Dianthosaponins A - F, triterpene saponins, flavonoid glycoside, aromatic amide glucoside and γ-pyrone glucoside from Dianthus japonicus

Article abstract of DOI:10.1248/cpb.59.1141

From aerial parts of Dianthus japonicus, six new and seven known oleanane-type triterpene saponins were isolated. The structures of the new saponins, named dianthosaponins A - F, were elucidated by means of high resolution mass spectrometry, and extensive inspection of one- and two-dimensional NMR spectroscopic data. A new C-glycosyl flavone, a glycosidic derivative of anthranilic acid amide and a maltol glucoside were also isolated.

Full text of DOI:10.1248/cpb.59.1141

September 2011
Regular Article
Chem. Pharm. Bull. 59(9) 1141—1148 (2011)
1141
Dianthosaponins A—F, Triterpene Saponins, Flavonoid Glycoside,
Aromatic Amide Glucoside and g-Pyrone Glucoside from
Dianthus japonicus
Takahiro NAKANO, Sachiko SUGIMOTO, Katsuyoshi MATSUNAMI, and Hideaki OTSUKA*
Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-
ku, Hiroshima 734–8553, Japan. Received May 16, 2011; accepted June 15, 2011; published online June 21, 2011
From aerial parts of Dianthus japonicus, six new and seven known oleanane-type triterpene saponins were
isolated. The structures of the new saponins, named dianthosaponins A—F, were elucidated by means of high
resolution mass spectrometry, and extensive inspection of one- and two-dimensional NMR spectroscopic data. A
new C-glycosyl flavone, a glycosidic derivative of anthranilic acid amide and a maltol glucoside were also iso-
lated.
Key words Dianthus japonicus; Caryophyllaceae; oleanane saponin; phenolic glycoside; maltol
Plants belonging to the Caryophyllaceae are rich sources
of oleanane-type saponins. From Dianthus superbus var.
1
)
longicalycinus, oleanane-type saponins dianosides A and B,
2)
3)
C, D, E and F, and G, H and I have been isolated, and the
isolation of a cytotoxic cyclic peptide, longicalcinin A, from
this plant has also been reported. The whole D. superbus
var. longicalycinus plant is medicinally used for antipyretic
4)
5)
and diuretic purposes. A flowering plant, D. caryophyllus,
known as the carnation, is globally cultivated for ornamental
purposes. A closely related species, D. japonicus THUNB.,
also blooms pink and showy flowers, and is currently avail-
able at flower shops as an ornamental plant. D. japonicus is a
perennial herb that grows wild in the coastal cliff areas of
Honshu, Shikoku, Kyushu, the Ryukyus and China. Since
phytochemical investigation of this plant has not been re-
ported so far, we performed isolation work on it.
Results and Discussion
From the 1-BuOH soluble fraction of a MeOH extract of
the aerial parts of D. japonicus, six triterpene saponins,
named dianthosaponins A—F (1—6) (Fig. 1), a flavonoid
glycoside (7), an anthranilic acid amide glycoside (8), and a
maltol glycoside (9) (Fig. 2), along with seven known triter-
pene saponins (10—16) and a flavonoid glucoside (17) were
isolated by a combination of various kinds of chromatogra-
phy. The known compounds were identified with dianversico-
6
)
7)
side G (10), chionaeosides B (11) and C (12), vaccaro-
8)
9)
sides A (13) and D (14), segetoside K (15), dianic acid 28-
10)
(
6ꢀ-O-b-D-galactopyranosy)-b-D-glucopyranosyl ester (16)
and saponarin (17) by comparison of their spectroscopic
data with those reported in the literature.
11)
Dianthosaponin A (1), [a]D ꢁ1.9, was isolated as an
amorphous powder and its elemental composition was deter-
mined to be C H O by high-resolution (HR) electrospray
Fig. 1. Structures of Dianthosaponins and a Reference Compound
5
3
84 24
ionization (ESI) mass spectrometry. The IR spectrum exhib- were detected on HPLC equipped with a optical rotation de-
ꢂ1
13
ited a strong absorption at 3388 cm for hydroxy groups, at tector. Of the 53 C-NMR signals observed, 17 signals were
ꢂ1
2
927 and 2865 cm for C–H stretching signals, and at 1739 assignable to one terminal arabinopyranoside (d 105.8,
C
ꢂ
1
6
)
and 1717 cm for carbonyl groups. Six singlet methyl sig- 72.7, 74.2, 69.1, 66.8), and two terminal glucopyranosides
nals (d 0.84ꢃ2, 0.93, 1.03, 1.17, 1.49), one olefinic proton (d 105.5, 105.2, 78.5, 78.3ꢃ2, 78.2, 75.4, 75.1, 71.6, 71.5,
H
C
signal (Table 1), and four anomeric proton signals [d 4.93 62.6, 62.4) (Table 3). Of the remaining 36 signals, six could
H
(
d, Jꢄ6 Hz), 4.95 (d, Jꢄ8 Hz), 5.20 (d, Jꢄ8 Hz), 6.14 (d, be assigned to those of a hexopyranoside, such as the inner
1
Jꢄ8 Hz)] (Table 2) were observed in the H-NMR spectrum, glucopyranoside, and the other 30 signals were comprised
and in the acid hydrolyzate of 1, L-arabinose and D-glucose those of six methyls, ten methylenes, three methines, six qua-
∗
To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp
© 2011 Pharmaceutical Society of Japan

1
142
Vol. 59, No. 9
1
Table 1. H-NMR Data for the Aglycone Regions of Dianthosaponins
A—F (1—6) (400 MHz, Pyridine-d , J in Hz)
5
a)
C
1
2
3
4
5
6
1
1.18 m
1.10 m
1.58 m
1.91 m
2.19 m
4.57 m
1.15 m
1.61 m
1.90 m
2.19 m
4.58 dd
1.10 m
1.56 m
1.89 m
2.18 m
4.54 dd
11, 4
1.03 m
1.59 m
1.88 m
2.02 m
4.04 m
1.28 m
1.87 m
1.95 m
2.22 m
4.55 dd
12, 4
1
.57 m
1.88 m
.16 m
2
3
2
4.56 m
1
2, 4
4
5
6
—
1.88 m
1.85 m
.96 m
1.08 m
.27 m
—
1.74 m
—
1.73 m
.89 m
—
1.86 m
1.97 m
2.19 m
1.10 m
1.28 m
—
1.76 m
—
1.91 m
2.01 m
—
—
—
1.39 m
0.92 m
1.39 m
1.16 m
1.42 m
—
1.70 m
—
1.86 m
1.92 m
—
1.92 m
1.52 m
1.67 m
0.87 m
2.38 m
—
1.93 m
—
1.91 m
1.97 m
1.87 m
1.93 m
2.13 m
1.24 m
1.54 m
—
1.73 m
—
1.73 m
1.89 m
3.74 d 3
1.46 m
1.71 m
0.96 m
1.29 m
—
1.78 m
—
3.12 br d 4
1
7
1
8
9
0
1
1
1
1
1
1
1
1
2
3
4
5
5.39 br s 5.40 br s 5.60 br s 5.44 br s 5.40 br s 3.59 d 4
—
—
—
—
—
—
—
—
—
—
—
—
1.06 m
1.06 m
2.23 m
1.58 m
1.19 m
—
1.64 m
2.28 m
5.16 br s 1.93 m
2.13 m
1.05 m
2.22 m
1.07 m
2.16 m
1.86 m
2.00 m
—
0.92 m
1.63 m
1.28 m
2.23 m
—
2
.16 m
1
6
1.51 m
2
.15 m
—
1
1
7
8
—
—
3.12 br d 3.14 br d 3.56
3.27 br d 3.14 br d 2.64 br d
1
4
13
14
13
13
1
9
1.19 m
1.19 m
1.31 m
2.76 dd
1.84 m
1.21 m
2.33 br d
1
3
1
.70 m
—
1.69 m
2.47 dd
14, 14
—
1.69 m
2.13 m
1.84 m
2.00 m
—
1.49 s
0.93 s
1.05 s
1.17 s
—
1.71 m
2.73 dd
13, 13
—
1.83 m
2.12 m
1.67 m
1.76 m
—
1.48 s
0.98 s
1.12 s
1.19 s
—
1
4, 14
2
2
0
1
—
1.03 m
1.24 m
0.85 m
1.78 m
—
1.52 s
0.94 s
1.06 s
1.18 s
—
—
—
1.09 m
1.27 m
1.69 m
1.80 m
9.57 s
1.32 s
0.92 s
1.02 s
1.18 s
—
1.06 m
.25 m
0.84 m
1.33 m
2.44 m
1.31 m
2.20 m
—
1.49 s
0.93 s
0.99 m
1.76 m
—
1
2
2
Fig. 2. Structures of New Phenolic Compounds
1
.83 m
—
2
2
2
2
2
3
4
5
6
7
ternary carbons, one trisubstituted double bond, two carbonyl
carbons and an oxymethine carbon. These functionalities
were the same as those of the aglycone of co-occurring dian-
1.49 s
0.93 s
1.03 s
1.17 s
—
1
3
versicoside G (10), and the C-NMR chemical shifts were
superimposable on those of 10 (Table 3). Thus, diantho-
28
12,13)
2
9
0.84 s
0.83 s
0.84 s
1.01 s
1.14 s
—
1.41 s
0.85 s
0.86 s
—
1.36 s
saponin A (1) was expected to be a gypsogenic acid
bis-
1
1
30 0.84 s
desmoside, and the H– H correlation spectroscopy (COSY)
and heteronuclear single quantum correlation (HSQC) spec-
tra were used for extensive assignment of the signals. The
a) At 600 MHz. m: multiplet or overlapped signals. s: singlet. d: doublet. br: broad.
