Cycloartane glycosides from the rhizomes of Cimicifuga racemosa and their cytotoxic activities

Article abstract of DOI:10.1248/cpb.50.121

Phytochemical analysis of the rhizomes of Cimicifuga racemosa (Ranunculaceae) resulted in the isolation of twelve cycloartane glycosides (1-12), including four new ones (4-6, 12). The structures of the new compounds were determined by spectroscopic analys

Full text of DOI:10.1248/cpb.50.121

January 2002
Notes
Chem. Pharm. Bull. 50(1) 121—125 (2002)
121
Cycloartane Glycosides from the Rhizomes of Cimicifuga racemosa and
Their Cytotoxic Activities
a
,a
b
a
Kazuki WATANABE, Yoshihiro MIMAKI,* Hiroshi SAKAGAMI, and Yutaka SASHIDA
a
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432–1
Horinouchi, Hachioji, Tokyo 192–0392, Japan and Department of Dental Pharmacology, Meikai University School of
b
Dentistry, 1–1 Keyaki-dai, Sakado, Saitama 350–0283, Japan. Received July 23, 2001; accepted September 14, 2001
Phytochemical analysis of the rhizomes of Cimicifuga racemosa (Ranunculaceae) resulted in the isolation of
twelve cycloartane glycosides (1—12), including four new ones (4—6, 12). The structures of the new compounds
were determined by spectroscopic analysis, including two-dimensional (2D) NMR data, and chemical methods.
The isolated compounds were evaluated for their cytotoxic activities against human oral squamous cell carci-
noma (HSC-2) cells and normal human gingival fibroblasts (HGF).
Key words Cimicifuga racemosa; Ranunculaceae; cycloartane glycoside; cytotoxic activity; HSC-2 cell
Cimicifuga racemosa NUTT., commonly going by the name nals, and the results of elemental analysis were consistent
of black cohosh, is a herb indigenous to North America and with the deduced formula. The IR spectrum showed an ab-
Europe, and its rhizomes have long been used for the treat-
ment of a variety of ailments such as diarrhea, sore throat,
1
)
and rheumatism by Native Americans. Now, black cohosh
has become a well-known alternative herbal medicine with
health benefits in treating painful menstrual periods and
menopausal disorders not only in the United States but also
2
)
in European countries. Black cohosh has been revealed to
contain triterpene monoglycosides with the cycloartane
3
)
4)
5)
6)
skeleton, isoflavones, alkaloids, and phenylpropanoids,
among which the cycloartane glycosides are its main sec-
ondary metabolites and are considered to partially contribute
7
)
to the pharmacological effects of this herbal medicine. The
present investigation is part of a series of studies on the
8
)
chemical constituents of the herbal medicines. As a result,
a total of twelve cycloartane glycosides (1—12), including
four new compounds (4—6, 12), were isolated from the
MeOH extract of C. racemosa. This paper deals with the
structural assignments of the new glycosides on the basis
of spectroscopic analysis, including two-dimensional (2D)
NMR data, and chemical methods. The cytotoxic activities of
the isolated compounds against human oral squamous cell
carcinoma (HSC-2) cells and normal human gingival fibro-
blasts (HGF) are also reported.
Results and Discussion
The rhizomes of C. racemosa (dry weight of 5.2 kg) was
extracted with hot MeOH. The MeOH extract was subjected
to column chromatography over porous-polymer polysty-
rene resin (Diaion HP-20), silica gel, and octadecylsilaniz-
ed (ODS) silica gel, as well as preparative HPLC, to give
compounds 1—12. Compounds 1—3 and 7—11 were identi-
9
)
fied as cimigenol 3-O-a-L-arabinopyranoside (1), 25-O-
1
0)
methoxycimigenol 3-O-a-L-arabinopyranoside (2),
12b-
1
1)
hydroxycimigenol 3-O-a-L-arabinopyranoside (3), 27-de-
1
2)
13)
14)
oxyactein (7),
actein (8),
cimiracemoside F (9),
1
4)
14)
cimiracemoside G (10), and cimiracemoside H (11), re-
spectively.
