Seven New Acyl Glycosides from Erycibe obtusifolia

Article abstract of DOI:10.1002/hlca.201500143

Seven new acyl glycosides, benzyl 5-O-vanilloyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (1), 4-hydroxy-3-methoxyphenyl 5-O-syringoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (2), isopentyl 5-O-syringoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (3), 3,4,5-trimethoxyphenyl 5-O-sinapoyl-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (4), 6-methoxy-7-[(6-O-sinapoyl-β-d-glucopyranosyl)oxy]coumarin (5), 6-methoxy-7-[(2-O-sinapoyl-β-d-glucopyranosyl)oxy]coumarin (6), and isopentyl β-d-apiofuranosyl-(1→6)-[5-O-syringoyl-β-d-apiofuranosyl-(1→2)]-β-d-glucopyranoside (7), were isolated from Chinese folk herb Erycibe obtusifolia. Their structures were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, MS, and 1D- and 2D-NMR techniques. Further, these compounds were evaluated against HCT-8 (human colon carcinoma), Bel-7402 (human liver carcinoma), BGC-823 (human stomach carcinoma), A549 (human lung carcinoma), and A2780 (human ovarian carcinoma) cell lines, however, none of them exhibited a significant bioactivity (IC₅₀ > 10 μm).

Full text of DOI:10.1002/hlca.201500143

Helv. Chim. Acta 2016, 99, 197 – 203
197
FULL PAPER
Seven New Acyl Glycosides from Erycibe obtusifolia
by Zi-Ming Fenga), Zhao-Zhen Liua)b), Kuo Xua), Ya-Nan Yanga), Jian-Shuang Jianga), and Pei-Cheng Zhang*a)
a) State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P. R. China (phone: +86-10-63165231;
e-mail: pczhang@imm.ac.cn)
b) Beijing Institute for Drug Control, Beijing 100035, P. R. China
Seven new acyl glycosides, benzyl 5-O-vanilloyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyranoside (1), 4-hydroxy-3-
methoxyphenyl 5-O-syringoyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyranoside (2), isopentyl 5-O-syringoyl-b-D-apiofuranosyl-
(1?6)-b-D-glucopyranoside (3), 3,4,5-trimethoxyphenyl 5-O-sinapoyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyranoside (4),
6-methoxy-7-[(6-O-sinapoyl-b-D-glucopyranosyl)oxy]coumarin (5), 6-methoxy-7-[(2-O-sinapoyl-b-D-glucopyranosyl)oxy]coumarin
(6), and isopentyl b-D-apiofuranosyl-(1?6)-[5-O-syringoyl-b-D-apiofuranosyl-(1?2)]-b-D-glucopyranoside (7), were isolated from
Chinese folk herb Erycibe obtusifolia. Their structures were elucidated on the basis of extensive spectroscopic analysis, including
UV, IR, MS, and 1D- and 2D-NMR techniques. Further, these compounds were evaluated against HCT-8 (human colon
carcinoma), Bel-7402 (human liver carcinoma), BGC-823 (human stomach carcinoma), A549 (human lung carcinoma), and
A2780 (human ovarian carcinoma) cell lines, however, none of them exhibited a significant bioactivity (IC50 > 10 lM).
Keywords: Erycibe obtusifolia, Acyl glycosides, Cytotoxic activity.
(human stomach carcinoma), A549 (human lung carci-
noma), and A2780 (human ovarian carcinoma) cell lines.
Introduction
Erycibe obtusifolia BENTH. (Convolvulaceae) is a Chinese
folk herb, which is mainly distributed in Asia and Aus-
Results and Discussion
tralia. In China, its roots and stems are used to relieve
symptoms of rheumatoid arthritis, and to treat some myo-
tic and neural dysfunctions, and other immune-related
diseases [1]. Previous chemical investigation revealed that
the plant contains various compounds including flavo-
Compound 1 was obtained as white powder, [a]D20 = ꢀ71.3
(c = 0.05, MeOH). Its molecular formula was determined
as C26H32O13 based on the pseudomolecular ion in HR-
ESI-MS spectrum (m/z 575.1741 ([M
+
Na]+; calc.
