C21 steroidal glycosides from the roots of Cynanchum paniculatum

Article abstract of DOI:10.1016/j.fitote.2016.07.001

As a part of our continuing research for bioactive constituents from Cynanchum plants, four new C₂₁ steroidal glycosides, cynapanoside D-G (1–4), together with six known compounds (5–10) were isolated from the roots of Cynanchum paniculatum (Bge.) Kitag. Their structures were elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. Compound 8 exhibited potent inhibitory activities against HL-60, HT-29, PC-3 and MCF-7 cell lines with IC₅₀ values of 8.3, 7.5, 34.3 and 19.4?μM, respectively and compounds 1–4 and 9 displayed moderate cytotoxicity against the four cell lines. The in vitro antioxidant activities of compounds 1–4, 8 and 9 were assayed by DPPH radical scavenging activity. Antibacterial and antifungal activities of compounds 1–4, 8 and 9 were also tested.

Full text of DOI:10.1016/j.fitote.2016.07.001

Fitoterapia 113 (2016) 51–57
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
C21 steroidal glycosides from the roots of Cynanchum paniculatum
Dan Zhao a,d, Baomin Feng b, Shaofei Chen a,d, Gang Chen a,d, Zhifeng Li c, Xiaojie Lu a,d, Xianan Sang a,d
,
a,d,
Xiao An a,d, Haifeng Wang a,d, , Yuehu Pei
⁎
⁎
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
School of Life Sciences and Biotechnology, Dalian University, Dalian 116622, China
Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
b
c
d
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our continuing research for bioactive constituents from Cynanchum plants, four new C21 steroidal gly-
cosides, cynapanoside D-G (1–4), together with six known compounds (5–10) were isolated from the roots of
Cynanchum paniculatum (Bge.) Kitag. Their structures were elucidated on the basis of 1D- and 2D-NMR spectro-
scopic data as well as HR-ESI-MS analysis. Compound 8 exhibited potent inhibitory activities against HL-60, HT-
29, PC-3 and MCF-7 cell lines with IC50 values of 8.3, 7.5, 34.3 and 19.4 μM, respectively and compounds 1–4 and 9
displayed moderate cytotoxicity against the four cell lines. The in vitro antioxidant activities of compounds 1–4, 8
and 9 were assayed by DPPH radical scavenging activity. Antibacterial and antifungal activities of compounds 1–
4, 8 and 9 were also tested.
Received 27 May 2016
Received in revised form 28 June 2016
Accepted 1 July 2016
Available online 2 July 2016
Keywords:
Cynanchum paniculatum
Steroidal glycosides
Bioactivities
© 2016 Published by Elsevier B.V.
NMR data
1. Introduction
2. Experimental
C21 steroidal and their glycosides have established themselves as an
important class of biologically active compounds. They possess a wide
range of pharmacological activities including antitumor, antifungal
and cytotoxic activities [1–5]. Cynanchum paniculatum, is a perennial
herb belonging to the genus Cynanchum in the family Asclepiadaceae,
which is chiefly distributed in China, Japan and Korea. Its radix and rhi-
zome have been used for rheumatic arthralgia, epigastric pain and dis-
tension, toothache, lumbago, traumatic injuries, urticaria and eczema
in traditional Chinese medicine [6–8]. It has been reported to be rich
in C21 steroidal and their glycosides [9], whose chemical structures are
classified into normal four-ring C21 steroid type and aberrant
13,14:14,15-diseco-pregnane-type [10,11]. As our current interest in
the biologically active and structurally unique natural products, the
EtOH extract of C. paniculatum was investigated, and four new steroidal
glycosides, cynapanoside D (1), cynapanoside E (2), cynapanoside F (3)
and cynapanoside G (4), together with six known ones (5–10) were
isolated.
2.1. General experiment procedure
Optical rotations were determined using a WZZ-2A (Shanghai base
solid Instrument Co., Ltd., Shanghai, China). UV spectra were recorded
on a Shimadzu-2201 (Kyoto, Japan). The IR spectrum was obtained
from a Bruker IFS-55 spectrophotometer (Karlsruhe, Germany) using
KBr pellet. HR-ESI-MS data were measured on
a Micro-mass
Autospec-Untima TOF mass spectrophotometer (Waters, USA). NMR
spectra were run on a Bruker AVANCE-400/-600 spectrometer (Karlsru-
he, Germany). Analytical HPLC was carried out on a Shimadzu LC-10AT
(Kyoto, Japan) liquid chromatography and preparative HPLC separation
was performed on a YMC-Pack ODS-A column(10 × 250 mm, 5 μm;
YMC-Pack, Japan), equipped with a Shimadzu LC-8A pump (Kyoto,
Japan) and a Shimadzu SPD-10A UV–vis detector (Kyoto, Japan). Sugars
analytical HPLC was carried out on a Jasco PU-4180 pump (Kyoto,
Japan) and a OR-4090 detector (Kyoto, Japan). HPLC was performed
with an Asahipak NH2P-50 4E column (4.6 mm × 250 mm, 5 μm, Japan).
