Fitoterapia 127 (2018) 293–300

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Cardenolides from the leaves of Nerium oleander T

a,1 a,1 a b,⁎ c
Yuan-Lin Cao , Meng-Han Zhang , Yun-Fang Lu , Chen-Yang Li , Jin-Shan Tang ,
⁎
Miao-Miao Jianga,
a
Tianjin State Key Laboratory of Modern Chinese Medicine, Pharmacology of Traditional Chinese Medicine Formulae, Tianjin University of Traditional Chinese Medicine,
Tianjin 300193, China
b
Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen 518060, China
c
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China

A R T I C LE I N FO A B S T R A C T

Keywords: Six new cardenolides (1–6), including three 14-hydroxylated cardenolides and three 14-carbonylated carde-
Nerium oleander nolides were isolated from the dried aerial parts of Nerium oleander Linn in addition to twenty-seven known
Cardenolides compounds (7–33). Their structures were elucidated on the basis of extensive spectroscopic evidences and
Cytotoxicity single-crystal X-ray diffraction analysis. Compounds 1, 4, 7–10 and 13 exhibited significant cytotoxicity against
four colon cancer cell lines (HCT116, HT29, SW620, RKO), one gastric cancer cell line (GT) and one cervical
cancer cell line (HeLa) in vitro.

1. Introduction the MTT method.

Nerium oleander Linn. (Apocynaceae) is a highly toxic ornamental 2. Experimental

shrub widely cultivated all over the world, especially in tropical and
subtropical regions [12,13]. The flowers and leaves are commonly used 2.1. General experimental procedures
in folk medicine for the treatment of heart failure, leprosy [25], an-
algesic [4], malaria, ringworms, anti-inflammatory [19] and indiges- Optical rotations were measured with sodium D line (590 nm) on a
tion [11]. Chemical investigations have demonstrated that aerial parts JASCO P-1020 digital polarimeter. HRESIMS data were determined on
of the plant are rich in a mixture of potent cardenolides, such as a Waters Xevo G2-Q-TOF mass spectrometer. NMR spectra were re-
oleandrin, which is responsible for strong heart clinical effect [31]. corded on Bruker AV 400/600 spectrometers at room temperature.
Recent in vitro evidences highlight the growth inhibition and apoptosis Preparative HPLC was run on a Agilent 1260 liquid chromatography
induction of oleandrin against a variety of human tumor cells [10], such system equipped with a UV detector using a Agilent Eclipse XDB-C18
as colon cancer [15], non-small cell lung cancer [17,31], leukemia, column (10 × 250 mm, 7 μm). Column chromatography was performed
pancreas, melanoma and prostate cell lines [26]. Oleander has been using D101 macroporous adsorption resin (Tianjin Hai guang Chemical
used in the Arab folk-medicine as powder and decoction for treating Company, Tianjin, China), silica gel (200–300 mesh, Qingdao Marine
solid tumors and for skin diseases. With the principal active constituent Chemical Factory, Qingdao, China), Sephadex LH-20 (GE Healthcare,
as oleandrin, the botanical drug candidate Anvirzel™ and PBI-05204 Sweden) and ODS (50 μm, YMC Co., Kyoto, Japan) were used.
have been into phase I clinical trials for a treatment of solid tumors Precoated silica gel GF254 plates (Qingdao Marine Chemical Factory,
[22,24,30]. Qingdao, China) were used for TLC analyses.
Due to the potential anticancer effects of oleandrin and analogs, we
investigated the chemical constituents in 95% ethanol extraction of the 2.2. Plant material
aerial parts of N. oleander. In the present work, six new cardenolides
(1–6) along with the fourteen known cardenolides (7–20), ten known The leaves of N. oleander were collected in July 2014 at an inter-
triterpenoids (21−30), two pregnanes (31−32) [18] and β-sitosterol national garden of Hefei, Anhui province, China, and were authenti-
(33) were isolated and elucidated by NMR, MS and X-ray techniques. cated by Associate Professor Hong-Hua Wu in Tianjin University of
These cardenolides were evaluated for the anticancer activities by using Traditional Chinese Medicine. A voucher specimen (TP201407) was

⁎
Corresponding authors.
E-mail addresses: [email protected] (C.-Y. Li), [email protected] (M.-M. Jiang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.fitote.2018.03.004
Received 23 January 2018; Received in revised form 6 March 2018; Accepted 9 March 2018
Available online 11 March 2018
0367-326X/ © 2018 Published by Elsevier B.V.
Y.-L. Cao et al. Fitoterapia 127 (2018) 293–300

Table 1
1 13
H-NMR (600 MHz) and C-NMR (150 MHz) data in Pyridine-d5 for compound 1, 4, 9 and 12.

Position 1 9 4 12

δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)