Compound 4 was isolated as an amorphous solid. Its mo-
lecular formula was derived as C H O by data from the
3
7
58 11
ϩ
positive-ion FAB-MS, which showed an [MϩNa] ion at m/z
01. The C-NMR spectrum, with a total of 37 carbon sig-
1
3
7
∗
To whom correspondence should be addressed. e-mail: mimakiy@ps.toyaku.ac.jp
© 2002 Pharmaceutical Society of Japan

1
22
Table 1. H-NMR Chemical Shift Assignments of 4—6, 5a, 12, and 12a in Pyridine-d5
5a
Vol. 50, No. 1
1
H
4
5
6
12
12a
1
2
1.58
1.59 ddd
13.6, 13.3, 3.1)
1.28
2.33
1.91
1.52
1.61 ddd
(13.7, 13.5, 3.1)
1.25
2.40
1.97
1.49
1.47
(
1
.24
2.32
.90
1.23
2.06
1.80
1.10
2.27
1.85
1.07
1.91
1.80
1
3
5
6
3.48 dd (11.7, 4.4)
1.32
1.54
3.49 dd (11.7, 4.3)
1.32 dd (12.4, 4.1)
1.53
0.78 qd-like
(12.5, 1.8)
2.09
3.48 dd (11.6, 4.4)
1.24 dd (12.4, 5.5)
1.52
0.77 qd-like
(12.5, 2.1)
3.52 dd (11.7, 4.3)
1.39
1.60 dd (13.0, 1.6)
0.76 qd-like
(13.0, 1.6)
2.09
3.44 dd (11.7, 4.3)
1.23 dd (12.3, 4.2)
1.45
0.68 qd-like
(12.3, 1.8)
3.48 dd (11.5, 4.4)
1.23 dd (12.6, 4.3)
1.50
0.75 qd-like
(12.7, 2.0)
0
.77 qd-like
12.7, 1.9)
2.14
.27
(
7
2.07
1.19
1.26
0.92
1.26
0.94
1
1.22
1.28
8
1
1.82
2.08
1.85 dd (12.0, 5.0)
2.68 dd (15.5, 9.1)
1.46 dd (15.5, 3.7)
4.36 dd (9.1, 3.7)
4.46 s
1.87 dd (12.5, 4.5)
2.13
1.15
1.79
4.36 s
1.56 dd (11.8, 5.3)
2.71 dd (16.0, 9.0)
1.15 dd (16.0, 3.7)
5.13 dd (9.0, 3.7)
1.90 dd (12.3, 7.9)
1.58 dd (12.1, 5.2)
2.76 dd (16.0, 9.0)
1.15 dd (16.0, 3.5)
5.15 dd (9.0, 3.5)
1.82 dd (10.7, 8.5)
1.75
1
2.79 dd (15.6, 9.0)
2.69 dd (15.5, 9.0)
1.47 dd (15.5, 3.8)
4.36 dd (9.0, 3.8)
4.49 s
1
.45 dd (15.6, 2.6)
1
1
2
5
4.13 dd (9.0, 2.6)
4.44 s
1
.71
1
1
1
1
6
7
8
9
—
1.83
1.42 s
0.62 d (4.1)
—
—
—
5.00 q-like (7.9)
1.81 dd (10.6, 7.9)
1.35 s
0.53 d (4.1)
0.19 d (4.1)
2.23
5.01 q-like (7.9)
1.82 dd (10.7, 8.5)
1.38 s
0.57 d (4.2)
0.23 d (4.2)
2.25
2.41 d (9.0)
1.52 s
0.65 d (4.1)
0.34 d (4.1)
2.23
2.38 d (9.0)
1.49 s
0.63 d (4.1)
0.35 d (4.1)
2.18
4.13 dd (10.7, 4.5)
3.98 dd (10.7, 5.8)
2.41 ddd
(13.6, 8.4, 8.4)
1.50
2.34 br d (6.5)
1.37 s
0.58 d (4.0)
0.32 d (4.0)
2.13
0
.38 d (4.1)
2
2
0
1
1.83
1.39 d (5.8)
4.11 dd (10.6, 4.5)
1.26 d (6.6)
1.32 d (6.3)
1.33 d (6.3)
4
.00 dd (10.6, 5.9)
2
2
2
3
2.39
2.46 ddd
2.65
3.88 d (10.5)
3.89 d (10.5)
(
13.5, 8.5, 8.4)
1
.10
1.46
4.83 br d (8.3)
1.75
5.40 ddd
4.62 br d (9.0)
4.79 br d (8.6)
(
10.7, 8.5, 2.5)
2
2
2
2
2
3
4
6
7
8
9
0
4.17 br s
1.69 s
1.71 s
1.21 s
1.27 s
3.92 d (0.6)
1.49 s
1.49 s
1.23 s
1.28 s
3.92 d (0.8)
1.45 s
1.47 s
1.21 s
1.14 s
3.03 d (8.5)
1.25 s
1.40 s
1.21 s
1.30 s
4.22 s
1.68 s
1.75 s
0.85 s
1.28 s
0.96 s
4.22 s
1.19 s
1.75 s
0.87 s
1.68 s
1.03 s
1.00 s
1.00 s
1.01 s
1.05 s
Ara 1Ј
4.79 d (6.9)
4.44 dd (7.7, 6.9)
4.17 br d (6.9)
4.32 br s
4.78 d (7.0)
4.43 dd (8.7, 7.0)
4.16 dd (8.7, 3.4)
4.32 br s
4.29 dd (11.8, 2.8)
3.79 dd (11.8, 1.1)
4.82 d (7.0)
4.47 dd (8.8, 7.0)
4.19 dd (8.8, 3.3)
4.34 br s
4.33 dd (12.9, 2.8)
3.82 br d (10.6)
2.06 s
4.77 d (7.0)
4.43 dd (8.8, 7.0)
4.16 dd (8.8, 3.3)
4.31 br s
4.29 dd (11.7, 2.7)
3.78 br d (10.9)
2.07 s
2
3
4
5
Ј
Ј
Ј
Јa 4.29 dd (12.4, 2.3)
b
3.79 br d (12.4)
1.98 s
Ac
2.09 s
Ϫ1
1
sorption band for hydroxyl groups at 3419 cm . The H- hydroxycimigenol 3-O-a-L-arabinopyranoside.
