Acyl glycosides with rare β-d-apiofuranosyl-β-d-glucopyranosyl- β-d-apiofuranosyl from Erycibe hainanesis

Article abstract of DOI:10.1016/j.carres.2013.07.011

Three new acyl glycosides with rare β-d-apiofuranosyl-(1 → 2)-[β-d-apiofuranosyl-(1 → 6)]-β-d-glucopyranosyl moieties, 2-O-[2,6-O-bis(5-O-syringoyl-β-d-apiofuranosyl)-β-d-glucopyranosyl] -isopropyl alcohol (1), 1-O-[2,6-O-bis(5-O-syringoyl-β-d-apiofuranos

Full text of DOI:10.1016/j.carres.2013.07.011

Carbohydrate Research 380 (2013) 59–63
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Acyl glycosides with rare b-D-apiofuranosyl-b-D-glucopyranosyl-b-D-
apiofuranosyl from Erycibe hainanesis
Zi-ming Feng a, Shuang Song a,b, Jun He c, Ya-nan Yang a, Jian-shuang Jiang a, Pei-cheng Zhang a,
⇑
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical
Sciences, Beijing 100050, PR China
b Dalian Polytechnic University, Dalian 116034, Liaoning, PR China
c Department of Pharmacy, China-Japan Friendship Hospital, Beijing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 May 2013
Received in revised form 25 July 2013
Accepted 27 July 2013
Available online 2 August 2013
Three new acyl glycosides with rare b-
D
-apiofuranosyl-(1 ? 2)-[b-
-apiofuranosyl)-b-
-apiofuranosyl)-b- -glucopyranosyl]-isoamyl alcohol (2), and
-apiofuranosyl)-b- -glucopyranosyl]-3,4,5-trimethoxyphenol (3) were
obtained from Erycibe hainanesis. Their structures were determined by UV, IR, HRESIMS, 1D and 2D NMR
data, and by chemical methods. Compounds 1–3 were evaluated against -galactosamine induced toxicity
D
-apiofuranosyl-(1 ? 6)]-b-
D-glucopyr-
anosyl moieties, 2-O-[2,6-O-bis(5-O-syringoyl-b-
(1), 1-O-[2,6-O-bis(5-O-syringoyl-b-
1-O-[2,6-O-bis(5-O-vanilloyl-b-
D
D-glucopyranosyl]-isopropyl alcohol
D
D
D
D
D
Keywords:
in WB-F344 rat hepatic epithelial stem-like cells, and compounds 1 and 3 showed moderate hepatoprotec-
tive activities.
b-D-Apiofuranosyl-b-D-glucopyranosyl-b-D-
apiofuranosyl
Acyl glycoside
Convolvulaceae
Erycibe hainanesis
Ó 2013 Published by Elsevier Ltd.
Hepatoprotective activity
1. Introduction
copyranosyl]-3,4,5-trimethoxyphenol (3) by UV, IR, HRESIMS,
1D and 2D NMR data, and by chemical methods. To the best of our
Erycibe hainanesis, a climbing shrub, belongs to the genus Erycibe
(Convolvulaceae). This genus comprises 66 species, of them 11 were
distributed in China, mainly in Guangdong, Guangxi, Yunnan, and
Hainan provinces. Previous chemical investigations on Erycibe
obtusifolia, Erycibe schmidtii, E. hainanesis, Erycibe expansa, and
Erycibe elliptilimba have indicated the presence of coumarins, chlor-
ogenic acids derivatives, alkaloids, and flavonoids, further these con-
stituents have been shown to exhibit anti-inflammatory, muscarinic
agonistic, and cytotoxic activities.1–8 However, in this genus, only
Erycibe obtusifolia has been used in traditional Chinese Medicine to
relieve symptoms of rheumatoid arthritis, function of other species
are scarcely reported. In our study on the constituents of E. hainane-
sis, its potent hepatoprotective activities were accidentally found.1
As a continuation of our efforts to explore new structures with hepa-
toprotective activities from E. hainanesis, three new acyl glycosides
knowledge, only two heptanol compounds with api-glc-api moiety,
but no acyl group, were isolated in the nature.9,10 In vitro bioassay,
compounds 1 and 3 showed moderate hepatoprotective activities
against D-galactosamine induced toxicity in WB-F344 rat hepatic
epithelial stem-like cells.