noids, coumarins, chlorogenic acid derivatives, alkaloids, 575.1735)). The IR spectrum suggested the presence of
and monoterpene glycosides [2 – 4]. In our continuing OH (3397 cm–1), C=O (1692 cm–1), and benzene groups
exploration for new bioactive agents from the plant, seven
(1510 cm–1). In the 1H-NMR spectrum, the H-atom sig-
new acyl glycosides were further obtained, including nals at d(H) 7.43 (br. s, 1 H), 6.85 (d, J = 8.0 Hz, 1 H),
benzyl 5-O-vanilloyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyr- 7.48 (d, J = 8.0 Hz, 1 H), and 3.78 (s, 3 H) could be
anoside (1), 4-hydroxy-3-methoxyphenyl 5-O-syringoyl-b- attributed to a vanilloyl moiety in comparison with eryci-
D-apiofuranosyl-(1?6)-b-D-glucopyranoside (2), isopentyl
boside D also isolated from this genus [5][6]. Besides, the
5-O-syringoyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyranoside signals of a monosubstituted benzene ring at d(H) 7.34
(3), 3,4,5-trimethoxyphenyl 5-O-sinapoyl-b-D-apiofuranosyl- (d, J = 7.5 Hz, 2 H), 7.30 (t, J = 7.5 Hz, 2 H), and 7.25
(1?6)-b-D-glucopyranoside (4), 6-methoxy-7-[(6-O-sinapoyl-
(t, J = 7.5 Hz, 1 H) together with a pair of geminal cou-
b-D-glucopyranosyl)oxy]coumarin (5), 6-methoxy-7-[(2- pling H-atom signals at d(H) 4.74 (d, J = 12.0 Hz, 1 H)
O-sinapoyl-b-D-glucopyranosyl)oxy]coumarin (6), and and 4.53 (d, J = 12.0 Hz, 1 H) were observed, implying the
isopentyl b-D-apiofuranosyl-(1?6)-[5-O-syringoyl-b-D-apio-
existence of the benzyloxy moiety. Further, two anomeric
furanosyl-(1?2)]-b-D-glucopyranoside (7) (Fig. 1), and their H-atoms at d(H) 4.20 (d, J = 7.5 Hz, 1 H) and 4.97 (br. s, 1
structures were determined on the basis of chemical and H), and the overlapped signals (d(H) 3.02 – 4.23) indi-
spectroscopic evidence, including UV, IR, MS, and 1D- and cated the presence of two sugar moieties. The 13C-NMR
2D-NMR. Subsequently, the cytotoxicities of these com- spectrum displayed 26 C-atom signals in which the
pounds were evaluated against HCT-8 (human colon carci- benzyloxy moiety was further confirmed based on the sig-
noma), Bel-7402 (human liver carcinoma), BGC-823 nals at d(C) 138.5 (C(1)), 128.3 (C(2)/C(6)), 128.7 (C(3)/C
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
DOI: 10.1002/hlca.201500143

198
Helv. Chim. Acta 2016, 99, 197 – 203
Fig. 1. Structures of isolated compounds 1 – 7
(5)), 127.9 (C(4)), and 70.1 (C(7)). Also, the vanilloyl moi- and benzene groups (1514 cm–1). The molecular formula
ety was determined according to the resonances at d(C) C27H34O16 was deduced from the positive pseudomolecu-
120.8 (C(1000)), 113.3 (C(2000)), 147.4 (C(3000)), 151.8 (C(4000)), lar ion (m/z 637.1735 ([M + Na]+; calc. 637.1739)) in the
115.8 (C(5000)), 124.3 (C(6000)), 166.1 (C(7000)), and 56.2 HR-ESI-MS spectrum. Comparing its NMR data with
(MeO–C(3000)). The remaining 11 C-atom signals indicated those of obtusifoside H from this plant [2], a 6-O-(5-O-sy-
the existence of one hexose and one pentose. D-Apiofura-
nose and D-glucopyranose were determined by NMR data be deduced. In addition, the H-NMR spectrum of 2 pre-
comparison [7], and gas chromatographic analysis after sented set of ABX system signals (d(H) 6.61
hydrolysis and derivatization with L-cysteine methyl (d, J = 2.5 Hz, 1 H), 6.63 (d, J = 8.5 Hz, 1 H), 6.45 (dd,
ester [8]. Furthermore, anomeric chemical shifts and J = 8.5, 2.5 Hz, 1 H)) and an additional MeO resonance
coupling constants (Glc: J = 7.5 Hz; Api: J = br. s) con- at d(H) 3.70 (s, 3 H). Further, the 13C-NMR signals con-
ringoyl-b-apiofuranosyl)-b-glucopyranosyl moiety could
1
a
firmed b-configurations [9]. The interglycosidic linkage was
established as
ranose based on the HMBC correlation H–C(100)/C(60)
(d(C) 67.7) (Fig. 2). In the HMBC spectrum, the correla-
tions H–C(7)/C(10) and H–C(10)/C(7) proved the connectiv-
firmed the aforementioned benzene ring moiety and the
MeO signal (d(C) 55.5). According to the above informa-
tion, the aglycone was determined to be 4-hydroxy-3-
methoxyphenyl [10]. The HMBC correlations (Fig. 2)
from H–C(100) to C(60) and from H–C(500) to C(7000) con-
b-D-apiofuranosyl-(1?6)-b-D-glucopy-
ity between benzyl alcohol and glucose, the correlation of firmed the connectivities between the syringoyl, apiofura-
H–C(500)/C(7000) confirmed the vanilloyl residue to be con-
nosyl, and glucopyranosyl moieties. The correlations from
nected to the position C(500) of apiofuranosyl (Fig. 2). H–C(10) to C(1) and from H–C(2) to C(10) proved the
Thus, the structure of compound 1 was determined to be sugar chain to be attached to position C(1) of the ben-
benzyl 5-O-vanilloyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyr-
anoside.
zene ring. The D-configurations of the sugars were deter-
mined by the same method as for compound 1.
Therefore, the structure of compound 2 was elucidated as
4-hydroxy-3-methoxyphenyl 5-O-syringoyl-b-D-apiofurano-
syl-(1?6)-b-D-glucopyranoside.