2.2. Plant material
The dried roots (10 kg) of C. panticulatum were bought from Anhui
Economy People Pharmaceutical Co., Ltd. A voucher specimen was iden-
tified by Prof. Jincai Lu of Shenyang Pharmaceutical University and has
been deposited in the School of Traditional Chinese Materia Medica of
Shenyang Pharmaceutical University (no. 20120913).
⁎
Corresponding authors at: School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang 110016, China.
E-mail addresses: wanghaifeng0310@163.com (H. Wang), peiyueh@vip.163.com
(Y. Pei).
http://dx.doi.org/10.1016/j.fitote.2016.07.001
0367-326X/© 2016 Published by Elsevier B.V.

52
D. Zhao et al. / Fitoterapia 113 (2016) 51–57
2.3. Extraction and isolation
2. Fr. D (6.8 g) was applied to a silica gel column to give eight fractions
(Fr. D1-D8). Fr. D2 (860 mg) was subjected to Sephadex LH-20 eluting
with MeOH to afford five fractions (Fr. D2-1-D2-5). Fr. D2-3 (218 mg)
was purified by semi-preparative HPLC eluting with 70% MeOH-H2O
(v = 4 mL/min) to yield compound 2 (26 mg, tR 40.0 min) and com-
pound 8 (45.3 mg, tR 70.2 min). Fr. D4 (1.4 g) was further separated
over a silica gel column using a solvent system of CH2Cl2-MeOH
(100:0–0:100, v/v) to give five subfractions (Fr. D4-1-D4-5). Fr. D4-2
(863 mg) was subjected to Sephadex LH-20 eluting with MeOH to af-
ford five fractions (Fr. D4-2-1-D4-2-5). Fr. D4-2-2 (215 mg) was puri-
fied by preparative reversed-phase HPLC using 68% MeOH-H2O (v =
4 mL/min) and then purified by preparative TLC using a solvent system
of cyclohexane-acetone (3:2, v/v) to obtain compound 9 (26.5 mg). Fr. E
(17.2 g) was separated into subfractions (Fr. E1-E7) by silica gel column
The dried roots (10 kg) of C. panticulatum were extracted three times
with 95% EtOH (each 2 h) and the combined solution evaporated to dry-
ness by a vacuum rotary evaporator to afford a syrup (1300 g). The
crude extract was successively partitioned with petroleum ether, ethyl
acetate and n-butanol to yield three layers of extracts. The ethyl acetate
extract (170 g) was fractionated by silica gel column chromatography
eluting with CH2Cl2-MeOH (100:0–0:100, v/v) to obtain twelve frac-
tions (Fr. A-L) based on TLC analyses. Fr. B (18.3 g) was separated into
subfractions (Fr. B1-B9) by silica gel column using PE-EtOAc (100:0–
0:100, v/v) as the eluent. Fr. B4 (476 mg) was purified via Sephadex
LH-20 eluting with CH2Cl2-MeOH (1:1, v/v) to afford four fractions (Fr.
B4-1-Fr. B4-4). Compound 10 (6.8 mg) was recrystallized from Fr. B4-
Table 1
1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in C5D5N for compounds 1–4.
1
2
3
4
No.