2. Results and discussion
The n-BuOH fraction of the ethanol extract was subjected to col-
umn chromatography on macroporous resin, silica gel, Sephadex
LH-20, and reversed-phase preparative HPLC, respectively, to
afford three acyl glycosides 1–3 (Fig. 1).
Compound 1 was obtained as a white powder, ½a D20
ꢁ44.2
ꢀ
(c 0.10, MeOH). UV (MeOH) kmax (log e) 280 (4.31) nm; In the IR
spectrum, its absorbance at 3368, 1701, 1610, 1514, 1462,
763 cmꢁ1 suggested the existence of hydroxyl, carbonyl, and ben-
zene ring; The positive HRESIMS data showed an [M+Na]+ ion at
m/z 869.2689 (calcd 869.2686) corresponding to a molecular for-
mula C37H50O22. In the 1H NMR spectrum, four aromatic proton
signals at dH 7.22 (4H, s) and four aromatic methoxyl signals at
3.80 (12H, s) implied two sets of syringoyl moieties existed in 1,
and two methyls at dH 1.01 (3H, d, J = 6.0 Hz, H-1) and 0.95 (3H,
d, J = 6.0 Hz, H-3) and one methine at 3.82 (1H, m, H-2) were ob-
served, which suggested the presence of an isopropyl alcohol
with rare b-
b- -glucopyranosyl moieties were isolated. Their structures were
determined as 2-O-[2,6-O-bis(5-O-syringoyl-b- -apiofuranosyl)-b-
-glucopyranosyl]-isopropyl alcohol (1), 1-O-[2,6-O-bis(5-O-
syringoyl-b- -apiofuranosyl)-b- -glucopyranosyl]-isoamyl alcohol
D-apiofuranosyl-(1 ? 2)-[b-D-apiofuranosyl-(1 ? 6)]-
D
D
D
D
D
(2), and 1-O-[2,6-O-bis(5-O-vanilloyl-b-D-apiofuranosyl)-b-D-glu-
⇑
Corresponding author. Tel.: +86 10 63165231; fax: +86 10 63017757.
E-mail address: pczhang@imm.ac.cn (P. Zhang).
0008-6215/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.carres.2013.07.011

60
Z. Feng et al. / Carbohydrate Research 380 (2013) 59–63
(approximately
Ddc + 3.0), and it was also supported by the corre-
OCH3
lations of H-500 with C-7000, and H-50000 with C-700000 in the HMBC
HO
4'''''
experiment. So far the sugar chain was determined as 2,6-O-
bis(5-O-syringoyl-b-D-apiofuranosyl)-b-D-glucopyranosyl. In addi-
1
6'
O
O
O
H3CO
4''''
1''''
O
2
tion, the HMBC spectrum also showed the correlations from H-10
to C-2 and from H-2 to C-10, which gave the aglycone linkage posi-
tion. Other 1H NMR and 13CNMR data were assigned by HSQC
experiment (Table 1). Finally, 1 was assigned as 2-O-[2,6-O-bis(5-
O
O
O
3''''
1'
OH
OH OH
OCH3
2'
OH
HO
4'''
O
7'''
O-syringoyl-b-D-apiofuranosyl)-b-D-glucopyranosyl]isopropyl
O
H3CO
4''
1''
alcohol.
