Minor cytotoxic cardenolide glycosides from the root of Streptocaulon juventas

Article abstract of DOI:10.1016/j.steroids.2014.10.005

In order to determine new minor natural cardenolide glycosides as cytotoxic candidates, we isolated six new cardenolide glycosides together with four known ones, which had never previously been reported in the genus, by bioassay-guided separation from the 75% ethanol extract of Streptocaulon juventas (Asclepiadaceae). Their structures were elucidated on the basis of spectroscopic analysis, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC). The cytotoxic activities of these compounds were evaluated against A549 and NCI-H460 cell lines by MTT assay and compound 7 exhibited inhibitory activity against the two cell lines, while other compounds displayed a range of inhibitory activity against NCI-H460 and A549 cells. Their structure-activity relationships were also discussed.

Full text of DOI:10.1016/j.steroids.2014.10.005

Steroids 93 (2015) 39–46
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Minor cytotoxic cardenolide glycosides from the root of Streptocaulon
juventas
Chun Ye a, Hua Wang a, Rui Xue a, Na Han a, Lihui Wang b, Jingyu Yang b, Yu Wang c, Jun Yin a,
⇑
a Development and Utilization Key Laboratory of Northeast Plant Materials, Key Laboratory of Northeast Authentic Materials Research and Development in Liaoning Province, School
of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China
c The People’s Liberation Army 463 Hospital, Shenyang 110042, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2014
Received in revised form 16 October 2014
Accepted 29 October 2014
Available online 18 November 2014
In order to determine new minor natural cardenolide glycosides as cytotoxic candidates, we isolated six
new cardenolide glycosides together with four known ones, which had never previously been reported in
the genus, by bioassay-guided separation from the 75% ethanol extract of Streptocaulon juventas
(Asclepiadaceae). Their structures were elucidated on the basis of spectroscopic analysis, including
homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC). The cytotoxic activities
of these compounds were evaluated against A549 and NCI-H460 cell lines by MTT assay and compound 7
exhibited inhibitory activity against the two cell lines, while other compounds displayed a range of
inhibitory activity against NCI-H460 and A549 cells. Their structure–activity relationships were also
discussed.
Keywords:
Asclepiadaceae
Streptocaulon juventas
Cardenolide glycoside
Antitumor
Ó 2014 Elsevier Inc. All rights reserved.
Structure–activity relationship
1. Introduction
in the Streptocaulon, Saussutea, Euonymus and Acokanthera genus
[5–8].
Streptocaulon is a small genus of the Asclepiadaceae family
which contains only five species. It has not been studied in detail
until recently when it attracted interest because it exhibited strong
anti-proliferative activity [1]. A survey of the literature showed
that cardenolide glycosides were the biologically active com-
pounds in this genus along with the discovery of a class of cardiac
glycosides with preventive and therapeutic effects on proliferative
diseases such as cancer [2–5]. Moreover, these cardenolide
glycosides were associated with typical genins, such as digitoxi-
genin, acovenosigenin A, periplogenin, 17b-H-periplogenin, ole-
andrigenin, 16-O-acetylacovenosigenin, 16-O-acetylperiplogenin,
3b,5b,14b-trihydroxyl-card-16,20(22)-dienolide, 1b,3b,14b-trihy-
Streptocaulon juventas, has a root which has been used as a folk
medicine for the treatment of rheumatism, neurasthenia and dys-
pepsia patients since ancient times in the Guangxi and Yunnan
provinces of China [9]. In 2002, Ueda et al. reported that the
MeOH extract of the roots of S. juventas possessed potent antipro-
liferative activity against the human HT-1080 fibrosarcoma cell
line in a study involving 231 extracts from 77 Vietnamese medic-
inal plants [3]. We have previously reported that the most active
fraction, the 75% ethanol extract, obtained from the roots of S.
juventas strongly inhibited the A549 cell line in vitro (IC50 value,
0.89 lg/mL) and also tumor growth in A549 tumor-bearing mice
(inhibition rate: 58%) [2], 39 cardenolide glycosides have already
been isolated from this plant [4,5]. It appears to be a good raw
medicinal material due to the fact that it has a total product yield
of cardenolides up to 2.35 mg/g (analyzed by the content of digi-
toxigenin) [10]. As part of our continuing search to discover novel
cardenolides, a further phytochemical analysis has been carried
out on the minor bioactive compounds with interesting structures
obtained from the active fraction of the plant, so that 1-hydroxyl-
ated cardenolides present in trace amounts (< 0.01%) were iso-
lated and identified, illustrating the structural diversity of
cardiac glycosides [11].
droxy-5b-card-16,20(22)-dienolide,
20(22)-enolide and evonogenin, and their sugar moieties included
b- -cymarose, b- -digitoxose, b- -digitalose and b- -glucose [3–5].
1a,14b-dihydroxy-5b-card-
D
D
D
D
Among these compounds, most of the novel cardenolide glycosides
contained the skeleton of acovenosigenin A. Despite this, statistical
data from SciFinder showed that only ten cardenolides with the
acovenosigenin A moiety have been identified in the natural world,
⇑
Corresponding author at: School of Traditional Chinese Materia Medica 48#,
Shenyang Pharmaceutical University, Wenhua Road 103, Shenhe District, Shenyang
110016, China. Tel./fax: +86 24 23986491.
In this paper, TLC, MPLC and HPLC techniques were mainly used
and these allowed us to discover six new cardenolides (1–6),
E-mail address: yinjun2002@yahoo.com (J. Yin).
http://dx.doi.org/10.1016/j.steroids.2014.10.005
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

40
C. Ye et al. / Steroids 93 (2015) 39–46
together with four known ones (7–10) which had never been found
in the genus before (Fig. 1). Using bioactivity-guided isolation, we
obtained four new compounds possessing the aglycone of
acovenosigenin A. The structures of the new cardenolides were
elucidated by analysis of 1 and 2D NMR and MS data. Compounds
1–3, 5–10 exhibited potent anti-proliferative activity in the
NCI-H460 and A549 cell lines.
