Tareciliosides A-G: Cycloartane glycosides from leaves of Tarenna gracilipes (HAY.) OHWI

Article abstract of DOI:10.1248/cpb.56.1153

From the 1-BuOH-soluble fraction of a MeOH extract of leaves of Tarenna gracilipes, collected in Okinawa, seven new cycloartane glycosides, named tareciliosides A-G (4-10), were isolated together with three known compounds, D-mannitol (1), (R)-linalool 6-O-α-L-arabinopyranosyl-β-D- glucopyranoside (2), and mussaenoside (3). Their structures were elucidated through a combination of spectroscopic analyses.

Full text of DOI:10.1248/cpb.56.1153

August 2008
Chem. Pharm. Bull. 56(8) 1153—1158 (2008)
1153
Tareciliosides A—G: Cycloartane Glycosides from Leaves of Tarenna
gracilipes (HAY.) OHWI
a
a
,a
b
Zhimin ZHAO, Katsuyoshi MATSUNAMI, Hideaki OTSUKA,* Takakazu SHINZATO, and
c
Yoshio TAKEDA
a
Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-
b
ku, Hiroshima 734–8553, Japan: Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1
Senbaru, Nishihara-cho, Nakagami-gun, Okinawa 903–0213, Japan: and Faculty of Integrated Arts and Sciences, The
c
University of Tokushima; 1–1 Minamijosanjima-cho, Tokushima 770–8502, Japan.
Received April 3, 2008; accepted May 20, 2008; published online May 21, 2008
From the 1-BuOH-soluble fraction of a MeOH extract of leaves of Tarenna gracilipes, collected in Okinawa,
seven new cycloartane glycosides, named tareciliosides A—G (4—10), were isolated together with three known
compounds, D-mannitol (1), (R)-linalool 6-O-a-L-arabinopyranosyl-b-D-glucopyranoside (2), and mussaenoside
(3). Their structures were elucidated through a combination of spectroscopic analyses.
Key words Tarenna gracilipes; Rubiaceae; tarecilioside; cycloartane glycoside
Rubiaceous plants are trees, shrubs, or infrequently herbs H O mixtures (1 : 4, 2 : 3, 3 : 2, 4 : 1, each 6 l), and 100%
2
comprising about 600 genera and more than 10000 species, MeOH (6 l) to give seven fractions. Then, fractions 2 and 5
including some lianous forms. Many plants belonging to were further subjected to silica gel column chromatography
the Rubiaceous family have been used as drugs in tradi- separately, using a CHCl –MeOH–H O solvent system with
3
2
tional medicine, such as Cinchona succirubra, Cephaelis increasing polarity. The fractions eluted from these columns
ipecacuanha and so on. The biological and chemical con- were combined and re-subjected to octadecylsilanized silica
stituents of these plants have been isolated and identified. gel column chromatography (ODS CC), droplet counter-cur-
However, there are still numerous plants of which the biolog- rent chromatography (DCCC), and HPLC, successively, to
ical and chemical constituents are unknown. In a search for afford ten compounds (1—10).
biological and chemical constituents, we conducted a study
on the plant Tarenna gracilipes (HAY.) OHWI.
T. gracilipes is distributed from the Kyushu district of ranosyl-b-D-glucopyranoside and mussaenoside, respec-
Japan to Taiwan, with the following botanical characters: it is tively, by comparison with the data reported in the literature.
Compounds 1, 2 and 3 were found to be known, and were
2)
identified as D-mannitol, (R)-linalool 6-O-a-L-arabinopy-
3) 4)
an evergreen shrub with slightly pubescent sprigs, and itsel-
liptical-acute leaves are opposite, glossy, usually 6—18 cm in phous powder and its molecular formula was determined to
length, and 3—8 cm in width. In summer, the white flowers be C H O on negative-ion HR-FAB-MS. The absorption
are borne in corymbs at the tips of the branches. Each flower band at 3370 cm in the IR spectrum indicated the presence
has a 5-toothed, bell-shaped calyx and a tubular, 4 or 5 of hydroxyl groups. The H-NMR spectrum of 4 (Table 1)
Tarecilioside A (4), [a] ꢀ14.5°, was isolated as an amor-
D
3
6
62 10
ꢁ1
1
petaled corolla. The petals are oval and elliptical, and longer showed highly shielded signals characteristic of methylene
than the tube. They twist in bud. The fruits are black and glo-
1)
bose drupes.
Isolation work on the 1-BuOH-soluble fraction of a MeOH
extract of leaves of T. gracipes afforded seven new cycloar-
tane glycoside, named tareciliosides A—G (4—10), together
2)
with three known compounds, D-mannitol (1), (R)-linalool
3
)
6
-O-a-L-arabinopyranosyl-b-D-glucopyranoside (2),
and
4)
mussaenoside (3). The structures of the new compounds
were elucidated from spectroscopic evidence and those of
known compounds were determined by comparison of the
physical data with those reported.
Results and Discussion
Air-dried leaves (9.90 kg) of Tarenna gracilipes were ex-
tracted at room temperature with methanol and then concen-
trated in vacuo. The concentrated methanol extract was ex-
tracted with n-hexane and then the MeOH fraction was con-
centrated. The concentrated MeOH layer was suspended in
H O, and then extracted with EtOAc and 1-BuOH, succes-
2
sively.