terminal arabinopyranoside was placed on the hydroxy group sorption maxima were similar to those of 1, and L-arabinose
at the 3-position, since significant correlation peak was ob- and D-glucose were detected in the acid hydrolyzate. The
served between H-1ꢀ (d 4.93) and C-3 (d 85.1) in the het- NMR data were also similar to those for 1, however, a set of
H
C
eronuclear multiple bond connectivity (HMBC) spectrum signals for only one terminal glucpyranose was observed in
1
3
(
2
Fig. 3). Further HMBC correlations, H-1ꢅ (d 6.14) and C- the C-NMR spectrum. The ester linked glucopyranose was
H
8 (d 176.3), and H -24 (d 1.49) and C-23 (d 180.4) es- expected to carry only one glucose unit at the 6ꢅ-position (d
tablished the position of the ester linkage, and correlations 69.2), and the position of the terminal glucopyranose was es-
C
3
H
C
C
H-1ꢆ (d 5.20) and C-3ꢅ (d 88.3), and H-1ꢅꢅ (d 4.98) and tablished to the hydroxyl group at the 2ꢆ-position by the
H
C
H
C-6ꢅ (d 68.9) substantiated the sequence of the sugar chain HMBC correlation H-1ꢅꢅ (dH 5.25) and C-2ꢆ (d 84.4).
C
C
to be as shown in Fig. 1. The mode of sugar linkage was de- Therefore, the structure of dianthosaponin B (2) was eluci-
termined from the coupling constants of anomeric protons in dated to be gypsogenic acid 3-O-a-L-arabinopyranoside 28-
1
the H-NMR spectrum (Table 2). Therefore, the structure of b -D-(2ꢆ-O-b -D-glucopyranosyl)(6ꢅ-O-b -D-glucopyra-
dianthosaponin A (1) was elucidated to be gypsogenic acid nosyl)glucopyranosyl ester, as shown in Fig. 1.
3
-O-a-L-arabinopyranoside 28-b-D-(3ꢅ,6ꢅ-di-O-b-D-glucopy-
Dianthosaponin C (3), [a]D ꢁ12.6, was isolated as an
amorphous powder and its elemental composition was deter-
ranosyl)glucopyranosyl ester.
Dianthosaponin B (2), [a]D ꢁ3.1, was isolated as an mined to be C H O by HR-ESI-MS. The NMR spectra
35
54 18
amorphous powder and its elemental composition, as deter- exhibited only one anomeric proton (d 4.91) and one carbon
H
mined by HR-ESI-MS, was the same as that of 1. The IR ab- (d 105.8) signal, and sugar analysis revealed the presence of
C

September 2011
1143
1
13
Table 2. H-NMR Data for the Sugar Region of Dianthosaponins A—F
Table 3.
C-NMR Data for Dianthosaponins A—F (1—6) and a Refer-
(
1—6) (400 MHz, Pyridine-d , J in Hz)
ence Compound, Dianversicoside G (10) (100 MHz, Pyridine-d5)
5
a)
C
1
2
3
4
5
6
C
1
10
2
3
4
5
6
1
2
ꢀ
ꢀ
4.93 d 6
4.34 m
4.92 m
4.35 m
4.91 d 7
4.32 dd
4.96 d 6
4.33 m
4.90 d 7
4.34 dd
9, 7
4.05 dd
9, 4
4.24 m
3.72 br d
12
4.27 dd
12, 3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.8
26.2
85.1
53.4
52.0
23.3
32.8
40.2
48.2
36.6
23.8
122.7
144.1
42.0
28.2
21.3
47.0
41.6
46.1
30.7
33.9
32.4
180.4
12.6
16.0
17.3
26.0
176.3
33.0
23.6
105.8
72.7
74.2
69.1
66.8
95.0
72.7
88.3
68.8
77.5
68.9
105.5
75.4
78.2
71.6
78.3
62.6
105.2
75.1
78.3
71.5
78.5
62.4
38.8
26.2
85.0
53.3
52.1
23.3
32.8
40.2
48.2
36.6
23.8
122.7
144.0
42.1
28.2
21.3
47.0
41.7
46.2
30.7
33.9
32.3
nd
12.6
16.0
17.3
26.0
176.3
33.0
23.6
105.8
72.7
74.2
69.2
66.4
95.8
73.1
88.0
69.1
76.8
68.9
105.7
76.5
78.1
71.2
78.4
62.5
102.6
73.5
78.0
70.9
78.2
62.2
105.7
75.6
78.0
71.3
78.5
62.4
38.9
25.9
85.0
53.2
52.0
23.3
32.9
40.2
48.3
36.7
23.8
122.7
144.0
42.1
28.2
21.4
46.9
41.7
46.2
30.7
33.9
32.3
180.4
12.6
16.0
17.3
26.0
176.5
33.1
23.6
105.9
75.8
74.2
69.2
66.4
95.6
74.0
78.5
71.0
77.1
69.2
102.7
84.4
78.2
70.7
78.3
62.1
106.1
76.3
78.2
70.4
78.7
62.2
38.8
26.2
85.0
53.3
52.1
21.3
33.7
40.2
47.7
36.7
23.8
122.1
145.1
42.0
36.05
74.6
48.8
41.3
47.2
30.9
36.13
33.17
180.4
12.5
16.0
17.3
27.1
179.9
33.2
24.7
105.8
72.7
74.1
69.0
66.3
38.8
26.1
85.0
53.3
52.1
23.4
32.7
40.2
48.2
36.6
23.8
123.3
143.6
42.0
28.1
21.2
46.9
40.8
40.7
42.3
29.0
31.6
180.3
12.6
16.0
17.3
25.9
176.3
180.6
19.9
105.8
72.7
74.1
69.1
66.3
95.7
73.8
78.6
70.8
77.9
69.3
105.1
75.1
78.33
71.5
78.30
62.6
38.5
27.0
71.7
56.2
47.4
21.0
32.5
40.2
48.0
36.1
23.8
122.6
144.1
42.2
28.2
21.2
47.0
41.7
46.2
30.7
33.9
32.3
207.3
9.6
38.4
25.8
84.8
53.2
51.3
20.3
31.1
40.8
51.2
36.1
52.6
57.1
87.6
42.0
27.0
21.5
44.0
48.7
33.1
42.7
29.6
26.8
180.8
11.9
17.6
20.4
18.8
178.6
180.4
19.8
105.9
72.6
74.1
69.0
66.3
8
, 7
4.03 dd
, 3
4.22 m
3
ꢀ
4.06 m
4.21 m
4.06 m
4.21 m
4.04 m
4.23 m
8
4
5
ꢀ
ꢀ
3.72 br d 3.73 m
3.72 br d 3.71 br d
12
4.26 dd
1
2
11
4.26 m
4
.27 m
4.27 m
1
2, 3
1
2
3
4
5
6
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
6.14 d 8
4.10 m
4.20 m
4.21 m
3.99 m
4.22 m
6.19 d 7
4.21 m
4.24 m
4.15 m
4.01 m
4.19 m
6.19 d 8
4.10 m
4.22 m
4.27 m
4.06 m
4.32 m
4.68 dd
11, 2
6.17 d 6
4.25 m
4.24 m
4.24 m
4.10 m
4.30 m
4.49 m
4
.55 br d 4.52 m
1
0
1
2
ꢆ
ꢆ
5.20 d 8
3.98 m
4.91 d 8
4.01 m
4.98 d 8
3.96 dd
5.28 d 8
4.05 m
8
, 8
3
4
5
6
ꢆ
ꢆ
ꢆ
ꢆ
4.13 m
4.14 m
3.81 m
4.21 m
4.21 m
4.43 m
3.75 m
4.29 m
4.55 m
4.14 m
4.13 m
3.84 m
4.29 m
4.42 dd
12, 2
4.24 m
4.12 m
3.76 m
4.28 m
4.48 m
15.8
17.4
26.0
176.3
33.1
23.6
4
.47 dd
2, 2
1
1
2
3
4
5
6
ꢅꢅ 4.95 d 8
ꢅꢅ 3.95 m
ꢅꢅ 4.17 m
