Cardiac glycosides from the bark of Antiaris toxicaria

Article abstract of DOI:10.1016/j.fitote.2014.05.013

Five new cardiac glycosides (1-5, namely antiaroside Y-ZC) together with 19 known compounds were obtained from the bark of Antiaris toxicaria. Their chemical structures were determined by IR, HR-ESI-MS, 1D and 2D NMR (HSQC, ¹H-¹H COS

Full text of DOI:10.1016/j.fitote.2014.05.013

Fitoterapia 97 (2014) 71–77
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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Cardiac glycosides from the bark of Antiaris toxicaria
Xiao-San Li a, Meng-Jie Hu b, Jie Liu b, Qian Liu a, Zhi-Xing Huang a, Shun-Lin Li c, Xiao-Jiang Hao c,
Xiao-Kun Zhang b, Xin-Sheng Yao a, Jin-Shan Tang a,d,
⁎
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China
b
School of Pharmaceutical Science, Xiamen University, Xiamen 361005, China
c
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2014
Accepted in revised form 20 May 2014
Available online 28 May 2014
Five new cardiac glycosides (1–5, namely antiaroside Y-ZC) together with 19 known
compounds were obtained from the bark of Antiaris toxicaria. Their chemical structures were
determined by IR, HR-ESI-MS, 1D and 2D NMR (HSQC, 1H–1H COSY, HMBC, ROESY). The
absolute configuration of sugar unit was defined by acid hydrolysis and appropriate
derivatization. Compound 1 was rare 5β-H-10β-H-19-nor-cardenolide, which might derive
from decarboxylative derivative of 19-COOH cardenolide. The inhibitory effects of cardiac
glycosides 1–11 on the viability of NIH-H460 lung cancer cells and their induction of Nur77
expression were evaluated and preliminary structure–activity relationship (SAR) was also
discussed.
Keywords:
Antiaris toxicaria
Chemical constituents
Cardiac glycoside
Cytotoxicity
© 2014 Elsevier B.V. All rights reserved.
Expression of Nur77
1. Introduction
to effectively induce apoptosis and Nur77 protein expression
in human NIH-H460 lung cancer cells. In addition, the cardiac
Cardiac glycosides are clinically used in the treatment of
congestive heart failure and as anti-arrhythmic agents due to
their strong inhibitory activity toward the ubiquitous cell
surface enzyme Na+/K+-ATPase. Recently, analysis of epide-
miological data along with results arising from in vitro and in
vivo studies demonstrate that cardiac glycosides exhibit
potent antiproliferative and apoptotic effects on cancer cells
through complex signal transduction mechanisms, and the
first cardiac glycoside-based substances are now undergoing
clinical trials for cancer treatment [1–9]. The exact mecha-
nisms underlying these effects of cardiac glycosides are not
yet fully elucidated.
glycosides found to induce Nur77 expression were also
examined for their modulation of the subcellular localization
of Nur77 protein. Our studies revealed that both induction of
Nur77 expression and its subsequent translocation from the
nucleus to the cytoplasm are critical events in apoptosis
induction by cardiac glycosides in cancer cells [10–12].
Herein, we reported the isolation and structural elucidation
of 5 new (1–5) (see Fig. 1.) and 19 known cardiac glycosides
from the bark of A. toxicaria. In addition, the inhibitory effects
of cardiac glycosides 1–11 on the viability of NIH-H460 lung
cancer cells and their induction of Nur77 expression were
evaluated and preliminary structure–activity relationship
(SAR) was also discussed.
In our previous search for anticancer agents from plants,
about 40 cardiac glycosides were isolated from the latex and
stem of Antiaris toxicaria by bioassay and chemical guided
fractionation [10,11]. Some cardiac glycosides were observed
2. Experimental
2.1. General experimental procedures
⁎
Corresponding author at: Institute of Traditional Chinese Medicine and
Optical rotations were obtained on a P-1020 digital
polarimeter (Jasco Corporation). IR spectra were recorded
on a JASCO FTIR-480 plus spectrometer. NMR spectra were
Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632,
China. Tel.: +86 20 85220785; fax: +86 20 85221559.
E-mail address: gztangjinshan@126.com (J.-S. Tang).
http://dx.doi.org/10.1016/j.fitote.2014.05.013
0367-326X/© 2014 Elsevier B.V. All rights reserved.