The 1-BuOH-soluble extract (149 g) was subjected to a
column of Diaion HP-20, using stepwise-gradient MeOH– Fig. 1. Structures
∗
To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp
© 2008 Pharmaceutical Society of Japan

1
154
Vol. 56, No. 8
1
Table 1. H-NMR Data for Tareciliosides A—G (4—10) (400 MHz, Pyridine-d5)
H
4
5
6
7
8
9
10
1
1.17 m
.50 m
1.85 m
.38 m
3.51 dd 12, 4
1.57 dd 13, 3
1.22 m
1.20 m
1.52 m
1.87 m
2.44 m
3.53 dd 12, 4
1.57 dd 12, 3
1.22 m
2.03 m
3.80 ddd 9, 9, 4
2.04 d 9
1.25 m
1.89 m
1.67 m
1.83 m
2.32 dd 14, 5
2.74 dd 14, 8
4.76 ddd 8, 8, 5
1.82 dd 11, 8
1.48 s
1.20 m
1.50 m
1.86 m
2.40 m
3.52 dd 12, 4
1.55 dd 13, 4
1.21 m
1.10 m
1.47 m
1.83 m
2.37 m
3.51 dd 12, 4
1.61 m
1.10 m
1.23 m
1.58 m
1.86 m
2.43 m
3.54 dd 12, 4
1.61 m
1.23 m
1.20 m
1.50 m
1.83 m
2.40 m
3.53 dd 11, 4
1.50 m
1.20 m
2.02 m
3.80 m
2.04 d 9
1.20 m
1.86 m
1.66 m
1.85 m
2.32 dd 14, 4
2.74 dd 14, 8
4.74 m
1.76 dd 11, 8
1.44 s
1.24 m
1
1.53 m
1.86 m
2.35 m
3.45 dd 12, 4
1.52 m
1.24 m
2.02 m
3.80 m
2.04 d 9
1.22 m
1.85 m
1.63 m
2
2
3
5
6
2
.00 m
2.02 m
1.98 m
2.02 m
7
8
1
3.79 ddd 10, 10, 4
2.02 d 9
1.21 m
3.78 ddd 10, 10, 4 3.79 br dd 10, 10
2.03 d 10
1.22 m
1.87 m
1.66 m
3.80 br dd 9, 9
2.04 d 9
1.24 m
1.86 m
1.66 m
2.31 d 10
1.59 m
2.02 m
1.82 2H, m
1.82 m
2.72 2H m
2.74 dd 13, 8
4.70 ddd 7, 7, 5
1.95 dd 10, 8
3.88 d 11
1
1
1
1
.85 m
1.66 m
.83 m
2.31 dd 14, 5
.73 dd 14, 8
2
5
1
1.80 m
1.83 m
2.29 dd 14, 5
2.71 dd 14, 8
4.71 ddd 8, 8, 5
1.77 dd 11, 8
1.44 s
2.32 dd 13, 4
2.73 dd 14, 8
4.74 m
1.80 dd 10, 7
1.48 s
2.32 dd 14, 5
2
1
1
1
6
7
8
4.75 ddd 8, 8, 5
1.80 dd 11, 8
1.46 s
4.76 m
1.75 m
1.45 s
4
.57 d 11
1
9
0.22 d 4
0.22 d 4
0.75 d 4
2.40 m
1.13 d 7
1.43 m
2.50 m
1.89 m
2.42 m
4.18 dd 10, 2
4.08 d 10
0.22 d 4
0.75 d 4
2.30 m
1.10 d 5
1.34 m
2.46 m
1.68 m
1.96 m
3.90 m
1.49 s
0.21 d 4
0.64 d 4
2.31 m
1.14 d 7
1.44 m
2.60 m
1.67 m
2.00 m
3.88 br d 11
1.52 s
0.23 d 4
0.76 d 4
2.41 m
1.12 d 6
1.33 m
2.50 m
1.66 m
2.00 m
3.88 br d 10
1.51 s
0.22 d 4
0.75 d 4
2.20 m
1.04 d 6
1.53 m
2.42 m
3.16 ddd 18, 8, 3
3.30 ddd 18, 8, 8
0.20 d 4
0.74 d 4
2.20 m
1.05 d 5
1.52 m
2.40 m
3.11 ddd 18, 8, 5
3.18 ddd 18, 8, 8
0
.74 d 4
2
2
2
0
1
2
2.34 m
1.12 d 6
1.40 m
2
.48 m
1.72 m
.15 m
2
3
2
2
2
4
6
3.78 dd 10, 1
1.47 s
1.55 s
1.51 s
4
.25 d 10
2
2
2
3
1
2
3
4
5
6
7
8
9
0
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
1.49 s
1.05 s
1.32 s
1.12 s
4.91 d 8
4.00 dd 8, 9
4.20 dd 9, 9
4.16 dd 9, 9
3.93 m
1.61 s
1.07 s
1.34 s
1.12 s
1.50 s
1.06 s
1.33 s
1.11 s
1.51 s
0.98 s
1.31 s
1.20 s
1.50 s
1.07 s
1.34 s
1.13 s
1.58 s
1.07 s
1.34 s
1.12 s
4.93 d 8
4.06 dd 8, 8
4.21 dd 8, 8
4.20 dd 8, 8
3.83 m
1.52 s
1.17 s
1.34 s
1.11 s
4.92 d 8
4.20 dd 8, 8
4.29 dd 8, 8
4.25 dd 8, 8
3.86 m
a)
4.90 d 8
4.92 d 8
4.00 dd 8, 8
4.18 dd 8, 8
4.14 dd 8, 8
3.92 m
4.36 dd, 12, 5
4.53 dd, 12, 2
5.15 d 8
3.95 dd 8, 8
4.22 m
4.82 d 8
4.94
4.04 dd 8, 8
4.21 dd 8, 8
4.20 dd 8, 8
3.97 m
4.40 dd 12, 5
4.56 dd 12, 2
4.00 dd 8, 8
4.21 dd 8, 8
4.18 dd 8, 8
3.95 m
4.36 dd 11, 5
4.53 dd 11, 2
5.05 d 8
3.93 dd 8, 8
4.12 m
4.13 m
4.04 dd 8, 8
4.25 dd 8, 8
4.20 dd 8, 8
3.98 m
4.38 dd 11, 5
4.56 br d 11
5.05 d 8
3.96 dd 8, 8
4.13 m
4.17 m
4.35 dd 12, 5
4.28 m
4.27 m
4
.52 dd 12, 2
4.42 dd 12, 2
4.99 d 8
4.10 dd 8, 8
4.24 m
4.22 m
3.69 m
4.52 dd 12, 2
5.36 d 8
4.08 dd 8, 8
4.23 m
4.15 m
3.89 m
1
2
3
4
5
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
4.20 m
3.94 m
3.70 dd 10, 10
3.70 dd 10, 10
4.27 dd 10, 5
4
.23 dd 10, 5
6
ꢄ
4.22 m
4.38 dd 12, 5
4.56 dd 12, 2
4.21 m
4.40 dd 12, 3
4
.47 dd 12, 2
Assignments were performed by means of COSY, HSQC and HMBC experiments. s: singlet, d: doublet, m: multiplet or overlapped. a) Position was assigned from the
HSQC spectrum, but was overlapped by the H O signal.