1 31.1 1.79 (1H, m), 1.36 (1H, m) 31.1 1.76 (1H, m), 1.65 (1H, m) 31.3 1.53 (1H, m), 1.31 (1H, m) 31.3 1.61 (1H, m), 1.34 (1H, m)
2 27.9 1.97 (1H, m), 1.68 (1H, m) 27.5 1.73 (1H, m), 1.37 (1H, m) 30.2 1.75 (1H, m), 1.30 (1H, m) 27.5 1.74 (1H, m), 1.52 (1H, m)
3 73.5 4.35 (1H, brs) 73.7 4.34 (1H, brs) 73.0 3.45 (1H, dd, 12.0, 4.0) 73.7 4.37 (1H, brs)
4 32.6 1.94 (1H, m), 1.66 (1H, m) 31.2 1.93 (1H, m), 1.48 (1H, m) 30.5 1.81 (1H, m), 1.31 (1H, m) 31.1 1.75 (1H, m), 1.59 (1H, m)
5 37.6 1.93 (1H, m) 37.6 1.80 (1H, m) 37.3 1.57 (1H, m) 37.2 1.62 (1H, m)
6 28.6 1.92 (1H, m), 1.72 (1H, m) 27.6 1.92 (1H, m), 1.37 (1H, m) 25.2 1.81 (1H, m), 1.56 (1H, m) 27.6 2.11 (1H, m), 1.33 (1H, m)
7 23.8 2.49 (1H, t, 13.8), 1.24 (1H, m) 22.2 2.14 (1H, m), 1.37 (1H, m) 23.4 2.28 (1H, m), 1.76 (1H, m) 22.2 1.74 (1H, m), 1.52 (1H, m)
8 77.1 42.4 1.70 (1H, m) 132.3 42.1 1.73 (1H, m)
9 37.2 1.99 (1H, d, 8.4) 36.4 2.34 (1H, dd, 11.6, 3.2) 137.7 37.6 1.99 (1H, m)
10 36.1 35.9 37.8 35.9
11 18.3 1.92 (1H, m), 1.43 (1H, m) 21.7 1.54 (1H, m), 1.21 (1H, m) 22.8 2.03 (1H, m), 1.80 (1H, m) 20.8 1.38 (1H, m), 1.22 (1H, m)
12 40.1 1.58 (1H, m), 1.48 (1H, m) 39.4 1.48 (1H, m), 1.37 (1H, m) 32.2 1.78 (2H, m) 41.5 1.96 (1H, m), 1.46 (1H, m)
13 57.6 51.0 52.1 53.1
14 85.0 83.9 82.9 85.3
15 43.7 2.99 (1H, dd, 15.6, 9.6) 41.7 2.85 (1H, dd,10.4, 6.4) 45.1 2.92 (1H, dd, 18.4, 2.8) 39 2.77 (1H, d, 18)
2.25 (1H, d, 15) 1.76 (1H, m) 2.81 (1H, dd, 18.4, 2.0) 2.56 (1H, dd, 18, 3.2)
16 74.8 5.71 (1H, m) 73.7 5.71 (1H, m) 135.3 6.17 (1H, t, 2.8) 134.2 6.19 (1H, brs)
17 57.9 3.42 (1H, d, 5.6) 57.3 3.42 (1H, m) 142.6 144.9
18 19.4 1.30 (3H, s) 16.8 1.10 (3H, s) 20.1 1.49 (3H, s) 17.4 1.51 (3H, s)
19 26.6 1.40 (3H, s) 24.4 0.92 (3H, s) 26.7 1.05 (3H, s) 24.6 0.99 (3H, s)
20 170.6 170.7 160.2 160.2
21 76.7 5.45 (1H, d, 18.6) 76.7 5.45 (1H, dd, 18.0, 1.2) 72.4 5.06 (2H, s) 72.4 5.11 (1H, dd, 16.4, 1.2)
5.21 (1H, d, 18.0) 5.26 (1H, dd, 18.0, 1.6) 4.81 (1H, dd, 16.4, 1.2)
22 122.1 6.23 (1H, brs) 122.1 6.36 (1H, brs) 112.2 6.41 (1H, brs) 112.4 6.32 (1H, brs)
23 174.6 174.6 175.2 175.2
24 170.2 170.3
25 21.1 1.84 (3H, s) 21.2 1.88 (3H, s)
1′ 99.5 4.79 (1H, d, 9.0) 99.6 4.78 (1H, d, 9.2) 99.6 4.80 (1H, dd, 9.6, 2.0) 99.5 4.81 (1H, dd, 9.6, 1.2)
2′ 33.7 2.36 (1H, dd, 10.2, 1.2) 33.7 2.34 (1H, dd, 11.0, 6.0) 33.7 2.33 (1H, dd, 12.0, 4.0) 33.7 2.36 (1H, dd, 22.0, 12.0)
2.16 (1H, d, 11.4) 2.13 (1H, d, 6.0) 2.17 (1H, ddd, 12.4, 4.8, 1.6) 2.17 (1H, dd, 12.0, 4.0)
3′ 79.7 3.44 (1H, ddd, 12.0, 4.4, 2.8) 79.7 3.44 (1H, ddd, 15.2, 4.8, 2.8) 79.6 3.45 (1H, dd, 12.0, 4.0) 79.7 3.45 (1H, dd, 12.0, 4.0)
4′ 67.6 3.92 (1H, s) 67.6 3.92 (1H, brs) 67.5 3.93 (1H, brs) 67.6 3.93 (1H, brs)
5′ 71.9 3.59 (1H, q, 6.0) 71.8 3.58 (1H, q, 6.4) 71.8 3.61 (1H, q, 6.4) 71.8 3.59 (1H, q, 6.4)
6′ 18.2 1.58 (3H, d, 6.6) 18.1 1.57 (3H, d, 6.4) 18.2 1.57 (3H, d, 6.4) 18.2 1.57 (3H, d, 6.4)
-OMe 55.7 3.40 (3H, s) 55.7 3.41 (3H, s) 55.7 3.41 (3H, s) 55.7 3.41 (3H, s)

