Medicinal flowers. XV. The structures of noroleanane- and oleanane-type triterpene oligoglycosides with gastroprotective and platelet aggregation activities from flower buds of Camellia japonica

Article abstract of DOI:10.1248/cpb.55.606

The methanolic extract from the flowers buds of Camellia japonica L. (Theaceae) were found to exhibit potent inhibitory activities on ethanol- or indomethacin-induced gastric mucosal lesions in rats. Through bioassay-guided separation, 28-noroleanane-type

Full text of DOI:10.1248/cpb.55.606

606
Chem. Pharm. Bull. 55(4) 606—612 (2007)
Vol. 55, No. 4
Medicinal Flowers. XV.1) The Structures of Noroleanane- and Oleanane-
Type Triterpene Oligoglycosides with Gastroprotective and Platelet
Aggregation Activities from Flower Buds of Camellia japonica
Masayuki YOSHIKAWA,* Toshio MORIKAWA, Yasunobu ASAO, Emi FUJIWARA, Seikou NAKAMURA, and
Hisashi MATSUDA
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received December 15, 2006; accepted January 23, 2007; published online February 1, 2007
The methanolic extract from the flowers buds of Camellia japonica L. (Theaceae) were found to exhibit po-
tent inhibitory activities on ethanol- or indomethacin-induced gastric mucosal lesions in rats. Through bioassay-
guided separation, 28-noroleanane-type triterpene oligoglycosides, camelliosides A, B, and C, and an oleanane-
type triterpene oligoglycoside, camellioside D, were isolated from the methanolic extract together with five
known compounds. The absolute stereostructures of camelliosides were determined on the basis of chemical and
physicochemical evidence, which included the structure revision of the nortriterpene aglycons (camellenodiol
and camelledionol). The principal oligoglycosides, camelliosides A and B, showed platelet aggregation activity in
addition to the gastroprotective effects on ethanol- or indomethacin-induced gastric mucosal lesions in rats.
Key words camellioside; gastroprotective effect; platelet aggregation activity; Camellia japonica; camellenodiol; camelledionol
The Theaceae plant, Camellia (C.) japonica L. (Japanese and absolute stereostructure elucidations of four then new
name “Tsubaki”) is widely cultivated as an ornamental or oligoglycosides (1—4).11) In addition, we describe the gastro-
garden tree in Japan. The flower buds of C. japonica have protective effects of principal camelliosides (1, 2) on gastric
been used for the treatment of blood vomiting and bleeding lesions induced by ethanol or indomethacin in rats as well as
due to internal and external injury, and also as an antiinflam- the inhibitory effects on rabbit platelet aggregation.
matory, tonic, and stomatic in Japanese folk medicine and
Isolation of Camelliosides The fresh flower buds of C.
Chinese traditional medicine. As chemical constituents of japonica (collected in Idzu-ohshima island, Tokyo, Japan)
this natural medicine, several triterpenes and flavonoids were were extracted with methanol under reflux. The methanolic
reported,2,3) but the constituents responsible for the tradi- extract (8.0% from the fresh flower buds) was partitioned
tional medicinal uses have not been identified. We have al- into an EtOAc–H2O mixture to furnish an EtOAc-soluble
ready reported the isolation and structure elucidation of acy- fraction and aqueous layer. The aqueous layer was further ex-
lated polyhydroxyoleanane-type triterpene oligoglycosides, tracted with n-BuOH to give n-BuOH and H2O-soluble frac-
camelliasaponins A1, A2, B1, B2, C1, and C2, from the seeds tions. As shown in Table 1, the methanolic extract and
of C. japonica and these oligoglycosides were found to in- n-BuOH-soluble fraction (the saponin fraction) were found
hibit alcohol absorption in rats.4,5) During the course of our to show potent protective activities on ethanol- or in-
studies on medicinal flowers,1,6—10) we found that the domethacin-induced gastric lesions in rats, whereas the
methanolic extract from the flower buds of C. japonica EtOAc-soluble and H2O-soluble fractions did not exhibit the
showed potent gastroprotective effects on ethanol- or in- activity.
domethacin-induced gastric mucosal lesions in rats. Through
The active fraction (the n-BuOH-soluble fraction) was
bioassay-guided separation, we isolated three then new 28- subjected to ordinary and reversed-phase silica-gel column
noroleanane-type triterpene oligoglycosides, camelliosides A chromatographies and finally HPLC to furnish camelliosides
(1), B (2), and C (3), and an oleanane-type triterpene oligo- A (1, 0.10%), B (2, 0.067%), C (3, 0.0052%), and D (4,
glycoside, camellioside D (4), together with five known con- 0.0023%) together with (ꢀ)-epicatechin (0.030%). Through
stituents. This paper presents a full account of the isolation the similar procedure, oleanolic acid (0.0006%), benzyl b-D-
Table 1. Effects of the MeOH Extract, AcOEt-, n-BuOH, and H2O-Soluble Fractions from the Flowers of C. japonica on Gastric Lesions Induced by EtOH
or Indomethacin in Rats
EtOH-induced gastric lesions
Lesion index (mm) Inhibition (%)
Indomethacin-induced gastric lesions
Dose
(mg/kg, p.o.)
Treatment
N
N
Lesion index (mm)
Inhibition (%)
Control
MeOH ext.
—
100
200
50
25
50
12
8
8
8
8
115.8ꢁ8.7
71.3ꢁ12.6
—
38.4
90.5
12.1
49.3
18
8
8
8
8
102.0ꢁ10.4**
60.9ꢁ10.4**
37.3ꢁ5.4**
84.8ꢁ11.6
51.4ꢁ13.5**
38.9ꢁ7.9**
87.2ꢁ6.8
—
40.3
63.5
16.9
49.6
61.9
14.6
11.0ꢁ5.3**
101.8ꢁ27.1
58.7ꢁ15.8**
37.6ꢁ13.0**
149.6ꢁ12.1
AcOEt-soluble fraction
n-BuOH-soluble fraction
8
8
67.6
ꢀ29.2
8
8
H2O-soluble fraction
50
Each value represents the meanꢁS.E.M. Significantly different from the control group, ∗∗ p<0.01.
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2007 Pharmaceutical Society of Japan

April 2007
607
Chart 1
glucopyranoside12) (0.0004%), methyl gallate (0.0003%),
(ꢂ)-catechin (0.0011%), and (ꢀ)-epicatechin (0.038%) were
isolated from the EtOAc-soluble fraction.
