Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cynanchum atratum

Article abstract of DOI:10.1016/j.steroids.2017.03.004

Two new (1–2) and two known C₂₁ steroids (3–4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MTT assay showed that compounds 1–4 displayed obvious cytotoxic activities against HepG2 cells with IC₅₀ values ranging from 10.19?μM to 76.12?μM. Compounds 1–3 also exhibited cytotoxic effects in A549 cells with IC₅₀ values of 30.87–95.39?μM. Compound 3 showed the antiproliferative activity via G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis. Western blotting analysis revealed that compound 3 could induce HepG2 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 expression, upregulating Bax protein expression, and activating caspase-9 and caspase-3.

Full text of DOI:10.1016/j.steroids.2017.03.004

Accepted Manuscript
Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cyn-
anchum atratum
Jian Zhang, Lin Ma, Zheng-Feng Wu, Shu-Le Yu, Lei Wang, Wen-Cai Ye,
Qing-Wen Zhang, Zhi-Qi Yin
PII:
S0039-128X(17)30039-9
http://dx.doi.org/10.1016/j.steroids.2017.03.004
STE 8086
DOI:
Reference:
Steroids
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25 November 2016
21 February 2017
13 March 2017
Please cite this article as: Zhang, J., Ma, L., Wu, Z-F., Yu, S-L., Wang, L., Ye, W-C., Zhang, Q-W., Yin, Z-Q.,
Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cynanchum atratum, Steroids (2017),
doi: http://dx.doi.org/10.1016/j.steroids.2017.03.004
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Cytotoxic and apoptosis-inducing activity of C21 steroids from the
roots of Cynanchum atratum
Jian Zhanga,b,1, Lin Maa,c,1, Zheng-FengWua,c, Shu-Le Yua,c, Lei Wangd, Wen-Cai Ye
d, Qing-Wen Zhange, Zhi-Qi Yinc*
aLaboratory of Translational Medicine, Jiangsu Province Academy of Traditional
Chinese Medicine, Nanjing, P.R.China
bAffiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing
University of Chinese Medicine, Nanjing210028, China,
cDepartment of Natural Medicinal Chemistry & State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing, P.R.China
dInstituteof Traditional Chinese Medicine and Natural Products & Guangdong
Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese
Medicine and New Drugs Research, Jinan University, Guangzhou, P.R.China
Corresponding authors at: Department of Natural Medicinal Chemistry & State Key
Laboratory of Natural Medicines, China Pharmaceutical University, No. 24,
Tongjiaxiang, Gulou District, Nanjing 210009, Jiangsu Province, China (Z.-Q. Yin).
E-mail addresses: cpu-yzq@cpu.edu.cn(Z.-Q Yin)
ABSTRACT
Two new(1-2) and two known C21steroids (3-4) were isolated from the roots of
Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D
spectroscopic.The MTT assay showed that compounds 1-4 displayed obvious
cytotoxic activities against HepG2 cells with IC50 values ranging from 10.19µM to

76.12µM. Compounds 1-3 also exhibited cytotoxic effects in A549 cells with IC50
values of 30.87-95.39 µM. Compound 3 showed the antiproliferative activity via
G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis.
Western blotting analysis revealed that compound 3 could induce HepG2 cell
apoptosis via the mitochondrial pathway by downregulating Bcl-2expression,
upregulating Bax protein expression, and activating caspase-9 and caspase-3.
Keywords
Cynanchum atratum, C21 steroids, cytotoxic activity, apoptosis, mitochondrial
pathway