2
5
—10)
protons of a cyclopropane ring as an AX system (d 0.22, side.
The assignments of the proton and carbon signals,
H
0
.74, each d, Jꢂ4 Hz, H -19), and signals due to six tertiary and the positions of the hydroxyl groups of 4 were estab-
methyls (d 1.05, 1.12, 1.32, 1.46, 1.47, 1.49) and a second- lished by analyses of the H– H chemical shift correlation
ary methyl (d 1.12, d, Jꢂ6 Hz) group. The C-NMR spec- (COSY), heteronuclear single quantum coherence (HSQC),
2
1
1
H
1
3
H
trum of tarecilioside A (4) exhibited signals of 36 carbons, of and heteronuclear multiple bond connectivity (HMBC) spec-
which 30 accounted for the aglycone moiety. The remaining tra (Fig. 2). In the HMBC spectrum, the correlation between
signals were in good accordance with the presence of a glu- H -28 and 29 (d 1.05, 1.32, respectively) and d 88.6
3
H
C
cose unit (Table 2). The resonances assigned to the aglycone placed one of the hydroxyl groups at the 3-position, and the
moiety consisted of seven methyls, nine methylenes, eight position of the second hydroxyl was determined to be at the
methines and six quaternary carbons. The signals at d 70.3, 7-position from the coupling pattern of the H-5 (d 1.57, dd,
C
H
7
2.2, 72.8, 80.5 and 88.6 indicated the presence of carbons Jꢂ13, 3 Hz) and H-8 (d 2.02, d, Jꢂ9 Hz). The hydroxyl
H
each bearing a hydroxyl group. Thus, tarecilioside A (4) was group at the 16-position was proved by the HMBC correla-
considered to be a cycloartane-type triterpene monoglyco- tions of H-8 with C-15 (d 50.7) and then H-15 (dH 2.31)
C

August 2008
1155
13
Table 2.
C-NMR Data for Tareciliosides A—G (4—10) (100 MHz, Pyri-
dine-d5)
C
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
31.8 t
29.8 t
88.6 d
40.9 s
46.4 d
31.9 t
70.3 d
55.3 d
20.3 s
26.9 s
26.8 t
33.2 t
46.1 s
47.1 s
50.7 t
72.2 d
56.8 d
18.8 q
28.6 t
31.6 d
18.9 q
35.0 t
29.4 t
80.5 d
72.8 s
26.1 q
25.8 q
15.3 q
25.8 q
20.0 q
31.8 t
29.8 t
88.6 d
41.0 s
46.4 d
32.0 t
70.3 d
55.3 d
20.3 s
26.9 s
26.8 t
33.2 t
46.2 s
47.1 s
50.8 t
72.2 d
56.8 d
18.8 q
28.6 t
31.6 d
18.9 q
35.0 t
29.1 t
77.9 d
74.8 s
69.3 t
20.0 q
15.4 q
25.9 q
20.2 q
31.9 t
29.7 t
88.6 d
40.9 s
46.4 d
31.8 t
70.3 d
55.1 d
20.3 s
26.9 s
26.8 t
33.1 t
46.1 s
47.0 s
50.6 t
72.1 d
56.9 d
18.7 q
28.5 t
31.5 d
18.9 q
35.1 t
29.3 t
79.0 d
80.9 s
21.5 q
24.2 q
15.3 q
25.8 q
20.0 q
32.3 t
29.8 t 29.8 t
31.9 t
31.8 t
29.8 t
88.6 d
41.0 s
46.4 d
32.0 t
70.3 d
55.2 d
20.3 s
26.9 s
26.8 t
33.2 t
46.2 s
47.0 s
50.5 t
71.9 d
56.9 d
18.8 q
28.6 t
30.5 d
18.4 q
30.9 t
34.4 t
31.8 t
29.8 t
88.7 d
41.0 s
46.4 d
31.9 t
70.3 d
55.1 d
20.2 s
26.9 s
26.8 t
33.2 t
46.2 s
47.1 s
50.3 t
72.1 d
56.8 d
18.7 q
28.5 t
30.5 d
18.3 q
30.8 t
34.1 t
a
88.5 d
41.0 s
47.0 d
32.0 t
70.6 d
55.3 d
20.3 s
27.1 s
26.7 t
30.1 t
47.2 s
51.4 s
52.1 t
72.6 d
55.5 d
88.6 d
41.0 s
46.4 d
31.8 t
70.3 d
55.2 d
20.3 s
26.9 s
26.8 t
33.2 t
46.2 s
47.1 s
50.8 t
72.1 d
56.9 d
18.9 q
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
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
1
2
3
4
5
6
Fig. 2. HMBC and COSY Correlations of Tarecilioside A (4)
65.0 t
a
29.9 t 28.5 t
31.6 d
19.1 q
34.4 t
29.1 t
78.9 d
81.0 s
21.4 q
24.0 q
15.4 q
25.8 q
22.7 q
31.6 d
18.8 q
35.0 t
29.3 t
Fig. 3. Phase-Sensitive ROESY Correlations of Tarecilioside A (4)
79.1 d 215.5 s 218.8 s
81.0 s
21.2 q
24.2 q
15.3 q
25.8 q
20.0 q
82.9 s
23.8 q
24.7 q
15.4 q
25.8 q
20.0 q
76.9 s
27.2 q
27.2 q
15.3 q
25.8 q
20.0 q
group at C-7. In the case of H-16 (dH 4.75), the large cou-
pling constant of H-16 with H-15ax-like (d 2.73, d,
H
Jꢂ8 Hz) and the small coupling constant of H-16 with H-
15eq-like (Jꢂ5 Hz), and also the large coupling constant
(Jꢂ11 Hz) with H-17 indicated the b configuration for the
hydroxyl group at this position. These observations were fur-
ther confirmed by the correlations observed in the PS-
ROESY experiment, in which H -30 (d 1.12) showed corre-
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
106.7 d 106.8 d 106.7 d 106.7 d 106.8 d 106.8 d 104.8 d
75.7 d
78.7 d
71.8 d
78.1 d
63.0 t
75.8 d
78.8 d
71.9 d
78.2 d
63.1 t
75.7 d
75.7 d
75.8 d
75.8 d
83.3 d
a
a
a
78.6 d 78.7 d 78.8 d
78.8 d 78.4 d
b
3
H
71.8 d 71.9 d
71.9 d
78.2 d
63.1 t
99.4 d
75.2 d
71.8 d
78.2 d
63.1 t
71.8 d
78.0 d
lations with H-7 (d 3.79) and H-16 (d 4.75) (Fig. 3). The
78.1 d
63.0 t
98.6 d
78.2 d
63.0 t
99.4 d
H
H
a
62.9 t
configuration of the C-24 hydroxyl group was indicated to be
4R on comparison with the chemical shift values of cy-
99.6 d 105.9 d
75.3 d 77.0 d
78.8 d 78.2 d
2
75.3 d
75.2 d
6)
clounifolioside C (24R, C-24: d 80.3) and cyclocantogenin
a
a
a
C
78.7 d 78.6 d 78.6 d
6)
b
(24S, C-24: d 77.0). On acid hydrolysis of 4, an aglycone
71.8 d 71.1 d
71.1 d
67.0 t
72.1 d
78.2 d
62.8 t
71.6 d
78.0 d
C
(4a) and D-glucose were isolated. Additionally, the resonance
78.1 d
62.8 t
67.0 t
a
62.8 t
for one anomeric proton (H-1ꢃ) was observed at d 4.91 (d,
H
Jꢂ8 Hz), which indicated the presence of one sugar moiety,
and the mode of linkage is b and the position of the sugar
linkage was confirmed to be to the hydroxy group at C-3 by
q: CH , t: CH , d: CH, s: C. The same superscripts may be interchangeable in each
column.
