Triterpene saponins from Silene gallica collected in North-Eastern Algeria

Article abstract of DOI:10.1016/j.phytochem.2020.112274

Eleven previously undescribed triterpene saponins, named silenegallisaponin A-K (1–11), were isolated from the aerial parts of Silene gallica L. Their structures were elucidated by analysis of 1D and 2D-NMR spectroscopic data and mass spectrometry (HR-ESI-MS). The saponins comprised caulophyllogenin, echinocystic acid, or quillaic acid substituted at C-3 by a β-D-glucuronic acid or β-D-galactopyranosyl-(1 → 3)-β-D-glucuronopyranoside and at C-28 by a β-D-fucopyranose substituted at C-2 by a β-D-glucose and at C-3 by a β-D-glucose or a β-D-quinovose.

Full text of DOI:10.1016/j.phytochem.2020.112274

Phytochemistry172(2020)112274
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpene saponins from Silene gallica collected in North-Eastern Algeria
Sara Bechkria,b, Abdulmagid Alabdul Magidb, Charlotte Sayaghb, Djemaa Berrehala,
Dominique Harakatb, Laurence Voutquenne-Nazabadiokob, Zahia Kabouchea,
Ahmed Kabouchea,∗
a Université des frères Mentouri-Constantine 1, Département de chimie, Laboratoire d’Obtention des Substances Thérapeutiques (LOST), Campus Chaabet-Ersas, 25000,
Constantine, Algeria
b Université de Reims Champagne Ardenne, CNRS, ICMR UMR 7312, 51097, Reims, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Silene gallica
Caryophyllaceae
Triterpene saponins
Eleven previously undescribed triterpene saponins, named silenegallisaponin A-K (1–11), were isolated from the
aerial parts of Silene gallica L. Their structures were elucidated by analysis of 1D and 2D-NMR spectroscopic data
and mass spectrometry (HR-ESI-MS). The saponins comprised caulophyllogenin, echinocystic acid, or quillaic
acid substituted at C-3 by a β-D-glucuronic acid or β-D-galactopyranosyl-(1 → 3)-β-D-glucuronopyranoside and at
C-28 by a β-D-fucopyranose substituted at C-2 by a β-D-glucose and at C-3 by a β-D-glucose or a β-D-quinovose.
1. Introduction
2. Results and discussion
The genus Silene, belonging to the family Caryophyllaceae, contains
more than 700 species mainly distributed in temperate regions of the
Northern Hemisphere of Eurasia and America, but also in Africa
(Mamadalieva et al., 2014). The genus consists mainly of herbaceous
plants and, more rarely, small shrubs or subshrubs (Mamadalieva et al.,
2014). Phytochemical investigations of the genus Silene have led to the
isolation of several phytoecdysteroids (Mamadalieva et al., 2014), tri-
terpene saponins (Gaidi et al., 2002), benzenoids, flavonoids
(Darmograi, 1977), anthocyanins, N-containing compounds (Dötterl
et al., 2005), sterols, and vitamins (Arnetoli et al., 2008; Eshmirzaeva
et al., 2005). The abundance and widespread occurrence of triterpene
saponins are typical features of the family Caryophyllaceae. Previous
investigations of Silene plants led to isolation of approximately 52 tri-
terpenoid saponins (Böttger and Melzig, 2011; Mamadalieva et al.,
2014). Silene gallica L., native to central Europe, is an annual species
growing to 0.4 m in height (Asai and Fujimoto, 2010). To our knowl-
edge, saponins have not been isolated from S. gallica however, ten cyclic
fatty acyl glycosides were previously reported from the glandular tri-
chome exudate (Asai and Fujimoto, 2010). Here, we report the isolation
and structure elucidation of eleven undescribed triterpene saponins
from the aerial parts of S. gallica L. Their structures were elucidated by
spectroscopic methods including 1D and 2D-NMR experiments (1H, 13C,
HSQC, HMBC, COSY, ROESY), in combination with HR-ESI-MS.
An ethanol/water (80/20 v/v) extract of the aerial parts of S. gallica
was suspended in H2O and partitioned successively with petroleum
ether, CHCl3, EtOAc and n-BuOH. The n-BuOH-soluble fraction was
purified by repeated chromatography on normal and reversed phase
RP-18 silica gel yielding eleven triterpenoid saponins (1–11).
Compound 1 exhibited, in the HR-ESI-MS (positive ion mode), a
quasimolecular ion peak at m/z 1157.5367 [M+Na]+, consistent with
the molecular formula C54H86O25. The presence of six tertiary methyl
groups (δH 0.72, 0.79, 0.91, 0.97, 1.02, and 1.37) and one tri-sub-
stituted olefinic proton (δH 5.32, t, J = 3.7 Hz, H-12) in the 1H-NMR
spectrum (Table 1) and six sp3carbons δC 12.0, 15.2, 16.5, 23.7, 26.0,
and 31.9 with two sp2 olefinic carbons (δC 121.9 and 143.5) in the 13C-
NMR indicated that compound 1 possesses an olean-12-ene skeleton
(Boutaghane et al., 2013). Furthermore, signals for one oxygenated
methylene (δC 63.3/δH 3.29 and 3.63), two oxygenated methines (δC
82.0/δH 3.66, and δC 72.7/δH 4.78) and one carboxyl group (δC 175.7)
were observed in 13C-NMR and 1H-NMR spectra. Through an extensive
2D-NMR study, the aglycone was identified as 3β,16α,23-trihydrox-
yolean-12-en-28-oic acid, commonly named caulophyllogenin, which is
in good agreement with literature data (Table 1) (Matsuo et al., 2009).
The chemical shift values of C-3 (δC 82.0) and C-28 (δC 175.7) sug-
gested that the saponin was a bisdesmosidic glycoside with saccharide
units attached to these positions (Lehbili et al., 2017, 2018). The HMBC
spectrum showed cross-peaks between H-16 signal with C-14, C-15, C-
∗ Corresponding author.
E-mail address: ahmedkabouche6@gmail.com (A. Kabouche).
https://doi.org/10.1016/j.phytochem.2020.112274
Received 13 September 2019; Received in revised form 7 January 2020; Accepted 13 January 2020
0031-9422/©2020PublishedbyElsevierLtd.

