Triterpene glycosides from Silene odontopetala

Article abstract of DOI:10.1016/j.phytochem.2020.112404

Silene species are known for their use in traditional medicine in treating several diseases. To the authors’ knowledge there is no report on the chemical composition of S. odontopetala. Therefore, the phytochemical investigation of the methanol extract of S. odontopetala was carried out, leading to the isolation of six undescribed oleanane-type glycosides along with the known saponin azukisaponin IV. Their structures were elucidated by the analysis of 1D and 2D-NMR experiments, along with mass spectrometry analysis. The cytotoxic activity of oleanane-type saponins was evaluated against a small panel of cancer cell lines, including PC-3 (prostate carcinoma cells), MCF-7 (breast cancer cells), A549 (alveolar basal carcinoma cells), and HeLa (cervical carcinoma cells). Furthermore, the activity of isolated compounds against a normal cell line HEK-293, used for assessing their cytotoxicity, was evaluated.

Full text of DOI:10.1016/j.phytochem.2020.112404

Phytochemistry176(2020)112404
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpene glycosides from Silene odontopetala
Rabia Nur Üna, Milena Masullob,∗, Tamer Karayildirima, Ayşe Nalbantsoyc, Ozgen Alankusa,
Sonia Piacenteb
a Ege University Faculty of Science Chemistry Department, Bornova, İzmir, 35100, Turkey
b Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II, 84084, Salerno, Italy
c Ege University Engineering Faculty Department of Bioengineering, Bornova, İzmir, 35100, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Silene species are known for their use in traditional medicine in treating several diseases. To the authors’
knowledge there is no report on the chemical composition of S. odontopetala. Therefore, the phytochemical
investigation of the methanol extract of S. odontopetala was carried out, leading to the isolation of six un-
described oleanane-type glycosides along with the known saponin azukisaponin IV. Their structures were elu-
cidated by the analysis of 1D and 2D-NMR experiments, along with mass spectrometry analysis. The cytotoxic
activity of oleanane-type saponins was evaluated against a small panel of cancer cell lines, including PC-3
(prostate carcinoma cells), MCF-7 (breast cancer cells), A549 (alveolar basal carcinoma cells), and HeLa (cervical
carcinoma cells). Furthermore, the activity of isolated compounds against a normal cell line HEK-293, used for
assessing their cytotoxicity, was evaluated.
Silene odontopetala
Caryophyllaceae
NMR analysis
Oleanane-type saponins
1. Introduction
Moreover, the cytotoxic activity of oleanane-type saponins (1–7)
has been evaluated against a small panel of cancer cell lines, including
The Caryophyllaceae family comprises 104 genera and more than
2000 species and is represented in the Flora of Central, Eastern and
South-Eastern Anatolia by 32 genera and about 500 species (Horo et al.,
2015; Kilinc et al., 2019). Caryophyllaceae are named ‘pink family’ for
the colour of the flowers and plants belonging to this family are gen-
erally perennial. Silene is a large genus with more than 700 species
growing in various temperate regions of the world (Golea et al., 2017).
This genus is represented by 150 taxa in the Flora of Turkey, of which
67 endemic. Several phytochemical studies have been carried out on
Silene species (Kilinc et al., 2019), highlighting the occurrence of ec-
dysteroids (Shakhmurova et al., 2012). A number of Silene species are
used in Traditional Medicine to treat inflammation, bronchitis, cold,
and infections or as a diuretic, antipyretic, analgesic, and emetic
(Mamadalieva et al., 2014). Furthermore fungicidal activity has been
reported for some Silene species (Mamadalieva et al., 2014). To the
authors' knowledge there is no report on the chemical composition of
Silene odontopetala Fenzl. Thus on the basis of the biological activities
reported for Silene species, the phytochemical investigation of S.
odontopetala was carried out. Herein, we report the isolation and
structural characterization by NMR experiments of six undescribed
oleanane-type saponins (1–6), in addition to the known azukisaponin
IV.
PC-3 (prostate carcinoma cells), MCF-7 (breast cancer cells), A549
(alveolar basal carcinoma cells), HeLa (cervical carcinoma cells) along
with a normal cell line HEK-293, used for assessing the selective cyto-
toxicity of the compounds.
2. Results and discussion
The MeOH extract of S. odontopetala was dissolved in H2O and
successively partitioned with n-Hexane, CH2Cl2 and n-BuOH; the n-
BuOH extract was chromatographed by MPLC on silica gel. The ob-
tained fractions were submitted to further chromatographic steps to
afford six underscribed triterpene saponins (Fig. 1), along with azuki-
saponin IV. Their structures were elucidated by extensive spectroscopic
methods including 1D- (1H and 13C) and 2D-NMR (DQF-COSY, HSQC,
HMBC, and TOCSY) experiments as well as ESIMS analysis. The agly-
cones of the isolated compounds were identified as oleanane-type tri-
terpenes by 1H NMR and 13C NMR analysis (Table 1) (Gulcemal et al.,
2013; Koz et al., 2010).
The HR-ESI-MS spectrum of 1 (m/z 793.4006 [M-H]-, calcd for
C41H61O15, 793.4010) supported a molecular formula of C41H62O15
.
The MS/MS spectrum of this ion showed fragment ions at m/z 643.3461
[M-H-132-18]-, due to the loss of a pentosyl unit with a H2O molecule
∗ Corresponding author.