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
36.3
30.3
77.2
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
1
2
3
4
5
6
7
8
0.91 (m), 1.79a
1.67a, 2.06a
3.74 (m)
2.27a, 2.53a
–
5.38 (m)
1.36a, 1.99a
2.47 (m)
1.20a
36.2
29.8
77.3
38.8
140.4
120.2
29.7
40.4
53.0
38.4
23.7
28.2
118.3
175.2
67.5
0.88 (m), 1.80a
1.54a, 2.09a
3.71 (m)
2.28a, 2.52a
–
0.88 (m), 1.79a
1.52a, 2.11a
3.70 (m)
2.26a, 2.52a
–
36.3
30.1
77.2
38.8
140.2
120.3
29.9
40.5
52.7
38.5
20.7
28.3
118.8
175.4
67.0
0.99 (m), 1.69 (m)
1.79a, 2.17 (m)
3.95 (m)
2.64 (m), 2.79a
–
5.40 (m)
1.86a, 2.51 (m)
1.81a
37.1
30.0
78.5
38.8
139.4
122.2
27.6
36.8
46.0
37.2
20.8
38.5
49.1
84.7
34.3
38.8
140.2
120.3
29.8
40.8
52.7
38.5
20.6
28.3
5.37 (m)
1.65a, 2.03a
2.51a
5.37 (m)
1.65a, 2.03a
2.50a
9
1.25a
−
1.22a
−
1.10 (m)
−
10
11
12
13
14
15
−
1.33a, 2.55a
2.11a, 2.60a
–
1.83a, 2.49a
2.14a, 2.64a
–
1.82a, 2.50a
2.13a, 2.63
–
1.28a, 1.35a
1.18a, 1.36a
–
118.7
175.3
66.9
–
–
–
–
3.92 (dd, 9.3, 9.0)
4.22 (dd, 8.5, 7.3)
5.41 (m)
3.53a
6.45 (s)
0.82 (s)
–
3.97 (t, 9.3)
4.29 (dd, 9.3, 7.5)
5.99 (t, 8.0)
–
6.63 (s)
0.85 (s)
–
3.97 (t, 9.3)
4.29 (dd, 9.3, 7.5)
5.97 (dd, 8.5, 7.5)
–
6.65 (s)
0.83 (s)
–
1.79a, 1.99a
16
17
18
19
20
21
75.3
55.9
143.6
17.6
114.1
24.5
81.8,
92.2
144.4
17.7
119.5
20.4
81.8
92.2
144.4
17.8
119.5
20.5
1.85a, 2.00a
2.80a
1.05 (s)
0.93 (s)
–
24.2
62.8
15.2
19.3
216.5
32.1
1.51 (s)
1.73 (s)
1.73 (s)
2.17 (s)
β-D-Ole
4.77 (br.d, 9.8)
1.78a, 2.40a
3.55a
β-D-Ole
4.75 (dd, 9.8, 1.8)
1.74a, 2.40a
3.52a
β-D-Ole
4.77 (br.d, 9.8)
1.77a, 2.40a
3.55a
β-D-Glc
5.05 (d, 7.5)
4.10a
1′
2′
3′
4′
5′
6′
97.9
37.7
78.9
82.9
71.5
18.6
97.9
37.7
78.9
82.7
71.4
18.5
97.9
37.8
78.9
83.0
71.5
18.6
101.1
84.5
77.6
71.2
78.0
62.3
4.21a
3.55a
3.52a
3.51a
4.20a
3.53a
1.44 (s)
3.53a
1.42 (s)
3.52a
1.41 (s)
3.87a
4.30 (m),
4.48 (dd, 11.9, 2.4)
–
3′-OMe
3.53 (s)
57.2
3.40 (s)
57.1
3.53 (s)
57.3
–
β-D-Dig
β-D-Cym
β-D-Dig
β-D-Glc
1″
2″
3″
4″
5″
6″
5.52 (brd, 9.8)
1.97a, 2.41a
4.42 (m)
98.5
39.5
68.5
73.8
70.3
18.8
5.23 (dd, 9.8, 1.8)
1.73a, 2.06a
3.82 (m)
3.40a
98.1
36.7
77.6
82.0
69.2
18.3
5.51 (brd, 9.8)
1.96a, 2.39a
4.37 (m)
3.50a
4.57 (br.s)
1.41 (s)
98.5
39.8
68.8
82.2
67.6
18.4
5.26 (d, 7.5)
4.11a
106.4
76.8
77.8
71.3
78.8
62.5
4.21a
3.57a
4.28a
4.26 (dd, 8.3, 7.0)
1.55 (s)
4.13a
3.85a
1.32 (s)
4.40 (m),
4.54 (dd, 11.9, 2.4)
−
3″-OMe
−
−
3.50
58.1
−
−
−
α-L-Cym
4.98 (d, 3.3)
1.75a, 2.39a
3.67 (m)
3.56a
4.50 (m)
1.50 (s)
3.34 (s)
α-L-Ole
5.21 (d, 3.0)
1.69a, 2.44a
3.79a
1‴
2‴
3‴
4‴
5‴
6‴
3‴-OMe
98.7
31.8
76.1
73.1
66.1
18.4
56.3
100.0
35.5
78.6
76.6
69.3
18.2
56.8
3.50a
4.37a
1.48 (s)
3.30 (s)
δ in ppm, J values are in parentheses and reported in Hz. The assignments were based on NOESY, HSQC and HMBC experiments. Ole = oleandropyranose, Dig = digitoxopyranose, Cym =
cymaropyranose, Glc = glucopyranose.
a
Overlapped with other signals.