ꢅꢅ 4.17 m
ꢅꢅ 3.93 m
ꢅꢅ 4.29 m
5.25 d 7
4.02 m
4.12 m
4.13 m
3.89 m
4.33 m
4.53 m
4.96 d 8
4.03 m
4.28 m
4.15 m
3.91 m
4.23 m
4.43 m
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅ
1ꢆ
4
.42 dd
2, 2
1
1
2
3
4
5
6
ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅꢆ
5.26 d 8
4.02 m
4.23 m
4.13 m
3.89 m
4.32 m
95.8
73.1
88.0
69.2
76.9
68.9
105.7
76.2
78.1
71.3
78.4
62.5
102.6
83.5
78.0
70.9
78.2
62.2
105.7
75.6
78.0
71.2
78.5
62.4
4
.52 m
2
ꢆ
a) At 600 MHz. m: multiplet or overlapped signals. br: broad. d: doublet.
3ꢆ
4
5
ꢆ
ꢆ
1
3
L-arabinose. The C-NMR chemical shifts for rings A and B,
and the sugar were essentially the same as those for 1 and 2,
and those of rings C, D and E were superimposable on those
of quillaic acid (olea-12-ene-3b,16a-diol-23-al-28-oic
acid). The H-NMR chemical shift and coupling pattern of
H-16 [d 5.16 (br s)] of 3 were also essentially the same as
those [d 5.24 (br s)] of quillaic acid. This finding also sup-
ported the orientation of the hydroxy group at the 16-position
to be a. Therefore, the structure of dianthosaponin C (3) was
elucidated to be 16a-hydroxygypsogenic acid 3-O-a-L-ara-
binopyranoside, as shown in Fig. 1.
6ꢆ
1ꢅꢅ
2ꢅꢅ
3ꢅꢅ
4ꢅꢅ
5ꢅꢅ
14)
1
6ꢅꢅ
1ꢅꢆ
H
H
2
3
4
5
ꢅꢆ
ꢅꢆ
ꢅꢆ
ꢅꢆ
6ꢅꢆ
Dianthosaponin D (4), [a] ꢂ2.7, was also isolated as an
D
nd: signal not observed.
amorphous powder and its elemental composition was deter-
mined to be C H O . From the spectroscopic data, 4 was
4
7
72 21
expected to be an analogous compound to 1, 2 and 3. In the B and C rings were essentially the same as those of 1 and 2,
1
H-NMR spectra, only five singlet methyl signals were ob- the one of the geminal methyls at the 20-position must be
1
3
served, and the C-NMR spectrum exhibited one less methyl replaced by a carboxyl group. On phase-sensitive (PS) rotat-
signal than for 1—3, instead, three carboxy carbon signals ing Overhauser enhancement and exchange spectroscopy
1
3
were observed. Since the C-NMR chemical shifts of the A, (ROESY), a distinct nuclear Overhauser effect (NOE) corre-

1
144
Vol. 59, No. 9
prime number.
Dianthosaponin F (6), [a]D ꢁ15.5, was isolated as an
amorphous powder and its elemental composition was deter-
mined to be C H O . IR spectrum exhibited a strong ab-
3
5
50 12
ꢂ1
sorption band at 1770 cm , arising from g-lactone, along
with the absorptions for carboxyl groups. The NMR spectro-
scopic data indicated the presence of a terminal arabinopyra-
noside and acid hydrolysis of 6 liberated L-arabinose as a
sugar component. The chemical shifts of rings A and B in-
cluding the sugar moiety were essentially the same as those
of 1—4. The signals for a trisubstituted double bond, which
were seen in the NMR spectra of 1—5, had disappeared and,
instead, two oxymethine carbons (d 52.6, 57.1) and one
C
oxygenated tertiary carbon (d 87.6) were assumed to be
C
present in the ring C, D and E regions of the molecule. From
the above evidence, the formation of an epoxy ring between
C-11 and C-12 (d 52.6, 57.1, respectively), and g-lactoniza-
C
tion between the 28-carboxyl group and a hydroxyl group on
the C-13 carbon atom (d 87.6) were expected to be the most
C
1
1
Fig. 3. Diagnostic HMBC Correlations of Dianthosaponin A (1)
plausible modification. H– H COSY correlations H-11 [d
H
3
.12 (br d, Jꢄ4 Hz)] and H-12 [d 3.59 (d, Jꢄ4 Hz)] sup-
H
lation cross peak between H-18 (d 3.27) and a methyl (d
ported the the presence of an oxirane ring, and HMBC corre-
H
H
1
.41) on the 20-position placed the third carboxyl group on lations H-9 (d 1.78), and C-12 and C-13 (Fig. 4a) substanti-
H
an a-face. Acid hydrolysis of 4 liberated L-arabinose and D- ated the position of oxirane ring. The PS-ROESY correla-
glucose, and three anomeric proton (d 4.96, 4.98, 6.19) and tions of H -25 (d 0.98) and H-11, and H -26 (d 1.12) and
H
3
H
3
H
three carbon (d 95.7, 105.1, 105.8) signals were observed in H-12 placed the epoxy ring in the a-face (Fig. 4b). Mean-
C
1
3
the NMR spectra. The C-NMR and HMBC spectroscopic while, those between H-18 and H -30, and H -24 and H -25
3
3
3
data indicated the terminal arabinopyranose was on the hy- confirmed the carboxyl groups, C-23 and C-29, were in the
droxyl group at the 3-position, and the terminal glucopyra- a-orientation. Therefore, the structure of 6 was established to
nose on the hydroxyl group at the 6ꢅ-position, and the inner be as shown in the Fig. 1.
glucopyranose was attached at the carboxyl group at the 28-
position through an ester bond. Therefore, the structure of di- amorphous powder and its elemental composition was deter-
Compound 7, [a] ꢂ78.8, was isolated as a pale yellow
D
anthosaponin D (4) was elucidated to be 29-carboxygypso- mined to be C H O . The IR spectrum exhibited absorp-
4
3
50 22
ꢂ1
genic acid 3-O-a-L-arabinopyranoside 28-b-D-(6ꢅ-O-b-D- tions for hydoxy groups (3405 cm ), carbonyl groups (1731,
ꢂ1
ꢂ1
glucopyranosyl)glucopyranosyl ester, as shown in Fig. 1.