72
X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
Fig. 1. Chemical structures of compounds 1–5.
measured on Bruker AV 300 and 400. The chemical shifts
were given in ppm relative to chemical shifts of solvent
resonances (pyridine-d5: 7.58 and 135.9 ppm). HR-ESI-MS
spectra were obtained on a Micromass Q-TOF mass spec-
trometer. Analysis HPLC was performed on a SHIMADZU
LC-20AB Liquid Chromatograph with SPD-M20A Detector
using a cosmosil C18 column (4.6 × 250 mm, 5 μm). Prepar-
ative HPLC was performed on a SHIMADZU LC-6AD Liquid
Chromatograph with SPD-20A Detector using an ODS column
[YMC-Pack ODS-A (10.0 × 250 mm, 5 μm, 220 and 254 nm)].
Open column chromatography (CC) was performed using
silica gel (200–300 mesh, Qingdao Haiyang Chemical Group
Corp, Qingdao), ODS (50 μm, YMC), and Sephadex LH-20
(Pharmacia). TLC analysis was performed on pre-coated silica
gel GF254 plates (Qingdao Haiyang Chemical Group Corp,
Qingdao).
fractions (C1–C12). Fraction C7 (3.53 g) was subjected to open
silica gel CC (ϕ 3.3 × 25 cm) using a CHCl3-MeOH gradient to
give 6 subfractions (C7-1–C7-6). Subfraction C7-5 (1.06 g) was
chromatographed over ODS (ϕ 3.3 × 25 cm) MPLC using
MeOH-H2O gradient to give 12 subfractions (C7-5-1–C7-5-12).
Subfraction C7-5-6 (65.3 mg) was subjected to preparative
RP-HPLC (52% MeOH-H2O, a flow rate of 3.0 mL/min) to afford
compound 6 (tR = 13.5 min, 32.7 mg). Subfraction C7-5-7
(25.5 mg) was applied to preparative RP-HPLC (58%
MeOH-H2O, a flow rate of 3.4 mL/min) to obtain compound
4 (tR = 10.0 min, 10.1 mg). Subfraction C7-5-9 (26.6 mg)
was applied to preparative RP-HPLC (58% MeOH-H2O, a flow rate
of 3.2 mL/min) to obtain compound 1 (tR = 15.5 min, 5.0 mg).
Fraction C8 (3.33 g) was subjected to ODS (ϕ 3.5 × 13 cm)
MPLC eluting with MeOH-H2O gradient to give 5 subfractions
(C8-1–C8-5). Subfraction C8-5 (172.0 mg) was applied to
preparative RP-HPLC (35% MeOH-H2O,
a flow rate of
2.2. Plant material
3.0 mL/min) to obtain compound 12 (tR = 17.0 min,
25.7 mg). Fraction C9 (4.18 g) was chromatographed over
ODS (ϕ 3.5 × 13 cm) MPLC eluted with MeOH-H2O (48%)
The bark of A. toxicaria was collected from Xishuangbanna
Tropical Botanical Garden, Chinese Academy of Sciences,
Yunnan province, P.R. China in March 2011. The plant was
authenticated by Professor Yu Chen of Kunming Institute of
Botany, Chinese Academy of Sciences. A voucher specimen
(ANTO201103) was deposited in the Institute of Traditional
Chinese Medicine & Natural Products, Jinan University.
to give
7 subfractions (C9-1–C9-7). Subfraction C9-4
(339.1 mg) was applied to preparative RP-HPLC (45%
MeOH-H2O, a flow rate of 3.0 mL/min) to obtain compounds
13 (tR = 16.2 min, 73.5 mg) and 14 (tR = 20.1 min, 53.9 mg).
Subfraction C9-6 (468.3 mg) was subjected to RP-HPLC (50%
MeOH-H2O, a flow rate of 3.0 mL/min) to yield compound 7
(tR = 35.0 min, 3.0 mg). Fraction C11 was subjected to ODS
(ϕ 3.3 × 25 cm) MPLC eluted with MeOH-H2O gradient to give
8 subfractions (C11-1–C11-8). Subfraction C11-3 (779.0 mg)
was subjected to ODS (ϕ 3.3 × 25 cm) MPLC eluted with
MeOH-H2O gradient to give 6 subfractions (C11-3-1–C11-3-6).
Subfraction C11-3-4 (132.1 mg) was applied to preparative
RP-HPLC (20% MeOH-H2O, a flow rate of 3.2 mL/min) to
obtain compound 15 (tR = 19.7 min, 16.7 mg). Subfraction
C11-3-4-3 (26.3 mg) was applied for semipreparative
2.3. Extraction and isolation
The bark of A. toxicaria (3.25 kg) was extracted by 95%
(v/v) EtOH-H2O (4 × 30 L) under reflux condition for 2 h
every time. The combined ethanol extracts were concen-
trated under vacuum to obtain the crude extract (135.0 g).