S. Bechkri, et al.
Phytochemistry172(2020)112274
Table 1
13C NMR and 1H NMR spectroscopic data of the aglycone moieties of compounds 1–6 in CD3OD.a, b
1
2
3
4
5
6
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
1
2
3
38.2
25.0
82.0
1.01, m 1.65
38.2
24.9
81.0
1.02, m 1.63, m
38.2
24.9
1.02, m 1.64, m
38.1
25.0
1.00, m 1.64,
m
1.76, m
1.91
3.67, dd (12.0, 89.6
4.3)
38.4
25.6
1.03, m 1.63
37.8
24.2
81.3
1.18, m
1.71, td ( 13.6, 3.4)
1.80
1.78
1.89
3.66, dd (12.5,
5.0)
1.78, m
1.97, m
3.67, dd (11.5, 4.7) 81.0
1.78, m
1.98
3.69, dd (12.1, 4.3) 81.9
1.72, m
1.90
3.20, dd (11.7,
4.3)
2.04
3.98, dd (11.7, 4.3)
4
5
6
42.5
46.8
17.4
–
42.5
46.8
17.5
–
1.28
42.5
46.9
17.6
–
42.5
46.9
–
1.27
1.40 1.52, m
38.8
55.7
18.0
–
55.0
47.4
–
1.38
0.98 1.59
1.26
1.39
1.51
1.33 1.68
1.28, br d (12.9)
1.43, td (12.5, 3.5) 17.5
1.55, br d (11.5)
0.82, br d (12.3)
1.46, td (12.3, 3.5) 20.0
1.60
1.39, m 1.56, td
(12.5,3.3)
–
1.42, td (12.1, 3.6)
1.52, br d (12.3)
1.33 1.68
7
32.4
32.4
32.3
1.37, m 1.68
32.4
1.32 1.68
33.0
32.2
1.32 1.58, m
8
9
39.5
46.8
–
1.70
39.5
46.9
–
39.5
46.8
–
39.5
46.8
–
39.5
46.8
39.8
46.7
–
1.80
1.72, br t (9.3)
1.73, t (13.3)
1.72, br t
(10.0)
–
1.68, br t (9.3)
10 36.3
11 23.1
–
36.3
23.1
–
36.3
23.1
–
36.3
23.1
36.5
23.1
–
35.6
23.0
–
1.91 1.93
1.91 1.93
1.90 1.92
1.89 1.91
1.91 1.93
1.95 1.97
12 121.9 5.32, t (3.7)
121.9 5.32, t (3.3)
122.0 5.32, t (3.5)
122.0 5.31, t (3.5)
122.0 5.32, t (3.1)
121.7 5.33, t (3.5)
13 143.5
14 41.2
15 35.5
–
–
143.5
41.1
35.7
–
–
1.33
1.80
143.4
41.2
35.6
–
–
143.7
41.2
–
–
1.33
1.93
143.5
41.1
35.7
–
–
143.6
41.1
35.8
–
–
1.32 1.79
1.35 1.81, dd (14.5, 35.4
3.5)
4.72, t (3.5)
1.32 1.80, dd
(15.0, 3.6)
4.78, t (3.5)
–
2.99, dd (14.4,
4.1)
1.31 1.79
16 72.7
17 48.6
18 40.6
4.78, t (3.5)
–
2.98, dd (14.3,
4.0)
72.7
48.5
40.5
4.78, t (3.4)
–
2.99, dd (14.5, 4.2) 40.6
72.8
48.4
73.1
47.5
4.62, br s
–
2.99, dd (13.5, 40.5
4.2)
72.7
48.5
72.7
47.5
40.4
4.77, br s
–
2.99, dd (13.3, 4.0)
–
2.99, dd (14.4, 4.0) 40.5
19 46.3
1.08, dd (12.7,
3.2) 2.28, t (13.8)
–
46.3
1.08, dd (13.0, 3.1) 46.3
2.27, t (13.4)
1.08, dd (14.1, 3.6) 46.3
2.26, t (14.1)
1.05, m 2.30,
br t (13.5)
–
46.3
1.08, m 2.27, t
(13.9)
–
46.2
1.09, dd (13.3, 5.2)
2.28, t (13.5)
–
20 30.0
21 35.0
29.9
35.0
–
29.9
34.9
–
29.9
35.1
29.9
35.0
29.9
35.0
1.16, m 1.91
1.16, br d (12.7)
1.92
1.17, m 1.91
1.16, m 1.91
1.16, m 1.93
1.18 1.95
22 29.9
23 63.3
1.70 2.01, dt
(14.0, 3.7)
3.29, d (11.5)
3.63, d (11.5)
0.72, s
1.02, s
0.79, s
1.37, s
–
30.0
63.4
1.70 2.02, dt (13.8, 29.9
3.9)
1.71 2.00
30.7
63.4
1.69 1.94
29.9
27.1
1.69 2.02 dt (13.2, 30.3
3.5)
1.69, td (13.5, 3.7)
2.01, m
3.28, d (11.7)
3.65, d (11.7)
0.79, s
63.4
3.24, d (11.7)
3.64, d (11.7)
0.72, s
1.01, s
0.80, s
1.38, s
–
0.91, s
0.97, s
3.30, d (12.0)
3.64, d (12.0)
0.73, s
1.02, s
0.78, s
1.40, s
–
0.91, s
0.97, s
1.08, s
207.5 9.44, s
24 12.0
25 15.2
26 16.5
27 26.0
28 175.7
29 31.9
30 23.7
12.1
15.2
16.6
26.0
175.6
31.9
23.7
12.1
15.2
16.6
26.0
175.7
31.9
23.8
12.0
15.2
16.5
25.9
175.7
31.9
23.5
15.6
14.7
16.5
25.9
175.6
31.9
23.6
0.88, s
0.98, s
0.79, s
1.37, s
–
8.9
1.12, s
1.03, s
0.79, s
1.39, s
–
1.01, s
14.8
16.4
25.9
175.5
31.9
23.5
0.79, s
1.38, s
–
0.91, s
0.97, s
0.91, s
0.91, s
0.97, s
0.91, s
0.97, s
0.97, s
a
Overlapped signals are reported without designated multiplicity.
in ppm, J in parentheses in Hz.
b
17, C-18 and C-22, thus unambiguously locating the hydroxyl group at
C-16 (δC 72.7). The 16α-configuration of hydroxyl group was evident
from the small J values of H-16 and H-15 (J = 3.5 Hz), characteristic of
an equatorial proton (Lehbili et al., 2017). Moreover, ROESY correla-
tion was observed between Me-26 (δH 0.79) and H-16 β-oriented while
no one was observed between Me-27 (δH 1.37) and H-16 confirming
that H-16 had β-configuration. The α-configuration of H-3 (δH 3.66) was
indicated by the coupling constants of proton H-3 with the protons H-2
(J = 12.5 and 5.0 Hz), characteristic of an axial proton. This bas-
sumption was confirmed by the ROESY correlation between H-3 (δH
3.66)/H-5 (δH 1.26) and H-5/H-9 (δH 1.70) α-oriented. Furthermore, an
HMBC cross-peak of δH-24 0.72 with δC-23 63.3 and a ROESY cross-peak
between H-3 and H2-23 (δH 3.29, 3.63) suggested the location of the
primary alcoholic function at C-23.
coupled with H-5 (δH 3.81, d, J = 9.1 Hz) and H-4 (δH 3.52, t,
J = 9.3 Hz) of the same sugar in the HMBC. The 13C-NMR signals of the
glucuronic acid of 1 were fully determined in the HSQC experiments
and revealed it to be in terminal position as summarized in Table 2
(Alabdul Magid et al., 2006). In the same manner, two β-D-glucopyr-
anose units in terminal positions were identified starting from their
anomeric protons (glc1, δH-1 4.88, d, J = 8.0 Hz; glc2, δH-1 4.63, d,
J = 7.5 Hz) and according to the large coupling constants (JH-1,H-2, JH-
2,H-3, JH-3,H-4, JH-4,H-5 ≥ 7.5 Hz) and their carbon chemical shift values
(Table 2) (Boutaghane et al., 2019). The last sugar unit was identified
as β-D-fucopyranose (fuc) (δH-1 5.38, d, J = 8.2 Hz) based on the large
coupling constants JH-1,H-2 and JH-2,H-3 (≥8.2 Hz) and the small cou-
pling constant between H-3 and H-4 (J = 3.0 Hz) and the doublet
methyl proton signal at δH 1.27 (d, J = 6.4 Hz, H-6), as summarized in
Table 2 (Voutquenne-Nazabadioko et al., 2013). The β-D-fucopyranose
unit was found to be substituted at the C-2 (δC 73.3) and C-3 (δC 84.0)
positions (Table 2). The anomeric configurations (β) were determined
by the JH-1-H-2 coupling constants and the comparison of 13C-NMR data
with those in the literature (Table 2) (Agrawal, 1992). The steric series
(D) were determined after acid hydrolysis of the saponin mixture (see
experimental). The rOe interactions observed in the ROESY spectrum
between H-1, H-3 and H-5 of each sugar unit confirmed the α-axial
The presence of four sugar moieties in 1 was evidenced by the 1H
NMR spectrum which displayed anomeric protons at δH 4.48, 4.63,
4.88, and 5.38, giving correlations with the corresponding anomeric
carbons at δC 104.5, 103.9, 102.3, and 92.6, respectively in the HSQC
spectrum. A β-D-glucuronopyranose unit (glcA) was identified starting
from the anomeric proton at δH 4.48 (d, J = 7.8 Hz), and characterized
by a five spin system possessing large coupling constants (J ≥ 7.8 Hz)
in the COSY spectrum and by a carbonyl (C-6) resonating at δC 171.3
2

