1
0. Yue, J. M.; Lin, Z. W.; Wang, D. Z.; Sun, H. D. Phytochemistry 1994, 36, 717–719.
1. Takeda, Y.; Ooiso, Y.; Masuda, T.; Honda, G.; Otsuka, H.; Sezik, E.; Yesilada, E.
Phytochemistry 1998, 49, 787–791.
(
No. 11HXYJS03) from the Chemistry and Biotechnology School.
12. Takara, K.; Matsui, D.; Wada, K.; Ichiba, T.; Chinen, I.; Nakasone, Y. Biosci.,
Biotechnol., Biochem. 2003, 67, 376–379.
Supplementary data
1
1
3. Jiang, Z. Y.; Zhang, X. M.; Zhou, J.; Chen, J. J. Helv. Chim. Acta 2005, 88, 297–303.
4. Podolak, I.; Koczurkiewicz, P.; Michalik, M.; Galanty, A.; Zajdel, P.; Janeczko, Z.
Carbonhydr. Res. 2013, 375, 16–20.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
1
5. Kitagawa, I.; Hori, K.; Sakagami, M.; Hashiuchi, F.; Yoshikawa, M.; Ren, J. Chem.
Pharm. Bull. 1993, 41, 1350–1357.
0
9.008.
16. Ma, J. P.; Tan, C. H.; Zhu, D. Y. Helv. Chim. Acta 2007, 90, 158–163.
1
1
1
2
7. Abe, F.; Yamauchi, T. Phytochemistry 1989, 28, 1737–1741.
8. Burke, B.; Nair, M. Phytochemistry 1986, 25, 1427–1430.
9. Achenbach, H.; Hemrich, H. Phytochemistry 1991, 30, 1957–1962.
0. Yoshimoto, K.; Itatani, Y.; Tsuda, Y. Chem. Pharm. Bull. 1980, 28, 2065–2076.
References
1
.
Duan, X. X.; Yang, X. S.; Tong, L. H.; Yang, B.; Hao, X. J. Chin. Tradit. Herb. Drugs
zhong-cao-yao) 2007, 38, 342–344.
21. Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
(