1.Introduction
Apoptosis is a polygenetically control process of programmed cell death that may
occur in multicellular organisms[1].Both Bcl-2 and caspase family proteins are key
regulators of apoptosis[2, 3]. Generally, the normal development of organizations is
maintained by the balance between cell proliferation and apoptosis. However, the
normal apoptosis process inhibition may lead to the cancer occurrence [4].
Accumulated evidences have supported that anticancer drugs exert their cytotoxic
effects mainly by inducing tumor cell apoptosis[5]. Nowadays over 60% anticancer
drugs are from natural resources [6]. Therefore, more research are focused on finding
natural products with apoptosis-inducing activity.
Cynanchum atratum (Asclepiadaceae) is a perennial herb native to East Asia. Its
roots, commonly called ‘BaiWei’, have been used as an anti-febrile and diuretic
herbal medicine in China [7]. C. atratum has been reported to be rich in C21 steroids
[8-10]. The pharmacological investigations indicate that C21 steroidal glycosides have
antineoplastic, immunomodulating, neuroprotective and appetite suppressing
activities[11-13]. Furthermore, cynanosides L, M, R1, R2 isolated from C.atratum
displayed significant cytotoxic activities against HL-60 cell lines, as well as
cynaversicoside A against 212 cell lines [14-15]. However, the antineoplastic
mechanism of C21 steroids from C.atratum is not clear yet.
In the present study, we report the isolation and structural elucidation of two new
and two known C21steroids (1-4) from C.atratum. Their cytotoxic activities were
further evaluated in A549 and HepG2 cell lines. We also explored its potential
apoptosis-inducing mechanisms in HepG2 cells.
2.Experimental
2.1.General methods
Optical rotations were determined by a Jasco P-1020 digital polarimeter in a 0.1
dm length cell. The UV spectra were recorded on a Beckman DU-640
spectrophotometer in MeOH.IR spectrawere obtained on a Nicolet Impact 410 FT-IR
spectrophotometer with KBr disc. NMR spectra were measured on a BrukerAvance

DRX-500(1H NMR 500 MHz,13C NMR 125 MHz) and DRX-300(1H NMR 300
MHz,13C NMR 75 MHz) . HR-ESI-MS analyses were carried out on performed on a
SynaptTMQ-TOF(Waters, USA). Analytical HPLC was performed on a Agilent 1260
chromatograph equipped with a Alltech 3300 evaporative light-scattering
detector(ELSD). Semipreparative HPLC was performed on a Alliance system
consisting of a Waters-Pack ODS column (250 × 20 mm, dp 5 µm). Column
chromatography was performed with silica gel (200-300 mesh, Qingdao Marine
Chemical, Inc.) and octade-cylsilanizedsilica gel (ODS) (YMC Co. Ltd.).
2.2.Plant material
The roots of C.atratum were collected in Hubei, China in March 2013, and
authenticated by Prof. Minjian Qin (China Pharmaceutical University). A voucher
specimen (No.20130328) has been deposited in the herbarium of China
Pharmaceutical University, Nanjing, P. R. China.
2.3.Extraction and isolation
The air-dried root barks of C. atratum (10 kg) were pulverized and extracted with
80% EtOH (3×10L, each 2 h) under reflux, then concentrated under vacuum to give a
residue (2.01 kg). This residue was suspended in water (5L) and successively
extracted with petroleum ether (5 L×3) and EtOAc (5 L×3). The EtOAc solution was
concentrated to afford a residue (290 g), which was separated by silica gel column
chromatography（2.5 kg,column:100×8cm）eluted with CHCl2-CH3OH (100:1, 80:1,
60:1, 40:1, 20:1, 10:1, 1:1, 2L each) to obtain five fractions (Fr.A-E) based on TLC
analysis. Fraction D (80 g) was chromatographied on silica gel (1.6 kg,
column:50×8cm) and eluted with CHCl3-CH3OH (20:1, 15:1, 10:1, 1:1,1L each) to
yield four subfractions (Fr.D1-D4). Fraction D3 (13g) was separated by ODS column
chromatography (250g, column:30×4cm) and eluted with CH3OH-H2O (40:60,5L) to
yield compounds 1 (35 mg, between 4400 and 4600 mL), 2 (20 mg, between 2600 and
2800 mL) and 3 (24 mg, between 400 and 700 mL). Fraction C (16 g) was first
separated by ODS column chromatography (250g, column:30×4cm) eluting with