Structure of Camellioside A (1) Camellioside A (1)
was isolated as colorless fine crystals of mp 226—229 °C
from aqueous MeOH with negative optical rotation ([a]D26
ꢀ32.1°). The IR spectrum of 1 showed strong absorption
bands at 3453 and 1078 cmꢀ1 suggestive of an oligoglyco-
sidic structure and weak bands at 1736, 1719, and 1656 cmꢀ1
ascribable to carbonyl, carboxyl, and olefin functions. In the
negative-ion FAB-MS of 1, a quasimolecular ion peak was
observed at m/z 1103 [MꢀH]ꢀ, together with fragment ion
peaks at 941 [MꢀC6H11O5]ꢀ, and 779 [MꢀC12H21O10]ꢀ,
which were derived by cleavage of the glycoside linkages of
the terminal hexose and dihexose parts. The positive-ion
FAB-MS of 1 showed a quasimolecular ion peak at m/z 1127
[MꢂNa]ꢂ and the molecular formula C53H84O24 of 1 was de-
termined by high-resolution MS measurement of the quasi-
molecular ion peak [MꢂNa]ꢂ. Acid hydrolysis of 1 with 5%
aqueous sulfuric acid (H2SO4)–1,4-dioxane (1 : 1, v/v) liber-
ated D-glucuronic acid, D-galactose, and D-glucose, which
were identified by GLC analysis of their trimethylsilyl thia-
zolidine derivatives.13—15) Enzymatic hydrolysis of 1 with
glycyrrhizic acid hydrolase gave an aglycon, camellenodiol
(5),2) which was derived to the 3-O-acetate (5a) by acetyla-
tion with acetic anhydride (Ac2O)-pyridine and camelle-
dionol (6) by oxidation with chromium trioxide (CrO3)-pyri-
dine.2) Camellenodiol and camelledionol were isolated from
the same plant material and their structures were reported as
Fig. 1. 2D NMR Correlations of 1—5 and 5a
1
5ꢃ and 6ꢃ, respectively, by means of detail H-NMR and CD
analysis.2)
Fig. 2. X-Ray Crystallographic Analysis of 5
From the biogenetic consideration on noroleanane-type
triterpene aglycons,16) we had a considerable doubt as to the 1H–1H COSY experiment on 5 and 5a indicated the presence
position of the tertiary hydroxyl group in the previous for- of partial structures written in bold lines as shown in Fig. 1.
mula (5ꢃ, 6ꢃ) of camellenodiol and camelledionol. The previ- The connectivities of the quaternary carbons were elucidated
ous structures of camellenodiol and camelledionol were fi- by HMBC experiment, which showed long-range correla-
nally revised as 5 and 6, respectively, on the basis of the tions between the following protons and carbons: H2-15 and
1H–1H correlation spectroscopy (1H–1H COSY) and het- C-16, 27; H-18 and C-14, 17, 19; H-22 and C-16, 17. This
eronuclear multiple-bond correlations (HMBC) experiments evidence led us to elucidate the position of the tertiary hy-
(Fig. 1) and X-ray crystallographic analysis (Fig. 2). Namely, droxyl group in 5. Furthermore, the structure of camellen-
the 1H-NMR (pyridine-d5) and 13C-NMR (Table 1) spectra of odial (5) was confirmed by the X-ray crystallographic analy-
5, which were assigned by various NMR experiment,17) sis as shown in Fig. 2. Consequently, the structure of camel-
showed signals due to seven tertiary methyls [d 0.80, 0.89, lenodiol was revised from 5ꢃ to 5. Next, the absolute stereo-
0.95, 0.96, 1.00, 1.04, 1.19 (H3-24, 29, 25, 30, 23, 26, 27)], chemistry of 5 was determined by application of modified
an allylic proton [d 2.72 (dd, Jꢄ4.0, 14.0 Hz, H-18)], a meth- Mosher’s method.18) That is, treatment of 5 with (R)- or
ylene adjacent to a ketone group [d 1.81 (d, Jꢄ14.0 Hz, H2- (S)-a-methoxy-a-trifluoromethylphenylacetic acid [(R)- or
15)], and an olefin proton [d 5.51 (dd-like, H-12)]. The (S)-MTPA] in the presence of 1-ethyl-3-(3-dimethylamino-

608
Vol. 55, No. 4
bands at 1744, 1726, and 1655 cmꢀ1 ascribable to carbonyl,
carboxyl, and olefin functions and strong broad bands at
3453 and 1078 cmꢀ1 suggestive of an oligoglycoside struc-
ture. In the positive- and negative-ion FAB-MS of 2, quasi-
molecular ion peaks were observed at m/z 1169 [MꢂNa]ꢂ
and m/z 1145 [MꢀH]ꢀ together with fragment ion peaks at
m/z 983 [MꢀC6H11O5]ꢀ and m/z 821 [MꢀC12H21O10]ꢀ, and
high resolution MS analysis revealed the molecular formula
of 2 to be C55H86O25. Acid hydrolysis with 5% H2SO4–1,4-
dioxane (1 : 1, v/v) of 2 liberated D-glucuronic acid, D-galac-
tose, and D-glucose, which were identified by GLC analysis
Fig. 3. Results of the Modified Mosher’s Method for 5b and 5c
1
of their trimethylsilyl thiazolidine derivatives.13—15) The H-
NMR (pyridine-d5) and 13C-NMR (Table 2) spectra17) of 2
showed signals assignable to a b-D-glucuronopyranosyl moi-
ety [d 4.89 (1H, d, Jꢄ7.6 Hz, H-1ꢃ)], a b-D-glucopyranosyl
moiety [d 5.16 (1H, d, Jꢄ7.6 Hz, H-1ꢅꢅ)], and two b-D-galac-
topyranosyl moieties [d 5.70 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.87
(1H, d-like, H-1ꢅ)] together with an acetyl group [d 2.13
(3H, s, –OCOCH3)] and an aglycon moiety [d 0.87, 0.90,
0.98, 1.10, 1.27, 1.34, 1.37 (3H each, all s, H3-25, 29, 30, 24,
26, 27, 23), 3.08 (1H, dd-like, H-18), 3.30 (1H, dd, Jꢄ3.5,
11.0 Hz, H-3), 5.45 (1H, br s, H-12)]. The proton and carbon
1
signals in the H- and 13C-NMR spectra of 2 were superim-
posable on those of 1, except for the signals due to an acetyl
group. Treatment of 2 with 0.1% sodium methoxide
(NaOMe)–MeOH liberated 1, and comparison of the 13C-
Chart 2
propyl)carbodiimide hydrochloride (EDC·HCl) and 4-di- NMR data of 2 with those of 1 indicated the presence of an
methylaminopyridine (4-DMAP) to yielded the (R)- and (S)- acetylation shift19) around the 4ꢅ-position. Finally, the HMBC
MTPA esters (5b, 5c), respectively. As shown in Fig. 3, the experiment on 2 showed a long-range correlation between
signals due to protons attached to C-5, C-6, C-23 and C-24 in the 4ꢅ-proton [d 5.93 (1H, br s)] and acetyl carbonyl carbon
the (S)-MTPA ester (5c) were observed at lower fields com- (dC 170.9). Consequently, the structure of camellioside B
pared with those of the (R)-MTPA ester (5b) [Dd: positive], was determined as the 4ꢅ-acetyl derivative (2) of camellio-
while the signals due to protons of C-1, C-2, and C-25 in 5c side A.