S. Bechkri, et al.
Phytochemistry172(2020)112274
Table 2
13C NMR and 1H NMR spectroscopic data in CD3OD of the sugar moieties of compounds 1–6.a,b
1
2
3
4
5
6
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
GlcA-
1
2
104.5 4.48, d (7.8)
103.9 4.46, d (7.8)
103.9 4.45, d (7.9)
104.4 4.48, d (7.8)
105.6 4.40, d (7.8)
103.2 4.22, d (7.8)
73.8
3.25, dd (8.9,
7.8)
73.7
3.26, t (8.8)
73.7
3.25, t (8.2)
73.7
3.21, t (8.8)
73.9
3.26, t (8.7)
73.7
3.1 3, t (7.8)
3
4
5
6
76.3
71.8
75.1
171.3
3.38, t (8.9)
3.52, t (9.3)
3.81, d (9.1)
–
76.6
72.2
75.2
172.1
3.39, t (8.9)
3.46, t (9.1)
3.78, d (9.1)
–
76.7
72.2
75.2
171.7
3.38
3.45, t (8.9)
3.78
–
76.3
71.9
75.2
172.1
3.39, t (8.9)
3.50, t (9.1)
3.78, d (9.1)
–
76.3
71.9
76.5
171.1
3.37, t (8.7)
3.51, t (9.0)
3.75
76.3
72.2
76.2
172.1
3.33
3.43, t (9.5)
3.42
–
–
Fuc-
1
2
92.6
73.3
5.38, d (8.2)
4.08, dd (9.4,
8.2)
92.4
73.1
5.40, d (8.2)
4.11, dd (9.6, 8.2) 74.7
92.6
5.35, d (8.2)
4.03, dd (9.4, 8.2) 73.3
92.5
5.40, d (8.2)
4.11, dd (9.6,
8.2)
92.4
73.1
5.40, d (8.3)
4.11, dd (9.7, 8.3) 73.0
92.4
5.40, d (8.3)
4.10, dd (9.5, 8.3)
3
84.0
3.88, dd (9.9,
3.0)
84.2
3.87, dd (10.0,
3.1)
74.3
3.78, dd (9.6, 3.5) 84.3
3.87, dd (10.0,
3.1)
84.2
3.87, dd (9.7, 3.1) 84.2
3.87, dd (9.5, 3.0)
4
5
6
71.3
71.2
15.2
3.95, d (3.0)
3.78, m
1.27, d (6.4)
71.4
71.3
15.1
3.95, d (3.1)
3.78, m
1.28, d (6.4)
71.8
71.5
15.2
3.62, d (3.5)
3.74, m
1.27, d (6.4)
71.2
71.1
15.2
3.95, d (3.1)
3.78, m
1.28, d (6.4)
71.4
71.3
15.1
3.95, d (3.1)
3.78, m
1.27, d (6.4)
71.4
71.3
15.1
3.95, d (3.0)
3.79
1.27, d (6.4)
Glc1
1
102.3 4.88, d (8.0)
101.9 5.03, d (8.0)
102.3 4.88 d (8.0)
102.1 4.93, d (8.0)
101.9 5.03, d (8.1)
101.8 5.03, d (8.1)
2
3
4
5
74.5
77.0
70.5
76.5
61.6
3.13, t (8.5)
3.36, t (8.5)
3.30
3.30
3.77, dd (12.0,
5.1)
72.9
78.3
68.7
76.2
61.4
3.27, t (8.0)
4.92
3.47, t (9.0)
3.38 m
3.79, dd (12.4,
5.9)
3.93, dd (12.4,
1.8)
72.7
77.9
68.7
76.4
61.4
3.36
4.94
3.45, t (9.0)
3.37
3.77, dd (12.0,
5.6)
3.93, dd (12.0,
1.8)
2.14, s
–
74.4
76.8
70.9
73.8
64.3
3.15, t (8.0)
3.38, t (8.3)
3.22, t (9.3)
3.51, m
4.35, dd (11.7,
2.0)
4.39, dd (11.7,
5.2)
2.17, s
73.0
78.3
68.7
76.2
61.4
3.27, t (9.5)
4.93
3.48, t (9.4)
3.38
3.78
3.93, dd
(12.5.,2.2)
73.0
78.3
68.7
76.2
61.4
3.25, dd (9.3, 8.1)
4.90, t (9.3)
3.44, t (9.3)
3.36
3.79, dd (12.1,
6.4)
3.92, dd (12.1,
1.8)
2.12, s
6
3.93, dd (12.0,
1.8)
Ac-CH3
COO
19.8
171.4
2.14, s
–
19.8
171.2
19.8
172.3
19.8
171.3
2.18, s
–
19.8
171.4
–
–
Glc2
1
2
3
4
5
6
103.9 4.63, d (7.5)
104.0 4.61, d (7.2)
104.0 4.61, d (7.2)
104.0 4.60, d (7.2)
104.0 4.60, d (7.0)
73.9
76.8
69.7
76.5
60.9
3.32
3.31
3.32
3.30
3.70, dd (12.0,
5.0)
74.0
76.8
69.6
76.5
60.9
3.32
3.33
3.33
3.30
3.70, dd (12.4,
5.1)
73.9
76.8
69.8
76.5
60.9
3.33
3.35
3.34
3.31
3.70, dd (12.4,
5.1)
74.0
76.8
69.6
76.5
60.9
3.32
3.33
3.32
3.29
74.0
76.8
69.6
76.5
3.31
3.32
3.31
3.28
3.69 dd (12.1,5.1) 60.9
3.85 dd (12.1,1.9)
3.69 dd (12.0,
5.0)
3.85, dd (12.0,
1.7)
3.85, dd (12.4,
1.8)
3.85, dd (12.4,
1.8)
3.85 (12.0, 1.9)
a
Overlapped signals are reported without designated multiplicity.
in ppm, J in parentheses in Hz.
