A new and 23 known cardenolide glycosides from Thevetia neriifolia seeds and their cytotoxic activities against human oral carcinoma cell lines

Article abstract of DOI:10.1080/14786419.2020.1716352

Phytochemical analysis of Thevetia neriifolia seeds resulted in the isolation of one new (1) and 23 known (2－24) cardenolide glycosides. The structure of 1 was determined based on one- and two-dimensional NMR spectroscopic analysis and acid hydrolytic cleavage reaction. The effect of the cytotoxic activity of 1－24 on three human oral carcinoma cell lines was assessed. The cell lines included Ca9-22 human gingival carcinoma cells, HSC-2 human mouth carcinoma cells, HSC-4 human tongue carcinoma cells, and HGF human gingival fibroblast cells. The isolated compounds had a cytotoxic effect on the carcinoma cells with IC₅₀ values ranging from 0.004 μM to 64.9 μM. The structure-activity relationship is also discussed.

Full text of DOI:10.1080/14786419.2020.1716352

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new and 23 known cardenolide glycosides
from Thevetia neriifolia seeds and their cytotoxic
activities against human oral carcinoma cell lines
Satoru Tatsuno, Tomoki Iguchi, Minpei Kuroda, Mai Ishihara, Hiroshi
Sakagami & Yoshihiro Mimaki
To cite this article: Satoru Tatsuno, Tomoki Iguchi, Minpei Kuroda, Mai Ishihara, Hiroshi Sakagami
& Yoshihiro Mimaki (2020): A new and 23 known cardenolide glycosides from Thevetiaꢀneriifolia
seeds and their cytotoxic activities against human oral carcinoma cell lines, Natural Product
Research, DOI: 10.1080/14786419.2020.1716352
To link to this article: https://doi.org/10.1080/14786419.2020.1716352
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1716352
A new and 23 known cardenolide glycosides from
Thevetia neriifolia seeds and their cytotoxic activities
against human oral carcinoma cell lines
Satoru Tatsunoa, Tomoki Iguchia, Minpei Kurodaa, Mai Ishiharaa, Hiroshi
Sakagamib and Yoshihiro Mimakia
aDepartment of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and
b
Life Sciences, Hachioji, Japan; Research Institute of Odontology (M-RIO), Meikai University,
Sakado, Japan
ABSTRACT
ARTICLE HISTORY
Received 15 December 2019
Accepted 9 January 2020
Phytochemical analysis of Thevetia neriifolia seeds resulted in the
isolation of one new (1) and 23 known (2－24) cardenolide glyco-
sides. The structure of 1 was determined based on one- and two-
dimensional NMR spectroscopic analysis and acid hydrolytic cleav-
age reaction. The effect of the cytotoxic activity of 1－ 24 on
three human oral carcinoma cell lines was assessed. The cell lines
included Ca9-22 human gingival carcinoma cells, HSC-2 human
mouth carcinoma cells, HSC-4 human tongue carcinoma cells, and
HGF human gingival fibroblast cells. The isolated compounds had
a cytotoxic effect on the carcinoma cells with IC50 values ranging
from 0.004 lM to 64.9 lM. The structure-activity relationship is
also discussed.
KEYWORDS
Thevetia neriifolia;
Apocynaceae; seed;
cardenolide glycoside;
cytotoxic activity; oral
carcinoma cell
1. Introduction
Thevetia neriifolia Juss. ex Steud. is a small ornamental tree belonging to the
Apocynaceae family (Siddiqui et al. 1992). The plant is distributed from tropical
America to the West Indies and has been used as a natural remedy for treating skin
CONTACT Tomoki Iguchi
iguchit@toyaku.ac.jp
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1716352.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
S. TATSUNO ET AL.
diseases and fever as a purgative and emetic (Siddiqui et al. 1992; Gulati et al. 2000).
Phytochemical analyses of T. neriifolia leaves were extensively conducted in the past,
and various cardenolide glycosides have been reported (Siddiqui et al. 1992; Abe et al.