D. Zhao et al. / Fitoterapia 113 (2016) 51–57
53
using CH2Cl2-MeOH (100:0–0:100, v/v) as the eluent. Fr. E3 (3 g) was
subjected to Sephadex LH-20 eluting with MeOH to afford five fractions
(Fr. E3-1-E3-5). Fr. E3-2 (1.1 g) was chromatographed on a silica gel col-
umn and eluted with CH2Cl2-MeOH (100:0–0:100, v/v) to yield four
fractions (Fr. E3-2-1-E3-2-4). Fr. E3-2-2 (323 mg) was purified by
semi-preparative HPLC eluting with 68% MeOH-H2O (v = 4 mL/min)
to yield compound 1 (29 mg tR 111 min). Fr. E4 (1.1 g) was purified
via silica gel column eluting with CH2Cl2-MeOH (100:0–0:100, v/v) to
afford ten fractions (Fr. E4-1-Fr. E4-10). Fr. E4-8 (780 mg) was purified
via Sephadex LH-20 eluting with MeOH to afford four fractions (Fr. E4-
8-1-Fr. E4-8-4). Fr. E4-8-2 (260 mg) was purified by semi-preparative
HPLC eluting with 61% MeOH-H2O (v = 4 mL/min) to yield compound
3 (32.3 mg, tR 15.0 min), compound 5 (8.1 mg, tR 21.3 min), compound
6 (7.6 mg, tR 40.5 min) and compound 7 (6.4 mg, tR 31.3 min), respec-
tively. The n-butanol fraction (128 g) was subjected to a silica gel col-
umn eluted with CH2Cl2-MeOH (100:0–0:100, v/v) to obtain thirteen
fractions (Fr. A-M). Compound 4 (25.3 mg) was recrystallized from frac-
tion E.
Cynapanoside F (3): light yellow amorphous gum, [α]2D0 −24.3 (c
0.226, MeOH); UV (MeOH) λmax: 216 nm; IR (KBr) νmax cm−1: 3419,
2935, 1724, 1656, 1453, 1383, 1382, 1304, 1274, 1157, 1058, 987; 1H
NMR and 13C NMR (C5D5N) data see Table 1; HR-ESI-MS m/z:
817.4011 [M + Na]+ (Calcd for C41H62NaO15, 817.3996).
Cynapanoside G (4): white needle crystal (MeOH), [α]2D0 +26.7 (c
0.071, MeOH); UV (MeOH) λmax: 227, 255 nm; IR (KBr) νmax cm−1
:
3444, 2938, 2920, 1675, 1641, 1452, 1384, 1368, 1170, 1075, 1020; 1H
NMR and 13C NMR (C5D5N) data see Table 1; HR-ESI-MS m/z:
679.3312 [M + Na]+ (Calcd for C33H52NaO13, 679.3306).
2.4. Acid hydrolysis of compounds 1–4
Each solution of 1, 2 and 3 (6 mg) in MeOH (3 mL) were treated sep-
arately with 0.1 N H2SO4 (3 mL) at 50 °C for 30 min. Then the mixture was
diluted with 6 mL of water and concentrated to 6 mL. The solution was
kept at 60 °C for another 30 min, then neutralized with aqueous saturated
Ba(OH)2 and the precipitates were filtered off. The filtrate was concen-
trated and analyzed by TLC with three solvent systems: solvent A,
CHCl3-CH3OH (9:1); solvent B, CH2Cl2-C2H5OH (9:1); and solvent C, PE-
acetone (3:2). The Rf values of cymarose, oleandrose, digitoxose in the
order of 0.51, 0.45 and 0.27 with solvent A; 0.58, 0.49, and 0.32 with sol-
vent B; and 0.41, 0.36 and 0.28 with solvent C, respectively [10,12].
A solution of 10 mg of 4 in dioxane (5 mL) was refluxed with 10% HCl
(5 mL) at 80 °C for 3 h. After the removal of dioxane, the solution was
diluted with H2O (20 mL) and extracted with CH2Cl2 (25 mL × 3), neu-
tralized with 1 N NaOH and then concentrated. Glucose was identified
by TLC comparison with authentic sample, respectively.
