Dianthosaponins G–I, triterpene saponins, an anthranilic acid amide glucoside and a flavonoid glycoside from the aerial parts of Dianthus japonicus and their cytotoxicity

Article abstract of DOI:10.1007/s11418-016-1019-8

Extensive isolation work on the 1-BuOH-soluble fraction of a MeOH extract of the aerial parts of Dianthus japonicus afforded three further triterpene glycosyl estsers, termed dianthosaponins G–I, an anthranilic acid amide glucoside and a C-glycosyl flavonoid along with one known triterpene saponin. Their structures were elucidated from spectroscopic evidence. The cytotoxicity of the isolated compounds toward A549 cells was evaluated.

Full text of DOI:10.1007/s11418-016-1019-8

J Nat Med
DOI 10.1007/s11418-016-1019-8
NOTE
Dianthosaponins G–I, triterpene saponins, an anthranilic acid
amide glucoside and a flavonoid glycoside from the aerial parts
of Dianthus japonicus and their cytotoxicity
1
2
1
1
•
Yuka Kanehira
Hideaki Otsuka
•
Susumu Kawakami
•
Sachiko Sugimoto
•
Katsuyoshi Matsunami
1
,2
Received: 25 February 2016 / Accepted: 10 June 2016
Ó The Japanese Society of Pharmacognosy and Springer Japan 2016
Abstract Extensive isolation work on the 1-BuOH-soluble
fraction of a MeOH extract of the aerial parts of Dianthus
japonicus afforded three further triterpene glycosyl estsers,
termed dianthosaponins G–I, an anthranilic acid amide
glucoside and a C-glycosyl flavonoid along with one
known triterpene saponin. Their structures were elucidated
from spectroscopic evidence. The cytotoxicity of the iso-
lated compounds toward A549 cells was evaluated.
(6) (Fig. 1) [2]. The structures of the new compounds were
elucidated by means of spectroscopic analysis and that of
the known compound by comparison with the reported data
in the literature. The cytotoxicity of the compounds iso-
lated toward human lung carcinoma cell line A549 was
evaluated.
Results and discussion
Keywords Dianthus japonicus Á Caryophyllaceae Á
Triterpene saponin Á Oleanane Á Anthranilic acid amide
glucoside Á Flavonoid glycoside
Further chromatographic isolation work on the fractions
obtained on Diaion HP-20 column chromatography (CC)
afforded new compounds 1–5 and a known compound, 6,
as described in the experimental section. The structures of
the new compounds were elucidated mainly from spec-
troscopic evidence.
Introduction
2
5
In a previous study, six oleanane-type saponins, named
dianthosaponins A–F, an anthranilic amide glycoside and a
C-glycosyl flavonoid were isolated from D. japonicus
collected in Hiroshima [1]. Further intensive phytochemi-
cal study on the BuOH-soluble fraction of a MeOH extract
of D. japonicus resulted in the isolation of three new
saponins, named dianthosaponins G, H and I (1–3), an
anthranilic acid amide glucoside (4) and a C-glycosyl fla-
vonoid (5) together with one known saponin, dianoside G
Compound 1, [a]D ?22.8, was isolated as an amorphous
powder, and its elemental composition was determined to be
C H O from the observation of a quasi-molecular ion at
4
8 76 20
?
m/z: 995.4797 [M ? Na] on high-resolution (HR)-elec-
trospray ionization (ESI) mass spectrometry (MS). The IR
–
spectrum exhibited a large absorption band at 3392 cm
1
assignable to a hydroxy group and absorptions due to two
–
types of carbonyl groups (1746 and 1718 cm ). Six singlet
1
1
methyl signals were observed in the H-NMR spectrum
together with three anomeric proton and one olefinic proton
1
3
signals (Table 1). The C-NMR signals of the aglycone
moiety comprised those of one trisubstituted double bond,
six methyls, ten methylenes, three methines and six qua-
ternary carbons along with one oxygenated methine and two
carbonyl carbons (dC 176.5 and 180.7) (Table 2). The
remaining 18 carbons were expected to form one terminal
glucopyranose and two hexopyranoses, whose analysis by
HPLC with an optical rotation detector revealed that only D-
glucose was present in the molecule. From the results of
&
Hideaki Otsuka
hotsuka@hiroshima-u.ac.jp; otsuka-h@yasuda-u.ac.jp
1
Department of Pharmacognosy, Graduate School of
Biomedical and Health Sciences, Hiroshima University, 1-2-
3
Kasumi, Minami-ku, Hiroshima 734-8553, Japan
2
Department of Natural Product Chemistry, Faculty of
Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi,
Asaminami-ku, Hiroshima 731-0153, Japan
123

J Nat Med
0
glucopyranosyl-(1 ? 6 )-b-D-glucopyranosyl ester and ter-
med dianthosaponin G (1).
2
5
Compound 2, [a]D -0.6, was isolated as an amorphous
powder, and its elemental composition was determined to
be C H O . The NMR spectral data were similar to
4
8 76 21
those of 1. In particular, those of the sugar region were
essentially the same as those of 1, and those of the A, B and
C rings were also like those of 1. An oxygenated methine
signal at d 74.2 with d 5.19 indicated one hydroxy group
C
H
must be located in the C, D and E ring region. The HMBC
correlation from H-18 (d 3.46) to d 74.2 suggested the
H
C
position of the hydroxy group was C-16, like in 16a-hy-
droxygypsogenic acid [4], which was a part of dianthos-
1
3
aponin C [1]. In fact, the C-NMR data of the aglycone
region were superimposable on those of dianchinenoside D
[
5] and dianversicoside C [6] (3b,16a-dihydroxyolean-12-
ene-23,28-dioic acid 28-O-glycosyl esters). Therefore, the
structure of compound 2 was elucidated to be 3b,16a-di-
hydroxyolean-12-ene-23,28-dioic acid (16a-hydroxygyp-
0
0
sogenic acid) 28-O-b-D-glucopyranosyl-(1 ? 2 )-b-D-
0
glucopyranosyl-(1 ? 6 )-b-D-glucopyranosyl ester and
termed dianthosaponin H (2). Alkaline hydrolysis of 2 gave
1
3
its aglycone, 16a-hydroxygypsogenic acid (2a). Since C-
NMR data were not readily available in the literature, they
were included in the experimental section.
