Oleanane-type triterpenoid saponins from Silene armeria

Article abstract of DOI:10.1016/j.phytochem.2016.07.011

Twelve triterpenoid saponins, including seven compounds (i.e., armerosides A–G) hitherto unknown, were isolated from whole plants of Silene armeria. Their structures were established based on extensive spectroscopic analyses and chemical methods. From a biosynthetic perspective, C-23 oxidation of the sapogenin appears to be a key factor in the glycosylation pathway.

Full text of DOI:10.1016/j.phytochem.2016.07.011

Phytochemistry xxx (2016) 1e9
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Oleanane-type triterpenoid saponins from Silene armeria
Nobuyuki Takahashi, Wei Li*, Kazuo Koike
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve triterpenoid saponins, including seven compounds (i.e., armerosides AeG) hitherto unknown,
were isolated from whole plants of Silene armeria. Their structures were established based on extensive
spectroscopic analyses and chemical methods. From a biosynthetic perspective, C-23 oxidation of the
sapogenin appears to be a key factor in the glycosylation pathway.
Received 23 January 2016
Received in revised form
15 July 2016
Accepted 18 July 2016
Available online xxx
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Sweet William Catchfly
Silene armeria
Caryophyllaceae
Triterpenoid saponin
Armeroside
1. Introduction
Catchfly, is an annual herbaceous plant that is native to Central and
Northern Europe and widely distributed in temperate regions of
The genus Silene belongs to the family Caryophyllaceae, which
contains more than 700 species. These species include many her-
baceous plants and are primarily distributed in temperate regions
of the Northern Hemisphere (Mamadalieva et al., 2014). Silene
plants contain triterpenoid saponins, and their surfactant proper-
ties have been exploited in some Silene plants as detergents since
ancient times. However, phytochemical studies of triterpenoid sa-
ponins in plants of the Silene genus remain limited. Chemical in-
vestigations have been previously conducted on only six Silene
species, including Silene fortunei (Lacaille-Dubois et al., 1999; Gaidi
et al., 2002), Silene jenisseensis (Lacaille-Dubois et al., 1995, 1997),
Silene viscidula (Xu et al., 2010, 2012), Silene vulgaris (Glensk et al.,
1999; Bouguet-Bonnet et al., 2002; Larhsini et al., 2003), Silene
rubicunda (Fu et al., 2005; Wu et al., 2015), and Silene cucubalus
(Larhsini et al., 2003). These studies resulted in isolation and
structural elucidation of approximately thirty types of triterpenoid
saponins. As part of an ongoing phytochemical investigation of
triterpenoid saponins from plants in the family Caryophyllaceae,
isolation and structural elucidation of eight triterpenoid saponins
from the roots of S. rubicunda was previously reported (Fu et al.,
2005).
Western Asia and America (Bajpai et al., 2008). This plant was
introduced into Japan as an ornamental in the Edo period (1603-
1868 CE). The chemical constituents of S. armeria are unknown,
except for some essential oil compositions (Bajpai et al., 2008).
Herein, the first phytochemical investigation of triterpenoid sapo-
nins from S. armeria is reported. Twelve triterpenoid saponins were
isolated, including seven new compounds that were named
armerosides A (3), B (5), C-F (7e10), and G (12).
2. Results and discussion
A methanol extract of whole plants of S. armeria was suspended
in H2O and then partitioned successively with EtOAc and n-BuOH.
The n-BuOH-soluble fraction was fractionated on a Diaion HP-20
column and eluted successively with water, aqueous MeOH (1:1)
and MeOH. The MeOH eluate was concentrated to afford a crude
saponin fraction. Further separation of this fraction by reversed-
phase (RP) and normal-phase (NP) column chromatography, fol-
lowed by repeated preparative HPLC purification, resulted in
isolation of twelve triterpenoid saponins (1e12).
The known compounds were identified as 3-O-
anosyl-(1/2)- -D-glucuronopyranosyl-quillaic acid (1) (Lacaille-
Dubois et al., 1995), 3-O- -D-galactopyranosyl-(1 / 2)-[ -D-xylo-
pyranosyl-(1 / 3)]- -D-glucuronopyranosyl-quillaic acid (2) (Guo
et al., 1998), dianchinenoside D [3 ,16 -dihydroxy-olean-12-en-
23,28-dioic acid 28-O- -D-glucopyranosyl-(1 6)- -D-
b-D-galactopyr-
Silene armeria L., which is commonly known as Sweet William
b
b
b
b
b
a
* Corresponding author.
E-mail address: liwei@phar.toho-u.ac.jp (W. Li).
b
/
b
http://dx.doi.org/10.1016/j.phytochem.2016.07.011
0031-9422/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

2
N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
glucopyranosyl ester] (4) (Li et al., 1994), sinocrassuloside I {3
dihydroxy-olean-12-en-23,28-dioic acid 28-O- -D-glucopyr-
anosyl-(1 / 3)-[ -D-glucopyranosyl-(1 / 6)]- -D-glucopyranosyl
ester} (6) (Zhao et al., 2004), and saponarioside K {3,16 -dihy-
droxy-3,4-seco-olean-4(24),12-dien-23,28-dioic acid 28-O- -D-
glucopyranosyl-(1 / 3)-[ -D-glucopyranosyl-(1 / 6)]- -D-glu-
copyranosyl ester} (11) (Koike et al., 1999) based on detailed NMR
spectroscopic analyses and comparison with literature data (Fig. 1).
Most of the known compounds (1, 2, 4, 6) were reported in other
Silene species, i.e.,1 from S. jenisseensis (Lacaille-Dubois et al.,1995),
2 from S. cucubalus (Larhsini et al., 2003), and 4 and 6 from
S. viscidula (Xu et al., 2012; Zhao et al., 2004). Saponin 11, which was
only isolated from Saponaria officinalis (Caryophyllaceae), was re-
ported for the first time from a Silene species (Koike et al., 1999).
The seven previously unknown compounds (3, 5, 7e10, and 12)
were given trivial names (i.e., armerosides A-G), and their chemical
structures were elucidated by detailed spectroscopic analyses and
b
,16
a
-
chemical methods.
b
Armeroside A (3) was obtained as an amorphous powder. Its
molecular formula was established as C62H96O30, based on the
positive-ion high-resolution (HR)-FAB-MS data that contained a
[MþNa]þ pseudo-molecular peak at m/z 1343.5846. In the 1H- and
13C-NMR spectra, characteristic resonances for the olean-12-ene
skeleton were observed, including six tertiary methyl groups at
dH 0.87, 0.96, 1.00, 1.10, 1.38, and 1.78 (each s) and dC 32.9, 26.8, 24.3,
17.2, 15.7, and 10.7 as well as an olefin moiety at dH 5.56 (br s) and dC
121.9 (C-12) and 144.5 (C-14) (Table 1 and Table 3). Other important
NMR resonances, which were assigned to the sapogenin moiety,
included: an aldehyde at dH 9.78 (s) and dC 209.7; an esterified
carboxyl moiety at dC 175.8; and two oxygenated methines at dH
4.07 and dC 83.3, as well as dH 5.23 (br s) and dC 73.4, respectively.
These data are in good agreement with those of 3,28-bisdesmosidic
b
b
a
b
b
b
quillaic acid (3
b,16a-dihydroxy-23-oxo-olean-12-en-28-oic acid;
17) (Jia et al., 2002).