Cynapanoside D (1): light yellow amorphous gum, [α]2D0 +43.5 (c
0.069, MeOH); UV (MeOH) λmax: 216 nm; IR (KBr) νmax cm−1: 3423,
2927, 2852, 1774, 1651, 1449, 1384, 1308, 1161, 1125, 1100, 1065; 1H
NMR and 13C NMR (C5D5N) data see Table 1; HR-ESI-MS m/z:
657.3174 [M + Na]+ (calcd for C34H50NaO11, 657.3251).
Cynapanoside E (2): light yellow amorphous gum, [α]2D0 −36.9 (c
0.244, MeOH); UV (MeOH) λmax: 215 nm; IR (KBr) νmax cm−1: 3437,
2933, 1726, 1655, 1450, 1383, 1303, 1274, 1107, 1061; 1H NMR and
13C NMR (C5D5N) data see Table 1; HR-ESI-MS m/z: 6831.4009
[M + Na]+ (Calcd for C42H64NaO15 831.4143).
Fig. 1. The structures of compounds 1–10.

54
D. Zhao et al. / Fitoterapia 113 (2016) 51–57
2.5. Determination of absolute configurations of glucose moieties in 1–4
(tR 12.1 min, negative polarity), D-cymarose (tR 18.6 min, positive
polarity), L-cymarose (tR 15.8 min, negative polarity), D-digitoxose
(tR 20.7 min, positive polarity) and D-glucose (tR 12.5 min, positive
polarity).
The absolute configurations of the glucoses and deoxysugars obtain-
ed from water layer of the hydrolysis solution of the compounds by
HPLC under the following conditions: column, Asahipak NH2P-50 4E
column (4.6 mm × 250 mm, 5 μm, Shodex); flow rate, 0.8 mL/min; sol-
vent, 75% MeCN-H2O; detection, OR (Jasco OR-4090) detector. Identifi-
cation of D-oleandrose, L-oleandrose, D-cymarose, L-cymarose, D-
digitoxose and D-glucose in each sugar fraction was carried out by a
comparison of the retention times and polarities with those of authentic
samples (the source of the standards of D-oleandrose, L-oleandrose, D-
cymarose, L-cymarose, D-digitoxose were obtained by acid hydrolysis
of the known compounds with the same method of those new com-
pounds). D-oleandrose (tR 15.1 min, positive polarity), L-oleandrose
2.6. Biological activity assays
2.6.1. Cytotoxicity assay
A MTT assay was used to determine the cytotoxicity of each com-
pound against four cultured human cancer cell lines. The cell lines
used were HL-60 (human leukemic promyelocytic cell), A549 (non-
small cell lung adenocarcinoma), PC-3 (prostate cancer cell) and MCF-
7 (breast cancer cell). All the cell lines (American Type Culture Collec-
tion) was maintained in RPMI-1640 medium (Gibco) with 10% fetal
Fig. 2. Key HMBC correlations of compounds 1–4.

D. Zhao et al. / Fitoterapia 113 (2016) 51–57
55
Fig. 3. Proposed the biological cleavage of the ring C/D junction for 13,14:14,15-diseco-pregnane-type steroid.
bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and
1 mmol/L L-glutamine, and 10% (v/v) heat-inactivated FBS in a humidi-
fied atmosphere of 5% CO2 at 37.0 °C. Logarithmic phase cells were used
for experiments. Appropriate dilutions of the test samples were added
to the cultures. The growth inhibition was calculated comparing with
control cells. 5-Fluorouracil was used as a positive control. The cytotox-
icity of 5-Fluorouracil against the HL-60, HT-29, PC-3 and MCF-7 cell
lines were estimated by their IC50 values, 6.38, 7.90, 7.77, and
17.01 μM, respectively, for these 4 cell lines.
2.6.2. 1-Diphenyl-2-picrylhydrazyl free radical (DPPH·) scavenging assay
The DPPH·scavenging activity was assessed according to the meth-
od described with minor modifications with lower DPPH concentration
(0.2 mM) and different sample to DPPH ratio (1:1). To a 100 μL aliquot
of the sample with different concentrations was added 100 μL of DPPH
solution (0.2 mM) in a 96-well microplate [13]. The mixture was shaken
vigorously and incubated in darkness for 30 min. The absorbance of the
reaction solution at 517 nm was recorded using a varioskan flash multi-
mode reader. Ascorbic acid was used as a positive control. The percent-
age of scavenging DPPH versus concentration was plotted using the
following equation: DPPH scavenging activity (%) = [1-(S-SB)/(C-
CB)] × 100%, where S, SB, C and CB are the absorption of the sample,
the blank sample, the control and the blank control, respectively. All ex-
periments were tested in triplicate.
