Two new glycosides and one new neolignan from the roots of Cynanchum stauntonii

Article abstract of DOI:10.1016/j.phytol.2015.07.015

Three new compounds including one C₂₁-steroidal glycoside, one methylglycoside, and one neolignan, named as Deoxyamplexicogenin A-3-O-yl-4-O-(4-O-α-L-cymaropyranosoyl-β-D-digitoxopyranosoyl)-β-D-canaropyranoside (1), Methyl-O-α-L-cymaropyranosoyl-(1 → 4)-β-D-digitoxopyranoside (2), and (+)-(7S, 8R, 7′E)-5-hydroxy-3, 5′-dimethoxy-4′, 7-epoxy-8, 3′-neolign-7′-ene-9, 9′-diol 9′-ethyl ether (3), respectively, were isolated from the roots of Cynanchum stauntonii. The structure elucidations were achieved by in-depth spectroscopic examination, mainly including the experiments and analyses of multiple 1D- and 2D-NMR and HRESIMS and CD analysis and qualitative chemical tests. Cytotoxicity activities of compounds 1-3 were evaluated against five tumor cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) in cell based assays where they were found to be inactive.

Full text of DOI:10.1016/j.phytol.2015.07.015

Phytochemistry Letters 13 (2015) 355–359
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Two new glycosides and one new neolignan from the roots of
Cynanchum stauntonii
b
Jin-Qian Yu a, , Lei Zhao
*
a Shandong Analysis and Test Center, Shandong Academy of Sciences, Jinan 250014, P. R. China
b Reyoung Pharmaceutical Co., Ltd, No. 44 Cultural West Road, Jinan 250012, P. R. China
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new compounds including one C21-steroidal glycoside, one methylglycoside, and one neolignan,
Received 3 April 2015
Received in revised form 7 July 2015
Accepted 10 July 2015
Available online
named as Deoxyamplexicogenin A-3-O-yl-4-O-(4-O-
a
-
L
-cymaropyranosoyl-
b-D-digitoxopyranosoyl)-
b
-
D
-canaropyranoside (1), Methyl-O-
a
-
L
-cymaropyranosoyl-(1 ! 4)- -D-digitoxopyranoside (2), and
b
(+)-(7S, 8R, 70E)-5-hydroxy-3, 50-dimethoxy-40, 7-epoxy-8, 30-neolign-70-ene-9, 90-diol 90-ethyl ether (3),
respectively, were isolated from the roots of Cynanchum stauntonii. The structure elucidations were
achieved by in-depth spectroscopic examination, mainly including the experiments and analyses of
multiple 1D- and 2D-NMR and HRESIMS and CD analysis and qualitative chemical tests. Cytotoxicity
activities of compounds 1–3 were evaluated against five tumor cell lines (HCT-8, Bel-7402, BGC-823,
A549, and A2780) in cell based assays where they were found to be inactive.
Keywords:
Cynanchum stauntonii
Asclepiadaceae
Steroidal glycoside
Methylglycoside
Neolignan
ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
C21-steroids and in continuation of our interest in the chemistry of
genus Cynanchum, a detailed investigation of this species was
carried out. One C21-steroidal glycoside (1), one methylglycoside
(2), and one neolignan (3), were isolated from the roots of C.
stauntonii. In this article, cytotoxicity activities of compounds 1–3
were evaluated against five tumor cell lines (HCT-8, Bel-7402, BGC-
823, A549, and A2780) by using the MTT in vitro assay, but with
none being found to be active. Described herein is a full account of
the isolation and structural elucidation of these new compounds,
as well as the cytotoxic activities of the compounds tested.
