Five new nortriterpenoid glycosides from the bulbs of Scilla scilloides

Article abstract of DOI:10.1248/cpb.c13-00116

Five new norlanostane-type triterpenoid glycosides were isolated from the bulbs of Scilla scilloides DRUCE (Liliaceae). Their chemical structures were determined on the basis of spectroscopic data as well as chemical evidence.

Full text of DOI:10.1248/cpb.c13-00116

5
92
Note
Chem. Pharm. Bull. 61(5) 592–598 (2013)
Vol. 61, No. 5
Five New Nortriterpenoid Glycosides from the Bulbs of Scilla scilloides
,
a
a
a
b
b
Masateru Ono,* Tetsuya Ochiai, Shin Yasuda, Yoichiro Nishida, Toshiharu Tanaka,
c
c
d
d
Masafumi Okawa, Junei Kinjo, Hitoshi Yoshimitsu, and Toshihiro Nohara
a
b
School of Agriculture, Tokai University; 5435 Minamiaso, Aso, Kumamoto 869–1404, Japan: Aso Pharmaceutical Co.,
c
Ltd.; 91–1 Tsukure, Kikuyo, Kumamoto 869–1101, Japan: Faculty of Pharmaceutical Sciences, Fukuoka University;
8
4
d
–19–1 Nanakuma, Jonan-ku, Fukuoka 814–0180, Japan: and Faculty of Pharmaceutical Sciences, Sojo University;
–22–2 Ikeda, Nishi-ku, Kumamoto 860–0082, Japan.
Received February 5, 2013; accepted March 7, 2013
Five new norlanostane-type triterpenoid glycosides were isolated from the bulbs of Scilla scilloides
Druce (Liliaceae). Their chemical structures were determined on the basis of spectroscopic data as well as
chemical evidence.
Key words Scilla scilloides; triterpenoid; norlanostane; glycoside; Liliaceae
Scilla scilloides Druce is a perennial herb belonging to the tion spectroscopy (COSY), heteronuclear multiple-quantum
Liliaceae family. The bulb of this plant has been used as a coherence (HMQC), and heteronuclear multiple-bond correla-
foodstuff and as a traditional medicine for promoting blood tion (HMBC) techniques, and the planar structure of 1 could
circulation, as an anti-inflammatory agent, and as an analge- be determined as illustrated in Fig. 1.
1)
sic. Homoisoflavones and norlanostane- and lanostane-type
On acidic hydrolysis, 1 afforded D-apiose, l-arabinose, and
triterpenoids are recognized chemical constituents of this D-glucose, which were identified by optical rotation using
2
–6)
7–9)
bulb, as previously reported.
In our prior studies,
a
chiral detection during HPLC analysis, along with several un-
1
13
new homostilbene, two new homoisoflavones, a new phenyl- identified artificial aglycones. The H- and C-NMR spectral
propanoid glycoside, five new norlanostane-type triterpenoid data of 1 indicated that the sugar moiety of 1 was composed
1
glycosides, and two new lanostane-type triterpenoid glyco- of 1mol each of α-l-arabinopyranose with C conformation
4
sides were isolated from the methanol (MeOH) extract of and β-D-apiofuranose and 2mol of β-D-glucopyranose with
4
9,10)
fresh bulbs of S. scilloides and were structurally characterized
C conformation
(Tables 1–4). The HMBC spectrum of 1
1
along with 16 known compounds comprising a homostilbene, showed key correlations between H-1 of the first glucosyl unit
seven homoisoflavones, a xanthone, a lignan, two alkaloids, (Glc) and C-3 of the aglycone moiety (Agl), H-1 of the arabi-
three norlanostane-type triterpenoids, and a norlanostane-type nosyl unit (Ara) and C-6 of Glc, H-1 of the second glucosyl
triterpenoid glycoside. As part of an ongoing study of this unit (Glc′) and C-2 of Ara, and H-1 of the apiosyl unit (Api)
13
plant, the isolation and structural characterization of five new and C-2 of Glc′ (Fig. 1). Furthermore, the assigned C-NMR
norlanostane-type triterpenoid glycosides from the MeOH ex- spectral data of Agl and the sugar moiety were consider-
8)
tract of S. scilloides are reported herein.
The MeOH extract of fresh bulbs of S. scilloides was sus- epoxy-3β,29-dihydroxy-27-nor-lanost-8-ene-15,24-dione
pended in H O and successively extracted with ethyl acetate β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-α-l-
ably similar to those of scillanostaside C and (23R)-17α,23-
3-O-
2
11)
(
EtOAc) and n-butanol (BuOH). Repeated chromatography of arabinopyranosyl-(1→6)-β-D-glucopyranoside,
respectively.
the aqueous layer using Diaion HP20, silica gel, and Chroma- The structure of 1 was thus concluded to be 15-deoxyeucosterol
torex octadecyl silica (ODS) column chromatography as well 3-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-
as HPLC on ODS led to the isolation of five compounds (1–5). α-l-arabinopyranosyl-(1→6)-β-D-glucopyranoside (Fig. 2).