3
2
with C-16 (d 72.2). The remaining two hydroxyl groups the HMBC correlation between H-1ꢃ and C-3 (d 88.6).
C
C
were placed at the 24 and 25-positions based on the HMBC Therefore, the structure of tarecilioside A (4) was elucidated
correlations for H -26 and 27 (d 1.47, 1.49, respectively) to be cycolartane-3b,7b,16b,24R,25-pentaol 3-O-b-D-glu-
3
H
with C-24 (d 80.5) and 25 (d 72.8). Other HMBC correla- copyranoside.
C
C
tions were also supportive of the positions of hydroxyl
groups on the cycloartane skeleton (Fig. 2). The long-range phous powder and its molecular formula was determined to
correlation observed between the anomeric proton of the glu- be C H O on negative-ion HR-FAB-MS. All the spectral
Tarecilioside B (5), [a] ꢀ10.8°, was isolated as an amor-
D
3
6
62 11
cose unit (d 4.91) and C-3 (d 88.6) of the aglycone moiety data for 5 were the same as those for 4, except for the pres-
H
C
confirmed the position of the sugar linkage. The stereochem- ence of one more hydroxyl functional group than observed in
istry of 4 was determined by analysis of the phase-sensitive 4. In the NMR spectra of 5, only five tertiary methyl carbons
rotating frame nuclear Overhauser and exchange spec- were observed, with a new primary alcohol signal [d 69.2
C
1
1
troscopy (PS-ROESY) spectra, and the H– H coupling con- with d 4.08 (d, Jꢂ10 Hz) and 4.25 (d, Jꢂ10 Hz)]. Thus, one
stants (Fig. 3, Table 1). The orientation of the hydroxyl group of the methyl groups was expected to be modified to a pri-
at C-3 was concluded to be b on the basis of the coupling mary alcohol. Since the C-NMR data for the ring portion,
constant of H-3, since the H-3 (d 3.51) signal was observed and C-20 and 21 of 5 were essentially the same as those for 4
H
1
3
H
as a doublet of doublets due to 1,2-diaxial (Jꢂ12 Hz) and (Table 2), one of the geminal methyl groups at C-25 must be
axial-equatorial coupling (Jꢂ4 Hz) with H-2ax and H-2eq, oxidized to a carbinol. This was further supported by the
respectively. The 1,2-diaxial coupling of H-8 (dH 2.02, d, HMBC correlation cross peak between H-24 (dH 4.18) and
Jꢂ9 Hz) suggested the b configuration for the hydroxyl the carbinol carbon signal. The absolute configuration of the

1
156
Vol. 56, No. 8
newly formed chiral center remains to be determined. There-
fore, the structure of tarecilioside B (5) was tentatively eluci-
dated to be cycolartane-3b,7b,16b,24R,25x,26-hexaol 3-O-
b-D-glucopyranoside.
Tarecilioside C (6), [a] ꢀ13.1°, was isolated as an amor-
D
phous powder and its molecular formula was determined to
be C H O on positive-ion HR-ESI-TOF-MS. From the
4
2
72 15
various spectral data, 6 was also expected to be a derivative
of a cycloartane glucoside and in the NMR spectra, two
anomeric carbon (d 106.7, 98.6) and proton (d 4.92, 5.15,
C
H
respectively) signals of glucoses were observed. Because the
1
3
C-NMR chemical shifts of the ring nucleus, and C-20 to 22
for 6 were not distinguishable from those of 4 and the rela-
tively shielded chemical shift of the anomeric carbon (d
Fig. 4. HMBC Correlations of Tarecilioside D (7)
C
9
8.6), a new glucoside bond was formed through the tertiary
hydroxyl group at C-25. This was further confirmed by the be C H O on negative-ion HR-FAB-MS. Tarecilioside F
4
2
70 15
HMBC correlation cross peak between d 5.15 on C-1ꢄ (d
(9) was also a cycloartane 3,25-O-diglucoside analogous to
H
C
9
8.6) and C-25 (d 80.9), and a significant downfield shift 6. A highly deshielded signal at d 215.5 was expected to
C
C
was observed from C-25 of 4 to that of 6 (d 72.8→d 80.9). represent an isolated ketone functional group, and its loca-
C
C
On hydrolysis of 5, only D-glucose was detected. Therefore, tion was determined to be at C-24 judging from the cross
the structure of 6 was elucidated to be cycolartane- peaks in the HMBC and PS-ROESY spectra. Therefore, the
3
b,7b,16b,24R,25-pentaol 3,25-di-O-b-D-glucopyranoside.
structure of 9 was elucidated to be 24-oxo-tarecilioside C, as
Tarecilioside D (7), [a] ꢀ17.7°, was isolated as an amor- shown in Fig. 1.