were observed at higher fields compared with those of 5b
Camellioside C (3), which was isolated as colorless fine
[Dd: negative]. Consequently, the absolute configuration at crystals of mp 224—227 °C (from aqueous MeOH) with
the 3-position of 5 was determined to be S and the absolute negative optical rotation ([a]D26 ꢀ15.8°), showed absorption
stereostructure of 5 was elucidated as shown. Moreover, the bands at 3453, 1736, 1665, 1655, and 1078 cmꢀ1 ascribable
structures of related compounds of 5, 3-acetylcamellenodiol to hydroxyl, carbonyl, carboxyl, olefin, and ether functions in
and camelledionol,2) were also revised from 5ꢃ and 6ꢃ to 5a the IR spectrum. In the UV spectrum of 3, the absorption
and 6, respectively (Chart 2).
maximum was observed at 299 (log e 4.0) nm, suggestive of
The H-NMR (pyridine-d5) and 13C-NMR (Table 2) spec- an a,b-unsaturated ketone group with extended conjuga-
tra17) of 1 showed signals assignable to a camellenodiol moi- tion.2) The molecular formula C53H82O23 of 3 was also deter-
ety [d 0.87, 0.90, 0.98, 1.10, 1.27, 1.34, 1.37 (3H each, all s, mined from the quasimolecular ion peaks at m/z 1109
H3-25, 29, 30, 24, 26, 23, 27), 3.10 (1H, dd, Jꢄ3.5, 14.5 Hz, [MꢂNa]ꢂ, m/z 1087 [MꢂH]ꢂ, and m/z 1085 [MꢀH]ꢀ in the
H-18), 3.27 (1H, dd, Jꢄ4.5, 11.8 Hz, H-3), 5.49 (1H, br s, H- positive-ion and negative-ion FAB-MS and by high-resolu-
12)] together with a b-D-glucuronopyranosyl moiety [d 4.90 tion MS measurement. By the acid hydrolysis of 3, D-glu-
(1H, d, Jꢄ7.4 Hz, H-1ꢃ)], a b-D-glucopyranosyl moiety [d curonic acid, D-galactose, and D-glucose were identified by
5.18 (1H, d, Jꢄ7.7 Hz, H-1ꢅꢅ)], and two b-D-galactopyra- GLC analysis of their trimethylsilyl thiazolidine deriva-
nosyl moieties [d 5.70 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.76 (1H, d, tives.13—15) Enzymatic hydrolysis of 3 with glycyrrhizic acid
Jꢄ7.6 Hz, H-1ꢅ)]. The oligoglycosidic structure of 1 was de- hydrolase furnished maragenin II (7) as its aglycon.2,20) The
1
1
termined by H–1H COSY, homo-nuclear Hartmann-Hahn 1H- and 13C-NMR (pyridine-d5, Table 1) spectra17) of 3
spectroscopy (1H–1H HOHAHA) and HMBC experiments showed the presence of a b-D-glucuronopyranosyl moiety [d
(Fig. 1). In the HMBC experiment of 1, long-range correla- 4.90 (1H, d, Jꢄ7.4 Hz, H-1ꢃ)], a b-D-glucopyranosyl moiety
tions were observed between the following protons and car- [d 5.17 (1H, d, Jꢄ8.1 Hz, H-1ꢅꢅ)], and two b-D-galactopyra-
bons (H-1ꢃ and C-3, H-1ꢅ and C-2ꢃ, H-1ꢆ and C-3ꢃ, H-1ꢅꢅ nosyl moieties [d 5.69 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.76 (1H, d,
and C-2ꢆ). On the basis of those findings, the structure of Jꢄ7.7 Hz, H-1ꢅ)] together with a maragenin II part.2,20) The
camellioside A (1) was characterized as shown.
oligoglycosidic structure of 3 was elucidated by the HMBC
Structures of Camelliosides B—D (2—4) Camellioside experiment as shown in Fig. 1, which showed the same long-
B (2) was obtained as colorless fine crystals of mp 221— range correlations as those of 1. This evidence led us to elu-
224 °C from aqueous MeOH with negative optical rotation cidate the structure of camellioside C (3) to be as shown.
([a]D23 ꢀ30.0°). The IR spectrum of 2 showed absorption
Camellioside D (4), obtained as colorless fine crystals of

April 2007
609
Table 2. 13C-NMR Data for Camelliosides A (1), B (2), C (3), and D (4) and Camellenodiol (5)
1a)
2a)
3a)
4a)
5a)
5b)
1a)
2a)
3a)
4a)
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.5
26.4
89.5
39.4
55.7
18.3
33.2
40.1
46.9
36.8
23.8
124.1
142.3
48.1
43.3
214.9
76.3
52.7
48.2
30.9
37.2
31.5
28.1
16.7
15.3
17.7
27.0
38.7
26.7
89.9
39.8
55.9
18.6
33.5
40.3
47.1
37.1
24.1
124.2
142.6
48.4
43.5
214.8
76.5
53.0
48.3
31.1
37.4
31.8
28.2
16.9
15.5
17.9
27.3
38.9
26.6
89.5
39.6
55.8
18.4
33.8
39.0
46.3
36.8
24.4
127.3
139.2
45.1
40.4
199.1
129.0
146.3
44.5
29.2
34.7
21.3
28.1
16.9
15.6
18.0
23.3
38.9
26.6
89.5
39.6
55.9
18.6
33.3
40.1
47.1
36.9
23.9
122.4
145.2
42.1
34.9
74.3
41.1
42.6
48.4
31.3
37.2
30.4
28.2
16.9
15.8
17.1
27.4
70.2
33.5
24.9
38.9
28.0
78.0
39.3
55.7
18.7
33.4
40.2
47.1
37.3
23.9
123.8
142.4
48.3
43.3
214.8
76.4
52.8
48.1
30.9
37.2
31.5
28.7
16.5
15.5
17.8
27.1
38.5
27.2
79.0
38.8
55.4
18.3
32.8
39.9
46.8
37.1
23.7
125.7
140.5
47.4
43.0
213.5
76.5
52.6
47.2
30.8
36.5
30.4
28.2
15.6
15.3
17.3
27.1
GlcA-1ꢃ
-2ꢃ
105.5
79.0
83.7
70.9
76.9
171.9
105.8
78.5
84.8
71.2
77.1
171.7
105.6
79.1
84.0
71.0
77.0
171.7
105.7
79.3
84.6
71.1
77.0
171.8
-3ꢃ
-4ꢃ
-5ꢃ
-6ꢃ
Gal-1ꢅ
-2ꢅ
103.0
73.7
74.7
69.8
76.3
61.8
102.8
73.2
74.0
72.0
75.1
61.4
103.1
73.9
74.9
69.9
76.4
61.9
103.3
74.0
75.0
70.0
76.4
61.8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
-3ꢅ
-4ꢅ
-5ꢅ
-6ꢅ
4ꢅ-Ac
21.2
170.9
Gal-1ꢆ
-2ꢆ
101.4
83.0
74.7
69.8
76.3
61.9
101.9
83.5
75.1
69.9
76.5
62.5
101.6
83.2
74.9
69.9
76.4
62.1
101.8
83.5
75.0
69.9
76.4
62.1
-3ꢆ
-4ꢆ
-5ꢆ
-6ꢆ
Glc-1ꢅꢅ
-2ꢅꢅ
106.2
75.8
78.1
71.0
78.2
62.2
106.8
76.2
78.5
71.4
78.5
61.8
106.5
76.0
78.3
71.2
78.4
62.4
106.7
76.1
78.4
71.2
78.5
62.4
-3ꢅꢅ
-4ꢅꢅ
-5ꢅꢅ
-6ꢅꢅ
32.6
23.6
32.9
23.9
28.6
28.2
32.7
23.7
32.4
23.7
Measured in a) pyridine-d5 and b) CDCl3 at 125 MHz. GlcA: b-D-glucopyranosiduronic acid; Gal: b-D-galactopyranosyl; Glc: b-D-glucopyranosyl.