Table 2
Cytotoxicity data of compounds 1–5a.
Compound
HL-60
HT-29
PC-3
MCF-7
1
2
3
4
7
8
9
13.4
28.9
33.2
N40
N40
8.3
N40
21.9
N40
N40
N40
7.5
N40
N40
N40
N40
N40
34.3
36.6
7.77
N40
N40
N40
N40
N40
19.4
28.9
17.0
10.1
6.38
25.4
7.90
2.6.3. Antimicrobial bioassay
5-Fluorouracil
The MIC values for all compounds were determined by the dilution
method. For sample preparation, each of the test compounds was dis-
solved in DMSO and then diluted with sterile broth to a concentration
HL-60, human leukemic promyelocytic cell, HT-29, human colon cancer cell, PC-3, human
prostate cancer cell and MCF-7, human breast cancer cell.
a
Data expressed as IC50 values (μM).

56
D. Zhao et al. / Fitoterapia 113 (2016) 51–57
of 500 μg/mL. Further dilutions of the compounds in the test medium
were prepared at the required quantities of 250, 125, etc. down to
3.9 μg/mL. Chloramphenicol and fluconazole were used as positive con-
trols for bacteria and fungus, respectively. The in vitro antimicrobial ac-
tivity of the compounds was tested by tube-dilution technique using
individually pack-aged, flat bottomed, 96-well microtiter plates
(NCCLS. 2000). Bacterial strains were maintained on LB medium for
48 h at 37 °C and fungal strains were on PDA medium for 48 h at 28 °
C. The cell density for bacteria was 2–4 × 107 CFU/mL and 2–
4 × 105 CFU/mL for fungus. A serial dilution of compounds were per-
formed in the microplates and incubated for 12 h. The last tube with
no growth of microorganism was recorded to represent the MIC value
expressed in μg/mL.
H-1‴)] as β-, β-, and α-form, respectively. In the HMBC spectrum, corre-
lations of H-1′ (δ 4.75) with C-3 (δ 77.2), H-1″ (δ 5.23) with C-40 (δ
82.7) and H-1‴ (δ 4.93) with C-40 (δ 82.0) were observed, indicating
the sugar moiety of 2 was deduced to be α-cymaropyranosyl-(1 → 4)-
β-cymaropyranosyl-(1 → 4)-β-oleandropyranosyl group by compari-
son with data in the literature as shown in Fig. 2 [16]. Acid hydrolysis
of 2 gave oleandrose and cymarose and they were identified by compar-
ison of their spectroscopic data and Rf values with those reported in the
literatures [10,15,13,17]. Identification of D-oleandrose, D-cymarose
and L-cymarose were also analyzed by HPLC with the detection by
using the optical rotation (OR) detectors. The HPLC analysis exhibited
a positive peak for the oleandrose, a positive and a negative peaks for
the cymaroses, suggesting D-, L-form of the cymaroses and D-form of
the oleandrose. In addition, the NMR data of the two cymaroses were
identical to those of Sublanceoside G3 [18], so the outer cymarose was
determined as α-L-cymarose, and the inner one was β-D-cymarose. In
the NOESY spectrum, correlations of H-19 to H-4 endo and H-11 endo,
indicated that H-19, H-4 endo and H-11 endo are on the same face of
the molecule. Consequently, all the aforementioned evidences charac-
terized 2 as neocynapanogenin F 3-O-α-L-cymaropyranosyl-(1 → 4)-
β-D-cymaropyranosyl-(1 → 4)-β-D-oleandropyranoside, and was des-
ignated cynapanoside E.