Compound 1, tentatively named scillanostaside H, was ob-
Compound 2, tentatively named scillanostaside I, was ob-
tained as an amorphous powder. The negative-ion FAB-MS tained as an amorphous powder, and it furnished the same
−
of 1 exhibited an [M−H] ion peak at m/z 1045 along with monosaccharides as those obtained from 1 on acidic hydro-
fragment ion peaks at m/z 913 [1045−132 (C H O , pento- lysis. The negative-ion FAB-MS of 2 was characterized by
5
8
4
−
−
−
syl unit)] , 751 [913−162 (C H O , hexosyl unit)] , and 619 an [M−H] ion peak at m/z 1061 accompanied by fragment
751−132] . The molecular formula of 1 was determined as ion peaks at m/z 929 [1061−132] , 767 [929−162] , and 635
C H O based on high-resolution (HR)-positive-ion FAB- [767−132] , all of which were 16 mass units greater than
MS. The H-NMR spectrum of 1 was characterized by signals those of 1. HR-positive-ion FAB-MS indicated that the mo-
6
10
5
−
−
−
[
−
51
82 22
1
1
due to four tertiary methyl groups [δ 1.54 (s), 1.51 (s), 0.92 (s), lecular formula of 2 was C H O . The H-NMR spectrum
51
82 23
0.89 (s)], one secondary methyl group [δ 1.01 (d, J=7.0Hz)], of 2 was analogous to that of 1; in particular, the signals due
one primary methyl group [δ 1.05 (dd, J=7.0, 7.0Hz)], and to the sugar moiety were almost superimposable. Further-
13
four anomeric protons [δ 6.36 (s), 5.19 (d, J=4.0Hz), 5.09 (d, more, the C-NMR spectrum of 2 was also similar to that
13
J=7.0Hz), 4.96 (d, J=7.5Hz)]. The C-NMR spectrum of 1 of 1, with the exception of the appearance of a signal due
exhibited signals due to one keto carbonyl carbon (δ 212.5), to one additional oxymethine carbon and the disappearance
two olefinic carbons (δ 135.3, 134.6), and four anomeric car- of the signal due to one methylene carbon. Detailed assign-
bons (δ 111.1, 106.1, 103.6, 101.4). A detailed analysis of these ment of these NMR spectral signals was performed with the
1
1
NMR spectral signals was performed using the H– H correla- aid of 2D-NMR techniques, as described for 1 (Tables 1–4).
1
1
The H– H COSY spectrum of 2 indicated correlation of the
signal due to the oxygenated methine proton at δ 4.72 (brdd,
The authors declare no conflict of interest.
To whom correspondence should be addressed. e-mail: mono@agri.u-tokai.ac.jp
*
© 2013 The Pharmaceutical Society of Japan

May 2013
593
1
13
Fig. 1. H– C Long-Range Correlations Observed in the HMBC Spectra of 1–5 (in Pyridine-d , 500MHz)
5
J=7.0, 7.0Hz) with the signals due to the methylene protons relations were observed between H-3 of Agl and H -28 of Agl,
3
at δ 2.23 (dd, J=7.0, 12.5Hz) and 2.00, with which the signal H-5 of Agl and H -28 of Agl, H-16 of Agl and H -30 of Agl,
3
3
due to the methylene carbon at δ 44.4 exhibited cross-peaks H -18 of Agl and H-20 of Agl, H -18 of Agl and H -19 of Agl,
3
3
3
in the HMQC spectrum. In addition, a long-range correlation H -19 of Agl and Ha,b-29 of Agl, and H -21 of Agl and H-23
3
3
1
between the signal due to the methylene carbon at δ 44.4 and of Agl (Fig. 3). Furthermore, the H-NMR spectral profile of
the signal due to the methyl protons at δ 1.56 (s) assignable to Agl of 2 exhibited downfield shifts of H -18 (0.31ppm) and
3
H -30 of Agl was observed in the HMBC spectrum (Fig. 1). H-20 (1.12ppm) relative to the corresponding peaks of 1; these
3
On the basis of the foregoing data, it was proposed that 2 is a shifts were attributed to deshielding effects of the hydroxyl
derivative of 1, in which a hydroxyl group is attached at C-16 group at C-16. Based on these effects, it was deduced that the
of Agl of 1. The stereochemistry was defined on the basis of hydroxyl group at C-16 assumed the β-configuration. Conse-
the nuclear Overhauser and exchange spectroscopy (NOESY) quently, the structure of 2 was concluded to be (23S)-17α,23-