The crude extract was subjected to open silica gel CC
(ϕ 3.3 × 53 cm) using a CHCl3-MeOH gradient to give 12

X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
73
RP-HPLC (11% CH3CN-H2O, a flow rate of 3.0 mL/min) to afford
2.4. Acid hydrolysis and sugar analysis
compounds 16 (tR = 22.5 min, 5.2 mg) and 17 (tR = 25.2 min,
11.7 mg). Subfraction C11-4 (1.036 g) was applied to
Acid hydrolysis reactions of compounds 1–3 [13]. The
cardiac glycosides (0.5 mg each) were hydrolyzed using
2 mL of 2 N HCl for 1 h at 80–90 °C. The resulting mixtures
were extracted with EtOAc (2 × 2 mL). The aqueous layers
were concentrated and heated with L-cysteine methyl ester
in 1 mL pyridine at 60 °C for 1 h. Sugar (D/L) standards were
also derivatized using L-cysteine methyl ester in the same
manner. Then arylisothiocyanates were added to the
reaction mixtures and heated for 1 h at 60 °C. The reaction
mixtures were analyzed using C18 HPLC (25% MeOH-H2O (0.01%
HCOOH), a flow rate of 0.8 mL/min) with a UV detector
(250 nm). The retention times (min) of the derivatized stan-
dards were as follows: D-glucose (21.05), L-glucose (18.34), and
L-rhamnose (34.67). By comparing retention times with those of
the standards, the rhamnoses in compounds 1 and 3 were
determined to be L-configurations; the glucoses in 2, 3, and 5
were determined to be D-configurations.
preparative RP-HPLC (30% MeOH-H2O,
3.2 mL/min) to obtain compounds 18 (tR = 10.8 min,
154.5 mg), 19 (tR = 15.0 min, 27.8 mg), and 20 (tR
a flow rate of
=
19.2 min, 42.7 mg). Subfraction C11-6 (763.0 mg) was
subjected to ODS (ϕ 3.3 × 25 cm) MPLC eluted with
MeOH-H2O gradient to give 6 subfractions (C11-6-1–C11-6-6).
Subfraction C11-6-4 (27.0 mg) was applied to semipreparative
RP-HPLC (25% MeOH-H2O, a flow rate of 3.2 mL/min) to obtain
compound 8 (tR = 26.0 min, 2.1 mg). Subfraction C11-6-5
(60.5 mg) was applied to semipreparative RP-HPLC (30%
MeOH-H2O, a flow rate of 3.0 mL/min) to obtain compounds
21 (tR = 12.5 min, 1.9 mg) and 22 (tR = 21.0 min, 9.4 mg).
Subfraction C11-6-6 (186.8 mg) was applied to semipreparative
RP-HPLC (20% CH3CN-H2O, a flow rate of 3.0 mL/min) to obtain
compounds 23 (tR = 12.6 min, 8.0 mg),
2.7 mg), 10 (tR = 19.5 min, 5.5 mg),
7.7 mg), 24 (tR = 23.8 min, 4.2 mg),
9
2
5
(tR =14.5 min,
(tR =21.0 min,
(tR =28.6 min,
10.0 mg), 3 (tR = 33.0 min, 6.8 mg) and 11 (tR = 34.5 min,
2.5. MTT assay
38.0 mg).
Cells were counted by using a hemocytometer, equally
distributed in 96-well plates (5 × 103 cells per well) and treated
with cardiac glycosides 1–11 and digoxin for 48 h, and cell
proliferation was evaluated with an MTT assay procedure as
previously described [14,15]. To determine cell viability, the
medium was removed and cells were incubated with 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) at a final concentration of 0.5 mg/mL in RPMI
1640 medium containing 10% FBS for 2 h in the dark at
37 °C. Then 100 μL DMSO was added to the wells. Cultures
were incubated at room temperature (RT) for 5 min and
read at 492 nm.
2.3.1. Antiaroside Y (1)
1.5 of dry wt. (mg/kg) colorless syrup; [α]2D6 −14.3 (c 2.6,
MeOH); IR (KBr) νmax 3418, 2928, 1736, 1618, 1066 cm−1
;
1H NMR and 13C NMR (see Table 1); ESI-MS m/z 1035
[2 M + Na]+, 541 [M + Cl]−; HR-ESI-MS: m/z 507.2962
[M + H]+ (calcd. for C28H43O8, 507.2958).
2.3.2. Antiaroside Z (2)
2.4 of dry wt. (mg/kg) colorless powder; [α]2D6 −6.2 (c 3.1,
MeOH); IR (KBr) νmax 3298, 2882, 1648, 1361, 1050 cm−1; 1H
NMR and 13C NMR (see Table 1); ESI-MS m/z 589 [M + Na]+,
601 [M + Cl]−; HR-ESI-MS m/z 589.2616 [M + Na]+ (calcd.
for C29H42O11Na, 589.2625).