Compound 2 was obtained as white powder, [a]D20
=
ꢀ78.5 (c = 0.05, MeOH). The IR spectrum showed
absorptions due to OH (3421 cm–1), C=O (1700 cm–1),
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com

Helv. Chim. Acta 2016, 99, 197 – 203
199
Fig. 2. Selected HMBC correlations of compound 1 – 4
Compound 3 was obtained as white powder, [a]D20
=
(C(1)), 94.3 (C(2/6)), 153.1 (C(3/5)), and 132.5 (C(4)), as
ꢀ67.9 (c = 0.05, MeOH). The IR spectrum suggested the well as three MeO signals at d(C) 55.7 (MeO–C(3)/(5))
existence of OH (3413 cm–1), C=O (1708 cm–1), and ben- and 60.0 (MeO–C(4)) were presented in the 13C-NMR
zene groups (1516 cm–1). The HR-ESI-MS gave the spectrum. According to the above information, the agly-
molecular formula C25H38O14 (m/z 585.2158 ([M + Na]+); cone of compound 4 was confirmed to be 3,4,5-trimethoxy-
calc. 585.2154)). Similar to compound 2, the 6-O-(5-O-sy- phenyl. The HMBC correlations H–C(10)/C(1), H–C(100)/C
ringoyl-b-apiofuranosyl)-b-glucopyranosyl moiety was also
readily deduced by the 1H- and 13C-NMR spectrum
(Table 1). The remaining H-atom signals included two
(60), and H–C(500)/C(9000) (Fig. 2) confirmed the connectiv-
ity between these moieties. The D-configurations of the sug-
ars were determined by the same method as for compound
oxygenated CH2 groups at d(H) 3.85 and 3.43, two CH2 1. Therefore, the structure of compound 4 was identified as
group at d(H) 1.33 (m, H–C(2), 2 H), a CH proton at
d(H) 1.58 – 1.64 (m, H–C(3), 1 H), and two Me groups at
3,4,5-trimethoxyphenyl 5-O-sinapoyl-b-D-apiofuranosyl-(1?
6)-b-D-glucopyranoside.
d(H) 0.81 (d, J = 6.5 Hz, H–C(4),
3
H) and 0.80
Compound 5 was obtained as yellow powder, [a]D20
=
(d, J = 6.5 Hz, H–C(5), 3 H). Taking also into the consid-
eration that the remaining five 13C-NMR signals at d(C)
67.7 (C(1)), 38.0 (C(2)), 24.2 (C(3)), 22.4 (C(4)), and 22.5
ꢀ94.9 (c = 0.05, MeOH). In the UV spectrum, absorption
maxima were observed at 280 and 340 nm. The IR spec-
trum suggested the presence of OH (3386 cm–1), C=O
(C(5)), the aglycone of compound 3 was confirmed as iso- (1702 cm–1), and benzene ring (1515 cm–1). The HR-ESI-
amyl alcohol. In the HMBC experiment, the correlations MS indicated the molecular formula to be C27H28O13 on
of H–C(1) with C(10), H–C(10) with C(1), H–C(60) with
C(100), and H–C(500) with C(7000) confirmed the connectivi-
ties between these moieties (Fig. 2). The D-configurations
of the sugars were determined by the same method as for
the basis of pseudomolecular ion peak (m/z 561.1612
1
([M + H]+; calc. 561.1603)). The H-NMR (Table 2) spec-
trum showed characteristic coumarin resonances at d(H)
6.23 (d, J = 9.5 Hz, 1 H) and 7.90 (d, J = 9.5 Hz, 1 H),
compound 1. Therefore, the structure of compound 3 was while two aromatic singlets at d(H) 7.27 (s, 1 H) and 7.16
determined to be isopentyl 5-O-syringoyl-b-D-apiofurano-
(s, 1 H) suggested that the oxygen-substituted positions
were at C(6) and C(7). In addition, 1H- and 13C-NMR
data (Table 2) displayed the resonances at d(H) 6.91 (s,
H–C(200)/(600)), 6.43 (d, J = 16.0 Hz, H–C(700)), 7.47 00(d,
J = 16.0 Hz, H–C(800)), and 3.77 (s, MeO–C(300)/(5 ));
syl-(1?6)-b-D-glucopyranoside.
Compound 4 was obtained as white powder, [a]D20
=
ꢀ76.1 (c = 0.05, MeOH), the maximum absorptions of
UV spectrum were at 260 and 330 nm. The IR spectrum
gave the absorptions of OH (3418 cm–1), C=O (1695 cm–1), d(C) 124.1 (C(100)), 106.2 (C(200/600)), 148.0 (C(300/500)),
and benzene groups (1507 cm–1). The molecular formula 138.5 (C(400)), 145.7 (C(700)), 114.3 (C(800)), and 166.5
was determined to be C31H40O17 by the HR-ESI-MS (m/z (C(900)), which were corresponding to a sinapoyl moiety.