D
phous powder and its molecular formula was determined to
be C H O by positive-ion HR-ESI-TOF-MS. Tarecilioside amorphous powder and its molecular formula was deter-
Tarecilioside G (10), [a]D ꢀ15.6°, was isolated as an
4
1
70 15
D (7) was also a cycloartane diglycoside analogous to the mined to be C H O on positive-ion HR-ESI-TOF-MS.
4
2
70 15
aforementioned compounds. Glucopyranose and xylopyra- Tarecilioside G (10) was also expected to be a cycloartane
nose were present in the molecule, and judging from the diglucoside with a ketone functional group at C-24. On acid
HMBC correlation cross peaks, the former was located on hydrolysis, only D-glucose was detected as a sugar compo-
the hydroxyl group at C-3 and the latter on that at C-25 (Fig. nent. However, one of the glucoses was not at the hydroxyl
4
). The results of HR-MS and NMR experiments [d 65.0 group at C-25, but at the 2ꢃ-position of the other glucose
C
with d 3.88 (d, Jꢂ11 Hz) and 4.57 (d, Jꢂ11 Hz)] indicated moiety. This was confirmed by comparison with reported
that tarecilioside D (7) possessed one more hydroxyl group
H
1
3
11)
C-NMR data for Glc(2←1)Glc and the HMBC experi-
on one of the methyl groups than in tareciliosiode C (6). ment, in which the H-1ꢄ proton signal (d 5.36) showed a
H
The HMBC correlations of the new carbinol protons with cross peak with the C-2ꢃ carbon signal (d 83.3). The glyco-
C
C-13 (d 47.2) and 17 (d 55.5), and H-17 (d 1.95) with sylation shift trend also supported the above fact that when
C
C
H
1
3
the carbinol carbon signal supported that the C-18 methyl the C-NMR spectra of 9 and 10 were compared, the C-2ꢃ
must be oxidized to a primary alcohol. This was further carbon signal was shifted downfield by 7.5 ppm and the
supported by a correlation peak between H -18 and H-20 anomeric carbon was shifted upfield by 1.9 ppm. Further
2
in the PS-ROESY experiments. On acid hydrolysis of 7, D- confirmation was provided by the acetylation experiment
glucose and D-xylose were obtained. Therefore, the structure with acetic anhydride and pyridine, which gave a nanoacetate
of tarecilioside D (7) was elucidated to be cycolartane- (10a). In the H–H COSY spectrum of 10a, the H-2ꢃ proton
3
b,7b,16b,18,24R,25-hexaol 3-O-b-D-glucopyranosyl 25-O- remained intact (dH 3.79, dd, Jꢂ8, 9 Hz). Therefore, the
b-D-xylopyranoside.
structure of tarecilioside G (10) was elucidated to be cycol-
Tarecilioside E (8), [a] ꢀ0.6°, was isolated as an amor- artane-3b,7b,16b,18,25-pentaol 3-O-(2ꢄ-O-b-D-glucopyra-
D
phous powder and its molecular formula was determined to nosyl)-b-D-glucopyranoside.
be C H O on positive-ion HR-ESI-TOF-MS. The NMR
From leaves of Tarenna gracilipes (Rubiaceae), seven new
4
1
70 14
data were essentially the same as those of 6, except for the cycloartane glycosides, named tareciliosides A—G, were
presence of xylopyranose. Thus, one of the glucopyranoses isolated. Cycloartane glycosides are often found in many
present in 6 was replaced by xylopyranose. On acid hydroly- plant sources, for example, Leguminosae plants, Astragalus
6)
12)
8,13)
sis of 8, D-glucose and D-xylose were obtained. The positions unifoliolatus, A. verrucosus and A. oleifolius.
From
of sugar linkages were also the same as those in 7, and the Rubiaceae plant, Mussaenda pubescens, cycloartane glyco-
glucopyranose moiety was also located at the hydroxyl group sides have also been isolated as mussaendosides, which are
at C-3 in the b-mode, since a HMBC correlation of the glu- characteristic in having an amide bond to the terminal car-
1
4—16)
cose anomeric proton (H-1ꢃ) (d 4.94) with C-3 (d 88.6) boxylic acid.
However, tareciliosides do not have such
H
C
was observed, although H-1ꢃ (d 4.49) was overlapped by a amide bond, but they have a b-hydroxyl group at the 7-posi-
huge water signal. Therefore, the structure of tarecilioside E tion. The cytotoxicity of tareciliosides was assayed by the
H
(
8) was elucidated to be cycolartane-3b,7b,16b,24R,25-pen- MTT method using KB cells. But they did not show any sig-
taol 3-O-b-D-glucopyranoside, 25-O-b-D-xylopyranoside.
nificant activity.
Tarecilioside F (9), [a] ꢀ7.1°, was isolated as an amor-
It is noteworthy that from the MeOH extract, 415 g of D-
D
phous powder and its molecular formula was determined to mannitol was obtained. This accounted for 4% of the dried

August 2008
1157
weight of leaves. Although no medical use of T. gracilipes of 4 from the peak at 14 min.
The residue (2.50 g in fractions 49—58) of the 17.5—20% MeOH in
has been reported, D-mannitol has been shown to reduce the
extent of ischemic injury, and to improve myocardial, renal
and cerebral function.
CHCl eluate obtained on silica gel CC was subjected to PRCC. The residue
3
(
534 mg in fractions 213—229) was separated by DCCC (105 mg in frac-
17,18)
tions 46—65) and then by HPLC with 60% MeOH to give 10.8 mg of 5
from the peak at 22 min. The residue (634 mg in fractions 230—249) was
similarly separated by DCCC to give two fractions, 99.4 mg in fractions
Experimental
General Experimental Procedures IR spectra were obtained on a 52—61 and 261 mg in fractions 62—68. The former was finally purified by
Horiba Fourier Transform Infrared spectrophotometer FT-710. Optical rota- HPLC with 65% MeOH to give 37.7 mg of compound 6 from the peak at
1
13
tion data were measured on a JASCO P-1030 polarimeter. H- and C-NMR 8.1 min, and the latter gave 32.7 mg of 8 by HPLC purification with 65%
spectra were recorded on a JEOL JNM a-400 spectrometer at 400 and
00 MHz, respectively, with tetramethylsilane (TMS) as an internal standard.
MeOH from the peak at 10 min (flow rate: 1.3 ml/min).
