C21 steroidal glycosides from Cynanchum stauntonii induce apoptosis in HepG2 cells

Article abstract of DOI:10.1016/j.steroids.2015.12.008

Two new (1–2) and three known (3–5) C₂₁ steroidal glycosides were isolated from Cynanchum stauntonii. Their structures were elucidated on the basis of 1D and 2D-NMR spectroscopic data as well as HRTOFMS analysis. The cytotoxicity of the compoun

Full text of DOI:10.1016/j.steroids.2015.12.008

Steroids xxx (2015) xxx–xxx
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Steroids
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C21 steroidal glycosides from Cynanchum stauntonii induce apoptosis
in HepG2 cells
Zhi-Qi Yin a, ,1, Shu-Le Yu a,b,1, Yu-Jian Wei c, Lin Ma a,b, Zheng-Feng Wu a,b, Lei Wang d, Qing-Wen Zhang e,
⇑
Ming Zhao f, Wen-Cai Ye d, Chun-Tao Che f, Jian Zhang b,
⇑
a Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
b Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, China
c The First Clinical Medical Institute, Nanjing Medical University, Nanjing 210000, China
d Institute of Traditional Chinese Medicine and Natural Products & Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and
New Drugs Research, Jinan University, Guangzhou 510632, China
e State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China
f Department of Medicinal Chemistry and Pharmacognosy, and WHO Collaborating Center for Tradition Medicine, College of Pharmacy, University of Illinois at Chicago, Chicago,
IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new (1–2) and three known (3–5) C21 steroidal glycosides were isolated from Cynanchum stauntonii.
Their structures were elucidated on the basis of 1D and 2D-NMR spectroscopic data as well as HRTOFMS
analysis. The cytotoxicity of the compounds against A549, HepG2, and 4T1 cell lines were evaluated by
MTT assay. Compound 4 exhibited good inhibitory activities with the IC50 values 26.82, 12.24, and
Received 10 August 2015
Received in revised form 27 November 2015
Accepted 10 December 2015
Available online xxxx
44.12 lM, respectively. Furthermore, compound 4 could induce G1 phase arrest, upregulate the
expression levels of caspases-3, -9, and Bax, and downregulate the expression level of Bcl-2. These results
indicated that compound 4 might be valuable to anticancer drug candidates.
Ó 2015 Elsevier Inc. All rights reserved.
Keywords:
Cynanchum stauntonii
C21 steroidal glycoside
Apoptosis
Caspase
1. Introduction
therapeutic strategies for cancers [7]. However, necrosis is typi-
cally described as a ‘nonspecific’ form of cell death, with conse-
Apoptosis, first demonstrated by Kerr in 1972 [1], is mediated
by genes resulting in orderly cell death to maintain the stability
of the environment in the body, also referred to as ‘programmed
cell death’ or ‘cell suicide’ [2]. There are extrinsic and intrinsic sig-
naling pathways [3], the former is mediated by death receptor on
the cell membrane, activating caspase-8, -10 [4]; the latter works
by activating casapse-9, which is regulated by gene family Bcl-2
[5]. Generally, both ways can lead to the activation of caspase-3,
DNA fragmentation, and finally cell death [6].
quent injury to surrounding cells and tissues [8]. By contrast,
apoptotic cell removal is more efficient and non-inflammatory
[9]. Given the above reasons, more and more studies were focused
on the tumor cell apoptosis, in order to achieve good control of
cancer while with limited detriment to normal cell function.
In recent years, C21 steroidal glycosides have been receiving
increasing attentions because of their bioactivities, including
anti-tumor effects. Extensive evidences confirmed that many C21
steroidal glycosides resist cancer by inducing apoptosis [10–15].
Cynanchum stauntonii is a perennial herb belonging to the genus
Cynanchum in the family Asclepiadaceae. It has been reported to
be rich in C21 steroidal glycosides. Cytotoxicity evaluation showed
Tumor cells can be eliminated by a number of alternative
mechanisms including necrosis and apoptosis, which can be the
that glaucogenin C-mono-
D-thevetoside and neocynapanogenin
⇑
Corresponding authors at: Department of Natural Medicinal Chemistry & State
F-mono- -thevetoside had obvious cytotoxicity in HeLa and
D
Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24,
Tongjiaxiang, Gulou District, Nanjing 210009, Jiangsu Province, China (Z.-Q. Yin).
Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional
Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing 210028, Jiangsu
Province, China (J. Zhang).
Bel-7402 cell lines, respectively [16]. However, there are few
reports about the cytotoxicity of C21 steroidal glycosides from
C. stauntonii, and the potential mechanism is also not clear.
In the present report, we describe the investigation of the C21
steroidal glycosides from C. stauntonii, activity evaluation against
E-mail addresses: cpu-yzq@cpu.edu.cn (Z.-Q. Yin), zjwonderful@hotmail.com
(J. Zhang).
1
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.steroids.2015.12.008
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
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Z.-Q. Yin et al. / Steroids xxx (2015) xxx–xxx
Table 1
A549, HepG2 and 4T1 cell lines, as well as the possible mechanism
of inducing apoptosis.
1H and 13C NMR data for aglycone of compounds 1 (CD3OD, J in Hz) and 2 (CDCl3, J in
Hz).
Position
1
2
2. Experimental
a
b
a
b
dC
dH
dC
dH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
37.54
30.54
79.92
39.70
141.69
121.31
30.85
54.47
41.70
39.40
24.62
29.07
119.43
176.99
68.51
76.50
57.10
144.48
18.33
115.60
24.87
1.07 (m), 2.04c
1.68 (m), 1.94 (m)
3.52 (m)
36.60
29.54
78.04
38.89
140.40
120.36
29.54
53.05
40.56
38.74
23.77
28.13
118.25
175.84
67.70
75.23
55.90
143.46
18.10
114.06
24.71
1.09 (m), 1.95 (m)
1.61 (m), 2.03c
3.57 (m)
2.1. General methods
1.65 (m), 2.20 (m)
1.65 (m), 2.20c
IR spectra were recorded on a Nicolet Impact 410 spectropho-
tometer. UV spectra were recorded using a UV-2500PC spectrome-
ter (Shimadzu, Japan). Optical rotations were measured with a
JASCY-1020 polarimeter. 1H and 13C NMR spectra were obtained
on a Bruker Avance-300 (1H NMR 300 MHz, 13C NMR 75 MHz)
and Bruker Avance-500 (1H NMR 500 MHz, 13C NMR 125 MHz).
The HR-ESI-MS analysis were performed on a SynaptTM Q-TOF
(Waters, USA). ESI-MS spectra were measured on a HP-1100
LC/EST spectrometer. Silica gel (100–200 mesh, 200–300 mesh,
Qingdao Marine Chemical Group Co., China) and Sephadex LH-20
(Pharmacia, Sweden) were employed for column chromatography.
All of the solvents and materials were of reagent-grade and
purified as required.
5.42 (d, 5.1)
1.35, 2.00c
1.17
5.38 (d, 4.6)
1.23c, 2.06c
1.33c
2.43 (m)
2.44 (m)
1.25c, 1.29c
1.25c, 1.31c
2.04c, 2.45 (m)
2.04c, 2.44 (m)
3.72 (m), 4.18 (t, 7.3)
5.27 (dd, 9.6, 7.1)
3.41 (d, 7.6)
6.30 (s)
3.84 (m), 4.15 (m)
5.31 (dd, 9.7, 7.7)
3.44 (d, 7.86)
6.25 (s)
0.95 (s)
0.92 (s)
1.47 (s)
1.53 (s)
2.2. Plant material
a
b
c
Measured at 75 MHz.
Measured at 300 MHz.
Overlapped signals.
The whole fresh plants of C. stauntonii were collected in Hubei,
China in September 2013, and authenticated by Prof. Minjian Qin
(China Pharmaceutical University).
A
voucher specimen
(No. 20130911) was deposited in the herbarium of China Pharma-
ceutical University, Nanjing, China.
Table 2
1H and 13C NMR data for sugars of compounds 1 (CD3OD, J in Hz) and 2 (CDCl3, J in
Hz).