Cynanchum stauntonii (Decne.) Schltr. ex Levl. (Asclepiadaceae),
known as a perennial medicinal herb, distributes broadly in south-
central region of China. The roots, along with another species of the
same genus, C. glaucescens (Decne.) Hand.-Mazz., have been used
widely in traditional Chinese medicine (TCM) as antitussive and
expectorant agent (China National Corp. of Traditional & Herbal
Medicine, 1995). Both of them are documented in the Chinese
Pharmacopoeia as ‘Bai Qian’. Previous investigations of phyto-
chemistry on this title plant have indicated the main native organic
compounds isolated from Cynanchum species are steroidal glyco-
sides, sharing aglycones assignable to either the normal four-ring
C21 steroid skeleton or the aberrant 13,14:14,15-disecopregnane-
type skeleton or the equally abnormal 14,15-secopregnane-type
skeleton (Qiu et al., 1989; Sugama et al., 1986; Yu et al., 2013).
These steroidal glycosides have been accepted, generally and
chemotaxonomically, as the most effective and characteristic
ingredients existing in the Cynanchum species. The potential
medicinal importance and our interest in the chemistry of
structurally unique natural products prompted us to investigate
the roots of C. stauntonii. In order to find more structurally unique
2. Experimental
2.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241 digital
polarimeter at 20 8C. IR spectra were recorded on a Nicolet
5700 spectrometer. CD spectrum was obtained using a Jasco J715
spectropolarimeter. 1D and 2D NMR spectra were taken on either a
Bruker 500 spectrometer or a Varian 500 spectrometer with
tetramethylsilane as internal standard. ESI-MS and HRESI-MS were
obtained using an Agilent 1100 series LC/MSD Trap SL mass
spectrometer. Preparative HPLC was performed on a Shimadzu LC-
6AD system equipped with a SPD-10A detector, and a reversed-
phase C18 column (YMC-Pack ODS-A U 20 ꢀ 250 mm, 10
mm) was
*
Corresponding author. Tel.: +86 0531 82605319; fax: +86 531 8296 4889.
employed. GC analyses were conducted on an Agilent 7890A
instrument. Column chromatography (CC) was undertaken over
E-mail addresses: yujinqian87528@126.com (J.-Q. Yu),
zhaolei2008boy@163.com (L. Zhao).
http://dx.doi.org/10.1016/j.phytol.2015.07.015
1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

356
J.-Q. Yu, L. Zhao / Phytochemistry Letters 13 (2015) 355–359
silica gel (200–300 mesh). TLC was carried out with glass plate
precoated silica gel G. Spots were visualized under UV light and by
spraying with 10% H2SO4 in 95% EtOH, followed by heating at
100 8C. Acetonitrile and methanol used in preparative HPLC
procedure were in HPLC grade, and other solvents were of
analytical grade.
2.3.2. Methyl-O-
a
-
L
-cymaropyranosoyl-(1 ! 4)-
b
-D-
digitoxopyranoside (2)
White amorphous powder; ½a D20
ꢂ
-11.6 (c = 0.12, CH3OH, 20 8C);
IR (KBr) n ;
max: 3395, 2924, 2850, 1451, 1379, 1158, 1060, 992 cmꢁ1
ESI-MS (positive mode) m/z: 329.5 [M + Na]+; 1H NMR (500 MHz,
pyridine-d5): dH 5.41 (dd, J = 10.2, 1.8 Hz, H-10), 5.06 (br s, H-100),
4.46 (2H, overlapped, H-30, 50), 4.10 (m, H-500), 3.70 (m, H-300), 3.60
(m, H-400), 3.54 (s, OMe-10), 3.38 (s, OMe-300), 3.41 (m, H-40), 2.38
(m, Ha-20), 2.35 (m, Ha-200), 1.91 (m, Hb-20), 1.82 (m, Hb-200), 1.43 (d,
J = 6.6 Hz, H-60), 1.36 (d, J = 5.4 Hz, H-600); 13C NMR (125 MHz,
2.2. Plant material
The roots of C. stauntonii were collected from Hebei Province of
China. It was identified by associate Prof. L Ma from Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College. A voucher specimen (ID-S-2426) was
deposited in the Herbarium of Institute of Materia Medica, Chinese
Academy of Medical Sciences, Beijing, P. R. China.