707.2168 ([M + Na]+; calc. 707.2158)). When comparing The other H-NMR data at d(H) 3.08 – 5.28 including the
1
with obtusifoside E [2], the 1H- and 13C-NMR data anomeric H-atom signals at d(H) 5.19 (d, J = 7.0 Hz,
(Table 1) showed the signals corresponding to 6-O-(5-O- H–C(10)), as well as the 13C-NMR signals at d(C) 99.2
sinapoyl-b-apiofuranosyl)-b-glucopyranosyl. Additionally, a (C(10)), 73.0 (C(20)), 76.4 (C(30)), 70.0 (C(40)), 73.7 (C
pair of aromatic protons (d(H) 7.02 (s, H–C(2)/(6), 2 H))
(50)), and 63.1 (C(60)) implied that 5 contained a glucose
and three MeO signals (d(H) 3.72 (s, MeO–C(3)/(5), 6 H), fragment. The D-configuration of the glucose was deter-
1
3.56 (s, MeO–C(4), 3 H)) were observed in the H-NMR mined by the same method as for compound 1. In the
spectrum. The benzene ring C-atom signals at d(C) 153.9 HMBC experiment (Fig. 3), the correlation of H–
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com

200
Helv. Chim. Acta 2016, 99, 197 – 203
Table 1. 1H- and 13C-NMR Data (500 and 125 MHz, resp.; (D6)DMSO) of 1 – 4. d in ppm, J in Hz
Position
1
2
3
4
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
1
2
138.5
150.7
3.85 – 3.86 (m),
3.43 – 3.44 (m)
1.30 – 1.33 (m),
1.33 – 1.36 (m)
1.58 – 1.64 (m)
0.81 (d, J = 6.5)
0.80 (d, J = 6.5)
67.7
153.9
7.34 (d, J = 7.5)
128.3
6.61 (d, J = 2.5)
102.4
38.0
6.33 (s)
6.33 (s)
94.3
3
4
5
6
7
7.30 (t, J = 7.5)
7.25 (t, J = 7.5)
7.30 (t, J = 7.5)
7.34 (d, J = 7.5)
4.74 (d, J = 12.0),
4.53 (d, J = 12.0)
4.20 (d, J = 7.5)
3.01 (overlap)
3.12 (t, J = 8.5)
3.02 (overlap)
3.25 – 3.26 (m)
3.92 (d, J = 9.5),
3.44 – 3.45 (m)
4.97 (br. s)
128.7
127.9
128.7
128.3
70.1
147.8
141.5
115.3
107.9
24.2
22.4
22.5
153.1
132.5
153.1
94.3
6.63 (d, J = 8.5)
6.45 (d, J = 8.5, 2.5)
10
20
30
40
50
60
102.5
74.0
77.2
70.9
76.2
67.7
5.12 (d, J = 9.0)
3.18 (t, J = 9.0)
3.24 (t, J = 9.0)
3.07 (t, J = 9.5)
3.42 – 3.43 (m)
3.91 (d, J = 9.5),
3.44 – 3.45 (m)
4.89 (br. s)
101.6
73.2
76.5
70.0
75.4
67.7
4.06 (d, J = 7.5)
2.90 – 2.92 (m)
3.07 – 3.11 (m)
2.95 – 2.97 (m)
3.24 (t, J = 8.5)
3.67 – 3.69 (m),
3.38 – 3.39 (m)
4.92 (br. s)
102.7
73.3
76.7
70.3
75.4
66.8
4.80 (d, J = 7.5)
3.20 (t, J = 9.0)
3.27 (t, J = 9.0)
3.08 (t, J = 9.0)
3.50 – 3.51 (m)
3.92 (t, J = 10.0),
3.41 – 3.43 (m)
4.86 (br. s)
100.8
73.1
76.4
70.0
75.4
67.7
100
200
300
400
109.6
77.4
77.7
74.0
108.9
77.1
77.1
73.4
109.0
76.8
77.1
73.3
108.5
76.6
77.0
73.3
3.83 – 3.85 (m)
3.80 (overlap)
3.81 (overlap)
3.70 (overlap)
3.95 (d, J = 9.5),
3.75 (d, J = 9.5)
4.23 (br. s)
3.94 (d, J = 9.5),
3.77 (d, J = 9.5)
4.23 (br. s)
3.93 (d, J = 9.5),
3.77 (d, J = 9.5)
4.22 (br. s)
3.90 (d, J = 9.5),
3.72 (d, J = 9.5)
4.11 (br. s)
500
67.0
120.8
113.3
147.4
151.8
115.8
124.3
166.1
66.6
119.7
107.1
147.8
141.3
147.8
107.1
165.8
66.7
119.0
107.1
147.5
140.1
147.5
107.1
166.5
65.7
124.1
106.3
148.0
138.1
148.0
106.3
145.7
114.6
166.5
55.7
1000
2000
7.43 (br. s)
7.24 (s)
7.24 (s)
3.70 (s)
7.22 (s)
7.02 (s)
3000
4000
5000
6.85 (d, J = 8.0)
7.48 (d, J = 8.0)
6000
7.22 (s)
7.02 (s)
7.57 (d, J = 16.0)
6.52 (d, J = 16.0)
7000
8000
9000
MeO–C(3,5)
MeO–C(40)00
55.5
3.72 (s)
3.56 (s)
3.79 (s)
3.79 (s)
60.0
56.1
MeO–C(3
)
)
3.78 (s)
56.2
3.80 (s)
3.80 (s)
56.2
56.2
3.79 (s)
3.79 (s)
56.1
56.1
MeO–C(5000
56.1
C(60)/C(900) confirmed the sinapoyl to be connected with
(d(H) 3.67 (3 H, s)) and C(7)/H–C(10) confirmed that the
C(60) of the glucose moiety. The correlations of MeO–C MeO group and sugar chain were located at the positions
(6) (d(H) 3.80 (s, 3 H))/C(6), and H–C(10)/C(7) indicated
that the MeO group and glucose were connected on the
positions C(6) and C(7) of the coumarin, respectively.