NMR spectrum of 4 showed signals for a cyclopropane Compound 5 was shown to have the molecular formula
methylene group at d 0.62 and 0.38 (each 1H, d, Jϭ4.1 Hz), C H O on the basis of the positive-ion FAB-MS (m/z 675
3
5
56 11
ϩ
13
six tertiary methyl groups at d 1.71, 1.69, 1.42, 1.27, 1.21, [MϩNa] ), C-NMR spectrum, and elemental analysis. The
1
and 1.00 (each 3H, s), a secondary methyl group at d 1.39
H-NMR spectrum of 5 contained signals for a cyclopropane
(
3H, d, Jϭ5.8 Hz), and an anomeric proton at d 4.79 (1H, d, methylene group at d 0.65 and 0.34 (each 1H, d, Jϭ4.1 Hz),
1
13
Jϭ6.9 Hz). These H-NMR data and C-NMR spectral fea- six tertiary methyl groups at d 1.52, 1.49ϫ2, 1.28, 1.23, and
tures of 4 were quite similar to those of 3. In addition, the 1.00 (each 3H, s), and an anomeric proton at d 4.78 (1H, d,
presence of an acetyl group in 4 was shown by the IR Jϭ7.0 Hz). Enzymatic hydrolysis of 5 with naringinase fur-
Ϫ1
1
13
(
1734 cm ), H-NMR [d 1.98 (3H, s)], and C-NMR [d nished the genuine aglycon (C H O : 5a) and L-arabinose.
70.2 (CϭO), 22.3 (Me)] spectra. Alkaline hydrolysis of 4 Comparsion of the H- and C-NMR spectra of 5a with
with Na CO solution yielded 3, indicating that 4 was a those of 12b-hydroxycimigenol showed their considerable
monoacetate of 3. When the C-NMR spectrum of 4 was structural similarity. However, the molecular formula of 5a
compared with that of 3, the signal due to C-25 was shifted was higher by one oxygen atom than that of 12b-hydroxy-
to a lower field by 12.1 ppm, whereas the signals assignable cimigenol, and complete acetylation of 5a gave the corre-
to C-24, C-26, and C-27 occurred, respectively, at higher sponding pentaacetate (5b). This indicated the presence of
fields by 3.3, 2.4, and 3.7 ppm. Thus, the acetyl moiety was one more hydroxyl group in addition to the C-3b, C-12b, C-
revealed to be linked to the aglycon C-25 hydroxyl group, 15a, and C-25 hydroxyl groups. When the H-NMR spec-
and the structure of 4 was formulated as 25-O-acetyl-12b- trum of 5a was compared with that of 12b-hydroxy-
3
0
48
7
1
13
1
1
1)
2
3
1
3
1

January 2002
123
1
3
cimigenol, the three-proton doublet signal observed at d 1.39 Table 2. C-NMR Chemical Shift Assignments of 4—6, 5a, 12, and 12a
in Pyridine-d5
(
d, Jϭ6.0 Hz) in 12b-hydroxycimigenol, was displaced by
the hydroxymethylene signals at d 4.13 (dd, Jϭ10.7, 4.5 Hz)
and 3.98 (dd, Jϭ10.7, 5.8 Hz). All other signals appeared at
almost the same positions between the two compounds. Fur-
thermore, tracing out the spin-coupling correlations, starting
from H-17 at d 2.38 in the H– H shift correlation spectro-
scopy (COSY) spectrum of 5a, revealed the structural frag-
C
4
5
5a
6
12
12a
1
2
3
4
5
6
7
8
9
32.5
30.0
88.5
41.3
47.3
20.9
26.1
47.3
20.7
26.6
40.8
72.7
47.8
48.2
79.8
112.8
59.6
12.0
30.7
23.9
21.0
38.5
71.6
86.7
83.1
21.6
23.3
11.8
25.7
15.3
107.4
72.9
74.6
69.5
66.7
170.2
32.4
29.9
88.4
41.2
47.2
20.9
25.9
47.0
20.7
26.9
39.4
73.2
47.9
48.6
80.0