deposited in Tianjin Key Laboratory of Modern Chinese Medicine, H2O to obtain compounds 32 (4 mg, tR = 29.9 min) and 17 (20 mg,
Taida, Tianjin, China. tR = 43.0 min). OlW-13 (12 g) was subjected to silica gel open column
chromatography using a CH2Cl2-MeOH gradient (100:0–0:100) to give
3 subfractions (OlW-131 to 133). OlW-133 was further separated by
2.3. Extraction and isolation ODS column chromatography and was subsequently purified by PR-
HPLC eluted with 80% MeOH-H2O to yield compounds 21 (5 mg,
Powered leaves of N. oleander (20 kg) were extracted with 95% tR = 46.0 min) and 30 (30 mg, tR = 24.9 min).
ethanol (450 L) for two weeks at room temperature. The crude extract OlW-2(85 g) was chromatographed over silica gel open column
was subsequently evaporated in vacuum and then partitioned between chromatography using a CH2Cl2-MeOH gradient (100:0–0:100) to af-
petroleum ether and H2O (1:1), to remove grease (petroleum ether ford 8 subfractons (OlW-21 to 28). OlW-21 (13 g) was chromato-
fraction), to yield a water-soluble fraction (600 g), The water-soluble graphed over silica gel open column chromatography using a EtOAc
fraction was subjected to macroporous D101 resin column chromato- –P.E gradient (100:0–0:100) to afford 8 subfractons (OlW-121 to 128).
graphy using a gradient elution of EtOH-H2O (0, 30, 60 and 95%) to OlW-211 afforded compound 26(9 mg) by separation using Sephadex
afford four subfractions (O1W-1 to 4). LH-20 column chromatography and crystallized from EtOAc. OlW-212
Fraction OlW-1 (163 g) was chromatographed over silica gel open was subjected to RP-HPLC (75% MeOH-H2O) to obtain compounds 23
column chromatography using a CH2Cl2-MeOH gradient (100:0–0:100) (7 mg, tR = 8.80 min) and 22 (31 mg, tR = 11.0 min). OlW-213 af-
to afford 11 subfractons (OlW-11 to 111). Subfraction OlW-11(6 g) was forded compound 27 (9 mg). OlW-214 was further separated by silica
further subjected to silica gel open column chromatography using a gel column chromatography eluted with a CH2Cl2-MeOH gradient
CH2Cl2-MeOH gradient (100:0–0:100), which yielded 4 subfractions (100:0–0:100) to two fractions (OlW-2141 to 2142). OlW-2142 was
(OlW-111 to 114). Subfraction OlW-111 and OlW-113 were separated crystallized from EtOAc to give compound 10 (300 mg) and OlW-2142
by Sephadex LH-20 open column chromatography eluted with 50% afforded compounds 31 (2 mg, tR = 15.3 min) by further separation
MeOH-CH2Cl2 to separately obtain compound 28 (60 mg) and com- using PR-HPLC (55% MeOH). OlW-216 was subjected to silica gel
pound 33 (10 mg). OlW-114 was separated by Sephadex LH-20 column column Chromatography and crystallized to give compound 2 (5 mg)
chromatography and was crystallized from EtOAc to give compound 25 and compound 15 (118 mg). OlW-217 was further separated by silica
(26 mg). OlW-12(8 g) was chromatographed over silica gel open gel column chromatography and then separation using HPLC (75%
column chromatography using a EtOAc-P.E (petroleum ether) gradient MeOH-H2O), followed by Preparative TCL (P.E-MeOH-
(100:0–0:100) to afford 3 subfractons (OlW-121 to 123). OlW-121 and EtOAc = 1:0.2:1) to give compound 12 (18 mg). OlW-218 was sepa-
OlW-122 were crystallized from EtOAc to give compounds 20 (20 mg) rated by silica gel column chromatography, followed by PR-HPLC (70%
and 24 (25 mg), OlW-123 was subjected to Sephadex LH-20 column MeOH–H2O) to give compound 18 (163 mg, tR = 32.3 min). OlW-22
chromatography and then purified RP-HPLC eluted with 70% MeOH-

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Y.-L. Cao et al. Fitoterapia 127 (2018) 293–300