2.6. Western blotting analysis for Nur77 expression
Equal amounts of the lysates were electrophoresed on
an 8% SDS-PAGE gel and transferred onto polyvinylidene
difluoride membranes, as reported previously [16,17], which
were then blocked with 5% nonfat milk in TBST [50 mmol/L
Tris–HCl (pH 7.4), 150 mmol/L NaCl, and 0.1% Tween 20]
for 1 h, incubated with various primary antibodies overnight
at 4 °C and incubated with secondary antibodies for 1 h.
Immunoreactive products were detected by using chemilumi-
nescence with an enhanced chemiluminescence system (ECL,
Amersham Biosciences). The dilutions of the primary anti-
bodies were anti-Nur77 (Cell signal, 3960) in 1: 1,000, anti-
PARP (BD Biosciences, 556494) in 1: 1,000. The blots were
reprobed with anti-β-actin antibody for loading control.
2.3.3. Antiaroside ZA (3)
2.1 of dry wt. (mg/kg) colorless syrup; [α]2D6 −13.0 (c 3.1,
MeOH); IR (KBr) νmax 3305, 2934, 1725, 1036 cm−1; 1H NMR
and 13C NMR (see Table 2); ESI-MS m/z 721 [M + Na]+, 743
[M + Cl]−; HR-ESI-MS m/z 721.3412 [M + Na]+ (calcd. for
C35H54O14Na, 721.3411).
2.3.4. Antiaroside ZB (4)
3.1 of dry wt. (mg/kg) colorless syrup; [α]2D6 −22.4
(c 5.1, MeOH); IR (KBr) νmax 3419, 2973, 2921, 1750, 1373,
1211 cm−1; 1H NMR and 13C NMR (see Table 2); ESI-MS m/z
1091 [2 M + Na]+
,
579 [M + Cl]−
;
HR-ESI-MS m/z
3. Results and discussion
535.2905 [M + H]+ (calcd. for C29H43O9, 535.2907).
Compounds 1–5 gave positive reaction with Keddle
reagent, indicating they were cardiac glycosides.
2.3.5. Antiaroside ZC (5)
Compound 1 was obtained as colorless syrup. The HR-ESI-
MS showed quasimolecular ion at m/z 507.2962 [M + H]+
(calcd. for 507.2958), indicating the molecular formula
of C28H42O8 and accounting for 8 degrees of unsaturations.
The IR spectrum of 1 displayed prominent absorption maxima
at 3418, 2928, 1736, 1618, and 1066 cm−1, indicating the
3.1 of dry wt. (mg/kg) colorless syrup; [α]2D6 −17.9
(c 4.1, MeOH); IR (KBr) νmax 3421, 2967, 2928, 1733, 1456,
1070 cm−1; 1H NMR and 13C NMR (see Table 2); ESI-MS m/z
719 [M + Na]+, 731 [M + Cl]−; HR-ESI-MS m/z 719.3250
[M + Na]+ (calcd. for C35H52O14Na, 719.3255).

74
X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
Table 1
1H and 13C NMR data for compounds 1 and 2 (pyridine-d5, δ in ppm, J in Hz).
Position
1
2
b
a
b
a
δC
δH
δC
δH
1a/b
2a/b
3
4a/b
5
6a/b
7a/b
8
27.0
26.5
73.4
30.6
30.9
32.7
22.5
42.6
34.6
48.8
22.7
40.3
50.6
84.4
33.7
27.8
51.9
16.7
–
1.84 m, 1.58 m
1.83 m, 1.27 m
4.20 br s
1.60 m
22.1
26.2
65.5
28.3
32.4
34.3
22.5
42.1
36.0
50.8
29.1
40.5
50.6
85.3
33.2
27.7
51.8
16.6
176.2
176.6
74.1
2.42 m, 1.56 m
2.92 m, 1.44 m
4.41 br s
2.13 m, 1.67 m
3.24 m
2.07 m, 1.67 m
2.13 m, 1.43 m
2.63 m
2.09 m
2.09 m, 1.54 m
2.14 m, 1.33 m
1.31 m
1.52 m
1.35 m
9
1.97 m
–
10
11
12
13
14
15
16
17
18
19
20
21
1.85 m, 1.68 m
1.41 m
1.83 m, 1.27 m
1.46 m
–
–
–
–
1.86 m, 1.46 m
2.11 m, 1.99 m
2.78 m
1.01 s
–
2.14 m, 1.89 m
2.11 m, 1.67 m
2.81 m
1.20 s
–
176.5
74.2
–
–
5.33 dd (18.1, 1.5)
5.33 dd (18.1, 1.8)
6.14 s
5.04 dd (18.1, 1.5)
5.05 dd (18.1, 1.8)
6.13 s
22
23
1′
2′
3′
4′
5′
6′
118.0
174.9
100.3
72.9
73.4
74.6
118.0
175.0
96.0
74.4
79.3
71.5
79.9
62.6
–
–
5.43 br s
4.55 m
4.55 m
4.32 m
4.30 m
6.39 d (8.2)
4.14 t (8.2)
4.27 m
4.30 m
4.04 m
70.6
19.1
1.69 d (5.6)
4.47 m, 4.36 m
The assignments of H and C signals are based on HSQC, 1H–1H COSY, and HMBC experiments.