3'O' H OH
O
Compound 2 was obtained as a white powder, ½a D20
ꢁ54.7 (c
ꢀ
1
0.08, MeOH). The positive HRESIMS ion of 2 at m/z 897.3018
([M+Na]+) proved the molecular formula to be C39H54O22. The
NMR data of 2 showed a close resemblance to those of 1 except
the aglycone moiety (Table 1), which suggested the same syringoyl
api-glc-api moiety also existed in 2. Similarly, this could be sup-
OCH3
HO
4'''''
ported by HMBC correlations of C-20 with H-100, C-60 with H-10000
,
6'
1
O
O
C-7000 with H-500, and C-700000 with H-50000 (Fig. 2 and Supplementary
data). The proton and carbon signals for the aglycone moiety of 2
[dH 3.24 (1H, m), 3.64 (1H, m), 1.23 (2H, m), 1.46 (1H, m), 0.66
(3H, d, J = 6.5 Hz), 0.65 (3H, d, J = 6.5 Hz); dC 67.4, 38.4, 24.8, 22.6,
22.8] were deduced to be an isoamyl alcohol instead of isopropyl
alcohol moiety in 1. The absolute configurations of these two apio-
furanosyls and glucopyranosyl were also determined as b-D config-
uration by the same gas chromagraph analysis method.11 Linkage
position of the sugar chain to the aglycone moiety was achieved
through the observation of HMBC correlations from H-10 to C-1
and from H2-1 to C-10 (Fig. 2). Furthermore, 1HNMR and 13CNMR
data of 2 were assigned by HSQC experiment (Table 1). Thus, the
O
H3CO
4''''
1''''
O
1'
O
O
O
3''''
OH
OH OH
OCH3
3
2'
OH
HO
4'''
O
7'''
O
H3CO
4''
1''
3'O' H OH
O
2
HO
OCH3
4'''''
6'
O
O
structure of 2 was deduced as 1-O-[2,6-O-bis(5-O-syringoyl-b-D-
O
1
H3CO 3'''''
4''''
O
1'
OCH3
1''''
4
O
O
apiofuranosyl)-b-D-glucopyranosyl]-isoamyl alcohol.
O
3''''
Compound 3 was obtained as a white powder, ½a D20
ꢀ
ꢁ68.5 (c
OH
O
OH OH
HO
OCH3
2'
OH
4'''
0.13, MeOH), and its molecular formula was determined to be
C
41H50O23 by the positive HRESIMS data ([M+Na]+, m/z found
O
4''
7'''
3'''
H3CO
1''
933.2629). According to the elucidation of above compound 1
and 2, NMR signals in the up-field region of 1H/13C NMR spectra
were assignable to two apiofuranosyl and a glucopyranosyl as well
as five aromatic methoxy singlets since those NMR signals in 1, 2,
and 3 were similar. The 1H NMR spectrum showed two sets of ABX
couplings in the down-field region at d 7.34 (1H, br s), 6.75 (1H, d,
J = 8.5 Hz), and 7.37 (1H, d, J = 8.5 Hz), and at d 7.44 (1H, br s), 6.85
(1H, d, J = 8.5 Hz), and 7.48 (1H, d, J = 8.5 Hz), respectively. In addi-
tion, HMBC correlations from the methoxy protons at d 3.79 (3H, s)
to C-3000and 3.76 (3H, s) to C-300000 were observed (Fig. 2 and Supple-
mentary data). All these indicated the existence of two vanilloyl
moieties in 3 instead of two syringoyl moieties in 1 and 2. Their
linkage positions were determined on the basis of the HMBC corre-
lations from H-100 to C-20, from H-10000 to C-60, from H-500 to C-7000, and
from H-50000 to C-700000(Fig. 2). Additionally, the 1H NMR spectrum
also showed a singlet at d 6.21 (2H, s) attributed to a symmetrical
1,3,4,5-tetrasubstituted aromatic ring, combining the HMBC exper-
iment, the correlations from the methoxy protons at d 3.61 (6H, s)
to C-3/5, from the methoxy protons at d 3.54 (3H, s) to C-4, and
from H-10 to C-1 (Fig. 2), demonstrated the aglycone of 3 as
3,4,5-trimethoxyphenol and the linkage position of the sugar chain
O
3'O' H OH
3
Figure 1. The structures of compounds 1, 2, and 3.