E-mail address: mmasullo@unisa.it (M. Masullo).
https://doi.org/10.1016/j.phytochem.2020.112404
Received 18 October 2019; Received in revised form 11 May 2020; Accepted 12 May 2020
0031-9422/©2020ElsevierLtd.Allrightsreserved.

R.N. Ün, et al.
Phytochemistry176(2020)112404
Fig. 1. Triterpene saponins from Silene odontopetala.
2

R.N. Ün, et al.
Phytochemistry176(2020)112404
Table 1
13C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 1–6 (600 MHz, δ ppm, in CD3OD).
1
2
3
4
5
6
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
38.7
25.1
82.2
1.73, 1.15, m
2.05, 1.82, m
3.98, dd (11.5,
4.6)
38.7
25.1
82.3
1.73, 1.15, m
2.08, 1.80, m
3.98, dd (11.5,
4.6)
39.3
26.4
90.5
1.64, 1.02, m
2.00, 1.72, m
3.22, dd (11.6,
4.4)
39.8
25.4
85.5
1.69, 1.10, m
2.03, 1.80, m
3.88, dd (11.5,
4.5)
39.6
26.4
91.8
1.60, 1.02, m
1.98, 1.75, m
3.21, dd (11.6,
4.4)
39.0
25.4
72.6
1.74, 1.17, m
2.05, 1.62, m
3.80, dd (11.6, 4.4)
4
5
6
7
8
9
56.0
48.6
21.0
33.0
40.4
48.9
–
56.1
48.7
21.0
33.2
40.0
48.7
36.9
24.0
–
40.0
56.7
19.3
33.9
40.3
48.7
37.9
24.2
–
56.4
48.6
21.3
33.3
40.8
48.8
36.8
24.7
–
40.0
56.9
19.4
33.7
40.9
48.8
37.8
24.5
–
56.7
48.6
21.7
33.1
40.8
48.8
36.8
24.8
–
1.37, m
1.37, m
0.82 brd (11.5)
1.53, 1.47, m
1.53, 1.35, m
–
1.34, m
0.78 brd (11.5)
1.56, 1.48, m
1.52, 1.33, m
–
1.35, m
1.55, 0.92, m
1.55, 1.28, m
–
1.60, 0.95, m
1.56, 1.28, m
–
1.60, 0.95, m
1.58, 1.28, m
–
1.51, 0.92, m
1.52, 1.24, m
–
1.75, m
1.75, m
1.63, m
1.69, m
1.58, m
1.74, m
–
10 36.6
11 24.0
–
–
–
–
–
1.97, 1.90, m
1.95, 1.90, m
1.96, 1.90, m
1.94, 1.90, m
1.96, 1.90, m
1.94, 1.89, m
12 121.8 5.26, t (3.5)
121.6 5.25, t (3.5)
122.2 5.25, t (3.5)
122.4 5.26, t (3.5)
122.3 5.26, t (3.5)
122.4 5.27, t (3.5)
13 145.2
14 43.6
15 28.5
16 18.1
17 53.0
18 43.1
–
145.2
43.6
28.5
18.1
53.1
43.1
–
145.0
43.4
28.5
18.2
53.0
42.9
–
145.4
42.8
29.2
24.9
50.8
42.5
–
145.4
43.0
28.9
24.9
50.9
42.4
–
145.4
42.8
29.0
24.9
50.8
42.4
–
–
–
–
–
–
–
1.70, 1.09, m
1.94, 1.69, m
–
1.70, 1.09, m
1.94, 1.69, m
–
1.71, 1.12, m
1.94, 1.70, m
–
1.87, m
1.69, 1.12, m
1.85, 1.79, m
–
1.87, m
1.85, 1.79, m
–
1.85, 1.78, m
–
2.84, dd (13.8,
3.7)
2.83, dd (13.8,
3.7)
2.83, dd (13.8,
3.7)
2.97, dd (13.8,
3.7)
2.98, dd (13.8,
3.7)
2.98, dd (13.8, 3.7)
19 47.7
20 31.8
21 41.8
22 72.0
1.76, 1.15, m
–
47.7
31.8
41.8
72.0
1.75, 1.13, m
–
47.5
31.9
41.7
72.0
1.76, 1.13, m
–
48.9
37.5
85.0
41.8
1.80, 1.19, m
–
49.0
37.0
85.1
41.0
1.82, 1.19, m
–
49.0
37.5
85.0
41.4
1.80, 1.18, m
–
1.41, 1.36, m
3.75, dd (11.6,
5.2)
1.39, 1.34, m
3.75, dd (11.6,
5.2)
1.41, 1.35, m
3.75, dd (11.6,
5.2)
3.48, m
3.50, m
3.50, m
1.93, 1.72, m
1.94, 1.75, m
1.95, 1.74, m
23 208.5 9.44, s
208.7 9.44, s
27.9
16.5
15.7
17.7
25.9
181.8
33.6
24.6
1.08, s
210.4 9.46, s
28.1
16.8
15.7
17.8
26.1
180.5
29.6
18.4
1.10, s
0.90, s
0.96, s
0.89, s
1.15, s
–
208.4 9.32, s
24 9.8
1.13, s
1.03, s
0.94, s
1.21, s
–
9.8
1.13, s
1.02, s
0.92, s
1.20, s
–
0.87, s
10.4
14.8
17.9
25.3
184.1
29.6
18.3
1.17, s
1.01, s
0.89, s
1.18, s
–
9.2
1.04, s
1.00, s
0.90, s
1.19, s
–
25 15.7
26 17.7
27 26.0
28 183.8
29 33.4
30 24.9
15.7
17.9
26.3
183.9
33.5
25.0
0.98, s
15.8
18.0
26.2
180.8
29.6
18.3
0.93, s
1.19, s
–
0.95, s
1.00, s
0.94, s
1.00, s
0.95, s
1.04, s
1.00, s
1.04, s
1.01, s
1.05, s
1.01, s
1.00, s
and at m/z 485.3253 [M-H-132-176]- attributable to the aglycone
moiety.