b
orientation of these protons and the β-anomeric configuration. Com-
plete assignments of the proton and carbon resonances of all the sugars
were achieved by extensive 1D and 2D NMR analyses (Table 2). The
sequence and the attachment of each saccharide were determined by
analysis of HMBC and ROESY spectra. The cross-peaks observed in the
HMBC spectrum between H-1-glc1/C-2-fuc, H-1-glc2/fuc-C-3, and H-1-
fuc/aglycone-C-28 indicated that a β-D-fucopyranose disubstituted at C-
2 and C-3 positions by two β-D-glucopyranose units was linked to C-28
of caulophyllogenin. The glycosylation at C-3 of the aglycone was
confirmed as a β-D-glucuronopyranose moiety, through HMBC correla-
tion of H-1-glcA to C-3-caulophyllogenin. In addition, ROESY correla-
tions, confirming the interglycosidic linkage of the trisaccharide and
the point of attachment of glcA at the C-3 of the aglycone, were ob-
served between H-1-glcA-/H-3-aglycone, H-1-glc1/H-2-fuc, and H-1-
glc2/H-3-fuc. Based on the above spectral data, the structure of 1 was
identified as 3-O-β-D-glucuronopyranosyl caulophyllogenin 28-O-β-D-
glucopyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyrano-
side, named silenegallisaponin A (Fig. 1).
were closely comparable to those of 1, except for the signals of the
glucose moiety (glc1) and additional signals of an acetyl group (δH 2.14
and δC 19.8, CH3) and 171.4 (CO) (Table 2). The β-D-glucopyranose
(glc1) possessed a deshielded proton H-3 at δH 4.92, indicating the
linkage of the acetyl group. This was readily confirmed by the HMBC
correlation between H-3-glc1 and the carbonyl signal (δC 171.4) of the
acetyl group. Full assignments of the proton and carbon resonances of
the aglycone and the sugar parts were achieved by analysis of the COSY,
HSQC, HMBC, and ROESY spectra (Tables 1 and 2). The sequence and
the attachment of the saccharide units in 2 were confirmed as in 1 by
HMBC and ROESY experiments. On the basis of the above analysis, the
structure of 2 was established as 3-O-β-D-glucuronopyranosyl caulo-
phyllogenin 28-O-β-D-glucopyranosyl-(1
→
3)-[3-O-acetyl-β-D-gluco-
pyranosyl-(1 → 2)]-β-D-fucopyranoside, named silenegallisaponin B
(Fig. 1).
Compound 3 displayed in the positive HR-ESI-MS a quasimolecular
ion peak [M+Na]+ at m/z 1137.4926 (calcd for C50H78O21Na,
1037.4933), suggesting the lack of one hexose unit compared to 2.
Furthermore, the appearance of three pairs of anomeric proton and
carbon signals in the 1H and 13C-NMR spectra (Table 2) confirmed the
presence of three sugar units. Comparison of the 1H and 13C-NMR va-
lues and the analysis of the HMBC and ROESY correlations showed that
2 and 3 contained the same aglycone (Table 1). The detailed analysis of
the 2D-NMR spectra led to the identification of caulophyllogenin as
Compound 2 gave, in the positive HR-ESI-MS, a quasimolecular ion
peak at m/z 1199.5454 [M+Na]+ (calcd for C56H88O26Na,
1199.5462), suggesting the presence of a supplementary acetyl group
(42 amu) compared to compound 1. The structural analysis revealed
that the NMR signals of the aglycone part of 2 were superimposable to
those of 1 (Table 1). The 1H and 13C-NMR data of the sugar portion of 2
3

S. Bechkri, et al.
Phytochemistry172(2020)112274
Fig. 1. Chemical structures of compounds 1–11 isolated from Silene gallica.
aglycone, an acetyl group (ac), and three sugar units: a terminal β-D-
glucuronopyranose (glcA), a β-D-glucopyranose (glc), and a β-D-fuco-
pyranose (fuc). The HMBC spectrum of 3 showed long-range correla-
tions between H-3-glc/C-1-ac, H-1-glc/C-2-fuc, H-1-fuc/C-28-aglycone,
H-1-glcA/C-3-aglycone. These findings led to the assignment of com-
pound 3 as 3-O-β-D-glucuronopyranosyl caulophyllogenin 28-O-[3-O-
acetyl-β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside, named silene-
gallisaponin C (Fig. 1).
chain glc(1 → 3)-[3-O-acetyl-glc(1 → 2)]-fuc- at C-28 of the aglycone
(Table 2). The difference was the absence of the hydroxymethylene
group (–CH2OH–23) of the aglycone. An extra methyl moiety was de-
tected in 5 (δH 1.08). The HMBC spectrum revealed proper correlations
for this methyl group with C-4 (δC 38.8), C-5 (δC 55.7), C-3 (δC 89.6), as
well as C-24 (δC 15.6). The chemical shifts values for all protons and
carbons of the aglycone moiety (Table 1) were in accordance with the
literature data of echinocystic acid (3β,16α-dihydroxyolean-12-en-28-
oic acid) (Lehbili et al., 2017), which was further confirmed by COSY,
HSQC, HMBC and ROESY experiments on 5. Consequently, the struc-
ture of 5 was concluded to be 3-O-β-D-glucuronopyranosyl echinocystic
acid 28-O-β-D-glucopyranosyl-(1 → 3)-[3-O-acetyl-β-D-glucopyranosyl
(1 → 2)]-β-D-fucopyranoside, named silenegallisaponin E (Fig. 1).
Compound 6 had a molecular formula of C56H86O26 according to the
HR-ESI-MS quasi molecular ion peak at m/z 1197.5298 [M+Na]+
(calcd for C56H86O26Na, 1197.5305). The 2D-NMR analysis showed
that compounds 5 and 6 differed only in the aglycone part at C-23
position (Tables 1 and 2). The methyl group CH3-23 signals (δH 1.08, δC
27.1) in 5 was replaced by an aldehyde function (δH 9.44, δC 207.5) in
6. Full assignments of the proton and carbon resonances of the aglycone
and the sugar parts were achieved by analysis of the COSY, HSQC and
HMBC spectra (Tables 1 and 2). The triterpene skeleton of 6 was thus
determined as the known quillaic acid (3β,16α-dihydroxy-23-oxoolean-
12-en-28-oic acid) (Takahashi et al., 2016). The sequence and the at-
tachment of the saccharide units in 6 were confirmed as in 5 by an
HMBC and ROESY experiments. Thus, the structure of 6 was elucidated
as 3-O-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-glucopyranosyl-
(1 → 3)-[3-O-acetyl-β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside,
named silenegallisaponin F (Fig. 1).
Compound 4 gave in the positive HR-ESI-MS a quasimolecular ion
peak at m/z 1199.5455 [M+Na]+ (calcd for C56H88O26Na, 1199.5462)
in agreement with a molecular formula of C56H88O26 which was an
isomeric with compound 2. Inspection of the spectroscopic data in-
dicated that saponin 4 had the same aglycone (caulophyllogenin) and
glycoside parts as saponin 2 (fuc: δH-1 5.40, glcA: δH-1 4.48, glc1: δH-1
4.93, glc2: δH-1 4.61) and an acetyl group (Table 2). The 1H and 13C-
NMR data of the sugar portion of 4 were closely comparable to those of
2, except for the signals of the glucose moiety (glc1). Glc1 possessed
deshielded protons H2-6 (δH 4.35 and 4.39) indicating the position of
the acetyl group. These results suggested that compound 4 is a regioi-
somer of 2 with the acetate attached to C-6-glc1 rather than to C-3-glc1
(Table 2). This was confirmed by the HMBC correlation between H2-6-
glc1 and the carbonyl signal (δC 172.3) of the acetyl group. The se-
quence and the attachment of the saccharide chains in 4 were con-
firmed as in 2 by HMBC and ROESY experiments. Thus, the structure of
compound 4 was established as 3-O-β-D-glucuronopyranosyl caulo-
phyllogenin 28-O-β-D-glucopyranosyl-(1
→
3)-[6-O-acetyl-β-D-gluco-
pyranosyl-(1 → 2)]-β-D-fucopyranoside, named silenegallisaponin D
(Fig. 1).