2
2
Compound 3, [a]D -3.3, was isolated as an amorphous
powder, and its elemental composition was determined to
be C H O . The IR spectrum exhibited a fairly strong
4
2 66 15
–
1
Fig. 1 New compounds isolated
absorption band at 1651 cm , which was ascribable to a
stretching signal of double bonds. In the NMR spectra, four
2
sp signals for di- and trisubstituted double bonds, two
heteronuclear single quantum coherence spectroscopy
carbonyl carbons, two anomeric proton and carbon signals
(
HSQC), one (dH 6.21) of the anomeric protons was
(
d 6.21 on d 95.7 and d 5.02 on d 105.3) and a primary
expected to be associated with a relatively shielded carbon
resonance at dC 95.6, which implied an ester linkage
between the aglycone and sugar. Other NMR spectroscopic
data of the aglycone were essentially the same as those for
gypsogenic acid 28-glycosyl ester, which is a part of acan-
japoside G (7), isolated from Acanthopanax japonicus [3].
The 6-position of one of the glucose molecules was obvi-
ously shifted downfield (dC 69.3). In the heteronuclear
multiple-bond correlation (HMBC) spectrum (Fig. 2), the
H
C
H
C
alcohol (d 3.80 and 3.92 on d 63.3) were observed. The
H
C
1
3
C-NMR signals assignable to the C, D and E rings were
superimposable on those of dianthosaponin G (1); thus,
structure modification must have occurred in the A or B
ring. The presence of a primary alcohol, a carboxylic acid
and an exocyclic-double bond in the A or B ring indicated
the C-3 and C-4 bond was cleaved, and the HMBC cor-
relations from the exo-cyclic double bond protons to C4,
C-5 and C-23 placed the double bond between C-4 and
anomeric proton at d 5.27 was correlated with the carbon at
H
1
3
C-24. The C-NMR resonances of the aglycone were
essentially the same as those of vaccaroside D (3,4-seco-
gypsogenic acid 28-O-glycosyl ester) isolated from Vac-
caris segetalis [7]. Sugar analysis of the hydrolyzate of 3
showed the presence of D-glucose, and the terminal glucose
d 84.4, and the proton on the latter was found to be cor-
C
00
1
1
related with the anomeric proton (H-1 , d 4.92) on H– H
H
correlation spectroscopy (COSY) (Fig. 2). The remaining
00
0
sugar linkage was found to be H-1 to C-6 from the HMBC
correlation shown in Fig. 2, and thus the sugar sequence was
finally established, as shown in Fig. 1. The mode of sugar
linkage was assigned as b from the coupling constants of the
0
was linked to the hydroxy group at C-6 of the inner glu-
copyranose judging from the obvious downfield shift (dC
1
6
1
9.5) and HMBC correlation from the anomeric proton (H-
0
anomeric proton in the H-NMR spectrum. Therefore, the
0
0
) to C-6 . Therefore, the structure of 3 was elucidated, as
structure of 1 was elucidated to be 3b-hydroxyolean-12-ene-
0
0
shown in Fig. 1, and termed dianthosaponin I (3).
2
3,28-dioic acid 28-O-b-D-glucopyranosyl-(1 ? 2 )-b-D-
1
23

J Nat Med
1
Table 1 H-NMR spectroscopic data for dianthosaponins G–I (1–3) (400 MHz, pyridine-d
5
)
H
1
1
2
3
1.57 (1H, m)
1.63 (1H, br d, J = 11.6 Hz)
1.17 (1H, br d, J = 11.6 Hz)
1.91 (1H, br d, J = 10.2 Hz)
1.88 (1H, m)
1.52 (1H, dd, J = 14.0, 5.4 Hz)
1.20 (1H, m)
1
.09 (1H, m)
1.93 (1H, m)
.73 (1H, m)
2
3
2.20 (1H, m)
1
1.62 (1H, m)
4.67 (1H, m)
4.62 (1H, m)
3.92 (1H, m)
3
.80 (1H, m)
5
6
2.24 (1H, m)
1.67 (1H, m)
2.07 (1H, br d, J = 8.5 Hz)
1.68 (1H, br d, J = 12.8 Hz)
1.57 (1H, m)
3.21 (1H, dd, J = 15.0, 4.4 Hz)
1.82 (1H, m)
1
.57 (1H, m)
1.29 (1H, br d, J = 11.8 Hz)
.11 (1H, m)
1.39 (1H, m)
7
9
1.76 (1H, m)
1.32 (1H, m)
1
1.39 (1H, dd, J = 13.2, 6.0 Hz)
1.97 (1H, m)
1.11 (1H, m)
1.73 (1H, m)
2.00 (1H, m)
2.21 (1H, dd, J = 12.3, 8.2 Hz)
2.01 (2H, m)
1
1
2.03 (1H, d, J = 9.6 Hz)
1.94 (1H, d, J = 9.6 Hz)
5.59 (1H, br s)
1
.91 (1H, m)
1
1
2
5
5.43 (1H, br s)
1.97 (1H, m)
5.41 (1H, dd, J = 3.7, 3.7 Hz)
2.27 (1H, m)