1717 cm ), and aromatic ring(s) (1608 cm ), and the UV
Dianthosaponin E (5), [a]D ꢁ10.2, was isolated as an spectrum also indicated the presence of aromatic ring(s)
1
amorphous powder and its elemental composition was deter- (271, 341 nm). In the H-NMR spectrum, three aromatic pro-
mined to be C H O . The NMR spectra indicated that 5 tons, coupled in an ABX system, four aromatic protons cou-
5
4
86 24
was also a similar compound to the previously mentioned pled in an AAꢀBBꢀ system, two singlet aromatic (olefinic)
saponins. Four anomeric proton (dH 4.96, 5.26, 5.28, 6.17) protons, and three anomeric protons (d 4.96, 5.05, 5.28)
H
1
3
and carbon (d 95.8, 102.6, 105.7ꢃ2) signals were observed were observed. The C-NMR spectrum exhibited 18 signals
C
in the NMR spectra, and acid hydrolysis of 5 gave D-glucose assignable to a terminal rhamnopyranose, parasubstituted and
only. The signal at dC 207.3 (d) with dH 9.57 (s) was as- 1,3,4 trisubstituted benzene rings, and two anomeric carbons.
1
3
signed as an aldehyde functional group and the C-NMR The HSQC spectrum revealed that one (dH 4.96) of the
chemical shifts of rings A, B and C were indistinguishable anomeric protons was associated with a fairly shielded car-
1
5)
from those of gypsogenin, and those of the remaining rings bon atom (d 73.0), this being indicated that the presence of
C
were essentially the same as those of gypsogenin 28-glycosyl C-glycoside, and the remaining signals comprised those of
ester. The aldehyde proton showed a cross peak with C-4 two methylenes, eight further sp carbons, eight oxygenated
d 56.2), whereas H-3 (d 4.04) and H -24 (d 1.32) with methines and two methylenes, probably arising from sugar
15)
2
(
H
H
3
H
the aldehyde carbon in the HMBC spectrum. The branching moieties, and two carbonyl and one methoxy functional
patterns of the four glucopyranoses were expected to be the group. On acid hydrolysis, D-glucose and L-rhamnose were
same as that of dianversicoside G (10) from the chemical liberated. The HMBC spectral data indicated that one of the
shift data in one-dimensional NMR spectra (Table 3), which carbonyl groups was involved in the formation of an acyl
was confirmed by inspection of the HMBC spectrum. The group along with two methylene and methoxy carbons, i.e.,
anomeric protons of the terminal glucopyranoses, H-1ꢅꢅ (d
7,8-dihydroferulic acid (Fig. 5). From the above evidence, 7
H
11)
4
.96) and H-1ꢅꢆ (d 5.26), showed cross peaks with C-3ꢅ (d
was expected to contain a saponarin skeleton, which co-oc-
H
C
1
1
8
8.0) and C-2ꢅꢅ (d 83.5), respectively, and that of the inner curred in this plant. Proton sequences, revealed by the H– H
C
glucopyranose, H-1ꢆ (d 5.28) with C-6ꢅ (d 68.9) of the in- COSY spectrum, indicated the location of an acyl moiety on
H
C
nermost glucopyranose. Therefore, the structure of 5 was the hydroxy group at the 6ꢅꢅ-position (C-6ꢅꢅ: d 65.3), and
C
elucidated to be as shown in Fig. 1. For the readers’ conve- the HMBC spectrum confirmed the attachment of this acyl
nience, the numbering of sugar moiety begins from a two- moiety and that the terminal rhamnopyranose was on the hy-

September 2011
1145
Fig. 6. Results of HMBC and Difference NOE Experiments for Com-
pound 8
The dual arrow curves for HMBC denote HMBC correlations were observed in both
directions.
ꢂ1
ꢂ1
droxy groups (3388 cm ), an ester bond (1703 cm ), an
ꢂ1
ꢂ1
amide bond (1649 cm ), and aromatic ring(s) (1582 cm ),
and the UV spectrum supported the presence of the aromatic
1
ring(s) (252, 278, 312 nm). In the H-NMR spectrum, two
anomeric protons (d 5.16, 5.29), three aromatic protons in
H
an ABX system, four aromatic protons on a 1,2-substituted
benzene ring and two methoxy signals were observed. The
1
3
C-NMR spectrum exhibited the signals for two anomeric
carbons (d 100.0, 102.6), two benzene rings with above
C
Fig. 4. Diagnostic HMBC Correlations (a) and PS-NOESY Correlations
mentioned substitution pattern and two methoxy groups and
(b) of Dianthosaponin F (6)
13
two carbonyl carbons. The C-NMR spectrum also indicated
The dual arrow curves for HMBC denote HMBC correlations were observed in both
directions.
the presence of a terminal rhamnopyranosyl moiety, and acid
hydrolysis of 8 liberated L-rhamnose and D-glucose as well.
The HMBC spectrum revealed the attachment of the rham-
nopyranose moiety on the hydroxyl group at the 2ꢀ-position.
The anomeric proton (dH 5.16) of the glucopyranoside was
correlated with d 162.9 (Fig. 6), and in the difference NOE
C
experiments, irradiation of the anomeric proton showed sig-
nificant enhancement of aromatic signals at dH 6.84 (dd,
Jꢄ9, 3 Hz) and 8.67 (d, Jꢄ3 Hz) (H-5, H-3, respectively).
The cross peaks of the aromatic proton signals at H-3 and H-
6
1
(d 8.02) with C-2 (d 143.7), the latter then with C-7 (d
H
C
C
68.7), and finally the amide proton (d 12.4 in pyridine-d )
H
5
with C-3 (d 109.3 in pyridine-d ) and C-7ꢆ (d 164.8 in
pyridine-d ) in the HMBC spectrum established the structure
C
5
C
5
of the 1,3,4-substituted aromatic ring to be a 4-hydroxyan-
thranilic acid methyl ester. The structure of the other aro-
matic ring was elucidated to be 2-methoxybenzoic acid by
extensive inspection of two-dimensional NMR spectra, and
the structure of 8 was comprising an amide of these two aro-
matic units, which was supported by the HMBC correlation
between the amide proton (d 12.4) and C-1 and C-3 (d
H
C
1
10.7, 109.3 in pyridine-d respectively), and C-7ꢆ (d 164.8
5
,
C
Fig. 5. Diagnostic HMBC Correlations of Compound 7
in pyridine-d ), as shown in Fig. 5. The methyl ester could be
an artifact, formed during the extraction and separation
processes.
5
The dual arrow curve for HMBC correlation denotes HMBC were observed in both
directions.
droxy group at the 2ꢅ-position (dC 77.1). Therefore, the
Compound 9, [a] ꢂ53.2, was isolated as an amorphous
D
structure of 7 was elucidated to be saponarin 2ꢅ-O-a-L- powder and its elemental composition was determined to be
rhamnopyranoside, 6ꢅꢅ-O-7,8-dihydroferulate, as shown in C H O . The IR spectrum exhibited the presence of hy-
Fig. 2.
1
9
20
9
ꢂ1
ꢂ1
droxy groups (3328 cm ), carbonyl groups (1717 cm ), a
ꢂ1
ꢂ1
Compound 8, [a] ꢂ84.3, was isolated as an amorphous double bond (1646 cm ), and aromatic ring(s) (1610 cm ),
D
powder and its elemental composition was determined to be and the UV absorption maximum bands at 252 and 228 nm
C H NO . The IR spectrum showed absorption for hy- also supported the presence of these functional groups. The
2
8
35
14

1
146
Vol. 59, No. 9
then extracted with EtOAc (6 l) to give 37.6 g of an EtOAc-soluble fraction.