CH3OH-H2O (50:50,3L) and collection started from 1400 mL to 1800 mL to give
Fraction C1(1.6g). Compound 4 (20.6 mg) were purified from fraction C1 by using
semipreparative HPLC with an isocratic gradient of 30% CH3OH in H2O over 30.0
min (flow rate: 5 mL/min; conpound4: tR = 20.1 min).
2.3.1.compound 1
3β,5α,6β-trihydroxyl-15,20:18,20-diepoxy-13,14:14,15-disecopregna-13(18)-eno-
14-oicacid-16β-lactone (1): colorless amorphous powder; [α]2D7 : -11.8(c0.1,MeOH); IR
( KBr)νmax: 3420, 2938, 1724, 1654, 1077 cm-1; 1H NMR and 13C NMR date see
Tables 1 and 2; HR-TOF–MS m/z:395.2064 [M + H]+ (calcd. for C21H32O7,
395.2069).
2.3.2 .compound 2
2α, 3β, 13α, 18α-tetrahydroxyl-15,20:18,20-diepoxy-13,14:14,15-disecopregna-5-
eno-14-oicacid-16β-lactone3-O-α-D-oleandropyranosyl-(1→4)-β-D-digitoxo-pyranos
yl-(1→4)-β-D-oleandropyranoside(2): colorless amorphouspowder; [α]2D7 :
-13.6(c0.1,MeOH); IR (KBr)νmax: 3439, 2934, 1740, 1637, 1061 cm-1; 1H NMR and
13C NMR date see Tables 1 and 2; HR-TOF–MS m/z: 827.4068 [M - H]- (calcd. for
C41H63O17, 827.4065).
2.3.3.compound 3
GlaucogeninC-3-O-β-D-glucopyranosyl-(1→4)-α-D-oleandropyranosyl-(1→4)-β
-D-digitoxopyranosyl-(1→4)-β-D-oleandropyranoside (3):White needle crystal; [α]2D7 :
-18.6(c1.0,MeOH)[lit. 16:-20.0°(c 1.5, CH3OH)].
2.3.4.compound 4
→2
3β,14β-dihydroxy-pregn-5-en-20-one3-O-β-D-(6-sinapoyl)-glucopyranosyl-(1
)-β-D-glucopyranoside (4):White needle crystal; [α]2D7 : +6.8(c0.1,MeOH)[lit.
17:+8.7(c0.229, CH3OH)].

2.4. Acid hydrolysis of compound 2
Compound 2 (1 mg) was hydrolyzed within MeOH (0.5 mL) and 0.05 M
H2SO4(0.5 mL) at 60°C for 1.5 h. The mixture was evaporated under vacuum and the
residue was partitioned between CHCl3 andH2O. The aqueous phase was concentrated
and dissolved in 1-(trimethylsilyl)-imidazole and pyridine(0.1mL). Then, the solution
was stirred at 60 °C for 5 min and dried with a stream of N2.The residue was
partitioned betweenCHCl3and H2O. The organic layer was analyzed by GC using an
I-Chirasil-Val column. The sugar moietiesof2 were identified as D-oleandrose and
D-digitoxose in a ratio of 2:1 by comparing their GC retention times of the
corresponding trimethylsilylated hydrolysate with the authentic samples. Retention
times for authentic samples were 11.24 min (D-oleandrose) and 12.56 min
(D-digitoxose).
2.5. Cell cytotoxicity assay
2.5.1. Cells and cell culture
Lung (A549) and hepar (HepG2) cancer cells lines were purchased from KeyGEN
and cultured in RPMI Medium 1640 (GIBCO Industries, Inc.), 10% heat-inactivated
fetal bovine serum (FBS), at 37 °C in a humidified atmosphere of 95% air and 5%
CO2.
2.5.2.Cell proliferation assay
Cell viability was assessed through MTT assay. The cells were seeded in 96-well
plates at 5 × 103 cells/100 µL/well. The day after, different concentrations of
compounds 1-4 (1, 10, 20, 40, 60, 80 and 100 µM) and cisplatin (25 µg/mL) used as
positive control were added to the cells and incubated at 37°C in a humidified
atmosphere of 5% CO2 for 48 h. At the end of incubations, 10 µL of MTT (10 mg/ml)
was added to the wells and incubated for at 37°C for 3h. Absorbance was recorded at
490 nm using Synergy H1 hybrid reader (Biotek). All the experiments were repeated
at least thrice independently. The growth inhibition was assessed using the following
formula：Inhibition rate (%) = (1 - ODexp/ODcon) × 100% where ODexp and ODcon are
the optical densities of the experimental groups and normal control group,