112.4
54.1
12.1
30.5
31.6
66.3
32.8
71.9
88.8
70.9
25.3
27.1
11.9
25.7
15.3
107.4
72.9
74.6
69.5
66.7
32.4
31.0
77.8
40.8
46.9
20.8
25.9
47.0
20.8
27.0
39.4
73.0
47.8
48.4
79.9
112.3
53.8
11.9
30.7
31.4
66.0
32.8
71.8
88.7
70.9
25.3
26.7
12.0
26.0
14.6
32.2
30.0
88.4
41.3
47.5
21.0
26.7
48.2
20.1
26.8
26.0
33.0
41.5
46.1
82.9
220.0
60.0
19.8
30.5
28.0
20.3
37.0
72.1
65.2
58.5
24.7
19.3
12.0
25.7
15.4
107.4
72.9
74.6
69.5
66.7
170.6
21.0
31.9
29.8
88.1
41.1
47.0
20.4
25.7
45.6
19.9
26.7
36.7
76.9
49.3
48.1
43.0
72.1
52.4
13.7
29.6
34.4
17.4
86.7
105.5
83.3
83.5
24.7
27.8
19.6
25.7
15.2
107.3
72.9
74.6
69.5
66.7
170.5
21.6
32.2
31.0
77.7
40.9
46.9
20.8
25.9
45.9
19.8
26.9
36.9
77.1
49.4
48.1
43.0
72.1
52.3
13.7
30.0
34.4
17.4
86.7
105.5
83.3
83.5
26.1
27.8
19.7
24.8
14.7
1
1
ment of –C(17)H–C(20)H–(C(21)H O–)–C H –C(23)H(–O–)–
2
(22)
2
C(24)H(–O–)– constituting 5a. Thus, the presence of a C-21
hydroxyl group was evident. The C-20a configuration was
ascertained by nuclear Overhauser effect (NOE) correlations
from H-20 (d 2.18) to Me-18 (d 1.49) and H-22b (d 2.41) in
the phase-sensitive NOE correlation spectroscopy (NOESY)
1
1
1
0
1
2
1
spectrum. In the H-detected heteronuclear multiple-bond
13
1
1
1
1
4
5
6
7
connectivities (HMBC) spectrum of 5, a long-range correla-
tion was observed from the anomeric proton of the a-L-ara-
binopyranosyl group at d 4.78 to the aglycon C-3 carbon at d
8
8.4. Accordingly, the structure of 5a, a new triterpene sa-
18
19
pogenin, was shown to be (20R,23R,24S)-16a,23:16a,24-di-
epoxy-9,19-cyclolanostane-3b,12b,15a,21,25-pentol (12b,21-
dihydroxycimigenol), and consequently, the structure of 5
was established as 12b,21-dihydroxycimigenol 3-O-a-L-
arabinopyranoside.
2
2
2
2
0
1
2
3
24
25
26
Compound 6 was analyzed for C H O by combined
3
7
58 10
ϩ
13
positive-ion FAB-MS (m/z 685 [MϩNa] ), C-NMR spec-
2
2
2
7
8
9
trum with distortionless enhancement by polarization trans-
1
fer (DEPT) data, and elemental analysis. The H-NMR spec-
trum of 6 showed a pair of cyclopropane methylene proton
signals at d 0.58 and 0.32 (each 1H, d, Jϭ4.0 Hz), six ter-
tiary methyl proton signals at d 1.40, 1.37, 1.30, 1.25, 1.21,
and 1.05 (each 3H, s), a secondary methyl proton signal at d
30
Ara 1Ј
2
3
4
5
Ј
Ј
Ј
Ј
1
.26 (3H, d, Jϭ6.6 Hz). In addition, a secondary hydroxyl
group, a carbonyl group, an acetyloxy group, and an epoxy
ring, as well as an a-L-arabinopyranosyl moiety, were re-
vealed to be included in the structure of 6 by analysis of its
Ac
170.5
21.6
2
2.3
1
13
H- and C-NMR spectra. Thus, 6 was presumed to be an a-
L-arabinopyranoside of a shengmanol derivative. Chemical Table 3. Cytotoxic Activities of Compounds 1—12 and Etoposide against
HSC-2 Cells and HGF
conversion of 6 to the corresponding cimigenol saponin was
carried out according to the method reported by Kusano et
IC50 (mM)
1
5)
al. After deacetylation of 6 with Na CO solution, the hy-
2
3
Compounds
drolysate was treated with 2.5% AcOH at 95 °C to furnish 1.