pyridine-d5): d
C 98.6 (C-10, d), 98.4 (C-100), 80.9 (C-40, d), 76.6 (C-300,
d), 72.8 (C-400, d), 69.2 (C-50, d), 67.9 (C-500, d), 67.2 (C-30, d), 57.5
(OMe-10, q), 56.8 (OMe-300, q), 38.6 (C-20, t), 32.3 (C-200, t), 18.6 (C-
600, q), 18.4 (C-60, q); HRESI-MS (positive mode) m/z:
329.4577 [M + Na]+ (calcd for C14H26O7Na, 329.4575).
2.3. Extraction and isolation
2.3.3. (+)-(7S, 8R, 7E0)-5-hydroxy-3, 50-dimrthoxy-40, 7-epoxy-8, 30-
neolign-70-ene-9, 90-diol 90-ethyl ether(3)
The dried-up and powdered roots of C. stauntonii (30 Kg) were
extracted three times under reflux with 95% EtOH (2 h, 1 h, 1 h).
The EtOH extract was evaporated under reduced pressure to yield a
dark-brown residue (5000 g). The residue was suspended in 80%
aqueous ethanol (10,000 mL) and then extracted with petroleum
ether, and EtOAc, respectively and successively, each for three
times in separatory funnel. The combined EtOAc solution was
washed with 5% aqueous solution of NaHCO3 (3 ꢀ 1000 mL) and
then H2O (2 ꢀ 1000 mL), respectively, to pH 7. After removal of the
organic solvent, 190 g of brown residue was obtained. This
resulting residue was fractionated by CC over silica gel eluted
with gradient solvents of CHCl3-MeOH (100:0 ! 1:1) to yield
13 fractions (designated as F1 to F13) based on their TLC analysis.
Fraction 3 (24.0 g) was applied to Flash C18 column chromatogra-
phy eluted with CH3OH/H2O (40% ! 100%) to give six subfractions
(F3-1 to F3-6). Fraction 3-4 (2.0 g) was applied to Flash C18 column
chromatography eluted with CH3OH/H2O (40% ! 100%) to give
five subfractions (F3-4-1 to F3-4-5). Fraction 3-4-5 (100 mg) was
applied to preparative HPLC system [mobile phase: CH3OH/H2O
(75:25, v/v); flow rate: 5 mL minꢁ1; UV detection at 210 nm]
resulting in the isolation of compound 2 (35 mg). Fraction 5
(12.0 g) was applied to Flash C18 column chromatography eluted
with CH3OH/H2O (40% ! 100%) to give five subfractions (F5-1 to
F5-5). Fraction 5-2 (2.5 g) was applied to Flash C18 column
chromatography eluted with CH3OH/H2O (40% ! 100%) to give
five subfractions (F5-2-1 to F5-2-5). Fraction 5-2-3 (400 mg) was
applied to preparative HPLC system [mobile phase: CH3CN/H2O
(45:55, v/v); flow rate: 5 mL minꢁ1; UV detection at 280 nm]
resulting in the isolation of compound 1 (80 mg). Fraction 6 (7.0 g)
was further separated by CC over silica gel using gradient solvents
of petroleum ether/acetone (25:1 ! 1:1) as eluents to yield four
further subfractions (F6-1 to F6-7). Fraction 6-3 (5.0 g) was applied
to Flash C18 column chromatography eluted with CH3OH/H2O
(40% ! 100%) to give five subfractions (F6-3-1 to F6-3-5). Fraction
6-3-3 (90 mg) was applied to preparative HPLC system [mobile
Brown jelly; ½a 2D0
ꢂ
+ 1.4 (c = 0.10, CH3OH, 20 8C); IR (KBr) nmax:
3285, 2939, 1646, 1534, 1447, 1400, 1239, 1076 cmꢁ1; ESI-MS
(positive mode) m/z: 409.1 [M + Na]+; 1H NMR (400 MHz, DMSO-
d6): 6.96 (br s, H-4), 6.95 (br s, H-20), 6.89 (br s, H-2), 6.75 (d,
J = 1.2 Hz, H-60), 6.74 (d, J = 1.2 Hz, H-6), 6.50 (d, J = 16.0 Hz, H-70),
6.19 (dt, J = 16.0, 6.0 Hz, H-80), 5.45 (d, J = 6.6 Hz, H-7), 4.03 (dd,