agreement with a β orientation of H-18. Thus the α-orientation of the
alcoholic function at C-22 was deduced from the ROESY correlations
between H-18 (δH 2.84), H-22 (δH 3.75) and Me-30 (δC 1.00), in
agreement with literature data on triterpene derivatives with alcoholic
functions on the E-ring (De Freitas et al., 2017) (Fig. 2). Thus, the
aglycone of 1 was identified as 3β,22α-dihydroxyolean-12-en-23-al-28-
oic acid. To the best of our knowledge, this is the first report of this
aglycone in literature. The downfield shift of C-3 (δC 82.2) suggested
that this carbon was a glycosidation site. The 1H NMR spectrum dis-
played in the sugar region signals corresponding to two anomeric
protons at δH 4.60 (d, J = 7.0 Hz) and 4.26 (d, J = 7.5 Hz). On the
basis of 1D-TOCSY, DQF-COSY and HSQC analysis, the presence of a β-
glucuronopyranosyl unit (δH 4.26) and an α-arabinopyranosyl unit (δH
4.60) was deduced. The configurations of glucuronic acid and arabinose
units were established as D and L, respectively, after hydrolysis of 1
with 1 N HCl, trimethylsilation and determination of retention time by
GC (Polat et al., 2010). An unambiguous determination of the sequence
and linkage sites was obtained from the HMBC spectrum which showed
key correlation peaks between the proton signal at δH 4.26 (H-1glcA)
and the carbon resonance at δC 82.2 (C-3), the proton signal at δH 4.60
(H-1ara) and the carbon resonance at δC 85.5 (C-3glcA). On the basis of
the above evidences, the structure of compound 1 was established as
3β,22α-dihydroxyolean-12-ene-23-al-28-oic acid 3-O-α-L-arabinopyr-
anosyl-(1 → 3)-β-D-glucuronopyranoside.
The 13C NMR spectrum showed 41 carbon signals, of which 30 were
assigned to the aglycone moiety and 11 to a sugar portion made up of
two sugar units (Tables 1 and 2). The 1H NMR spectrum displayed
signals for an aldehyde proton at δH 9.44, six tertiary methyl groups at
δH 0.94, 0.95, 1.00, 1.03, 1.13, and 1.21, an olefinic proton at δH 5.26
(t, J = 3.5 Hz), and two oxygen-bearing methine protons at δH 3.98
(dd, J = 11.5, 4.6 Hz, H-3) and 3.75 (dd, J = 11.6, 5.2 Hz, H-22)
(Table 1). These signals along with the carbon resonances for an alde-
hyde function at δC 208.5, a carboxyl group at δC 183.8, six methyl
groups at δC 9.8, 15.7, 17.7, 24.9, 26.0, and 33.4, two olefinic carbons
at δC 121.8, 145.2 in the 13C NMR spectrum, suggested that the agly-
cone of compound 1 was a 3-hydroxyolean-12-en-28-oic acid with an
aldehyde function and an additional secondary alcoholic function
(Masullo et al., 2014). The HMBC correlations between the proton
signal at δH 9.44 and the carbon resonances at δC 82.2 (C-3), 56.0 (C-4),
48.6 (C-5) and 9.8 (C-24), along with the ROESY correlations between
the proton signal at δH 9.44 and the proton signals at δH 3.98 (H-3) and
1.37 (H-5) allowed to locate the aldehyde group at C-23. The additional
secondary alcoholic function was placed at C-22 on the basis of the
HMBC correlations between the proton signals at δH 1.01 (Me-30) and
0.95 (Me-29) with the carbon resonance at δC 41.8 (C-21) and on the
basis of the COSY correlations of H2-21 at δH 1.41 and 1.36 with the
proton signal at δH 3.75 (H-22). In case of an oleanane-type triterpene
the configuration of C-18 can be assigned on the basis of the chemical
shift of C-18, which appears 7–8 ppm upfield shifted in the H-18α series
if compared with the H-18β series, and of the basis of the chemical shift
of C-28, which is reported to resonate at higher field of about
10–11 ppm in the H-18α series (Mahato and Kundu, 1994). The 13C
NMR resonances of C-18 (δC 43.1) and C-28 (δC 182.0) were in
The HR-ESI-MS spectrum of 2 (m/z 661.3574 [M-H]-, calcd for
C
36H53O11, 661.3588) supported a molecular formula C36H54O11. The
MS/MS spectrum of this ion showed a fragment ion at m/z 485.3254
[M-H-176]-, due to the loss of a hexosyluronic acid and corresponding
to the aglycone.
The 1H and 13C NMR chemical shifts of the aglycone moiety of 2
were superimposable on those of 1 (Table 1). In the sugar region, the 1H
3

R.N. Ün, et al.
Phytochemistry176(2020)112404
Table 2
13C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–6 (600 MHz, δ ppm, in CD3OD).