This chemical evidence allowed the structural determination
of 6 as 23-O-acetylshengmanol 3-O-a-L-arabinopyranoside.
Compound 12 was deduced as C H O from its FAB-
HSC-2
HGF
1
2
3
4
5
6
7
8
9
Ͼ400
30
Ͼ400
54
3
7
58 11
74
352
271
265
267
276
141
275
280
294
261
Ͼ400
1
3
1
MS, C-NMR spectral, and elemental analysis data. The H-
NMR spectrum showed signals characteristic of the 9,19-cy-
142
222
63
211
44
80
18
171
170
24
1
3
cloartane derivative. Analysis of the C-NMR spectrum of
2 and comparison with that of 11 implied that the aglycon
1
of 12 was identical to that of 11, but differed from 11 in
terms of the monosaccharide constituent. Instead of the sig-
nals for a xylose moiety, five signals assignable to an a-L-
arabinopyranosyl residue were observed at d 107.3 (CH),
1
0
11
12
Etoposide
7
2.9 (CH), 74.6 (CH), 69.5 (CH), and 66.7 (CH ). Hydrolysis
2
of 12 with naringinase gave L-arabinose and an aglycon
12a), which was identical to the product obtained by enzy-
(
matic hydrolysis of 11. The glycosidic linkage of the arabi- acetyloxy-16b,23:22,25-diepoxy-23,24-dihydroxy-9,19-
nosyl group to C-3 of the aglycon was ascertained by an cyclolanostan-3b-yl a-L-arabinopyranoside.
HMBC correlation from the H-1 arabinose proton at d 4.77
The isolated compounds were evaluated for their cytotoxic
d, Jϭ7.0 Hz) to the C-3 aglycon carbon at d 88.1. Thus, activities against HSC-2 cells and HGF. The results are
(
the structure of 12 was shown to be (22R,23R,24R)-12b- shown in Table 3. It is suggested that slight differences in the

1
24
Vol. 50, No. 1
aglycon structures exerted some effects on the cytotoxic ac- by passing it through an Amberlite IR-120B (Organo, Tokyo, Japan) column
and purified by silica gel column chromatography eluting with
tivities. Although 1 did not exhibit any apparent cytotoxicity
CHCl –MeOH (9 : 1) to afford 3 (1.3 mg).
3
against HSC-2 cells even at the sample concentration of
00 mM, the 25-O-methyl derivative (2) of 1 dose-depen-
27
Compound 5: Amorphous solid, [a]D ϩ10.0° (MeOH, cϭ0.10). FAB-MS
positive mode) m/z: 675 [MϩNa] . Anal. Calcd for C H O ·1/2H O: C,
4
ϩ
(
3
5
56 11
2
Ϫ1
dently reduced the viable cell number and its IC value was 63.52; H, 8.68. Found: C, 63.65; H, 8.36. IR n (film) cm : 3384 (OH),
5
0
max
2
9
946 and 2924 (CH), 1404, 1365, 1236, 1141, 1077, 1037, 1021, 1006, 977,
calculated to be 30 mM. The C-27 hydroxy derivative (8) of 7,
which is the main secondary metabolite of C. racemosa, was
more cytotoxic than 7. In the cimiacerogenin derivatives (9—
1
13
47. H-NMR, see Table 1. C-NMR, see Table 2.
Enzymatic Hydrolysis of 5 Compound 5 (20.0 mg) was treated with
naringinase (Sigma, EC 232-962-4) (80 mg) in AcOH/AcOK buffer (pH 4.3,
0 ml) at room temperature for 72 h. The reaction mixture was passed
1
2), the cytotoxicity of the 7(8)-dehydro saponins (9, 10) was
1
more potent than that of the corresponding saturated through a combination of Sep-Pak C18 cartridge (Waters, Milford, MA,
U.S.A.) and Toyopak IC-SP M cartridge (Tosoh) eluting with 20% MeOH
followed by MeOH. The MeOH eluate fraction was purified by silica gel
saponins (11, 12). It is notable that 10 showed about 15-fold
higher cytotoxic activity against HSC-2 tumor cells than
against normal HGF.
column chromatography eluting with CHCl –MeOH (19 : 1) to afford 5a
3
(
8.6 mg). The 20% MeOH eluate fraction was analyzed by HPLC under
the following conditions: column, Capcell Pak NH2 UG80 (4.6 mm
Experimental
i.d.ϫ250 mm, 5 mm, Shiseido, Tokyo, Japan); solvent, MeCN–H O (17 : 3);
2
1
NMR spectra were recorded on a Bruker DRX-500 (500 MHz for H- flow rate, 1.0 ml/min; detection, reflactive index (RI) and optical rotation
NMR, Karlsruhe, Germany) spectrometer using standard Bruker pulse pro- (OR). Identification of L-arabinose was carried out by comparison of its re-
grams. Diaion HP-20 (Mitsubishi-Kasei, Tokyo, Japan), Sephadex LH-20 tention time and OR with those of an authentic sample; t (min): 9.93 (posi-
R
(
Pharmacia, Uppsala, Sweden), silica gel (Fuji-silysia Chemical, Aichi, tive optical rotation).
Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan) were used for Compound 5a: Amorphous solid, [a] ϩ6.0° (MeOH, cϭ0.10). High-res-
column chromatography. HPLC was performed using a system comprised of olution electron impact (HR-EI)-MS m/z: 520.3397 [M] (C H O , Calcd
2
6
D
ϩ
3
0
48
7
Ϫ1
a Tosoh CCPM pump (Tokyo, Japan), a Tosoh CCP PX-8010 controller, a
Tosoh RI-8010 detector, a Shodex OR-2 detector (Showa-Denko, Tokyo, 1384, 1288, 1169, 1071, 1026, 984, 948. H-NMR, see Table 1. C-NMR,
Japan), and Rheodyne injection port. A Capcell Pak C18 UG80 column see Table 2.
for 520.3400). IR nmax (film) cm : 3358 (OH), 2935 and 2889 (CH), 1454,
1
1
3
(
10 mm i.d.ϫ250 mm, ODS, 5 mm, Shiseido, Tokyo, Japan) was employed
Complete Acetylation of 5a Compound 5a (3.3 mg) was acetylated
for preparative HPLC. The following reagents were obtained from the indi- with a mixture of Ac O (1.0 ml) and pyridine (1.0 ml) in the presence of 4-
2
cated companies: Dulbecco’s modified Eagle medium (DMEM) (Gibco, (dimethylamino)pyridine (1.8 mg) as catalyst, and the crude acetate was
Grand Island, NY, U.S.A.); fetal bovine serum (FBS) (JRH Biosciences, chromatographed on silica gel eluting with hexane–Me CO (4 : 1) to afford
2
Lenexa, KS, U.S.A.); penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)- the corresponding pentaacetate (5b) (1.6 mg).
Ϫ1
2
,5-diphenyl-2H-tetrazolium bromide (MTT), and a-minimum essential
Compound 5b: Amorphous solid, IR nmax (film) cm : 2917 and 2849
1
medium (a-MEM) (Sigma, St. Louis, MO, U.S.A.). All other chemicals (CH), 1736 (CϭO), 1367, 1242, 1139, 1025, 985. H-NMR (pyridine-d ) d:
5
used were of biochemical reagent grade.
5.90 (1H, s, H-15), 5.24 (1H, dd, Jϭ11.6, 3.4 Hz, H-12), 4.76 (1H, dd,
Plant Material The plant material defined as the rhizomes of C. race- Jϭ14.5, 5.6 Hz, H-3), 4.66 (1H, d, Jϭ10.7 Hz, H-23), 4.53 (1H, dd, Jϭ13.6,
mosa was provided by Tokiwa Phytochemical Co., Ltd., Chiba, Japan. A 4.7 Hz, H-21a), 4.17 (1H, d, Jϭ10.7 Hz, H-24), 3.99 (1H, dd, Jϭ13.6,
small amount of the sample is preserved in our laboratory (00-CR-011).
8.7 Hz, H-21b), 2.27, 2.25, 2.09, 2.07, 2.00 (each 3H, s, Ac), 1.69, 1.58,
Extraction and Isolation The plant material (dry weight, 5.2 kg) was 1.42, 1.28, 0.91, 0.88 (each 3H, Me), 0.56 (1H, d, Jϭ5.3 Hz, H-19a), 0.30
extracted with hot MeOH (21 l) for 3 h twice. The MeOH extract was con- (1H, d, Jϭ5.3 Hz, H-19b).
2
6
centrated under reduced pressure, and the viscous concentrate (445 g) was
Compound 6: Amorphous solid, [a] Ϫ26.0° (MeOH, cϭ0.10). FAB-MS
D
ϩ
passed through a Diaion HP-20 column, successively eluting with 30% (positive mode) m/z: 685 [MϩNa] . Anal. Calcd for C H O ·3/2H O: C,
MeOH, 50% MeOH, MeOH, EtOH, and EtOAc. Column chromatography of 64.42; H, 8.91. Found: C, 64.75; H, 8.91. IR nmax (film) cm 3408 (OH),
the 50% MeOH eluate portion (14 g) on silica gel and elution with stepwise 2935 and 2889 (CH), 1739 (CϭO), 1456, 1381, 1241, 1068, 991, 953. H-
3
7
58 10
2
Ϫ1
1
1
3
gradient mixtures of CHCl –MeOH–H O (19 : 1 : 0; 9 : 1 : 0; 40 : 10 : 1; NMR, see Table 1. C-NMR, see Table 2.