The aqueous layer was extracted with 1-BuOH (6 l) to give a 1-BuOH-solu-
ble fraction (114 g), and the remaining water layer was concentrated to fur-
nish 631 g of a water-soluble fraction. The 1-BuOH-soluble fraction (114 g)
was subjected to Diaion HP-20 CC (Fꢄ60 mm, Lꢄ40 cm), using
H O–MeOH (4 : 1, 3 l), (3 : 2, 3 l), (2 : 3, 3 l), and (1 : 4, 3 l), and MeOH (3 l),
2
500 ml fractions being collected. The residue (19.8 g in fractions 9—15) of
the 40—60% MeOH eluate obtained on HP-20 CC was subjected to silica
gel (500 g) CC with increasing amounts of MeOH in CHCl [CHCl (6 l),
3
3
and CHCl –MeOH (49 : 1, 3 l), (24 : 1, 3 l), (23 : 2, 3 l), (9 : 1, 3 l), (17 : 3, 3 l),
3
Fig. 7. Diagnostic HMBC Correlations of Compound 9
(
4 : 1, 3 l), (3 : 1, 3 l), and (7 : 3, 3 l)], and CHCl –MeOH–H O (35 : 15 : 2,
3 2
3
5
1
l), 500 ml fractions being collected. The residue (1.88 g) in fractions 51—
7 was separated by ODS open CC to give 69.0 mg of 16 in fractions 118—
21. The residue (1.99 g) in fractions 58—63 was separated by ODS open
1
H-NMR spectrum exhibited signals assignable to one
methyl protons, one anomeric proton (d 5.39), a cis-double
H
CC to give three residues. The first residue (477 mg) in fractions 65—86 was
purified by DCCC to give 8.63 mg of 17 in fractions 45—57. The second
residue (377 mg) in fractions 87—97 was purified by DCCC and then the
bond [d 6.39 (d, Jꢄ6 Hz) and 7.71 (d, Jꢄ6 Hz)], and five
H
aromatic protons for a monosubstituted benzene ring. The
1
3
C-NMR spectrum revealed the presence of one methyl, one residue (48.6 mg) in fractions 9—12 was finally purified by HPLC
2
(MeOH–H O containing 1% CH COOH, 45 : 55) to yield 7.69 mg of 4 from
anomeric, ten sp and two carbonyl carbon signals. On acid
hydrolysis, 9 liberated D-glucose, whose 6-position in the
original compound must carry an acyl group (H-6: d 4.90,
5
2
3
the peak at 37 min. The third residue (239 mg) in fractions 98—105 was sep-
arated by DCCC to give two fractions. The former (27.8 mg) in fractions 4—
9 was purified by HPLC (MeOH–H O containing 1% CH COOH, 45 : 55) to
H
2
3
.08, and C-6: d 64.9). Thus, the aglycone moiety com- yield a further amount (11.9 mg) of 4 from the peak at 37 min. The latter
C
prised of six carbons and three oxygen atoms with four de- (96.4 mg) in fractions 16—25 was purified by HPLC (MeOH–acetone–H
O,
2
2
: 1 : 7) to give 18.4 mg of 7 from the peak at 53 min.
The residue (17.9 g) in fractions 16—20 obtained on Diaion HP-20 CC
grees of unsaturation. The correlations observed in the
HMBC spectrum (Fig. 7) suggested that the aglycone of 9
was a g-pyrone derivative, such as maltol. Therefore, the
was subjected to silica gel CC (500 g) with increasing amounts of MeOH in
CHCl [CHCl (6 l), and CHCl –MeOH (49 : 1, 3 l), (24 : 1, 3 l), (23 : 2, 3 l),
16)
3
3
3
structure of 9 was elucidated to be maltol b-D-glucopyra- (9 : 1, 3 l), (17 : 3, 3 l), (4 : 1, 3 l), (3 : 1, 3 l), and (7 : 3, 3 l)], and
noside, 6ꢀ-O-benzoate, as shown in Fig. 2. Structurally re- CHCl
–MeOH–H O (35 : 15 : 2, 3 l), 500 ml fractions being collected. The
3
2
residue (1.80 g out of 2.72 g) in fractions 49—56 was separated by ODS
open CC, and the residue (287 mg) in fractions 121—125 was purified by
silica gel CC (70 g) with CHCl3 (50 ml), CHCl –MeOH [(9 : 1, 100 ml),
lated compounds have been isolated from some Caryopyl-
laceae plants. From D. superbus var. longicalycinus, 2-
methyl-3,4-dihydropyran and its monoglucoside have been
3
(4 : 1, 200 ml), and (7 : 3, 200 ml)], and MeOH (200 ml), 5 ml fractions being
17)
isolated, tunicoside, methoxytunicoside and dianthoside collected, to give 0.93 mg of 9 and 2.77 mg of 8 in fractions 20—23 and
18)
5
8—65, respectively. The residue (1.80 g out of 3.24 g) in fractions 57—64
was separated by ODS open CC to give four residues. The first residue
60.3 mg) in fractions 163—170 was purified by HPLC (MeOH–H O, 7 : 13)
from Tunica profifera, and 2-hydroxy-3-methyl-4H-pyran-
4
lene vulgaris.
-one b-D-glucopyranoside, 6ꢀ-O-trans cinnamate from Si-
(
19)
2
to afford 4.03 mg of 9 from the peak at 55 min. The second residue (291 mg)
in fractions 213—221 was purified by silica gel CC (70 g) with CHCl3
Experimental
(50 ml), CHCl –MeOH [(9 : 1, 100 ml), (4 : 1, 200 ml), and (7 : 3, 200 ml)]
3
General Experimental Procedures Optical rotations were measured on
a JASCO P-1030 digital polarimeter. IR and UV spectra were measured on
Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively.
and MeOH (200 ml), 5 ml fractions being collected, to give 11.8 mg of 8 in
fractions in 57—71. The forth residue (88.0 mg) in fractions 267—277 was
separated by DCCC and the residue (31.5 mg) in fractions 23—28 was puri-
1
13
H- and C-NMR spectra were taken on JEOL JNM a-400 and ECA-600
fied by HPLC (MeOH–H O, 7 : 3) to give 8.75 mg of 13 from the peak at
2
spectrometers at 400 MHz or 600 MHz, and 100 MHz or 150 MHz, respec-
tively, with tetramethylsilane as an internal standard. Positive-ion HR-ESI-
28 min.
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), and CHCl –MeOH (49 : 1, 3 l), (24 : 1, 3 l), (23 : 2, 3 l),
®
MS was performed with an Applied Biosystems QSTAR XL NanoSprayTM
System.
3
3
3
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 octadecyl silica (ODS) open
CC on Cosmosil 75C -OPN (Nacalai Tesque, Kyoto, Japan) [Fꢄ50 mm,
(9 : 1, 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 collected. The
3
2
resulting residue (1.72 g) was separated by ODS open CC to give two
residues. The former (127 mg) in fractions 151—164 was purified by HPLC
(MeOH : H O containing 1% CH COOH, 13 : 7) to give 15.4 mg of 5 from
1
8
Lꢄ25 cm, linear gradient: MeOH–H O (1 : 9, 500 ml)→(1 : 1, 500 ml), (1 : 1,
2
2
3
5
00 ml)→(7 : 3, 500 ml), and then (7 : 3, 500 ml)→(9 : 1, 500 ml), fractions the peak at 67 min, and the latter (173 mg) in fractions 165—183 was sepa-
of 10 g being collected]. The DCCC (Tokyo Rikakikai, Tokyo, Japan) was
rated by DCCC to give 32.2 mg of 12 and 73.7 mg of 3 in fractions 124—
equipped with 500 glass columns (Fꢄ2 mm, Lꢄ40 cm), the lower and 172 and 173—221, respectively. The residue (2.06 g) in fractions 49—60
upper layers of solvent mixture of CHCl –MeOH–H O–n-PrOH was separated by ODS open CC to give three residues. The first residue
a
3
2
(
9 : 12 : 8 : 2) being used as the stationary and mobile phases, respectively. (91.2 mg) in fractions 64—81 was purified by HPLC (MeOH : H O contain-
2
Five-gram fractions were collected and numbered according to their order of
elution with the mobile phase. HPLC was performed on an ODS column
ing 1% CH COOH, 13 : 7) to give 7.72 mg of 6 from the peak at 21 min. The
3
second residue (165 mg) in fractions 113—124 was separated by DCCC and
then the residue (54.8 mg) in fractions 10—23 was purified by HPLC
(MeOH : H O containing 1% CH COOH, 13 : 7) to give 5.07 mg of 14 from
(Inertsil; GL Science, Tokyo, Japan; Fꢄ20 mm, Lꢄ250 mm, 6 ml/min), and
the eluate was monitored with a UV detector, at 254 nm, and a reflective
index monitor.