2.2. Plant material
The roots of S. juventas were collected in Yunnan Province,
China, in 2009. The plant material was identified by Prof. Jun Yin
(Division of Pharmacognosy, Shenyang Pharmaceutical University),
and a voucher sample (SPU 2162–2165) was deposited at the her-
barium of Shenyang Pharmaceutical University, Shenyang, China.
2.3. Extraction and isolation
2. Experimental
The milled roots of S. juventas (30 kg, air-dried) were extracted
with 75% EtOH (300 L, reflux, 1.5 h ꢀ 3). The residue was concen-
trated under reduced pressure, then separated on a HPD100 mac-
roporous resin column and eluted with a gradient mixture of
H2O/EtOH (100:0, 70:30, 40:60, 10:90 v/v) to give four fractions
(H2O, 30%, 60%, 90% EtOH). Fr. 60% EtOH (315 g) which showed sig-
nificant activity in the human lung A549 adenocarcinoma cell line
2.1. General
Optical rotations were determined on a Perkin–Elmer 241MC
polarimeter using a 0.1 dm cell and using MeOH as a blank.
NMR spectra were obtained on Bruker ARX-300 and ARX-600
spectrometers, using TMS as an internal standard. HR-TOFMS
was obtained on a Bruker microTOF mass spectrometer, equipped
with an electrospray ionization (ESI) ion source. Ultraviolet–visi-
ble spectra was recorded on UV-2201 Shimadzu UV–vis scanning
spectrophotometer using MeOH as a blank. Column chromatogra-
phy was peformed with macroporous resin HPD100 (Tianjin Resin
Co., Ltd.), silica gel (100–200 mesh and 200–300 mesh; Qingdao
with an IC50 value of 1.44 lg/mL [10], was subjected to open-col-
umn chromatography on a silica gel column (200–300 mesh,
8.5 ꢀ 20 cm, 650 g) with stepwise gradient elution with a mixture
of petroleum ether and EtOAc (5:1, 2:1, 1:1) followed by CH2Cl2/
MeOH (30:1, 20:1, 10:1, 5: 1, 3:1, 1:1, each 5 L) to give five frac-
tions (Fr. 1-Fr. 5). Fr. 5 (95.5 g) was further separated into three
fractions (Fr. 5-1- Fr. 5–3) by MPLC on a silica gel column (200–
300 mesh, 6.5 ꢀ 32 cm, 530 g), eluting with CH2Cl2/MeOH/H2O
(5:1:1, 3:1:1, 1:1:1; 3 L each). A portion of Fr. 5–1 (8.0 g) was fur-
ther chromatographed on an ODS column (120 g) using a MeOH/
H2O gradient as the mobile phase to give fraction A [MeOH/H2O
(3:7)] and fraction B [MeOH/H2O (7:13)] by MPLC. The fractions
collected were monitored and recombined based on their TLC pro-
files. Fr. A (634 mg) was purified using a Sephadex LH-20 column
and elution with MeOH followed by preparative HPLC (MeOH/
H2O 50:50, 9.0 mL/min) to yield 5 (10 mg, tR 27.0 min). Fr. B
(3.1 g) was loaded onto a Sephadex LH-20 column and eluted with
MeOH to obtain six fractions (Fr. B1–B6). Fr. B1 (102 mg) was puri-
fied by preparative HPLC (27% Acetonitrile–H2O, 9.0 mL/min) to
yield 1 (48 mg, tR 100.0 min). Fr. B2 (148 mg) was purified by pre-
parative HPLC (23% Acetonitrile–H2O, 9.0 mL/min) and silica gel
column chromatography (EtOAc/MeOH/H2O 5:1:0.9) to yield 2
(6 mg). Fr. B3 (112 mg) was purified by preparative HPLC (23% Ace-
tonitrile–H2O, 9.0 mL/min) to yield 3 (32 mg, tR 73.0 min). Fr. B4
(38 mg) was purified by silica gel column chromatography
(EtOAc/MeOH/H2O 5:1:0.9) to yield 4 (26 mg). Fr. B5 (108 mg)
was purified by preparative HPLC (23% Acetonitrile–H2O, 9.0 mL/
min) to yield 6 (10 mg, tR 224 min) and 7 (14 mg, tR 94 min). Fr.
B6 (111 mg) was purified by preparative HPLC (25% Acetonitrile–
H2O, 9.0 mL/min) followed by silica gel column chromatography
(EtOAc/MeOH/H2O 5:1:0.9) to yield 8 (11 mg). A portion of Fr.
5–2 (7.0 g) was subjected to ODS column chromatography and gra-
dient elution with MeOH/H2O to give fraction C [MeOH/H2O
60:40], and Fr. C (480 mg) was purified by preparative HPLC (50%
MeOH–H2O, 9.0 mL/min) and silica gel column chromatography
(EtOAc/Acetone/H2O 1:1:0.1) to yield 9 (4.2 mg) and 10 (1 mg).
Marine Chemical Co., Ltd.), ODS-A (50
LH-20 (GE Healthcare). Preparative HPLC was conducted on a
M) equipped with a
lM; YMC), and Sephadex
YMC ODS-A column (250 ꢀ 30 mm I.D., 5
l
pump and a single-wavelength UV detector. Analytical HPLC
was carried out on a Shimadzu LC10-A HPLC system with an
LC-10ATVP pump, a Shimadzu LC-10AVP UV–vis detector (Shima-
dzu Co., Ltd.), and an N-2000 chromatographic work station
(Intelligent Information Engineering Co., Ltd.) using a C18 column
(250 ꢀ 4.6 mm).
Cisplatin was purchased from Sigma Co., Ltd. with a purity of
99%.
All chemical reagents used in the studies were produced by
Yuwang Chemicals Industries, Ltd.
The human lung A549 adenocarcinoma cell line and large cell
carcinoma NCI-H460 cell line were purchased from American Type
Culture Collection (ACTT, Manassas, U.S.A.).