a
Recorded at 300 MHz (for 1H).
b
Recorded at 400 MHz (for 1H).
presence of hydroxyl and carbonyl functionalities. The 1H and
13C NMR signals for 1 were assigned using 1D and 2D NMR
experiments (see Table 1), which were similar to those of
antiaroside I [18]. The 1H NMR spectrum for 1 showed
characteristic of butenolide ring protons at δ 6.14 (1H, s,
H-22), 5.33 (1H, dd, 18.1, 1.5, H-21a), and 5.04 (1H, dd, 18.1,
1.5, H-21b), two methyl protons at δ 1.01 (3H, s, H-18) and δ
1.69 (3H, d, 5.6, H-6′), and one anomeric proton at δ 5.43 (1H,
br s, H-1′). The 13C NMR resonances at δ 100.3 (C-1′), 72.9
(C-2′), 73.4 (C-3′), 74.6 (C-4′), 70.6 (C-5′), and 19.1 (C-6′)
revealed the presence of rhamnose moiety in 1. Analyses of 13C
NMR and DEPT 135 signals revealed that 1 possessed four
quaternary carbons, having one quaternary carbon less than
antiaroside I. The presence of a carbon signal at δ 30.9 (CH, C-5
in 1) and the absence of a quaternary carbon signal at δ 72.2
(C-5 in antiaroside I) and a downfield shift of C-10 from δ 37.4
to δ 48.8 in 1 suggested that 1 was a 5-deoxy derivative of
antiaroside I. The L-configuration of rhamnose unit in 1 was
defined via acid hydrolysis and appropriate derivatization of
the resulting sugar [13]. Key HMBC correlation between δ 5.43
(1H, br s, H-1′) and δ 73.4 (d, C-3) confirmed that the sugar
moiety was located at C-3 position of the aglycone. Thus,
compound 1 was identified as 19-nor-digitoxigenin 3β-O-α-L-
rhamnopyranoside named antiaroside Y.
[M + Na]+ (calcd. for 589.2625), suggesting the molecular
formula of C29H42O11 and accounting for degrees of
unsaturations. The IR spectrum of 2 displayed prominent
absorption maxima at 3298, 2882, 1648, 1361, 1050 cm−1
9
,
indicating the presence of hydroxyl and carbonyl functionali-
ties. The 1H and 13C NMR signals for 2 were assigned using 1D
and 2D NMR experiments (see Table 1) which were similar to
those of antiaroside R [11], except for having one oxygen atom
less than antiaroside R. The 1H NMR spectrum for 2 showed
characteristic of butenolide ring protons at δ 6.13 (1H, s, H-22),
5.33 (1H, dd, 18.1, 1.8, H-21a), and 5.05 (1H, dd, 18.1, 1.8,
H-21b), one methyl protons at δ 1.20 (3H, s, H-18), and one
anomeric proton at δ 6.39 (1H, d, 8.2, H-1′). The 13C NMR
spectrum showed two carbonyl carbon signals at δ 175.0
(s, C-23) and 176.2 (s, C-19), indicating that sugar moiety was
attached at C-19 position of aglycone. The 13C NMR resonances
at δ 96.0 (C-1′), 74.4 (C-2′), 79.3 (C-3′), 71.5 (C-4′), 79.9 (C-5′),
and 62.6 (C-6′) revealed the presence of glucose moiety in 2.
The presence of a carbon signal at δ 32.4 (C-5 in 2) and the
absence of a quaternary carbon signal at δ 75.1 (C-5 in
antiaroside R) suggested that 2 was a 5-deoxy derivative of
antiaroside R. The large J value of H-1′ (J = 8.2 Hz) indicated
that the anomeric proton in
2 was β-orientated. The
D-configuration of glucose unit in 2 was defined via acid
hydrolysis and appropriate derivatization of the resulting sugar
[13]. Key HMBC correlation between H-1′ (δ 6.39, 1H, d, 8.2)
Compound 2 was obtained as a colorless powder. The
HR-ESI-MS showed quasimolecular ion at m/z 589.2616

X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
75
Table 2
1H and 13C NMR data for compounds 3–5 (pyridine-d5, δ in ppm, J in Hz).