Thus, compound 5 was elucidated to be 6-methoxy-7-[(6-
O-sinapoyl-b-D-glucopyranosyl)oxy]coumarin.
C(6) and C(7) of coumarin, respectively. Therefore, com-
pound 6 was determined to be 6-methoxy-7-[(2-O-sina-
poyl-b-D-glucopyranosyl)oxy]coumarin.
Compound 7 was obtained as white powder, [a]D20
=
ꢀ53.9 (c = 0.05, MeOH). In the UV spectrum, absorption
Compound 6 was obtained as yellow powder, [a]D20
=
maximum was observed at 278 nm. The IR spectrum
ꢀ40.8 (c = 0.05, MeOH). The molecular formula C27H28O13 showed the absorptions of OH (3393 cm–1), C=O
was determined by the HR-ESI-MS (m/z 561.1602 ([M + H]+; (1692 cm–1), and benzene group (1512 cm–1). The HR-
calc. 561.1603)). Similar to compound 5, the 1H- and ESI-MS gave the pseudomolecular ion (m/z 717.2585
13C-NMR (Table 2) also showed the signals of coumarin,
glucose, and sinapoyl. Careful comparison of the 13C-
NMR data with those of compound 5 revealed that the
resonances of H–C(20) (Dd(H) +1.62) and C(60) (Dd(C)
([M + Na]+; calc. 717.2576)), corresponding to the molec-
ular formula C30H46O18. Comparing the NMR data with
those of 1-O-[2,6-O-bis(5-O-syringoyl-b-D-apiofuranosyl)-
b-D-glucopyranosyl]isoamyl alcohol [11], compound 7 also
ꢀ2.5) were shifted in compound 6, indicating that the showed two sets of apiofuranosyl signals and a set of glu-
attachment position of glucose in 6 was not on position
C(60) but on C(20) of the glucose. The HMBC experiment
(Fig. 3) also supported this conclusion by the correlations of
cose signals (Table 3). The D-configurations of the sugars
were determined by the same method as for compound 1.
The other H-atom signals at d(H) 3.66 – 3.67 (m, H–C(1)),
H–C(20)/C(900). Additional correlations C(6)/MeO–C(6) 3.21 – 3.22 (m, H–C(1)), 1.21 – 1.35 (2 m, H–C(2)), 1.46 –
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com

Helv. Chim. Acta 2016, 99, 197 – 203
201
Table 2. 1H- and 13C-NMR Data (500 and 125 MHz, resp.;
(D6)DMSO) of 5 and 6. d in ppm, J in Hz
Experimental Part
Position
5
6
General
d(H)
d(C) d(H)
d(C)
Column chromatography (CC): macroporous resin (Dia-
ion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan),
Rp-18 (50 lm, YMC, Kyoto, Japan), Sephadex LH-20
(Pharmacia Fine Chemicals, Uppsala, Sweden), silica gel
(200 – 300 mesh, Qingdao Marine Chemical Inc. Qingdao,
P. R. China). Prep. HPLC: Shimadzu LC-6AD instrument
with an SPD-20A detector; YMC-Pack ODS-A column
(250 9 20 mm, 5 lm). HPLC-DAD Analysis: Agilent 1260
series system (Agilent Technologies, Waldbronn, Ger-
many); Apollo C18 column (250 9 4.6 mm, 5 lm, Grace
Davison, IL, USA); D-apiose (International Laboratory,
San Francisco, CA, USA), D-glucose (J&K, Beijing,
China). Optical rotations: JASCO P-2000 polarimeter. UV
Spectra: JASCO V-650 spectrophotometer; kmax (log e) in
2
160.3
160.3
113.9
144.1
110.4
146.3
149.5
104.5
148.8
113.2
98.5
3
6.23 (d, J = 9.5)
7.90 (d, J = 9.5)
7.27 (s)
113.3 6.33 (d, J = 9.5)
144.0 7.93 (d, J = 9.5)
109.7 7.25 (s)
145.9
4
5
6
7
149.5
8
7.16 (s)
102.9 7.24 (s)
148.8
112.3
9
10
10
20
30
40
50
60
5.19 (d, J = 7.0)
3.33 (overlap)
3.33 (overlap)
3.26 – 3.28 (m)
3.77 (overlap)
4.38 (d, J = 11.0)
4.18 – 4.25 (m)
99.2 5.28 (d, J = 8.0)
73.0 4.95 (t, J = 8.0)
76.4 3.52 – 3.54 (m)
70.0 3.26 – 3.30 (m)
73.7 3.54 – 3.56 (m)
63.1 3.74 – 3.76 (m),
3.50 – 3.51 (m)
124.1
73.1
77.5
70.0
74.2
60.6
nm. IR Spectra: Nicolet 5700 spectrometer by a FT-IR
–1
microscope transmission method; v in cm . GC: Agilent
100
124.3
106.2
148.1
138.5
148.1
106.2
~
200
6.91 (s)
106.2 7.00 (s)
148.0
138.5
300
7890A instrument. 1D- and 2D-NMR spectra: INOVA 500
spectrometers. HR-ESI-MS: Agilent 1100 series LC/MSD
ion trap mass spectrometer; in m/z. ESI-MS: Agilent 1100
series LC/MSD TOF (Agilent Technologies); in m/z.