2.26 (1H, d, J = 12.7 Hz)
1.65 (1H, m)
1
.03 (1H, m)
1.96 (1H, m)
.83 (1H, m)
1.13 (1H, m)
1
6
5.19 (1H, br s)
1.99 (1H, m)
1
1.91 (1H, dd, J = 14.2, 4.1 Hz)
3.17 (1H, dd, J = 13.6, 4.0 Hz)
1.73 (1H, br d, J = 13.6 Hz)
1.25 (1H, m)
1
8
9
3.17 (1H, dd, J = 13.4, 5.2 Hz)
3.46 (1H, dd, J = 13.8, 2.9 Hz)
2.71 (1H, dd, J = 13.8, 13.8 Hz)
1.33 (1H, dd, J = 13.8, 4.6 Hz)
2.45 (1H, br d, J = 12.5 Hz)
1.25 (1H, m)
1
1.69 (1H, m)
1
.19 (1H, m)
1.23 (1H, m)
.03 (1H, m)
1.77 (1H, m)
.67 (1H, m)
21
22
24
1.35 (1H, dd, J = 14.8, 4.1 Hz)
1.10 (1H, br d, J = 11.6 Hz)
1.86 (1H, m)
1
2.29 (1H, br t, J = 12.8 Hz)
2.05 (1H, m)
1
1.76 (1H, br d, J = 10.6 Hz)
6.51 (1H, s)
1.63 (3H, s)
1.60 (3H, s)
5
.54 (1H, s)
2
2
2
2
3
1
2
3
4
5
6
5
6
7
9
1.01 (3H, s)
1.03 (3H, s)
0.93 (3H, s)
1.11 (3H, s)
1.12 (3H, s)
1.17 (3H, s)
1.18 (3H, s)
1.74 (3H, s)
1.17 (3H, s)
0.83 (3H, s)
0.92 (3H, s)
0.89 (3H, s)
0
0
0.85 (3H, s)
0.99 (3H, s)
0.86 (3H, s)
6.21 (1H, d, J = 7.8 Hz)
4.21 (1H, o)
6.18 (1H, d, J = 7.4 Hz)
4.13 (1H, o)
6.21 (1H, d, J = 8.0 Hz)
4.13 (1H, m)
0
0
0
0
0
4.25 (1H, br t, J = 9.0 Hz)
4.14 (1H, o)
4.02 (1H, br d, J = 8.5 Hz)
4.14 (1H, br d, J = 8.5 Hz)
4.00 (1H, ddd, J = 8.5, 5.4, 3.2 Hz)
4.52 (1H, br d, J = 9.8 Hz)
4.27 (1H, dd, J = 9.8, 5.4 Hz)
4.89 (1H, d, J = 7.8 Hz)
4.21 (1H, br t, J = 8.9 Hz)
4.48 (1H, br t, J = 8.5 Hz)
3.75 (1H, dd, J = 8.5, 7.8 Hz)
4.45 (1H, dd, J = 12.6, 2.2 Hz)
4.35 (1H, br d, J = 12.6 Hz)
5.24 (1H, d, J = 7.8 Hz)
3.99 (1H, br t, J = 8.2 Hz)
4.20 (1H, br t, J = 9.3 Hz)
4.29 (1H, o)
4.05 (1H, o)
4.10 (1H, br d, J = 8.3 Hz)
4.68 (1H, br d, J = 11.2 Hz)
4.19 (1H, dd, J = 11.2, 5.1 Hz)
5.02 (1H, d, J = 8.1 Hz)
4.16 (1H, dd, J = 9.0, 7.5 Hz)
4.18 (1H, br t, J = 9.0 Hz)
3.86 (1H, o)
4.53 (1H, o)
4
.19 (1H, br d, J = 10.1 Hz)
0
0
0
0
0
0
0
0
0
0
1
2
4
5
6
4.92 (1H, d, J = 8.0 Hz)
4.02 (1H, o)
4.40 (1H, o)
3.77 (1H, o)
4.45 (1H, br d, J = 10.1 Hz)
4.43 (1H, dd, J = 13.1, 2.1 Hz)
4.32 (1H, dd, J = 13.1, 4.5 Hz)
4
.27 (1H, br d, J = 10.1 Hz)
0
0
00
00
1
2
5.27 (1H, d, J = 7.9 Hz)
4.01 (1H, o)
123

J Nat Med
Table 1 continued
H
1
2
3
0
0
0
0
0
3
4
5
6
4.13 (1H, o)
4.12 (1H, br t, J = 9.3 Hz)
4.10 (1H, dd, J = 9.3, 8.6 Hz)
3.88 (1H, o)
00
00
00
4.13 (1H, o)
3.91 (1H, o)
4.54 (1H, br d, J = 12.1 Hz)
4.50 (1H, br d, J = 10.7 Hz)
4.35 (1H, dd, J = 10.7, 4.2 Hz)
4
.35 (1H, dd, J = 12.1, 4.0 Hz)
m miltiplets, o overlapped signals
1
3
Table 2 C-NMR spectroscopic data for dianthosaponins G–I (1–3)
100 MHz, pyridine-d
Table 2 continued
(
5
)
a
C
1
7
2
3
a
C
1
7
2
3
0
0
2
84.4
78.2
70.7
78.3
62.1
84.3
78.2
70.4
78.5
62.2
75.2
78.4
71.6
78.4
62.7
1
2
3
4
5
6
7
8
9
39.1
27.8
75.5
54.4
51.9
21.7
33.0
40.3
48.4
36.9
23.8
39.0
27.7
75.5
54.4
51.9
21.7
33.0
40.1
48.4
36.9
23.8
39.2
27.7
75.7
54.4
51.9
21.7
33.3
40.5
47.5
36.9
23.9
37.0
27.0
63.3
00
3
4
5
6
1
2
3
4
5
6
a
00
00
146.2
43.5
26.0
32.1
39.8
38.4
39.7
24.4
122.9
144.1
42.8
28.3
23.5
47.2
41.8
46.4
30.8
34.0
32.5
171.5
124.3
18.9
17.7
26.0
176.6
33.1
23.7
95.7
73.9
78.8
71.0
78.0
69.5
105.3
00
000
000
000
000
000
000
106.2
76.3
78.2
70.4
78.8
62.2
106.1
76.2
78.2
70.7
78.9
62.2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Data taken from Ref. [3] at 150 MHz
122.7
144.2
42.2
28.3
23.3
47.0
41.7
46.2
30.7
33.9
32.3
180.7
12.2
16.1
17.5
26.0
176.5
33.0
23.6
95.6
74.0
78.6
71.1
77.1
69.3
102.7
122.7
144.1
42.1
28.2
23.3
47.0
41.6
46.2
30.7
33.9
32.4
180.7
12.2
16.1
17.4
26.0
176.5
33.0
23.6
122.5
144.5
42.1
36.1
74.2
49.1
41.3
47.2
30.7
35.9
32.1
180.5
12.2
16.3
17.5
27.2
176.0
33.0
24.6
95.7
74.0
78.6
71.1
77.1
69.3
102.7
2
2
Compound 4, [a]D -34.1, was isolated as an amorphous
powder, and its elemental composition was determined to
1
be C H O N. In the C-NMR spectrum, six signals
3
2
8 25 14
were assignable as those of the terminal glucopyranoside,
and the remaining signals including those of two aromatic