Position
3
4
5
b
a
b
a
b
a
δC
δH
δC
δH
δC
δH
1a/b
2a/b
3
4a/b
5
6a/b
7a/b
8
26.5
26.6
75.0
35.1
74.0
35.8
24.7
41.3
39.5
41.5
22.4
40.3
50.4
85.2
33.5
27.6
51.7
16.5
17.6
176.3
74.1
1.71, m/1.44, m
2.10, m/1.83, m
4.29, br s
2.16, m/1.70, m
–
29.0
26.0
73.5
30.1
30.3
22.4
22.6
42.6
36.4
51.7
21.7
40.2
50.5
84.7
32.8
27.6
51.7
16.5
207.1
176.3
74.1
1.44, m/1.22, m
2.11, m/1.58, m
4.33, (br s) overlap
1.78, m/1.47, m
2.55, m
2.05, m/1.27, m
2.08, m/1.60, m
2.15, m
28.9
26.0
73.6
30.1
30.2
22.3
22.6
42.6
35.5
51.7
21.7
40.2
50.5
84.8
32.8
27.6
51.7
16.5
207.0
176.3
74.1
1.47, m/1.22, m
2.08, m/1.59, m
4.27, br s
1.80, m/1.75, m
2.59, m
2.06, m/1.27, m
2.07, m/1.57, m
2.15, m
1.88, m/1.49, m
2.27, m/1.31, m
1.86, m
1.63, m
–
9
1.87, m
–
1.84, m
–
10
11
12
13
14
15
16
17
18
19
20
21
2.14, m/1.41, m
1.46, m
2.03, m/1.87, m
1.45, m/1.32, m
–
2.08, m/1.87, m
1.44, m/1.34, m
–
–
–
–
–
2.08, m/1.89, m
2.11, m/1.96, m
2.83, m
1.05, s
1.07, s
2.05, m/1.82, m
2.08, m/1.96, m
2.78, m
1.10, s
9.57, s
2.05, m/1.81, m
2.06, m/1.95, m
2.78, m
1.10, s
9.57, s
–
–
–
5.33, dd (18.1, 1.2)
5.31, dd (18.0, 1.6)
6.16, s
5.05, dd (18.1, 1.2)
5.03, dd (18.0, 1.6)
6.13, s
5.30, dd (18.0, 1.7)
5.03, dd (18.0, 1.7)
6.13, s
22
23
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
118.1
174.9
100.2
72.3
73.1
85.0
69.2
18.8
107.2
76.8
78.9
71.8
78.9
63.0
118.1
174.9
101.1
72.9
73.7
74.8
118.1
174.9
100.8
72.4
72.9
83.9
69.2
18.6
106.7
75.6
78.7
72.0
78.6
62.9
–
–
–
5.43, br s
4.51, m
4.56, m
4.41,m
4.18, m
1.71, d (6.2)
5.23, d (7.6)
4.13, t (8.1)
4.20, m
4.25, br s
3.81, m
5.33, d (8.0)
4.00, dd (7.9, 2.9)
4.68, t (2.9)
3.71, dd (9.3, 2.4)
4.32, m
5.29, d (7.8)
3.96, dd (8.0, 3.0)
4.35, m
3.84, dd (9.5, 2.4)
4.47, m
1.70, d (6.3)
5.07, d (8.0)
3.97, m
4.25, m
4.23, m
3.94, m
4.45, m/4.32, m
70.8
19.2
1.63, d (6.1)
4.44, m/4.39, m
The assignments of H and C signals are based on HSQC, 1H–1H COSY, and HMBC experiments.
a
Recorded at 300 MHz (for 1H).
b
Recorded at 400 MHz (for 1H).
and C-19 (δ 176.2) confirmed that the sugar moiety was
located at C-19 position of the aglycone. Thus, compound 2 was
named antiaroside Z.
rhamnose unit and D-configuration of glucose unit were
determined via acid hydrolysis and appropriate derivati-
zation of the resulting sugar [13]. The β-orientation of
anomeric proton for glucose unit was defined by large
coupling value of H-1″ (J = 7.6 Hz). The HMBC correlation
between δ 5.23 (1H, d, 7.6, H-1″) and δ 85.0 (d, C-4′)
suggested that glucose unit was linked to C-4 of rhamnose
unit; HMBC correlation between 5.43 (1H, br s, H-1′) and δ
75.0 (d, C-3) revealed that the sugar chain was attached to
C-3 position of aglycone. Thus, compound 3 was iden-
tified as periplogenin 3β-O-β-D-glucopyranosyl (1–4)-α-L-
rhamnopyranoside named antiaroside ZA.