Compound 5 exhibited in the positive HR-ESI-MS a quasimolecular
ion peak at m/z 1183.5502 [M+ Na]+ (calcd C56H88O25Na,
1183.5512), suggesting one oxygen less than that of 2. Comparison of
the 13C-NMR data of 5 to those of 2 and analysis of the 2D-NMR spectra
of 5 showed that both possessed a glcA at C-3 and the trisaccharide
Compound 7 showed, in the positive HR-ESI-MS, a quasimolecular
ion peak at m/z 1319.5870 [M
+
Na]+ (calcd C60H96O30Na,
1319.5884). Comparison of the NMR data of 7 with 1 and detailed
analysis of the 2D-NMR spectra showed that they possessed the same
4

S. Bechkri, et al.
Phytochemistry172(2020)112274
Table 3
13C NMR and 1H NMR spectroscopic data of the aglycone moieties of compounds 7–11 in CD3OD.a,b
7
8
9
10
11
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
1
2
38.2
24.9
1.01
1.63, m
1.77
38.1
24.9
1.01, m
1.63
1.78
38.1
25.0
1.01, m
1.63, m
1.77, m
38.2
25.0
1.00
1.63, m
1.78, m
38.2
24.9
1.01, m 1.63, m
1.78
1.94
1.92
1.90, m
1.90, m
1.92, m
3
4
5
6
81.4
42.5
46.8
17.4
3.68 dd (12.0, 4.5)
–
1.26
1.40
1.52
1.33
1.68
–
81.5
42.5
46.7
17.5
3.67, dd (12.3, 4.7)
–
1.26, m
1.39
1.51
1.33
1.68
–
82.0
42.5
46.8
17.5
3.66, dd (12.0, 4.1)
–
81.9
42.5
46.8
17.4
3.66, dd (12.1, 4.1)
–
81.4
42.5
46.7
17.5
3.68, dd (12.1, 4.1)
–
1.25, m
1.39, m
1.52, m
1.33
1.68
–
1.70, t (10.3)
–
1.90 1.92
1.27
1.41, m
1.52, m
1.33
1.69
–
1.71
–
1.91 1.93
1.27
1.39, m
1.52, m
1.32
1.66
–
1.70
–
1.91 1.93
7
32.4
32.4
32.4
32.4
32.0
8
9
39.5
46.9
39.4
46.9
36.3
23.1
39.5
46.8
36.3
23.1
39.5
46.8
36.3
23.1
39.5
46.2
36.3
23.1
1.70, t (9.9)
–
1.91 1.92
1.72, t (10.3)
–
1.90 1.92
10 36.3
11 23.1
12 121.9 5.31, t (3.9)
122.0 5.31, t (3.1)
121.9 5.32, t (3.1)
121.9 5.32, t (3.5)
121.9 5.32, t (3.5)
13 143.5
14 41.2
15 35.0
16 72.7
17 48.5
18 40.6
19 46.3
–
–
143.7
41.2
–
–
143.5
41.2
–
–
143.5
41.2
–
–
143.5
41.2
–
–
1.32 1.80, dd (14.7, 3.5) 35.4
4.77, t (3.8)
–
2.98, dd (14.2, 4.2)
1.08, m
2.28, t (13.5)
–
1.15, m 1.91
1.69 1.99, dt (14.2, 4.4) 30.7
3.28, d (11.5)
3.64, d (11.5)
0.72, s
1.01, s
0.79, s
1.38, s
–
0.91, s
0.97, s
1.32 1.83, dd (14.1, 3.5) 35.7
4.62, br s
–
1.33 1.80, dd (15.1, 3.6) 35.5
4.77, t (3.2)
–
2.99, dd (14.5, 4.1)
1.08, m
2.28, t (13.5)
–
1.17, m 1.92
1.69 2.01
3.29, d (11.6)
3.65, d (11.6)
0.73, s
1.01, s
0.79, s
1.38, s
–
0.91, s
0.97, s
1.33 1.79, dd (15.2, 3.3) 35.5
4.77, t (3.5)
–
2.98 dd (14.5, 4.3)
1.08 m 2.26 t (13.4)
1.33 1.78, dd (11.5, 3.5)
4.77, t (3.5)
–
2.98, dd (14.2, 4.6)
1.08, m 2.28,t (13.9)
73.0
48.5
40.5
46.3
72.7
48.5
40.5
46.3
72.7
48.5
40.6
46.3
72.7
48.5
40.6
46.3
2.99, dd (14.0, 4.0)
1.08, m
2.30, t (13.0)
–
1.16, m 1.95
1.69 1.94
3.28, d (11.5)
3.64, d (11.5)
0.72, s
1.01, s
0.78, s
1.40, s
–
20 29.8
21 35.1
22 30.7
23 63.5
29.9
35.1
29.9
35.0
30.0
63.3
29.9
35.0
29.9
63.3
–
29.8
35.0
29.8
63.5
–
1.17, m 1.92
1.69 2.01
3.29, d (11.6)
3.64, d (11.6)
0.72, s
1.01, s
0.79, s
1.38, s
–
1.16, m 1.93
1.69 2.01
3.28, d (11.4)
3.64, d (11.4)
0.72, s
1.01, s
0.79, s
1.38, s
–
63.5
24 12.0
25 15.2
26 16.5
27 26.0
28 175.7
29 31.7
30 23.7
12.0
15.2
16.5
25.9
175.6
31.9
23.5
12.0
15.2
16.6
26.0
175.6
31.9
23.7
12.0
15.2
16.6
26.0
175.7
31.9
23.7
12.0
15.2
16.6
25.9
175.7
31.9
23.7
0.91, s
0.97, s
0.91, s
0.97, s
0.91, s
0.97, s
a
Overlapped signals are reported without designated multiplicity.
in ppm, J in parentheses in Hz.,
b
saccharide and aglycone (caulophyllogenin) pattern but with the pre-
sence of one supplementary sugar moiety in 7 (Tables 3 and 4). Analysis
of COSY and HSQC spectra, allowed assignment of this additional
monosaccharide as a terminal β-D-galactopyranose unit (gal) (δH-1 4.57,
d, J = 7.8 Hz; δC-1 104.0), characterized by the large coupling constants
JH-1,H-2 and JH-2,H-3 (> 7.8 Hz) and the small coupling constant between
H-3 and H-4 (JH-3,H-4 = 3.4 Hz) as summarized in Table 4 (Lehbili et al.,
2018). The deshielded signals of C-3-glcA (δC 84.9) indicated that ga-
lactopyranose moiety was attached to C-3 of the glucuronopyranose
unit. This was confirmed by the HMBC correlation between H-1-gal and
C-3-glcA. Thus, the structure of 7 was established as 3-O-β-D-galacto-
pyranosyl-(1 → 3)-β-D-glucuronopyranosyl caulophyllogenin 28-O-β-D-
glucopyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyrano-
side, named silenegallisaponin G (Fig. 1).