moiety. Moreover, three anomeric protons at dH 4.23 (1H, d,
J = 7.5 Hz, H-10), 5.33 (1H, br s, H-100), and 4.91 (1H, br s, H-10000) to-
gether with the remaining proton signals at dH 2.97–4.36, combin-
ing the corresponding 16 sugar carbon signals in the 13C NMR
spectrum, attributed to the existence of one hexose and two pen-
toses. Besides above 16 sugar carbon signals, the 13C NMR spec-
trum (Table 1) also displayed two carbonyl carbon signals at dc
165.4 (C-700000) and 165.5 (C-7000), four methoxyl carbons, 12 aro-
matic carbons, two methyl carbons, and one methine carbon sig-
nal. Thus, an isopropyl alcohol and two syringoyl moieties could
be confirmed in 1. Subsequently,
D-configuration apiofuranosyl
and glucopyranosyl were determined by NMR data comparison10
and gas chromagraph analysis after acid hydrolysis and derivatiza-
tion of 1.11 Furthermore, the b anomeric configurations were de-
duced on the basis of their chemical shifts and coupling
constants (glc: J = 7.5 Hz; api: J = br s).9 The linkage positions of
to the aglycone moiety was at C-1. Subsequently, 1H NMR and 13
C
NMR data of 3 were also carefully assigned by HSQC experiment
(Table 1). Based on these information, 3 was established as 1-O-
three sugar moieties were established as b-
(1 ? 2)-[b- -apiofuranosyl-(1 ? 6)]-b- -glucopyranosyl according
to the 13C NMR data shift value (approximately
dC-glc-
6 + 6.0 ppm; dC-glc-2 + 3.0 ppm) and the HMBC experiment, in
D-apiofuranosyl-
D
D
[2,6-O-bis(5-O-vanilloyl-b-
D
-apiofuranosyl)-b-
D
-glucopyranosyl]-
D
3,4,5- trimethoxyphenol.
D
The hepatoprotective activities against
D-galactosamine in-
which the key correlations of C-20 (dc 75.7) with H-100 and C-60 (dc
67.7) with H-10000 were observed (Fig. 2 and Supplementary data).
Moreover, esterifications were formed at position C-api-5 of two
apiofuranosyls due to the changes of chemical shifts here
duced toxicity of compounds 1–3 were examined in WB-F344 cells.
Compounds 1 and 3 showed moderate hepatoprotective activities
(Table 2) compared with the positive control bicyclol at concentra-
tions of 1 ꢂ 10ꢁ5 M.

Z. Feng et al. / Carbohydrate Research 380 (2013) 59–63
61
Table 1
1H and 13C NMR spectroscopic data of compounds 1–3a
Position
1
1
2
3
dH (J in Hz)
dC
23.4
dH (J in Hz)
dC
67.4
dH (J in Hz)
dC
1.01 d (6.0)
3.24 m,
153.6
3.64 m
2
3
4
5
3.82 m
0.95 d (6.0)
69.7
21.3
1.23 m
1.46 m
0.66 d (6.5)
0.65 d (6.5)
38.4
24.8
22.6
22.8
6.21 s
94.0
153.0
132.5
153.0
94.0
99.0
75.9
76.9
70.3
6
6.21 s
10
4.23 d (7.5)
3.18 m
3.32 m
2.97 m
3.63 m
3.83 Overlapped
3.42 m
5.33 br s
99.0
75.7
77.1
70.7
75.2
67.7
4.12 d (7.5)
3.24 m
3.24 m
2.99 m
3.80 Overlapped
3.84 m
3.44 m
5.33 br s
3.80 Overlapped
101.5
76.0
77.6
71.1
75.8
68.1
4.90 d (8.0)
3.34 Overlapped
3.47 m
3.09 m
3.61 m
3.75 Overlapped
3.78 Overlapped
5.39 br s
20
30
40
50
75.2
67.6
60a
60b
100
108.1
77.2
77.2
73.9
108.6
77.6
78.0
74.3
108.7
76.8
77.5
73.9
200
300
3.76 Overlapped