1
2
4
δC
δH (J in z)
δC
δH (J in Hz)
δC
δH (J in Hz)
β-D-GlcA (at C-3)
β-D-GlcA (at C-3)
β-D-GlcA (at C-3)
1
2
3
4
5
6
103.7
4.26, d (7.5)
3.32, dd (7.5, 9.0)
3.57, dd (9.0, 9.0)
3.53, dd (9.0, 9.0)
3.58, d (9.0)
–
103.7
75.4
77.5
73.8
76.3
176.0
4.20, d (7.5)
3.12, dd (7.5, 9.0)
3.32, dd (9.0, 9.0)
3.43, dd (9.0, 9.0)
3.52, d (9.0)
–
103.9
4.37, d (7.0)
3.70, dd (7.5, 9.0)
3.68, dd (9.0, 9.0)
3.57, dd (9.0, 9.0)
3.59, d (9.0)
–
74.5
78.5
85.2
85.8
71.0
71.8
76.0
77.4
176.0
176.1
α-L-Ara (at C-3glcA) (
β-D-Gal (at C-2glcA
)
1
2
3
4
5
104.8
72.2
73.8
69.3
66.7
4.60, d (7.0)
103.3
73.3
75.0
69.5
76.3
4.81, d (8.0)
3.64, dd (8.0, 7.0)
3.58, dd (8.0, 3.0)
3.86, m
3.48, dd (8.5, 8.0)
3.45, dd (8.5, 3.0)
3.81, dd (3.0, 1.2)
3.51, m
3.96, dd (12.5, 3.0)
3.62, dd (12.5, 2.6)
61.8
3.77, dd (12.0, 2.5)
3.74, dd (12.0, 4.5)
1
2
3
4
5
6
α-L-Ara (at C-3glcA
)
104.4
72.4
74.3
70.1
67.0
4.64, d (7.0)
3.60, dd (8.0, 7.0)
3.52, dd (8.0, 3.0)
3.81, m
3.91, dd (12.5, 3.0)
3.57, dd (12.5, 2.6)
β-D-Xyl (at C-21)
1
2
3
4
5
106.2
74.4
87.4
69.5
65.6
4.32, d (7.5)
3.33, dd (7.5, 9.2)
3.42, t (9.2)
3.52, m
3.85, dd (5.2, 11.7)
3.23, t (11.7)
β-D-Qui (at C-3xyl
)
1
2
3
4
5
6
104.9
74.1
77.3
76.0
73.4
17.0
4.51, d (8.2)
3.30, dd (8.2, 9.5)
3.36, dd (9.5, 9.5)
3.06, dd (9.5, 9.5)
3.40, m
1.31, d (6.5)
a The chemical shift values of the sugar moieties of 3 were superimposable to those of 1.
The chemical shift values of the sugar moieties of 5 were superimposable to those of 4.
The chemical shift values of the sugar chain at C-21 of 6 were superimposable to those of the sugar chain at C-21 of 4.
NMR spectrum displayed a unique anomeric proton signal at δH 4.20 (d,
J = 7.5 Hz), assigned on the basis of HSQC, HMBC, COSY and 1D-
TOCSY experiments to a β-glucuronopyranosyl unit. The configuration
of the glucuronic acid unit was established as D after hydrolysis of 2
with 1 N HCl, trimethylsilation and determination of retention time by
GC (Horo et al., 2012). The linkage of the sugar unit to C-3 was es-
tablished by the HMBC correlation between the proton signal at δH 4.20
with the carbon resonance at δC 82.2. Therefore, the structure of
compound 2 was elucidated as 3β,22α-dihydroxyolean-12-en-23-al-28-
oic acid 3-O-β-D-glucuronopyranoside.
detailed comparison of NMR data of the sugar portion of compounds 3
and 1 demonstrated that the two compounds possessed the same sugar
chain. Consequently, the structure of compound 3 was established as
3β,22α-dihydroxyolean-12-en-28-oic acid 3-O-α-L-arabinopyranosyl-
(1 → 3)-β-D-glucuronopyranoside.
The HR-ESI-MS spectrum of 4 (m/z 1233.5518 [M-H]-, calcd for
C
58H89O28, 1233.5540) supported the molecular formula C58H90O28.
The MS/MS spectrum of this ion showed fragment ions at m/z
1083.4966 [M-H-132-18]-, corresponding to the loss of a pentosyl unit
with a H2O molecule and at m/z 763.4247 [M-H-132-162-176]-, cor-
responding to the further loss of a hexosyl unit and a hexosyluronic
acid.
The HR-ESI-MS spectrum of 3 (m/z 779.4189 [M-H]-, calcd for
C
41H63O14, 779.4218) supported the molecular formula C41H64O14. The
MS/MS spectrum of this ion showed a fragment ion at m/z 471.3454
[M-H-176-132]-, corresponding to the loss of a sugar chain made up of a
hexosyluronic acid and a pentosyl unit.