spectrum, in which key nuclear Overhauser effect (NOE) cor- epoxy-3β,16β,29-trihydroxy-27-nor-lanost-8-ene-24-one 3-O-β-

5
94
Vol. 61, No. 5
Fig. 2. Structures of 1–5
1
Table 1. H-NMR Spectral Data for Aglycone Moiety of 1–3 (in Pyridine-d , 500MHz)
5
1
2
3
a)
1
1
2
2
3
5
6
6
7
7
1a
1b
2a
2b
5a
5b
6a
6b
8
9
0
1
2a
2b
3
5a
5b
6
a
b
a
b
1.68
1.71 m
1.15
1.71 m
1.15
a)
a)
1.15 ddd (3.5, 14.5, 14.5)
2.26 m
2.28 m
2.28 m
a)
a)
a)
1.98
2.07
2.04
3.57 dd (4.0, 12.0)
1.26 d (12.5)
3.59 dd (4.5, 11.5)
1.28 d (12.5)
1.79 m
3.58 dd (4.5, 12.0)
1.28 d (12.5)
1.80 m
a)
a
b
a
b
1.80
1.47
2.04
1.98
2.13
1.96
a)
1.49 m
1.49 m
a)
a)
a)
2.08
2.08
2.07
2.07
a)
a)
a)
a)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
2.18 m
2.15 m
a)
a)
a)
2.02
2.47
1.56
2.04
2.48
1.57
a)
a)
2.41 ddd (8.5, 8.5, 13.0)
a)
a)
a)
1.45
1.66
a)
2.23 dd (7.0, 12.5)
2.23 dd (8.0, 12.5)
a)
a)
1.38 dd (9.5, 9.5)
2.00
2.00
4.73
a)
a)
2.16
1.61
4.72 brdd (7.0, 7.0)
a)
0.89 s
0.92 s
1.20 s
0.94 s
1.21 s
0.94 s
a)
2.04
3.16 dq (7.0,7.0)
1.16 d (7.0)
3.16 dq (7.0, 7.0)
1.16 d (7.0)
1.01 d (7.0)
a)
a)
a)
2.00
2.54
2.53
1.76 dd (7.5, 12.0)
4.61 dd (7.5, 10.5)
1.89 dd (7.5, 12.0)
4.75 dd (7.5, 11.0)
1.92 dd (7.5, 11.5)
a)
4.75
2.51
2.51
a)
a)
a)
2.56
2.53
2.51
2.51
a)
a)
a)
1.05 dd (7.0, 7.0)
1.54 s
1.00 dd (7.5, 7.5)
1.55 s
1.00 dd (7.5, 7.5)
1.55 s
8
9a
9b
0
4.41 d (11.0)
3.64 brd (11.0)
1.51 s
4.42 d (11.0)
3.64 brd (11.0)
1.56 s
4.43 d (11.0)
3.64 d (11.0)
1.57 s
δ in ppm from tetramethylsilane (TMS) (coupling constants (J) in Hz are given in parentheses). a) Signals were overlapped with other signals.
−
−
D-apiofuranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-α-l- (C H O , deoxyhexosyl unit)] , 767 [929−162] , and 635
6
10
4
−
arabinopyranosyl-(1→6)-β-D-glucopyranoside (Fig. 2).
[767−132] . The molecular formula of 3 was determined to
Compound 3, tentatively named scillanostaside J, was be C H O using HR-positive-ion FAB-MS. The H-NMR
1
52
84 23
obtained as an amorphous powder. Acidic hydrolysis of 3 spectrum of 3, which was analogous to that of 2, showed
afforded l-rhamnose, l-arabinose, and D-glucose. The nega- signals due to four tertiary methyl groups, two secondary
−
tive-ion FAB-MS of 3 exhibited an [M−H] ion peak at m/z methyl groups, one primary methyl group, and four anomeric
13
1
075 along with fragment ion peaks at m/z 929 [1075−146 protons. The C-NMR spectrum of 3, which was also similar

May 2013
595
1
Table 2. H-NMR Spectral Data for Sugar Moiety of 1–3 (in Pyridine-d , 500MHz)
5
1
2
3
Glc-1
4.96 d (7.5)
4.97 d (7.5)
4.97 d (7.0)
a)
2
3
4
5
6
6
3.96 dd (7.5, 8.5)
3.98 dd (7.5, 8.5)
3.98
a)
a)
a)
4.18
4.18
4.18
4.19
4.18
4.18
a)
a)
a)
a)
4.02 ddd (4.0, 4.0, 8.5)
4.04 ddd (4.0, 4.0, 9.5)
3.99
a)
a)
a
b
4.51
4.24
4.52
4.27
4.50 dd (4.0, 10.5)
4.23 dd (4.5, 10.5)
5.34 d (3.0)
a)
a)
Ara-1
5.19 d (4.0)
5.21 d (3.5)
a)
a)
a)
2
3
4
5
5
4.53
4.50
4.51
4.32
4.54
4.53
4.53
4.35
4.64
4.67
4.63
a)
a)
a)
a)
a)
a)
a)
a)
a
b
4.40 dd (7.5, 11.0)
3.94 dd (3.5, 11.0)
5.17 d (7.5)
3.80 dd (2.5, 12.0)
5.09 d (7.0)
3.82 dd (3.0, 11.5)
5.11 d (7.5)
Glc′-1
a)
2
3
4
5
6
6
4.12
4.13
4.13
4.12 dd (7.5, 9.0)
4.24 dd (7.5, 8.5)
a)
a)
a)
4.15
4.15
4.18
4.18
a)
a)
a)
3.68 m
3.71 m
3.69 m
4.34 dd (2.5, 12.0)
4.27 dd (4.0, 12.0)
a)
a)
a
b
4.35
4.25
4.38
4.27
a)
a)
Api-1
6.36 s
4.77 s
4.77 d (9.5)
6.38 d (1.5)
4.79 d (1.5)
2
4
4
a)
4.79
4.35
4.26
a)
a)
4.32
4.25
4.24
a)
a)
5
a
a)
5
b
4.23 d (11.5)
Rha-1
6.38 s
a)
2
3
4
5
6
4.75
4.66
a)
4.31 dd (9.0, 9.0)
4.90 dq (9.0, 6.0)
1.76 d (6.0)
δ in ppm from TMS (coupling constants (J) in Hz are given in parentheses). Glc, glucopyranosyl; Ara, arabinopyranosyl; Api, apiofuranosyl; Rha, rhamnopyranosyl. a)
Signals were overlapped with other signals.