3.86 br s
400a
400b
500a
500b
1000
2000
3000
4000
5000
6000
7000
10000
20000
30000
4.08 d (9.5)
3.72 d (9.5)
4.36 d (10.5)
4.19 d (10.5)
4.03 d (9.5)
3.76 d (9.5)
4.31 d (11.5)
4.19 d (11.5)
4.10 d (9.5)
3.82 d (9.5)
4.21 d (11.5)
4.18 Overlapped
68.0
68.3
66.8
119.5
107.1
147.5
141.0
147.5
107.1
165.5
109.0
76.5
119.5
107.6
148.0
141.4
148.0
107.6
166.0
109.5
77.1
120.1
112.7
147.5
151.0
115.1
123.6
165.3
108.7
76.9
7.22 s
7.23 s
7.34 br s
6.75 d (8.5)
7.37 d (8.5)
7.22 s
7.23 s
4.91 br s
3.76 Overlapped
4.92 d (2.0)
3.80 Overlapped
4.86 d (3.0)
3.74 overlapped
76.8
77.3
77.0
40000
40000
50000
50000
a
b
a
b
3.91 d (9.5)
3.79 Overlapped
4.22 s
73.3
3.92 d (9.5)
3.72 d (9.5)
4.22 s
73.8
3.91 d (9.5)
3.72 d (9.5)
4.18 s
73.3
66.6
67.2
66.2
4.22 s
4.22 s
4.18 s
100000
119.5
107.1
147.5
141.0
147.5
107.1
165.4
119.5
107.5
148.0
141.4
148.0
107.5
165.9
120.1
112.7
147.3
151.0
115.2
123.7
165.5
55.5
200000
7.22 s
7.22 s
7.22 s
7.22 s
7.44 br s
300000
400000
500000
6.85 d (8.5)
7.48 d (8.5)
600000
700000
3/5-OCH3
4-OCH3
3000-OCH3
5000-OCH3
300000-OCH3
500000-OCH3
3.61 s
3.54 s
3.79 s
60.0
55.6
3.80 s
3.80 s
3.80 s
3.80 s
56.1
56.1
56.1
56.1
3.80 s
3.80 s
3.80 s
3.80 s
56.6
56.6
56.6
56.6
3.76 s
55.6
a
NMR data (d) were measured in DMSO-d6 at 500 MHz for 1H NMR, and at 125 MHz for 13C NMR. Coupling constants (J) in Hz are given in parentheses.
a Shimadzu LC-6AD instrument with an SPD-20A detector, using
m). HPLC-DAD
analysis was performed using an Agilent 1200 series system (Agi-
3. Experimental section
a YMC-Pack ODS-A column (250 mm ꢂ 20 mm, 5
l
3.1. General experimental procedures
lent Technologies, Waldbronn, Germany) with an Apollo C18 col-
umn (250 mm ꢂ 4.6 mm,
5
l
m; Grace Davison).
L
-Cysteine
-Api-
The optical rotations were measured on a Jasco P-2000 polarim-
eter. The UV spectra were scanned by a Jasco V650 spectrophotom-
eter. IR spectra were recorded on an IMPACT 400 (KBr)
spectrometer. GC was conducted on an Agilent 7890A instrument.
1H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR spectra were
run on INOVA 500 MHz spectrometer. HRESIMS were performed
on a Finnigan LTQ FT mass spectrometer. The ESI mass spectra
were recorded on an Agilent 1100 series LC/MSD TOF from Agilent
Technologies. Column chromatography was performed with
Macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp. To-
methyl ester hydrochloride, N-trimethylsilylimidazole, and
D
ose (J&K, Beijing, China). Bicyclol (Beijing Union Pharmaceutical
Factory, Beijing, China). WB-F344 cells (Jining Shiye Co., Ltd,
Shanghai, China).
3.2. Plant material
The roots and stems of E. hainanesis were collected in Hainan
Province, People’s Republic of China, in March 2008. The plant
material was identified by Mr. Huanqiang Chen (Jianfengling
National Nature Reserve of Hainan Province). A voucher specimen
(ID-21741) was deposited at the Institute of Materia Medica,
Chinese Academy of Medical Sciences, Beijing 100050, China.