The residue (2.51 g in fractions 59—65) of the 25% MeOH in CHCl elu-
1
3
HR-FAB mass spectra (negative-ion mode) and HR-ESI-TOF mass spectra ate obtained on silica gel CC was subjected to PRCC and the residue
positive-ion mode) were taken on a JEOL JMS-SX 102 mass spectrometer (220 mg in fractions 186—204) was purified by DCCC to give 10.9 mg of 2
(
and Applied Biosystems QSTAR XL System, respectively.
Highly-porous synthetic resin Diaion HP-20 (Fꢂ60 mm, Lꢂ65 cm) was
in fractions 73—90.
Tarecilioside A (4): Amorphous powder. [a] ꢀ14.5° (cꢂ2.48, pyri-
2
2
D
ꢁ1
purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Silica gel dine); IR n (film) cm : 3370, 2935, 1459, 1379, 1252, 1161, 1076, 1036;
max
1
13
column chromatography (CC) was performed on silica gel 60 [(E. Merck,
H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Ta-
5
5
Darmstadt, Germany) 70—230 mesh]. Reversed-phase [octadecyl silica gel bles 1 and 2, respectively; HR-FAB-MS (negative-ion mode) m/z: 653.4239
ꢁ
(
ODS)] open CC (RPCC) was performed on Cosmosi1 75C -OPN (Nacalai [MꢁH] (Calcd for C H O : 653.4206).
18
36 61 10
2
0
Tesque, Kyoto) [Fꢂ50 mm, Lꢂ25 cm, linear gradient: MeOH–H O (1 : 9,
Tarecilioside B (5): Amorphous powder, [a] ꢀ10.8° (cꢂ0.827, pyri-
D
ꢁ1
2
1
.5 l)→(7 : 3, 1.5 l), 10 g fractions being collected]. The droplet counter-cur-
rent chromatograph (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was equipped
with 500 glass columns (Fꢂ2 mm, Lꢂ40 cm), and the lower and upper lay- bles 1 and 2, respectively; HR-FAB-MS m/z: 669.4184 [MꢁH] (Calcd for
ers of a solvent mixture of CHCl –MeOH–H O–1-PrOH (9 : 12 : 8 : 2) were
C H O : 669.4155).
dine); IR n (film) cm : 3367, 2938, 1442, 1379, 1256, 1163, 1076, 1034;
max
1 13
H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Ta-
5
5
ꢁ
3
2
36 61 11
2
0
used as the mobile and stationary 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 (Inertsil; GL Science,
Tarecilioside C (6): Amorphous powder, [a] ꢀ13.1° (cꢂ2.51, pyri-
D
ꢁ1
dine); IR n (film) cm : 3367, 2939, 1591, 1441, 1370, 1157, 1076, 1033;
max
1
13
H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Ta-
5
5
Tokyo, Japan; Fꢂ6 mm, Lꢂ5 cm, flow rate 1.6 ml/min) column. Precoated bles 1 and 2, respectively; HR-ESI-MS (positive-ion mode) m/z: 839.4739
ꢀ
silica gel 60 F2 TLC plates (E. Merck; 0.25 mm in thickness) were used for [MꢀNa] (Calcd for C H O Na: 839.4763).
54
42 72 15
2
0
identification.
Tarecilioside D (7): Amorphous powder, [a] ꢀ17.7° (cꢂ2.18, pyri-
D
ꢁ1
Plant Material Leaves of T. gracilipes OHWI were collected in Oki-
nawa, Japan, in July 2002, and a voucher specimen was deposited in the
Herbarium of Pharmaceutical Sciences, Graduate School of Biomedical Sci-
ences, Hiroshima University (02-TG-Okinawa-0705).
dine); IR n (film) cm : 3395, 2940, 1591, 1441, 1382, 1160, 1075, 1039;
max
13
1
H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Ta-
5
5
bles 1 and 2, respectively; HR-ESI-MS (positive-ion mode) m/z: 825.4604
ꢀ
[MꢀNa] (Calcd for C H O Na: 825.4606).
4
1
70 15
2
0
Extraction and Isolation Air-dried leaves of Tarenna gracilipes
9.90 kg) were extracted three times with MeOH (45 lꢅ3) for one week at IR nmax (film) cm : 3395, 2930, 1650, 1458, 1381, 1161, 1075, 1038; H-
room temperature, and then the MeOH extract was concentrated to 6 l in
Tarecilioside E (8): Amorphous powder, [a] ꢀ0.6° (cꢂ0.45, pyridine);
D
ꢁ1 1
(
13
NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Tables
5 5
vacuo. On evaporation of the MeOH extract, a colorless precipitate formed 1 and 2, respectively; HR-ESI-MS (positive-ion mode) m/z: 809.4648
ꢀ
(
1), which was collected by suction (415 g). A portion of the precipitates was [MꢀNa] (Calcd for C H O Na: 809.4657).
41 70 14
2
0
recrystallized from EtOH to give colorless prisms. The extract was washed
Tarecilioside F (9): Amorphous powder, [a] ꢀ7.1° (cꢂ0.693, pyridine);
D
ꢁ1 1
with n-hexane (6 l) and then the methanolic layer was concentrated to a vis- IR nmax (film) cm : 3368, 2937, 1705, 1461, 1381, 1163, 1076, 1033; H-
cous gum (n-hexane-soluble fraction: 58.8 g). The gummy residue was sus- NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Tables
1
3
5
5
pended in H O (6 l) and then extracted with EtOAc (6 l) to give 172 g of an 1 and 2, respectively; HR-FAB-MS (negative-ion mode) m/z: 813.4643
2
ꢁ
EtOAc-soluble fraction. The aqueous layer was extracted with 1-BuOH (6 l) [MꢁH] (Calcd for C H O : 813.4636).
4
2
69 15
2
0
to give 149 g of a 1-BuOH-soluble fraction. The remaining water-layer was
concentrated to furnish 248 g of a water-soluble fraction.