Compound
3 was obtained as colorless syrup. The
molecular formula of C35H54O14 was determined by the
HR-ESI-MS at m/z 721.3412 [M + Na]+ (calcd. for 721.3411),
and accounting for
9 degrees of unsaturations. The IR
spectrum of 3 displayed prominent absorption maxima at
3305, 2934, 1725, and 1036 cm−1, indicating the presence of
hydroxyl and carbonyl functionalities. The 1H and 13C NMR
signals for 3 were assigned using 1D and 2D NMR ex-
periments (see Table 2). The 1H NMR spectrum showed
resonance characteristic of butenolide ring protons at δ 6.16
(1H, s, H-22), 5.33 (1H, dd, 18.1, 1.2, H-21a), and 5.05 (1H,
dd, 18.1, 1.2, H-21b). The 1H NMR spectrum also displayed
three methyl protons at δ 1.05 (3H, s, H-18), 1.07 (3H, s,
H-19), and 1.71 (3H, d, 6.2, H-6′), two anomeric protons at δ
5.43 (1H, br s, H-1′) and 5.23 (1H, d, 7.6, H-1″). Analyses of
1H–1H COSY, HSQC, and HMBC spectra indicated that the
aglycone for 3 was periplogenin [18]. The sugar units for 3
were identified as glucose and rhamnose units according
to their 1H and 13C NMR resonances. The L-configuration of
Compound
4 was obtained as colorless syrup. The
HR-ESI-MS showed quasimolecular ion at m/z 535.2905
[M + H]+ (calcd. for 535.2907), suggesting the molecular
formula of C29H42O9 and accounting for 9 degrees of
unsaturations. The IR spectrum of 4 displayed prominent
absorption maxima at 3419, 2973, 2921, 1750, 1373, and
1211 cm−1
, indicating the presence of hydroxyl and
carbonyl functionalities. The 1H and 13C NMR signals for 4
were assigned using 1D and 2D NMR experiments (see
Table 2). The 1H NMR spectrum for 4 showed characteristic

76
X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
Table 3
were defined by large coupling value of H-1′ (J = 7.8 Hz)
and H-1″ (J = 8.0 Hz). The D-configuration of glucose unit in
5 was defined via acid hydrolysis and appropriate derivati-
zation of the resulting sugar [13]. The HMBC correlation
between δ 5.29 (1H, d, 7.8, H-1′) and δ 73.6 (d, C-3) revealed
that the sugar chain was attached to C-3 position of
aglycone; HMBC correlation between δ 5.07 (1H, d, 8.0,
H-1″) and δ 83.9 (d, C-4′) suggested that glucose unit was
linked to C-4 of 6-deoxy-D-allose. Thus, compound 5 was
identified as cannogenin 3-O-β-D-glucopyranosyl (1–4)-β-
D-allopyranoside named antiaroside ZC.
The antiproliferative effects of compounds 1–11 on NIH-H460 cancer cells.a
Compound
IC50 (nM)
Compound
IC50 (nM)
1
2
3
4
5
6
978.2
N10 μM
1212
158.4
34.18
27.03
7
8
9
10
11
Digoxin
140.2
253.2
229.3
50.03
26.82
712.2
a
NIH-H460 cells were incubated for 48 h with compounds 1–11 and cell
viability was then evaluated by the MTT assay. Digoxin was used as positive
control.
Nineteen known compounds were identified as
malayoside (6) [10,20], peripalloside (7) [21], toxicario-
of butenolide ring protons at δ 6.13 (1H, s, H-22), 5.31 (1H,
dd, 18.0, 1.6, H-21a), and 5.03 (1H, dd, 18.0, 1.6, H-21b),
one methyl protons at δ 1.10 (3H, s, H-18), and one
anomeric proton at δ 5.33 (1H, d, 8.0, H-1′). The 13C NMR
resonances at δ 101.1 (C-1′), 72.9 (C-2′), 73.7 (C-3′), 74.8
(C-4′), 70.8 (C-5′), and 19.2 (C-6′) revealed the presence
of 6-deoxy-D-allose moiety in 4. The aglycone for 4 was
identified as cannogenin by comparison of 13C NMR
resonance with that of malayoside (6), which was con-
firmed by analyses of 1H–1H COSY, HSQC, and HMBC
spectra [10,19,20]. Key HMBC correlation between δ 5.33
(1H, d, 8.0, H-1′) and δ 73.5 (d, C-3) confirmed that the
sugar moiety was located at C-3 position of the aglycone.