rings, two carbonyl carbons and two methoxy groups were
0
0
essentially the same as those of 4-O-b-D-(2 -O-a-L-
rhamnopyranosyl)glucopyranoside reported in the previous
paper [1]. Therefore, the structure of 4 was determined to
be as shown in Fig. 1, and the formation of a methyl ester
may have occurred during the isolation and purification
procedures.
22
Compound 5, [a]D -122, was isolated as a pale yellow
amorphous powder, and its elemental composition was
determined to be C H O . The NMR spectra of 5 were
4
1 46 22
similar to those of a C-glycosyl flavonoid reported in the
preceeding paper [1], except for the signals assignable as
1
those of the acyl moiety (Table 3). In the H-NMR spec-
0
0
trum, three aromatic protons coupled in an ABX system
and methoxy methyl signals were observed for the acyl
13
0
0
0
0
0
moiety. The C-NMR signals comprised three aromatic
signals with a proton, three substituted aromatic signals and
a carbonyl carbon. The HMBC correlations of d 7.55 (d,
H
J = 1.9 Hz) and 7.56 (dd, J = 8.3, 1.9 Hz) with the car-
0
0
bonyl carbon (dC 168.1) established a 1,3,4-substitution,
1
23

J Nat Med
1
13
respectively. H- and C-NMR spectra were taken on JEOL
JNM a-400 or Brucker Avance III 600 spectrometer at 400
MHz or 600 MHz, and 100 MHz or 150 MHz, respectively,
with tetramethylsilane as an internal standard. Positive-ion
HR-ESI-MS was performed with an Applied Biosystems
Ò
TM
QSTAR XL NanoSpray System.
A highly porous synthetic resin (Diaion HP-20) was
purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica
gel CC was performed on silica gel 60 (E. Merck, Darmstadt,
Germany) and ODS open CC on Cosmosil 75C -OPN
1
8
(Nacalai Tesque, Kyoto) [U = 50 mm, L = 25 cm, linear
gradient: MeOH–H O (1:9, 500 ml) ? (1:1, 500 ml), (1:1,
2
5
5
00 ml) ? (7:3, 500 ml) and then (7:3, 500 ml) ? (9:1,
00 ml), fractions of 10 g being collected]. The droplet
counter-current chromatograph (DCCC) (Tokyo Rikakikai,
Tokyo, Japan) was equipped with 500 glass columns
(
U = 2 mm, L = 40 cm), the lower and upper layers of a
solvent mixture of CHCl –MeOH–H O-n-PrOH (9:12:8:2)
3
2
being used as the stationary and mobile phases, respectively.
Five-gram fractions were collected and numbered according
to their order of elution with the mobile phase. HPLC was
performed on an ODS column (Inertsil; GL Science, Tokyo,
Japan; U = 6 mm, L = 250 mm, 1 ml/min), and the eluate
was monitored with a UV detector, at 254 nm, and a
refractive index monitor.
1
Fig. 2 H- H COSY and HMBC correlations for dianthosaponin G
1
(
1)
and the difference nuclear Overhauser effect experiment
00000
enhancement of the H-2
proton on irradiation of the
methoxy proton substantiated that the acyl group was
vanillic acid. Alkaline hydrolysis of 5 gave deacylated
0000
compound (5a) and vanillic acid (5b). The H-6 protons
dH 4.30 and 4.87) were correlated with the carbonyl car-
bon in the HMBC spectrum and were sequentially corre-
Plant material
(
Aerial parts of D. japonicus were collected in Toyota-gun,
Hiroshima, Japan, in August 1999, and a voucher specimen
was deposited in the Herbarium of Pharmaceutical Sci-
ences, Graduate School of Biomedical Sciences, Hiroshima
University (99-DJ-Hiroshima-0806). D. japonicas was
identified by Prof. Takakazu Shinzato of Faculty of Agri-
culture, University of the Ryukyus.
lated up to the anomeric proton [d 5.10 (d, J = 7.3 Hz)]
H
in the COSY spectrum. Therefore, the structure of 5 was
established to be saponarin 2 -O-a-L-rhamnopyranoside
00
0
000
6
-O-vanillate, as shown in Fig. 1. It is known that the C-
6
substituents of 6-C-glycosylflavones with a bulky group
at the 7-positon obstructed its free rotation because of steric
hindrance [8]. Thus, all the NMR signals appeared with
minor satellite peaks in a ratio of 1:2 (Table 3).