respectively.
2.5.3.Cellcycle analysis by flow cytometry
Cell cycle parameters were analyzed by flow cytometry. After induction treatment
under 0, 1, 10, 20 µM of compound 3, HepG2 cells were fixed with ethanol and then
stained with propidiumiodide after removing the RNA in the cells by RNase
treatment,and the fluorescence of individual nuclei (about 10,000 events)was
analyzed by flow cytometry (Becton Dickinson FACScanTM). The percentage of
cells in the G0/G1, S, G2 and subG1 phases of the cell cycle was determined.
2.5.4 .In vitro apoptosis analysis by flow cytometry
Apoptosis was detected by annexin V-FITCbinding assay. Normal, early apoptotic,
late apoptosis and necroticcells were distinguished by using the annexin V-propidium
iodide(PI) kit according to the manufacturer instructions (Becton Dickinson). HepG2
cells were exposed to differentdoses of compound 3 (0, 1, 10, 20 µM) for
48h.Afterwashing in PBS buffer, cells were resuspended for 10 min inthe staining
solution and analyzed by flow cytometry.
2.5.5. Western blotting analysis
Western blotting assay was performed to detect the apoptosis-related proteins of
caspase-9, -8, -3, and the Bcl family. HepG2 cells were cultured in RPMI 1640
medium at themid-log phase and then incubated with compound 3 at 0, 1, 10 and 20
µM for 48 h. The treated cells were washed twice with cold PBS and lysed with RIPA
lysis buffer. The cells lysates were quantified with the BCA Protein Assay
Kit(Sunshinebio) according to the manufacturer′s instructions. In total, 50 µg of each
sample was electrophoresed using a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred onto Hybond-P polyvinylidene fluoride
membranes (Millipore). After being washed briefly with TBS-T and blocked with 5%
nonfat milk for 1 h, the membranes were washed and probed with the appropriate
antibody overnight at 4°C with shaking. After washing with TBS-T, the membranes
were incubated with 1:2000 dilutions of the appropriate secondary antibody at room
temperature for 1 h. The proteins were visualized using ECL chemiluminescence
reagents (Sunshinebio) and exposed to Kodak X-ray films.

2.6 .Statistics analysis
Statistical calculations were carried out with the SPSS 19.0 for Windows
software.All the results were expressed as mean SD. Differences were analyzed by
Student’s t-test.Differences were considered significant at * p< 0.05 and more
significant at ** p< 0.01.
3.Results and Discussion
3.1. Structural elucidation of isolated compounds
The ethanol extract from the roots of C.atratum was successively subjected to CC
over silica gel, ODS gel and semipreparative HPLC to afford four
compounds(1-4)(Fig.1) and a chromatographic comparisons of low temperature
extract and refluxing extract showed they are not the products of secondary
reactions(Fig.21S). Comparing their MS and NMR data and physical properties
with the literature,twoknown compounds were determined as glaucogenin C-
3-O-β-D-glucopyranosyl-(1→4)-α-D-oleandropyranosyl-(1→4)-β-D-digitoxopyranos
yl-(1→4)-β-D-oleandropyranoside(3)[16] and 3β, 14β-dihydroxy-pregn-5-en-20-
→2)
one3-O-β-D-(6-sinapoyl)-glucopyranosyl-(1
-β-D-glucopyranoside(4)[17].
Compound 1 was obtained as colorless crystals (MeOH). Its molecularformula
C21H30O7 was deduced from the quasi-molecularion peak atm/z395.2064 [M+H]+
(C21H31O7, calcd. 395.2069) in the HR‑ESI‑MS.The 1HNMR spectrum (Table 1) of 1
displayed signals of two angular methyl proton signals at δ1.07, 1.47(each 3H, s,
Me-19, 21),one olefinic proton signals atδ 6.28 (1H, s, H-18), three
oxygen-substituted methine protons atδ4.04 (1H, m, H-3), 3.53(1H, t, J =2.7 Hz,
H-6)and 5.33(1H, dd, J=16.9, 7.6 Hz, H-16), two oxygen-substituted methylene
protons atδ4.16(1H, m,H-15α)and 3.78 (1H, t, J =9.1 Hz, H-15β).The 13C NMR
spectra of 1 exhibited the presence of 21 carbons including one lactone
carbonylcarbon, two olefinic bonds，two methyl groups, seven methylenes, six
methines and three quaternary carbons(Table 2).These data suggestedthat the
structure of 1 was similar to that of glaucogenin C isolated from C.atratum[7], except