Tarecilioside G (10): Amorphous powder. [a] ꢀ15.6° (cꢂ0.43, pyri-
D
ꢁ1
dine); IR n (film) cm : 3376, 2935, 1702, 1458, 1370, 1165, 1075, 1034;
max
1
13
The 1-BuOH-soluble extract (149 g) was applied to highly porous syn-
thetic resin (Diaion HP-20) CC (Fꢂ60 mm, Lꢂ65 cm), using a stepwise-
H-NMR (400 MHz, pyridine-d ) and C-NMR (100 MHz, pyridine-d ): Ta-
5
5
bles 1 and 2, respectively; HR-ESI-MS (positive-ion mode) m/z: 837.4613
ꢀ
gradient of MeOH–H O [(1 : 4, 6 l), (2 : 3, 6 l), (3 : 2, 6 l), (4 : 1, 6 l), and
[MꢀNa] (Calcd for C H O Na: 837.4606).
2
42 70 15
MeOH (6 l)], 500 ml factions being collected. The residue eluted with the
Known Compounds Isolated D-Mannitol (1): Colorless prisms, mp
2
0
20% MeOH (50.7 g in fractions 2—4) eluate obtained on HP-20 CC was
160—160 °C (EtOH), [a] ꢀ19.6° (cꢂ10.0, 2 ml of H O containing
D 2
2)
subjected to silica gel (1.0 kg) CC using CHCl (6 l), CHCl –MeOH [(99 : 1,
256 mg of Borax).
(R)-Linalool 6-O-a-L-Arabinopyranosyl-b-D-glucopyranoside (2): Amor-
3
3
6
l), (97 : 3, 6 l), (19 : 1, 6 l), (37 : 3, 6 l), (9 : 1, 6 l), (7 : 17, 6 l), (17 : 3, 6 l),
2
0
3)
(
33 : 7, 6 l), (4 : 4, 6 l), (7 : 3, 6 l)], and CHCl –MeOH–H O (35 : 15 : 2, 3 l),
phous powder, [a]D ꢁ24.0° (cꢂ1.00, MeOH).
26
3
2
4)
1
1
l fractions being collected. The residue (2.25 g in fractions 39—49) of the
Mussaenoside (3): Amorphous powder, [a] ꢁ77.8° (cꢂ1.26, MeOH).
D
Acid Hydrolysis of Tarecilioside A (4) A solution of tarecilioside A (4)
2.5—15% MeOH in CHCl eluate was subjected to PRCC and then the
3
residue (79.4 mg in fractions 105—114) was purified by HPLC with 40%
(10.0 mg) in 2 ml of 1 N HCl–dioxane (1 : 1) was heated at 100 °C for 1 h
MeOH to afford 18.9 mg of compound 3 from the peak at 28 min (flow rate: under a N atmosphere. After cooling, the reaction mixture was neutralized
2
0.4 ml/min). The residue (228 mg in fractions 66—73) of the 25% MeOH in
by the addition of Amberlite IRA-96SB, and then chromatographed on silica
CHCl eluate afforded 20.0 mg of 1.
gel (7.0 g), with elution with a linear gradient mixture of CHCl –MeOH
3
3
The residue eluted with the 60—80% MeOH (29.6 g in fractions 14—19)
eluate obtained on HP-20 CC was subjected to silica gel (500 g) CC using
CHCl (3 l), CHCl –MeOH [(99 : 1, 3 l), (97 : 3, 3 l), (19 : 1, 3 l), (37 : 3, 3 l),
(20 : 1, 100 ml) to (1 : 1, 100 ml), 5 ml fractions being collected. An aglycone
(4a) and D-glucose were recovered in fractions 12—18 (2.62 mg, 38%) and
19—23 (1.74 mg, 63 %), respectively. Aglycone (4a), amorphous powder,
3
3
2
0
1
(9 : 1, 3 l), (7 : 1, 3 l), (17 : 3, 3 l), (33 : 7, 3 l), (4 : 1, 3 l), (3 : 1, 3 l), (7 : 3, 3 l)],
[a] ꢀ23.8° (cꢂ0.17, pyridine); H-NMR (400 MHz, pyridine-d ) d: 0.29
D
5
and CHCl –MeOH–H O (35 : 15 : 2, 3 l), fractions of 500 ml being collected.
(1H, d, Jꢂ4 Hz, H-19a), 0.76 (1H, d, Jꢂ4 Hz, H-19b), 1.05 (3H, s, H -28),
3
2
3
The residue (2.00 g in fractions 42—48) of the 15% MeOH in CHCl eluate
1.10 (3H, s, H -30), 1.10 (3H, d, Jꢂ6 Hz, H -21), 1.17 (3H, s, H -29), 1.44
3
3
3
3
was subjected to PRCC. The residue (30.8 mg in fractions 211—225) was
separated by DCCC to afford 9.1 mg of 9 in fractions 60—78 and 6.8 mg of
compound 7 in fractions 79—88. The residue (35.9 mg in fractions 96—
(3H, s, H -26), 1.45 (3H, s, H -18), 1.48 (3H, s, H -27), 2.00 (1H, d,
3
3
3
Jꢂ10 Hz, H-8), 2.29 (1H, dd, Jꢂ13, 4 Hz, H-15a), 2.71 (1H, dd, Jꢂ13,
8 Hz, H-15b), 3.50 (1H, dd, Jꢂ11, 4 Hz, H-3), 3.74 (1H, dd, Jꢂ10, 2 Hz, H-
1
3
1
21) of the eluate obtained on DCCC was purified by HPLC with 57%
MeOH to afford 7.20 mg of 10 from the peak at 12 min (flow rate: d: 14.7 (C-28), 18.8 (C-18), 18.9 (C-21), 20.0 (C-30), 20.3 (C-9), 25.7 (C-
.9 ml/min). The residue (60.7 mg in fractions 122—163) of the eluate ob-
29), 26.0 (C-27), 26.2 (C-26), 26.9 (C-11), 27.2 (C-10), 28.9 (C-19), 29.5
tained on DCCC was purified by HPLC with 65% MeOH to afford 33.2 mg (C-23), 30.0 (C-1), 31.0 (C-2), 31.5 (C-20), 32.2 (C-6), 33.2 (C-12), 35.0
24), 4.71 (1H, ddd, Jꢂ8, 8, 5 Hz, H-16); C-NMR (100 MHz, pyridine-d5)
0

1
158
Vol. 56, No. 8
(
(
(
C-22), 40.8 (C-4), 46.2 (C-13), 46.4 (C-5), 47.0 (C-14), 50.6 (C-15), 55.3
inal Resources and the Takeda Science Foundation for the financial supports.