The 13C NMR spectrum showed 58 carbon signals, of which 30 were
assigned to the aglycone moiety and 28 to a sugar portion made up of
five sugar units (Table 2). The 1H NMR spectrum showed signals for an
aldehyde proton at δH 9.46, six tertiary methyl groups at δH 0.89, 1.00,
1.01, 1.04, 1.17, and 1.18, an olefinic proton at δH 5.26 (t, J = 3.5 Hz),
and two oxygen-bearing methine protons at δH 3.48 (m, H-21) and 3.88
(dd, J = 11.5, 4.5 Hz, H-3) (Table 1). These signals along with the
carbon resonances in the 13C NMR spectrum suggested that the agly-
cone of compound 4 showed the same chemical features of compound
1, except for the difference observed in the chemical shift of an oxygen-
bearing methine proton (δH 3.48 in 4), instead of the signal at δH 3.75
attributed to H-22 in 1. This secondary alcoholic function was located
The 1H NMR spectrum displayed signals for seven tertiary methyl
groups at δH 0.87, 0.93, 0.95, 0.98, 1.00, 1.08 and 1.19, an olefinic
proton at δH 5.25 (t, J = 3.5 Hz), two oxygen-bearing methine protons
at δH 3.22 (dd, J = 11.6, 4.4 Hz, H-3), and 3.75 (dd, J = 11.6, 5.2 Hz,
H-22) (Table 1). A detailed analysis of NMR data revealed that the
aglycone of compound 3 differed from the aglycone of 1 for the re-
placement of a 23-CHO with a methyl function (Table 1). The 1H NMR
spectrum displayed in the sugar region signals corresponding to two
anomeric protons at δH 4.26 (d, J = 7.5 Hz), and 4.60 (d, J = 7.0 Hz). A
4

R.N. Ün, et al.
Phytochemistry176(2020)112404
Fig. 2. (A) 1H–1H COSY and key HMBC correlations for 1; (B) Selected ROESY correlations for 1.
at C-21 on the basis of the HMBC correlations between the proton
signals at δH 1.00 (Me-30) and 1.04 (Me-29) with the carbon resonance
at δC 85.0 (C-21), and the COSY correlations between the proton signal
at δH 3.48 and the proton signals at δH 1.93 and 1.72 (H2-22). The β-
orientation of the alcoholic function was deduced from the correlations
observed in the ROESY spectrum between H-21 (δH 3.48) and Me-29
(δH 1.04) and between H-18 (δH 2.97) and Me-30 (δH 1.00). Thus, the
aglycone of 4 was identified as 3β,21β-dihydroxyolean-12-en-23-al-28-
oic acid. The downfield shifts of C-3 (δC 85.5) and C-21 (δC 85.0) of the
aglycone suggested that compound 4 was a bidesmosidic glycoside. The
1H NMR spectrum displayed in the sugar region signals corresponding
to five anomeric protons at δH 4.32 (d, J = 7.5 Hz), 4.37 (d,
J = 7.0 Hz), 4.64 (d, J = 7.0 Hz), 4.51 (d, J = 8.2 Hz), and 4.81 (d,
J = 8.0 Hz). On the basis of 1D-TOCSY, DQF-COSY and HSQC analysis,
the presence of a β-xylopyranosyl unit (δH 4.32), a β-glucuronopyr-
anosyl unit (δH 4.37), a β-quinovopyranosyl unit (δH 4.51), a α-arabi-
nopyranosyl unit (δH 4.64), and a β-galactopyranosyl unit (δH 4.81) was
deduced. The configuration of glucuronic acid, galactose, xylose and
quinovose was established as D, while the configuration of arabinose
was established as L, after hydrolysis of 4 with 1 N HCl, trimethylsi-
lation and determination of retention time by GC (De Marino et al.,
2003; Horo et al., 2012; Montoro et al., 2013). An unambiguous de-
termination of the sequence and linkage sites was obtained from the
HMBC spectrum, which showed key correlation peaks between the
proton signal at δH 4.81 (H-1gal) and the carbon resonance at δC 78.5 (C-
pentosyl and hexosyl units and hexosyluronic acid.
The 1H NMR spectrum displayed signals for seven tertiary methyl
groups at δH 0.89, 0.90, 0.96, 1.01, 1.04, 1.10 and 1.15, an olefinic
proton at δH 5.26 (t, J = 3.5 Hz), and two oxygen-bearing methine
protons at δH 3.50 (m, H-21) and 3.21 (dd, J = 11.6, 4.4 Hz, H-3)
(Table 1). The NMR data of the aglycones of compounds 4 and 5 were
superimposable except for an additional methyl signal in 5, replacing
the 23-CHO group of 4 (Table 1). A detailed comparison of NMR
spectroscopic data of the sugar region of compounds 5 and 4 demon-
strated that they possessed the same sugar chain. Therefore, the
structure of compound 5 was established as 21-O-β-D-quinovopyr-
anosyl-(1
→ 3)-β-D-xylopyranosyl-3β,21β-dihydroxyolean-12-en-28-
oic acid 3-O-α-L-arabinopyranosyl-(1
→ 3)-[β-D-galactopyranosyl-
(1 → 2)]-β-D-glucuronopyranoside.
The HR-ESI-MS spectrum of 6 (m/z 763.4247 [M-H]-, calcd for
C41H63O13, 763.4269) supported a molecular formula of C41H64O13
.
The MS/MS spectrum of this ion showed a fragment ion at m/z
485.3253 [M-H-146-132]-, corresponding to the loss of pentosyl and
hexosyl units, attributable to the aglycone moiety.
A detailed analysis of NMR spectroscopic data of compound 6
showed that compounds 6 and 4 share the same aglycone moiety,
namely 3β,21β-dihydroxyolean-12-en-23-al-28-oic acid. The downfield
shift exhibited only by C-21 (δC 85.4) of the aglycone suggested that
compound 6 was a monodesmosidic glycoside. The comparison of NMR
spectroscopic data of the sugar region of compounds 6 and 4 demon-
strated that they possessed the same sugar chain linked to C-21.