Extraction and isolation
The cytotoxic activity of the isolated compounds toward
lung adenocarcinoma A549 cells was evaluated (Table 4).
Among the compounds tested, dianthosaponins G (1) and I
Extraction and a part of the isolation were described in the
previous paper [1]. The residue (19.8 g in fractions 9–15)
of the 40–60 % MeOH eluate obtained on Diaion HP-20
CC was subjected to silica gel (500 g) CC with increasing
amounts of MeOH in CHCl [CHCl (6 l), and CHCl –
(
3) showed fairy strong cytotoxicity toward A549 cells
with the IC50 values of 18.1 ± 0.08 and 13.0 ± 0.33 lM,
respectively.
3
3
3
MeOH (49:1, 3 l), (24:1, 3 l), (23:2, 3 l), (9:1, 3 l), (17:3,
3
l), (4:1, 3 l), (3:1, 3 l) and (7:3, 3 l)] and CHCl –MeOH–
3
Experimental
H
O (35:15:2, 3 l), 500-ml fractions being collected. The
2
residue (1.99 g) in fractions 58–63 was separated by
octadecyl silica (ODS) open CC, and the residue (377 g) in
fractions 87–97 was purified by DCCC to give 5 (58.6 mg)
in fractions 27–30.
General experimental procedures
Optical rotations were measured on a JASCO P-1030 digital
polarimeter. IR and UV spectra were measured on Horiba FT-
The residue (17.9 g) in fractions 16–20 obtained on
Diaion HP-20 CC was subjected to silica gel CC (500 g)
7
10 and JASCO V-520 UV/vis spectrophotometers,
123

J Nat Med
13
1
Table 3 C- and H-NMR
spectroscopic data for
compound 5 (100 and 400 MHz
C
H
–
C
H
000
2
3
4
5
6
7
8
9
1
1
2
3
4
1
2
3
4
5
6
166.7
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
7
–
101.7
101.4
72.4
5.26 (1H, d, J = 1.6 Hz)
3.84 (1H, o)
at 35 °C, respectively, CD
3
OD)
1
66.7
104.5
04.0
184.1
84.3
161.1
61.0
111.8
12.1
164.0
0
0
0
0
0
0
0
0
00
00
6.63 (1H, s)
1
72.3
–
71.8
3.49 (1H, o)
1
71.7
00
–
73.4
3.08 (1H, o)
1
73.8
00
–
70.0
2.41 (1H, dq, J = 9.8, 6.2 Hz)
0.79 (3H, d, J = 6.2 Hz)
5.10 (1H, d, J = 7.3 Hz)
3.67 (1H, dd, J = 7.3, 7.1 Hz)
3.57 (1H, o)
1
69.9
00
–
18.2
1
64.0
95.7
17.9
000
000
000
6.80 (1H, s)
102.7
102.3
75.0
9
5.3
158.7
58.3
106.9
07.3
122.9
22.6
129.6
29.6
117.1
17.0
162.8
–
1
74.5
0
–
77.4
1
77.7
0
0000
–
72.2
3.46 (1H,o)
1
72.1
0
0
0
0000
,6
7.65 (2H, d, J = 8.1 Hz)
6.82 (2H, d, J = 8.1 Hz)
–
75.8
3.93 (1H, o)
1
75.4
0
0
0
0
0
0
0
0
0000
,5
65.5
4.30 (1H, dd, J = 11.9, 7.4 Hz)
1
65.1
4.87 (1H, dd, J = 11.9, 2.0 Hz)
00000
00000
00000
00000
00000
00000
00000
122.3
122.2
113.6
113.4
148.7
148.5
152.9
152.7
116.1
115.8
125.2
124.9
168.1
167.9
56.6
–
1
62.9
73.1
0
4.95 (1H, d, J = 9.9 Hz)
4.37 (1H, o)
7.55 (1H, d, J = 1.9 Hz)
7
3.2
77.1
6.9
81.6
1.3
71.2
1.1
82.0
2.3
62.0
3.4
0
0
0
0
0
–
7
3.60 (1H, o)
–
8
3.64 (1H, o)
6.57 (1H, d, J = 8.3 Hz)
7
3.36 (1H, o)
7.56 (1H, dd, J = 8.3, 1.9 Hz)
8
3.75 (1H, dd, J = 10.7, 5.1 Hz)
3.85 (1H, br d, J = 10.7 Hz)
–
6
OCH
3
3.76 (3H, s)
5
6.2
1
3
C-NMR data in the lower rows are from minor peaks
o overlapped
Table 4 Cytotoxicity toward A549 cell (IC50, lM)
Comp.
1
2
3
4
5
6
Dx
1
8.1 ± 0.08
37.7 ± 0.15
13.0 ± 0.33
58.2 ± 0.76
58.7 ± 0.22
50.0 ± 0.33
0.50 ± 0.20
Dx doxorubicin
1
23

J Nat Med
1 13
1026; H-NMR (400 MHz, pyridine-d ): Table 1; C-
with increasing amounts of MeOH in CHCl [CHCl (6 l),
3
3
5
and CHCl –MeOH (49:1, 3 l), (24:1, 3 l), (23:2, 3 l), (9:1,
3
NMR (100 MHz, pyridine-d ): Table 2; HR-ESI–MS
5
?
(positive-ion mode) m/z: 833.4280 [M ? Na] (Calcd for
3
l), (17:3, 3 l), (4:1, 3 l), (3:1, 3 l) and (7:3, 3 l)] and
CHCl –MeOH–H O (35:15:2, 3 l), 500-ml fractions being
3
C H O Na: 833.4294).