Compound 8 exhibited, in the positive HR-ESI-MS, a molecular ion
at m/z 1361.5981 [M+Na]+ (calcd for C62H98O31Na, 1361.5990),
suggesting a supplementary acetyl unit compared to compound 7. The
1H and 13C-NMR data of compound 8 were closely comparable to those
of 7 except for the signals of β-D-glucopyranose unit (glc1) and the
presence of one acetyl group (δH 2.18; δC 19.8, 172.3) (Tables 3 and 4).
The glc1 possessed two deshielded protons H2-6 (δH 4.35, 4.39), in-
dicating the position of the acetyl group. This was confirmed by the
HMBC correlation between H-6-glc1 and the carbonyl signal of the
acetyl group. Assignments of all proton and carbon resonances of 8
were completed by extensive analysis of the 2D-NMR spectra (Tables 3
and 4). The HMBC correlations showed that the sugars were attached in
the same way in both saponins 7 and 8. Consequently, the structure of 8
was concluded to be 3-O-β-D-galactopyranosyl(1
→
3)-β-D-glucur-
onopyranosyl caulophyllogenin 28-O-β-D-glucopyranosyl-(1 → 3)-[6-O-
acetyl-β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside, named silene-
gallisaponin H (Fig. 1).
Compound 9 exhibited, in the positive HR-ESI-MS, a molecular ion
peak at m/z 1183.5503 [M+Na]+ (calcd for C56H88O25Na,
1183.5512), suggesting a supplementary deoxyhexose unit compared to
saponin 3. The 1H and 13C-NMR resonances of the aglycone of 9 mat-
ched well with the signals of 3 indicating the same aglycone and one
acetyl group for both compounds. The 1H-NMR spectrum of 9 showed
indeed the occurrence of four anomeric signals at δH 4.48, 4.59, 5.04,
and 5.40. Complete assignments of each sugar were achieved by ex-
tensive 1D and 2D-NMR analyses, allowing the identification of one β-
glucuronopyranose, one β-fucopyranose, and one β-glucopyranose
units, as in 3. Th e fourth sugar unit (δH-1 4.59 d, J = 7.6 Hz) was
identified as terminal β-quinovopyranose unit (qui) as ascertained from
the analysis of 2D-NMR spectra, based on the large coupling constants
between JH-1,H-2, JH-2,H-3, JH3-,H-4, JH-4,H-5 (J ≥ 7.6 Hz) and the doublet
methyl proton signal at δH 1.28 (d, J = 6.4 Hz, H-6), as summarized in
Table 4 (Pertuit et al., 2014). The deshielded signals of C-3-fuc (δC 83.9)
indicated that the additional quinovopyranose moiety was attached to
C-3 of the fucopyranose unit. An HMBC cross-peak between the signals
of H-1-glcA (δH 4.48) and C-3-aglycone (δC 82.0) confirmed the pre-
sence of a β-D-glucuronopyranosyl unit linked at C-3 of the aglycone.
The sequence of the trisaccharide chain at C-28 was established by the
HMBC cross-peaks between H-1-fuc (δH 5.40)/C-28 (δC 175.6), H-1-glc
(δH 5.04)/C-2-fuc (δC 73.1), H-1-qui/C-3-fuc, and H-3-glc (δH 4.91)/C-
5

S. Bechkri, et al.
Phytochemistry172(2020)112274
Table 4
13C NMR and 1H NMR spectroscopic data in CD3OD of the sugar moieties of compounds 7–11.a,b
7
8
9
10
11
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
glcA-
1
103.7 4.52, d (7.9)
103.8 4.53, d (7.8)
104.5 4.48, d (7.9)
104.4 4.48, d (7.8)
103.6 4.52, d (7.9)
2
3
4
5
72.7
84.9
70.5
75.5
171.3
3.46, t (8.0)
3.64, dd (9.9, 8.0)
3.61
3.70
–
73.1
85.2
70.9
75.5
171.1
3.46, t (8.0)
3.63, t (8.5)
3.61
3.58
–
73.7
76.3
71.8
75.3
171.3
3.25, t (8.2)
3.39, t (8.5)
3.52
3.80
–
73.7
76.4
71.9
75.2
172.1
3.25, dd (9.0, 8.1)
73.2
85.0
70.5
75.9
171.1
3.46, t (8.2)
3.64, t (8.5)
3.64
3.58
–
3.39, t (9.0)
3.51, t (9.0)
3.79
6
–
gal
1
104.0 4.57, d (7.8)
104.1 4.56, d (7.8)
104.0 4.57, d (7.7)
2
3
4
5
71.4
73.3
69.1
75.9
61.3
3.63, t (8.1)
3.52, dd (9.6,3.4)
3.81, d (3.4)
3.58
71.5
73.3
69.0
75.9
61.3
3.63, t (8.1)
3.52, dd (9.7,3.0)
3.81, d (3.0)
3.57
71.4
73.3
69.1
75.9
61.3
3.63, t (8.5)
3.52, dd (9.2, 3.3)
3.88, d (3.3)
3.58
6
3.68 3.80
3.69 3.80
3.68 3.80
fuc-
1
2
3
4
5
6
92.6
73.3
84.0
71.3
71.1
15.2
5.37, d (8.3)
4.08, dd (9.6, 8.3)
3.88, dd (9.6, 3.3)
3.95, d (3.3)
3.78, m
92.4
73.3
84.3
71.2
71.1
15.1
5.40, d (8.2)
4.01, dd (9.5, 8.2)
3.88, dd (9.5, 3.0)
3.95, d (3.0)
3.78, m
92.4
73.1
83.9
71.7
71.2
15.2
5.40, d (8.2)
4.10, dd (9.7, 8.2) 73.4
3.84, dd (9.7, 3.1) 83.7
3.88, d (3.0)
3.79, m
1.27 d (6.4)
92.6
5.38, d (8.3)
4.07, dd (9.7, 8.3)
3.85, dd (9.7, 3.0)
3.88, d (3.0)
3.79, m
92.6
73.4
83.8
71.6
71.1
15.1
5.37, d (8.3)
4.07, dd (9.6, 8.3)
3.87, dd (9.6, 3.2)
3.88, d (3.2)
3.78, m
71.6
71.1
15.2
1.27, d (6.4)
1.27, d (6.4)
1.27, d (6.4)
1.27, d (6.4)
glc- at 2-fuc
1
2
3
4
5
6
102.3 4.90, d (8.1)
102.1 4.89, d (7.9)
101.9 5.04, d (8.1)
102.3 4.89, d (8.1)
3.26, dd (9.4, 8.1) 74.5 3.13, dd (8.7, 8.3)
3.35
102.3 4.90, d (8.1
74.4
77.0
70.5
76.5
61.5
3.14, t (8.6)
3.35
3.32
3.30
3.77, dd (11.7, 4.7) 3.91,
dd (11.7, 1.7)
74.4
76.8
70.8
73.8
64.2
3.15, t (9.0)
3.37, t (9.5)
3.23, t (9.4)
3.50
4.35, dd (11.7, 1.6) 4.39,
dd (11.7, 7.1)
2.18, s
73.0
78.3
68.7
76.2
61.4
74.2
76.8
70.5
76.5
61.6
3.13, t (8.4)
3.34
3.31
3.31
3.78 3.92, dd
(12.3, 1.8)
4.91
77.0
70.5
76.5
61.6
3.47, t (9.4)
3.37, m
3.80 3.93, dd
(11.2, 2.0)
2.14, s
3.30 m
3.31
3.78 3.92, dd
(12.3m 1.5)
Ac-CH3
COO
sugar at 3-fuc glc
19.8
172.3
glc
19.8
171.4
qui
–
–
qui
qui
1
2
3
4
5
6
103.9 4.63 d (7.5)
103.9 4.63, d (7.1)
103.9 4.59, d (7.6)
103.8 4.63, d (7.5)
103.8 4.63, d (7.1)
73.9
76.8
69.8
76.5
60.8
3.31
3.32
3.30
3.28
73.9
76.8
69.7
76.5
60.9
3.32
3.33
3.34
3.30
74.3
76.6
75.1
72.0
16.8
3.33
74.2
76.6
75.5
72.0
16.8
3.32
3.30
3.02, t (9.0)
4 m
1.28 d (6.4)
73.9
76.6
75.5
72.0
16.8
3.31
3.30, t (8.4)
3.02, t (9.0)
3.34
3.30, t (8.4)
3.03, t (9.1)
3.34
3.70, dd (12.1, 4.9)
3.85, dd (12.1, 1.8)
3.70, dd (12.1, 4.9)
3.85, dd (12.1, 1.8)
1.28, d (6.4)
1.28, d (6.4)
a
b
Overlapped signals are reported without designated multiplicity.