400
500
148.0
106.2 7.00 (s)
600
6.91 (s)
700
7.47 (d, J = 16.0) 145.7 7.55 (d, J = 16.0) 145.5
6.43 (d, J = 16.0) 114.3 6.52 (d, J = 16.0) 115.0
800
900
166.5
56.0 3.67 (s)
56.0 3.77 (s)
165.6
56.0
56.4
Plant Material
MeO–C(6)
MeO–C(300,500) 3.77 (s)
3.80 (s)
The stems of Erycibe obtusifolia BENTH. were collected in
Jianfengling National Nature Reserve of Hainan Pro-
vince, China, in March 2010. The plant material was iden-
tified by Prof. Huanqiang Chen (Jianfengling National
1.54 (m, H–C(3)), 0.71 (d, J = 5.0 Hz, MeO–C(4)), 0.70 Nature Reserve of Hainan Province). A voucher speci-
(d, J = 5.0 Hz, H–C(5)), and C-atom signals at d(C) 67.0 men (ID-21180) was deposited at the Institute of Materia
(C(1)), 38.1 (C(2)), 24.5 (C(3)), 22.5 (C(4)), 22.3 (C(5))
Medica, Chinese Academy of Medical Sciences, Beijing
were attributed to an isoamyloxy residue. The comparison 100050, China.
indicated that only one syringoyl moiety was present in
compound 7. The HMBC correlations (Fig. 3) H–C(500)/C
Extraction and Isolation
(7000) and H–C(100)/C(20) proved that the 5-O-syringoyl-b-
D-apiofuranosyl moiety was located on the position C(20)
of the glucose. The correlation of H–C(1″″)/C(60) proved
a b-D-apiofuranosyl connected on position C(60) of the
The dried stems of Erycibe obtusifolia BENTH. (18.5 kg)
were extracted with 95% EtOH (3 9 30 l, 1.5 h each)
under reflux. The extract was concentrated under reduced
glucose moiety. At the same time, the correlations H–C pressure to give a residue (0.96 kg), which was suspended
(10)/C(1) and H–C(1)/C(10) confirmed the connectivity in H2O (2.3 l) and sequentially partitioned with petro-
between glucose and isoamyl alcohol. Therefore, the struc- leum ether (3 9 2.0 l), AcOEt (3 9 2.0 l), and n-BuOH
ture of compound 7 was identified as isopentyl b-D-apiofura-
nosyl-(1?6)-[5-O-syringoyl-b-D-apiofuranosyl-(1?2)]-b-D-
glucopyranoside.
(3 9 2.0 l). After the solvent was evaporated under
reduced pressure, the n-BuOH extract (400 g) was sub-
jected to CC (macroporous resin HP-20; EtOH/H2O
0:100?95:5). After removing the solvent, the 50% EtOH
The cytotoxicities of these compounds were evaluated
against the A549, Bel7402, BGC-823, HCT-8, and A2780 fraction (50 g) was subjected to CC (Sephadex LH-20;
cell lines. However, they were inactive against these
tumor cell lines (IC50 > 10 lM).
MeOH/H2O 1:10?100:0) to obtain 10 fractions (A1 – A10)
through a HPLC-DAD analysis. Fraction A1 (500 mg)
was subjected to preparative HPLC (YMC-Pack ODS;
The research described in this publication was supported MeOH/H2O 40:60; flow rate 4 ml/min) to give 4 (23 mg;
by the Research Fund for the Doctoral Program of Higher
tR = 39 min), 5 (16 mg; tR = 41 min), and 6 (17 mg; tR
Education of China (No. 20111106110031) and National 35 min). Fraction A5 (300 mg) was further separated by
=
Science and Technology Project of China (No.
2012ZX09301002001003).
preparative HPLC (YMC-Pack ODS; MeOH/H2O 45:55;
flow rate 4 ml/min) to obtain 1 (15 mg; tR = 45 min), 2
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com

202
Helv. Chim. Acta 2016, 99, 197 – 203
Fig. 3. Selected HMBC correlations of compound 5 – 7
Table 3. 1H- and 13C-NMR Data (500 and 125 MHz, resp.; (D6)DMSO) of 7. d in ppm, J in Hz
Position
1
d(H)
d(C)
Position
500
d(H)
d(C)
3.66 – 3.67 (m),
3.21 – 3.22 (m)
1.21 – 1.35 (m)
1.46 – 1.54 (m)
0.71 (d, J = 5.0)
0.70 (d, J = 5.0)
4.14 (d, J = 8.0)
3.22 (overlap)
3.29 – 3.30 (m)
3.17 – 3.19 (m)
3.62 – 3.64 (m)
3.80 – 3.82 (m),
3.37 – 3.40 (m)
5.34 (br. s)
67.0
4.31 (d, J = 11.0),
4.19 (d, J = 11.0)
67.9
2
38.1
24.5
22.5
22.3
101.1
75.4
77.1
70.6
75.1
67.5
1000
119.2
107.3
147.7
141.2
147.7
107.3
165.6
56.2
3
2000
7.23 (s)
4
3000
5
4000
10
20
30
40
50
60
5000
6000
7.23 (s)
7000
MeO–C(3000,5000
)
3.78 (s)
4.84 (br. s)
3.72 – 3.74 (m)
1″″
2″″
3″″
4″″
109.2
75.9
78.8
100
200
300
400
108.1
76.7
77.5
73.9
3.82 (d, J = 9.0),
3.56 (d, J = 9.0)
3.26 – 3.28 (m),
3.32 – 3.35 (m)
73.4
3.78 (overlap)
5″″
63.2
4.03 (d, J = 9.5),
3.81 (d, J = 9.5)
(50 mg; tR = 46 min), and 3 (18 mg; tR = 48 min). The 30%
260 (4.06), 280 (4.16). IR: 3421, 1700, 1615, 1514, 1462,
1
EtOH fraction (200 g) was also applied to CC (Sephadex 1336, 1229, 1115, 1069, 802, 764. H- and 13C-NMR ((D6)
LH-20; MeOH/H2O 1:10?100:0) to yield 20 fractions (B1 –
DMSO): Table 1. ESI-MS (pos.): 614 ([M + Na]+). HR-
B20) through a HPLC-DAD analysis. Fraction B12 (3 g) ESI-MS (pos.): 637.1735 ([M + Na]+, C27H34NaO+16; calc.