to that of 2, was characterized by signals assignable to one cleaved (Tables 3–6). The coupling constants of the signals
keto carbonyl carbon, two olefinic carbons, and four anomeric due to the anomeric and methine protons of the sugar moiety
1
carbons. These signals were assigned by cross-comparison in the H-NMR spectral data indicated that all of the mono-
with 2D-NMR data as in the case of 1, and the data assign- saccharide units were of the pyranose form, and further, the
ments for Agl and the sugar moiety of 3 were quite similar modes of the glycosidic linkages of the two glucopyranosyl
4
to those of 2 and (23R)-17α,23-epoxy-3β,29-dihydroxy-27- units were β in the C conformations, and that of the arabi-
1
4
nor-lanost-8-ene-15,24-dione 3-O-α-l-rhamnopyranosyl-(1→ nopyranosyl unit was α in the C conformation, which was
1
2
6
)-O-β-D-glucopyranosyl-(1→2)-O-α-l-arabinopyranosyl-(1→ different from that of the arabinopyranosyl unit in 1–3. Com-
)-β-D-glucopyranoside, respectively (Tables 1–4). Thus, 3 parison of the data for 4 with the C-NMR spectral data of
11)
13
was determined to be a derivative of 2, in which the apiofura- methyl β-D-glucopyranoside and methyl α-l-arabinopyranoside
12)
13,14)
nosyl unit in 2 was replaced by an α-l-rhamnopyranosyl unit.
from the literature indicated glycosylation shifts
in the
Compound 4, tentatively named scillanostaside K, was ob- data of 4 at C-6 (+6.7ppm) of Glc and C-2 (+8.8ppm) of
tained as an amorphous powder, and afforded l-arabinose and Ara. Furthermore, key correlations were observed between
D-glucose upon acidic hydrolysis. The negative-ion FAB-MS H-1 of Glc and C-3 of Agl, H-1 of Ara and C-6 of Glc, and
−
of 4 was characterized by an [M−H] ion peak at m/z 913 H-1 of Glc' and C-2 of Ara in the HMBC spectrum, as illus-
−
accompanied by fragment ion peaks at m/z 751 [913−162]
trated in Fig. 2. Consequently, the structure of 4 was found to
−
and 619 [751−132] . Based on HR-positive-ion FAB-MS, the be 15-deoxyeucosterol 3-O-β-D-glucopyranosyl-(1→2)-O-α-l-
molecular formula of 4 was deduced to be C H O . The arabinopyranosyl-(1→6)-β-D-glucopyranoside.
H- and C-NMR spectra of 4 were similar to those of 1; the
4
6
74 18
1
13
Compound 5, tentatively named scillanostaside L, was ob-
signals due to Agl of the respective molecules were almost tained as an amorphous powder, and its negative-ion FAB-MS
−
superimposable, with the exception of the disappearance of exhibited an [M−H] ion peak at m/z 971 along with fragment
−
−
the signals due to the apiosyl unit in the case of 4. As in the ion peaks at m/z 911 [971−60 (CH COOH)] , 809 [971−162] ,
3
1
13
−
case of 1, these H- and C-NMR spectral signals were ex- and 677 [809−132] . The molecular formula of 5 was de-
amined in detail, and the structure of 4 was deduced to be a termined to be C H O using HR-positive-ion FAB-MS.
4
8
76 20
prosapogenol of 1 (Fig. 2), in which the apiosyl unit of 1 was Acidic hydrolysis of 5 furnished the same monosaccharides

5
96
Vol. 61, No. 5
13
Table 3.