2.3. Extraction and Isolation
Position
1
2
The air-dried whole fresh plant of C. stauntonii (10 kg) was
powdered and refluxed with 80% EtOH (3 ꢀ 10 L) for three times
to yield a semi-solid residue (450 g). The extract was dispersed
in H2O, partitioned with EtOAc (3 ꢀ 5 L) and n-BuOH (3 ꢀ 5 L), to
afford EtOAc fraction (130 g), n-BuOH fraction (210 g), and aqueous
fraction (110 g), respectively. The EtOAc fraction was subjected to a
silica gel column (100 ꢀ 8 cm) eluted with a gradient system of
CH2Cl2–MeOH (50:1 ? 0:1, 2 L each), to yield fraction A (52.3 g),
B (12.7 g), C (10.4 g), and D (11.9 g). Fraction A was further sub-
jected to silica gel (50 ꢀ 8 cm) eluted with petroleum ether-EtOAc
(20:1 ? 1:1, 1 L each), followed by a Sephadex LH-20 CC (eluted
with MeOH) and RP-HPLC (210 nm, MeOH:H2O, 80:20, 10.0 mL/
min) to afford compounds 1 (14.1 mg, tR 38.5 min), 3 (903 mg, tR
a
b
a
b
dC
dH
dC
dH
D-The
102.39
74.77
85.97
83.39
74.38
18.07
60.49
D-Canaro
98.05
38.70
69.91
88.62
70.55
10
4.33 (d, 7.6)
3.24 (m)
3.16c
4.57 (dd, 9.6, 1.2)
1.61c, 2.21c
3.61 (m)
2.94 (m)
3.28 (m)
20
30
40
3.14c
50
3.19c
60
1.27 (s)
3.63 (s)
17.98
1.26 (d, 6.0)
-OMe
D-Digit
99.88
39.79
69.27
72.34
70.90
18.62
D-Digit
99.36
37.82
68.17
72.77
69.87
17.98
100
200
300
400
500
600
4.95 (dd, 8.2, 1.4)
1.65c, 2.00c
4.00 (m)
3.19 (m)
3.77 (m)
4.82 (dd, 9.5, 0.8)
1.83c, 2.33c
4.10c
3.38 (m)
3.87 (m)
15.6 min), and
4 (53.2 mg, tR 82.5 min). Fraction B was
1.25 (s)
1.33 (d, 6.0)
chromatographed on a silica gel column (50 ꢀ 8 cm) eluted with
CH2Cl2–MeOH in gradient (50:1 ? 0:1, 1 L each), then a Sephadex
LH-20 CC (MeOH) and purified by RP-HPLC (210 nm, MeOH:H2O,
a
b
c
Measured at 75 MHz.
Measured at 300 MHz.
Overlapped signals.
82:18, 10.0 mL/min) to afford compounds
2 (31.5 mg, tR
32.1 min), and 5 (28.6 mg, tR 58.5 min).
2.3.1. Compound 1
2.3.3. Anhydrohirundignin-3-O-b- -monothevetoside (3)
D
Compound 1: white powder; [a]
20 + 18.1 (c 0.015, MeOH); UV
D
White needle crystal; [
(c 0.241, CH3OH)].
a]
20-26.1 (c 0.126, CH3OH) [lit. 17: ꢁ22.8
D
(MeOH) kmax: 205, 284 nm; IR mmax cmꢁ1: 3443, 2936, 1734,
1652, 1453, 1386, 1165, 1073; 1H NMR and 13C NMR (CD3OD) data
see Tables 1 and 2; HR-TOF-MS m/z: 685.3009 [M+Cl]ꢁ (calcd. for
2.3.4. Glaucogenin C-3-O-b-
D
-monothevetoside (4)
C34H50O12Cl, 685.2991).
White needle crystal; [a]
20 + 23.5 (c 0.126, CHCl3) [lit. 18:+27.4
D
(c 0.103, CHCl3)].
2.3.2. Compound 2
Compound 2: white needle crystal; [a]
20 + 20.1 (c 0.126, CHCl3);
D
UV (MeOH) kmax: 240, 273 nm; IR mmax cmꢁ1: 3409, 2932, 1740,
1660, 1437, 1371, 1160, 1063; 1H NMR and 13C NMR (CDCl3) data
see Tables 1 and 2; HR-TOF-MS m/z: 655.2909 [M+Cl]ꢁ (calcd. for
2.3.5. Glaucogenin C-3-O-b-
digitoxopyranosyl-(1 ? 4)-b-
D
-cymaropyranosyl-(1 ? 4)-b-
D-
D
-thevetopyranoside (5)
White amorphous powder; [a]
20 + 13.8 (c 0.126, CH3OH) [lit.