2
glcA), the proton signal at δH 4.64 (H-1ara) and the carbon resonance at
δC 85.8 (C-3glcA), the proton signal at δH 4.37 (H-1glcA) and the carbon
resonance at δC 85.5 (C-3); further correlations were observed between
the proton signal at δH 4.51 (H-1qui) and the carbon resonance at δC
87.4 (C-3xyl), the proton signal at δH 4.32 (H-1xyl) and the carbon re-
sonance at δC 85.0 (C-21). On the basis of these evidences, the structure
of compound 4 was established as 21-O-β-D-quinovopyranosyl-(1 → 3)-
β-D-xylopyranosyl-3β,21β-dihydroxyolean-12-en-23-al-28-oic acid 3-
O-α-L-arabinopyranosyl-(1 → 3)-[β-D-galactopyranosyl-(1 → 2)]-β-D-
glucuronopyranoside.
Therefore, compound 6 was determined as 21-O-β-D-quinovopyr-
anosyl-(1 → 3)-β-D-xylopyranosyl-3β,21β-dihydroxyolean-12-ene-23-
al-28-oic acid.
Additionally, azukisaponin IV, for the first time described in Vigna
angularis, was isolated and determined by comparison of its spectro-
scopic data with those present in literature (Kitagawa et al., 1983).
The abundance and widespread occurrence of triterpene saponins is
a typical feature of the family Caryophyllaceae (Mamadalieva et al.,
2014). Several oleanane saponins isolated from Silene genus show as
characteristic features an aldehyde or carboxyl group at C-23, a car-
boxyl group at C-28 and secondary alcoholic functions at C-16, and
rarely at C-11 (Mamadalieva et al., 2014; Takahashi et al., 2016). In
agreement with saponins previously reported in Silene spp., compounds
The HR-ESI-MS spectrum of 5 (m/z 1219.5714 [M-H]-, calcd for
C
58H91O27, 1219.5748) supported a molecular formula of C58H92O27.
The MS/MS spectrum of this ion showed a peak at m/z 749.4228 [M-H-
132-162-176]-, corresponding to the loss of a sugar chain made up of
5

R.N. Ün, et al.
Phytochemistry176(2020)112404
Table 3
IC50 (μM) values of compounds 1–6 and doxorubicin on treated cell lines.a.
Compound
A549
HEK293
Hela
MCF7
PC3
1
> 50
> 50
0.21
0.12
3.25
8.57
0.09
0.17
> 50
> 50
> 50
> 50
> 50
> 50
> 50
0.24
45.59
> 50
42.92
10.23
23.58
45.68
19.68
1.73
2.32
> 50
> 50
> 50
> 50
> 50
> 50
> 50
6.43
0.52
0.16
1.51
2
> 50
> 50
> 50
8.12
3
0.02
0.01
0.09
1.10
0.01
0.02
1.77
0.43
3.15
4.37
1.69
4
5
6
2.34 + 0.61
> 50
azukisaponin IV
Doxorubicin
0.05
0.08
0.38
14.88
0.23
a
Data are expressed as the mean values
SD of three experiments.
1, 2, 4 and 6 are characterized by the presence of an aldehyde group
and a carboxyl group at C-23 and C-28, respectively. Their peculiarity is
in the occurrence of further secondary alcoholic functions at positions
21 or 22.
3.3. Extraction and isolation procedure
The air-dried and powdered plant material (S. odontopetala; whole
plant, 500 g) was first extracted with n-Hexane (2 × 3 L), CH2Cl2
(2 × 3 L) and MeOH (3 × 4 L) at room temperature. After carrying out
filtration and evaporation procedures, n-Hexane (5.8 g), CH2Cl2 (8.4 g)
and MeOH (71.0 g) extracts were obtained, respectively. The MeOH
extract (71.0 g) was dissolved in H2O (500 mL) and successively par-
titioned with n-Hexane (2 × 200 mL), CH2Cl2 (2 × 200 mL) and n-
BuOH saturated with H2O (3 × 300 mL). The n-BuOH phase (15.0 g)
was subjected to a MPLC using reverse-phase material (Lichroprep. RP-
18, 25–40 mm, 300.0 g) eluting with H2O:MeOH (100:0–0:100) to give
5 main fraction (1–5).
Naturally occurring oleanane saponins have been reported to show
anticancer effect through various pathways, such as antimetastasis,
immunostimulation and chemoprevention (De et al., 2018;
Martucciello et al., 2018; Masullo et al., 2017b). In order to evaluate
the cytotoxic activity of isolated compounds against four cancer cell
lines, including PC-3 (prostate carcinoma cells), MCF-7 (breast cancer
cells), A549 (alveolar basal carcinoma cells), HeLa (cervical carcinoma
cells), an MTT assay, detecting the activity of mitochondrial reductase
of viable cells, was performed. The achieved results showed that com-
pounds 1–6 isolated from S. odontopetala inhibited cell viability in a
dose-dependent manner with IC50 values in a range of 0.09–45.68 μM.
The IC50 values for all affected cell lines are shown in Table 3. Azuki-
saponin IV and compound 4 showed significant activity as compared to
positive control, doxorubicin, against A549 cell line. All isolated com-
pounds did not exhibit any activity at the tested concentrations on
MCF7 cells and showed moderate cytotoxic activity against HeLa cells.