1992a, 1992b; Begum et al. 1993; Abe et al. 1994). However, a literature survey dis-
closed that no systematic phytochemical examination of T. neriifolia seeds has been
performed. Therefore, detailed chemical analysis with a focus on cardenolide glyco-
sides was carried out on T. neriifolia seeds. In this study, the structural characterization
of one new (1) and 23 (2－24) known isolated cardenolide glycosides from T. neriifolia
seeds is reported. The isolated compounds were evaluated for the effect of the cyto-
toxic activities on three human oral carcinoma cell lines, Ca9-22 human gingival car-
cinoma cells, HSC-2 human mouth carcinoma cells, and HSC-4 human tongue
carcinoma cells, along with HGF human gingival fibroblast cells. The structure-activity
relationship is also discussed.
2. Results and discussion
T. neriifolia seeds (2.0 kg) were extracted with hot MeOH (20 L) and concentrated using
an evaporator. The MeOH extract (250 g) was loaded on a porous polymer polystyrene
resin Diaion HP-20 column and successively eluted with MeOH/H2O (1:4), EtOH, and
EtOAc. The EtOH eluted portion was separated by silica gel column chromatography
(CC), octadecylsilanized (ODS) silica gel CC, and preparative HPLC to collect 24 com-
pounds (1－24). Compounds 2－24 were identified as the following cardenolide glyco-
sides: digitoxigenin 3-O-a-L-thevetopyranoside (neriifolin) (2) (Yamauchi et al. 1987a; Abe
et al. 1992b), uzarigenin 3-O-a-L-thevetopyranoside (thevefolin) (3) (Miyagawa
et al. 2009), digitoxigenin 3-O-(2-O-acetyl-a-L-thevetopyranoside) (cerberin) (4) (Zhang
et al. 2010), digitoxigenin 3-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (5)
(Abe et al. 1992a), uzarigenin 3-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (6)
(Abe et al. 1992a), cannogenin 3-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (7)
(Tian et al. 2016), cannogenol 3-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (8)
(Tian et al. 2016), digitoxigenin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-
(1!4)-a-L-thevetopyranoside (thevetin B) (9) (Yamauchi et al. 1987b; Kohls et al. 2012),
digitoxigenin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-a-L-acofrioside (10)
(Abe et al. 1992b), uzarigenin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-
(1!4)-a-L-thevetopyranoside (11) (Abe et al. 1992a), uzarigenin 3-O-b-D-glucopyranosyl-
(1!6)-O-b-D-glucopyranosyl-a-L-acofrioside (12) (Abe et al. 1992a), digitoxigenin 3-O-
b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-(1!4)-2-O-acetyl-a-L-thevetopyrano-
side (acetylthevetin B) (13) (Endo et al. 1997; Kohls et al. 2012; Tian et al. 2016), cannoge-
nin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside
(thevetin A) (14) (Abe et al. 1994; Kohls et al. 2012), cannogenin 3-O-b-D-glucopyranosyl-
(1!6)-O-b-D-glucopyranosyl-(1!4)-2-O-acetyl-a-L-thevetopyranoside (acetylthevetin A)
(15) (Kohls et al. 2012), cannogenol 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyrano-
syl-(1!4)-a-L-thevetopyranoside (16) (Tian et al. 2016), 19-nor-10-hydroxydigitoxigenin
3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (17)
(Tian et al. 2016), (20R,S)-18,20-epoxydigitoxigenin 3-O-a-L-thevetopyranoside (18) (Abe
et al. 1992a), (20R,S)-18,20-epoxydigitoxigenin 3-O-(2-O-acetyl-a-L-thevetopyranoside)

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of 1–24.