C-NMR Spectral Data for Aglycone Moiety of 1–5 (in Pyridine-d , 125MHz)
5
1
2
3
4
5
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
8
9
0
1
2
35.7
27.5
89.0
44.4
51.8
18.7
26.9
135.3
134.6
36.8
21.1
25.3
48.9
50.8
32.1
39.7
97.1
19.3
19.5
43.7
17.2
36.9
81.6
212.5
32.3
7.7
35.7
27.5
89.0
44.3
51.8
18.7
26.9
135.2
134.6
36.8
20.9
25.6
48.4
48.0
44.4
80.2
99.2
18.9
19.5
37.1
17.0
37.5
82.1
212.4
32.2
7.7
35.7
27.5
89.0
44.3
51.8
18.7
27.0
135.3
134.7
36.8
20.9
25.6
48.4
48.0
44.4
80.2
99.2
19.0
19.5
37.2
17.0
37.5
82.1
212.4
32.3
7.7
35.9
27.5
88.8
44.4
51.9
18.7
26.9
135.1
134.6
36.8
21.1
25.3
48.9
50.8
32.1
39.8
97.1
19.3
19.6
43.7
17.2
36.9
81.6
212.5
32.3
7.7
35.9
27.6
88.8
44.4
51.9
18.8
27.0
135.1
134.8
36.8
21.1
25.3
49.9
50.5
32.6
40.1
97.5
19.4
19.6
49.5
15.4
82.1
85.0
208.8
33.4
7.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
23.1
63.2
26.4
23.1
63.1
27.3
23.2
63.2
27.3
23.2
63.2
26.4
23.2
63.2
26.5
169.9
20.8
′
′
δ in ppm from TMS.
1
13
as those obtained from 4. The H- and C-NMR spectral data in H O and successively extracted with EtOAc and BuOH. The
2
of 5 were similar to those of 4, except for the appearance of aqueous layer was chromatographed over Diaion HP20 column
signals due to one each of acetoxy group and one oxygenated (Mitsubishi Chemical Industries Co., Ltd., Tokyo, Japan), eluted
methine group and the disappearance of the signals due to with H O, MeOH, and acetone. The MeOH eluate (76.7g) was
2
one methylene group. Furthermore, the NMR spectral data of further subjected to Diaion HP20 column chromatography
the sugar moiety and Agl were quite similar to those of 4 and using gradient mixtures of H O–MeOH (50% MeOH, 60%
2
15)
2
2R-acetoxy-15-deoxyeucosterol glycoside, respectively (Ta- MeOH, 70% MeOH, 80% MeOH, 90% MeOH, 100% MeOH)
bles 3–6). On the basis of these data, 5 was determined to be as eluents to give fractions 1–4. Fraction 2 (31.4g) was chroma-
2R-acetoxy-15-deoxyeucosterol 3-O-β-D-glucopyranosyl-(1→ tographed over silica gel column (Merck, Art. 1.09385; Merck,
2
2)-O-α-l-arabinopyranosyl-(1→6)-β-D-glucopyranoside.
Darmstadt, Germany) using gradient mixtures of CHCl3–
To the best of our knowledge, 1–5 are new compounds; no- MeOH–H O (8:2:0.2, 7:3:0.5, 6:4:1, 0:1:0) as eluents to
2
tably, the conformation of the α-l-arabinopyranosyl unit in 4 give fractions 2.1–2.6. Fraction 2.2 (5.08g) was subjected to
and 5 was different from that in 1–3.
silica gel column chromatography using gradient mixtures
of CHCl –MeOH–H O (10:2:0.1, 8:2:0.2, 7:3:0.5, 6:4:1,
3
2
Experimental
0:1:0) as eluents to afford fractions 2.2.1–2.2.20. Fraction
All instruments and materials used were the same as cited 2.2.7 (155mg) was subjected to Chromatorex ODS column (Fuji
16)
in a previous report unless otherwise specified.
Silysia Chemical, Ltd., Aichi, Japan) chromatography using
Plant Material The bulbs of S. scilloides were cultivated gradient mixtures of H O–MeOH (60% MeOH, 65% MeOH,
2
in Kumamoto prefecture, Japan, and were harvested in August 70% MeOH, 75% MeOH, 80% MeOH, 90% MeOH, 100%
2
005, and identified by one of authors (T. Nohara). A voucher MeOH) as eluents to afford fractions 2.2.7.1–2.2.7.6. Frac-
specimen (SCK2005) has been deposited at the laboratory tions 2.2.7.3 (30mg) and 2.2.7.4 (23mg) were each subjected
of Natural Products Chemistry, School of Agriculture, Tokai to HPLC [Nacalai Tesque, Inc., Kyoto, Japan, Cosmosil 5C18
University.