Table 3. Effects of Camelliosides A (1) and B (2) on Gastric Lesions Induced by EtOH or Indomethacin in Rats
EtOH-induced gastric lesions
Lesion index (mm) Inhibition (%)
Indomethacin-induced gastric lesions
Dose
(mg/kg, p.o.)
Treatment
N
N
Lesion index (mm)
Inhibition (%)
Control
Camellioside A (1)
—
5
10
20
5
9
6
6
6
6
6
6
7
141.4ꢁ9.0
93.8ꢁ13.3
85.1ꢁ30.2
32.0ꢁ10.6**
92.1ꢁ20.0
63.1ꢁ20.6**
11.6ꢁ3.1**
146.8ꢁ9.7
—
5
5
5
5
5
5
5
5
4
5
5
119.3ꢁ11.5
106.6ꢁ9.8
84.2ꢁ10.7
61.1ꢁ12.8**
104.7ꢁ6.5
68.0ꢁ10.9*
43.0ꢁ8.2**
88.7ꢁ9.6
—
33.7
39.8
77.3
34.9
55.4
91.8
—
10.7
29.4
46.2
12.2
43.0
63.9
—
8.8
57.2
94.7
Camellioside B (2)
10
20
—
2.5
5
10
15
20
—
12.5
25
50
100
Control
Omeprazole
80.9ꢁ12.4
38.0ꢁ16.5*
4.7ꢁ1.8**
7
7
7
8
5
7
7
7
78.3ꢁ15.9**
31.1ꢁ11.6**
15.9ꢁ3.1**
136.0ꢁ10.6
123.4ꢁ9.4
87.2ꢁ7.9**
72.9ꢁ10.1**
64.4ꢁ4.9**
46.7
78.8
89.2
—
Control
Cimetidine
6
6
6
6
85.2ꢁ2.4
—
9.3
61.2ꢁ5.5**
34.8ꢁ5.0**
17.6ꢁ3.2**
28.2
59.2
79.3
35.9
46.4
52.6
Each value represents the meanꢁS.E.M. Significantly different from the control group, ∗ p<0.05, ∗∗ p<0.01.
mp 222—225 °C (from aqueous MeOH) with negative opti- lution MS measurement. The acid hydrolysis of 4 liberated
cal rotation ([a]D26 ꢀ3.2°), showed absorption bands due to D-glucuronic acid, D-galactose, and D-glucose, which were
hydroxyl, carboxyl, olefin, and ether functions (3453, 1719, identified by GLC analysis,13—15) while enzymatic hydrolysis
1655, 1078 cmꢀ1) in the IR spectrum. The molecular formula of 4 with glycyrrhizic acid hydrolase furnished primulagenin
C54H88O24 of 4 was also determined from the quasimolecular A (8).21) The 1H- and 13C-NMR (pyridine-d5, Table 2)
ion peaks at m/z 1143 [MꢂNa]ꢂ and m/z 1119 [MꢀH]ꢀ in spectra17) of 4 showed the presence of a b-D-glucuronopyra-
the positive-ion and negative-ion FAB-MS and by high-reso- nosyl moiety [d 4.92 (1H, d, Jꢄ7.3 Hz, H-1ꢃ)], a b-D-glu-

610
Vol. 55, No. 4
Table 4. Platelet Aggregating Activities of Camelliosides A (1) and B (2)
coated TLC plates with Silica gel 60F254 (Merck, normal-phase), Silica gel
RP-18 WF254S (Merck, reversed-phase). Detection was done by spraying
with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Dose
Aggregation
Treatment
(mg/ml)
(% of control)
Extraction and Isolation The fresh flower buds of C. japonica (6.0 kg,
collected in Izu-ohshima island, Tokyo, Japan) was finely cut and extracted
three times with methanol under reflux for 3 h. Evaporation of the solvent
under reduced pressure gave the methanolic extract (480 g, 8.0% from flesh
flowers). The methanolic extract (400 g) was partitioned in an EtOAc–H2O
(1 : 1, v/v) mixture. The aqueous layer was extracted with n-BuOH and re-
moval of the solvent in vacuo from the EtOAc-, n-BuOH-, and H2O-soluble
portions yielded 25 g (0.5%), 100 g (2.0%), and 275 g (5.5%) of the residue,
respectively. The n-BuOH-soluble fraction (83.3 g) was subjected to ordi-
nary-phase silica gel column chromatography {3.0 kg, CHCl3–MeOH–H2O
[(7 : 3 : 0.5—6 : 4 : 1—5 : 5 : 1, v/v/v)–MeOH)] to afford 13 fractions [Fr. 1
(1.5 g), Fr. 2 (0.8 g), Fr. 3 (2.3 g), Fr. 4 (2.8 g), Fr. 5 (25.6 g), Fr. 6 (0.8 g), Fr.