2
3
the peak at 30 min. The third residue (495 mg) in fractions 137—155 was
Plant Material Aerial parts of D. japonicus were collected in Shimoka- separated by DCCC. The residue (93.2 mg) in fractions 12—16 was purified
magari-cho, Toyota-gun, Hiroshima, Japan, in August, 1999, and a voucher
specimen was deposited in the Herbarium of Pharmaceutical Sciences,
Graduate School of Biomedical Sciences, Hiroshima University (99-DJ-
Hiroshima-0806).
by HPLC (MeOH : H O containing 1% CH COOH, 3 : 2) to afford 4.82 mg
of 15, 6.74 mg of 2, 21.9 mg of 10, and 11.1 mg of 1 from the peaks at
69 min, 86 min, 94 min, and 99 min, respectively. The residue (24.0 mg) in
2
3
fractions 32—34 was purified by HPLC (MeOH : H O containing 1%
2
Extraction and Isolation Air-dried aerial parts of D. japonicus
CH COOH, 13 : 7) to give 5.62 mg of 1 from the peak at 44 min. The residue
3
(
5.16 kg) were extracted three times with MeOH (30 lꢃ3) at room tempera- (65.5 mg) in fractions 35—43 was purified by HPLC (MeOH : H O contain-
2
ture for one week and then concentrated to 6 l in vacuo. The concentrated ing 1% CH COOH, 13 : 7) to yield 20.6 mg of 11 from the peak at 53 min.
3
2
8
extract was washed with n-hexane (6 l, 45.1 g) and then the MeOH layer was
concentrated to a gummy mass. The latter was suspended in water (6 l) and
Dianthosaponin A (1): Amorphous powder; [a] ꢁ1.9 (cꢄ0.74, MeOH);
D
ꢂ1 1
IR nmax (film) cm : 3388, 2927, 2865, 1739, 1717, 1065, 1030; H-NMR

September 2011
400 MHz, pyridine-d ): Tables 1 and 2; C-NMR (100 MHz, pyridine-d ):
1147
1
3
13
(
Table 4.
C-NMR Data for Compounds 7, 8 and 9 (100 MHz, CD OD)
5
5
3
ꢁ
Table 3; HR-ESI-MS (positive-ion mode) m/z: 1127.5260 [MꢁNa] (Calcd
a)
b)
for C H O Na 1127.5245).
C
7
8
9
5
3
84 24
2
8
Dianthosaponin B (2): Amorphous powder; [a] ꢁ3.1 (cꢄ0.45, MeOH);
D
ꢂ1
1
IR n
(
(film) cm : 3384, 2927, 2861, 1733, 1715, 1065, 1029; H-NMR
1
2
3
4
5
6
7
8
112.2
143.7
110.1
162.9
112.1
133.7
168.7
max
1
3
400 MHz, pyridine-d ): Tables 1 and 2; C-NMR (100 MHz, pyridine-d ):
166.7
104.5
184.1
161.1
111.7
163.9
95.6
161.9
143.0
175.2
117.0
155.1
15.4
5
5
ꢁ
Table 3; HR-ESI-MS (positive-ion mode) m/z: 1127.5264 [MꢁNa] (Calcd
for C H O Na 1127.5245).
5
3
84 24
2
8
Dianthosaponin C (3): Amorphous powder; [a] ꢁ12.6 (cꢄ3.89,
D
ꢂ1
MeOH); IR n
(film) cm : 3388, 2931, 2868, 1715, 1701, 1061, 1022;
max
1
13
H-NMR (400 MHz, pyridine-d ): Tables 1 and 2; C-NMR (100 MHz, pyri-
5
ꢁ
dine-d ): Table 3; HR-ESI-MS (positive-ion mode) m/z: 657.3615 [MꢁNa]
5
(
Calcd for C H O Na 657.3609).
9
10
–OCH3
1ꢀ
158.7
106.9
35
54 18
2
4
Dianthosaponin D (4): Amorphous powder; [a] ꢂ2.7 (cꢄ0.51, MeOH);
D
ꢂ1
1
IR nmax (film) cm : 3392, 2929, 2877, 1734, 1713, 1701, 1057, 1016; H-
NMR (400 MHz, pyridine-d ): Tables 1 and 2; C-NMR (100 MHz, pyri-
dine-d ): Table 3; HR-ESI-MS (positive-ion mode) m/z: 995.4434 [MꢁNa]
52.5
100.0
79.2
79.2
71.3
78.3
62.4
102.6
72.3
72.3
74.1
70.1
18.3
1
3
122.9
129.5
117.0
162.8
117.0
129.5
73.0
77.1
81.6
71.1
81.9
105.4
75.5
78.1
71.3
75.9
5
ꢁ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
1ꢅ
2ꢅ
3ꢅ
5
(Calcd for C H O Na 995.4458).
47
72 21
2
8
Dianthosaponin E (5): Amorphous powder; [a] ꢁ10.2 (cꢄ1.02,
D
ꢂ1
MeOH); IR n
(film) cm : 3382, 2935, 2868, 1732, 1718, 1068, 1031;
max
1
13
H-NMR (400 MHz, pyridine-d ): Tables 1 and 2; C-NMR (100 MHz, pyri-
64.9
5
dine-d5): Table 3; HR-ESI-MS (positive-ion mode) m/z: 1141.5394
131.0
129.9
128.8
133.3
128.8
129.9
166.4
ꢁ
[MꢁNa] (Calcd for C H O Na 1141.5401).
54
86 24
2
8
Dianthosaponin F (6): Amorphous powder; [a] ꢁ15.5 (cꢄ0.51,
D
ꢂ1
MeOH); IR n
1
(film) cm : 3395, 2935, 2875, 1770, 1716, 1701, 1054,
4ꢅ
5ꢅ
max
1
13
019, 873; H-NMR (400 MHz, pyridine-d ): Tables 1 and 2; C-NMR
5
(
6
100 MHz, pyridine-d ): Table 3; HR-ESI-MS (positive-ion mode) m/z:
6ꢅ
7ꢅ
61.9
5
ꢁ
85.3196 [MꢁNa] (Calcd for C H O Na 685.3195).