AR-II, 20mm i.d.×250mm (column 1)] with 80% MeOH as
Extraction and Isolation The crushed fresh bulbs of S. eluent to give 5 (7mg) from fraction 2.2.7.3 and 4 (12mg) from
scilloides (18.5kg) were extracted with MeOH at room tem- fraction 2.2.7.4. Fraction 2.2.8 (848mg) was subjected to Chro-
perature, and the solvent was removed under reduced pressure matorex ODS column chromatography using gradient mixtures
to give a syrup (3521.7g). The MeOH extract was suspended of H O–MeOH (60% MeOH, 65% MeOH, 70% MeOH, 75%
2

May 2013
Table 4.
597
13
C-NMR Spectral Data for Sugar Moiety of 1–5 (in Pyridine-d , 125MHz)
5
1
2
3
4
5
Glc-1
106.1
75.3
78.3
72.4
75.6
68.8
101.4
79.8
72.1
67.0
63.3
103.6
79.6
78.4
71.2
78.1
62.0
111.1
77.9
80.3
75.3
65.9
106.1
75.3
78.3
72.4
75.6
68.8
101.4
79.6
72.1
66.9
63.3
103.6
79.8
78.4
71.2
78.1
62.0
111.1
77.9
80.3
75.3
65.9
106.1
75.4
78.3
72.7
75.4
68.6
100.9
78.2
71.5
66.4
62.2
103.1
77.7
79.3
71.4
78.2
62.1
106.0
75.5
78.4
72.0
76.1
69.2
102.3
81.0
73.1
67.8
65.0
105.9
75.9
78.3
71.2
78.7
62.3
106.0
75.5
78.4
72.0
76.1
69.2
102.3
81.0
73.1
67.8
65.0
105.9
75.9
78.3
71.2
78.7
62.3
2
3
4
5
6
Ara-1
2
3
4
5
Glc′-1
2
3
4
5
6
Api-1
2
3
4
5
Rha-1
101.9
72.3
72.6
74.2
69.7
18.7
2
3
4
5
6
δ in ppm from TMS. Glc, glucopyranosyl; Ara, arabinopyranosyl; Api, apiofuranosyl; Rha, rhamnopyranosyl.
−
−
1
7
51 [913−162] , 619 [751−132] . H-NMR spectral data: see
13
Tables 1 and 2. C-NMR spectral data: see Tables 3 and 4.
2
5
2
: Amorphous powder. [α]D −37.4° (c=2.0, pyridine).
Positive-ion FAB-MS m/z: 1085 [M+Na] . HR-positive-ion
FAB-MS m/z: 1085.5139 (Calcd for C H O Na: 1085.5144).
+
51
82 23
−
−
Negative-ion FAB-MS m/z: 1061 [M−H] , 929 [1061−132] ,
−
−
1
7
67 [929−162] , 635 [767−132] . H-NMR spectral data: see
13
Tables 1 and 2. C-NMR spectral data: see Tables 3 and 4.
2
5
3
: Amorphous powder. [α]D −28.2° (c=1.2, pyridine).
Positive-ion FAB-MS m/z: 1099 [M+Na] . HR-positive-ion
FAB-MS m/z: 1099.5286 (Calcd for C H O Na: 1099.5301).
+
52
84 23
−
−
Negative-ion FAB-MS m/z: 1075 [M−H] , 929 [1075−146] ,
−
−
1
7
67 [929−162] , 635 [767−132] . H-NMR spectral data: see
Fig. 3. Key NOE Correlations Observed in the NOESY Spectrum of 2
in Pyridine-d , 500MHz)
13
Tables 1 and 2. C-NMR spectral data: see Tables 3 and 4.
: Amorphous powder. [α]D −21.9° (c=2.4, pyridine).
Positive-ion FAB-MS m/z: 937 [M+Na] . Negative-ion FAB-
(
5
24
4
+
−
−
−
MeOH, 80% MeOH, 85% MeOH, 90% MeOH, 100% MeOH) MS m/z: 913 [M−H] , 751 [913−162] , 619 [751−132] . HR-
as eluents to give fractions 2.2.8.1–2.2.8.11. HPLC (column 1) negative-ion FAB-MS m/z: 913.4786 (Calcd for C H O :
of fraction 2.2.8.8 (81mg) with 75% MeOH as eluent furnished 913.4797). H-NMR spectral data: see Tables 5 and 6.
C-NMR spectral data: see Tables 3 and 4.
5: Amorphous powder. [α]D −21.5° (c=1.2, pyridine).
of H O–MeOH (60% MeOH, 65% MeOH, 70% MeOH, 75% Positive-ion FAB-MS m/z: 995 [M+Na] . HR-positive-ion
MeOH, 80% MeOH, 85% MeOH, 90% MeOH, 100% MeOH) FAB-MS m/z: 995.4838 (Calcd for C H O Na: 995.4828).
4
6
73 18
1
13
1
(37mg). Fraction 2.2.9 (2368mg) was subjected to Chroma-
2
4
torex ODS column chromatography using gradient mixtures
+
2
4
8
76 20
−
−
as eluents to furnish fractions 2.2.9.1–2.2.9.10. Fraction 2.2.9.5 Negative-ion FAB-MS m/z: 971 [M−H] , 911 [971−60] ,
−
−
1
(
106mg) was subjected to HPLC (column 1) with 65% MeOH 809 [971−162] , 677 [809−132] . H-NMR spectral data: see
13
as eluent to afford 2 (70mg) and 3 (14mg).