7 (15.6 g), Fr. 8 (2.9 g), Fr. 9 (8.4 g), Fr. 10 (4.0 g), Fr. 11 (2.7 g), Fr. 12
(1.6 g), Fr. 13 (14.3 g)]. Fraction 5 (18.6 g) was separated by reversed-phase
silica gel column chromatography [560 g, MeOH–H2O (10 : 90—30 : 70—
50 : 50—70 : 30, v/v)–MeOH] to furnish 13 fractions [Fr. 5-1 (0.30 g), Fr. 5-
2 (0.12 g), Fr. 5-3 (2.10 g), Fr. 5-4 (1.40 g), Fr. 5-5 (0.58 g), Fr. 5-6 (1.30 g),
Fr. 5-7 (0.44 g), Fr. 5-8 (0.34 g), Fr. 5-9 (0.60 g), Fr. 5-10 (4.90 g), Fr. 5-11
(2.00 g), Fr. 5-12 (2.10 g), Fr. 5-13 (2.52 g)]. Fraction 5-4 (1.30 g) was fur-
ther separated by HPLC [YMC-pack ODS-A, 250ꢇ20 mm i.d., MeOH–1%
aqueous AcOH (25 : 75, v/v)] to give (ꢀ)-epicatechin (431 mg, 0.036%).
Fraction 5-10 (1.15 g) was separated by HPLC [MeOH–1% aqueous AcOH
(70 : 30, v/v)] to give camelliosides A (1, 260 mg, 0.10%) and B (2, 175 mg,
0.067%). Fraction 5-11 (1.15 g) was purified by HPLC [MeOH–1% aqueous
AcOH (75 : 25, v/v)] to furnish camelliosides C (3, 34 mg, 0.052%) and D
Camellioside A (1)
1
10
50
100
1
10
15
16
47
83
17
13
43
104
Camellioside B (2)
50
100
Each values represents mean of two experiments.
copyranosyl moiety [d 5.18 (1H, d, Jꢄ7.9 Hz, H-1ꢅꢅ)], and
two b-D-galactopyranosyl moieties [d 5.64 (1H, d, Jꢄ7.6 Hz,
H-1ꢆ), 5.76 (1H, d, Jꢄ7.6 Hz, H-1ꢅ)] together with a primu-
lagenin A part. The carbon signals due to the 3-O-tetraglyco-
side moiety in the 13C-NMR spectrum of 4 were superimpos-
able on those of 1 and 3. The HMBC experiment of 4 exhib-
ited long-range correlations as shown in Fig. 1, so that the
structure of camellioside D (4) was characterized to be as
shown.
Effects of Principal Saponin Constituents (1, 2) on Gas- (4, 15 mg, 0.0023%).
The EtOAc-soluble fraction (18.0 g) was subjected to ordinary-phase sil-
tric Lesions by Ethanol or Indomethacin in Rats Effects
of the principal oligoglycosides, camelliosides A (1) and B
(2), on ethanol- or indomethacin-induced gastric lesions were
examined.22,23) As shown in Table 3, 1 and 2 showed protec-
tive effects on both ethanol- and indomethacin-induced gas-
tric lesions [ethanol-induced: 1 (ED50ꢄ9.7 mg/kg), 2 (7.4
mg/kg); indomethacin-induced: 1 (21 mg/kg), 2 (13 mg/kg)]
and their gastroprotective effects were equivalent or stronger
than those of reference compounds, omeprazole (ethanol-in-
duced: 10 mg/kg; indomethacin-induced: 4.7 mg/kg), cimeti-
dine (ethanol-induced: 69 mg/kg; indomethacin-induced: 21
mg/kg).24—26)
Platelet Aggregation Activities of Principal Saponin
Constituents (1, 2) Furthermore, since this natural medi-
cine has been used as hemostatic, we examined the rabbit
platelet aggregation activities (in vitro).27) As shown in Table
4, the principal oligoglysides, camelliosides A (1) and B (2),
concentration-dependently caused the aggregation of the
platelets (10—100 mg/ml).
ica gel column chromatography [600 g, CHCl3–MeOH–H2O (15 : 3 : 1, lower
layer–10 : 3 : 1, lower layer–7 : 3 : 0.5, v/v/v)–MeOH) to afford nine fractions
[Fr. 1 (1.5 g), Fr. 2 (1.9 g), Fr. 3 (0.7 g), Fr. 4 (1.6 g), Fr. 5 (4.4 g), Fr. 6
(1.1 g), Fr. 7 (1.1 g), Fr. 8 (1.5 g), Fr. 9 (4.2 g)]. Fraction 2 (1.7 g) was puri-
fied by reversed-phase silica gel column chromatography [60 g, MeOH–H2O
(50 : 50—70 : 30—90 : 10, v/v)–MeOH] and finally HPLC [MeOH–1%
aqueous AcOH (90 : 10, v/v)] to give oleanolic acid (22 mg, 0.0006%). Frac-
tion 4 (1.3 g) was purified by reversed-phase silica gel column chromatogra-
phy [40 g, MeOH–H2O (30 : 70—50 : 50—70 : 30, v/v)–MeOH] and finally
HPLC [MeOH–1% aqueous AcOH (35 : 65, v/v)] to furnish benzyl b-D-glu-
copyranoside (14 mg, 0.0004%) and methyl gallate (12 mg, 0.0003%). Frac-
tion 5 (2.0 g) was subjected to reversed-phase silica gel column chromatog-
raphy [60 g, MeOH–H2O (10 : 90—20 : 80—70 : 30, v/v)–MeOH] and finally
HPLC [MeOH–1% aqueous AcOH (25 : 75, v/v)] to furnish (ꢀ)-epicatechin
(30 mg, 0.0017%) and (ꢂ)-catechin (19 mg, 0.0011%).
These known constituents were identified by comparison of their physical
data with commercially obtained sample or with reported values.12)
Camellioside A (1): Colorless fine crystals (mp 226—229 °C from aque-
ous MeOH); [a]D26 ꢀ32.1° (cꢄ1.80, pyridine); IR nmax (KBr) 3453, 2962,
1736, 1719, 1656, 1078 cmꢀ1 1H-NMR (pyridine-d5) d: 0.87, 0.90, 0.98,
;
1.10, 1.27, 1.34, 1.37 (3H each, all s, H3-25, 29, 30, 24, 26, 23, 27), 3.10
(1H, dd, Jꢄ3.5, 14.5 Hz, H-18), 3.27 (1H, dd, Jꢄ4.5, 11.8 Hz, H-3), 4.90
(1H, d, Jꢄ7.4 Hz, H-1ꢃ), 5.18 (1H, d, Jꢄ7.7 Hz, H-1ꢅꢅ), 5.49 (1H, br s,
H-12), 5.70 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.76 (1H, d, Jꢄ7.6 Hz, H-1ꢅ); 13C-
NMR (pyridine-d5) dC given in Table 2; Positive-ion FAB-MS m/z 1127
[MꢂNa]ꢂ; Negative-ion FAB-MS m/z 1103 [MꢀH]ꢀ, 941 [MꢀC6H11O5]ꢀ,
779 [MꢀC12H21O10]ꢀ; High-resolution MS m/z 1127.5244 (Calcd for
C53H84O24Na, 1127.5250).
The gastroprotective and platelet aggregation effects of
this natural medicine and the principal constituents may be
important evidence substantiating the traditional effect of this
natural medicine such as the treatment effects of blood vom-
iting and bleeding and stomatic effects.