for 5,6-double bond had disappeared and three hydroxyl groups located in rings A and
B. In the HMBC experiment(Fig. 2),correlations between H-19 at δ 1.07 and C-5 at
δ76.0, H-1at δ 1.67 and C-3 a tδ 68.1, H-4 at δ2.02 and C-6 at δ 75.7 confirmed three
hydroxyls locating at C-3, C-5and C-6.The stereo-configurations of rings A and B in
1were determined through the ROESY experiment (Fig.2). By comparing the NMR
data of 1 with those of 3,5,6-trihydroxy steroids Cynanosides O indicated that
trans-linkage between rings A/B and the hydroxyl groups at C-5 and C-6 were located
on the α and βface[18]. The conclusion was also supported by NOESY spectrum,
correlations between H-19 at δ1.07 and two axial proton signals both H-2β at δ 1.52
and H-8 at δ 2.74 confirmed the trans-linkage between rings A/B, suggesting the
α-orientation of 5-OH. Furthermore, the β-orientation of 3-OH and 6-OH was
deduced by the correlations between H-3 at δ 4.04 and H-2α, as well as H-6 at3.53
and H-4α at δ 1.59.The conclusion was also supported by their coupling constant
values of 11.0 Hz for H-3 and 2.7 Hz for H-6.Thus, compound 1was established as
3β,5α,6β-trihydroxyl-15,20:18,20-diepoxy-13, 14:14, 15-disecopregna-13(18)-eno-
14-oicacid-16β-lactone.
Compound 2 was isolated as colorless powder. The molecular formula was
determined to be C41H64O17by its HR-ESI-MS(m/z827.4068 [M-H]-).The 1H NMR
spectrum and 13C NMR spectrum could be assigned to the aglycone moiety of 21
carbon signals includingone lactone carbonyl carbon, two olefinic bonds, two methyl
groups, six methylenes, seven methines and three quaternary carbons(Table 1 and 2).
These NMR data of 2 was similar to the aglycone of glaucogenin A[19], except that
13,18-double bond was replaced by two hydroxyl groups. The location of two
hydroxyl groups at C-13 and C-18 was confirmed by the correlations of H-17 at δ
2.56 with both C-13 at δ 89.9 and C-18 at δ105.0 in the HMBC spectrum (Fig. 3).The
ROSEY spectrum showed no correlations from H-18 to H-17 and H-21, indicating
18-OH is α-orientation(Fig. 3). The orientation of 13-OH can be deduced by the in
situ dimolybdenum CD method developed by Snatzke[20]. The positive Cotton effect
at around 310 and 400 nm observed in the induced CD spectrum revealed a positive
sign of the HO-C-C-OH(Fig. 4), indicating the α-orientation of 13-OH. The sugar

moieties of 2 were determined to be D-oleandrose and D-digitoxose in a ratio of 2:1
by acidichydrolysis and comparing their GC retention times of the corresponding
trimethylsilylated hydrolysate with the authentic samples.The detailed NMR analysis
also revealedthe presence of a β-oleandrose, a β-digitoxse and a α-oleandrose moieties
with the anomeric proton resonances at 4.64 (d, J= 9.3 Hz), 5.03 (d, J= 9.1 Hz) and
4.98 (d, J= 2.7 Hz), respectively.The HMBC correlations from H-1′′′to C-4′′，H-1′′to
C-4′and H-1′to C-3 suggested that the connection of the three sugars wasthrough (1→
4) linkage and the sugar chain was located at C-3 of the aglycone. Thus, the structure
of 2 was determined as 2α, 3β,13α,18α-tetrahydroxyl-15,20:18,20-diepoxy-
13,14:14,15-disecopregna-5-eno-14-oicacid-16β-lactone3-O-α-D-oleandropyranosyl-(
1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-oleandropyranoside.
3.2. Cell cytotoxicity assay
3.2.1. MTT assay
The growth inhibitory effects of compound 1-4on A549 and HepG2 cells were
investigated by MTT assay and cisplatin was taken as a positive control. All showed a
dose-dependent inhibitory effect on HepG2 cells(Fig. 5).However, as shown in Table
3,compounds1-3with seco-pregnane aglycone displayed significant cytotoxic
activities against A549 cellswith IC50 values of 30.87-95.39µM,whilecompound 4
with pregnane aglycone showed no activity. These findings indicated that steroids
with seco-pregnane aglycone showed better cytotoxic activity than common pregnane
steroids in A549 cells. As compound 3 showed the best inhibitory activity with an
IC50 of 9.70 µM in HepG2 cells,HepG2 cells were chosen for further mechanism
experiments.
3.2.2. Compound3 induces apoptosis in HepG2 Cells
Targeting apoptosis pathways is believed to be an efficient strategy for cancer
prevention and treatment [21]. An increasing number of evidence suggest that C21
steroids with cytotoxic effects can trigger the cancer cell apoptosis[22, 23,24].
Tenacissoside C, C21 steroid isolated from Marsdenia tenacissima, can induce
apoptosis of human hematologic neoplasm K562 cells via the mitochondrial pathway