3
2
2
0 : 10 : 1), and finally with MeOH alone, gave 7 fractions (frs. I—VII). Fr.
Transformation of 6 into 1 Compound 6 (1.5 mg) was dissolved in
IV was subjected to column chromatography on silica gel eluting with MeOH (0.2 ml) and added to 1% Na CO solution (1.0 ml), which was
2
3
CHCl –MeOH (30 : 1; 10 : 1; 5 : 1) and ODS silica gel with MeOH–H O stirred at room temperature for 12 h. The reaction mixture was neutralized
3
2
(
8 : 5) to give 4 (12.5 mg). Fr. V was refined using ODS silica gel column by addition of 5% AcOH (0.6 ml) and extracted with EtOAc (3.0 mlϫ3).
chromatography eluting with MeOH–H O (8 : 5) to give 3 (67.0 mg). Fr. VII After removal of EtOAc, the residue was dissolved in a mixture of 1,4-diox-
2
was chromatographed on silica gel eluting with CHCl –MeOH (9 : 1) and ane (0.6 ml) and 5% AcOH (0.6 ml), and was heated at 95 °C for 2 h under
3
ODS silica gel with MeOH–H O (4 : 3) to give 5 (55.3 mg). The MeOH elu- an Ar atmosphere. The reaction mixture was concentrated and subjected to
2
ate portion (181 g) was chromatographed on silica gel and elution with step- column chromatography on silica gel eluting with CHCl –MeOH (19 : 1) to
3
wise gradient mixtures of CHCl –MeOH (19 : 1; 9 : 1; 4 : 1; 2 : 1), and finally give 1 (0.6 mg).
3
2
6
with MeOH alone, gave 4 fractions (frs. VIII—XI). Fr. IX was subjected to
Compound 12: Amorphous solid, [a] Ϫ20.0° (MeOH, cϭ0.10). FAB-
D
ϩ
column chromatography on silica gel eluting with CHCl –MeOH (19 : 1) MS (positive mode) m/z: 701 [MϩNa] . Anal. Calcd for C H O ·H O: C,
3
37 58 11
2
Ϫ1
and further divided into two fractions (frs. IXa, IXb). Fr. IXa was suspended 63.77; H, 8.68. Found: C, 63.74; H, 8.80. IR nmax (film) cm : 3417 (OH),
in MeOH and the insoluble solid was filtered off. The filtrate was subjected 2966 and 2936 (CH), 1731 (CϭO), 1456, 1381, 1246, 1145, 1060, 986, 950.
1
13
to column chromatography on silica gel eluting with CHCl –MeOH (30 : 1;
H-NMR, see Table 1. C-NMR, see Table 2.
3
1
9 : 1), ODS silica gel with MeCN–H O (1 : 1) and MeOH–H O (8 : 3), and
Enzymatic Hydrolysis of 12 Compound 12 (7.6 mg) was subjected to
2
2
on Sephadex LH-20 with MeOH to give 1 (39.0 mg), 2 (102 mg), 6 (9.8 mg), enzymatic hydrolysis using naringinase as described for 5 to give an aglycon
(100 mg), and 8 (810 mg). Fr. IXb was separated by subjecting it to a silica (12a) (4.3 mg) and a sugar fraction. HPLC analysis of the sugar fraction
gel column eluting with CHCl –MeOH (19 : 1; 9 : 1; 4 : 1), an ODS silica gel under the same conditions as in the case of that of 5a showed the presence of
7
3
column with MeCN–MeOH–H O (2 : 2 : 3; 1 : 1 : 1; 8 : 8 : 5) and MeOH–H O
L-arabinose. Compound 12a was identical to the aglycon obtained by enzy-
2
2
1
13
(
8 : 5), and to preparative HPLC using MeCN–MeOH–H O (8 : 8 : 11) to
matic hydrolysis of 11. Since the H- and C-NMR spectral data for 12a
have not been reported previously, they are shown in Tables 1 and 2.
Cell Culture HSC-2 cells were maintained as monolayer cultures at
2
yield 9 (39.8 mg), 10 (10.2 mg), 11 (45.8 mg), and 12 (17.3 mg).