Camellioside B (2): Colorless fine crystals (mp 221—224 °C from aque-
Experimental
ous MeOH); [a]D23 ꢀ30.0° (cꢄ0.80, pyridine); IR nmax (KBr) 3453, 2962,
1
The following instruments were used to obtain physical data: melting 1744, 1726, 1655, 1380, 1078 cmꢀ1; H-NMR (pyridine-d5) d: 0.87, 0.90,
points, Yanagimoto micro-melting point apparatus MP-500D (values are 0.98, 1.10, 1.27, 1.34, 1.37 (3H each, all s, H3-25, 29, 30, 24, 26, 27, 23),
uncorrected); specific rotations, Horiba SEPA-300 digital polarimeter 2.13 (3H, s, –OCOCH3), 3.08 (1H, dd-like, H-18), 3.30 (1H, dd, Jꢄ3.5,
(lꢄ5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shi- 11.0 Hz, H-3), 4.89 (1H, d, Jꢄ7.6 Hz, H-1ꢃ), 5.16 (1H, d, Jꢄ7.6 Hz, H-1ꢅꢅ),
madzu FTIR-8100 spectrometer; 1H-NMR spectra, JNM-LA500 (500 MHz);
5.45 (1H, br s, H-12), 5.70 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.87 (1H, d-like, H-1ꢅ),
13C-NMR spectra, JNM-LA500 (125 MHz) spectrometer with tetramethylsi- 5.93 (1H, br s, H-4ꢅ); 13C-NMR (pyridine-d5) dC given in Table 2; Positive-
lane as an internal standard; MS and high-resolution MS, JEOL JMS-GC-
MATE mass spectrometer, JEOL JMS-SX 102A mass spectrometer.
ion FAB-MS m/z 1169 [MꢂNa]ꢂ; Negative-ion FAB-MS m/z 1145
[MꢀH]ꢀ, 983 [MꢀC6H11O5]ꢀ, 821 [MꢀC12H21O10]ꢀ; High-resolution MS
The following experimental conditions were used for chromatography: or- m/z 1169.5352 (Calcd for C55H86O25Na, 1169.5356).
dinary-phase column chromatography; Silica gel BW-200 (Fuji Silysia
Camellioside C (3): Colorless fine crystals (mp 224—227 °C from aque-
Chemical, Ltd., 150—350 mesh), reversed-phase column chromatography; ous MeOH); [a]D26 ꢀ15.8° (cꢄ0.10, MeOH); UV (MeOH) lmax (log e) 299
Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100—200 (4.0) nm; IR nmax (KBr) 3453, 2926, 1736, 1665, 1655, 1078 cmꢀ1; 1H-NMR
mesh); TLC, pre-coated TLC plates with Silica gel 60F254 (Merck, normal-
phase) and Silica gel RP-18 F254S (Merck, reversed-phase); HPTLC, pre- 30, 29, 24, 27, 23), 3.30 (1H, dd, Jꢄ4.5, 11.7 Hz, H-3), 4.90 (1H, d,
(pyridine-d5) d: 0.84, 0.87, 0.88, 0.90, 1.10, 1.20, 1.30 (3H, all s, H3-25, 26,

April 2007
611
the mixture was stirred at room temperature for 3.5 h. The reaction mixture
was poured into ice-water and the whole reaction mixture was extracted with
EtOAc. The EtOAc extract was successively washed with 5% aqueous HCl,
saturated aqueous NaHCO3, and brine, then dried over MgSO4 powder and
filtered. Removal of the solvent from the filtrate under reduced pressure fur-
nished a residue, which was by ordinary-phase silica gel column chromatog-
raphy [0.5 g, n-hexane–EtOAc (5 : 1, v/v)] to give 5b (5.4 mg, 95%). Using a
similar procedure, (S)-MTPA esters [5c (4.6 mg, 91%)] were obtained from
5 (3.9 mg), using (S)-MTPA (3.5 mg), EDC·HCl (3.0 mg), and 4-DMAP
(2.5 mg).
Jꢄ7.4 Hz, H-1ꢃ), 5.17 (1H, d, Jꢄ8.1 Hz, H-1ꢅꢅ), 5.69 (1H, d, Jꢄ7.6 Hz, H-
1ꢆ), 5.76 (1H, d, Jꢄ7.7 Hz, H-1ꢅ), 6.07 (1H, br s, H-12); 13C-NMR
(pyridine-d5) dC given in Table 2; Positive-ion FAB-MS m/z 1109 [Mꢂ
Na]ꢂ, 1087 [MꢂH]ꢂ; Negative-ion FAB-MS m/z 1085 [MꢀH]ꢀ, 923
[MꢀC6H11O5]ꢀ, 761 [MꢀC12H21O10]ꢀ; High-resolution MS m/z 1109.5137
(Calcd for C53H82O23Na, 1109.5145).
Camellioside D (4): Colorless fine crystals (mp 222—225 °C from aque-
ous MeOH); [a]D26 ꢀ3.2° (cꢄ0.10, MeOH); IR nmax (KBr) 3453, 2926,
1719, 1655, 1078 cmꢀ1 1H-NMR (pyridine-d5) d: 0.85, 0.94, 1.05, 1.09,
;
1.12, 1.30, 1.81 (3H each, all s, H3-25, 26, 29, 24, 30, 23, 27), 2.47 (1H, dd-
like, H-18), 3.32 (1H, dd, Jꢄ4.3, 11.9 Hz, H-3), 4.59 (1H, br s, H-16), 4.92
(1H, d, Jꢄ7.3 Hz, H-1ꢃ), 5.18 (1H, d, Jꢄ 7.9 Hz, H-1ꢅꢅ), 5.37 (1H, br s, H-
12), 5.64 (1H, d, Jꢄ7.6 Hz, H-1ꢆ), 5.76 (1H, d, Jꢄ7.6 Hz, H-1ꢅ); 13C-NMR
(pyridine-d5) dC given in Table 2; Positive-ion FAB-MS m/z 1143 [MꢂNa]ꢂ;
Negative-ion FAB-MS m/z 1119 [MꢀH]ꢀ, 957 [MꢀC6H11O5]ꢀ, 795 [Mꢀ
C12H21O10]ꢀ; High-resolution MS m/z 1143.5538 (Calcd for C54H88O24Na,
1143.5563).