by downregulating Bcl-2 and Bcl-xL protein expression, upregulating Bax and Bak
protein expression, and activating caspase-9 and caspase-3[25]. To investigate the
potential apoptosis-inducing mechanism of C21 steroids from C.atratum, compound 3
with different concentrations and cisplatin(25 µg/mL) as positive control was tested in
HepG2 cells,and Annexin V-FITC/PI fluorescence assays were performed to evaluate
necrotic cells as well as early stage and late stage apoptotic cells. As shown in Fig.6A,
a significant accumulation in the early and late stage apoptotic cells of compound 3(0,
1, 10, 20 µM) and cisplatin treated HepG2 cells in a dose dependent manner. The
percentage of apoptotic tumor cells was 13.29, 42.08, 58.75, 65.86 and 78.44%,
respectively(Fig.6B). These results suggested that compound 3can mediate the growth
inhibition of HepG2 cells, which is closely related with the enhanced apoptosis in
tumor cells.
3.2.3. Compound 3 induced cell cycle arrest in G0/G1 phase in HepG2 cells
Cell cycle arrestis another way for cell growth inhibition. Previous studies have
demonstrated that cell cycle is linked to cell proliferation and apoptosis[26,27].The
cell cycle results from flow cytometry showed that compound 3arrested the
HepG2cells progression in G0/G1 phase(Fig. 7). After 48 h, cells treated with 1 µM
compound 3 did not show a considerable difference from the control cells. However,
after treated with compound 3 at higher concentration, a significantly increased
amount of cells in the G0/G1-phase was observed. A subset of cells treated with 10
and 20 µM of compound 3 had undergone apoptosis, as indicated by the increase of
sub-G1 peak. Apparently, the effect of G0/G1-phase arrest is contributed to the
growth inhibition of HepG2 cells caused by compound 3.
3.2.4. Western blot analysis
The Bcl-2 family proteins significantly regulates apoptosis either as an activator
(Bax, Bak, Bad, Mtd, Bok and Diva) or as an inhibitor (Bcl-2, Bcl-xL, Bcl-w, Mcl-1,
Bfl-1 and Boo)[28]. The ratio of Bax/Bcl-2 determines the survival or death of cells
following an apoptotic stimulus[29]. Thus, an elevated Bax/Bcl-2 ratio directs the
cells to death by apoptosis. In this study, western blotting analysis showed that the
expression of Bcl-2 was down-regulated, while Bax was up-regulated in a