D
C33H48O11Cl, 655.2885).
19:+17.7 (c 1.14, CH3OH)].
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3
the
digit
21
Me
Me
O
O
O
HO
HO
O
Ra =
Rb =
Rc =
Rd =
18
20
16
O
17
MeO
19
OH
15
OH
digit
H
H
14
O
canaro
O
Me
3
H
Me
O
RO
O
HO
6
1 R = Ra
2 R = Rb
4 R = Rc
5 R = Rd
OH
Me
the
O
HO
MeO
18
O
21
OH
17
20
19
O
O
H
16
H
14 O
digit
O
cym
O
the
O
15
Me
Me
H
Me
H
3
HO
O
O
RO
6
MeO
OMe
OH
OH
3 R = Rc
Fig. 1. Chemical structures of compounds 1–5.
O
O
O
O
O
O
H
H
O
H
H
O
H
H
H
Me
Me
Me
Me
O
O
O
O
O
HO
O
HO
O
O
HO
MeO
H
OH
H
OH
H
H
OH
Fig. 2. Key HMBC correlations of compounds 1 (left)-2 (right).
H
H
Me
Me
O
O
Ra =
Rb =
HO
O
O
O
17
H
MeO
M1e9
H
H
H
H
OH
O
H
OH
H
H
H
H
Me
16
H1
H
O
15
H
O
H
H
H
4
H
Me
H
H
O
Me
O
O
H
HO
O
O
H
HO
H
H
OH
H
H
H
Fig. 3. Key NOESY correlations of compounds 1–2.
8:2, v/v) to give the monosaccharides,
ose (3 mg) and
tion of these sugars was determined and compared to the
literature values for [
20 + 43.5 (c 0.25, H2O) for D-digit (lit 20,
21:+45),+25 (c 0.10, H2O) for D-canaro (lit 20, 21:+22.8) and +55
(c 0.43, H2O) for D-the (lit 22:+42.3).
D
-digitoxose (9 mg),
D
-canar-
Table 3
Cytotoxicity of compounds 1–5 on three tumor cells.
D
-thevtose (7 mg), respectively. The specific rota-
Cell lines
IC50
a
]
D
1a
2a
3a
4a
5a
4T1
A549
HepG2
Nsb
Ns
Ns
Ns
Ns
Ns
Ns
Ns
Ns
44.12
26.82
12.24
Ns
Ns
Ns
a
2.5. Cell cytotoxicity assay
l
M.
b
Not significant: >100
l
M.
2.5.1. Cell lines and cell culture
Human lung cancer cells A549, human liver cancer cells HepG2
and rat breast cancer cells 4T1, were obtained from BD (USA). The
cell lines were cultured in medium (RPMI1640) with 10% FBS at
37 °C in a humidified incubator with 5% CO2.
2.4. Acid hydrolysis of compounds 1ꢁ2
The glycosides (500 mg) were hydrolyzed within MeOH (50 mL)
at 60 °C for 5 h, and 0.05 M H2SO4 (50 mL). Then the mixture was
neutralized with saturated Ba(OH)2 in H2O. After the precipitate
was removed by filtration, the filtrate was partitioned with CHCl3.
The aqueous layer was concentrated and separated by silica gel CC
eluting with CHCl3–MeOH (8:1, v/v) and then further fractionated
by RP-18 CC using a stepwise gradient of MeOH–H2O (6:4, 7:3 and
2.5.2. MTT assay
Briefly, cells were seeded in 96-well plate at a density of
5 ꢀ 103 cells/well. After overnight incubation, a serial of com-
pounds 1–5 (0.1, 1, 10, 20, 40, 60, 80, 100 lM) were added to wells
(triplicate for each concentration). A blank control (wells without
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Z.-Q. Yin et al. / Steroids xxx (2015) xxx–xxx
cells), a negative control (cells treated with culture medium), and a
positive control (cells employed cisplatin), were included. After
48 h of incubation, all cells were cultured with MTT (5 mg/mL in
48 h treatment, the cells were washed twice with PBS, fixed in
70% ethanol, then stayed overnight. The samples were finally trea-
ted with 1% (v/v) Triton X-100 and 0.01% RNase and stained with
0.05% propidium iodide for 30 min in darkness.