Compounds 1, 5 and 6 showed more than twenty-eight fold, twice-fold
and six fold activity than doxorubicin against PC3 cell line, respec-
tively. In addition, the cytotoxic activity of compounds 1–6 against a
normal cell line HEK-293 was tested. None of the tested compounds
exhibited activity against this cell line.
Fraction 1 (3.0 g) was subjected to silica gel (Merck. 7734, 250.0 g)
column chromatography with the solvent system CH2Cl2:MeOH:H2O
(90:10:1) to give 7 sub-fractions (1a-1g). The sub-fraction 1 b (30.0 mg)
was subjected to a MPLC using reverse-phase material (RP-18, 50.0 g)
eluting with H2O:MeOH (1:1) to give compound 6 (1.7 mg). Fraction 1e
(95.8 mg) was submitted to silica gel (Merck. 7734, 250 g) column
chromatography with the following solvent system CH2Cl2:MeOH:H2O
(80:20:2–70:30:3) to give sub-fraction 1e1 and 1e1 (37.0 mg) was
subjected to MPLC (RP-18, 5.0 g) eluting with H2O:MeOH (1:1) to give
azukisaponin IV (2.5 mg). Fraction 1 d (339.0 mg) was applied to silica
gel (Merck. 7734, 250.0 g) column chromatography with the solvent
system CH2Cl2:MeOH:H2O (80:20:2) to give sub-fraction 1d1 and 1d1
(27.0 mg) was subjected to MPLC (RP-18, 5 g) eluting with H2O:MeOH
(3:2) to give compounds 1 (5.9 mg) and 3 (1.4 mg). Fraction 1f
(43.7 mg) was applied to silica gel (Merck. 7734, 90.0 g) column
chromatography with the solvent system CH2Cl2:MeOH:H2O (70:30:3)
to give sub-fraction 1f2 and 1f2 (27.0 mg) was subjected to MPLC (RP-
18, 5.0 g) eluting with H2O:MeOH (7:3) to give compounds 4 (2.3 mg)
and 5 (3.5 mg).
3. Materials and methods
3.1. General procedures
Optical rotations were measured on a Autopol IV (Rudolph Research
Analytical) polarimeter. IR measurements were obtained with a Bruker
IFS-48 spectrometer. NMR experiments were acquired in methanol-d4
(99.95%, Sigma-Aldrich) with a Bruker DRX-600 spectrometer (Bruker
BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm
TCI CryoProbe at 300 K. Data processing were carried out with Topspin
3.2 software. The ROESY spectra were acquired with tmix = 400 ms. GC
analyses were performed on a Termo Finnigan Trace GC apparatus
using a l-Chirasil-Val column (0.32 mm × 25 m). HRESIMS data were
acquired on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher
Scientific, San Jose, CA, USA) operating in negative ion mode.
Fraction 3 (590.0 mg) was subjected to silica gel (Merck. 7734,
30.0 g) column chromatography with the solvent system
CH2Cl2:MeOH:H2O (90:10:1–80:20:2) to give 4 sub-fractions (3a-3d).
The sub-fraction 3 d (21.0 mg) was subjected to a Sephadex (LH-20
Amersham Biosciences) column with MeOH to give compound 2
(1.0 mg).
The extraction and isolation procedure has been repeated to afford
the required amount of each compound for further chemical and bio-
logical analyses.
3.4. 3β,22α-dihydroxyolean-12-en-23-al-28-oic acid 3-O-α-L-
3.2. Plant material
arabinopyranosyl-(1 → 3)-β-D-glucuronopyranoside (1)
Whole plants of Silene odontopetala Fenzl. (Caryophyllaceae) were
collected from Malatya Darende Ağılbaşı, 1600 m, Turkey (GPS co-
ordinates 38°32′52.0"N; 37°50′49.3"E) in May 2010, and identified by
Serdar G. Şenol (Department of Biology, Faculty of Science, Ege
University, İzmir, Turkey). In the Herbarium of Ege University (EGE-
HERB 42722) a voucher specimen has been deposited.
Amorphous white solid; C41H62O15; [α]2D5 +8.7 (c 0.1 MeOH); IR
KBr
ν
cm−1: 3430, 2925, 1650, 1615; for 1H and 13C NMR (methanol-d4,
max
600 MHz and 150 MHz) data, see Tables 1 and 2; HR-MS [M-H]−m/z
793.4006 (C41H61O15, 793.4010).
6

R.N. Ün, et al.
Phytochemistry176(2020)112404
3.5. 3β,22α-dihydroxyolean-12-en-23-al-28-oic acid 3-O-β-D-
VA, U.S.A.). The cell lines were maintained in Dulbecco's modified
Eagle's medium F12 (DMEM/F12), (10% FBS, 1% L-glutamin, 1%
gentamicin, and 1 mM HEPES). The cells were incubated at 37 °C in a
humidified atmosphere of 5% CO2. During the experiments, ex-
ponential growth phase were used and these cells were sub-cultured
twice a week.
glucuronopyranoside (2)
Amorphous white solid; C36H54O11; [α]2D5 +27.3 (c 0.1, MeOH); IR
νKBrmax cm−1: 3420, 2935, 1715, 1650; for 1H and 13C NMR (me-
thanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2; HR-MS m/z
661.3574 [M-H]- (calcd for C36H53O11, 661.3588).