in ppm, J in parentheses in Hz.
1-Acetyl (δC 171.4). Therefore, compound 9 was identified as 3-O-β-D-
glucuronopyranosyl caulophyllogenin 28-O-β-D-quinovopyranosyl-
(1 → 3)-[3-O-acetyl-β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside,
named silenegallisaponin I (Fig. 1).
TOCSY, HSQC, HMBC and ROESY spectra of 11 compared with those of
10 revealed that they share the same trisaccharide sequence at C-28 of
the caulophyllogenin [β-D-qui-(1 → 3)-[β-D-glc-(1 → 2)]-β-D-fuc-], and
differed only by the saccharide chain at C-3, which was identified by
comparison of NMR spectra of 11 and 7 as the disaccharide β-D-gal-
(1 → 3)-β-D-glcA- (Tables 3 and 4). Assignments of all proton and
carbon resonances of 11 were achieved by analysis of the 2D NMR
spectra (Tables 3 and 4). Thus, the structure of 11 was established as 3-
O-β-D-galactopyranosyl-(1 → 3)-β-D-glucuronopyranosyl caulophyllo-
genin 28-O-β-D-quinovopyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 →
2)]-β-D-fucopyranoside, named silenegallisaponin K (Fig. 1).
Compound 10 showed, in the positive HR-ESI-MS, a molecular ion
peak at m/z 1141.5413 [M+Na]+ (calcd for C54H86O24Na,
1141.5407). The 1H and 13C-NMR spectra of 10 displayed many simi-
larities with those of 9, especially for the resonances assigned to cau-
lophyllogenin and β-glucuronopyranosyl, β-fucopyranosyl, β-glucopyr-
anosyl and β-quinovopyranosyl units. However, those attributed to the
acetyl group were absent (Tables 3 and 4). The identities of the
monosaccharides were determined by detailed analysis of the 2D-NMR
spectra. An HMBC experiment made clear all interglycosidic con-
nectivities showing correlations between H-1-glcA (δH 4.48)/C-3-agly-
cone (δC 81.9), H-1-fuc (δH 5.38)/C-28-aglycone (δC 175.7), H-1-glc (δH
4.63)/C-2-fuc (δC 83.7), and H-1-qui (δH 4.89)/C 3-fuc (δC 73.4).
Consequently, the structure of compound 10 was concluded to be 3-O-
β-D-glucuronopyranosyl caulophyllogenin 28-O-β-D-quinovopyranosyl-
(1 → 3)-[β-D-glucopyranosyl-(1 → 2)]-β-D-fucopyranoside, named sile-
negallisaponin J (Fig. 1).
3. Conclusions
Plants of the genus Silene have proved to be a rich source of tri-
terpenoid saponins with oleanane type skeleton. Approximately 54
triterpenoid saponins were isolated from 9 Silene species till date: S.
jenisseensis (Lacaille-Dubois et al., 1995, 1997, S. vulgaris (Bouguet-
Bonnet et al., 2002; Glensk et al., 1999), S. fortunei (Gaidi et al., 2002;
Lacaille-Dubois et al., 1999), S. rubicunda (Fu et al., 2005; Wu et al.,
2014, 2015), S. nutans (Bukharov and Karneeva, 1971), S. szechuensis
(Zou et al., 1999), S. viscidula (Xu et al., 2010, 2012; Liao et al., 2013),
S. cucubalus (Larhsini et al., 2003), and S. armeria (Takahashi et al.,
2016). Most of them are based on oleanolic acid skeleton with C-23
Compound 11 had the molecular formula C60H96O29, deduced from
the molecular ion peak, observed in its HR-ESI-MS at m/z 1303.5925
[M+Na]+ (calcd for C60H96O29Na, 1303.5935), suggesting a supple-
mentary hexose unit compared to saponin 10. The 1D-NMR, COSY,
6

S. Bechkri, et al.
Phytochemistry172(2020)112274
exhibiting different degrees of oxidation (CH3, CHO or COOH) and a
hydroxyl group or a ketone group at C-16. A β-D-glucuronic acid linked
to C-3 and a β-D-fucose linked to C-28 of the aglycone, seems to be
typical for this genus. For the first time, eleven previously undescribed
triterpenoid saponins (1–11) were isolated from the aerial plant of Si-
lene gallica L. These bisdesmosidic saponins contain saccharide moieties
at C-3 and C-28 and their aglycones were identified as caulophyllogenin
(3β,16α,23-trihydroxyolean-12-en-28-oic acid) for compounds 1–4,
7–11, echinocystic acid (3β,16α-dihydroxyolean-12-en-28-oic acid) for
compound 5, and quillaic acid (3β,16α-dihydroxy-23-oxoolean-12-en-
28-oic acid) for compound 6. The sugar moiety linked at C-3 was a β-D-
glucuronic acid (1–11), substituted at C-3 by a β-D-galactose only for
compounds 7–8 and 11. The other one linked to C-28 was determined
as β-D-fucose substituted at C-2 by free or acylated β-D-glucoses at po-
sition 3 or 6 for all compounds. However, the fucose was substituted at
C-3 by the glucose (1–2 and 4–8) or by the quinovose (9–11). Furter-
more, according to the literature, the present study confirms that sa-
ponins of Silene, identified as 3β,16α-dihydroxyolean-12-en-28-oic acid
with different degrees of oxidation of C-23 (CH3, CH2OH, CHO, COOH),
and linked at C-3 by a β-D-glucuronic acid and at C-28 by a β-D-fucose,
are the predominent.saponin pattern. To the best of our knowledge,
they represent 46 compounds, including the herein decribed ones,
among 65 of these specialized metabolites, reported until now. Thus,
they can be considered as a chemotaxonomic marker for the genus Si-
lene. The unusual echinocystic and caulophyllogenin acids were pre-
viously reported in the Caryophyllaceae family (Böttger and Melzig,
2011; Koz at al., 2010; Timité et al., 2011). It is very interesting to note
that saponins isolated from S. gallica provide new features with others
types of aglycones such as caulophyllogenin and echinocystic, de-
scribed for the first time in this genus. In addition, the presence, in our
plant, of quinovose moiety, attached to the β-D-fucose, was peviously
reported only from Silene rubicunda but it was linked at C-4 of the fucose
(Wu et al., 2015). Compounds 9–11, with caulophyllogenin and qui-
novose moiety at C-3 of fucose, could be considered as chemotax-
omomic markers of S. gallica.
the herbarium of LOST Laboratory, University frères Mentouri-
Constantine, Algeria.