(19) (Cruz et al. 1979), (20R,S)-18,20-epoxydigitoxigenin 3-O-b-D-glucopyranosyl-(1!6)-
O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (thevetin C) (20) (Kohls et al. 2012),
(20R,S)-18,20-epoxydigitoxigenin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-
(1!4)-2-O-acetyl-a-L-thevetopyranoside (acetylthevetin C) (21) (Kohls et al. 2012), theve-
tiogenin 3-O-a-L-thevetopyranoside (thevetioside A) (22) (Abe et al. 1992b), thevetioge-
nin 3-O-b-D-glucopyranosyl-(1!4)-a-L-thevetopyranoside (thevetioside C) (23) (Abe
et al. 1992b), and thevetiogenin 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-
(1!4)-a-L-thevetopyranoside (thevetioside F) (24) (Abe et al. 1992b) (Figure 1).
Compound 1 was obtained as an amorphous powder. The molecular formula was
identified as C41H64O19, based on the high resolution-electrospray ionization-time of
flight-mass spectroscopy (HR-ESI-TOF-MS; m/z: 883.3914 [M þ Na]þ, calculated for
C41H64NaO19: 883.3940) and 13C-NMR data. The IR spectrum of 1 showed absorption
bands for hydroxy groups at 3363 cmꢀ1 and the carbonyl group of an a,b-unsaturated
1
c-lactone moiety at 1737 cmꢀ1. The H- and 13C-NMR spectra of 1 exhibited signals for
a tertiary methyl group [dH 0.88 (s, Me-18); dC 16.4 (C-18)], an oxygenated methine

4
S. TATSUNO ET AL.
group [dH 3.93 (m, H-3); dC 73.4 (C-3)], an oxygenated methylene group [dH 3.84 (d,
J ¼ 11.5 Hz, H-19a) and 3.39 (d, J ¼ 11.5 Hz, H-19b); dC 65.7 (C-19)], an a,b-unsaturated
c-lactone group [dH 5.90 (br s, H-22), 5.03 (br d, J ¼ 18.4 Hz, H-21a), and 4.91 (br d,
J ¼ 18.4 Hz, H-21b); dC 178.4 (C-20), 177.2 (C-23), 117.8 (C-22), and 75.3 (C-21)], and
three anomeric protons and carbons [dH 4.78 (br s), 4.60 (d, J ¼ 7.9 Hz), and 4.38 (d,
J ¼ 7.8 Hz); dC 105.3, 104.9, and 99.7]. The above spectroscopic data suggest that 1 is a
1
cardenolide glycoside. The H- and 13C-NMR spectra of 1 were related to those of 16,
except for the signals arising from the sugar moiety. Acid hydrolysis of 1 with 1 M HCl
(dioxane/H2O, 1:1) exposed D-glucose and L-rhamnose as the carbohydrate moieties.
The monosaccharides and their absolute configurations were identified by direct HPLC
1
analysis of the hydrolysate. Analysis of the H-1H COSY and HSQC spectra of 1 dis-
closed that the sugar moiety comprised a 4-substituted a-L-rhamnopyranosyl unit
[Rha: dH 4.78 (br s, H-10); dC 99.7, 72.8, 72.5, 83.3, 68.5, and 18.3 (C-10－60)], a 6-substi-
tuted b-D-glucopyranosyl unit [Glc (I): dH 4.60 (d, J ¼ 7.9 Hz, H-100); dC 104.9, 76.0, 78.1,
71.6, 77.2, and 70.2 (C-100－600)], and a terminal b-D-glucopyranosyl unit [Glc (II): dH
4.38 (d, J ¼ 7.8 Hz, H-1000); dC 105.3, 75.2, 77.9, 71.6, 78.0, and 62.8 (C-1000－6000)]. The
1
3
large JC-1,H-1 value (166 Hz) and intense JC,H correlations between H-10 and C-30/C-50
implied that the anomeric configuration of Rha was a (Bock et al. 1973; Jia et al.
1998). The b-orientation of the anomeric centers of Glc (I) and Glc (II) were confirmed
3
by the relatively large JH-1,H-2 values [Glc (I): 7.9 Hz; Glc (II): 7.8 Hz]. In the HMBC spec-
trum of 1, long-range correlations were observed between H-1000 of Glc (II) (dH 4.38)
and C-600 of Glc (I) (dC 70.2), H-100 of Glc (I) (dH 4.60) and C-40 of Rha (dC 83.3), and
between H-10 of Rha (dH 4.78) and C-3 of the aglycone (dC 73.4). Compound 1 was
defined as cannogenol 3-O-b-D-glucopyranosyl-(1!6)-O-b-D-glucopyranosyl-(1!4)-
a-L-rhamnopyranoside.