42 66 15
2
collected. The residue (1.80 g out of 2.72 g) in fractions
9–56 was separated by ODS open CC, and the residue
197 mg) in fractions 246–266 was purified by DCCC. The
4
Compound 4
(
2
D
2
residue (47.4 mg) in fractions 8–19 was finally purified by
An amorphous powder, [a] –34.1 (c = 0.97, MeOH); IR
mmax (film) cm : 3388, 2929, 2875, 1701, 1650, 1588, 1072,
–
1
HPLC (H O–MeOH, 7:13) to give 5.27 mg of 2 from the
2
peak at 35 min. The residue (59.7 mg) in fractions 20–25
1047; UV kmax (MeOH) nm (log e): 313 (3.88), 281 (3.92),
1
was finally purified by HPLC (H O–MeOH, 7:13) to give a
2
252 (4.04), 218 (4.01); H-NMR (600 MHz, CD OD): d
3
further amount of 2 (27.0 mg) from the peak at 35 min.
DCCC fractions 62–72 gave 5.87 mg of 3.
8.58 (1H, d, J = 2.5 Hz, H-3), 7.95 (1H, dd, J = 8.6,
0
1.6 Hz, H-6 ), 7.94 (1H, d, J = 8.9 Hz, H-6), 7.47 (1H, ddd,
0
The residue (10.9 g) in fractions 21–25 obtained on
Diaion HP-20 CC was subjected to silica gel CC (500 g)
with increasing amounts of MeOH in CHCl [CHCl (6 l),
J = 8.4, 7.3, 1.6 Hz, H-4 ), 7.10 (1H, br d, J = 8.4 Hz,
0
0
H-3 ), 7.00 (1H, br dd, J = 8.6, 7.3 Hz, H-5 ), 6.79 (1H, dd,
0
0
J = 8.9, 2.4 Hz, H-5), 4.96 (1H, d, J = 7.4 Hz, H-1 ), 3.96
3
3
and CHCl –MeOH (49:1, 3 l), (24:1, 3 l), (23:2, 3 l), (9:1,
3
(3H, s, –OCH ), 3.82 (3H, s, –COOCH ), 3.45 (1H, ddd,
3
3
00 00
3
l), (17:3, 3 l), (4:1, 3 l), (3:1, 3 l) and (7:3, 3 l)] and
J = 9.5, 5.2, 2.3 Hz, H-5 ), 3.42 (1H, m, H-3 ), 3.86 (1H,
0
0
CHCl –MeOH–H O (35:15:2, 3 l), 500-ml fractions being
3
dd, J = 12.2, 2.3 Hz, H-6 a), 3.68 (1H, dd, J = 12.2,
2
00 00
collected. The residue (2.06 g) in fractions 49–60 was sep-
arated by ODS open CC to give a residue. The residue
5.2 Hz, H-6 b), 3.40 (1H, m, H-2 ), 3.37 (1H, dd, J = 9.5,
0
0
13
9.5 Hz, H-4 ); C-NMR (150 MHz, CD OD): d 168.8 (C-
3
0
0
(
397 mg) in fractions 156–171 was purified by DCCC, and
7), 166.6 (C-7 ), 163.2 (C-4), 159.1 (C-2 ), 143.7 (C-2),
0 0 0
134.9 (C-4 ), 133.7 (C-6), 132.9 (C-6 ), 123.4 (C-1 ), 121.9
the residue (76.7 mg) in fractions 41–54 was subjected to
prep. HPLC (67 % MeOH in H O containing 1 % CH
0
0
(C-5 ), 112.9 (C-3 ), 112.2 (C-1), 112.1 (C-5), 110.3 (C-3),
2
3-
00 00 00 00 00
COOH) to give 6 (14.5 mg) and 1 (5.84 mg) from the peaks
at 35 and 38 min, respectively. The residue (1.12 g)
obtained on silica gel CC was separated by ODS open CC to
give a residue (142 mg) in fractions 221–245, which was
101.7 (C-1 ), 78.0 (C-3 and 5 ), 74.2 (C-2 ), 71.2 (C-4 ),
00
62.4 (C-6 ), 56.3 (CH O–), 52.5 (–COOCH ); HR-ESI-MS
3
3
?
(positive-ion mode) m/z: 486.1367 [M ? Na] (Calcd for
C H O NNa: 486.1371).
28 25 14
then purified by HPLC (55 % MeOH in H O containing 1 %
2
CH COOH) to afford 4 (15.0 mg) from the peak at 78 min.
3
Compound 5
A pale yellow amorphous powder, [a] -122 (c = 2.49,
2
D
2
Dianthosaponin G (1)
–
1
MeOH); IR mmax (film) cm : 3395, 2938, 2837, 1712,
1607, 1070, 1026; UV kmax (MeOH) nm (log e): 413 (2.25),
355 (3.70), 250 (4.08), 217 (4.13); H-NMR (400 MHz,
2
5
An amorphous powder, [a] ?22.8 (c = 0.35, MeOH); IR
D
–
1
1
mmax (film) cm : 3392, 2935, 2875, 1746, 1718, 1389,
1
367, 1069, 1030; H-NMR (400 MHz, pyridine-d5):
13
1
CD OD): Table 3;
3
C-NMR (100 MHz, CD OD):
3
1
Table 1; C-NMR (100 MHz, pyridine-d ): Table 2; HR-
3
Table 3; HR-ESI-MS (positive-ion mode) m/z: 913.2369
?
[M ? Na] (Calcd for C H O Na: 913.2373).
5
?
ESI–MS (positive-ion mode) m/z: 995.4797 [M ? Na]
4
1 46 22
(
Calcd for C H O Na: 995.4822).