(7 g) was chromatographed over silica gel open column chromato- 2.4. Cytotoxic assay
graphy using a CH2Cl2-MeOH gradient (100:0–0:100) to afford 5 sub-
fractions (OlW-221 to 225). Subfraction OlW-222 afforded compound Human colon cancer cells, HCT116, HT29, SW620, RKO; human
11 (3 mg, tR = 30.06 min) by successive separation using PR-HPLC cervical cancer cells, HELA cells were maintained in RPMI 1640
(55% and 53% MeOH-H2O). Fraction OlW-223 afforded compounds7 medium containing 10% FBS and 1% (penicillin and streptomycin),
(43 mg, tR = 18.5 min), 8 (87 mg, tR = 19.8 min), 9 (121 mg, human gastric cancer cells (GT) was maintained in RPMI 1640 medium
tR = 15.7 min) and 13 (220 mg, tR = 16.5 min) by separation using PR- containing 10% FBS and 1% (penicillin and streptomycin). By the
HPLC (60% MeOH-H2O) and crystallized. Fraction OlW-225 was sub- standard trypsinization method, the cells were transferred into 96-well
jected by ODS MPLC eluted with a MeOH-H2O and then followed suc- plates (1 × 105 cells per well) and incubated at 37 °C in a 5% CO2 in-
cessive separation by Preparative HPLC (65% and 55% MeOH-H2O) to cubator for 12 h. The test samples were dissolved in DMSO to obtain
give compound 3 (18 mg, tR = 22.2 min) and compound 1 (12 mg, stock solutions. Different dilutions of the test samples were added to the
tR = 34.7 min). Fraction OlW-224 was further separated by ODS MPLC cell cultures. The medium was removed and replaced by the growth
and was subsequently purified by PR-HPLC eluted with 60% MeOH- medium containing various compounds of each sample. The cells were
H2O to yield compounds 4 (5 mg, tR = 41.4 min), 16 (12 mg, incubated for a further 48 h, and then cell proliferation was evaluated
tR = 83.5 min) and 19 (6 mg, tR = 4.60 min), and follow separated by with an MTT assay procedure as previously described [14]. 50 μL of 3-
PR-HPLC (55% MeOH-H2O) to give compound 14 (4 mg, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
tR = 22.2 min). OlW-23 (5 g) was chromatographed over silica gel open solution (1 mg/mL) was added to each well and incubated at 37 °C for
column chromatography using a CH2Cl2-MeOH gradient (100:0–0:100) 4 h. The formed formazan crystals were dissolved in 100 mL DMSO for
to afford 3 subfractions (OlW-231 to 233). OlW-232 was separated by 10 min with shaking, Its UV/vis absorbance at 570 nm was measured by
silica gel column chromatograph (P.E-EtOAc), followed by PR-HPLC microplate reader, and IC50 was calculated using GraphPad Prism 5
(55% MeOH-H2O) to give compound 6 (20 mg, tR = 48.2 min). OlW- software. Cytotoxicity was expressed as the concentration of the extract
233 was separated by silica gel column chromatography, followed by required to inhibit cellular growth 50% (IC50).
PR-HPLC (55% MeOH-H2O) to give compound 5 (9 mg, tR = 69.0 min).
3. Results and discussion
2.3.1. 8-hydroxy-oleandrigenin-3-O-β-D-diginoside (1)
3.1. Chemistry
White crystals; [α]25 D-0.800° (c 0.10, MeOH);1H and 13C NMR
data (Table 1); HRESIMS (negative): m/z 637.3229 [M + COOH]−
A crude extract of the aerial parts of N. oleander was separated by
(calculated for C33H49O12, 637.3224); 591.3161 [M-H]−; HRESIMS
macroporous adsorption resin, sephadex and silica gel chromatographic
(positive): m/z593.3326 [M + H]+; 431.2425 [M + H-162]+;
columns, preparative TLC and preparative C18 reversed-phase HPLC to
371.2209 [M + H-162-60]+.
furnish six new cardenolides (1–6), and the other twenty-seven com-
pounds (7–33).The known structures were elucidated by comparing
2.3.2. 5α-oleaside A (2) physical and spectroscopic data with reported data, and further iden-
White, amorphous powder; [α]25 D+ 13.72° (c 0.29, MeOH); 1H tified as odoroside A (7) [6], odoroside B (8) [16], oleandrigenin-3-O-β-
and 13C NMR data (Table 3); HRESIMS (negative): m/z561.3066 D-diginopyranoside (9) [9], oleandrin (10) [3],16-O-acetylgitoxigenin
[M + COOH]− (calculated for C31H45O9, 561.3064); 515.3007 [M- (11), 3β-O-(β-D-diginosyl)-14β-hydroxy-5β,14β-card-16,20(22)-dieno-
H]−; HRESIMS (positive) m/z517.3162 [M + H]+; 373.2370 lide (12) [27], 3β-O-(β-D-diginosyl)-8,14-dihydroxy-5β,14β-card-
[M + H + H2O-162]+. 20(22)-enolide(13) [2], deacetyloleandrin(14), adynerin (15) [7], 5α-
adynerin (16) [1], △16-Adynerigenin-β-D-sarmentosie (17) [33], ner-
iaside (18) [7,34], neriagenin (19) [33], oleaside A (20) [8,15], un-
2.3.3. 14-carbonyl-neriaside (3)
carinic acid C (21), uncarinic acid A (22), uncarinic acid A (23), be-
White, amorphous powder; [α]25 D-38.49° (c 0.70, MeOH); 1H and
13 tulinic acid methyl ester (24), oleanolic acid (25), myricerol (26) [29],
C NMR data (Table 2); HRESIMS (negative): m/z577.3015
ursolic acid (27), ursolic aldehyde (28) [20], cis-karenin (29), trans-
[M + COOH]− (calculated for C31H45O10, 577.3015); HRESIMS(posi-
karenin (30), 12β-hydroxypregna-4,6-diene-3,20-dione (31), 20-R-hy-
tive) m/z551.3187 [M + H2O + H]+; 371.2183 [M + H-162]+.
droxypregna-4,6-diene-3,20-dione (32) and β-sitosterol (33) [32].
Cardenolide 1 was isolated as white or colorless crystal (CHCl3/
2.3.4. 3β-O-(β-D-diginosyl)-14β-hydroxy-5β, 14β-card-8, 16,20(22)- MeOH), and negative HRESIMS provided an ion at 637.3229
dienolide(4) [M + COOH]−, corresponding to a molecular formula of C33H48O10.
White, amorphous powder; [α]25 D+ 68.99° (c 0.30, MeOH); 1H Kedde's reagent reaction was positive, suggesting an existing α, β-un-
and 13C NMR data (Table 1); HRESIMS (negative): m/z513.2847 [M- saturated-γ-lactone. The 1H and 13C NMR signal assignments for 1 were
H]−,559.2903 [M + COOH]− (calculated for C31H43O9, 559.2907); achieved by employing 1D and 2D NMR experiments (Table 1). The 1H
HRESIMS (positive) m/z553.3317 [M + H2O + H]+. 371.2227 NMR spectrum of 1 showed characteristic signals of a butenolactone
[M + H + H2O-162]+. moiety at δH 5.45 and 5.21 (2H, d, J = 18.0 Hz, H-21a and 21b), 6.23
(1H, brs, H-22) and two angular methyl singlets at δH 1.30 (3H, s, H-18)
and 1.40 (3H, s, H-19). 13C NMR and DEPT-135 spectra revealed the
2.3.5. 21-Hydroxy-neriaside (5)
compound was consisted of two acyl groups, six quaternary carbons,
White, amorphous powder; [α]25 D-15.85° (c 0.32, MeOH);1H and
13 ten methine groups, ten methylene groups, two methyl groups and two
C NMR data (Table 2); HRESIMS (negative): m/z595.3117
methoxycarbon signals. Through analyses of the HMBC data, A methyl
[M + COOH]− (calculated for C31H47O11, 595.3117); HRESIMS (posi-
proton signal at δH1.84 (3H, s) and an oxygenated methine proton
tive): m/z569.3513 [M + H2O + H]+; 373.2367 [M + H-162]+.
signal δH5.71 (1H, m) are associated with δC74.8 (C-16), based on the
HMBC spectrum, H-16 (δH 5.71) showed a correlation with the carbonyl
2.3.6. 16-Hydroxy-oleaside A (6) at C-24 (δ 170.2), suggesting the presence of an acetoxy group at C-16.
White, amorphous powder; [α]25 D+ 4.73° (c 0.17, MeOH); 1H and Two D2O exchangeable protons at δH 5.89 and 4.31 were related to C-
13
C NMR data (Table 3); HRESIMS (negative): m/z577.3015 14 (δ 85.0) and C-8 (δ 77.1) respectively, indicating C-14 and C-8 were
[M + COOH]− (calculated for C31H45O10, 577.3013); HRESIMS (posi- hydroxylated. A comparison of the 1H and 13C NMR data of 1 with those
tive): m/z533.3115 [M + H]+; 389.2320 [M + H + H2O-162]+. of oleandrigenin-3-O-β-D-diginopyranoside (9) [9] demonstrated the