2
6
Compound 4: Amorphous solid, [a] ϩ26.0° (MeOH, cϭ0.10). FAB-MS
D
ϩ
(
positive mode) m/z: 701 [MϩNa] . Anal. Calcd for C H O ·2H O: C, 37 °C in DMEM supplemented with 10% heat-inactivated FBS in a humidi-
37 58 11 2
Ϫ1
16)
6
2
1
2.16; H, 8.74. Found: C, 62.44; H, 8.47. IR nmax (film) cm : 3419 (OH),
fied 5% CO2 atmosphere. HGF were isolated, as described previously.
936 and 2869 (CH), 2869 (CH), 1734 (CϭO), 1456, 1372, 1249, 1141,
Briefly, gingival tissues were obtained from healthy gingival biopsies from a
1
13
088, 1044, 1024, 988, 947. H-NMR, see Table 1. C-NMR, see Table 2.
10-year-old girl, undergoing periodontal surgery. The tissue was cut into 1 to
3
Alkaline Hydrolysis of 4 Compound 4 (2.5 mg) was dissolved in 2 mm pieces, washed twice with phosphate-buffered saline (PBS, 0.01 M
MeOH (0.5 ml) and added to 2% Na CO solution (2.0 ml), which was
stirred at room temperature for 24 h. The reaction mixture was neutralized cillin and 100 mg/ml streptomycin, and placed into 25 cm tissue culture
phosphate buffer, 0.15 M NaCl, pH 7.4) supplemented with 100 U/ml peni-
2
3
2

January 2002
125
flask. The explants were incubated in a-MEM supplemented with 30% FBS
and antibiotics. When outgrowth of the cells was observed, the medium was
replaced twice until the cells reached confluence. The cells were detached
3) Radics L., Kajtar-peredy M., Corsano S., Standioli L., Tetrahedron
Lett., 1975, 4287—4290.
4) Struck D., Tegtmeier M., Harnischfeger G., Planta Med., 63, 289
(1997).
2
from the monolayer by trypsinizaition and recultured in 100 cm tissue cul-
ture flasks until confluent monolayers were again obtained. Cells between
the fifth and seventh passages were used.
5) Crum J. D., Cassady J. M., Olmstead P. M., Picha N. J., Proc. West Va.
Acad. Sci., 37, 143—147 (1965).
Assay for Cytotoxic Activity Cells were trypsinized and inoculated at
ϫ10 per each 96-microwell plate (Falcon, flat bottom, treated polystyrene,
6) Kruse S. O., Lohning A., Pauli G. F., Winterhoff H., Nahrstedt A.,
Planta Med., 65, 763—764 (1999).
3
6
Becton Dickinson, San Jose, CA, U.S.A.), and incubated for 24 h. After
washing once with PBS, they were treated for 24 h without or with test com-
pounds. They were washed once with PBS and incubated for 4 h with
7) Liske E., Adv. Ther., 15, 45—53 (1998).
8) Kuroda M., Mimaki Y., Harada H., Sakagami H., Sashida Y., Natural
Medicines, 55, 134—138 (2001).
0
.2 mg/ml MTT in DMEM supplemented with 10% FBS. After the medium
9) Ye W., Zhang J., Che C. T., Ye T., Zhao S., Planta Med., 65, 770—772
(1999).
10) Bedir E., Khan I. A., Pharmazie, 56, 268—269 (2001).
11) Kusano A., Shibano M., Kusano G., Chem. Pharm. Bull., 43, 1167—
1170 (1995).
was removed, the cells were lysed with 0.1 ml dimethyl sulfoxide (DMSO),
and the relative viable cell number was determined by measuring the
absorbance at 540 nm of the cell lysate, using Labsystems Multiskan
®
(
Biochromatic, Helsinki, Finland) connected to a Star/DOT Matrix printer
JL-10. The IC50 value, the concentration that reduces the viable cell number 12) Koeda M., Aoki Y., Sakurai N., Nagai M., Chem. Pharm. Bull., 43,
by 50%, was determined from the dose–response curve.
771—776 (1995).
1
3) Kusano A., Takahira M., Shibano M., In Y., Ishida T., Miyase T., Ku-
sano G., Chem. Pharm. Bull., 46, 467—472 (1998).
Acknowledgments We are grateful to Dr. Y. Shida and Mr. H. Fukaya,
Tokyo University of Pharmacy and Life Science, for the measurements of 14) Shao Y., Harris A., Wang M., Zhang H., Cordell G. A., Bowman M.,
the MS and elemental analysis.
Lemmo E., J. Nat. Prod., 63, 905—910 (2000).
5) Kusano A., Shibano M., Kusano G., Miyase T., Chem. Pharm. Bull.,
44, 2078—2085 (1996).
1
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Liberman S., J. Womens Health, 7, 525—529 (1998).
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