O
23
O
21
18
22
20
17
12
11
19
R1
13
14
9H
R4
1
16
2
10H 8 OH15
3
5
7
6
R2O
R1
H
H
4
R3
R2
R3
OH
OH
H
R4
H
1
2
(2-O-Ac-Dtl)4-Glc6-Glc
Dig4-Glc4-Glc
H
3
OH
OH
OH
OH
H
Cym4-Glc6-Glc
(2-O-Ac-Dtl)4-Glc6-Glc
(2-O-Ac-Dtl)4-Glc6-Glc
Cym4-Dtl4 -Glc6-Glc
Dtl4-Glc6-Glc
H
H
4
H
2.4. Periplogenin 3-O-[O-b-
D-glucopyranosyl-(1?6)-O-b-
D-
5
H
OAc
H
glucopyranosyl-(1?4)-2-O-acetyl-b-
D-digitalopyranoside] (1)
6
H
7
H
H
White powder (MeOH); ½a D23
ꢂ5.6 (c 0.49, MeOH); UV (MeOH)
ꢁ
8
H
Glc4-Glc
H
H
kmax 218; HR–ESI-MS m/z 939.4183 [M+Na]+ (calcd for C44H68O20-
9
H
(3-O-Ac-Dig)4-Glc
H
H
H
10
H
OH
OH
Na, 939.4196); 1H NMR and 13C NMR, see Table 1.
2.5. Periplogenin 3-O-[O-b-
D-glucopyranosyl-(1?4)-O-bD-
Cym:β-
Dig: β-
D
D
-cymarose
-digitoxose
glucopyranosyl-(1?4)-b- -digitoxopyranoside] (2)
D
Dtl: β-
Glc: β-
D
-digitalose
-glucose
White powder (MeOH); ½a D23
ꢁ
+17.2 (c 0.29, MeOH); UV (MeOH)
D
kmax 213; HR–ESI–MS m/z 889.4030 [M+HCOO]ꢂ (calcd for
Fig. 1. Chemical structures of compounds 1–10.
C
42H65O20, 889.4075); 1H NMR and 13C NMR, see Table 1.

C. Ye et al. / Steroids 93 (2015) 39–46
41
Table 1
1H and 13C NMR data of compounds 1, 2 and 3 (CD3OD).
Position
1a
2b
3a
dH (J in Hz)
dC
26.5
dH (J in Hz)
dC
26.6
dH (J in Hz)
dC
74.0
1
2
3
4
5
6
7
8
1.35 m, 1.70 m
1.64 m, 1.77 m
4.13 br s
1.39 m, 1.72 m
1.66 m, 1.78 m
4.16 br s
3.70 br s
1.90 m, 2.09 m
4.17 br s
1.51 m, 1.97 m
2.10 m
1.35 m, 1.85 m
1.29 m, 1.34 m
1.68 m
26.9
78.6
36.1
75.0
35.6
24.8
41.6
40.1
41.8
22.6
40.9
50.9
86.3
33.3
28.0
51.9
16.3
17.2
178.3
75.3
26.8
77.3
35.9
75.7
35.5
24.8
41.7
40.2
41.8
22.7
40.9
50.9
86.3
33.3
28.0
51.9
16.3
17.2
178.4
75.3
32.1
76.6
31.2
32.1
27.3
22.3
42.7
38.4
41.2
22.1
40.8
50.9
86.2
33.3
27.3
52.0
16.4
19.2
178.3
75.3
1.51 m, 2.19 m
1.58 m, 2.18 m
1.31 m, 1.71 m
1.19 m, 1.92 m
1.61 m
1.34 m, 1.71 m
1.21 m, 1.95 m
1.64 d (3.1)
1.62 m
9
1.61 m
1.59 m
10
11
12
13
14
15
16
17
18
19
20
21
1.31 m, 1.45 m
1.48 m, 1.51 m
1.32 m, 1.47 m
1.52 m
1.81 m
1.29 m, 1.49 m
1.71 m, 2.13 m
1.87 m, 2.16 m
2.83 m
0.88 s
0.92 s
1.71 m, 2.15 m
1.87 m, 2.16 m
2.84 m
0.88 s
0.93 s
1.73 m, 2.17 m
1.35 m, 1.85 m
2.81 m
0.88 s
1.07 s
4.91 dd (1.5, 18.3)
5.00 dd (1.3, 18.6)
5.90 s
4.91 dd (1.7, 18.5)
5.03 dd (1.5, 18.5)
5.90 s
4.91 dd (1.5, 17.1)
5.03 dd (1.5, 18.5)
5.90 s
22
117.8
177.2
102.5
72.7
83.4
75.4
71.8
17.4
58.7
172.2
21.1
104.6
75.8
77.8
71.8
77.4
70.4
117.8
177.2
98.0
38.9
68.4
84.2
69.7
18.5
117.8
177.2
97.8
36.9
78.6
83.8
70.4
18.6
58.7
23
10
4.53 d (8.0)
5.09 dd (8.0, 10.1)
3.47 dd (2.9, 10.1)
4.25 d (2.4)
3.69 m
4.94 dd (1.8, 9.7)
1.71 m, 1.97 m
4.29 dd (2.9, 6.5)
3.27 m
3.88 dt (2.1, 11.8)
1.29 d (6.2)
4.82 (overlap)
1.65 m, 2.08 m
3.98 d (2.8)
3.36 m
3.88 m
1.31 d (6.2)
3.46 s
20
30
40
50
60
1.31 d (6.4)
3.44 s
3-O-Me
2-O-Ac
2.09 s
4.54 d (7.5)
3.26 m
3.37 m
3.29 m
3.46 m
3.76 dd (6.6, 11.8)
4.15 m
100
200
300
400
500
600
4.41 d (7.8)
3.28 dd (2.4, 9.6)
3.50 t (9.0)
105.6
74.8
76.3
80.0
76.2
62.4
4.35 d (7.7)
3.24 m
3.34 m
3.25 m
3.49 m
106.2
75.3
77.9
71.9
76.9
70.6
3.60 t (9.2)
3.41 dt (9.6, 3)
3.65 dd (5.6, 11.9)
3.87 dt (2.1, 11.8)
3.71 m, 4.17 m
1000
2000
3000
4000
5000
6000
4.40 d (7.7)
3.19 m
3.33 m
3.27 m
3.24 m
105.1
75.2
78.1
71.7
78.1
62.9
4.41 d (7.8)
3.22 m
3.36 m
104.6
74.9
77.8
71.4
78.1
61.4
4.35 d (7.7)
3.19 m
3.31 m
3.32 m
3.25 m
105.1
75.2
78.1
71.7
78.1
62.9
3.30 m
3.33 dd (2.2, 5.7)
3.82 dd (2.3, 12.0)
3.90 m
3.66 m, 3.87 m
3.65 m, 3.87 m
a
1H NMR data were measured at 300 MHz, and 13C NMR data were measured at 75 MHz.