1
Compound 5b: H-NMR (CDCl3): d: 0.82, 0.84, 0.89, 0.96, 0.99, 1.04,
1.19 (3H each, all s, H3-24, 23, 29, 30, 25, 26, 27), 0.87 (1H, d-like, H-5),
1.11 (1H, m, H-1), 1.32 (3H, m, H-7, 19, 21), 1.41 (1H, m, H-22), 1.48 (1H,
m, H-19), 1.51 (1H, m, H-7), 1.54 (1H, m, H-21), 1.56 (3H, m, H2-6), 1.57
(1H, m, H-9), 1.69 (1H, m, H-1), 1.77 (1H, m, H-2), 1.81 (1H, d,
Jꢄ14.7 Hz, H-15), 1.84 (1H, m, H-2), 1.99 (2H, m, H2-11), 2.10 (1H, m, H-
22), 2.72 (1H, dd, Jꢄ4.2, 14.0 Hz, H-18), 3.17 (1H, d, Jꢄ14.7 Hz, H-15),
3.56 (3H, s, –OCH3), 4.72 (1H, dd, Jꢄ4.9, 11.6 Hz, H-3), 5.51 (1H, dd,
Jꢄ3.6, 3.7 Hz, H-12), 7.38—7.56 (5H, m, Ph-H).
Acid Hydrolysis of Camelliosides A (1), C (3), and D (4) A solution
of 1, 3, or 4 (5 mg each) in 5% aqueous H2SO4–1,4-dioxane (1 : 1, 2 ml) was
heated under reflux for 1 h. After cooling, the reaction mixture was neutral-
ized with Amberlite IRA-400 (OHꢀ form) and the resin was removed by fil-
tration. After removal of the solvent from the filtrate under reduced pressure,
the residue was transferred to a Sep-Pak C18 cartridge with H2O and MeOH.
The H2O-eluted fraction was concentrated in vacuo to give a residue, which
was treated with L-cysteine methyl ester hydrochloride (4 mg) in pyridine
(0.5 ml) at 60 °C for 1 h. After the reaction, the solution was treated with
N,O-bis(trimethylsilyl)trifluoroacetamide (0.2 ml) at 60 °C for 1 h. The su-
pernatant of the reaction mixture was then subjected to GLC analysis to
identify the derivatives of D-glucose (i), D-galactose (ii), and D-glucuronic
acid (iii) from 1, 3, and 4. GLC conditions: Supelco STBTM-1, 30 mꢇ
0.25 mm (i.d.) capillary column; injector temperature, 230 °C; detector tem-
perature, 230 °C; column temperature 230 °C; He flow rate 15 ml/min; tR: (i)
24.4 min, (ii) 25.6 min, and (iii) 26.5 min.
Enzymatic Hydrolysis of Camelliosides A (1), C (3), and D (4) A so-
lution of 1 (100 mg) in 0.2 M acetate buffer (pH 4.4, 5.0 ml) was treated with
glycyrrhizinic acid hydrolase (Maruzen Pharmaceutical Co., Ltd., Hi-
roshima, Japan, 5.0 ml) and the mixture was stirred at 44 °C for over night.
After treatment of the reaction mixture with EtOH, the solvent was evapo-
rated to dryness under reduced pressure and the residue was purified by ordi-
nary-phase silica gel column chromatography [2.0 g, n-hexane–EtOAc (3 : 1,
v/v)] to give camellenodiol (5, 30 mg, 76%). Using a similar procedure, a
solution of 3 (13 mg) or 4 (2 mg) in 0.2 M acetate buffer (pH 4.4, 2.0 or
0.5 ml) was treated with glycyrrhizinic acid hydrolase (2.0 or 0.5 ml) and the
mixture was stirred at 44 °C for over night. After EtOH was added to the re-
action mixture, the solvent was removed under reduced pressure and the
residue was purified by ordinary-phase silica gel column chromatography
[1.0 g, n-hexane–EtOAc (5 : 1, v/v)] to furnish 7 (from 3: 4 mg, 78%) or 8
(from 4: 0.6 mg, 73%), which were identified by comparison of physical data
([a]D, IR, 1H-NMR, 13C-NMR) with reported values.20,21)
1
Compound 5c: H-NMR (CDCl3): d: 0.83, 0.89, 0.92, 0.96, 0.96, 1.03,
1.19 (3H each, all s, H3-24, 29, 23, 30, 25, 26, 27), 0.88 (1H, d-like, H-5),
1.08 (1H, m, H-1), 1.32 (3H, m, H-7, 19, 21), 1.41 (1H, m, H-22), 1.48 (1H,
m, H-19), 1.51 (1H, m, H-7), 1.54 (1H, m, H-21), 1.56 (1H, m, H-9), 1.57
(2H, m, H2-6), 1.66 (1H, m, H-1), 1.69 (1H, m, H-2), 1.76 (1H, m, H-2),
1.81 (1H, d, Jꢄ14.4 Hz, H-15), 1.99 (2H, m, H2-11), 2.10 (1H, m, H-22),
2.72 (1H, dd, Jꢄ4.2, 14.0 Hz, H-18), 3.17 (1H, d, Jꢄ14.4 Hz, H-15), 3.53
(3H, s, –OCH3), 4.72 (1H, dd, Jꢄ4.9, 11.6 Hz, H-3), 5.51 (1H, dd, Jꢄ3.3,
3.7 Hz, H-12), 7.39—7.54 (5H, m, Ph-H).
Acetylation of 5 A solution of 5 (5 mg) in pyridine (1.0 ml) was treated
with acetic anhydride (Ac2O, 0.8 ml) and the mixture was stirred at room
temperature for 8 h. The reaction mixture was poured into ice-water and the
whole reaction mixture was extracted with EtOAc. Work-up of the EtOAc
extract as described above gave a product, which was purified by ordinary-
phase silica gel column chromatography [0.5 g, n-hexane–EtOAc (5 : 1, v/v)]
to furnish 5a (6 mg, quant.) which was identified by comparison of the phys-
ical data ([a]D, IR, 1H-NMR, MS) with reported values.2)
Oxidation of 5 A solution of 5 (5 mg) was treated with chromium triox-
ide (CrO3)–pyridine suspension mixture (3.0 mg: 0.5 ml), and the whole
mixture was stirred at room temperature for 8 h. The reaction mixture was
poured into saturated aqueous NaCl and the whole was extracted with
EtOAc. The EtOAc extract was washed with brine then dried over MgSO4.
Removal of the solvent under reduced pressure gave a crude product, which
was purified by ordinary-phase silica gel column chromatography [0.5 g, n-
hexane–EtOAc (5 : 1, v/v)] to furnish 6 (2 mg, 41%), which was identified by
comparison of the physical data ([a]D, IR, UV, 1H-NMR, MS) with reported
values.
Alkaline Hydrolysis of Camellioside B (2) A solution of 2 (20 mg) in
0.1% NaOMe–MeOH (5.0 ml) was stirred at room temperature for 1 h. The
reaction mixture was neutralized with Dowex HCR W2 (Hꢂ form) and the
residue was removed by filtration. After removal of the solvent from the fil-
trate in vacuo, the residue was separated by normal-phase silica gel column
chromatography [0.4 g, CHCl3–MeOH–H2O (7 : 3 : 1, lower layer, v/v/v)] to
give 1 (13 mg, 66%).