Tables 5 and 6. C-NMR spectral data: see Tables 3 and 4.
Sugar Analysis Compounds 1 (5mg), 2 (5mg), 3 (5mg),
Positive-ion FAB-MS m/z: 1069 [M+Na] . HR-positive-ion 4 (5mg), and 5 (5mg) were each heated in 2m HCl (1mL)
FAB-MS m/z: 1069.5203 (Calcd for C H O Na: 1069.5196). at a temperature of 95°C for 2h. The reaction mixture was
Negative-ion FAB-MS m/z: 1045 [M−H] , 913 [1045−132] , extracted with EtOAc (1mL). The aqueous layer was neutral-
2
5
1
: Amorphous powder. [α]D −55.7° (c=1.6, pyridine).
+
51
82 22
−
−

5
98
Vol. 61, No. 5
1
1
Table 5. H-NMR Spectral Data for Aglycone Moiety of 4 and 5 (in Table 6. H-NMR Spectral Data for Sugar Moiety of 4 and 5 (in Pyri-
Pyridine-d , 500MHz)
dine-d , 500MHz)
5
5
4
5
4
5
a)
1
1
2
2
3
5
6
6
7
7
1a
1b
2a
2b
5a
5b
6a
6b
8
9
0
1
2a
2b
3
5a
5b
6
a
b
a
b
1.70
1.71 ddd (4.5, 4.5, 13.0)
1.24 m
Glc-1
5.05 d (8.0)
5.06 d (8.0)
1.22 m
2
3
4
5
4.02 dd (8.0, 9.0)
4.22 dd (9.0, 9.0)
4.26 dd (9.0, 9.0)
4.02 dd (8.0, 9.0)
4.22 dd (9.0, 9.0)
4.26 dd (9.0, 9.0)
2.30 dddd (4.5, 4.5, 4.5, 13.5)
2.32 dddd (4.5, 4.5, 4.5, 14.5)
a)
a)
2.03
2.04
a)
a)
3.70 dd (4.5, 11.5)
1.31 brd (12.5)
1.82 brdd (6.0, 13.5)
1.51 m
3.72 dd (4.5, 11.5)
1.33 dd (1.0, 11.5)
1.83 brdd (6.0, 13.5)
4.15
4.15
6a
6b
Ara-1
4.64 dd (3.0, 12.5)
4.65 dd (2.5, 10.5)
a)
a)
a
b
a
b
4.30
4.30
a)
1.50
5.12 d (5.5)
5.13 d (5.5)
a)
2.05
1.99
2.18
1.96
2.10 m
2
3
4
5a
5b
4.59 dd (5.5, 7.5)
4.36 dd (3.0, 7.5)
4.40 ddd (3.0, 3.0, 4.5)
4.59 dd (5.5, 7.5)
4.37 dd (2.5, 7.5)
4.40 ddd (2.5, 2.5, 4.5)
a)
a)
2.04
a)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
2.19 ddd (1.0, 8.5, 16.5)
a)
a)
a)
a)
1.98
2.38
4.32
4.31
a)
2.42 ddd (9.0, 9.0, 13.0)
1.44 dd (9.0, 13.0)
3.76 dd (2.5, 12.5)
5.11 d (8.0)
3.76 dd (2.5, 12.0)
5.11 d (8.0)
1.44 dd (9.5, 11.5)
1.76 m
Glc′-1
a)
1.68
2
3
4
5
6a
6b
4.03 dd (8.0, 9.0)
4.13 dd (9.0, 9.0)
4.18 dd (9.0, 9.0)
3.81 ddd (2.5, 5.0, 9.0)
4.48 dd (2.5, 11.5)
4.04 dd (8.0, 9.0)
4.13 dd (9.0, 9.0)
4.18 dd (9.0, 9.0)
3.81 ddd (2.5, 4.5, 9.0)
4.49 dd (2.5, 11.5)
a)
1.39 dd (10.0, 10.0)
1.51
a)
2.17
1.64
2.62 m
a)
a)
2.01
0.91 s
0.95 s
0.91 s
0.96 s
2.41 q (7.0)
1.09 d (7.0)
5.43 d (5.5)
a)
a)
4.32
4.32
a)
2.05
δ in ppm from TMS (coupling constants (J) in Hz are given in parentheses). Glc,
glucopyranosyl; Ara, arabinopyranosyl. a) Signals were overlapped with other signals.
1.02 d (7.0)
a)
2.00
1.78 dd (7.5, 12.0)
4.62 dd (7.5, 10.5)
FAB-MS.