PBS) for 4 h. After adding 150 lL of DMSO, the OD value of
each well was determined at a wave length of 490 nm. Relative
cell proliferation inhibition rate was calculated using the
following equation: rate (%) = (ODcontrol group ꢁ ODexperimental group)/
(ODcontrol group ꢁ ODblank group) ꢀ 100%.
2.5.5. Western blot analysis
Apoptosis related protein analysis was analyzed by western
blotting to detect the proteins of caspase-3, -8, -9, Bcl-2, Bax. The
control and different concentrations of compound 4 (0, 1, 10,
2.5.3. Cell apoptosis analysis
The Annexin V-PI kit was used according to the manufacturer
instructions (Bipec, USA). HepG2 cells (1 ꢀ 106) were seeded into
24-well culture plates and the cells were treated with medium
overnight, followed by a treatment of compound 4 (0, 1, 10,
20 lM) treated HepG2 were used. Cell lysates were prepared in
RIPA lysis buffer (Beyotime Institute of Biotechnology, China) and
protein concentrations were determined by using a BCA Protein
Assay Kit (Thermo, USA) according to the manufacturer’s instruc-
20 lM) and cisplatin (20 lM). After 48 h of treatment, all cells
were Cells were analyzed by flow cytometry within 1 h
(FACSCalibur, Becton Dickinson, USA).
tions. Samples containing 50 lg proteins were electrophoresed
on SDS–PAGE, transferred onto PVDF membranes (Millipore,
USA) and blocked in 5% non-fat milk. The membranes were subse-
quently incubated with the corresponding primary antibodies for
caspase-3, 8, 9, Bcl-2, and Bax (Abcam, Britain). After washing with
TBS-T, the membranes were incubated with the secondary anti-
body for 1 h. The proteins were visualized using Supersignal West
Pico Trial kit (Pierce, USA) and exposed to Kodak X-ray films.
2.5.4. Cell cycle analysis
Cell cycle analysis was carried out by a flow cytometry (Becton
Dickinson FACScanTM, CA, USA). Briefly, HepG2 were seeded,
harvested and treated with compound 4 (0, 1, 10, 20 lM). After
Fig. 4. Compound 4 decreases the cell viability in HepG2. HepG2 cells were treated with different concentrations of compound 4 for 48 h.
Fig. 5. Compound 4 induces apoptosis in HepG2 cells. (A) The HepG2 cells were treated with different concentrations of 4 for 48 h and the Annexin V/PI assay was used to
detect apoptosis using flow cytometry. (B) Representative histograms for apoptotic rate in HepG2 cells.
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Fig. 6. Compound 4 induced cell cycle arrest at G1 phase in HepG2 cells. (A) HepG2 cells were treated with indicated concentrations of 4 for 48 h. Cell cycle distribution was
measured by flow cytometry. (B) Representative histograms for cell cycle distribution in HepG2 cells.
2.6. Statistical analysis
Compound 1 was obtained as white powder (CH3OH). The
molecular formula was determined as C34H50O12 by HR-TOF-MS
(m/z 685.3009 [M+Cl]ꢁ, calcd for C34H50O12Cl, 685.2991). The IR
spectrum exhibited absorptions at 3444 cmꢁ1 (hydroxyl),
1734 cmꢁ1 (carbonyl) and 1652 cmꢁ1 (olefinic). The 1H NMR spec-
trum showed characteristic shift values of a aglycone of glauco-
Experimental values were expressed as mean SD. A two-tailed
Student’s-t-test was used to examine the significance of the data. A
statistically significant difference was considered at *P < 0.05,
**P < 0.01 and ***P < 0.001. All calculations were performed using a
JMP 5.1 program (SPSS Statistics 17.0 (SPSS Inc., Ireland)).
genin C-3-O-b-D-monothevetoside [16] with two olefinic protons
at dH 5.42 (1H, d, J = 5.1 Hz), 6.30 (1H, s) and two methyls at dH
0.95 (3H, s) and 1.47 (3H, s). Furthermore, the 13C NMR spectrum
also exhibited the corresponding carbon signals from the aglycone.