3.12. Analysis of cell viability
3.6. 3β,22α-dihydroxyolean-12-en-28-oic acid 3-O-α-L-arabinopyranosyl-
(1 → 3)-β-D-glucuronopyranoside (3)
The cytotoxicity of pure compounds was determined using a mod-
ified MTT assay, which detects the activity of mitochondrial reductase
of viable cells (Hamed et al., 2006; Masullo et al., 2017a; Mosmann,
1983). The assay principle is based on the cleavage of MTT that forms
formazan crystals by cellular succinate-dehydrogenases in viable cells.
Briefly, all the cell lines were cultivated for 24 h in 96-well microplates
with an initial cell number of 1 × 105 cells/mL in a humidified at-
mosphere with 5% CO2 at 37 °C. Hence, the cultured cells were treated
with different concentrations of compounds (0.05, 0.1, 0.5, 5, 50 μM),
followed by incubation for 48 h at 37 °C. Doxorubicin (Sigma, St. Louis,
MO, U.S.A.) was used as a positive control. After 48 h, MTT (2.5 mg/
mL) stock solution was added as 25 μL at the end of each incubation
period and incubated for 4 h at 37 °C. Dissolved formazan crystals in
150 μL DMSO were measured at 570 nm (reference filter 620 nm) with
a UV visible spectrophotometer. The viability (%) was determined using
the following formula:
Amorphous white solid; C41H64O14; [α]2D5 +9.6 (c 0.1, MeOH); IR
νKBrmax cm−1: 3430, 2945, 1725, 1650; for 1H and 13C NMR (me-
thanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2; HR-MS m/z
779.4189 [M-H]- (calcd for C41H63O14, 779.4218).
3.7. 21-O-β-D-quinovopyranosyl-(1→3)-β-D-xylopyranosyl-3β,21β-
dihydroxyolean-12-en-23-al-28-oic acid 3-O-α-L-arabinopyranosyl-(1 →
3)-[β-D-galactopyranosyl-(1 → 2)]-β-D-glucuronopyranoside (4)
Amorphous white solid; C58H90O28; [α]2D5 +16.4 (c 0.1, MeOH); IR
νKBrmax cm−1: 3425, 2935, 1725, 1650; for 1H NMR and 13C NMR
(methanol-d4, 600 MHz and 150 MHz) data see Tables 1 and 2; HR-MS
m/z 1233.5518 [M-H]- (calcd for C58H89O28, 1233.5540).
3.8. 21-O-β-D-quinovopyranosyl-(1→3)-β-D-xylopyranosyl-3β,21β-
dihydroxyolean-12-en-28-oic acid 3-O-α-L-arabinopyranosyl-(1 → 3)-[β-
D-galactopyranosyl-(1 → 2)]-β-D-glucuronopyranoside (5)
Absorbance of treated cells Absorbance of blank
Absorbance of control Absorbance of blank
%Viable cells =
× 100
The mean IC50 represents the concentration of an agent that reduces
cell growth by 50% under the experimental conditions, and it is the
average calculated from at least three independent measurements that
would be reproducible and statistically significant. The IC50 values were
reported with 95% confidence intervals ( 95% CIs). This analysis
was performed using Graph Pad Prism 5 (San Diego, CA, U.S.A.).
Amorphous white solid; C58H92O27; [α]2D5 +14.3 (c 0.1, MeOH); IR
νKBrmax cm−1: 3430, 2940, 1725, 1650; for 1H and 13C NMR (me-
thanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2; HR-MS m/z
1219.5714 [M-H]- (calcd for C58H91O27, 1219.5748).
3.9. 21-O-β-D-quinovopyranosyl-(1 → 3)-β-D-xylopyranosyl-3β,21β-
Declaration of competing interest
dihydroxyolean-12-en-23-al-28-oic acid (6)
Amorphous white solid; C41H64O13; [α]2D5 +14.6 (c 0.1, MeOH); IR
νKBrmax cm−1: 3415, 2935, 1720, 1645; for 1H and 13C NMR (me-
thanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2; HR-MS m/z
763.4274 [M-H]- (calcd for C41H63O13, 763.4269).
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
3.10. Acid hydrolysis
This work was supported by the project “Fighting Cancer resistance:
Multidisciplinary integrated Platform for a technological Innovative
Approach to Oncotherapies (Campania Oncotherapies)”. The authors
are grateful to TUBITAK (114Z226), and to the Ege University Research
Foundation (2012 Fen 033) for financial support.
The configuration of sugar units was established by hydrolysis of
compounds 1–6 with 1 N HCl, trimethylsilation and determination of
the retention times by GC operating in the experimental conditions
previously reported (Altunkeyik et al., 2012). The peaks of D-glu-
curonic acid (15.84 min) was detected in the hydrolysate of compounds
1–5. The peaks of L-arabinose (8.94 and 9.80 min) were detected in the
hydrolysate of 1, 3–6. The peaks of D-xylose (10.98 and 12.01 min),
and D-quinovose (11.67, 12.72) were detected in the hydrolysate of
4–6. The peak of D-galactose (13.99 min) was detected in the hydro-
lysate of 4 and 5. Retention times for authentic samples after being
treated in the same manner with 1-(trimethylsilyl)-imidazole in pyr-
idine were detected at 15.81 min (D-glucuronic acid), 13.99 (D-ga-
lactose), D-quinovose (11.65, 12.70), 10.98 and 12.0 (D-xylose), 8.90
and 9.78 (L-arabinose) (De Marino et al., 2003; Horo et al., 2012;
Montoro et al., 2013; Santos et al., 2017; Savran et al., 2012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112404.
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