Compounds 1－24 were evaluated for the effect of the cytotoxic activities on three
oral carcinoma cell lines including Ca9-22, HSC-2, and HSC-4 cells, and HGF oral nor-
mal cells using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay method (Table 1) (Sakagami et al. 2015; Sakagami et al. 2017).
Etoposide was used as a positive control. Compounds 2－4 exhibited a particularly
potent cytotoxic activity toward three carcinoma cell lines, with IC50 values ranging
from 0.004 lM to 0.066 lM. The average IC50 values of 18 and 19 for the three carcin-
oma cell lines was ten times lower than for HGF normal cells, indicating that 18 and
19 were selectively cytotoxic to the carcinoma cell lines. Comparison of the IC50 values
of 2, 4, 9, and 13 with those of 18, 19, 20, and 21, respectively, revealed that the for-
mation of an ether linkage between C-18 and C-20 of the aglycone decrease the cyto-
toxic effect. Furthermore, 22, 23, and 24 are the corresponding C-nor-D-homo type
steroids of 2, 5, and 9, respectively, and this structural rearrangement significantly low-
ered the cytotoxic effect of 2, 5, and 9. The following structure-activity relationships
corresponded to previously reported results (Tian et al. 2016): 1) the cardenolide gly-
cosides with the A/B cis ring fusion (2, 5, 9, and 10) exhibited stronger cytotoxic activ-
ities than those with the A/B trans ring fusion (3, 6, 11, and 12); 2) comparison of the
cytotoxic activities of 9, 14, and 16 implied that the oxidation of Me-19 of the agly-
cone to hydroxymethyl and aldehyde decreased the cytotoxic activity; 3) comparison
of the cytotoxic activities of 2, 5, and 9, or 3, 6, and 11 revealed that an increase in

NATURAL PRODUCT RESEARCH
5
Table 1. Cytotoxic activity of 1–24 and etoposide toward Ca9-22, HSC-2, HSC-4, and HFG cells
Ca9-22
HSC-2
HSC-4
HGF
Compounds
IC50 (lM)
IC50 (lM)
IC50 (lM)
IC50 (lM)
1
2
3
4
5
6
7
8
12.1 4.2
6.5 0.51
7.6 0.47
0.0095 0.0021
0.022 0.011
0.0061 0.00014
0.13 0.02
1.3 0.08
0.24 0.03
1.1 0.04
0.85 0.20
1.7 0.37
6.4 0.77
7.3 1.2
25.7 4.6
0.030 0.045
0.021 0.0055
0.011 0.0023
0.066 0.0045
0.004 0.0002
0.40 0.05
3.4 0.29
0.58 0.13
2.8 0.14
5.1 1.6
8.7 0.77
39.9 12.5
30.1 0.88
8.4 0.46
0.0077 0.00055
0.043 0.015
0.0073 0.0012
0.12 0.01
1.3 0.08
0.21 0.09
1.1 0.11
1.1 0.06
1.6 0.40
6.5 0.71
7.9 0.41
3.8 0.87
2.3 0.28
3.6 0.43
7.6 2.3
23.4 0.65
0.28 0.14
0.35 0.03
31.9 3.2
6.9 2.1
52.8 1.0
0.0029 0.0002
0.89 0.06
4.2 0.21
1.1 0.06
10.7 0.14
14.0 10.0
7.2 2.6
51.6 25.9
36.8 18.1
28.5 2.3
14.9 3.9
19.6 4.4
47.5 10.3
96.2 15.2
7.7 4.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Etoposide
3.1 0.06
2.6 0.30
4.7 1.3
5.6 0.50
9.4 1.1
19.3 1.3
64.9 8.7
6.4 1.6
18.5 3.6
0.37 0.04
0.49 0.03
32.7 2.8
7.8 1.1
0.80 0.26
0.99 0.20
> 100
30.2 1.6
> 100
7.0 0.86
> 100
74.2 28.6
> 100
11.5 2.6
70.1 7.6
> 100
> 100
1.9 0.76
26.8 0.07
7.0 2.8
0.95 0.06
12.1 3.6
1.6 0.94
1.2 0.03
9.7 0.46
2.3 0.69
Data are represent the mean value S.E.M. of three experiments performed in triplicate.