Fig. 1. Structures of the triterpenoid saponins isolated from S. armeria.
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
3
Table 1
Table 2
13C NMR spectroscopic data (
(125 MHz in pyridine-d5).
d
) for the aglycone moieties of 3, 5, 7e10, 12, and 10a
13C NMR spectroscopic data (
d
) for the sugar moieties of 3, 5, 7e10, and 12 (125 MHz
in pyridine-d5).
Position
3
5
7
8
9
10
12
10a
Position
3
5
7
8
9
10
12
1
2
3
4
5
6
7
8
38.1
24.8
83.3
54.9
48.3
20.3
31.6
40.2
46.8
36.1
23.6
121.9
144.5
42.1
36.1
73.4
49.4
41.4
47.4
30.5
35.8
32.6
209.7
10.7
15.7
17.2
26.8
175.8
32.9
24.3
37.8
26.4
74.1
53.1
50.6
20.4
32.0
39.0
46.2
35.5
22.5
120.6
143.5
40.6
34.8
72.7
47.6
40.0
46.0
29.3
34.7
30.2
179.2
10.8
14.8
16.1
25.6
174.4
31.7
23.1
39.2
27.8
75.6
54.7
52.0
21.8
33.4
40.5
47.6
36.9
23.9
122.0
144.7
42.0
36.2
74.0
49.0
41.3
47.4
30.7
36.0
31.6
180.6
12.2
16.3
17.5
27.0
175.9
33.1
24.5
39.0
27.5
75.2
55.2
52.3
21.3
33.1
40.4
47.5
37.0
23.9
122.7
144.5
42.0
36.1
74.2
49.1
41.2
47.2
30.8
35.9
32.1
177.6
12.0
16.3
17.5
27.1
175.8
33.2
24.7
39.3
27.9
75.6
54.5
52.1
21.8
33.3
40.5
47.6
37.0
23.9
122.7
144.4
42.1
36.2
74.2
49.2
41.3
47.2
30.8
35.9
32.1
180.6
12.2
16.3
17.6
27.2
175.8
33.1
24.8
39.0
27.6
75.6
54.3
51.7
21.6
32.8
40.7
47.2
36.8
23.9
124.8
140.9
48.6
46.3
208.4
59.0
46.8
46.7
30.6
34.9
27.1
181.2
12.4
16.1
17.5
27.4
171.7
32.8
23.3
37.3
27.6
63.4
146.3
43.7
26.1
32.6
39.8
37.6
39.9
24.5
122.5
144.8
42.6
36.3
75.6
49.2
41.4
47.5
30.8
31.9
36.0
171.5
124.2
19.1
17.8
27.2
176.0
33.2
24.7
38.6
27.6
75.2
54.2
51.9
21.5
33.0
39.1
47.7
36.9
23.4
117.6
142.7
42.8
44.0
213.4
49.6
40.0
42.8
30.5
38.5
23.7
180.2
11.9
15.8
16.7
25.4
3-O-sugar
GlcA-1
2
3
4
5
6
Gal-1
2
3
4
5
6
103.1
82.3
77.5
72.7
77.3
172.1
106.2
74.4
77.0
70.0
74.7
62.0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
28-O-sugar
Fuc
Glc
Glc 1
93.8
79.3
78.2
70.5
77.8
69.0
GlcA
105.2
75.7
77.7
73.2
77.8
173.6
Glc 1
95.2
72.7
88.5
69.0
77.7
69.1
Glc 2
105.7
75.1
78.3
71.6
78.6
62.5
Glc 1
95.1
72.7
88.5
69.0
77.6
69.1
Glc 2
105.2
75.0
78.1
71.6
78.5
62.5
Glc 1
95.6
72.5
88.2
68.7
77.7
68.8
Glc 2
105.6
75.4
78.2
71.7
78.5
62.5
Glc 1
93.5
79.4
78.6
70.5
77.8
69.2
GlcA
105.2
75.6
77.6
73.0
77.3
171.0
52.3
Glc 2
105.4
75.2
78.4
71.7
78.4
62.8
1
2
3
4
5
6
94.3
72.2
82.7
74.1
70.4
16.3
Rha
101.4
72.1
72.2
73.7
69.9
18.6
92.5
78.4
77.1
69.5
77.8
60.7
GlcA
103.9
74.4
76.3
71.8
76.4
173.2
1
2
3
4
5
6
33.2
24.6
OCH3
Glc
Glc 2
105.3
75.7
78.3
71.6
78.3
62.6
Glc 3
105.4
75.4
78.3
71.7
78.4
62.8
Glc 3
105.6
75.2
78.2
71.7
75.4
64.7
Glc 3
105.4
75.1
78.3
71.7
78.3
62.8
1
2
105.4
75.0
78.2
71.0
78.2
62.5
3
4
5
6
For the sugar moiety, the 1H- and 13C-NMR spectra of 3 con-
tained resonances that were assigned to -fucopyranosyl (Fuc),
rhamnopyranosyl (Rha), -glucopyranosyl (Glc), -galactopyr-
anosyl (Gal), and -glucuronopyranosyl (GlcA) moieties with
b
a-
b
b
23-O-sugar
Glc-4-1
b
96.6
74.4
78.7
71.3
79.3
62.4
anomeric proton resonances at dH 6.03 (d, J ¼ 7.8 Hz, Fuc-H-1), 6.47
(br s, Rha-H-1), 5.07 (d, J ¼ 7.7 Hz, Glc-H-1), 5.23 (d, J ¼ 7.5 Hz, Gal-
H-1) and 4.94 (d, J ¼ 7.5 Hz, GlcA-H-1), respectively. In addition,
corresponding anomeric carbon resonances were observed at dC
94.3, 101.4, 105.4, 106.2 and 103.1 (Table 2 and Table 4). All of the
NMR resonances that corresponded to sugars were assigned based
on careful analyses of the 1H-1H COSY, TOCSY, HSQC, HSQC-TOCSY,
HMBC and NOESY data. The sugar chain at C-3 of the sapogenin was
established by HMBC correlations from dH 5.23 (Gal-H-1) to dC 82.3
(GlcA-C-2) and dH 4.94 (GlcA-H-1) to dC 83.3 (C-3). Similarly, the
sugar chain at C-28 was established by HMBC correlations from dH
6.47 (Rha-H-1) to dC 72.2 (Fuc-C-2), dH 5.07 (Glc-H-1) to dC 82.7
(Fuc-C-3), and dH 6.03 (Fuc-H-1) to dC 175.8 (C-28). The sugar
sequence was also confirmed by observation of NOESY correlations
between the anomeric proton and the proton attached to the car-
bon where the sugar was linked. In addition to the resonances due
to sapogenin and component sugars, the 1H- and 13C-NMR spectra
also indicated presence of an acetyl moiety at dH 1.91 (s) and dC 171.1
and 20.6. Its position was deduced as Fuc-C-4 based on an HMBC
correlation from the downfield shifted Fuc-H-4 at dH 5.85 to dC
171.1.