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Table 2
1 13
H NMR (600 MHz) and C NMR (150 MHz) data in Pyridine-d5 for compound 3, 5 and 18.

Position 3 18 5

δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)

1 31.3 1.95 (1H, m), 1.81 (1H, m) 31.4 1.95 (1H, m), 1.80 (1H, m) 31.3 1.95 (1H, m), 1.81 (1H, m)
2 23.4 2.09 (1H, m), 1.85 (1H, m) 28.2 2.09 (1H, m), 1.85 (1H, m) 28.8 2.08 (1H, m), 1.87 (1H, m)
3 73.7 4.34 (1H, brs) 73.4 4.35 (1H, brs) 73.4 4.27 (1H, brs)
4 28.0 1.96 (1H, m), 1.67 (1H, m) 31.6 1.96 (1H, m), 1.68 (1H, m) 31.5 1.90 (1H, m), 1.58 (1H, m)
5 37.3 2.39 (1H, m) 37.3 2.18 (1H, m) 37.2 1.92 (1H, m)
6 36.1 1.52 (1H, m), 1.07 (1H, m) 29.2 1.52 (1H, m), 1.31 (1H, m) 29.2 1.86 (1H, m), 1.47 (1H, m)
7 37.9 2.58 (1H, m), 2.32 (1H, m) 38.6 2.56 (1H, ddd, 13.4, 13.4, 6.8), 2.36 (1H, ddd, 38.6 2.43 (1H, m), 2.26 (1H, m)
13.4, 4.9, 2.7)
8 213.8 216.8 213.8
9 51.9 2.73 (1H, d, 9.6) 52.4 2.84 (1H, d, 10.0) 52.5 2.73 (1H, m)
10 43.0 43.2 43.1
11 18.0 1.81 (1H, m), 1.07 (1H, m) 18.8 1.87 (1H, m), 1.31 (1H, m) 19.0 1.87 (1H, m), 1.51 (1H, m)
12 31.6 1.66 (1H, m), 1.43 (1H, m) 35.7 1.76 (1H, m), 1.48 (1H, m) 36.1 1.94 (1H, m), 1.53 (1H, m)
13 53.1 51.8 51.9
14 219.9 79.9 4.20 (1H, d, 4.8) 80.2 4.27 (1H, m)
15 38.6 2.47 (1H, m), 2.28 (1H, m) 27.7 2.17 (1H, m), 2.02 (1H, m) 28.3 2.34 (1H, m), 2.09 (1H, m)
16 29.1 2.10 (1H, m), 1.43 (1H, m) 31.2 2.16 (1H, m), 1.58 (1H, m) 31.2 2.15 (1H, m), 1.58 (1H, m)
17 42.3 3.37 (1H, m) 46.8 3.05 (1H, t, 9.6) 45.3 3.53 (1H, m)
18 20.1 0.78 (3H, s) 18.1 0.72 (3H, s) 18.4 0.81 (3H, s)
19 24.4 0.72 (3H, s) 24.4 0.78 (3H, s) 24.4 0.77 (3H, s)
20 170.6 172.9 172.1
21 74.3 5.16 (1H, d, 18.6), 5.10 (1H, d, 18.0) 74.5 4.90 (1H, dd, 17.6, 1.6), 4.77 (1H, dd, 17.6, 1.6) 101.4 6.60 (1H, brs)
22 118.1 6.34 (1H, brs) 117.3 6.06 (1H, brs) 119.5 6.26 (1H, brs)
23 174.4 174.6 173.1
1′ 99.9 4.77 (1H, dd, 8.8, 2.4) 99.8 4.78 (1H, d, 9.8) 99.8 4.78 (1H, d, 9.6)
2′ 33.7 2.35 (1H, dd, 12.0, 4.0), 2.15 (1H, ddd, 33.7 2.35 (1H, dd, 12.2, 9.8), 2.17 (1H, dd, 12.2, 4.4) 33.7 2.35 (1H, dd, 12.0, 4.0), 2.17 (1H, ddd, 12.4,
12.4, 4.8, 1.6) 4.8, 1.6)
3′ 79.6 3.45 (1H, ddd, 12.0, 4.4, 2.8) 79.6 3.45 (1H, ddd, 15.2, 4.8, 2.8) 79.6 3.45 (1H, ddd, 15.2, 4.8, 2.8)
4′ 67.5 3.93 (1H, d, 1.6) 67.5 3.93 (1H, d, 2.4) 67.5 3.93 (1H, d, 2.4)
5′ 71.9 3.60 (1H, q, 6.0) 71.9 3.59 (1H, q, 6.8) 71.9 3.59 (1H, q, 6.4)
6′ 18.1 1.57 (3H, d, 6.4) 18.1 1.58 (3H, d, 6.4) 18.1 1.57 (3H, d, 6.0)
-OMe 55.7 3.42 (3H, s) 55.8 3.42 (3H, s) 55.8 3.41 (3H, s)

Table 3
1 13
H NMR (600 MHz) and C NMR (150 MHz) data in Pyridine-d5 for compound 2, 6 and 20.