the number of sugar units attached to C-3 of the aglycone lowered the cyto-
toxic activity.
3. Experimental
3.1. Compound 1
25
Amorphous powder; [a]D ꢀ151.9 (c 0.049, MeOH); IR (film) mmax: 3363 (OH), 2928
1
(CH), 1737 (C ¼ O) cmꢀ1; UV kmax (MeOH) nm (log e): 270 (3.43), 218 (3.80); H-NMR
spectral data of the aglycone moiety (500 MHz, CD3OD) dH: 5.90 (1 H, br s, H-22), 5.03
(1 H, br d, J ¼ 18.4 Hz, H-21a), 4.91 (1 H, br d, J ¼ 18.4 Hz, H-21b), 3.93 (1 H, m H-3), 3.84
(1 H, d, J ¼ 11.5 Hz, H-19a), 3.39 (1 H, d, J ¼ 11.5 Hz, H-19b), 0.88 (3 H, s, Me-18); 13C-
NMR spectral data of the aglycone moiety (125 MHz, CD3OD) dC: 24.9, 27.3, 73.4, 30.8,
30.1, 27.5, 22.4, 42.5, 36.5, 40.4, 22.2, 41.3, 51.0, 86.5, 33.2, 28.0, 52.1, 16.4, 65.7, 178.4,
75.3, 117.8, 177.2 (C-1－ 23); 1H-NMR spectral data of the sugar moiety (500 MHz,
CD3OD) dH: 4.78 (1 H, br s, H-10), 4.60 (1 H, d, J ¼ 7.9 Hz, H-100), 4.38 (1 H, d, J ¼ 7.8 Hz,
H-1000), 4.13 (1 H, dd, J ¼ 11.7, 1.7 Hz, H-600a), 3.95 (1 H, dd, J ¼ 9.5, 3.2 Hz, H-30), 3.87
(1 H, dd, J ¼ 11.5, 1.8 Hz, H-6000a), 3.81 (1 H, br d, J ¼ 3.2 Hz, H-20), 3.78 (1 H, dd, J ¼ 11.7,
5.8 Hz, H-600b), 3.75 (1 H, m, H-50), 3.68 (1 H, dd, J ¼ 11.5, 5.2 Hz, H-6000b), 3.64 (1 H, dd,
J ¼ 9.5, 9.5 Hz, H-40), 3.47 (1 H, m, H-500), 3.36 (1 H, m, H-300), 3.35 (1 H, m, H-3000), 3.33
(1 H, m, H-400), 3.29 (1 H, m, H-4000), 3.27 (1 H, m, H-5000), 3.21 (1 H, dd, J ¼ 9.0, 7.9 Hz, H-
200), 3.20 (1 H, dd, J ¼ 9.2, 7.8 Hz, H-2000), 1.31 (3 H, d, J ¼ 6.1 Hz, H-60); 13C-NMR spectral
data of the sugar moiety (125 MHz, CD3OD) dC: 99.7, 72.8, 72.5, 83.3, 68.5, 18.3 (C-10－

6
S. TATSUNO ET AL.
60), 104.9, 76.0, 78.1, 71.6, 77.2, 70.2 (C-100－600)þ, 105.3, 75.2, 77.9, 71.6, 78.0, 62.8 (C-
1000－6000). HR-ESI-TOF-MS m/z: 883.3914 [M þ Na] (calcd for C41H64NaO19: 883.3940).
See supplemental material for others.
Disclosure statement
No potential conflict of interest was reported by the authors.
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