was subjected to CC (Sephadex LH-20; MeOH/H2O 637.1739).
0:100?100:0), resulting in four subfractions (B12-1 – B12-4).
Using the mobile phase (MeOH/H2O 45:55), subfraction
B12-3 was separated by preparative HPLC (YMC-Pack
Isopentyl 5-O-Syringoyl-b-D-apiofuranosyl-(1 ? 6)-b-
D-glucopyranoside (= 3-Methylbutyl 5-O-(4-Hydroxy-3,5-
dimethoxybenzoyl)-b-D-apiofuranosyl-(1 ? 6)-b-D-gluco-
ODS; MeOH/H2O 35:65; flow rate 4 ml/min) to yield 7 pyranoside; 3). White powder. [a]2D0 = –67.9 (c = 0.05,
(25 mg; tR = 45 min).
MeOH). UV (MeOH): 277 (4.07). IR: 3413, 1708, 1612,
1516, 1463, 1336, 1220, 1115, 764. 1H- and 13C-NMR
Benzyl 5-O-Vanilloyl-b-D-apiofuranosyl-(1 ? 6)-b-D-
glucopyranoside (= Benzyl 5-O-(4-Hydroxy-3-methoxy- ((D6)DMSO): Table 1. HR-ESI-MS (pos.): 585.2158 ([M
benzoyl)-b-D-apiofuranosyl-(1 ? 6)-b-D-glucopyranoside;
1). White powder. [a]2D0 = –71.3 (c = 0.05, MeOH). UV
(MeOH): 260 (4.08), 290 (4.02). IR: 3397, 1692, 1595, 1510,
1342, 1283, 1109, 827, 766. 1H- and 13C-NMR ((D6)
DMSO): Table 1. HR-ESI-MS (pos.): 575.1741 ([M + Na]+,
C26H32NaO+13; calc. 575.1735).
+ Na]+, C25H38NaO+14; calc. 585.2154).
3,4,5-Trimethoxyphenyl 5-O-Sinapoyl-b-D-apiofurano-
syl-(1 ? 6)-b-D-glucopyranoside (= 3,4,5-Trimethoxyphe-
nyl 5-O-[(2E)-3-(4-Hydroxy-3,5-dimethoxyphenyl)prop-2-
enoyl]-b-D-apiofuranosyl-(1 ? 6)-b-D-glucopyranoside; 4).
Yellow powder. [a]2D0 = –76.1 (c = 0.05, MeOH). UV
(MeOH): 260 (4.06), 330 (3.85). IR: 3418, 1695, 1602,
1507, 1461, 1256, 1120, 832. 1H- and 13C-NMR ((D6)
4-Hydroxy-3-methoxyphenyl 5-O-Syringoyl-b-D-apio-
furanosyl-(1 ? 6)-b-D-glucopyranoside (= 4-Hydroxy-3-
methoxyphenyl 5-O-(4-Hydroxy-3,5-dimethoxybenzoyl)-b- DMSO): Table 1. ESI-MS (pos.): 707 ([M + Na]+). HR-
D-apiofuranosyl-(1 ? 6)-b-D-glucopyranoside; 2). White
ESI-MS (pos.): 707.2168 ([M + Na]+, C31H40NaO+17; calc.
powder. [a]2D0 = –78.5 (c = 0.05, MeOH). UV (MeOH): 707.2158).
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com

Helv. Chim. Acta 2016, 99, 197 – 203
203
6-Methoxy-7-[(6-O-sinapoyl-b-D-glucopyranosyl)oxy]
coumarin (= 7-({6-O-[(2E)-3-(4-Hydroxy-3,5-dimethoxy- column, (60 m 9 0.25 mm, with a 0.25 lm film, Dikma);
phenyl)prop-2-enoyl]-b-D-glucopyranosyl}oxy)-6-methoxy- detection, FID; detector temperature, 280 °C; injection
2H-1-benzopyran-2-one; 5). Yellow powder. [a]2D0 = –94.9 temperature, 260 °C; initial temperature 160 °C, raised to
extract was subjected to GC analysis on a capillary HP-5
(c = 0.05, MeOH). UV (MeOH): 280, 340. UV (MeOH):
280 (4.22), 340 (4.75). IR: 3386, 1702, 1616, 1567, 1515,
280 °C at 5 °C/min and final temperature maintained for
10 min; and carrier N2 gas. D-Glucose and D-apiose were
1461, 1281, 1116, 826. 1H- and 13C-NMR ((D6)DMSO): confirmed in the hydrolysates by comparing their reten-
Table 2. ESI-MS (pos.): 561 ([M + Na]+). HR-ESI-MS
(pos.): 561.1612 ([M + H]+, C27H29O1+3; calc. 561.1603).