4.3. Extraction and isolation
The dried aerial part of S. gallica (1 Kg) was macerated in 80% EtOH
(3 × 5 L, 24h) at room temperature. After filtration, the solvent was
removed under reduced pressure, the EtOH extract was diluted with
H2O (700 mL), then successively extracted with petroleum ether (PE),
chloroform, ethyl acetate, and n-butanol (3 × 300 mL, each). After
evaporation of the solvents, 4 g of PE, 0.8 g of CHCl3, 2.3 g of EtOAc
and 33.2 g of n-BuOH extracts were obtained. A part of n-BuOH extract
(10.80 g) was subjected to vacuum liquid chromatography over silica
gel (9 cm × 5.5 cm) eluted with the system CH2Cl2–MeOH–H2O
(95:5:0, 9:1:0, 8:2:0, 7:3:0, 7:3:0.5, 6:4:0.7, 1:1:0, 0:1:0) to obtain 8
fractions B1–B8, respectively. The combined fraction B6-7 (1.54 g) was
fractionated by flash chromatography over silica gel, eluted by a gra-
dient system of 20%–50% of MeOH in CH2Cl2, in 40 min to afford 17
sub-fractions. Fraction B6-7-13 (220 mg) was submitted to a flash
chromatography over RP-C18, eluted by a gradient system of 20%–80%
MeOH, in 30 min. Sub-fractions B6-7-13[46-50] (18.9 mg) was purified by
semi-prep. HPLC using a gradient (30–50% CH3CN, in 15 min) to give
compounds 3 (1.5 mg, tR 8.5 min) and 6 (1.5 mg, tR 7.8 min) whereas
the purification of the B6-7-13[55-60] (24 mg) in the same condition gave
compounds 5 (3.6 mg, tR 8.7 min) and 9 (6.9 mg, tR 10.1 min). Fraction
B6-7-14 (158 mg) was flash chromatographed over RP-C18, eluted with
MeOH:H2O (35%–80% MeOH, in 24 min). Sub-fraction B6-7-14[15-16]
(35 mg) was purified by semi-prep HPLC eluted by gradient system
30%–43% CH3CN, in 15 min to yield compound 2 (4.6 mg, tR 7.3 min).
The purification by semi-prep HPLC of B6-7-16 (80 mg) using a gradient
from 30% to 45% CH3CN, in 20 min led to compound 4 (9.1 mg, tR
11.4 min). Fraction B6-7-17 (288 mg) was submitted to a flash chro-
matography over RP-C18, eluted by a gradient system of 20–80%
MeOH, in 30 min. Sub-fraction B6-7-17[56-60] (35 mg) was purified by
semi-prep HPLC using a gradient from 30% to 45% CH3CN, in 15 min,
to afford compounds 1 (11.2 mg, tR 7.6 min), 7 (1.6 mg, tR 6.8 min) and
8 (2.9 mg, tR 9.2 min). Sub-fraction B6-7-17[66-78] (43 mg) was also
purified by semi-prep HPLC using a gradient from 30% to 45% CH3CN,
in 15 min, to yield compounds 10 (6.5 mg, tR 11.0 min) and 11 (1.8 mg,
tR 10.6 min).
4. Experimental
4.1. General experimental procedures
Optical rotations were measured in MeOH using a PerkinElmer 341
Polarimeter. 1H, 13C-NMR and 2D-NMR measurements were recorded
in CD3OD on a Bruker Avance III 600 spectrometer (1H at 600 MHz and
13C at 150 MHz) equipped with a 5 mm TCI cryoprobe. 2D-NMR ex-
periments were performed using standard Bruker microprograms
(TopSpin 3.5 software). HR-ESI-MS analysis was conducted using a
Micromass Q-TOF micro instrument. Flash chromatography was carried
out on a Grace Reveleris system equipped with dual UV and ELSD de-
tection using Grace® cartridges (Silica gel or RP-C18). HPLC separations
were performed on a Dionex apparatus equipped with an ASI-100 au-
tosampler, an Ultimate 3000 pump, a STH 585 column oven, a diode
array detector UVD 340S and a Chromeleon software. A prepacked RP-
C18 column (Phenomenex 250 × 10 mm, Luna 5 μ) was used for semi-
preparative HPLC. The eluting mobile phase consisted of H2O with TFA
(0.0025%) and CH3CN with a flow rate of 5 mL/min and the chroma-
togram was monitored at 205 and 210 nm. Thin-layer chromatography
(TLC) was carried out using silica gel 60 F254 pre-coated aluminium
plates (0.2 mm, Merck). After developing with solvent systems, spots
were visualized by spraying with 50% H2SO4 followed by heating.
4.3.1. Silenegallisaponin A (1)
Amorphous white powder; [a]2D5-9.4 (c 0.93, MeOH); 1H (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data; see Tables 1 and 2. HR-
ESI-MS m/z 1157.5367 [M+Na]+ (calcd for C54H86O25Na,
1157.5356).
4.3.2. Silenegallisaponin B (2)
Amorphous white powder; [a]2D5-9.2 (c 0.38, MeOH); 1H (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data; see Tables 1 and 2. HR-
ESI-MS m/z 1199.5454 [M+Na]+ (calcd for C56H88O26Na,
1199.5462).
4.3.3. Silenegallisaponin C (3)
Amorphous white powder; [a]2D5-13.3 (c 0.12, MeOH); 1H (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data; see Tables 1 and 2. HR-
ESI-MS m/z 1037.4926 [M+Na]+ (calcd for C50H78O21Na,
1037.4933).
4.2. Plant material
4.3.4. Silenegallisaponin D (4)
The aerial parts of Silene gallica were collected from Djebel El-
Ouahch, Constantine (North-Eastern Algerian (GPS: x = 6.671694,
y = 36.394611, z = 888 m) in May 2016, and identified by Mr. Kamel
Kabouche. A voucher specimen (LOST Sg.05/16) has been deposited at
Amorphous white powder; [a]2D5-10.7 (c 0.75, MeOH); 1H (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data; see Tables 1 and 2. HR-
ESI-MS m/z 1199.5455 [M+Na]+ (calcd for C56H88O26Na,
1199.5462).
7

S. Bechkri, et al.
Phytochemistry172(2020)112274
4.3.5. Silenegallisaponin E (5)
Appendix A. Supplementary data
Amorphous white powder; [a]2D5-7.7 (c 0.3, MeOH); 1H (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data; see Tables 1 and 2. HR-
ESI-MS m/z 1183.5502 [M+Na]+ (calcd for C56H88O25Na, 1183.5512)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112274.
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and glucuronic acid [1.8 mg, Rf = 0.10]. The absolute configurations of
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The authors are grateful to MESRS Algeria for the Profas grant to Ms
Sara Bechkri, to CNRS, Conseil Regional Champagne Ardenne, Conseil
General de la Marne, Ministry of Higher Education and Research
(MESR) in France, and to the PlANET CPER project for financial sup-
port.
Zou, C., Jiang, J., Chen, C., Zhou, J., Zhao, Q., 1999. Diacylated triterpenoid saponin from
Silene szechuensis. Chin. Chem. Lett. 10, 33–36.
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