J = 6.0, 1.2 Hz, H-90), 3.78 (s, OMe-50), 3.73 (s, OMe-3), 3.70 (m, Hb-
9), 3.62 (m, Ha-9), 3.44 (dd, J = 14.0, 7.2 Hz, H-110), 3.43 (m, H-8),
1.13 (t, J = 6.0 Hz, H-120); 13C NMR (100 MHz, DMSO-d6): 147.5 (C-
3, s), 147.4 (C-40, s), 146.4 (C-5, s), 143.7 (C-50, s), 132.3 (C-1, s),
131.6 (C-70, d), 130.0 (C-10, s), 129.5 (C-30, s), 124.0 (C-80, d), 118.5
(C-6, d), 115.3 (C-4, d), 115.3 (C-60, d), 110.4 (C-20, d), 110.2 (C-2, d),
87.3 (C-7, d), 70.4 (C-90, t), 64.6 (C-110, t), 62.9 (C-9, t), 55.7 (OMe-3,
t), 55.6 (OMe-50, q), 53.0 (C-8, d), 15.2 (C-120, q); HRESI-MS
(positive mode) m/z: 409.1631 [M + Na]+ (calcd for C22H26O6Na,
409.1622).
2.4. Acid hydrolysis of 1 and 2 and determination of the absolute
configurations of the monosaccharides
Each solution of 6 mg of compounds 1 and 2 was refluxed
within 10% HCl (3 ml) at 75 8C for 2.5 h. After cooling, the reaction
mixture was extracted thoroughly with CHCl3, the CHCl3 layer was
washed with water, and then the water fraction was combined
with the original aqueous layer. The aqueous layer was evaporated
under vacuum, then re-diluted with water and re-evaporated in
vacuo repeatedly to eliminate the surplus HCl and furnish a final
neutral residue. The monosaccharides obtained on acid hydrolysis,
as described above, were dissolved in pyridine and reacted with L-
cysteine methyl ester hydrochloride at 60 8C for 1 h. Equal volume
of acetic anhydride was added and heating was carried out for
another 1 h. Acetylated thiazolidine derivatives were injected into
GC system. The absolute configurations of the sugars were
determined by comparing the retention times with those of
acetylated thiazolidine derivatives synthesized from the known
sugars obtained through acid hydrolysis of the known compound
phase: CH3CN/H2O (38:62, v/v); flow rate: 5 mL minꢁ1
; UV
detection at 210 nm] resulting in the isolation of compound 3
(12 mg).
Glaucogenin-C-3-O-
a
b
-
-
L
-cymaropyranosoyl- (1 ! 4)-
-canaropyranoside (Fu et al., 2007). Also,
-canarose, -digitoxose, and -cymarose
b-D-digitoxo-
pyranosoyl-(1 ! 4)-
D
the retention times of
D
D
L
2.3.1. Deoxyamplexicogenin A-3-O-yl-4-O-(4-O-
a
-
-
L
-
were determined by interactive comparison among the known
compound Glaucogenin-C-3-O-
- cymaropyranosoyl-(1 ! 4)-
-digitoxopyranosoyl-(1 ! 4)- -canaropyranoside. GC condi-
m;
detection, FID; carrier gas, N2; injection temperature, 250 8C,
detection temperature, 280 8C, column temperature, 150 8C
(0 min), 10 8C/min to 250 8C (20 min). Retention times of the
cymaropyranosoyl- - digitoxopyranosoyl)-
b
-
D
b
-D
a
b-D
-
L
b-
canaropyranoside(1)
D
White amorphous powder; ½a D20
ꢂ
+ 2.21 (c = 0.09, CH3OH, 20 8C);
tions in the test: column, HP-5, 30 m ꢀ 0.25 mm, 0.25
m
IR (KBr)
n
max: 3432, 2934, 1679, 1452, 1379, 1163, 1063, 870 cmꢁ1
;
ESI-MS (positive mode) m/z: 769.4 [M + Na]+; 1H NMR (500 MHz,
pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) for aglycone and
sugar moiety, see Table 1; HRESI-MS (positive mode) m/z:
769.3775 [M + Na]+ (calcd for C40H58O13Na, 769.3770).