dose-dependent manner(Fig.8B).The ratio of Bax/Bcl-2 was increased significantly
under treatment with increased concentration of compound 3(Fig.8C). These findings
suggest that a Bax/Bcl-2 ratio increase may contribute to the apoptosis-inducing
activity of C21 steroids from the roots of Cynanchum atratum.
Apoptosis can be triggered in the target cells by two major signaling pathways.
One is the caspase-9-dependent mitochondria-mediated apoptosis pathway, and
another is the caspase-8-initiated death-receptor pathway[30]. Either activation of
caspase-8 by death-receptor pathway or activation of caspase-9 by mitochondrial
pathway can activate the expression of caspase-3[31]. To elucidate the underlying
apoptosis-inducing mechanisms of C21 steroids, we investigated the activities of
caspase-3, -8, and -9 in HepG2 cells treated by compound 3. Our results indicated that
compound 3 could significantly upregulate the expression of caspase-3 and caspase-9
in HepG2 cells, while the caspase-8 expression showed no significant difference when
compound 3 concentration was increased(Fig.8A).These findings support that
compound 3 can induce apoptosis through a mitochondria-mediated pathway.
4. Conclusion
In summary, our results indicated that C21 steroids from Cynanchum atratum
inhibited the proliferation ofHepG2and A549 cells. Compound 3can induce apoptosis
through a mitochondria-mediated pathway and cell cycle arrest in the HepG2 cells.
These findings suggest the plant Cynanchum atratum may be an alternative source
from of natural anticancer plant with apoptosis-inducing activity.
Acknowledgments
The authors thank Prof. Min-Jian Qin (China Pharmaceutical University) for
authenticating the plant material, Prof. Wen-Bin Shen (China Pharmaceutical
University) for NMR measurements, and Dr. Ping Zhou (China Pharmaceutical
University) for Flowcytometry analysis. This work was partially supported by grants
awarded from the Natural Science Foundation of Jiangsu Province(No. BK20161460),
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 Education Institutions.
Appendix A. Supplementary data
The original spectra of NMR and MSdata for the compounds 1-4 are available as
Supporting Information.
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Table and Figure Captions
Table 11H-NMR (500 MHz) data of compounds 1-2 in CD3OD (δ value, J in Hz).
Table 2 13C-NMR (125 MHz) data of compounds 1-2 in CD3OD.
Table 3 IC50 values of compounds1-4 in A549 and HepG2 cells.
Fig.1Chemical structures of compounds 1-4.
Fig.2 Key HMBC and selected ROESY correlations of compound 1.
Fig.3 Key HMBC and selected ROESY correlations of compound 2.
Fig.4 The spectrum of compound2 in DMSO containing Mo2(AcO)4 with the inherent
CD spectra subtracted.
Fig.5 Effect of compounds1-4 on proliferation of A549 (A) and HepG2 (B) cell. Cells
were incubated with increasing concentrations ofcompounds1-4for 48h, and the
growth inhibition was determined by MTT assay with cisplatin(25µg/ml) as a positive
control.

Fig.6 Compound 3 induced apoptosis in HepG2 cells. (A) A flow cytometric analysis
of annexin V-FITC/PI double-staining in HepG2 cells treated with compound 3 (0, 1,
10, 20 µM) for 48 h. (B)Representative histograms for apoptotic rate in HepG2 cells
Fig.7 Compound 3 induced G0/G1 phase cell cycle arrest in HepG2 cells. (A) HepG2
cells were treated with Compound 3 (0, 1, 10, 20 µM) for 48 h and the cell cycle
distribution was determined by a flow cytometric analysis. (B) Representative
histograms for cell cycle distribution in HepG2 cells.
Fig.8 Effect of Compound 3 on expression of apoptosis-related proteins in HepG2
cells. (A) The protein level of caspase-3, -8, -9, Bax and Bcl-2 was examined
bywestern blot assay, andβ-actin was used as a loading control. (B) Protein expression
of Bax/Bcl-2 ratio.

Table 11H-NMR (500 MHz) data of compounds 1-2 in CD3OD (δ value, J in Hz).
No
1
2
No
2
1
1.67 a , 1.51a
1.82 (m), 1.52 a
2.17 (m), 1.02 (m)
3.71(m)
3.33 a
3-O-β-D-Ole
4.64 (d, 9.25)
1.47a, 2.29 (m)
3.40 a
2
1
2
3
4
5
6
3
4.04 (m)
4
2.02 (m), 1.59 (dd,1.5, 5.0)
2.41a, 2.28 a
5
3.19 (m)
3.39 a
6
3.53 (t, 2.7)
5.47 (brs)
2.39 a, 2.15 a
2.89 (m)
7
2.49 (dt, 13.7, 3.5), 1.55 (m)
2.74 (dt, 3.4, 14.05)
1.70(m)
1.29 (d, 5.95)
8
OMe 3.44 (s)
9
1.66 (m)
β-D-Digit
10
11
1
2
5.03 (d, 9.10)
1.68 a , 2.02 (m)
2.07 (m),1.17 (m)
2.63 (dt, 3.8, 12.5),1.26 (dt, 3.4,
15.8)
1.34 a, 2.09 a
12
2.69 (m), 1.34a
3
4.11 (d, 2.0)
13
14
15
16
17
18
19
20
21
4
5
6
3.21 (m)
3.90a
4.16 (m), 3.78 (t, 9.1)
5.33 (dd, 16.9, 7.6)
3.37 (d, 7.6)
4.35 (t, 4.8), 3.92a
5.66 (m)
1,25 (d, 6.25)
α-D-Ole
2.56(d, 9.95)
4.92 (s)
1
2
3
4
5
6
4.98 (d, 2.65)
1.50a, 2.28a
3.56 (m)
6.28 (s)
1.07 (s)
1.13 (s)
3.03 (d, 9.15)
3.80 (m)
1.47 (s)
1.55 (s)
1.22 (d, 6.2)
OMe 3.42 (s)
Ole,oleandropyranosyl; Digit, digitoxopyranosyl.
aOverlapped signals.