Bioassay. Animals Male Sprague-Dawley rats weighing about 230—
250 g and male white rabbits weighting about 2.0—3.0 kg were purchased
from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. The animals
were housed at a constant temperature of 23ꢁ2 °C and were fed a standard
laboratory chows (MF for rats and RC-4 for rabbits, Oriental Yeast Co., Ltd.,
Japan). The rats were fasted for 24—26 h prior to the beginning of the ex-
periment, but were allowed free access to tap water.
Ethanol- or Indomethacin-Induced Gastric Mucosal Lesions in Rats
The acute gastric lesions were induced by oral administration of ethanol or
indomethacin according to the method described previously with slight
modifications.22,23) Briefly, 99.5% ethanol (1.5 ml/rat) or indomethacin
(20 mg/kg, dissolved in 5% sodium bicarbonate, and then diluted in water
and neutralized with 0.2 M HCl and adjusted to 1.5 ml/rat) was administered
to 24—26 h fasted rats using a metal orogastric tube. One hour after admin-
istration of ethanol or 4 h after administration of indomethacin, the animals
were killed by cervical dislocation under ether anesthesia and the stomach
was removed and inflated by injection of 10 ml 1.5% formalin to fix the
inner and outer layers of the gastric walls. Subsequently, the stomach was in-
cised along the greater curvature and the lengths of gastric lesions were
measured as previously described, and the total length (mm) was expressed
as a lesion index.
Camellenodiol (5): 1H-NMR (CDCl3): d: 0.75 (1H, d-like, H-5), 0.80,
0.89, 0.95, 0.96, 1.00, 1.04, 1.19 (3H each, all s, H3-24, 29, 25, 30, 23, 26,
27), 1.00 (1H, m, H-1), 1.30 (1H, m, H-19), 1.32 (2H, m, H-7 and 21), 1.41
(1H, m, H-22), 1.45 (1H, m, H-19), 1.48 (1H, m, H-7), 1.54 (1H, m, H-9 and
21), 1.58 (1H, m, H-2), 1.63 (4H, m, H-1, 2, and H2-6), 1.81 (1H, d,
Jꢄ14.0 Hz, H-15), 1.98 (2H, m, H2-11), 2.21 (1H, m, H-22), 2.72 (1H, dd,
Jꢄ4.0, 14.0 Hz, H-18), 3.17 (1H, d, Jꢄ14.0 Hz, 15-H), 3.22 (1H, dd-like, H-
3), 5.51 (1H, dd-like, H-12); 13C-NMR (CDCl3 and pyridine-d5) dC given in
Table 2. Camellenodiol (5) was identified by comparison of the physical data
[[a]D, IR, 1H-NMR (90 MHz), and MS] with reported values.2)
Crystal Data for 5 Colorless prismatic crystals, mp 222.8—223.2 °C
(from aqueous acetone), C29H46O3·(CH3)2CO, Mꢄ500.76, crystal dimen-
sions: 0.25ꢇ0.18ꢇ0.30 mm, crystal system: orthorhombic, lattice type:
primitive, lattice parameters: aꢄ13.237(2), bꢄ28.911(2), cꢄ7.649(2) Å,
Vꢄ2927.2(8) Å3, space group: P212121 (#19), Zꢄ4, Dcalcꢄ1.136 g/cm3,
F
000ꢄ1104.00, m(CuKa)ꢄ5.65 cmꢀ1, temperature: 23.0 °C, structure solu-
tion: TEXSAN (direct method: SAPI91), residuals: Rꢄ0.081, Rwꢄ0.140,
R1ꢄ0.046, goodness of fit indicator: 1.38. All measurements were made on
a Rigaku AFC7R diffractometer with graphite monochromated CuKa
(lꢄ1.54178 Å) radiation and a rotating anode generator.
Preparation of the (R)-MTPA Ester (5b) and (S)-MTPA Ester (5c)
from 5 A solution of 5 (3.8 mg) in CH2Cl2 (0.5 ml) was treated with (R)-
a-methoxy-a-trifluoromethylphenylacetic acid [(R)-MTPA, 3.5 mg] in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC·HCl, 3.0 mg) and 4-dimethylaminopyridine (4-DMAP, 2.5 mg), and

612
Vol. 55, No. 4
Test samples were suspended in 5% acacia solution. Cimetidine and
omeprazole were suspended in 0.5% CMC–Na. Test samples in vehicle and
10) Nakamura S., Sugimoto S., Matsuda H., Yoshikawa M., Heterocycles,
to be published.
vehicle only (control group) was administered orally at a dose of 5 ml/kg 1 h 11) This work was partly reported in our preliminary communications:
prior to the application of ethanol or indomethacin.
Yoshikawa M., Morikawa T., Fujiwara E., Ohgushi T., Asao Y., Ma-
Statistical Analysis Values were expressed as meansꢁS.E.M. For sta-
tsuda H., Heterocycles, 55, 1653—1658 (2001).
tistical analysis, one-way analysis of variance followed by Dunnett’s test was 12) Audichya T. D., Ingle T. R., Bose J. L., Indian J. Chem., 9, 282—283
used. Probability (p) values less than 0.05 were considered significant. ED50 (1971).
values were estimated based on linear regressions of probit-transformed val- 13) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 843—845
ues of inhibition (%). (1986).
Platelet Aggregation Activity Wash rabbit platelets were prepared as 14) Morikawa T., Matsuda H., Yoshikawa M., Heterocycles, 68, 1139—
described previously.25) The platelets suspension (5ꢇ105 cells/ml) in 225 ml
of an assay buffer (137 mM NaCl, 2.7 mM KCl, 3.8 mM HEPES, pH 7.4) and
25 ml of 12 mM CaCl2/saline were mixed and incubated at 37 °C for 1 min.
1148 (2006).
15) Nakamura S., Murakami T., Nakamura J., Kobayashi H., Matsuda H.,
Yoshikawa M., Chem. Pharm. Bull., 54, 1545—1550 (2006).
One microliter of test sample dissolved in DMSO was added to 24 ml of the 16) Murakami T., Oominami H., Matsuda H., Yoshikawa M., Chem.
assay buffer, and the solution was dropped into the platelets suspension with Pharm. Bull., 49, 741—746 (2001).
stirring. Aggregation responses were recorded by an aggregometer (Hema 17) The H- and 13C-NMR spectra of 1—4 were assigned with the aid of
1
Tracer 1, Nikko Bioscience, Japan). The aggregation (%) was calibrated
with the platelets suspension (0% transmission) and the assay buffer (100%
transmission).
distortionless enhancement by polarization transfer (DEPT), homo-
and hetero-correlation spectroscopy (1H–1H, 13C–1H COSY), homo-
and hetero-nuclear Hartmann-Hahn spectroscopy (1H–1H, 13C–1H HO-
HAHA), and HMBC experiments.
References and Notes
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