authentic samples: tR
D-digitoxose 13.09 min, tR L-cymarose
13.46 min, and tR -canarose 16.51 min).
D

J.-Q. Yu, L. Zhao / Phytochemistry Letters 13 (2015) 355–359
357
3. Results and discussion
categorized (Table 1). The above mentioned three olefin methine
carbons were corresponded to a 4,6-diene system at H 5.73 (1H, s,
d
Compound 1 was obtained as a white amorphous powder with
H-4), 6.03 (1H, d, J = 9.5 Hz, H-6) and 6.69 (1H, d, J = 9.5 Hz, H-7) by
the HSQC and HMBC spectra, which also provided solid evidence to
unambiguously assign all signals of 1. The 1H and 13C NMR
resonances from the aglycone moiety of 1 were very superimpos-
able to its counterparts in stauntoside C, a known steroidal
glycoside isolated previously from C. stauntoii (Yu et al., 2013),
confirming the presence of the same aglycone between 1 and
stauntoside C, and demonstrating the attachment of an oligosac-
charide chain to C-3 of the aglycone of 1 through oxygen atom.
Then, in order to determine the sugar moiety, acid hydrolysis of 1
½
a 2D0
+ 2.21 (c = 0.09, CH3OH, 20 8C) and showed positive
ꢂ
Libermann–Burchard and Keller–Kiliani reactions, suggesting its
glycosidic steroidal category with 2-deoxysugar units existing in
the sugar moiety (Chen et al., 2008). The positive-ion mode
HRESIMS experiment gave a [M + Na]+ quasi-molecular ion peak at
m/z 769.3775, consistent with the molecular formula of C40H58O13
and indicative of twelve unsaturation degrees. The IR spectrum
showed the absorption bands for hydroxy (3432 cmꢁ1) and olefin
(1679 cmꢁ1) functionalities. The 1H NMR spectrum, in conjunction
with the 1H, 1H-COSY and HSQC experiments, revealed the
diagnostic signals of a C21-steroidal glycoside, with an aglycone
of 14,15-secopregnane-type skeleton being exhibited typically by
two angular methyl groups actually at dH 0.83 (3H, s, H-19) and
was carried out and one
D-canaropyranose, one D-digitoxopyr-
anose, and one -cymaropyranose showing up the proportion of
L
1:1:1 were detected through derivatization reaction and GC
analysis (Section 2). The 1H, 1H-COSY experiment, in combination
with the HSQC spectrum, established the spin systems within the
sugar moiety which were compatible with the identified one
methylated 2,6-dideoxypyranose and two 2,6-dideoxypyranoses.