6-Methoxy-7-[(2-O-sinapoyl-b-D-glucopyranosyl)oxy]
coumarin (= 7-({2-O-[(2E)-3-(4-Hydroxy-3,5-dimethoxy-
phenyl)prop-2-enoyl]-b-D-glucopyranosyl}oxy)-6-methoxy-
tion times with those of authentic sugars derivatized fol-
lowing the same procedure.
Cytotoxicity Assay
2H-1-benzopyran-2-one; 6). Yellow powder. [a]2D0 = –40.8 Compounds 1 – 7 were tested for cytotoxicity against
(c = 0.05, MeOH). UV (MeOH): 280, 340. UV (MeOH): HCT-8 (human colon carcinoma), Bel-7402 (human liver
280 (4.25), 340 (4.76). IR: 3390, 1712, 1611, 1565, 1514, carcinoma), BGC-823 (human stomach carcinoma), A549
1460, 1280, 1118, 827. 1H- and 13C-NMR ((D6)DMSO): (human lung carcinoma), and A2780 (human ovarian car-
Table 2. ESI-MS (pos.): 561 ([M + H]+). HR-ESI-MS cinoma) by means of an MTT method described in the
(pos.): 561.1602 ([M + H]+, C27H29O1+3; calc. 561.1603).
Isopentyl b-D-Apiofuranosyl-(1 ? 6)-[5-O-syringoyl-b-D-
apiofuranosyl-(1 ? 2)]-b-D-glucopyranoside (= 3-Methylbutyl
b-D-Apiofuranosyl-(1 ? 6)-[5-O-(4-hydroxy-3,5-dimethoxy-
benzoyl)-b-D-apiofuranosyl-(1 ? 2)]-b-D-galactopyranoside;
7). White powder. [a]D20 = –53.9 (c = 0.05, MeOH). UV
(MeOH): 278 (4.09). IR: 3393, 2953, 1692, 1594, 1512, 1464,
literature [12].
REFERENCES
[1] H.-Y. Hsu, C.-C. Lin, J.-Y. Chen, J.-J. Yang, R. Zhang,
J. Ethnopharmacol. 1998, 62, 101.
[2] Z.-Z. Liu, Z.-L. Zhan, F. Liu, Y.-N. Yang, Z.-M. Feng,
J.-S. Jiang, P.-C. Zhang, Carbohydr. Res. 2013, 372, 47.
[3] Z. Liu, Z. Feng, Y. Yang, J. Jiang, P. Zhang, Fitoterapia 2014,
99, 109.
1
1286, 1224, 764. H- and 13C-NMR ((D6)DMSO): Table 3.
HR-ESI-MS (pos.): 717.2585 ([M + Na]+, C30H46NaO1+8; calc.
717.2576).
[4] T. Morikawa, F. Xu, H. Matsuda, M. Yoshikawa, Chem. Pharm.
Bull. 2006, 54, 1530.
[5] J. Wang, Y. Di, X. Yang, S. Li, Y. Wang, X. Hao, Phytochem-
istry 2006, 67, 486.
Acid Hydrolysis and Sugar Analysis
[6] S. Song, Y. Li, Z. Feng, J. Jiang, P. Zhang, J. Nat. Prod. 2010,
73, 177.
The procedure previously reported was used [11], com-
pounds 1 – 7 (2 mg each) were dissolved in 1M HCl 5 ml
and heated at 60 °C for 2 h in a H2O bath, respectively.
The mixture was concentrated under reduced pressure.
The residue was suspended in H2O and extracted with
EtOAc. The aqueous layer was evaporated, repeatedly
diluted with H2O and evaporated to give a neutral resi-
due. The residue was dissolved in anhydrous pyridine
(1 ml). L-Cysteine methyl ester hydrochloride (2 mg) was
then added and the mixture was incubated at 60 °C for
1 h. After evaporation to dryness, 0.5 ml of N-trimethylsi-
lylimidazole was added, and the mixture was further
incubated at 60 °C for 1 h. The products were partitioned
between n-hexane and H2O (2 ml each). The n-hexane
[7] S. H. Kim, J. H. Park, T. B. Kim, H. H. Lee, K. Y. Lee,
Y. C. Kim, S. H. Sung, Bioorg. Med. Chem. Lett. 2010, 20, 2824.
[8] S. Hara, H. Okabe, K. Mihashi, Chem. Pharm. Bull. 1987, 35,
501.
ꢀ
[9] E. Smite, L. N. Lundgren, R. Andersson, Phytochemistry 1993,
32, 365.
[10] M. J. Jung, S. S. Kang, Y. J. Jung, J. S. Choi, Chem. Pharm.
Bull. 2004, 52, 1501.
[11] Z.-M. Feng, S. Song, J. He, Y.-N. Yang, J.-S. Jiang,
P.-C. Zhang, Carbohydr. Res. 2013, 380, 59.
[12] Z.-F. Zheng, J.-F. Xu, Z.-M. Feng, P.-C. Zhang, J. Asian Nat.
Prod. Res. 2008, 10, 833.
Received June 8, 2015
Accepted January 5, 2016
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.helv.wiley.com