2
3
4
5
6
HMG-1
2
3
4
5
6
171.7
46.3
70.1
46.6
174.7
28.1
COCH3
COCH3
171.1
20.6
acetyl-a-methylbenzylamino]-1-deoxyalditol acetate derivatives
after acid hydrolysis and comparison to standard sugars (Li et al.,
2005). Therefore, the chemical structure of armeroside A (3) was
determined to be 3-O-
onopyranosyl-quillaic acid 28-O-
D-glucopyranosyl-(1 / 3)]-4-O-acetyl-
b
-D-galactopyranosyl-(1 / 2)-
-L-rhamnopyranosyl-(1 / 2)-[
-D-fucopyranosyl ester.
b-D-glucur-
a
b-
b
Armerosides B (5), C (7), D (8), and E (9) were isolated as
amorphous powders. Upon acid hydrolysis, these compounds
afforded the same sapogenin (i.e., 3b,16a-dihydroxy-olean-12-en-
23,28-dioic acid (18)) (Li et al., 1993) but different component
sugars.
The molecular formula of armeroside B (5) was determined to
be C42H64O17 based on the positive-ion HR-FAB-MS data. In the 1H-
and 13C-NMR spectra, in addition to the resonances assigned to the
b-glucopyranosyl and b-glucuronopyr-
anosyl moieties were observed with anomeric protons at dH 6.06 (d,
Acid hydrolysis of 3 with 1 M HCl in dioxaneeH2O (1:1) afforded
a partially hydrolyzed product, 3-O-b-D-glucuronopyranosyl-quil-
laic acid (21) (Bouguet-Bonnet et al., 2002), and with 2 M aqueous
trifluoroacetic acid (TFA) afforded the sapogenin, quillaic acid (17)
(Iwamoto et al., 1985). Acid hydrolysis of structurally related sa-
ponins 1 and 2 yielded identical results. The component sugars
were identified as D-galactose, L-rhamnose, D-glucose, D-fucose
and D-glucuronic acid based on HPLC analyses of their 1-[(S)-N-
sapogenin, resonances for
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

4
N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
Table 3
1H NMR spectroscopic data (
d
) for the aglycone moieties of 3, 5, 7e10, 12, and 1a (500 MHz in pyridine-d5).
Position 3
5
7
8
9
10
12
10a
1
2
3
0.90a
1.49a
1.18a
2.17a
4.07a
1.23 (m)
1.67a
1.22 (m)
1.68a
1.14 m
1.64a
1.87 m
1.25a
1.18a
1.19a
1.17a
1.69a
1.65 (br d, 13.4) 1.60 (m)
1.93 (2H, m)
1.61a
1.92a (2H)
1.92a (2H)
1.95a (2H)
1.81 (m)
1.91a(2H)
2.39a
4.64 (dd, 8.2, 6.8) 4.63 (dd, 9.4, 7.4) 4.63 (dd, 11.3, 5.3) 4.66 (dd, 10.2, 6.2) 4.59 (br d, 8.1)
3.78 (m)
3.90 (m)
4.62a
5
6
1.36a
1.98a
1.59a
1.98 (br d, 11.2) 1.92a
2.04 (br d, 11.4) 1.94 (br d, 11.4) 3.24 (dd, 12.9, 2.1) 1.97 (dd, 11.4, 1.7)
0.90a
1.62a
1.68a
1.72a 1.84a
1.50 (m) 1.57 (m)
1.36a
1.51 (br d, 12.0, 3.4) 1.51 (br d, 10.3) 1.56a
1.51 (br d, 12.0, 3.4) 1.51 (br d, 10.3) 1.28a
1.53 (br d, 11.8) 1.61a
1.44 (dd, 13.0, 2.2) 1.68 (ddd, 16.1, 12.6, 3.4)
1.09 (br d, 11.1) 1.64 (m)
1.15a
7
2.17a
1.34a
2.40a
1.69 (m)
1.68a
1.71a
1.72a
1.52a
1.71 (m)
1.41 (m)
9
11
1.82
1.92a
1.92a
1.90a
1.96a
1.71 (dd, 9.7, 7.7) 2.41a
1.80 (dd, 10.6, 6.8)
2.10 (m)
1.96a (2H)
2.04 (m)
1.93a
2.03 (m)
2.01 m
2.06a (2H, m)
2.06 (m)
2.01 (m)
1.19a
1.91a
2.11a
1.90a
12
15
5.56 (br s)
1.96a
5.61 (br s)
1.82 (br d, 15.1)
5.60 (br s)
5.56 (t, 3.3)
5.59 (br s)
1.65a
2.39 (dd, 14.8, 3.1) 3.39 (d, 14.6)
5.17 brs
5.62 (br s)
2.05 (d, 14.6)
5.61 (br s)
5.43 (t, 2.6)
1.82 (br d, 15.2) 1.62a
1.89 (dd, 15.2, 2.3) 2.08 (d, 15.4)
2.31 (dd, 14.9, 3.2) 2.58 (d, 16.0)
5.31 (br s)
2.19a
2.22 (dd, 16.3, 4.0) 2.20 (br d, 12.6) 2.33a
16
17
18
19
5.23 (br s)
5.49 (br s)
5.48 (br s)
5.15 (br s)
1.88a
3.37 (dd, 3.7, 140) 3.34 (dd, 4.0, 14.0) 3.35 (dd, 14.2, 3.9) 3.45 (dd, 14.4, 4.0) 3.47 (dd, 14.3, 3.7) 3.64 (dd, 14.3, 3.6) 3.44 (dd, 14.1, 4.6) 2.34 (m)
2.74 (t, 13.6)
2.64 (t, 13.5)
2.62 (t, 13.5)
2.72 (t, 13.5)
2.76 (t, 13.5)
1.56a
1.3a
1.36 (m)
2.72 (t, 13.5)
0.92a
1.34a
1.33a
1.32 (dd, 12.0, 3.2) 1.32a
1.34a
1.88a
21
22
1.28 (m)
2.40
2.26 (td, 13.1, 4.4) 2.23 (td, 12.8, 4.7) 2.33a
2.38 (td, 14.8, 3.1) 1.75 (td, 12.6, 3.8) 1.28 (m)
1.09 (dd, 12.9, 3.2)
1.35 (m)
2.27 (2H, m)
1.17 (m)
2.37 (br d, 12.6)
1.16 (br d, 13.4) 1.26a
2.37 (br d, 13.5) 2.33a
1.28a
1.18a
2.36a
1.49a
2.13 m
2.59 (dt, 13.2, 2.6) 2.11a
1.59 (br d, 12.0) 1.98a
9.78 (s)
2.02a
2.11a
2.33 (br d, 10.0) 1.66a
2.36a
23
24
1.38 (s)
1.59 (s)
1.59 (s)
1.58 s
1.63 s
5.44a
1.61 (s)
1.59 (s)
6.49 (s)
0.95 (s)
1.15 (s)
1.76 (s)
1.01 (s)
1.05 (s)
25
26
27
29
30
0.87 (s)
1.10 (s)
1.78 (s)
0.96 (s)
1.00 (s)
0.99 (s)
1.02 (s)
1.66 (s)
0.92 (s)
0.97 (s)
1.00 (s)
1.02 (s)
1.65 (s)
0.90 (s)
0.97 (s)
1.00 s
1.06 s
1.66 s
0.94 s
1.02 s
1.08 s
1.12 s
1.74 s
0.95 s
1.05 s
1.02 (s)
1.19 (s)
1.23 (s)
0.82 (s)
0.89 (s)
0.97 (s)
0.79 (s)
0.98 (s)
0.91 (s)
0.83 (s)
a
Overlapping signals.