1H NMR data were measured at 600 MHz, and 13C NMR data were measured at 150 MHz.
b
2.6. Acovenosigenin A 3-O-[O-b-
D
-glucopyranosyl-(1?6)-O-b-
D
-
2.8. 16-O-acetyl-hydroxyacovenosigenin 3-O-[O-b-
(1?6)-O-b- -glucopyranosyl-(1?4)-2-O-acetyl-b-
digitalopyranoside] (5)
D-glucopyranosyl-
D-
glucopyranosyl-(1?4)-b- -cymaropyranoside] (3)
D
D
White powder (MeOH);
(MeOH) kmax 216; HR–ESI–MS m/z 881.4141 [M+Na]+ (calcd
for
42H66O18Na, 881.4141); 1H NMR and 13C NMR, see
½
a 2D3
ꢁ
ꢂ15.6 (c 0.18, MeOH); UV
White powder (MeOH); ½a D23
ꢂ37.6 (c 0.19, MeOH); UV (MeOH)
ꢁ
C
kmax 212; HR–ESI–MS m/z 997.4228 [M+Na]+ (calcd for
Table 1.
C
46H70O22Na, 997.4251); 1H NMR and 13C NMR, see Table 2.
2.9. Acovenosigenin A 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b-
D-
2.7. Acovenosigenin A 3-O-[O-b-
glucopyranosyl-(1?4)-2-O-acetyl-b-
D
-glucopyranosyl-(1?6)-O-b-
-digitalopyranoside] (4)
D-
glucopyranosyl-(1?4)-O-b-
cymaropyranoside] (6)
D
-digitalopyranosyl-(1?4)-b-
D-
D
White powder (MeOH); ½a D23
ꢁ
ꢂ33.1 (c 0.16, MeOH); UV (MeOH)
White powder (MeOH); ½a D23
ꢂ16.2 (c 0.21, MeOH); UV (MeOH)
ꢁ
kmax 218; HR–ESI–MS m/z 939.4188 [M+Na]+ (calcd for C44H68O20-
kmax 216; HR–ESI–MS m/z 1041.4900 [M+Na]+ (calcd for C49H78O22-
Na, 939.4196); 1H NMR and 13C NMR, see Table 2.
Na, 1041.4877); 1H NMR and 13C NMR, see Table 2.

42
C. Ye et al. / Steroids 93 (2015) 39–46
2.10. Odoroside G (7)
incubation for 24 h at 37 °C at the appropriate density of 8 ꢀ 104/
mL in 96-well tissue culture plates (100 lL per well), compounds
White power (MeOH); ½a D20
ꢂ17.0 2 (c 1.060, MeOH)].
ꢁ
ꢂ11.0 (c 0.27, MeOH) [[12] ½a D24
ꢁ
1–3, 5–10 were added after being dissolved in dimethyl sulfoxide
(DMSO) and diluted at a concentration of 100 mol/L (the final
l
concentration of DMSO was 0.1%), then the cells continued to be
incubated with the nine compounds for 48 h. Cell growth was mea-
sured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay incubated for 3–4 h at 37 °C. Then, the med-
2.11. Digitoxigenin 3-O-b- -cellobioside (8)
D
White power (MeOH); ½a D20
ꢁ
+8.1 (c 0.12, MeOH); 1H NMR (CD3-
OD, 300 MHz): d 5.89 (1H, s, H-22), 5.00 (1H, d, J = 18.6 Hz, H-21),
4.91 (1H, dd, J = 18.3, 1.5 Hz, H-21), 4.41 (1H, d, J = 7.8 Hz, H-10),
4.35(1H, d, J = 7.7 Hz, H-100); 4.06 (1H, br s, H-3), 2.83 (1H, m, H-
17), 0.96 (3H, s, H-19), 0.88 (3H, s, H-18), 13C NMR (CD3OD,
75 MHz): d 178.5 (C-20), 177.3 (C-23), 117.8 (C-22), 104.6 (C-10),
102.6 (C-100), 86.5 (C-14), 80.9 (C-40), 78.1 (C-300), 77.9 (C-500),
76.6 (C-30), 76.4 (C-50), 75.8 (C-200), 75.4 (C-21), 75.0 (C-20), 74.9
(C-3), 71.4 (C-400), 62.5 (C-600), 62.0 (C-60), 52.2 (C-17), 51.1 (C-
13), 42.7 (C-8), 41.0 (C-12), 37.5 (C-5), 36.9 (C-9), 36.3 (C-10),
33.4 (C-15), 31.3 (C-1), 30.9 (C-4), 28.1 (C-16), 27.8 (C-2), 27.5
(C-6), 24.1 (C-19), 22.6 (C-11), 22.4 (C-7), 16.4 (C-18).
ium was removed, and the cells were dissolved in 100
sulfoxide and the absorbance at 492 nm was measured. Cisplatin
(100 mol/L) was used as a positive control. The percentage of cell
lL dimethyl
l
growth inhibition was calculated as follows:
Cell growth inhibition ð%Þ¼½A549ðcontrolÞꢂA549ðcompoundÞꢁ=A549ðcontrolÞ
ꢀ100
The IC50 values of compounds that inhibited cell viability by
over 50% at a concentration of 100 lM were calculated. All cyto-
toxic activity data were analyzed by SPSS (16.0) and expressed as
mean S.E.