The 1H NMR spectrum also displayed signals corresponding to two
anomeric protons at dH 4.33 (d, J = 7.6 Hz) and dH 4.95 (dd, J = 8.2,
1.4 Hz), which correlated with the anomeric carbon signals at dC
102.4 and dC 99.9 in the 13C NMR spectrum. The NMR data of the
sugar units (Table 2) suggested the presence of a digitoxopyranose
and a thevetopyranose, and the sugars were believed to be in the
D-form on the basis of their optical rotation values. Their anomeric
3. Results and discussion
In this study, two new (1–2) and three known (3–5) C21
steroidal glycosides were identified from C. stauntonii. Their anti-
proliferation activity and action mechanisms on three cancer cell
lines (A549, HepG2 and 4T1) were investigated.
3.1. Structural elucidation of isolated compounds
0
00
The 80% EtOH extract of C. stauntonii was suspended in H2O, and
partitioned into EtOAc and n-BuOH-fractions. Five steroid
glycosides (1–5) (Fig. 1) were obtained from EtOAc fraction.
configurations were all determined as b based on their JH-1 ,H-1
coupling constants and the 13C NMR data of the sugar units
(Table 2).
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Z.-Q. Yin et al. / Steroids xxx (2015) xxx–xxx
Fig. 7. Effect of compound 4 on protein expressions of apoptosis related genes of HepG2 tumor cells. (A) The protein level of caspase-3, -8, -9, Bax and Bcl-2 was examined by
western blot assay, and b-actin was used as a loading control. (B) The histogram shows that there were significant increases of caspase-3, -9, Bax, significant decrease of Bcl-2
in dose-dependent manners after drug treatment.
In the HMBC spectrum, correlations of The-H-10 (d 4.33) with C-
3 (d 79.9), and Digit-H-100 (d 4.95) with The-C-40 (d 83.4) were
observed, indicating the linkage as shown in Fig. 2. In addition,
the correlations observed in the NOESY experiment also supported
the proposed relative configuration of 1, in which NOESY correla-
tions of H-19 and H-1b and H-4b, of H-16 and H-17 and H-15,
H-10ax and H-30ax and H-50ax, of H-100ax and H-500ax indicated the ori-
entation of the aglycone and two sugars (Fig. 3). Thus, the structure
Thus, the structure of compound 2 was determined to be glauco-
genin C-3-O-b- -digitoxopyranosyl-(1 ? 4)-b- -canaropyranoside.
Additionally, the known compounds were identified to
be anhydrohirundignin-3-O-b- -monothevetoside (3) [17],
glaucogenin C-3-O-b- -monothevetoside (4) [16], glaucogenin
C-3-O-b- -cymaropyranosyl-(1 ? 4)-b- -digitoxopyranosyl-(1 ? 4)-
b- -thevetopyranoside (5) [19], on the basis of the NMR data
D
D
D
D
D
D
D
comparison.
of 1 was established as glaucogenin C-3-O-b-D-digitoxopyranosyl-
(1 ? 4)-b- -thevetopyranoside.
D
3.2. Cell cytotoxicity assay
Compound 2 was isolated as white needle crystals (CHCl3), mp
232–234 °C. The molecular formula was determined as C33H48O11
by HR-TOF-MS at m/z 655.2909 [M+Cl]ꢁ (calcd for C33H48O11Cl,
655.2885). The IR spectrum showed the absorptions of
3409 cmꢁ1 (hydroxyl), 1739 cmꢁ1 (carbonyl) and 1660 cmꢁ1 (olefi-
nic). A detailed NMR data comparison of compounds 2 with 1
revealed that they shared the same aglycone moiety. The 1H
NMR spectrum showed the anomeric proton of two 6-deoxysugar
at dH 4.57 (dd, J = 9.6, 1.2 Hz) and dH 4.82 (dd, J = 9.5, 0.8 Hz), which
correlated to the corresponding anomeric carbon signals at d 98.0
and d 99.4 in the 13C NMR spectrum. The 1H-1H COSY experiment,
combined with the HSQC spectrum, established the connection
3.2.1. MTT assay
The MTT assay was employed to evaluate the cytotoxicity of
compound 1–5 on tumor cells. The results were summarized in
Table 3 and Fig. 4. Compound 4 could obviously inhibit the cell
growth in a dose-dependent manner, and be most active against
HepG2 with a IC50 value of 12.24
lM, while the IC50 of cisplatin
is 21.51 M, indicating that HepG2 was slightly more sensitive to
l
compound 4. Therefore, compound 4 was selected for further
mechanism study on HepG2 cell line.