Position 2 20 6

δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)

1 38.9 1.67 (1H, m), 0.77 (1H, m) 31.6 1.53 (1H, m), 1.43 (1H, m) 32.6 1.61 (1H, m), 1.52 (1H, m)
2 35.0 1.79 (1H, m), 1.36 (1H, m) 24.2 1.14 (1H, m), 1.09 (1H, m) 25.2 2.40 (1H, m), 1.19 (1H, m)
3 76.6 3.96 (1H, brs) 72.4 4.05 (1H, brs) 73.3 4.27 (1H, brs)
4 35.9 2.17 (1H, m), 0.82 (1H, m) 29.2 1.92 (1H, m), 1.10 (1H, m) 30.1 2.05 (1H, m), 1.25 (1H, m)
5 44.2 1.64 (1H, m) 36.9 1.63 (1H, m) 37.8 1.83 (1H, m)
6 30.2 2.06 (1H, m), 1.59 (1H, m) 30.1 1.89 (1H, m), 1.66 (1H, m) 31.0 1.71 (1H, m), 1.57 (1H, m)
7 27.2 1.67 (1H, m), 1.18 (1H, m) 26.9 1.78 (1H, m), 1.58 (1H, m) 27.6 1.92 (1H, m), 1.53 (1H, m)
8 50.0 48.9 48.8
9 60.3 1.69 (1H, d, 8.4) 46.1 2.48 (1H, d, 8.4) 46.5 2.54 (1H, m)
10 38.9 37.4 37.9
11 22.2 2.23 (1H, m), 1.68 (1H, m) 21.4 2.03 (1H, m), 1.82 (1H, m) 21.7 2.30 (1H, m), 1.72 (1H, m)
12 42.7 1.95 (2H, m) 42.7 2.08 (2H, dd, 10.8, 4.8) 42.8 2.03 (1H, m), 1.93 (1H, m)
13 48.2 47.5 47.8
14 221.5 221.5 221.5
15 44.1 1.78 (1H, m), 0.91 (1H, m) 44.2 2.85 (1H, m), 1.75 (1H, m) 52.7 2.44 (1H, m), 2.04 (1H, m)
16 26.9 2.65 (1H, dt, 21.6, 14.4, 6.8), 1.38 (1H, m) 27.1 2.85 (1H, m), 1.72 (1H, m) 66.0 5.45 (1H, dd, 7.6, 4.4)
17 53.3 2.98 (1H, d, 6.4) 53.3 3.07 (1H, d, 6.8) 59.0 3.44 (1H, d, 6.8)
18 23.7 0.93 (3H, s) 23.4 0.93 (3H, s) 24.0 0.99 (3H, s)
19 14.0 0.62 (3H, s) 26.4 0.77 (3H, s) 26.7 0.78 (3H, s)
20 172.4 170.7 170.4
21 74.0 4.86 (1H, d, 18.8), 4.75 (1H, d, 17.6) 73.0 4.60 (1H, dd, 17.6, 2.0), 4.55 (1H, dd, 17.6, 76.1 5.13 (1H, dd, 11.6, 1.2), 4.98 (1H, dd, 11.6, 1.2)
1.6)
22 116.8 116.7 118.4
23 174.5 5.91 (1H, brs) 173.7 5.68 (1H, brs) 174.5 6.09 (1H, brs)
1′ 98.8 4.84 (1H, d, 10) 97.8 4.43 (1H, dd, 9.6, 2.0) 99.4 4.73 (1H, dd, 9.6, 2)
2′ 33.7 2.34 (1H, dd, 22.0, 1.2), 2.17 (1H, d, 11.4) 32.1 2.08 (1H, dd, 10.4, 4.4), 1.93 (1H, d, 12.0) 33.6 2.35 (1H, m), 2.15 (1H, d, 4.8)
3′ 79.6 3.50 (1H, d, 12.0) 78.1 3.34 (1H, ddd, 12.0, 5.0, 3.0) 79.6 3.41 (1H, ddd, 12.0, 5.0, 3.0)
4′ 67.6 3.93 (1H, m) 67.4 3.68 (1H, d, 2.8) 67.5 3.92 (1H, dd, 10.0, 4.4)
5′ 71.9 3.66 (1H, q, 6.4) 70.5 3.42 (1H, dd, 6.4, 1.0) 71.8 3.59 (1H, q, 6.0)
6′ 18.2 1.61 (3H, d, 6.4) 16.9 1.33 (1H, d, 6.4) 18.2 1.57 (1H, d, 6.4)
-OMe 55.8 3.40 (3H, s) 55.9 3.40 (3H, s) 56.0 3.42 (3H, s)