J ¼ 7.7 Hz, Glc-H-1) and 5.65 (d, J ¼ 7.7 Hz, GlcA-H-1). In addition,
the corresponding anomeric carbons were observed at dC 92.5 and
103.9, respectively. The component sugars were identified as being
in the D-form based on the same method used for compound 3. The
linkage of the sugar chain was deduced by the HMBC correlations
from GlcA-H-1 (dH 5.65) to Glc-C-2 (dC 78.4) and Glc-H-1 (dH 6.06)
to C-28 (dC 174.4), as well as from the NOESY correlation between
GlcA-H-1 and Glc-H-2. Therefore, the structure of armeroside B (5)
1H- and 13C-NMR spectra indicated resonances that were assigned
to four sets of -glucopyranosyl moieties, and two of these moieties
b
were attached to the sapogenin through ester linkages based on the
upfield-shifted anomeric carbon resonances at dC 95.2 and 96.6 and
downfield-shifted anomeric proton resonances at dH 6.13 and 6.36.
A comparison of the 13C-NMR data for 8 and 6 indicated that
compound 8 has one more glucose unit than 6, and a downfield
shift was observed at C-23 (þ2.9 ppm), suggesting that 8 was a 23-
glucosyl ester of 6. This assignment was further confirmed by
HMBC correlations from Glc4-H-1 (dH 6.36) to C-23 (dC 177.6) and
Glc1-H-1 (dH 6.13) to C-28 (dC 175.8). The remaining two gluco-
pyranosyl moieties were attached to the C-3 and C-6 of the C-28-
glucopyranosyl moiety based on the HMBC correlations from
Glc2-H-1 (dH 5.19) to Glc1-C-3 (dC 88.5) and Glc3-H-1 (dH 4.95) to
Glc1-C-6 (dC 69.1), as well as NOESY correlations between Glc2-H-1
and Glc1-H-3, and Glc3-H-1 and Glc1-Ha,b-6. Thus, the structure of
was elucidated to be 3
b
,16
a-dihydroxy-olean-12-en-23,28-dioic
acid 28-O-
b
-D-glucuronopyranosyl-(1 / 2)-b
-D-glucopyranosyl
ester.
The molecular formula of armeroside C (7) was determined to
be C48H74O22 based on the positive-ion HR-FAB-MS data. The 1H-
and 13C-NMR spectra indicated the presence of two
b-glucopyr-
anosyl and one
b-glucuronopyranosyl moieties, and their absolute
configurations were determined to be D by acid hydrolysis. The
linkage of the sugar chain was deduced by HMBC correlations from
GlcA-H-1 (dH 5.57) to Glc1-C-2 (dC 79.4), Glc2-H-1 (dH 4.93) to Glc1-
C-6 (dC 69.0), and Glc1-H-1 (dH 5.97) to C-28 (dC 175.9), as well as
NOESY correlations between GlcA-H-1 and Glc1-H-2, and Glc2-H-1
and Glc1-Ha,b-6. This conclusion was also confirmed by the glyco-
sylation shifts for Glc1-C-6 (þ8.3 ppm) compared to that for 5 and
for Glc1-C-2 (þ3.9 ppm) compared to that for 4. Therefore, the
armeroside D (8) was determined to be 23-O-
28-O- -D-(1 / 3)-[ -D-glucopyranosyl-(1 / 6)]-
anosyl 3 ,16 -dihydroxy-olean-12-en-23,28-dioic acid.
b-D-glucopyranosyl-
b
b
b-D-glucopyr-
b
a
The molecular formula of armeroside E (9) was determined to be
C
54H84O25 based on positive-ion HR-FAB-MS data. Acid hydrolysis
of 9 resulted in only D-glucose as the component sugar. In the 1H-
and 13C-NMR spectra, the resonances of the sugar moiety over-
lapped substantially, except for the anomeric resonances for Glc1
structure of armeroside C (7) was elucidated to be 3
droxy-olean-12-en-23,28-dioic acid 28-O- -D-glucuronopyr-
-D-glucopyranosyl-(1 / 6)]- -D-glucopyranosyl
b,16a-dihy-
b
(dH 6.14, dC 95.1), Glc2 (dH 5.18, dC 105.6), and Glc3 (dH 4.93, dC 105.2).
anosyl-(1 / 2)-[
b
b
The TOCSY and HMQC-TOCSY spectra enabled the assignment of
resonances for each glucose unit. The linkage of the sugar chain was
deduced based on the HMBC correlations from Glc3-H-1 (dH 4.93)
to Glc1-C-6 (dC 69.1), Glc2-H-1 (dH 5.18) to Glc1-C-3 (dC 88.5), and
Glc1-H-1 (dH 6.14) to C-28 (dC 175.8), as well as NOESY correlations
ester.
The molecular formula of armeroside D (8) was determined to
be C54H86O26 based on the positive-ion HR-FAB-MS data. Upon acid
hydrolysis, 8 afforded only D-glucose as the component sugar. The
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
5
Table 4
1H NMR spectroscopic data (
d
) for the sugar moieties of 3, 5, 7e10, and 12 (500 MHz in pyridine-d5).