2.12. Digitoxigenin-3-O-b-D-glucosyl-(1?4)-3-O-acetyl-b-D-
digitoxoside (9)
3. Results and discussion
White power (MeOH); ½a D20
ꢁ
+20.0 (c 0.17, MeOH) [(13) ½a D25
ꢁ
Compound 1, a white amorphous powder, with the formula
+29.1 (c 0.75, MeOH)]; 1H NMR (CD3OD, 300 MHz): d 5.89 (1H, s,
H-22), 5.49 (1H, m, H-10), 5.00 (1H, d, J = 18.3 Hz, H-21), 4.93
(1H,d, J = 1.5 Hz, H-21), 4.36 (1H, d, J = 7.7 Hz, H-100); 4.01 (1H, br
s, H-3), 2.83 (1H, m, H-17), 2.08 (3H, s, 3-O-Me), 1.32 (3H, d,
J = 6.2 Hz, H-10), 0.94 (3H, s, H-19), 0.88 (3H, s, H-18), 13C NMR
(CD3OD, 150 MHz): d 178.5 (C-20), 177.3 (C-23), 172.8 (C-2-O-
Ac), 117.8 (C-22), 105.8 (C-100), 97.6 (C-10), 86.5 (C-14), 81.6 (C-
40), 78.0 (C-300), 78.0 (C-500), 75.4 (C-200), 75.3 (C-21), 75.1 (C-3),
72.0 (C-30), 71.5 (C-400), 70.6 (C-50), 63.0 (C-600), 52.2 (C-17), 51.1
(C-13), 42.7 (C-12), 41.0 (C-8), 38.1 (C-9), 37.5 (C-5), 36.9 (C-10),
36.4 (C-20), 33.4 (C-15), 31.4 (C-1), 31.4 (C-4), 28.1 (C-16), 27.9
(C-6), 27.6 (C-2), 24.3 (C-19), 22.6 (C-7), 22.4 (C-11), 21.3 (C-3-O-
Me), 18.6 (C-60), 16.4 (C-18).
C
44H68O20 having an HR–ESI–MS at m/z 939.4183 [M+Na]+ ion
(calcd for 939.4196).
The 1H NMR and 13C NMR spectra displayed characteristic sig-
nals of a cardenolide [dH 5.90 (1H, s, H-22), 5.00 (1H, dd, J = 18.6,
1.3 Hz, H-21), 4.91 (1H, dd, J = 18.3, 1.5 Hz, H-21), dC 178.3 (C-
20), 75.3 (C-21), 117.8 (C-22), 177.2 (C-23)] representative of an
a
,b-unsaturated c-lactone, and two methyl groups [dH 0.88 (s,
18-Me), 0.92 (s, 19-Me), dC 16.3 (C-18), 17.2 (C-19)]. In addition,
one oxymethyl [dH 3.44 (s), dC 58.7] and one acetyl [dH 2.09 (s),
dC 172.2, 21.1] group were observed. The aglycone part of 1 was
identified as periplogenin by the above analysis and comparison
with the1H and 13C NMR resonances in the Ref. [15].
The presence of three sugar units in 1 was indicated by three
anomeric proton signals at dH 4.53 (1H, d, J = 8.0 Hz, H-10), dH
4.40 (1H, d, J = 7.7 Hz, H-1000) and dH 4.54 (1H, d, J = 7.5 Hz, H-100).
It was also evident from the 1H and 13C NMR data that one of the
sugar units was 6-deoxysugar due to the proton signal displayed
at 1.31 (d, J = 6.4 Hz, H-60) [16]. The HMBC correlation between
the methoxy protons (dH 3.44) and C0-3 (dC 83.4), showed that
the methoxy group was located at C0-3. The methyl protons (dH
2.10, dC 21.1) and the carbonyl carbon (dC 172.2) indicated the
presence of an acetyl group, which was located at C-2’ of digitalose
by the connectivity from dH 5.09 (H-20) to dC 172.2 in HMBC (Fig. 2).
Complete assignment of the sugar protons and carbons by 1H–1H
COSY and HSQC experiments showed characteristic signals of one
2-O-acetyl b-digitalopyranosyl unit (dC 102.5, 72.7, 83.4, 75.4,
71.8, 17.4, 58.7, 172.2, 21.1) and two b-glucopyranosyl (dC 104.6,
75.8, 77.8, 71.8, 77.4, 70.4; 105.1, 75.2, 78.1, 71.7, 78.1, 62.9) units
(Table 1). An unambiguous determination of the sugar sequencing
and linkage sites was made from the HMBC spectrum. The HMBC
correlations from H-1000 (dH 4.40) to C-600 (dC 70.4), H-100 (dH 4.54)
to C-40 and H-10 (dH 4.53) to C-3 (dC 78.6) of the aglycone moiety,
suggested the connections of the sugar units, and the connection
between the glycosides and aglycone. On the basis of this evidence,
the structure of compound 1 was established as periplogenin 3-O-
2.13. 5b-Hydroxygitoxigenin (10)
White power (MeOH);
+48.6 (c 0.62, MeOH)].
½
a 2D0
+13.7 (c 0.26, MeOH) [[14]
ꢁ
½ ꢁ
a 2D0
2.14. Identification of the monosaccharide of compounds 1–6
A solution containing compound 1 (1 mg) in 1, 4-dioxane
(0.5 mL) and 1 N HCl (4.5 mL) was heated at 90 °C for 6 h. After
cooling, the mixture was extracted with EtOAc (3 ꢀ 5 mL),
followed by neutralizing the aqueous layer with 1 N KOH.
Compounds 2–6 were also treated in the same way. The monosac-
charides were purified by preparative TLC, then identified using
n-BuOH-Me2CO-H2O (8:5:1) and the Rf values were 0.45, 0.60,
0.80 and 0.85 for glucose, digitalose, digitoxose and cymarose,
respectively. The absolute configurations were determined by
measurement of the optical rotations as
D
-glucose [½a 2D3
ꢁ
+51.3 (c
0.1, H2O)],
D
-digitalose [½a D23
ꢁ
+105.0 (c 0.1, H2O)], D-digitoxose
[½a 2D3
ꢁ
+42.1 (c 0.03, H2O)] and
D
-cymarose [½a 2D3
ꢁ
+ 55.9 (c 0.07,
H2O)]. We figured out the position of the sugar attachments by
the H–C long range correlations in the HMBC spectrum and 1H
and 13C NMR chemical shifts, and mentioned it in detail in the
structure analysis of results and discussion part.