48 76 20
Sugar analysis
Dianthosaponin H (2)
An amorphous powder, [a] -0.6 (c = 1.2, MeOH); IR
About 1 mg each of 1–5 was hydrolyzed with 1 M HCl
(0.1 ml) at 80 °C for 2 h. The reaction mixtures were
partitioned with an equal amount of EtOAc (0.1 ml), and
the water layers were analyzed with a chiral detector
(JASCO OR-2090plus) on an amino column [Asahipak
NH P-50 4E, CH CN–H O (4:1), 1 ml/min]. The
2
D
5
–
1
mmax (film) cm : 3398, 2930, 2878, 1746, 1714, 1060,
1
13
1
032; H-NMR (400 MHz, pyrdine-d ): Table 1; C-
5
NMR (100 MHz, pyridine-d ): Table 2; HR-ESI-MS
5
?
positive-ion mode) m/z: 1011.4765 [M ? Na] (Calcd for
(
2
3
2
C H O Na: 1011.4771).
8 76 21
hydrolyzates of 1–4 gave a peak for D-glucose at 12.4 min
with a positive optical rotation sign. The hydrolyzate of 5
gave peaks for L-rhamnose and D-glucose at 6.3 and
4
Dianthosaponin I (3)
An amorphous powder, [a] -3.3 (c = 0.29, MeOH), IR
12.3 min with negative and positive optical rotation signs,
2
D
2
respectively. Peaks were identified by co-chromatography
with authentic samples.
–
1
mmax (film) cm : 3378, 2928, 2865, 1714, 1651, 1066,
123

J Nat Med
Alkaline hydrolysis of 2
obtained. Vanillic acid (1.7 mg) (5b) was recovered from
the EtOAc fraction, which showed the same R value (0.61)
f
A solution of compound 2 (17.3 mg) in 50 % aqueous 1,4-
dioxane (0.5 ml) was treated with 10 % aqueous KOH
as that of an authentic sample on TLC (silica gel,
CHCl :MeOH:H O = 10:3:1).
2
3
00
26
(
0.5 ml) and stirred at 60 °C for 4 h. The reaction mixture
Isovitexin 7-O-b-D-glucopyranosyl-2 -O-a-L-rhamnopy-
?
was neutralized with Dowex HCR W2 (H form), and then
the resin was removed by filtration. The filtrate was
extracted with EtOAc, and the EtOAc layer was evaporated
ranoside (5a): pale yellow powder, [a]D -38.3 (c = 0.60,
1
MeOH); H-NMR (600 MHz, CD OD): d 6.71, 6.73 (1H, s,
3
H-3), 6.98, 7.02 (1H, s, H-8), 7.92, 7.94 (2H, d, J = 8.8 Hz,
0
0
0
0
under vacuum to give 16a-hydroxygypsogenic acid (2a,
H-2 , 6 ), 6.95, 6.98 (2H, d, J = 8.8 Hz, H-3 , 5 ), 4.99, 5.04
1
.6 mg): 2a: amorphous powder; H-NMR (600 MHz,
00
7
(1H, d, J = 9.4 Hz, 9.3 Hz, respectively, H-1 ), 5.12, 5.26
0
00
0000
pyridine-d ): d 5.65 (1H, br s, H-12), d 5.19 (1H, br s,
(1H, br s, H-1 ), 5.13, 5.14 (1H, d, J = 8.4 Hz, H-1 ) (the
5
H-16), 4.70 (1H, dd, J = 12.0, 5.0 Hz, H-3), 3.50 (1H, br
former figures are from minor peaks and the latter from major
1
peaks; sugar signals were omitted from assignment); C-
3
d, J = 10.0 Hz, H-18), 2.80 (1H, t, J = 13.5 Hz, H-19a),
2
.47 (1H, dd, J = 11.9, 4.4 Hz, H-21a), 2.41 (1H, brd,
J = 14.3 Hz, H-22a), 2.34 (1H, br d, J = 14.5 Hz, H-15a),
.25 (1H, dd, J = 14.3, 4.7 Hz, H-22b), 2.06 (1H, d,
NMR (150 MHz, CD OD): d 167.0, 167.1 (C-2), 104.6, 104.4
(C-3), 184.5, 184.8 (C-4), 161.5, 162.3 (C-5), 112.0, 112.8 (C-
3
2
6), 164.5 (C-7), 95.8, 96.4 (C-8), 159.2, 158.8 (C-9), 107.2,
0 0 0
107.8 (C-10), 123.3, 123.2 (C-1 ), 129.9, 130.0 (C-2 , 6 ),
J = 11.2 Hz, H-5), 2.06–1.95 (2H, o, H -11), 2.00 (1H, br
2
0 0 0 00
d, J = 9.0 Hz, H-2a), 1.97 (1H, m, H-9), 1.78 (3H, s, H3-
117.3, 117.4 (C-3 , 5 ), 163.3, 163.4 (C-4 ), 73.4, 73.6 (C-1 ),
00 00 00
78.7, 78.3 (C-2 ), 81.5, 81.7 (C-3 ), 71.4, 71.3 (C-4 ), 82.4,
2
7), 1.75 (1H, br d, J = 12.8 Hz, H-7a), 1.70 (1H, dd,
00 00 000
J = 10.1, 1.3 Hz, H-6a), 1.67 (1H, br d, J = 13.5 Hz,
82.5 (C-5 ), 62.9, 62.4 (C-6 ), 102.9, 102.6 (C-1 ), 72.6, 72.5
000 000 000
(C-2 ), 71.6, 71.7 (C-3 ), 73.9, 74.1 (C-4 ), 70.4, 70.2 (C-
H-15b), 1.63 (3H, s, H -24), 1.59 (1H, br d, J = 11.2 Hz,
3
000 000 0000
H-6b), 1.58 (1H, br d, J = 12.5 Hz, H-1a), 1.39 (1H, br d,
J = 11.2 Hz, H-7b), 1.34 (1H, dd, J = 13.5, 6.0 Hz,
H-19b), 1.26 (1H, dd, J = 11.9, 6.4 Hz, H-21b), 1.22 (1H,
br d, J = 12.0 Hz, H-2b), 1.19 (1H, br d, J = 10.0 Hz,
H-1b), 1.16 (3H, s, H -26), 1.05 (3H, s, H -25), 1.02 (3H, s,
5 ), 18.2, 18.3 (C-6 ), 103.0, 102.9 (C-1 ), 75.4, 75.2 (C-
0000
0
000
0000
0000
2 ),77.8, 77.3(C-3 ), 72.3, 72.1(C-4 ),79.2, 78.8(C-5 ),
0000
63.7, 63.8 (C-6 ) (the former figures are from minor peaks
and the latter from major peaks); HR-ESI-MS (negative-ion
-