Table 2 13C-NMR (125 MHz) data of compounds 1-2 in CD3OD.
No
1
2
No
2
1
32.6
31.8
68.1
41.3
76.0
75.7
32.1
41.5
49.4
40.8
30.7
25.7
119.7
177.7
68.6
76.1
57.5
144.3
16.0
115.9
24.7
46.3
70.9
85.7
37.5
141.0
122.0
29.0
40.5
51.4
40.9
23.2
30.1
89.9
177.7
68.1
72.3
55.2
105.0
21.7
118.6
27.1
3-O-β-D-Ole
99.8
2
1
3
2
37.9
4
3
80.0
5
4
83.9
6
5
72.6
7
6
18.5
8
OMe
57.8
9
β-D-Digit
99.6
10
11
12
13
14
15
16
17
18
19
20
21
1
2
3
4
5
6
39.9
68.7
82.6
69.9
18.5
α-D-Ole
101.2
36.0
1
2
3
79.0
4
77.5
5
69.9
6
18.1
OMe
57.4
Ole,oleandropyranosyl; Digit, digitoxopyranosyl.
Table 3 IC50 values of compounds1-4 in A549 and HepG2 cells.
Compounds
IC50(µM)
A549
HepG2
76.93 1.86
95.39 3.54
30.87 4.32
> 100
32.06 3.24
76.12 0.86
10.19 2.81
22.00 3.36
1
2
3
4

Fig.1Chemical structures of compounds 1-4.
Fig.2 Key HMBC and selected ROESY correlations of compound 1.
Fig.3 Key HMBC and selected ROESY correlations of compound2.

Fig.4 The spectrum of compound2 in DMSO containing Mo2(AcO)4 with the inherent
CD spectra subtracted.

Fig.5 Effect of compounds1-4 on proliferation of A549 (A) and HepG2 (B) cell. Cells
were incubated with increasing concentrations of compounds 1-4 for 48h, and the
growth inhibition was determined by MTT assay with cisplatin(25µg/ml) as a positive
control.

Fig.6 Compound 3 induced apoptosis in HepG2 cells. (A) A flow cytometric analysis
of annexin V-FITC/PI double-staining in HepG2 cells treated with compound 3 (0, 1,
10, 20 µM) for 48 h.(B)Representative histograms for apoptotic rate in HepG2 cells
Fig.7 Compound 3 induced G0/G1 phase cell cycle arrest in HepG2 cells. (A) HepG2
cells were treated with Compound 3 (0, 1, 10, 20 µM) for 48 h and the cell cycle

distribution was determined by a flow cytometric analysis. (B) Representative
histograms for cell cycle distribution in HepG2 cells.
Fig.8 Effect of Compound 3 on expression of apoptosis-related proteins in HepG2
cells. (A)The protein level of caspase-3, -8, -9, Bax and Bcl-2 was examined
bywestern blot assay, andβ-actin was used as a loading control. (B) Protein expression
of Bax/Bcl-2 ratio.

Highlights
•Two new and two known C21 steroids were isolated from Cynanchum atratum.
• All compounds showed obvious cytotoxic activities against HepG2 cells.
•One C21 steroids can induce apoptosis through a mitochondria-mediated pathway and
cell cycle arrest in the HepG2 cells.