[O-b-
D
-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-2-O-
acetyl-b-
D
-digitalopyranoside].
Compound 2, obtained as a white amorphous powder, had a
molecular formula of C41H64O18 from the HR–ESI–MS at m/z
889.4030 [M+HCOO]ꢂ ion in negative mode (calcd for 889.4075).
Compared with the 1D and 2D NMR spectroscopic data of com-
pound 1, the aglycone of 2 was identical to that of 1. The rest of the
2.15. In vitro anti-proliferation bioassay
A549 and NCI-H460 cells were cultured in RPMI-1640 medium
(GIBCO, NY, U.S.A.) and maintained at 37 °C in 5% CO2. After

C. Ye et al. / Steroids 93 (2015) 39–46
43
Table 2
1H and 13C NMR data of compounds 4, 5 and 6 (CD3OD).
Position
4a
5b
6b
dH (J in Hz)
dC
73.7
dH (J in Hz)
dC
73.7
dH (J in Hz)
dC
74.0
1
2
3.66 m
1.97 m
3.65 m
1.98 m
3.70 br s
1.89 m
32.4
32.4
32.1
2.09 dd (2.4, 15.0)
4.17 br s
1.50 m
3
4
4.22 br s
1.55 m, 1.94 m
75.8
29.9
4.23 br s
1.55 m, 1.94 m
75.8
29.9
76.6
31.2
1.95 dd (2.4, 13.8)
2.08 m
1.35 m, 1.84 m
1.34 m, 1.81 m
1.68 d (3.0)
1.59 d (3.6)
5
6
7
8
1.76 m
32.1
27.3
22.3
42.7
38.4
41.3
22.1
40.7
50.9
86.1
33.3
1.75 m
32.1
27.2
21.8
42.7
38.2
41.3
21.9
39.8
51.2
84.8
41.2
32.1
27.3
22.3
42.7
38.4
41.2
22.1
40.8
50.9
86.2
33.3
1.35 m, 1.85 m
1.33 m, 1.81 m
1.68 m
1.36 m, 1.85 m
1.25 m, 1.84 m
1.67 td (3.5, 11.9)
1.56 m
9
1.59 m
10
11
12
13
14
15
1.29 m, 1.84 m
1.45 m, 1.47 m
1.22 m, 1.35 m
1.41 m, 1.53 m
1.29 m
1.29 m, 1.47 m
1.73 m, 2.17 m
1.79 m
1.73 m, 2.17 m
2.76 dd (9.8, 15.5)
5.45 dt (2.3, 9.0)
3.25 d (9.0)
0.94 s
16
17
18
19
20
21
1.87 m, 2.17 m
2.82 m
0.88 s
28.1
52.0
16.4
19.3
178.3
75.3
75.9
57.3
16.4
19.3
171.5
77.5
1.87 m, 2.17 m
2.82 m
0.88 s
28.0
52.1
16.4
19.2
178.3
75.3
1.06 s
1.05 s
1.07 s
4.91 dd (1.8, 16.5)
5.03 dd (1.5, 18.0)
5.90 br s
4.95 dd (1.5, 18.5)
5.01 dd (1.5, 18.5)
5.98 br s
4.91 dd (1.2, 18.6)
5.03 dd (1.2, 18.6)
5.89 s
22
117.8
177.2
121.8
176.7
172.1
20.9
99.6
72.9
83.1
75.2
71.8
17.3
58.7
172.1
21.1
104.6
75.7
78.0
71.9
77.4
70.5
117.8
177.2
23
16-OAcc
1.94 s
10
4.49 d (8.1)
5.10 m
3.50 dd (2.9, 10.2)
4.27 d (2.5)
3.69 m
99.6
72.9
83.1
75.2
71.8
17.5
58.8
172.1
21.1
104.6
75.7
77.8
71.9
77.4
70.5
4.49 d (8.4)
5.09 t (9.3)
3.49 d (3.0)
4.26 d (3.0)
3.69 m
4.83 dd (1.8, 9.6)
1.55 m, 2.13 m
3.83 q (3.0)
3.30 m
3.89 m
1.31 d (6.0)
97.7
36.5
78.4
83.8
70.4
18.7
20
30
40
50
60
1.32 d (6.3)
3.45 s
1.32 d (6.4)
3.45 s
3-O-Me
2-O-Acc
2.06 s
4.54 d (7.5)
3.25 m
3.36 m
3.30 m
3.47 m
3.76 dd (6.6, 11.7)
4.16 dd (1.5, 11.7)
2.07 s
4.54 d (7.8)
3.25 m
3.25 m
3.29 m
3.46 d (1.8)
3.75 dd (6.8, 12.0) 4.15 dd (1.8, 12.0)
100
200
300
400
500
600
4.30 d (7.8)
3.64 m
3.23 m
4.15 m
3.63 m
106.5
71.7
85.5
76.1
71.4
17.7
1.29 d (6.6)
3-O-Me
1000
3.51 s
4.39 d (7.8)
3.19 m
3.32 m
3.27 m
3.25 m
3.65 m
3.86 dd (1.9, 12.0)
58.9
105.2
75.2
77.8
71.7
78.0
62.8
3.51 s
4.55 d (7.8)
3.22 m
3.35 m
3.28 m
3.45 m
3.77 dd (6.5, 11.8)
4.13 m
58.9
104.8
75.9
78.0
71.8
77.4
70.3
4.40 d (7.8)
3.20 m
3.30 m
3.30 m
3.26 m
105.1
75.2
78.1
71.7
78.0
62.8
2000
3000
4000
5000
6000
3.66 m
3.87 d (12.3)
10000
20000
30000
40000
50000
60000
4.39 d (7.8)
3.19 m
3.35 m
3.28 m
3.22 m
105.1
75.2
78.1
71.8
78.0
62.8
3.65 m
3.86 dd (2.0, 11.9)
a
b
c
1H NMR data were measured at 300 MHz, and 13C NMR data were measured at 75 MHz.
1H NMR data were measured at 600 MHz, and 13C NMR data were measured at 150 MHz.