C-8), 56.9 (C-17), 70.4 (C-7), 72.2 (C-16), 72.8 (C-25), 77.9 (C-3), 80.5
ꢀ
C-24); HR-ESI-MS (positive-ion mode) m/z: 515.3702 [MꢀNa] (Calcd References
2
0
for C H O Na: 515.3706). D-Glucose: [a] ꢀ26.9° (cꢂ0.12, 24 h after
being dissolved in H O).
1) Hatusima S., “Flora of Ryukyus, Added and Corrected,” The Biologi-
cal Society of Okinawa, Naha, Japan, 1975, pp. 578, 852.
2) “Merck Index,” 13th ed., Merck & Co., Inc., Whitehouse Station, N.J.,
U.S.A., 2001, p. 1026.
3
0
52
5
D
2
Analyses of the Sugar Moiety About 2 mg each of tareciliosides A—G
4—10) was hydrolyzed with 1 N HCl (0.1 ml) at 100 °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 NH2P-50 4E, CH CN–H O (80 : 20),
3) Watanabe N., Nakajima R., Watanabe S., Moon J. H., Inagaki J.,
Sakata Y., Yagi A., Phytochemistry, 37, 457—459 (1994).
4) Otsuka H., Watanabe E., Yuasa K., Ogimi C., Takushi A., Takeda Y.,
Phytochemistry, 32, 983—986 (1993).
3
2
1
ml/min]. Tareciliosides A (4), B (5), C (6), F (9), and G (10) each gave a
peak for D-glucose at the retention time at 14.2 min (positive optical rotation
sign), and tareciliosides D (7) and E (8) gave peaks for D-xylose and D-glu-
cose at the retention times of 9.3 min (positive optical rotation sign) and
5) Kaipnazarov T. N., Uteniyazov K. K., Saatov Z., Chem. Nat. Compd.,
40, 983—986 (2004).
6) Kucherbaev K. D., Uteniyazov K. K., Saatov Z., Shashkov A. S.,
Chem. Nat. Compd., 38, 447—449 (2002).
14.2 min (positive optical rotation sign), respectively. Peaks were identified
by co-chromatography with authentic D-xylose and D-glucose.
7) Gutierrez-Lugo M. T., Singh M. P., Maiese W. M., Timmermann B. N.,
J. Nat. Prod., 65, 872—875 (2002).
8) Özipek M., Dönmez A. A., C¸ ali ¸s Ì., Brun R., Rüedi P., Tasdemir D.,
Phytochemistry, 66, 1168—1173 (2005).
9) C¸ ali ¸s Ì., Yusufoglu H., Zerbe O., Sticher O., Phytochemistry, 50, 846—
847 (1999).
Acetylation of Tarecilioside G Tarecilioside G (10) (3.4 mg) was acety-
lated with 50 ml each of acetic anhydride and pyridine at 25 °C for 18 h. The
reagents were removed under a N stream, followed by drying up in vacuo to
2
1
give a nanoacetate (10a) in a quantitative yield. Nanoacetate (10a), H-NMR
(
400 MHz, CDCl ) d: 1.981 (3H, s), 1.998 (3H, s), 2.002 (3H, s), 2.010 (6H,
3
s); 2.045 (3H, s), 2.045 (3H, s), 2.055 (3H, s), 2.090 (3H, s) (CH COꢅ9), 10) Zhao W. M., Wang P., Xu R. S., Qin G. W., Jiang S. K., Wu H. M.,
3
3
1
6
.67 (2H, m, H-5ꢃ, 5ꢄ), 3.79 (1H, dd, Jꢂ9, 8 Hz, H-2ꢃ), 4.05 and 4.08 (each
H, each dd, each Jꢂ12, 2 Hz, H-6ꢃa, H-6ꢄa), 4.24 (2H, dd, Jꢂ12, 6 Hz, H- 11) Sun R.-Q., Jia Z.-J., Phytochemistry, 30, 3480—3482 (1991).
ꢃb, 6ꢄb), 4.46 (1H, d, Jꢂ8 Hz, H-1ꢃ), 4.70 (1H, d, Jꢂ8 Hz, H-2ꢄ), 4.89 (1H, 12) Pistelli L., Pardossi S., Flamini G., Bertoli A., Manunta A., Phyto-
Phytochemistry, 42, 827—830 (1996).
dd, Jꢂ9, 8 Hz, H-2ꢄ), 4.92 (1H, dd, Jꢂ9, 9 Hz, H-4ꢃ), 5.10 (1H, dd, Jꢂ9,
chemistry, 45, 585—587 (1997).
9
Hz, H-3ꢄ), 5.14 (1H, dd, Jꢂ9, 9 Hz, H-4ꢄ), 5.18 (1H, dd, Jꢂ9, 9 Hz, H-3ꢃ); 13) Bedir E., C¸ ali ¸s Ì., Khan I. A., Chem. Pharm. Bull., 48, 1081—1083
13
C-NMR (100 MHz, CDCl ) d: 169.42, 169.78, 169.98, 170.25, 170.29,
(2000).
3
1
1
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3
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6
0
88 24
1
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Acknowledgements The authors are grateful for access to the supercon-
ducting NMR instrument and ESI-TOF-MS at the Analytical Center of Mol- 16) Zhao W., Wang P., Xu R., Qin G., Jiang S., Wu H., Phytochemistry, 42,
ecular Medicine and the Analysis Center of Life Science, respectively, of the 827—830 (1996).
Graduate School of Biomedical Sciences, Hiroshima University. This work 17) Kostopanagiotou G., Pandazi A. K., Markantonis S. L., Niokou D.,
was supported in part by Grants-in-Aid from the Ministry of Education, Cul-
ture, Sports, Science and Technology of Japan, the Ministry of Health,
Labour and Welfare of Japan, and Japan Society for the Promotion of Sci-
ence. Thanks are also due to the Astellas Foundation for Research on Medic-
Costopanagiotou C., Arkadopoulos N., Smyrniotis V., J. Clin. Anesth.,
18, 570—574 (2006).
18) Larsen M., Webb G., Kennington S., Kelleher N., Sheppard J., Kuo J.,
Unsworth-White J., Perfusion, 17, 51—55 (2002).