kyo, Japan), Rp-18 (50 lm, YMC, Kyoto, Japan), Sephadex LH-20
(Pharmacia Fine Chemicals, Uppsala, Sweden), and silica gel
(100–200, 200–300 mesh, Qingdao Marine Chemical Inc. Qingdao,
People’s Republic of China). Preparative HPLC was carried out on

62
Z. Feng et al. / Carbohydrate Research 380 (2013) 59–63
OCH3
OCH3
HO
HO
O
O
O
O
O
O
H3CO
H3CO
O
O
O
O
O
O
O
O
OH
OH
OH
OH OH
OCH3
OH OH
OCH3
OH
HO
H3CO
HO
O
O
O
O
H3CO
O
O
OH OH
HO
OH OH
1
2
OCH3
OCH3
OCH3
O
O
O
H3CO
O
O
O
OH
OH OH
OH
HO
H3CO
O
O
O
O
OH OH
3
Figure 2. Selected HMBC correlations of compounds 1, 2, and 3.
Table 2
MeOH–H2O (46:54) as the mobile phase, to afford 2 (10 mg) and 3
(15 mg).
Hepatoprotective effects of compounds 1–3 against D-galactosamine-induced toxicity
in WB-F344 cellsa
Compound
OD means SD
Cell survival rate
(% of normal)
Inhibition
(% of control)
3.4. Identification
Normal
0.430 0.007
0.162 0.004
0.185 0.003
0.189 0.009
0.191 0.019
0.179 0.005
100%
30%
2-O-[2,6-O-Bis(5-O-syringoyl-b-
D
-apiofuranosyl)-b-
D
-glucopyr-
anosyl]-isopropyl alcohol (1): White powder; ½a D20
ꢀ
ꢁ44.2 (c 0.10,
Control(GalN)
Bicyclolb
36%⁄⁄
36%⁄
37%
8.6
10.1
10.8
6.3
MeOH); UV (MeOH) kmax (loge) 280 (4.31) nm; IR mmax 3368,
1
2
3
1701, 1610, 1514, 1462, 1335, 1219, 1114, 763 cmꢁ1 1H NMR
;
(DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see
Table 1; (ꢁ)-ESIMS m/z 845 [MꢁH]ꢁ; (+)-ESIMS m/z 869
[M+Na]+; (+)-HRESIMS m/z 869.2689 (calcd for C37H50O22Na,
869.2686).
34%⁄⁄
The compounds were tested at 1 ꢂ 10ꢁ5 M; results are expressed as means SD
a
(n = 3; for normal and control, n = 6); ⁄p <0.05, ⁄⁄p <0.01.
b
Positive control substance (Beijing Union Pharmaceutical Factory, purity is 98%
by HPLC).
1-O-[2,6-O-Bis(5-O-syringoyl-b-
D-apiofuranosyl)-b-
D
-glucopyr-
anosyl]-isoamyl alcohol (2): White powder; ½a D20
ꢀ
ꢁ54.7 (c 0.08,
MeOH); UV (MeOH) kmax (loge) 278 (4.34) nm; IR mmax 3415,
1701, 1610, 1515, 1461, 1334, 1213, 1111, 762 cmꢁ1 1H NMR
;
3.3. Extraction and isolation
(DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see
Table 1; (ꢁ)-ESIMS m/z 873 [MꢁH]ꢁ; (+)-HRESIMS m/z 897.3018
(calcd for C39H54O22Na, 897.2999).
The dried roots and stems of E. hainanesis (22.5 kg) were
extracted with 90.0 L 95% EtOH under reflux (3 ꢂ 1.5 h). The EtOH
extract was concentrated under reduced pressure to give a residue
(1.3 kg), which was suspended in H2O (7500 mL) with the suspen-
sion sequentially partitioned with petroleum ether (3 ꢂ 6000 mL),
EtOAc (3 ꢂ 6000 mL), and n-BuOH (3 ꢂ 5000 mL), successively.