3. Results and discussion
3.1. Structure elucidation
Cynapanoside D (1), was afforded as light yellow amorphous gum,
[α]2D0 +43.5 (c 0.069, MeOH). Its positive HR-ESI-MS showed an ion
peak at m/z 657.3174 [M + Na]+ (calcd. 657.3251), indicating a molec-
ular formula of C34H50O11. The 1H NMR spectrum of 1 revealed the pres-
ence of two tertiary methyl groups [δH 0.82 (3H, s, H-19) and 1.51 (3H, s,
H-21)], two oxygen-substituted methine protons [δH 3.74 (1H, m, H-3)
and 5.41 (1H, m, H-16)], two oxygen-substituted methylene protons [δH
3.92 (1H, dd, J = 9.3, 9.0 Hz, Hα-15) and 4.22 (1H, dd, J = 8.5, 7.3 Hz, Hβ-
15)], one olefinic proton [δH 5.38 (1H, m, H-6)], and one olefinic
deshielded proton [δH 6.45 (1H, s, H-18)] connected with the trisubsti-
tuted double bond. The 1H and 13C NMR data (Table 1) of the aglycone
part of 1 were almost identical to those of glaucogenin C [14], except
for the glycosidation shifts at C-2 (−2.5 ppm), C-3 (+6.2 ppm) and
C-4 (−4.2 ppm). Thus, the aglycone structure of 1 was confirmed to
be glaucogenin C by detailed 1D NMR and 2D NMR (HSQC, HMBC and
NOESY) spectral analysis, and the sugar chain was linked at its C-3 hy-
droxyl group. The proton signals showed two anomeric proton signals
at [δH 4.77 (1H, br.d, J = 9.8 Hz, H-1′) and 5.52 (1H, br.d, J = 9.8 Hz,
H-1″)], one methoxy group [δH 3.53 (3H, s, C-3′ –OCH3)], and two meth-
yl groups [δH 1.44 (3H, d, J = 6.0 Hz, H-6′) and 1.55 (3H, d, J = 6.0 Hz, H-
6″)] of the sugar moiety. The splitting patterns of anomeric proton sig-
nals indicated that 1 had two sugar units with β-linkages. The linkage
positions and sequence of the two sugars were ascertained by HMBC
correlations from δH 5.52 (H-1″ of β-digitoxopyranose) to δC 82.9 (C-
4′ of β-oleandropyranose) and from δH 4.77 (H-1′ of β-
oleandropyranose) to δC 77.3 (C-3). Acid hydrolysis of 1 afforded two
sugars (oleandrose and digitoxose). Identification of D-oleandrose and
D-digitoxose were performed by HPLC analysis with an optical rotation
detector and comparison of Rf values with the literature [15]. In addi-
tion, the correlations observed in the NOESY experiment also supported
the proposed relative configuration of 1, in which NOESY correlations of
H-19 to H-11β and H-8, of H-16 to H-17 and H-15α, of H-1ʹ to H-3ʹ and
H-5′, of H-1″ to H-5″ indicated the orientation of the aglycone and two
sugars. Thus, the structure of 1 was established as glaucogenin C-3-O-β-
D-digitoxopyranosyl-(1 → 4)-β-D-oleandropyranoside and named
cynapanoside D.
Cynapanoside F (3), [α]2D0 −24.3 (c 0.23, MeOH), was yielded as
light yellow amorphous gum. Its molecular formula was determined
as C41H62O15 by the [M + Na]+ ion peak at m/z 817.4011 (Calcd for
C41H62NaO15, 817.3996) in HR-ESI-MS. From its 13C NMR data (Table
1), it was apparent that 3 possessed the same aglycone as that of 2.
The 1H NMR spectrum of 3 showed three anomeric protons signals at
[δH 4.77 (1H, br.d, J = 9.8 Hz, H-1′), 5.21 (1H, d, J = 3.0 Hz, H-1‴),
and 5.51 (1H, br.d, J = 9.8 Hz, H-1″)] in its sugar moiety, indicating
the presence of one sugar with α-linkage and two sugars with β-link-
ages. The 1H and 13C NMR (Table 1) spectra of the deoxysugar units of
3 were similar to Cynanside A [19]. The HMBC experiments of 3 showed
correlations from δH 5.21 (H-1‴ of α-oleandrose) to δC 82.2 (C-4″ of β-
digitoxose), 5.51 (H-1″ of β-digitoxose) to δC 83.0 (C-4′ of β-
oleandrose) and δH 4.77 (H-1′ β-oleandrose) to δC 77.2 (C-3), which fur-
ther confirmed the linkage sequence. Acid hydrolysis of 3 yielded
oleandrose and digitoxose. The absolute configuration of oleandrose
and digitoxose were identified as D-, L-oleandrose and D-digitoxose ac-
cording to the HPLC analysis. Moreover, the NMR data sugar moiety of 3
were the same as those of
a previously reported compound,
amplexicosides B [12], which indicated that the inner oleandrose was
β-D-oleandrose, and the outer one was α-L-oleandrose. Therefore, the
structure of 3 was elucidated as neocynapanogenin F 3-O-α-L-
oleandropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-
oleandropyranoside and named cynapanoside F.