mode) m/z: 739.2086 [M - H] (Calcd for C H O :
3
3
33 39 19
1
739.2091). Vanillic acid (5b): H-NMR (400 MHz, CD OD):
1
3
H -30), 1.00 (3H, s, H -29); C-NMR (150 MHz, pyr-
3
3
3
00000
idine-d ): d 180.4 (C-23), 179.7 (C-28), 144.9 (C-13),
d 7.58 (1H, d, J = 1.8 Hz, H-2 ), 7.57 (1H, dd, J = 8.8,
5
00000 00000
1.8 Hz, H-6 ), 6.69 (1H, d, J = 8.8 Hz, H-5 ), 3.91 (3H,
1
22.0 (C-12), 75.3 (C-3), 74.4 (C-16), 54.3 (C-4), 51.8 (C-
1
3
00000
5
), 48.6 (C-17), 47.3 (C-9), 47.0 (C-19), 41.2 (C-18), 41.9
s,–OCH3); C-NMR (100 MHz, CD OD): d 168.6 (C-7 ),
3
00000 00000 00000 00000
(
C-14), 40.1 (C-8), 38.9 (C-1), 36.7 (C-10), 35.97 (C-15),
151.3 (C-4 ), 147.3 (C-3 ), 123.9 (C-6 ), 121.7 (C-1 ),
0
0000
00000
3
2
5.94 (C-21), 33.1 (C-7), 33.0 (C-29), 32.6 (C-22), 30.8 (C-
0), 27.6 (C-2), 26.9 (C-27), 21.5 (C-6), 23.7 (C-11), 24.5
114.4 (C-2 ), 112.4 (C-5 ), 55.0 (–OCH ); HR-ESI-MS
3
-
(negative-ion mode) m/z: 167.0352 [M - H] (Calcd for
(
(
C-30) 12.0 (C-24), 15.9 (C-25), 17.2 (C-26); HR-ESI-MS
-
negative-ion mode) m/z: 501.3222 [M - H] (Calcd for
C H O : 167.0350).
8 7 4
C H O : 501.3220).
3
0
45
6
Cytotoxicity analysis
Alkaline hydrolysis of 5
Cytotoxic activity toward lung adenocarcinoma cells was
determined by colorimetric cell viability assaying using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT). Lung adenocarcinoma cell line A549 was
purchased from the Japanese Collection of Research
Bioresources Cell Bank, Japan. A549 cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with
10 % heat-inactivated FCS and kanamycin (100 lg/ml)
and amphotericin B (5.6 lg/ml).
A solution of compound 5 (9.7 mg) in 50 % aqueous 1,4-
dioxane (0.5 ml) was treated with 10 % aqueous KOH
(
0.5 ml) and stirred at 60 °C for 10 h. The reaction mixture
?
was neutralized with Dowex HCR W2 (H form), and then
the resin was removed by filtration. The filtrate was
extracted with EtOAc. The remaining aqueous layer was
evaporated under vacuum, and then the residue was sub-
jected to silica gel CC: 0.5 g, CHCl :H O:MeOH [50:3:1
3
2
In a 96-well plate, 1-ll aliquots of sample solutions
3
(
10 ml) ? 7:3:1 (10 ml) ? 6:4:1 (10 ml) ? MeOH]. On
and the cancer cells (5 9 10 cells/well) in 100 ll
medium were added to each well, and then the plate was
evaporation of the 6:4:1 eluate, isovitexin 7-O-b-D-glu-
0
0
copyranosyl-2 -O-a-L-rhamnopyranoside (5a, 6.0 mg) was
incubated at 37 °C under a 5 % CO2 atmosphere for
1
23

J Nat Med
7
2 h. A solution of MTT (100 ll) was then added to
each well, and the incubation was continued for a further
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
1
5
h. The absorbance of each well was measured at
40 nm using a Molecular Devices Versamax tunable
microplate reader. DMSO was used as a negative control
and doxorubicin as a positive control. The cytotoxic
activity was calculated as:
References
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Chem Pharm Bull 59:1141–1148
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absorbance of the cell-free wells.
2
. Oshima Y, Ohsawa H, Hikino H (1984) Structures of dianosides G,
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3
Acknowledgments The authors are grateful for access to the super-
conducting NMR instrument (JEOL JNM a-400) at the Analytical
Center of Molecular Medicine of the Hiroshima University Faculty of
Medicine and an Applied Biosystem QSTAR XL system ESI (Nano
Spray)-MS at the Analysis Center of Life Science of the Graduate
School of Biomedical Sciences, Hiroshima University. The authors
are also grateful for the use of the NMR instrument (JEOL ECA-600)
at the Natural Science Center for Basic Research and Development,
Hiroshima University. This work was supported in part by Grants-in-
Aid from the Ministry of Education, Science, Sports, Culture and
Technology of Japan and the Japan Society for the Promotion of
Science (nos. 22590006, 23590130 and 25860078). Thanks are also
due to the Research Foundation for Pharmaceutical Sciences and the
Takeda Science Foundation for the financial support.
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123