The large J value of H-1′ (J = 8.0 Hz) indicated that the
anomeric proton for 6-deoxy-D-allose moiety in 4 was
β-orientated. Hence, compound 4 was established for
cannogenin 3β-O-6-deoxy-β-D-allopyranoside named
antiaroside ZB.
Compound 5 was obtained as colorless syrup. The HR-
ESI-MS showed the quasimolecular ion at m/z 719.3250
[M + Na]+ (calcd. for 719.3255), indicating the molecular
formula C35H52O14 and accounting for 10 degrees of un-
saturations. The IR spectrum of 5 displayed prominent
absorption maxima at 3421, 2967, 2928, 1733, 1456, and
1070 cm−1, indicating the presence of hydroxyl and carbonyl
functionalities. The 1H and 13C NMR signals for 5 were
assigned using 1D and 2D NMR experiments (see Table 2),
which were similar to those of 4 except for resonance signals
of sugar residues. The 1H NMR spectrum displayed two
anomeric protons at δ 5.29 (1H, d, 7.8, H-1′) and 5.07 (1H, d,
8.0, H-1″), indicating that it was a disaccharide glycoside. The
13C NMR resonances at δ 100.8 (C-1′), 72.4 (C-2′), 72.9 (C-3′),
83.9 (C-4′), 69.2 (C-5′), and 18.6 (C-6′) revealed the presence
of 6-deoxy-D-allose moiety; δ 106.7 (C-1″), 75.6 (C-2″), 78.7
(C-3″), 72.0 (C-4″), 78.6 (C-5″), and 62.9 (C-6″) revealed the
presence of glucose moiety in 5. The β-orientation of both
anomeric protons for 6-deoxy-D-allose and glucose units
side O (8) [22], convalloside (9) [23], periplogenin
glucoside (10) [24], 3-O-β-D-glucopyranosyl-(1 → 4)-
α-L-rhamnopyranosylcannogenin (11) [25], antiaroside J
(12) [11], deglucocheirotoxin (13) [26], strophalloside
(14) [27], antiarosideK (15) [11], antiaroside L (16) [11],
antiaroside M (17) [11], β-antiarin (18) [28], antialloside
(19) [26], α-antiarin (20) [21], glucostrophanthidin (21)
[23,27], antiaroside
R (22) [11], 19-(glucosyloxy)-
3β,5,14-trihydroxy-5β-card-20(22)-enolide (23) [29],
and convallaoxin (24) [27] by comparison of their
physical and spectroscopic data with those reported
previously.
The cytotoxicity of cardiac glycosides 1–11 toward
human NIH-H460 lung cancer cells were evaluated using
MTT assays (see Table 3). Digoxin was used as positive
control. Compounds 5, 6, 10 and 11 showed significant
inhibitory effects on the proliferation of NIH-H460 cells
with IC50 values of 25–50 nM. Preliminary structure–
activity relationship analysis revealed that cardiac glyco-
sides with both an aldehyde functional group at C-10 and
β-H substitution at C-5 in the aglycone (cardiac glycosides
5, 6, and 11) exhibited stronger cytotoxic activities. In
addition, α-L-rhamnose substitution at C-3 position of the
aglycone showed stronger cytotoxicity (cardiac glycosides
4/6 and 5/11), which was consistent with previous reports
[11,30].
Our previous studies suggested that the cardiac gly-
cosides exerted their apoptotic effect through the Nur77-
dependent apoptotic pathway [11]. In order to explore the
effects of cardiac glycosides 1–11 on expression of Nur77,
protein level induction of Nur77 was determined by using
Western blotting (see Fig. 2). The result demonstrated
that all compounds exhibited strong induction of Nur77
expression at concentrations of 50 nM in 3 h. Interestingly,
cardiac glycosides 1–3 and digoxin, which exhibited medi-
um cytotoxicity, also showed strong induction of Nur77
expression. This phenomenon suggested that cytotoxicity of
Fig. 2. Induction of Nur77 expression by 1–11.

X.-S. Li et al. / Fitoterapia 97 (2014) 71–77
77
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Sensitization of human renal cell carcinoma cells to cis-
cardiac glycosides was not proportional to their function of
induction of Nur77 expression, which might be interpreted
that both induction of Nur77 expression and its subsequent
translocation from the nucleus to the cytoplasm are critical
events in apoptosis induction by cardiac glycosides in
cancer cells.
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Acknowledgment
The authors are grateful to Mr. T. Shi, Mr. J. Geng, and
Dr. Y. Yu for the HRESIMS and NMR measurements. This
work was supported by grants from the National Natural
Science Foundation of China (81001373 and 91129302)
and the Fund of the State Key Laboratory of Drug Research
(SIMM1403KF-15).
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