The coupling states of the aforementioned three anomeric proton
1.57 (3H, s, H-21), two oxy-methine groups at
and 4.80 (1H, m, H-16), and two oxy-methylenes at
J = 11.0, 4.2 Hz, H -15) and 4.28 (1H, br d, J = 11.0 Hz, H -15), and
d
H 4.53 (1H, m, H-3)
d
H 3.82 (1H, dd,
b
a
at dH 4.01 (1H, d, J = 8.5 Hz, H-18a) and 4.06 (1H, d, J = 8.5 Hz, H-
18b), as well as three anomeric proton signals at dH 4.91 (1H, d,
J = 10.0 Hz, H-10), 5.26 (1H, d, J = 10.0 Hz, H-100), and 5.04 (1H, br s,
H-1000) correlating to the corresponding anomeric carbon signals at
signals delivered the evidences that the
digitoxopyranose were -linkages and the
-linkage. The sugar chain of 1 was substantiated unambiguously
by the HMBC correlations from H-1000 of
-cymaropyranose to C-
400 of -digitoxopyranose, from H-100 of
-digitoxopyranose to
C-40 of -canaropyranose, and from H-10 of
-canaropyranose
to C-3. Therefore, compound 1 was elucidated unequivocally as
Deoxyamplexicogenin A-3-O-yl-4-O-(4-O- -cymaropyranosoyl-
-digitoxopyranosoyl)- -canaropyranoside.
Compound 2 was obtained as a white amorphous powder with
-11.6 (c = 0.12, CH3OH, 20 8C). The pseudo-molecular ion peak
D-canaropyranose and
D-
b
L-cymaropyranose was
a
d
C 99.0 (C-10), 99.9 (C-100), and 98.6 (C-1000), respectively, and three
a-L
secondary methyls at dH 1.36 (3H, d, J = 6.0 Hz, H-60), 1.31 (3H, d,
J = 6.0 Hz, H-600), and 1.42 (3H, d, J = 6.0 Hz, H-6000) (Table 1), which
combined with the 1H, 1H-COSY and HSQC spectra suggested the
presence of three 2,6-dideoxypyranoses (Fig. 1). In addition, one
b
-D
b-D
b-
D
b-D
a-L
methoxyl at
d
H 3.36 (3H, s, OMe-3000) was also determined in the 1H
b-
D
b-D
NMR spectrum, which, along with the above mentioned three
secondary methyls, was compatible with one methylated 2,6-
dideoxypyranose among the aforementioned three 2,6-dideoxy-
pyranoses when examining the 13C and DEPT NMR data of 1 that
exhibited forty carbon signals, with six methyls, nine methylenes,
nineteen methines including three olefinic ones at dC 125.7 (d),
124.8 (d), and 122.8 (d), and six quaternary carbons being
½ ꢂ
a 2D0
at m/z 329.4577 [M + Na]+ in the positive-ion mode HRESIMS
experiment indicated the molecular formula of C14H26O7 with two
unsaturation degrees. The IR spectrum showed the absorption
band for hydroxy (3395 cmꢁ1) group. The 1H NMR spectrum of 2
displayed the diagnostic signals of two 2,6-dideoxypyranoses
Table 1
1H and 13C NMR data of the aglycone and sugar moieties for 1 (500/125 MHz, in pyridine-d5) (
d
in ppm, J values in Hz).
Aglycone moiety
1
Sugar moiety
1
dH
dC
dH
dC
1
1
2
2
3
4
5
6
7
8
9
a
b
a
b
1.33 ma
1.53 ma
1.92 m
2.24 m
4.53 m
5.73 s
33.6, t
b-D-can
10
20
30
40
50
60
4.91 d (10.0)
2.55 m, 2.01 m
3.98 m
99.0, d
40.1, t
70.1, d
88.6, d
71.0, d
18.1, q
27.7, t
74.7, d
124.8, d
144.9, s
125.7, d
122.8, d
108.1, s
44.2, d
35.6, s
3.30 m
3.54 m
1.36 d (6.0)
6.03 d (9.5)
6.69 d (9.5)
b-D-digt
100
200
300
400
500
600
5.26 d (10.0)
1.97 m, 2.43 m
4.49 m
99.9, d
38.2, t
67.6, d
80.6, d
69.4, d
18.1, q
2.14 dd (11.5, 6.0)
10
11
11
12
12
13
14
15
15
16
17
3.43 m
a
b
a
b
1.48 ma
1.29 ma
1.58 ma
1.21 ma
20.5, t
4.18 m
1.31 d (6.0)
30.8, t
a-L-cym
1000
5.04 br s
1.82 m, 2.33 m
3.69 m
98.6, d
32.2, t
76.4, d
72.5, d
67.4, d
18.4, q
56.7, q
54.9, s
155.4, s
72.1, t
2000
3000
a
b
4.28 br d (11.0)
3.82 dd (11.0, 4.2)
4.80 m
4000
3.62 m
5000
4.46 m
86.2, d
62.1, t
77.4, t
6000
1.42 d (6.0)
3.36 s
2.78 d (7.5)
4.01 d (8.5)
4.06 d (8.5)
0.83 s
OMe-3000
18a
18b
19
17.7, q
118.4, s
23.0, q
20
21
1.57 s
a
: Overlapped. can, canaropyranosyl; digt, digitoxopyranosyl; cym, cymaropyranosyl.