Position
3
5
7
8
9
10
12
3-O-sugar
GlcA-1
2
3
4
4.94 (d, 7.5)
4.22 (t, 8,5)
4.31 (t, 8.8)
4.54a
5
4.54a
Gal-1
2
3
4
5
6
5.23 (d, 7.5)
4.59a
4.15a
4.59a
4.16a
4.59a (2H)
28-O-sugar
Fuc
Glc
Glc 1
Glc 1
Glc 1
Glc 1
Glc 1
1
2
3
4
5
6
6.03 (d, 8.3)
4.67 (t, 8.8)
4.43 (dd, 9.7, 3.5)
5.85 (d, 3.2)
4.03a
6.06 (d, 7.7)
5.97 (d, 7.4)
6.13 (d, 8.3)
4.04 (t, 8.5)
4.16a
6.14 (d, 8.3)
4.02 (t, 8.7)
4.14 (t, 9.6)
4.18 (t, 9.1)
3.99a
4.26 (dd, 11.9, 4.8)
4.61 (br d, 10.1)
Glc 2
6.14 (d, 8.3)
3.93 (t, 8.3)
4.17 (t, 7.9)
4.18 (t, 9.2)
4.04 (m)
4.24 (dd, 12.0, 4.5)
4.55 (br d, 12.0)
Glc 2
6.05 (d, 7.5)
4.19 (t, 8.6)
4.20a
4.32a
4.20a
4.28 (t, 8.9)
4.21 (t, 9.2)
3.91 (ddd, 9.4, 4.3, 2.6)
4.30a
4.38 (dd, 11.8, 2.3)
GlcA
3.98a
4.23 (t, 9.2)
4.21a
4.20a
3.95a
4.01 (m)
3.96 (m)
4.26 (dd, 11.5, 4.6)
4.60 (dd, 11.4, 2.0)
GlcA
1.16 (d, 6.3)
4.27 (dd, 11.8, 4.6)
4.61 (br d, 10.3)
GlcA
4.17a
4.56 (br d 10.3)
Glc 2
Rha
1
2
3
4
5
6
6.47 (br s)
4.77 (br s)
4.47 (dd, 8.6, 3.2)
4.25a
5.65 (d, 7.7)
4.12 (t, 8.5)
4.25 (t, 9.2)
4.51 (t, 9.6)
4.54 (t, 9.3)
5.57 (d, 8.1)
4.09 (t, 8.5)
4.23 (t, 9.2)
4.51a
5.19 (d, 8.0)
3.97 (t, 8.2)
4.11 (t, 7.6)
4.14a
4.93 (d, 8.0)
5.18 (d, 7.7)
3.93 (t, 8.3)
4.15a
4.06 (t, 9.0)
3.92 (m)
4.20 (dd, 11.3, 5.0)
4.46 (dd, 11.8, 2.3)
Glc 3
5.60 (d, 7.8)
4.08 (t, 8.5)
4.20a
3.94a
4.12 (t, 9.6)
3.96 (t, 8.5)
3.92a
4.22 (dd, 11.4, 5.7)
4.46 (dd, 11.4, 2.6)
Glc 3
4.42a
4.50a
4.51a
3.94 (m)
4.43a
1.67 (d, 6.0)
4.27a
4.47 (dd, 11.9, 2.5)
Glc 3
3.84 (s)
Glc 2
Glc
Glc 2
1
2
3
4
5
6
5.07 (d, 7.7)
3.95 (t, 8.3)
4.13 (t, 9.5)
4.05 (t, 9.2)
3.89 m
4.93 (d, 8.8)
3.95 (t, 7.9)
4.13a
4.95 (d, 7.7)
3.94 (d, 7.6)
4.14a
5.18 (d, 7.7)
4.95 (d, 8.0)
3.95 (t, 8.0)
4.15a
4.95 (d, 7.7)
3.97 (t, 8.3)
4.17a
3.94a
4.12 (t, 9.6)
4.12 (t, 9.0)
3.95a
4.70 (dd, 11.4, 5.7)
4.96 (dd, 11.5, 1.4)
4.13a
4.15a
4.15a
4.16a
3.82 (m)
4.29 (dd, 11.9, 5.1)
4.42 (dd, 11.9, 2.4)
3.84 (m)
3.83 (m)
4.28 (dd, 12.0, 5.4)
4.42 (dd, 12.0, 2.6)
3.84 (m)
4.31 (dd, 12.3, 5.7)
4.44 (dd, 11.8, 2.0)
4.26a
4.30a
4.45a
4.43 (dd, 11.7, 2.3)
23-O-sugar
Glc 4-1
6.36 (d, 8.1)
4.13 (t, 8.4)
4.23a
2
3
4
5
6
4.25a
3.97a
4.27a
4.33 (dd, 12.0, 2.8)
HMG-2
4
3.10 (d, 14.2)
3.15 (d, 14.2)
3.12 (d, 14.9)
3.17 (d, 14.9)
1.73 (s)
6
COCH3
1.91 (s)
a
Overlapping signals.
between Glc3-H-1 and Glc1-Ha,b-6, and Glc2-H-1 and Glc1-H-3.
The 1H- and 13C-NMR data also indicated presence of a 3-
hydroxy-3-methylglutaryl (HMG) moiety, based on the character-
istic resonances of the tertiary methyl group [dH 1.73 (s), and dC
28.1], two methylenes [dH 3.10 and 3.15 (d, J ¼ 14.2 Hz), and dC 46.3;
dH 3.12 and 3.17 (d, J ¼ 14.9 Hz), and dC 46.6], two carbonyl groups
hydroxy-3-methylglutaryl)-glucopyranosyl-(1 / 3)]-[
b-D-gluco-
pyranosyl-(1 / 6)]- -D-glucopyranosyl ester.
b
Ameroside F (10) was isolated as an amorphous powder, and its
molecular formula was determined to be C48H74O21 based on the
positive-ion HR-FAB-MS data. A comparison of the 1H- and 13C-
NMR spectra of 10 and 6 indicated identical resonances for the
sugar moieties but differences in the sapogenin resonances. Further
detailed analyses of the NMR data indicated that the sugar chains of
10 and 6 were identical.
The sapogenin resonances of compounds 10 and 6 differed
because a resonance was observed for the ketone group (dC 208.4)
in 10, but hydroxylated methine resonances were also observed at
C-16 in 6. The ketone group was located at C-16 based on the HMBC
correlations from Ha,b-22, H-18, and Ha,b-15 to C-16. Rather than the
genuine sapogenin, acid hydrolysis of 10 afforded compound 10a,
(
dC 171.7, and 174.7), and a quaternary carbon (dC 70.1). The location
of the HMG moiety was deduced based on the HMBC correlations
from Glc2-Ha,b-6 to HMG-C-1 and the acylation shifts at Ha,b-6
(þ0.5 ppm) and C-6 (þ2.2 ppm) of Glc2 compared to those for 6. To
determine the absolute configuration of the HMG moiety, the
esterified carboxyl moiety was selectively reduced by LiEt3BH to
afford mevalonolactone (Fujimoto et al., 2001), which was identical
to 3R-mevalonolactone based on chiral GC-MS analysis. Therefore,
the structure of armeroside E (9) was elucidated to be 3
b,16a-
dihydroxy-olean-12-en-23,28-dioic acid 28-O-[ -D-6-O-((3S)-3-
b
and the structure of this compound was determined to be 3b-
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

6
N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
hydroxy-16-oxo-28-nor-olean-12-en-23-oic acid based on detailed
2D-NMR spectroscopic analysis (Fig. 2). The cis geometry of the D/E
ring fusion for 10a was deduced based on key NOESY correlations
1997; Glensk et al., 1999; Larhsini et al., 2003; Fu et al., 2005).
Silene saponins have been reported to have inhibitory activity
against lymphocyte proliferation (Gaidi et al., 2002). In this study,
all of the compounds were screened for their cytotoxicity against
human hepatocellular carcinoma HepG2 cells. None of the com-
between H-17 and H-15
The generation of 10a by decarboxylation upon acid hydrolysis is
consistent with the -keto carboxyl ester structure in 10. Therefore,
the structure of armeroside F (10) was determined to be 3 -hy-
droxy-16-oxo-olean-12-en-23,28-dioic acid 28-O- -D-glucopyr-
-D-glucopyranosyl
b, H-18 and H-12, and H-22b and H3-30.
b
pounds exhibited cytotoxicity at a final concentration of 10 mM.
b
b
3. Conclusions
anosyl-(1 / 3)-[b-D-glucopyranosyl-(1 / 6)]-b
ester.
This first phytochemical investigation of triterpenoid saponins
from S. armeria resulted in the isolation and structural elucidation
of twelve oleanane-type triterpenoid saponins, including seven
new compounds (armerosides AeG). Saponins 11 and 12 possess
cleavage structures at C-3 and C-4 of the oleanane-type sapogenin,
which extends the structural diversity of Silene saponins. From a
biosynthetic perspective, the substitution at C-23 of the sapogenin
appears to be a key factor in the glycosylation pathway. Further
investigation is required to identify the biosynthetic enzymes
involved in the biosynthesis of Silene saponins.