Assignments may be interchanged in each column.
1H and 13C NMR data of the sugar moieties were found to be similar
by comparison with previously published data [17], except for the
linkage between the two glucose units. The HMBC correlation from
dH 4.41 (H-1000) of the outer unit to dC 80.0 (C-400) of the inner glu-
cose unit, indicated that the two sugar moieties were (1?4) linked.
periplogenin 3-O-[O-b-
D-glucopyranosyl-(1?4)-O-b-D-glucopyr-
anosyl-(1?4) -b- -digitoxopyranoside].
D
Compound 3, obtained as a white amorphous powder, had a
molecular formula of C42H66O18 as determined by the HR–ESI–
MS at m/z 881.4141 [M+Na]+ ion in positive mode (calcd for
881.4141).
Thus, the structure of compound
2 was determined to be

44
C. Ye et al. / Steroids 93 (2015) 39–46
The 1H NMR spectrum of compound 3 showed two sets of three
one b-digitalopyranose and two b-glucopyranose units. The HMBC
correlation from dH 4.39 (H-10000) to dC 70.3(C-6000), dH 4.55 (H-1000) to
dC 76.1 (C-400) and dH 4.30 (H-100) to dC 83.8 (C-40), suggested a link-
age of the four sugars from C-100 to C-40, C-1000 to C-400, and C-10000 to
C-6000. The location of the sugar moiety was determined to be C-3
from an HMBC correlation between dH 4.83 (H-10) of the cymarose
unit and dC 76.6 (C-3) of the aglycone moiety. Thus, the structure of
proton singlet signals at 0.88 (3H, s, 18-Me), and 1.07 (3H, s, 19-
Me)], one olefinic proton signal at dH 5.90 (1H, s, H-22), and a pair
of double doublet signals at 5.03 (dd, J = 18.5, 1.5 Hz, H-21) and
4.91 (dd, J = 17.1, 1.5 Hz, H-21), which were characteristic of a car-
denolide skeleton. Two oxymethine signals at dH 3.70 (1H, br s),
and 4.17 (1H, br. s) indicated that the aglycone had two O-substi-
tuted protons. The aglycone of 3 was determined to be acovenosi-
genin A by comparison with the data in the Ref. [5]. Also, the three
resonances for the anomeric protons at dH 4.82 (overlap), dH 4.35
(d, J = 7.7 Hz) and dH 4.35 (d, J = 7.7 Hz) suggested the presence of
three sugar units, and the set of signals at dC 97.8, 36.9, 78.6,
83.8, 70.4, 18.6, and 58.7 suggested the presence of one b-cymaro-
pyranose and the rest of values matched the chemical shifts of two
b-glucopyranose units [18]. The HMBC correlations between H-1000
(dH 4.35) and C-600 (dC 70.6), H-100 (dH 4.35) and C-4 0 (dC 83.8), and
H-10 (dH 4.82) and C-3 (dC 76.6) confirmed the connectivity of the
sugar units. Thus, 3 was concluded to be acovenosigenin A 3-O-
compound 6 was determined to be acovenosigenin A 3-O-[O-b-
glucopyranosyl-(1?6)-O-b- -glucopyranosyl-(1?4)-O-b- -digi-
talopyranosyl-(1?4)-b- -cymaropyranoside].
D-
D
D
D
The monosaccharides of compounds 1–6 obtained by acid
hydrolysis of each compound were identified as glucose, cymarose,
digitalose and digitoxose by TLC comparison with authentic sam-
ples. Also, the absolute configurations of
D-glucose, D-cymarose,
D
-digitalose and -digitoxose were determined by measurement
D
of their optical rotations after separation by preparative TLC. The
3
relatively large JH-1,H-2 values of glucose, digitalose (7.8 Hz), digi-
toxose and cymarose (9.6 and 1.2 Hz) in their pyranose form indi-
cated their b anomeric orientation [3,19,20].
[O-b-
D-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-O-b-D-
cymaropyranoside].
The known compounds 8 and 9 were identified as digitoxigenin
Compound 4, obtained as a white amorphous powder, exhibited
an [M+Na]+ ion at m/z 939.4188 in positive mode of the HR–ESI–
MS, establishing that its molecular formula was C44H68O20 (calcd
for 939.4196).
3-O-b-
(1?4)-3-O-acetyl-b-
D
-cellobioside (8) [21] and digitoxigenin-3-O-b-
D-glucosyl-
D
-digitoxoside (9) [13] by analyses of their
1H and 13C NMR spectra, while the 1H and 13C NMR spectroscopic
data of these two compounds were reported for the first time. The
other two known compounds were identified as odoroside G (7)
[22] and 5b-hydroxygitoxigenin (10) [23] by comparing the 13C
NMR data with reference data.
Comparison of the 13C NMR data of compound 4 with that of 3
[C-1 (dC 73.7), C-5 (dC 32.1) C-19 (dC 19.3)], it was concluded that
the aglycone of 4 was acovenosigenin A. However, analyses of
the 13C NMR and 1H NMR signals of the sugar moiety of 4, showed
that it consisted of one 2-O-acetyl b-digitalopyranosyl unit and two
b-glucopyranosyl units, similar to the sugar moiety of 1. The (1?6)
linkage between the two glucoses, and the (1?4) linkage between
the inner glucose and digitalose was established by the correlation
between H-1000 (dH 4.40) and C-600 (dC 70.5), and the correlation
between H-100 (dH 4.54) and C-40 (dC 75.2) in the HMBC spectrum.
A correlation between H-10 (dH 4.49) of the digitalose unit and C-
3 (dC 75.8) of cardenolide aglycone was also identified in the HMBC
spectrum. Thus, 4 was determined to be acovenosigenin A 3-O-[O-
The anti-proliferative activities of all the isolated compounds,
except for compound 4 (it was not present in a high enough
amount for activity investigation), were evaluated in vitro against
human lung A549 adenocarcinoma and large cell carcinoma NCI-
H460 cell lines by MTT-assay at a concentration of 100 lM, while
cisplatin was used as a positive control (Fig. 3). The compounds
with the inhibition rate over 50% towards either of the cell lines
were measured and the IC50 values as shown in Table 3. Except
the compounds 1, 7, 9 and the positive control cisplatin, most com-
pounds displayed much stronger anti-proliferative activities
against A549 cell line than those against the NCI-H460 cell line.