358
J.-Q. Yu, L. Zhao / Phytochemistry Letters 13 (2015) 355–359
Fig. 1. The structures of compounds 1–3.
being shown by two anomeric proton signals at dH 5.41 (1H, dd,
J = 10.2, 1.8 Hz, H-10) and 5.06 (1H, br s, H-100), which correlated to
the corresponding anomeric carbon signals at dC 98.6 (C-10) and
98.4 (C-100), respectively, in the HSQC spectrum, and two secondary
J = 16.0, H-70), indicative of a Z-configuration, and dihydrofuran
protons at H 3.43 (1H, m, H-8) and 5.45 (1H, d, J = 6.6 Hz, H-7). In
addition, three oxymethines at H 3.62 (1H, m, H-9a), 3.70 (1H, m,
H-9b), 4.03 (2H, dd, J = 6.0, 1.2 Hz, H-90), and 3.44 (2H, dd, J = 14.0,
7.2 Hz, H-110), and two methoxyls at dH 3.73 (3H, s, OMe-3), and
3.78 (3H, s, OMe-50), and one methyl at
dH 1.13 (3H, t, J = 6.0 Hz, H-
d
d
methyls at d
H 1.43 (3H, d, J = 6.6 Hz, H-60), 1.36 (3H, d, J = 5.4 Hz, H-
600) (Section 2). According to the HMQC and HMBC spectra, the
carbon signals were assigned respectively. In addition, the 1H and
13C NMR spectra also revealed the presence of two methoxyls at dH
3.54 (3H, s) and 3.38 (3H, s), which were determined to be linked at
120), were also present in the 1H NMR spectrum. Analysis of the 13C
NMR and DEPT spectra (Section 2) showed resonances for twenty-
two carbons: seven aromatic quaternary carbons (including four
oxygenated ones), nine methines (including five aromatic and two
olefinic ones), three oxygenated methylenes, and three methyls
(including two methoxyls). The proton and carbon signals in the 1H
and 13CNMR spectra of 3 were superimposable on those of (+)-(7S,
8R, 70E)-4-hydroxy-3, 50-dimethoxy-40, 7-epoxy-8, 30-neolign-70-
ene-9, 90-diol 90-ethyl ether, a known neolignan isolated previously
from Sinocalamus affinis (Xiong et al., 2011), except that the
hydroxyl group of 3 was proposed to be linked at C-5 instead of C-4,
which was supported by the long-range 1H-13C correlations from
C-10 and C-300, respectively, by the HMBC correlations from
d d dC 76.6 (C-
300), indicating that compound 2 was a methyl disaccharide
d
H 3.54
(OMe-10) to C 98.6 (C-10) and from H 3.38 (OMe-300) to
glycoside. Then, in order to determine the sugar moiety, acid
hydrolysis of 2 was carried out and one
D-digitoxopyranose, and
one -cymaropyranose showing up the proportion of 1:1 were
L
detected through derivatization reaction and GC analysis (Section
2). The 1H, 1H-COSY experiment, in combination with the HSQC
spectrum, established the spin systems within the sugar moiety
which were compatible with the identified two 2,6-dideoxypyr-
anoses. The coupling states of the aforementioned two anomeric
d
H 6.89 (H-2) to
d
C 115.3 (C-4), 118.5 (C-6), 87.3 (C-7);
dH 6.96 (H-4)
to dC 110.2 (C-2), 118.5 (C-6);
d
H 6.74 (H-6) to C 110.2 (C-2), 115.3
d
proton signals delivered the evidences that the
was -linkage and the -cymaropyranose was
sequence of the sugar chain was confirmed by the HMBC
correlations from H-100 of -cymaropyranose to C-40 of
digitoxopyranose, from H-10 of -digitoxopyranose to OMe-10.