Armeroside G (12) was isolated as an amorphous powder, and
its molecular formula was determined to be C54H84O25 based on the
positive-ion HR-FAB-MS data. In the 1H- and 13C-NMR spectra of 12,
the resonances that were assigned to the sapogenin moiety were
identical to those in 11. Detailed analyses of the 2D-NMR spectro-
scopic data led to elucidation of the sapogenin as 3,16a-dihydroxy-
3,4-seco-olean-4(24),12-dien-23,28-dioic acid (Koike et al., 1999).
This conclusion was further supported by acid hydrolysis of 12 and
11, which afforded the same product. Detailed analyses of the 1H-,
13C- and 2D-NMR data also indicated the presence of two
b-glu-
copyranosyl and one methylated
b-glucuronopyranosyl moieties in
4. Experimental
12 with anomeric resonances at dH 6.05 and dC 93.5 for Glc1, dH 5.60
and dC 105.2 for GlcA, and dH 4.95 and dC 105.4 for Glc2. All of the
component sugars were determined to be in the D-form based on
acid hydrolysis results. The sugar linkage was deduced based on the
HMBC correlations from: Glc2-H-1 (dH 4.95) to Glc1-C-6 (dC 69.2);
GlcA-H-1 (dH 5.60) to Glc1-C-2 (dC 79.4); Glc1-H-1 (dH 6.05) to C-28
4.1. General
IR spectra were recorded on a JASCO FT/IR-4100 spectrometer
using the KBr disk method, and the optical rotations were
measured with a JASCO P-2200 digital polarimeter in a 0.5 dm cell.
The NMR spectra were measured with a JEOL ECA-500 NMR
spectrometer using TMS as the internal reference, and the chemical
(
dC 176.0), as well as NOESY correlations between Glc2-H-1 and
Glc1-Ha,b-6, and GlcA-H-1 and Glc1-H-2. Therefore, the structure of
armeroside G (12) was elucidated to be 3,16 -dihydroxy-3,4-seco-
olean-4(24),12-dien-23,28-dioic acid 28-O-[ -D-6-methyl-glucur-
onopyranosyl-(1 / 2)]-[ -D-glucopyranosyl-(1 / 6)]- -D-gluco-
a
shifts are expressed in d (ppm). HR-FAB-MS was conducted using a
b
JEOL JMS-700 MStation mass spectrometer. Diaion HP-20 resin
(Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (silica
gel 60, Merck), and ODS (Chromatorex, 100e200 mesh, Fuji Sylisia
Chemical, Ltd., Aichi, Japan) were used for column chromatography
(CC). Preparative HPLC was performed on a Waters 515 HPLC Pump
system equipped with a Shodex RI-101 differential refractometer
detector. A Shiseido CAPCELL PAC RP-C18 column (150 ꢀ 20 mm i.d.)
was used for RP HPLC, and an YMC-Pack SIL column (250 ꢀ 20 mm
i.d.) was used for NP HPLC. TLC was conducted on Kieselgel 60 F254
plates (E. Merck).
b
b
pyranosyl ester. Because methanol was used for extraction,
compound 12 may be a methylated artifact of corresponding
saponin 13.
A possible biogenesis pathway is shown in Figs. 3 and 4.
Oxidation at C-16, C-23, and C-28 of
acid (17; sapogenin of 1e3). Further oxidation at C-23 generates
,16 -dihydroxyolean-12-en-23,28-dioic acid (18; sapogenin of
4e9) followed by oxidation of the 16 -OH group to generate 3
hydroxy-16-oxo-olean-12-en-23,28-dioic acid (19; sapogenin of
10) or oxidative cleavage at C-3 and C-4 to generate 3,16 -dihy-
b-amyrin (16) affords quillaic
3
b
a
a
b-
a
4.2. Plant material
droxy-3,4-seco-olean-4(24),12-dien-23,28-dioic acid (20; sapo-
genin of 11, 12). In accordance with C-23 substitution by an
aldehyde or carboxyl group, glycosylation diverges based on the
component sugar types and sugar linkage. C-23 oxidation may be a
key step in the biosynthesis of Silene saponins, which is supported
by the triterpenoid saponins reported from other Silene species that
possess similar metabolic profiles (Lacaille-Dubois et al., 1995,
S. armeria plants were cultivated at the Botanical Garden of the
Faculty of Pharmaceutical Sciences, Toho University, and collected
in June 2005. The plants were identified by one of the authors (K.
K.) A voucher sample of the plant (TH-SA-1) is deposited at the
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences,
Toho University.
Fig. 2. Key COSY, HMBC, and NOESY correlations of 10a.
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
7
Fig. 3. Possible biosynthetic pathway for different aglycones of triterpenoid saponins isolated from S. armeria.
Fig. 4. Possible biosynthetic pathway for compounds 1e12 isolated from S. armeria.
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

8
N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
4.3. Extraction and isolation
4.8. Armeroside E (9)
22
Fresh whole plants of S. armeria (10 kg) were extracted with
MeOH (80 L) at room temperature for one day. The MeOH extract
was concentrated to afford an extract (580 g), which was then
suspended in H2O (1 L) and successively partitioned between EtOAc
(1 L each, ꢀ 5) and n-BuOH (1 L each, ꢀ 5), respectively. The n-
BuOH-soluble fraction (137 g) was applied to a Diaion HP-20 col-
umn (95 ꢀ 8.5 cm i.d.), eluted with H2O, MeOH-H2O (1:1), and
MeOH (each 10 L). The MeOH-eluted fraction (crude saponin frac-
tion, 34.6 g) was applied to an ODS column (50 ꢀ 5.5 cm i.d.) and
eluted with a MeOH-H2O gradient (4:6, 5:5, 6:4, 7:3, 8:2, 2 L each)
followed by MeOH (2 L) to afford ten fractions (F1-F10). Further
separation of fraction F4 (3.0 g) by preparative RP-HPLC using
MeCN-H2O containing 0.06% TFA (28:72, v/v) led to isolation of
compounds 11 (8 mg) and 12 (6 mg). The separation of fraction F5
(1.3 g) by preparative RP-HPLC using MeCN-H2O containing 0.06%
TFA (30:70, v/v) led to isolation of compounds 7 (76 mg) and 8
(8 mg). Fraction F6 (7.5 g) was subjected to silica gel CC and eluted
with CHCl3-MeOH-H2O (60:29:6, v/v/v) to afford seven sub-
fractions (S1eS7) and CHCl3-MeOH-H2O (6:4:1, v/v/v) to afford
three sub-fractions (S8eS10). Separation of sub-fraction S4 by
preparative NP-HPLC with CHCl3-MeOH-H2O (60:33:7, v/v/v) led to
isolation of compounds 4 (63 mg) and 10 (18 mg). Separation of
sub-fraction S6 by preparative RP-HPLC using MeCN-H2O con-
taining 0.06% TFA (32:68, v/v) led to isolation of compound 6
(1634 mg). Separation of sub-fraction S8 by preparative RP-HPLC
with MeOH-H2O (65:35, v/v) led to isolation of compound 5
(76 mg). Separation of sub-fraction S9 by preparative RP-HPLC us-
ing MeCN-H2O containing 0.06% TFA (35:65, v/v) led to isolation of
compounds 1 (48 mg), 2 (71 mg), 3 (11 mg), and 9 (21 mg).