Cynapanoside G (4), [α]2D0 +26.7 (c 0.071, MeOH), was obtained as
white needle crystal (MeOH). It gave the molecular formula as
C33H52O13 by the [M + Na]+ ion peak at m/z 679.3312 (Calcd for
C33H52NaO13 679.3306) according to the HR-ESI-MS. The 1H NMR spec-
trum of 4 revealed the presence of three tertiary methyl groups [δH 0.93
(3H, s, H-19), 1.05 (3H, s, H-18) and 2.17 (3H, s, H-21)], one olefinic pro-
ton [δH 5.40 (1H, m, H-6)] in its aglycone moiety. The 1H NMR and cor-
responding 13C NMR spectral data (Table 1) were similar to those of the
aglycone of Carumbelloside I [12]. The 1H NMR spectrum of 4 showed
two anomeric proton signals at [δH 5.05 (1H, d, J = 7.5 Hz, H-1ʹ), and
5.26 (1H, d, J = 7.5 Hz, H-1″)] in its sugar moiety, indicating the pres-
ence of two sugars with β-linkages. Acid hydrolysis of 4 yielded gluco-
pyranose, and identification of D-glucopyranose was performed by
HPLC analysis and co-TLC with standard samples. Compared with
Carumbelloside I [12], the sugar chain was also linked to the C-3 hy-
droxyl group of aglycone. The difference between the sugar compo-
nents of 4 and those of Carumbelloside I was that the sugar sequence
of 4 changed into β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl by
the HMBC correlations from δH 5.26 (H-1″ of terminal β-D-glucopyra-
nose) to δC 84.5 (C-2′ of β-D-glucopyranose). Thus, the structure of 4
Cynapanoside E (2) was obtained as light yellow amorphous gum,
[α]2D0 −36.9 (c 0.24, MeOH). The formula of 2 was determined to be
C42H64O15 by the [M + Na]+ ion peak at m/z 831.4009 (Calcd for
C42H64NaO15 831.4143) in HR-ESI-MS. The 1H and 13C NMR data
(Table 1) of the aglycone part of 2 were almost identical to those of
neocynapanogenin F [8], except for the glycosidation shifts at C-2
(−1.0 ppm), C-3 (+6.7 ppm) and C-4 (−3.1 ppm), so the sugar was
determined to be linked to the C-3 hydroxyl group of the aglycone.
The 1H NMR spectrum of 2 showed three secondary methyl and three
methoxyl methyl signals of deoxysugars. The anomeric configuration
of sugars was deduced by J values at [δH 4.75 (1H, dd, J = 9.8, 1.8 Hz,
H-1′), 5.23 (1H, dd, J = 9.8, 1.8 Hz, H-1ʹʹ) and 4.93 (1H, d, J = 3.3 Hz,

D. Zhao et al. / Fitoterapia 113 (2016) 51–57
57
was determined to be 3β, 14β-dihydroxypregn-5-en-20-one 3-O-β-D-
Appendix A. Supplementary data
glucopyranosyl-(1 → 2)-β-D-glucopyranoside (Fig. 1) and named
cynapanoside G.
The NMR and HR-ESI-MS spectra of compounds 1–4 were available
as supporting information in the Supplementary data. Supplementary
data associated with this article can be found in the online version, at
doi: http://dx.doi.org/10.1016/j.fitote.2016.07.001
The known compounds were identified as glaucoside A (5) [20],
neocynapanogenin F 3-O-β-D-oleandropyranoside (6) [21], glaucogenin A
3-O-β-D-digitoxopyranosyl-(1 → 4)-O-β-D-oleandropyranoside (7) [22],
cynatratoside B (8) [14], (3β,8β,9α,16α,17α)-14,16β:15,20α:18,20β-
triepoxy-16β:17α-dihydroxy-14-oxo-13,14:14,15-disecopregna-5,13(18)-
dian-3-yl α-cymaropyranosyl-(1 → 4)-β-digitoxopyransyl-(1 → 4)-β-
oleandropyranoside (9) [9] and 3β,14-dihydroxy-14β-pren-5-en-20-one
(10) [10] by comparison of their physico-chemical and spectroscopic prop-
erties with previously reported values.
13,14:14,15-Diseco-pregnane-type compounds are novel C21 steroi-
dals. Their main cleavage pattern were C/D ring broken and a series of
oxidation reactions. The possible mechanism of biological cleavage of
the ring C/D junction was proposed. (Fig. 3).
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Conflict of interests
The authors declare that there are no conflicting interests.
Acknowledgements
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The work was supported by National Natural Science Foundation of
China (no. 31370375).