Therefore, compound 2 was identified as Methyl-O- -cymar-
D
-digitoxopyranose
(C-4), 87.3 (C-7) in the HMBC spectrum (Fig. 2). In addition, the
absolute configurations of C-7 and C-8 were established by
analysis of the CD spectrum of 3, which displayed a typical Cotton
effect, positive at 230 nm and negative at 258 nm and 283 nm,
suggesting compound 3 had the 7S and 8R configurations (Xiong
et al., 2011). On the basis of the evidences above, compound 3 was
concluded to be (+)-(7S, 8R, 70E)-5-hydroxy-3, 50-dimethoxy-40, 7-
epoxy-8, 30-neolign-70-ene-9, 90-diol 90-ethyl ether.
Compounds 1–3 were tested for cytotoxicity activities against
HCT-8 (human colon cancer cell line), Bel-7402 (human hepatoma
cancer cell line), BGC-823 (human gastric cancer cell line), A549
(human lung epithelial cell line), and A2780 (human ovarian
cancer cell line) by means of the MTT method as described in the
literature (Ni et al., 2009) with fluorouracil as the positive control.
b
L
a-linkage. The
a-L
b-D-
b
-D
a-L
opyranosoyl-(1 ! 4)-
b-D-digitoxopyranoside.
Compound 3 was obtained as a brown jelly with ½a D20
ꢂ
+ 1.4
(c = 0.10, CH3OH, 20 8C). The positive-ion mode HRESIMS experi-
ment exhibited the pseudo-molecular ion peak at m/z
409.1631 [M + Na]+, indicative of the molecular formula of
C
22H26O6 with ten unsaturation degrees. The IR spectrum showed
the absorption bands for hydroxy (3285 cmꢁ1) and aromatic ring
(1646 cmꢁ1) groups. The 1H NMR spectrum, in combination with
the 1H,1H-COSY and HSQC experiments, allowed the identification
of the diagnostic signals assignable to five aromatic protons,
But none of them showed obvious activities (IC50 > 10
m
M for all
subjects, Table 2).
including three AB doublets at
d
H 6.74 (1H, d, J = 1.2 Hz, H-6), 6.89
(1H, br s, H-2), 6.96 (1H, br s, H-4) and two AB doublets at
d
H 6.95
Table 2
(1H, br s, H-20), 6.75 (1H, d, J = 1.2 Hz, H-60), two trans-olefinic
protons at dH 6.19 (1H, dt, J = 16.0, 6.0 Hz, H-80) and 6.50 (1H, d,
Cytotoxic activities of compounds 1-3 by the MTT method.
Sample
IC50 (mM)
HCT-8
Bel-7402
BGC-823
A549
A2780
1
>10
>10
>10
3.07
>10
>10
>10
4.06
>10
>10
>10
1.12
>10
>10
>10
>10
>10
>10
1.71
2
3
Fluorouracila
1.41
a
Fig. 2. Key HMBC correlations in 3.
Positive control.

J.-Q. Yu, L. Zhao / Phytochemistry Letters 13 (2015) 355–359
359
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21-steroidal glycoside from Cynanchum stauntoini. Chin. Chem. Lett. 18, 415–
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We thank associate Professor L Ma for the sample authentica-
tion. This work was supported by the grant from Science and
Technology development Project of Shandong Academy of Sciences
(2014QN02).
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