After evaporation of the solvent under reduced pressure, the n-
BuOH extract (450 g) was subjected to column chromatography
over macroporous resin, eluting successively with H2O, 15% EtOH,
30% EtOH, 50% EtOH, 70% EtOH, and 95% EtOH (20 L each). After re-
moval of solvent, the 50% EtOH fraction (89 g) was applied to a nor-
mal-phase silica gel column. Successive elution of the column with
a gradient of increasing MeOH (0–100%) in CHCl3 afforded five
fractions (A1–A5) on the basis of HPLC-DAD analysis. Fraction A4
(10 g) was subjected to chromatography over Sephadex LH-20
with a gradient of increasing MeOH (15–100%) in H2O as the mo-
bile phase, to give six subfractions (A4-1–A4-6). These subfractions
were separated by reversed-phase preparative HPLC, respectively,
for subfraction A4-4 (200 mg) using MeOH–H2O (48:52) as the mo-
bile phase, to afford 1 (15 mg), for subfraction A4-5 (200 mg) using
1-O-[2,6-O-Bis(5-O-vanilloyl-b-D-apiofuranosyl)-b- -glucopyr-
anosyl]-3,4,5-trimethoxyphenol (3): white powder; ½aDꢀD20 ꢁ68.5 (c
0.13, MeOH); UV (MeOH) kmax (loge) 265 (4.42), 295 (4.30) nm;
IR mmax 3414, 1697, 1597, 1507, 1280, 1219, 1110, 1015, 824,
763 cmꢁ1 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6,
;
125 MHz) data, see Table 1; (ꢁ)-ESIMS m/z 909 [MꢁH]ꢁ; (+)-ESIMS
m/z 933 [M+Na]+; (+)-HRESIMS m/z 933.2629 (calcd for C41H50O23-
Na, 933.2635).
3.5. Acid hydrolysis of the glycosides and determination of the
absolute configuration
Based on the reported method,11 compound 1 (2 mg) was dis-
solved in 1 M HCl 5 mL and heated at 60 °C for 2 h in a water bath.
The mixture was concentrated in a vacuum; the resulting residue
was suspended with H2O and extracted with EtOAc three times.
The aqueous layer was evaporated under a vacuum, diluted repeat-
edly with H2O, and evaporated in a vacuum to produce a neutral

Z. Feng et al. / Carbohydrate Research 380 (2013) 59–63
63
residue. The residue was dissolved in anhydrous pyridine (1 mL).
-Cysteine methyl ester hydrochloride (2 mg) was then added,
3.7. Statistical analysis
L
and the reaction mixture was incubated at 60 °C for 1 h. After the
mixture was concentrated to dryness under a vacuum, 0.5 mL of
N-trimethylsilylimidazole was added, and the mixture was
incubated at 60 °C for 1 h. The reaction mixture was partitioned
betweenn-hexane and H2O (2 mL each). Then-hexane extract was
subjected to GC under the following conditions: capillary column,
Student’s t-test for unpaired observations between normal or
control and tested samples was carried out to identify statistical
differences; p values less than 0.05 were considered as significantly
different.
Acknowledgments
HP-5 (60 m ꢂ 0.25 mm, with a 0.25
lm film, Dikma); detection,
FID; detector temperature, 280 °C; injection temperature, 260 °C;
initial temperature 160 °C, raised to 280 °C at 5 °C/min and final
temperature maintained for 10 min; and carrier N2 gas. Com-
pounds 2 and 3 were operated using the same method. From the
The research described in this publication was supported by the
Research Fund for the Doctoral Program of Higher Education of
China (No. 20111106110031) and National Science and Technology
Project of China (No. 2011ZX09307-002-01).
acid hydrolysates of 1–3, D-glucose and D-apiose were confirmed
by comparing the retention times of their derivatives with those
of authentic sugars, which showed the same retention times,
24.7 and 15.8 min, respectively.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
3.6. Protective effect on cytotoxicity induced by
amine in WB-F344 cells
D-galactos-
j.carres.2013.07.011.
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D
D
medium was changed into a fresh one containing 0.5 mg/mL
MTT. After 3.5 h incubation, the medium was removed and
150 lL of DMSO was added to dissolve formazan crystals. The opti-
cal density (OD) of the formazan solution was measured on a
microplate reader at 492 nm. Inhibition (%) was obtained by the
following formula:
Inhibitionð%Þ ¼ ½ðODðsampleÞ ꢁ ODðcontrolÞÞ=ðODðnormalÞ ꢁ ODðcontrolÞÞꢀ
ꢂ 100