3
5
50 12
Saponarin 2ꢅ-O-a-L-Rhamnopyranoside, 6ꢅꢅ-O-Dihydoferulate (7): Pale
1ꢆ
2ꢆ
3ꢆ
4ꢆ
5ꢆ
6ꢆ
7ꢆ
101.7
72.4
71.8
73.4
69.9
18.2
123.5
159.1
112.9
134.9
121.9
132.9
166.7
56.3
2
3
yellow amorphous powder; [a] ꢂ78.8 (cꢄ1.22, MeOH); IR nmax (film)
D
ꢂ1
cm : 3405, 2930, 1731, 1717, 1608, 1072, 1038; UV l
(MeOH) nm
max
1
(
log e): 341 (4.19), 271 (4.16); H-NMR (400 MHz, CD OD) d: 7.77 (2H, d,
3
Jꢄ8 Hz, H-2ꢀ, 6ꢀ), 6.85 (1H, s, H-8), 6.83 (2H, d, Jꢄ8 Hz, H-3ꢀ, 5ꢀ), 6.62
(
(
1H, s, H-3), 6.56 (1H, d, Jꢄ2 Hz, H-2ꢅꢆ), 6.55 (1H, d, Jꢄ8 Hz, H-5ꢅꢆ), 6.46
1H, dd, Jꢄ8, 2 Hz, H-6ꢅꢆ), 5.28 (1H, d, Jꢄ2 Hz, H-1ꢆ), 5.05 (1H, d,
Jꢄ7 Hz, H-1ꢅꢅ), 4.96 (1H, d, Jꢄ10 Hz, H-1ꢅ), 4.51 (1H, dd, Jꢄ12, 2 Hz, H-
ꢅꢅa), 4.30 (1H, dd, Jꢄ12, 5 Hz, H-6ꢅꢅb), 3.90 (1H, dd, Jꢄ12, 2 Hz, H-6ꢅa),
.85 (1H, dd, Jꢄ3, 2 Hz, H-2ꢆ), 3.81 (1H, m, H-5ꢅꢅ), 3.75 (1H, dd, Jꢄ12,
–OCH3
1ꢅꢅ
2ꢅꢅ
3ꢅꢅ
4ꢅꢅ
5ꢅꢅ
6ꢅꢅ
1ꢅꢆ
2ꢅꢆ
3ꢅꢆ
4ꢅꢆ
5ꢅꢆ
6ꢅꢆ
7ꢅꢆ
8ꢅꢆ
9ꢅꢆ
–OCH3
6
3
5
3
3
3
8
102.4
75.0
77.6
72.2
75.5
Hz, H-6ꢅb), 3.70 (3H, s, –OCH ), 3.63 (1H, m, H-2ꢅꢅ), 3.62 (1H, m, H-4ꢅ),
3
.60 (1H, m, H-3ꢅ), 3.56 (1H, m, H-3ꢅꢅ), 3.50 (1H, dd, Jꢄ10, 3 Hz, H-3ꢆ),
.45 (1H, dd, Jꢄ9, 9 Hz, H-4ꢅꢅ), 3.39 (1H, m, H-4ꢅꢅ), 3.35 (1H, m, H-5ꢅ),
.34 (1H, m, H-2ꢅ), 3.09 (1H, dd, Jꢄ10, 10 Hz, H-4ꢆ), 2.75 (1H, dd, Jꢄ16,
Hz, H-7ꢅꢆa), 2.67 (1H, dd, Jꢄ16, 8 Hz, H-7ꢅꢆb), 2.62 (1H, dd, Jꢄ16, 8 Hz,
65.3
133.2
112.9
148.7
145.7
116.2
121.6
31.5
37.4
174.5
56.4
H-8ꢅꢆa), 2.58 (1H, dd, Jꢄ16, 8 Hz, H-8ꢅꢆb), 2.41 (1H, dq, Jꢄ10, 6 Hz, H-5ꢆ),
1
3
0
.79 (3H, d, Jꢄ6 Hz, H -6ꢆ); C-NMR (100 MHz, CD OD): Table 4; HR-
3 3
ꢁ
ESI-MS (positive-ion mode) m/z: 941.2672 [MꢁNa] (Calcd for
C H O Na 941.2691).
4
3
50 22
Methyl 4-Hydroxy-2-(2ꢅ-methoxybenzamido)benzoate O-b-D-(2ꢀ-O-a-L-
2
4
Rhamnopyranosyl)glucopyranoside (8): Amorphous powder; [a] ꢂ84.3
D
ꢂ1
(
1
(
3
cꢄ0.78, MeOH); IR nmax (film) cm : 3388, 2927, 2840, 1703, 1649, 1582,
071, 1047; UV l (MeOH) nm (log e): 312 (3.88), 278 (4.01), 252
max
1
4.09), 218 (3.98); H-NMR (400 MHz, CD OD) d: 8.67 (1H, d, Jꢄ3 Hz, H-
3
), 8.04 (1H, dd, Jꢄ8, 2 Hz, H-6ꢆ), 8.02 (1H, d, Jꢄ9 Hz, H-6), 7.54 (1H,
ddd, Jꢄ8, 8, 2 Hz, H-4ꢆ), 7.18 (1H, dd, Jꢄ8, 2 Hz, H-3ꢆ), 7.08 (1H, ddd,
Jꢄ8, 8, 2 Hz, H-5ꢆ), 6.84 (1H, dd, Jꢄ9, 3 Hz, H-5), 5.29 (1H, d, Jꢄ2 Hz, H-
a) At 150 MHz. b) Recorded in pyridine-d .
5
1
6
6
ꢅ), 5.16 (1H, d, Jꢄ8 Hz, H-1ꢀ), 4.07 (3H, s, –OCH ), 3.99 (1H, dq, Jꢄ10,
Hz, H-5ꢅ), 3.96 (1H, dd, Jꢄ3, 2 Hz, H-2ꢅ), 3.93 (1H, dd, Jꢄ12, 2 Hz, H-
3ꢀ), 74.2 (C-4ꢅ), 72.7 (C-3ꢅ), 72.4 (C-2ꢅ), 71.1 (C-4ꢀ), 70.0 (C-5ꢅ), 62.1 (C-
6ꢀ), 55.7 (–OCH ), 51.8 (–COOCH ), 18.9 (C-6ꢅ); HR-ESI-MS (positive-ion
3
3
3
ꢁ
ꢀa), 3.92 (3H, s, –COOCH ), 3.75 (1H, dd, Jꢄ12, 5 Hz, H-6ꢀb), 3.72 (1H, mode) m/z: 632.1933 [MꢁNa] (Calcd for C H NO Na 632.1950).
3
28 35
14
23
dd, Jꢄ9, 8 Hz, H-2ꢀ), 3.63 (1H, dd, Jꢄ10, 3 Hz, H-3ꢅ), 3.63 (1H, dd, Jꢄ9,
9
H-4ꢀ), 3.40 (1H, dd, Jꢄ10, 10 Hz, H-4ꢅ), 1.34 (3H, d, Jꢄ6 Hz, H -6ꢅ); H- 1066; UV lmax (MeOH) nm (log e): 254 (3.81), 228 (3.97); H-NMR
NMR (400 MHz, pyridine-d ) 12.4 (CONH), 9.33 (1H, d, Jꢄ2 Hz, H-3),
Maltol b-D-Glucopyranoside 6ꢀ-O-Bezoate (9): Amorphous powder; [a]
D
ꢂ1
Hz, H-3ꢀ), 3.52 (1H, ddd, Jꢄ10, 5, 2 Hz, H-5ꢀ), 3.45 (1H, dd, Jꢄ10, 9 Hz, ꢂ53.2 (cꢄ0.27,MeOH); IR nmax (film) cm : 3328, 2903, 1717, 1646, 1610,
1
1
3
(400 MHz, CD OD) d: 8.11 (2H, dd, Jꢄ8, 1 Hz, H-2ꢅ, 6ꢅ), 7.71 (1H, d,
5
3
8
.33 (1H, dd, Jꢄ8, 2 Hz, H-6ꢆ), 7.87 (1H, d, Jꢄ9 Hz, H-6), 7.37 (1H, ddd, Jꢄ6 Hz, H-6), 7.48 (1H, tt, Jꢄ8, 1 Hz, H-4ꢅ), 7.35 (2H, dd. Jꢄ8, 8 Hz, H-3ꢅ,
Jꢄ8, 7, 2 Hz, H-4ꢆ), 7.10 (1H, dd, Jꢄ9, 2 Hz, H-5), 6.96 (1H, dd, Jꢄ8, 7, 5ꢅ), 6.39 (1H, d. Jꢄ6 Hz, H-5), 5.39 (1H, d, Jꢄ8 Hz, H-1ꢀ), 5.08 (1H, dd,
Hz, H-5ꢆ), 6.93 (1H, dd, Jꢄ8, 2 Hz, H-3ꢆ), 6.27 (1H, s, H-1ꢅ), 5.62 (1H, d, Jꢄ12, 2 Hz, H-6ꢀa), 4.90 (1H, dd, Jꢄ12, 6 Hz, H-6ꢀb), 4.18 (1H, dd, Jꢄ8,
Jꢄ8 Hz, H-1ꢀ), 4.76 (1H, m, H-5ꢅ), 4.70 (1H, m, H-2ꢅ), 4.50 (1H, br d, 8 Hz, H-3ꢀ), 4.09 (1H, dd, Jꢄ8, 8 Hz, H-2ꢀ), 4.04 (1H, m, H-5ꢀ), 4.03 (1H,
2
1
3
Jꢄ12 Hz, H-6ꢀa), 4.44 (1H, m, H-3ꢅ), 4.43 (1H, m, H-3ꢀ), 4.32 (1H, m, H- m, H-4ꢀ), 2.29 (3H, s, H -7); C-NMR (100 MHz, CD OD): Table 4; HR-
6
3
3
ꢁ
ꢀb), 4.27 (1H, m, H-2ꢀ), 4.25 (1H, m, H-4ꢅ), 4.20 (1H, m, H-4ꢀ), 4.07 (1H, ESI-MS (positive-ion mode) m/z: 415.0997 [MꢁNa] (Calcd for
m, H-5ꢀ), 3.91 (3H, s, –OCH , 3.72 (3H, s, –COOCH ), 1.83 (3H, d, C H O Na 415.1000).