3.2.2. Compound 4 induces apoptosis in HepG2 Cells
within the sugar moiety. The presence of a b-D-canaropyranose
Induction of apoptosis and cell cycle arrest are two main ways
for cell growth inhibition [23]. Annexin V/PI staining was
employed to discriminate among normal cells (Annexin Vꢁ/PIꢁ),
early apoptotic cells (Annexin V+/PIꢁ), late apoptotic cells
(Annexin V+/PI+), and necrosis cells (Annexin Vꢁ/PI+). As shown
in Fig. 5, compound 4 could increase the number of apoptosis cells
and a b- -digitoxopyranose were determined by their optical rota-
D
tion values, which were also supported by the NOE correlations of
H-10ax and H-30ax and H-50ax, of H-100 and H-500 in the NOESY
ax
ax
spectrum (Fig. 3). The linkage of the two sugars were deduced by
the correlations between Canaro-H-10 (d 4.57) and C-3 (d 78.0),
Digit-H-100 (d 4.82) and Canaro-C-40 (d 88.6) in the HMBC spectrum.
in
a concentration-dependent manner. After 48 h treatment,
Please cite this article in press as: Z.-Q. Yin et al., C21 steroidal glycosides from Cynanchum stauntonii induce apoptosis in HepG2 cells, Steroids (2015),
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Z.-Q. Yin et al. / Steroids xxx (2015) xxx–xxx
7
compound 4 at 1
late apoptosis, while 55.91% early apoptosis and 12.12% late apop-
tosis at 10 M, 63.18% early apoptosis and 15.39% late apoptosis at
20 M. These results suggested that the growth inhibitory effect of
l
M caused 52.60% early apoptosis and 11.89%
Wen-Bin Shen (China Pharmaceutical University) for NMR
measurements. This work was partially supported by grants
awarded from the National Natural Science Foundation of China
(No. 81001379), the Ninth Batch of ‘‘Six Talent Peaks” Project of
Jiangsu Province (No. 2012-YY-008), and A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Educa-
tion Institutions.
l
l
compound 4 was partly due to apoptosis of HepG2 cells.
3.2.3. Compound 4 induced cell cycle arrest at G1 phase in HepG2 cells
To determine whether the anti-proliferative effect is related to
cell cycle arrest, we examined the cell cycle changes after drug
treatment. As shown in Fig. 6, compound 4 induced cell cycle arrest
at G1 phase. The percentage of cells in G0-G1 phase increased from
68.80% as the control to 82.27% treated at the highest dose
Appendix A. Supplementary data
HRTOFMS and NMR spectra of compounds 1–5 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.2015.12.008.
(20 lM). Meanwhile, the apoptotic cell rate increased from 0.43%
to 12.62%. These were accompanied with a decreased percentage
of cells in S and G2-M phase. These results indicated that com-
pound 4 exerted its growth suppressive activity by perturbing cell
cycle progression.
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Compound 4 could significantly inhibit the cell growth, induc-
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HepG2 cells in vitro. The caspases also participate in this process.
The present data provides a scientific support for the potential uti-
lization of C. stauntonii as a new source of lead compounds for can-
cer therapy.
Acknowledgments
The authors thank Prof. Min-Jian Qin (China Pharmaceutical
University) for authenticating the plant material and Prof.
Please cite this article in press as: Z.-Q. Yin et al., C21 steroidal glycosides from Cynanchum stauntonii induce apoptosis in HepG2 cells, Steroids (2015),
http://dx.doi.org/10.1016/j.steroids.2015.12.008