4.96 d (5.5)
a)
a)
2.57
2.53
2.57
2.56
References
a)
a)
1) “Chuyaku Daijiten,” ed. by Koso Shin Igakuin, Shanghai Kagaku
Gijutsu Shuppansha, 1978, p. 2270.
1.07 dd (7.0, 7.0)
1.58 s
4.46 d (11.0)
3.66 brd (11.0)
1.53 s
1.10 dd (7.0, 7.0)
1.60 s
4.47 d (11.5)
3.67 brd (11.5)
1.59 s
8
2) Kouno I., Komori T., Kawasaki T., Tetrahedron Lett., 14, 4569–
4572 (1973).
3) Kouno I., Noda N., Ida I., Sholichin M., Miyahara K., Komori T.,
Kawasaki T., Liebigs Ann. Chem., 1982, 306–314 (1982).
4) Sholichin M., Miyahara K., Kawasaki T., Heterocycles, 17, 251–257
9a
9b
0
2
′
2.00 s
(
1982).
5) Sholichin M., Miyahara K., Kawasaki T., Chem. Pharm. Bull., 33,
756–1759 (1985).
δ in ppm from TMS (coupling constants (J) in Hz are given in parentheses). a)
Signals were overlapped with other signals.
1
ized with Amberlite MB-3 column (Organo Co., Tokyo, Japan,
6) Lee S.-M., Chun H.-K., Lee C.-H., Min B.-S., Lee E.-S., Kho Y.-H.,
1
3mm i.d.×230mm) and then evaporated under reduced pres-
Chem. Pharm. Bull., 50, 1245–1249 (2002).
Nishida Y., Eto M., Miyashita H., Ikeda T., Yamaguchi K., Yoshi-
mitsu H., Nohara T., Ono M., Chem. Pharm. Bull., 56, 1022–1025
7
)
sure to give a monosaccharide fraction. This fraction was
analyzed by HPLC under the following conditions: column,
Shodex RS-Pac DC-613 (Showa Denko, Tokyo, Japan, 6.0mm
i.d.×150mm; solvent, CH CN–H O (4:1); flow rate, 1.0mL/
min; column temperature, 70°C; detector, JASCO OR-2090
plus (JASCO Co., Tokyo, Japan); pump, JASCO PU-2080; and
(2008).
8)
Ono M., Toyohisa D., Morishita T., Horita H., Yasuda S., Nishida
Y., Tanaka T., Okawa M., Kinjo J., Yoshimitsu H., Nohara T.,
Chem. Pharm. Bull., 59, 1348–1354 (2011).
Ono M., Takatsu Y., Ochiai T., Yasuda S., Nishida Y., Tanaka T.,
Okawa M., Kinjo J., Yoshimitsu H., Nohara T., Chem. Pharm. Bull.,
3
2
9)
column oven, JASCO CO-2060. The retention time (t ) and
R
optical activity of each of the monosaccharides were detected
6
0, 1314–1319 (2012).
as follows. D-apiose [tR 5.7min; optical activity, positive], l- 10) Ono M., Sugita F., Shigematsu S., Takamura C., Yoshimitsu H., Mi-
arabinose [t , 8.1min; optical activity, positive], and D-glucose
yashita H., Ikeda T., Nohara T., Chem. Pharm. Bull., 55, 1093–1096
R
[
tR, 11.8min; optical activity, positive] for 1 and 2: l-rhamnose
(2007).
1
1) Kuroda M., Mimaki Y., Ori K., Sakagami H., Sashida Y., J. Nat.
[t , 4.8min; optical activity, negative], l-arabinose [t , 8.1min;
R
R
Prod., 67, 2099–2103 (2004).
optical activity, positive], and D-glucose [t , 11.8min; optical
activity, positive] for 3; l-arabinose [t , 8.1min; optical activ-
ity, positive] and D-glucose [t , 11.8min; optical activity, posi-
tive] for 4 and 5. D-Apiose was prepared by the acidic hydro-
lysis of benzyl β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside
R
1
2) Seo S., Tomita Y., Tori K., Yoshimura Y., J. Am. Chem. Soc., 100,
331–3339 (1978).
3) Kasai R., Suzuo M., Asakawa J., Tanaka O., Tetrahedron Lett., 18,
75–178 (1977).
4) Tori K., Seo S., Yoshimura Y., Arita H., Tomita Y., Tetrahedron
Lett., 18, 179–182 (1977).
R
3
R
1
1
1
10)
(icariside F2). However, the EtOAc extract exhibited several
spots by TLC, and the aglycones of 1–5 could not be obtained. 15) Amschler G., Frahm A. W., Müller-Doblies D., Müller-Doblies U.,
Phytochemistry, 47, 429–436 (1998).
Acknowledgment We express our appreciation to Mr. H. 16) Takigawa A., Muto H., Kabata K., Okawa M., Kinjo J., Yoshimitsu
H., Nohara T., Ono M., J. Nat. Prod., 74, 2414–2419 (2011).
Harazono of Fukuoka University for the measurement of the