3
3
19 20
9
13
13
Jꢄ6 Hz, H-6ꢅ);
C-NMR (100 MHz, CD OD): Table 4;
C-NMR
Sugar Analysis About 1 mg each of 1—9 was hydrolyzed with 1 M HCl
3
(
2
100 MHz, pyridine-d ) d: 167.6 (C-7), 164.8 (C-7ꢆ), 162.4 (C-4), 157.9 (C-
(0.1 ml) at 90 °C for 2 h. The reaction mixtures were each partitioned with
ꢀ), 143.7 (C-2), 133.7 (C-4ꢆ), 133.0 (C-6), 132.6 (C-6ꢆ), 123.5 (overlapped an equal amount of EtOAc (0.1 ml), and the water layers were analyzed with
5
with the solvent, C-1ꢆ), 121.1 (C-5ꢆ), 112.0 (C-3ꢆ), 110.9 (C-5), 110.7 (C- a chiral detector (JASCO OR-2090plus) on an amino column [Asahipak
), 109.3 (C-3), 102.5 (C-1ꢅ), 99.6 (C-1ꢀ), 79.2 (C-2ꢀ), 78.8 (C-5ꢀ), 78.2 (C- NH P-50 4E, CH CN–H O (4 : 1), 1 ml/min]. The hydrolyzates of 3 and 6
1
2
3
2

1
148
Vol. 59, No. 9
gave a peak for L-arabinose at 8.5 min with a positive optical rotation sign.
References
Those of 1, 2 and 4 gave peaks for L-arabinose and D-glucose at 8.5 min and
1) Oshima Y., Ohsawa T., Oikawa K., Konno C., Hikino H., Planta Med.,
50, 40—43 (1984).
12.5 min, respectively, with positive optical rotation signs. However, those of
5
and 9 gave a peak for D-glucose at 12.5 min with a positive optical rotation
2) Oshima Y., Ohsawa T., Hikino H., Planta Med., 50, 43—47 (1984).
3) Oshima Y., Ohsawa T., Hikino H., Planta Med., 50, 254—258 (1984).
4) Hsieh P.-W., Chang F.-R., Wu C.-C., Li C.-M., Wu K.-Y., Chen S.-L.,
Yen H.-F., Wu Y.-C., Chem. Pharm. Bull., 53, 336—338 (2005).
5) Su J., “Dictionary of Chinese Traditional Medicines,” Shanghai Peo-
ple’s Press, Shanghai, 1977, pp. 2701—2702.
sign. Ones of 7 and 8 gave peaks for L-rhamnose and D-glucose at 6.3 min
and 12.5 min, with negative and positive optical rotation signs, respectively.
The peaks were identified by co-chromatography with authentic samples.
Known Compounds Isolated Dianversicoside G (10): Amorphous
2
7
6)
powder; [a] ꢂ1.3 (cꢄ1.46, MeOH). Chionaeoside B (11): Amorphous
D
2
5
7)
powder; [a] ꢁ4.4 (cꢄ1.37, MeOH). Chionaeoside C (12): Amorphous
6) Ma L., Gu Y.-C., Luo J.-G., Wang J.-S., Huang X.-F., Kong L.-Y., J.
Nat. Prod., 72, 640—644 (2009).
D
2
6
7)
powder; [a] ꢁ10.8 (cꢄ1.16, MeOH). Vaccaroside A (13): Amorphous
D
2
7
8)
powder; [a] ꢁ4.1 (cꢄ0.58, MeOH). Vaccaroside D (14): Amorphous
7) Avunduk S., Lacaille-Dubois M. A., Miyamoto T., Bedir E., Senol S.
G., Caliskan O. A., J. Nat. Prod., 70, 1830—1833 (2007).
8) Koike K., Jia Z., Nikaido T., J. Nat. Prod., 62, 1655—1659 (1999).
9) Sang S.-M., Zou M.-L., Lao A.-N., Chen Z.-L., Uzawa J., Fujimoto Y.,
Chin. Chem. Lett., 11, 49—53 (2000).
D
2
6
8)
powder; [a] ꢂ19.2 (cꢄ0.34, MeOH). Segetoside K (15): Amorphous
D
28
9)
powder; [a] ꢂ12.8 (cꢄ0.32, MeOH). Dianic Acid 28-(6ꢀ-O-b-D-Galac-
D
2
7
topyranosy)-b-D-glucopyranosyl Ester (16): Amorphous powder; [a] ꢁ2.9
D
10)
25
(
(
cꢄ3.26, MeOH). Saponarin (17): Amorphous powder; [a]D ꢂ21.2
11)
cꢄ0.50, DMSO).
10) Elgmal M. H. A., Soliman H. S. M., Tóth G., Halász J., Duddeck H.,
Magn. Reson. Chem., 34, 697—702 (1996).
Acknowledgements The authors are grateful for access to the supercon- 11) Österdahl B.-G., Acta Chem. Scand. A, 32B, 93—97 (1978).
ducting NMR instrument (JEOL JNM a-400) at the Analytical Center of 12) Belous V. N., Ryabinin A. A., Khim. Prir. Soedin, 3, 95—97 (1967).
Molecular Medicine of the Hiroshima University Faculty of Medicine, and 13) Ruzicka L., Giacomello G., Helv. Chim. Acta, 20, 299—309 (1937).
an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS at the
Analysis Center of Life Science of the Graduate School of Biomedical Sci- 15) Kawata Y., Kizu H., Miyaichi Y., Tomimori T., Chem. Pharm. Bull.,
ences, Hiroshima University. The authors are also grateful for the use of the
49, 635—638 (2001).
NMR instrument (JEOL ECA-600) at the Natural Science Center for Basic 16) Wada H., Murakami T., Tanaka N., Nakamura M., Saiki Y., Chen C.
Research and Development, Hiroshima University. This work was supported
M., Yakugaku Zasshi, 106, 989—994 (1986).
in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, 17) Shimizu M., Hayashi T., Shimizu K., Morita N., Phytochemistry, 21,
14) Jia Z., Koike K., Nikaido T., J. Nat. Prod., 61, 1368—1373 (1998).
Science and Technology of Japan, the Japan Society for the Promotion of
Science (No. 22590006 and 23590130), and the Ministry of Health, Labour
and Welfare. Thanks are also due to the Research Foundation for Pharma-
245—247 (1982).
18) Plouvier V., Martin M.-T., Brouard J.-P., Phytochemsitry, 32, 1618—
1620 (1993).
ceutical Sciences and the Takeda Science Foundation for the financial sup- 19) Borris R. P., Seifert K., Johne S., Hesse M., Helv. Chim. Acta, 65,
port.
2481—2485 (1985).