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absence of a methine group and the presence of a hydroxyl proton and a carbonyl-neriaside.
hydroxyl group at C-8 in 1. The data also showed the deshielding of C-8 Cardenolide 4 was isolated as a white amorphous powder (CHCl3/
from δC 42.4 (9) to δC 77.1 (1) and C-7 from δC 23.8 (9) to δC 57.3 (1). MeOH) and positive to Kedde's reagent reaction. Combining with the
These observations suggested that the aglycone of 1 was 16β-acetoxy-8, NMR data, a negative ion at m/z 559.2903 [M + COOH]− in HR-ESI-
14-dihydroxy-5β,14β-card-20(22)-dienolide. An anomeric proton MS indicated the molecular formula C30H42O7 of 4. The 1H NMR
doublet at δH 4.79 (1H, d, J = 9 Hz, H-1′), four oxymethine protons spectrum of 4 showed characteristic signals of a butenolactone moiety
ranged from δH 3.44 to 4.79, a methyl doublet at δH 1.58 (3H, d, at δH 5.45 and 5.21 (2H, d, J = 18.0 Hz, H-21a and 21b) and 6.23 (1H,
J = 6.6 Hz), and a methoxy signal at δH 3.40 (3H, s) in the 1H NMR brs, H-22) and two angular methyl singlets at δH 1.30 (3H, s, H-18) and
spectrum suggested the presence of adeoxyhexose unit in 1, which was 1.40 (3H, s, H-19), indicating that 4 was a cardenolide. The 1H and13C
identified as diginose via comprehensive analysis of the 1H -1H COSY, NMR spectra of 4 showed significant similarities to those of neriumo-
HSQC and HMBC correlation information (Fig. 3). The coupling con- side (12) [27] except for the signals for C-8 and C-9, compound 12 had
stants H-1′ (J = 9.0 Hz), H-3′ (J = 12.0, 4.4, 2.8 Hz) and CH3–6′ one more H-atom than 4. The 13C NMR resonance of C-8 was shifted
(J = 6.6 Hz), and NOESY correlations (H-1'with H-2'a, H-3′ and H-5′; H- from δC42.1 (12) to δC132.3 (4) and C-9 was shifted from δC37.6 (12) to
4′ with H-3′, H-5′, 6′ and 3′-OMe) of a 2,6-dideoxyhexose sugar of 1 δC137.7 (4), indicating the presence of a carbon-carbon double bonds
suggested it to be diginose. The anomeric proton of β-D-diginose was between C-8 and C-9 for 4. An anomeric proton signal at δH 4.80 (1H,
identified as β-oriented due to the small coupling constant of 9.0 Hz. dd, 9.6, 2.0) in the 1H NMR spectrum and carbon signals at δC 99.6,
Key HMBC correlation betweenH-1′ (δH 4.79) and C-3 (δC 73.5) re- 33.7, 79.6, 67.5, 71.8, 18.2, and 55.7 revealed the presence of diginosyl
vealed that the sugar moiety was located at C-3 of the aglycone. The in 4, as observed in 1. The NMR characteristics suggested 4 had the
relative configuration of 1 was assigned by proton-proton coupling same monosaccharide moiety as 1 and 2. The coupling constant of
constants NOE correlationship. The Cu Ka X-ray crystallographic data 9.6 Hz indicated a β-substituted orientation for the anomeric proton (H-
confirmed its overall structure and established the absolute configura- 1′) of diginose. An HMBC correlation between H-1′ (δH 4.80) and C-3
tion of 1 (Fig. 1). Thus, compound 1 was established as 8-hydroxy- (δC 73.0) confirmed that the sugar moiety was located at C-3 of the
oleandrigenin-3-O-β-D-diginoside (Fig. 2). aglycone. Therefore, the structure of compound 4 was defined as 3β-O-
Cardenolide 2 was isolated as a white amorphous powder (CHCl3/ (β-D-diginosyl)-14β-hydroxy-5β,14β-card-8,16,20(22)-trienolide.
MeOH) and positive to Kedde's reagent reaction. Considering 30 carbon Cardenolide 5 was isolated as a colorless amorphous powder
signals in 13C NMR spectrum, a negative ion at m/z 515.3007 [M-H]− (CHCl3/MeOH). Kedde's reagent reaction was positive and had a mo-
in HR-ESI-MS indicated the molecular formula C30H44O7 of 2. A com- lecular formula of C30H46O9 (m/z 595.3117 [M + COOH]− calcd for
parison of the 1H and 13C NMR data with those of oleaside A (20) [8,15] C31H47O11, 595.3117), which was jointly determined by 13C NMR and
revealed that they had the same planar structures (Table 3). The ROESY HRESIMS data. The 1H and 13C NMR data of 5 showed significant si-
correlation between H-5 (δH1.64) and H-9 (δH 1.69) (Fig. 3), as well as milarities to that of neriaside (18) [7,34] except for the signals of C-21
the shielding effect on C-19 from δC 26.4 (20) to 14.0 (2) confirmed and compound 5 had one more oxygen atom than 18. The 13C re-
that H-5 was α-oriented, and the aglycone of 2 was a (8R)-3β-hydroxy- sonance signal of C-21 shifted from δC74.5 (18) to δC101.4 (5), in-
14-oxo-15(14 → 8)abeo-5α-card-20(22)-enolide [5,32]. The sugar dicating the presence of a hydroxy group at C-21 for 5 [21]. An
moiety of 2 was the same as 18 based on a comparison of their 1H and anomeric proton signal at δH 4.78 (1H, d, J = 9.6 Hz) in the 1H NMR
13
C NMR data. Therefore, compound 2 was identified as 5α-oleaside A. spectrum and the carbon signals at δC 99.8, 33.7, 79.6, 67.5, 71.9, 18.1,
Cardenolide 3 was isolated as a white amorphous powder (CHCl3/ and 55.8 revealed the presence of diginosyl in 5, as observed in 1. An
MeOH) and had a molecular formula C30H44O8 that was jointly de- HMBC (Fig. 3) correlation between H-1′ (δH4.78) and C-3 (δC 73.4)
termined by 13C NMR and HR-ESI-MS (m/z 577.3015 [M + COOH]−, confirmed that the sugar moiety was located at C-3 of the aglycone.
calcd for C31H47O10, 577.3015) data. The 1H and 13C NMR spectra of 3 Therefore, the structure of compound 5 was defined as 21-hydroxy-
showed significant similarities to those of 18 except for the signals at C- neriaside.
13, C-15, and C-14. The 13C NMR resonance of C-14 was shifted from δC Cardenolide 6 was isolated as colorless amorphous powder (CHCl3/
79.9 (18) to δC 219.9 (3), showing the presence of a carbonyl group at MeOH). Kedde's reagent reaction was positive. The molecular formula
C-14 for 3 [28]. An anomeric proton signal at δH 4.77 (1H, dd, J = 8.8, was assigned as C30H44O8 using 13C NMR and HRESIMS (m/z 577.3015
2.4 Hz) and the carbon signals at δC 99.9, 33.7, 79.6, 67.5, 71.9, 18.1 [M + COOH]− calculated for C31H45O10, 577.3013) data. The 1H and
13
and 55.7 revealed the presence of diginosyl in 3. The NMR character- C NMR spectra of 6 had significant similarities to those of 20 except
istics suggested 3 had the same monosaccharide moiety as 1 and 2. The for the signals for C-15, C-16, and C-17. The 13C NMR resonance of C-16
coupling constant of 8.8 Hz indicated a β-substituted orientation for the was shifted from δC 27.1 (20) to δC 66.0 (6), indicating the presence of a
anomeric proton (H-1′) of diginose. Key HMBC correlation between H- hydroxy group at C-16 for 6. An anomeric proton signal at δH 4.75 (1H,
1′ (δH 4.77) and C-3 (δC 73.7) confirmed that the sugar moiety was dd, J = 9.6, 2 Hz) in the 1H NMR spectrum and the carbon signals at δC
located at C-3 of the aglycone. Thus, compound 3 was identified as 14- 99.4, 33.6, 79.6, 67.5, 71.8, 18.2, and 56.0 revealed the presence of
diginosyl in 6, as observed in 1. An HMBC (Fig. 3) correlation between
H-1′ (δH4.75) and C-3 (δC 73.3) confirmed that the sugar moiety was
located at C-3 of the aglycone. Therefore, the structure of compound 6
was identified as 16-hydroxy-oleaside A.