Only compound 7 inhibited both of cancer cell lines, displaying
wide cytotoxicity and more prospective potential.
We previously reported the cytotoxicities of cardenolide glyco-
sides from S. juventas against the two above NSCLC cell lines and
have discussed their structure–activity relationships [24]. Compar-
ing with the previous report, some new structure–activity relation-
ships against A549 cells are summarized as far as the new
compounds were found and investigated in this manuscript. We
found that for 3b, 14b-dihydroxyl cardiac glycosides, the hydroxyl
group at C-1, C-5 or C-16 all decreased the inhibiting activities, the
acetyl group at C-16 could increase (4 < 1 < 5 < 17⁄) or decrease
(3 < 14⁄ < 40⁄ < 27⁄, 38⁄ < 15⁄ < 13⁄ < 21⁄) the inhibition depending
on whether there were of other substituents on the aglycone.
The decrease of cytotoxicity induced by the acetyl group at C-30
b-
D-glucopyranosyl-(1?6)-O-b-
D-glucopyranosyl-(1?4)-2-O-
acetyl-b-
D-digitalopyranoside].
Compound 5, obtained as a white amorphous powder, with an
[M+Na]+ ion at m/z 997.4228 in positive HR–ESI–MS, was assigned
the molecular formula of C46H70O22 (calcd for 997.4251).
By comparing the 1D and 2D NMR spectroscopic data, the agly-
cone of 5 was found to be similar to that of 4 except for one addi-
tional acetyl group (Table 2). Considering the low field shifting of
1H and 13C of C-16 [(dC 75.9, dH 5.45 dt (2.3, 9.0 Hz)], the acetyl
group was deduced to connected to C-16. The same coupling con-
stants of H-16 and H-17 (9.0 Hz) indicated that the acetyl group
connected at C-16 had a b-axial orientation, so the aglycone of 5
was deduced to be 16-O-acetyl-hydroxyacovenosigenin, which
was also confirmed by comparation with reference data [5]. Fur-
thermore, the 1H and 13C NMR data of the sugar portions were in
agreement with those of compounds 1 and 4. Thus, 5 was con-
cluded to be 16-O-acetyl-hydroxyacovenosigenin 3-O-[O-b-
D-glu-
copyranosyl-(1?6)-O-b-
digitalopyranoside].
D
-glucopyranosyl-(1?4)-2-O-acetyl-b-
D-
O
O
Compound 6, a white amorphous powder, showed an [M+Na]+
ion at m/z 1041.4900 by HR–ESI–MS in positive mode, correspond-
ing to a molecular formula of C49H78O22 (calcd for 1041.4877).
Comparing the 1H and 13C NMR data of 6 with those of 3, the
aglycone was confirmed as Acovenosigenin A. Then, four anomeric
proton signals [dH 4.83 (dd, J = 9.6, 1.8 Hz), dH 4.30 (d, J = 7.8 Hz), dH
4.55 (d, J = 7.8 Hz), and dH 4.39 (d, J = 7.8 Hz)] confirmed that 6 was
acovenosigenin A tetrasaccharide. By analysis of the HSQC and
HMBC spectra and comparison with the reference data [3], the
sugar moiety was deduced to consist of one b-cymaropyranose,
OH
O
O
HO
HO
H
OH
HO
O
O
H
OH
HO
O
OH
O
OH
H3CO
OCOCH3
Fig. 2. Selected COSY (1H–1H— and HMBC (1H–13Cy) correlations of compound 1.

C. Ye et al. / Steroids 93 (2015) 39–46
45
100
80
60
40
20
0
NCI-H460
A549
Fig. 3. The cell growth inhibition (%) of A549 and NCI-H460 cells after treatment with 9 compounds (100
(100 mol/L) was used as a positive control.
lmol/L), as measured by MTT-assay (mean S.E.). Cisplatin
l
Acknowledgements
Table 3
Antitumor activities of compounds 1–3, 5–10.
This work was financially supported by grants from the
National Natural Science and Technology Major Project ‘‘Key New
Drug Creation and Manufacturing Program’’ (No. 2010ZX09401-
304) and the Natural Science Foundation of China (No. 30973958).
Compounds
IC50 (lM)
NCI-H460
A549
1
2
3
38.308
–
–
–
0.016
0.38
5
6
7
8
–
–
21.497
3.159
9.048
6.876
–
Appendix A. Supplementary data
0.873
–
8.206
–
1H-NMR, 13C-NMR, COSY, HMBC, HR-ESI-MS and UV spectra for
compounds 1–6, as well as the 13C NMR data of compound 7 and
10 are available as Supporting Information.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2014.
10.005.
9
10
Cisplatin
1.845
35.635
2.564
‘‘–’’: Not tested, IC50 values of compounds were calculated if they inhibited a
tumour cell proliferation of over 50% at a concentration of 100
Using Cisplatin as a positive control.
lM.
References
reported by Xue would be weakened accompanied with the exten-
sion of carbohydrate chain (9 < 28⁄ < 29⁄ < 21⁄). The acetylation at
the C-20 of sugar moiety would enhance the inhibiting activity for
triglycosylation (7 < 17⁄). The connection way of saccharides also
affect the activity, the order of activity is 1?4 < 1?2 < 1?6 con-
nection (8 < 25⁄ < 23⁄) (⁄ means this compounds from Ref. [24]).
Forty years ago, when cardiac glycosides were first reported to
exhibit antitumor activity, their toxicity limited their development
[25,26]. Our results show that these compounds might be modestly
selective for different cancer cells. The possibility of using cardiac
glycosides to treat lung cancer depends on the identification of a
leading compound effective against a range of different types of
lung cancers, which would provide us with a new strategy to
develop in future studies.
[1] Ueda JY, Tezuka Y, Banskota AH, Tran QL, Tran QK, Harimaya Y, Saiki I, Kadota
S. Antiproliferative activity of Vietnamese medicinal plants. Biol Pharm Bull
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