Amorphous white powder. [
a
]
þ0.71 (c 0.50, EtOH). IR (KBr)
D
ymax cmꢁ1: 3406, 2925, 2856, 1710, 1678, 1458, 1384, 1204, 1074. For
1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-
d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (positive) m/z
1155.5224 [MþNa]þ (Calcd for C54H84O25Na, 1155.5199).
4.9. Armeroside F (10)
Amorphous white powder. [
a
]
17 -19.3 (c 0.50, EtOH-H2O ¼ 1:1).
D
IR (KBr) ymax cmꢁ1: 3431, 2924, 2854, 1699, 1636, 1559, 1458, 1376,
1159, 1063. For 1H-NMR (500 MHz, pyridine-d5) and 13C-NMR
(125 MHz, pyridine-d5) spectroscopic data, see Tables 1e4. HR-FAB-
MS (positive) m/z 1009.4613 [MþNa]þ (Calcd for C48H74O21Na,
1009.4620).
4.10. Armeroside G (12)
Amorphous white powder. [a]
22 -7.5 (c 0.50, EtOH). IR (KBr) ymax
D
cmꢁ1: 3416, 2925, 2858,1735,1683, 1431, 1381, 1204, 1136, 1078. For
1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-
d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (positive) m/z
1039.4701 [MþNa]þ (Calcd for C49H76O22Na, 1039.4726).
4.11. Acid hydrolysis
Individual solutions of 1e10 (each 5 mg) and 11e12 (each 3 mg)
in 1 M HCl (dioxane-H2O, 1:1, 10 mL) were separately heated until
reflux occurred, this being maintained for 1 h. After removal of
dioxane by evaporation, the solution was extracted with EtOAc
(10 mL ꢀ 3). The EtOAc fractions from compounds 1, 2, and 3 were
then separated by preparative NP-HPLC with CHCl3-MeOH-H2O
4.4. Armeroside A (3)
(60:29:6, v/v/v) as eluent to afford 3-O-b-D-glucuronopyranosyl-
quillaic acid (21). EtOAc fractions from compounds 4e9 were
separated by preparative NP-HPLC with CHCl3-MeOH (95:5, v/v) as
eluent to afford 3b,16a-dihydroxy-olean-12-en-23,28-dioic acid
(18). The EtOAc fraction from compound 10 was separated by
preparative NP-HPLC with CHCl3-MeOH (96:4, v/v) as the eluent to
afford 3b-hydroxy-16-oxo-28-nor-olean-12-en-23-oic acid (10a).
Amorphous white powder. [
a
]
19 þ 2.9 (c 0.22, EtOH-H2O ¼ 1:1).
D
IR (KBr) ymax cmꢁ1: 3410, 2924, 2855, 1734, 1636, 1420, 1376, 1160,
1060. For 1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz,
pyridine-d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (pos-
itive) m/z 1343.5846 [MþNa]þ (Calcd for C62H96O30Na, 1343.5884).
EtOAc fractions from compounds 11e12 were separated by pre-
4.5. Armeroside B (5)
parative NP-HPLC with CHCl3-MeOH (95:5, v/v) as eluent to afford
3,16
a
-dihydroxy-3,4-seco-olean-4(24),12-dien-23,28-dioic
acid
Amorphous white powder. [
a
]
21 -1.0 (c 0.50, EtOH). IR (KBr) ymax
(20). Acid hydrolysis of compounds 1e3 (each 5 mg) in 2 M aqueous
TFA using the same procedure afforded quillaic acid. Spectroscopic
data including NMR and MS for all of the compounds except 10a
were identical to those for authentic samples.
D
cmꢁ1: 3415, 2924, 2855,1703,1681,1456,1387,1260,1202,1077. For
1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-
d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (positive) m/z
863.4049 [MþNa]þ (Calcd for C42H64O17Na, 863.4041).
Compound 10a (3b-hydroxy-16-oxo-28-nor-olean-12-en-23-
21
oic acid): Amorphous white powder. [
a
]
-10.8 (c 0.33, EtOH).
For 1H NMR (500 MHz, pyridine-d5) and 13DC NMR (125 MHz, pyri-
dine-d5) spectroscopic data, see Tables 1 and 3. HR-FAB-MS (posi-
tive) m/z 479.3140 [MþNa]þ (Calcd for C29H44O4Na, 479.3137).
4.6. Armeroside C (7)
22
Amorphous white powder. [
a
]
-15.3 (c 0.50, EtOH). IR (KBr)
D
ymax cmꢁ1: 3412, 2925, 2856, 1677, 1636, 1457, 1377, 1204, 1077. For
1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-
d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (positive) m/z
1025.4592 [MþNa]þ (Calcd for C48H74O22Na, 1025.4569).
4.12. Determination of absolute configurations of sugars in
compounds 1e12
Aqueous layers obtained by each acid hydrolysis were neutral-
ized by passage over an ion-exchange resin (DIAION W20) column
and concentrated under reduced pressure to dryness to obtain
corresponding sugar fractions. These were individually dissolved in
H2O (1 mL), and (S)-(ꢁ)-1-phenylethylamine (5 mg) and NaBH3CN
(3 mg) in EtOH (1 mL) were added. After incubation at 40 ꢂC for 4 h,
glacial AcOH (0.2 mL) was added, with each reaction mixture
evaporated to dryness. Each resulting solid was acetylated with
Ac2O (0.3 mL) in pyridine (0.3 mL) for 24 h at room temperature,
4.7. Armeroside D (8)
Amorphous white powder. [a]
22 -1.7 (c 0.50, EtOH). IR (KBr) ymax
D
cmꢁ1: 3417, 2925, 2855, 1732, 1678, 1636, 1455, 1378, 1205, 1075.
For 1H-NMR (500 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyr-
idine-d5) spectroscopic data, see Tables 1e4. HR-FAB-MS (positive)
m/z 1173.5315 [MþNa]þ (Calcd for C54H86O26Na, 1173.5305).
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011

N. Takahashi et al. / Phytochemistry xxx (2016) 1e9
9
activity of essential oil and various extract of Silene armeria L. Bioresour.
Technol. 99, 8903e8908.
Bouguet-Bonnet, S., Rochd, M., Mutzenhardt, P., Henry, M., 2002. Total assignment
of 1H and 13C NMR spectra of three triterpene saponins from roots of Silene
vulgaris (Moench) Garcke. Magn. Reson. Chem. 40, 618e621.
with corresponding reaction mixture evaporated 5 times by adding
H2O to remove pyridine and passed through a Sep-Pak C18 car-
tridge (Waters) using CH3CN-H2O (20:80 and 50:50, v/v, each
10 mL) as solvents. Then, each CH3CN-H2O (1:1, v/v) eluate, which
Chang, X., Li, W., Jia, Z., Satou, T., Fushiya, S., Koike, K., 2007. Biologically active
triterpenoid saponins from Ardisia japonica. J. Nat. Prod. 70, 179e187.
Fu, H., Koike, K., Li, W., Nikaido, T., Lin, W., Guo, D., 2005. Silenorubicosides A-D,
triterpenoid saponins from Silene rubicunda. J. Nat. Prod. 68, 754e758.
Fujimoto, H., Nakamura, E., Kim, Y.P., Okuyama, E., Ishibashi, M., Sassa, T., 2001.