3.2. Cytotoxic activity

The cytotoxicities of all the cardenolides (1−20) were examined on

a panel of human cancer cell lines, including GT gastric cancer cells,
HCT116, HT29, SW620 and RKO colon cancer cells, and HELA cervical
cancer cells. The results demonstrated that the cardenolides 1, 4, 7, 8,
9, 10 and 13 exhibited significant inhibitory activities against the
proliferation of these human cancer cell lines and compound 10 has the
strongest cytotoxicity with IC50 < 0.4 μM against the six cancer cell
lines, while the other cardenolides primary IC50 value > 100 μM. The
Fig. 1. X-ray crystal structures of compounds 1.
research on antitumor activity of these compounds provided more data

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Fig. 2. Chemical structures of 1–33.

on structure–activity relationships (SAR) of cardenolides [23]. Cardiac structure of cytotoxic activity. Cardiac glycosides 1, 4, 7–10 and 13
glycosides 7, 9 and 10, which exhibited strong cytotoxic activities (Table 4) exhibited stronger inhibitory effects on the growth of the GT,
against GT cells (IC50 values of 0.07, 0.29 and 0.03 μM, respectively), HELA, HCT116, HT29, SW620, RKO cancer cell lines. These results
The C3 sugar species played an important role in their cytotoxicities demonstrated that cardiac glycosides might selectively inhibit the
against cancer cell lines (10 > 9), when the C16 hydrogen acetyl, it has proliferation of tumor cell lines.
little effect on the activity (7/9). 7, 9 and 10 shows that the steroids
structure, the A/B ring cis-fused, and C13 position unsubstituted cardiac Conflict of interest
glycosides, has a strong cytotoxic activity (7/9/10). In addition, the
orientation of H-5 in cardiac glycosides also influenced their cytotoxi- The authors declare that there is no conflict of interest.
cities toward cancer cell lines. Cardiac glycosides with H-5 in a β-or-
ientation showed stronger cytotoxicities than those with 5α-H (7 > 8).
Acknowledgments
Cytotoxic activities also could be remarkably altered after 8β-hydro-
xylation of aglycones (compounds 1/9 and 7/13). Additionally, activity
against cancer cell-growth increased after acetylation at 16-OH of This work was supported by the grants from Tianjin Science and
Technology Research Programs of Application Foundation and
aglycones (compounds 7/9 and 1/13), the double bond between C8/C9
and C16/C17 position will weaken the cytotoxicity (1/7). The activity Advanced Technology (14JCYBJC29000), Tianjin Technology
Innovation System and Platform Development Plan (15PTCYSY00030),
will disappear when the ring open or form an epoxy structure between
C14 and C8 position, it shows that the steroids structure is the essential the National Natural Science Foundation of China (81503215) and the
Science Foundation of Shenzhen (JCYJ20150525092941057).

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Fig. 3. Key HMBC 1H–1H COSY or NOESY correlations of compounds 1,2, 3, 4, 5and 6.

Table 4
Cytotoxicity of compounds (IC50 values expressed in μM).

Compound Cell lines

HCT116 HT29 SW620 RKO GT HELA

1 1.12 ± 0.51 2.76 ± 0.40 2.393 ± 0.90 13.79 ± 3.1 > 100 9.12 ± 0.32
4 30.86 ± 1.3 22.93 ± 1.0 28.52 ± 2.5 > 100 7.42 ± 1.21 > 100
7 0.21 ± 0.03 0.09 ± 0.01 0.10 ± 0.05 1.38 ± 0.88 0.07 ± 0.01 26.28 ± 1.56
8 7.77 ± 0.66 6.78 ± 0.86 8.74 ± 1.20 88.42 ± 2.65 3.21 ± 0.63 > 100
9 0.27 ± 0.01 0.25 ± 0.03 0.72 ± 0.22 1.31 ± 0.40 0.29 ± 0.44 1.38 ± 0.77
10 0.02 ± 0.01 0.04 ± 0.01 0.07 ± 0.05 0.10 ± 0.04 0.03 ± 0.05 0.33 ± 0.08
13 8.79 ± 0.10 5.70 ± 0.64 3.35 ± 0.21 18.09 ± 1.20 3.32 ± 0.19 0.81 ± 0.20

Appendix B. Supplementary data 14–18.

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