Immunomodulatory constituents from an ascomycete, Eupenicillium crusta-
ceum, and revised absolute structure of macrophorin D. J. Nat. Prod. 64,
1234e1237.
Gaidi, G., Miyamoto, T., Laurens, V., Lacaille-Dubois, M.A., 2002. New acylated tri-
terpene saponins from Silene fortunei that modulate lymphocyte proliferation.
J. Nat. Prod. 65, 1568e1572.
Glensk, M., Wray, V., Nimtz, M., Schӧpke, T., 1999. Silenosides A-C, triterpenoid
saponins from Silene vulgaris. J. Nat. Prod. 62, 717e721.
Guo, S., Kenne, L., Lundgren, L.N., Rӧnnberg, B., Sundquist, B.G., 1998. Triterpenoid
saponins from Quillaja saponaria. Phytochemistrv 48, 175e180.
Iwamoto, M., Okabe, H., Yamauchi, T., Tanaka, M., Rokutani, Y., Hara, S., Mihashi, K.,
Higuchi, R., 1985. Studies on the constituents of Momordica cochinchinensis
Spreng. I. Isolation and characterization of the seed saponins, momordica sa-
ponins I and II. Chem. Pharm. Bull. 33, 464e478.
Jia, Z., Koike, K., Sahu, N.P., Nikaido, T., 2002. Triterpenoid saponins from Car-
yophyllaceae family. Stud. Nat. Prod. Chem. 26, 3e61.
Koike, K., Jia, Z., Nikaido, T., 1999. New triterpenoid saponins and sapogenins from
Saponaria officinalis. J. Nat. Prod. 62, 1655e1659.
Lacaille-Dubois, M.A., Hanquet, B., Cui, Z.H., Lou, Z.C., Wagner, H., 1995. Acylated
triterpene saponins from Silene jenisseensis. Phytochemistry 40, 509e514.
Lacaille-Dubois, M.A., Hanquet, B., Cui, Z.H., Lou, Z.C., Wagner, H., 1997. Jenisseen-
sosides C and D, biologically active acylated triterpene saponins from Silene
jenisseensis. Phytochemstry 45, 985e990.
Lacaille-Dubois, M.A., Hanquet, B., Cui, Z.H., Lou, Z.C., Wagner, H., 1999. A new
biologically active acylated triterpene saponin from Silene fortunei. J. Nat. Prod.
62, 133e136.
Larhsini, M., Marston, A., Hostettmann, K., 2003. Triterpenoid saponins from the
roots of Silene cucubalus. Fitoterapia 74, 237e241.
Li, H.Y., Koike, K., Ohmoto, T., Ikeda, K., 1993. Dianchinenosides A and B, two new
saponins from Dianthus chinensis. J. Nat. Prod. 56, 1065e1070.
Li, H.Y., Koike, K., Ohmoto, T., 1994. Triterpenoid saponins from Dianthus chinensis.
Phytochemistry 35, 751e756.
Li, W., Asada, Y., Koike, K., Nikaido, T., Furuya, T., Yoshikawa, T., 2005. Bellisosides
AeF, six novel acylated triterpenoid saponins from Bellis perennis (Compositae).
Tetrahedron 61, 2921e2929.
Mamadalieva, N.Z., Lafont, R., Wink, M., 2014. Diversity of secondary metabolites in
the genus Silene L. (Caryophyllaceae)dStructures, distribution, and biological
properties. Diversity 6, 415e499.
Wu, Q., Tu, G.Z., Fu, H.Z., 2015. Silenorubicoside E-I, five new triterpenoid saponins
isolated from Silene rubicunda Franch. Mag. Reson. Chem. 53, 544e550.
Xu, W., Wu, J., Zhu, Z., Sha, Y., Fang, J., Li, Y., 2010. Pentacyclic triterpenoid saponins
from Silene viscidula. Helv. Chim. Acta 93, 2007e2014.
Xu, W., Fang, J., Zhu, Z., Wu, J., Li, Y., 2012. A new triterpenoid saponin from the roots
of Silene viscidula. Nat. Prod. Res. 26, 2002e2007.
consisted of a mixture of 1-[(S)-N-acetyl-a-methylbenzylamino]-1-
deoxy-alditol acetate derivatives of monosaccharides, was analyzed
by HPLC under the following conditions: column, YMC Triart C18
(150 ꢀ 4.6 mm i.d.); solvent: CH3CN-H2O (40:60, v/v); flow rate,
0.8 mL/min; column temperature, 40 ꢂC; detection, UV 230 nm.
Derivatives of D-xylose, D-glucuronic acid, D-galactose, D-fucose,
D-glucose and L-rhamnose were detected as follows: tR (min) 17.3
(derivative of D-xylose), 19.0 (derivative of D-glucuronic acid), 20.4
(derivative of D-galactose), 20.7 (derivative of D-fucose), 24.4 (de-
rivative of D-glucose), and 26.2 (derivative of L-rhamnose).
4.13. Determination of the absolute configuration of the 3-hydroxy-
3-methylglutaryl (HMG) moiety in 9
A solution of LiEt3-BH (1.0 M) in dry THF (100
mL) (Aldrich) was
added to a solution of 9 (3.0 mg) in dry THF (400
mL) in an ice bath.
The reaction mixture was stirred under N2 using ice-cooling for
30 min. After H2O (3.0 mL) was added to the reaction mixture, 0.1 M
HCl was added dropwise to adjust the pH to 3. The reaction mixture
was stirred under N2 for 48 h. Then, the reaction mixture was
partitioned with n-hexane (3 mL ꢀ 3). The n-hexane layer con-
taining mevalonolactone and standard compounds of (3S,3R)-
mevalonolactone and (3S)-mevalonolactone were analyzed by
chiral GC-FID. GC analysis was performed on a GC-2010 Plus (Shi-
madzu, Japan) equipped with a flame ionization detector (FID) and
an Agilent J&W chiral capillary column CYCLOSIL (30 m ꢀ 0.25 mm
i.d.). The column temperature was maintained at an initial tem-
perature of 40 ꢂC for 5 min and increased at 5 ꢂC/min to 230 ꢂC,
which was maintained for 20 min. He was used as carrier gas at a
constant flow rate of 2.11 mL/min. Authentic (3S)- and (3R)-
mevalonolactone possessed retention times of 33.65 min and
33.54 min, respectively. The retention time of the sample was
identical to that of (3R)-mevalonolactone, establishing that the
absolute configuration of the HMG moiety in 9 was 3S.
4.14. Cytotoxic assays
Cytotoxic assays using the human hepatocellular carcinoma cell
line HepG2 (RIKEN Cell Bank, Tsukuba, Japan) were performed
using an MTT assay as previously reported (Chang et al., 2007).
Zhao, J., Nakamura, N., Hattori, M., Yang, X., Komatsu, K., 2004. New triterpenoid
saponins from the roots of Sinocrassula asclepiadea. Chem. Pharm. Bull. 52,
230e237.
References
Bajpai, V.K., Shukla, S., Kang, S.C., 2008. Chemical composition and antifungal
Please cite this article in press as: Takahashi, N., et al., Oleanane-type triterpenoid saponins from Silene armeria, Phytochemistry (2016), http://
dx.doi.org/10.1016/j.phytochem.2016.07.011