J. Nat. Prod.

2004, 67, 1511-1516 1511

Triterpene Saponins from the Roots of Clematis chinensis

Yoshihiro Mimaki,*,† Akihito Yokosuka,† Mari Hamanaka,† Chiseko Sakuma,† Takao Yamori,‡ and
Yutaka Sashida†
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan,
and Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1, Kami-ikebukuro,
Toshima-ku, Tokyo 170-8455, Japan

Received April 1, 2004

A saponin-enriched fraction prepared from the MeOH extract of the roots of Clematis chinensis showed
cytotoxic activity against HL-60 promyelocytic leukemia cells, from which five new triterpene saponins
(1-5) based on oleanolic acid, along with three known saponins (6-8), were isolated. The structures of
the new saponins were determined on the basis of spectroscopic analysis, including extensive 1D and 2D
NMR data and hydrolysis followed by chromatographic and spectroscopic analysis. Among the isolated
saponins, monodesmosidic saponins exhibited cytotoxic activities against cultured tumor cells.

There are about 250 species belonging to the genus

Clematis (Ranunculaceae) in the world. Clematis chinensis
Osbeck is native to People’s Republic of China, and its roots
have long been used as an analgesic, a diuretic, an
antitumor, and an anti-inflammatory agent in traditional
Chinese medicine.1 Some pharmacological studies of this
crude drug showed that it had a significant hepatic
protective and hypotensive effects.2,3 The roots of C. chin-
ensis are rich in saponins, and Kizu and Tomimori isolated
more than 20 prosapogenins from the alkaline-treated
crude saponin fraction of the roots extract.4-8 Recently,
three new bisdesmosidic triterpene saponins named clem-
atichinenosides A-C have been isolated from the intact
saponin fraction of C. chinensis roots.9,10 During the course
of a systematic survey of biologically active triterpene
saponins from higher plants,11-22 we have found that a
saponin-enriched fraction prepared from the MeOH extract
of C. chinensis roots showed cytotoxic activity against HL-
60 human promyelocytic leukemia cells. Chromatographic
separation of the saponin fraction has resulted in the
isolation of five new triterpene saponins with up to a total
of nine monosaccharides (1-5), along with three known
triterpene saponins (6-8). This paper reports the struc-
tural determination of the new saponins (1-5) on the basis
of spectroscopic analysis, including extensive 1D and 2D
NMR data and hydrolysis followed by chromatographic and
spectroscopic analysis, and also the cytotoxic activities of
the isolated saponins.

Results and Discussion

The dry roots of C. chinensis (5.0 kg) were extracted with β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-R-
hot MeOH. The MeOH extract was passed through a L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-R-L-rham-
porous-polymer resin (Diaion HP-20) column and divided nopyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f6)-β-D-glucopy-
into 30% MeOH, 60% MeOH, 80% MeOH, MeOH, EtOH, ranosyl ester (7),10 and 3β-[(O-β-D-glucopyranosyl-(1f4)-
and EtOAc eluate fractions. The MeOH eluate fraction O-β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-
exhibited a potent HL-60 cell growth inhibitory activity R-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-R-L-
(98.6% inhibition at a sample concentration of 10 µg/mL; rhamnopyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f6)-β-D-
IC50 1.4 µg/mL) and was subjected to column chromatog- glucopyranosyl ester (8),10 respectively.
raphy over silica gel and octadecylsilanized (ODS) silica Compound 1 was obtained as an amorphous solid, [R]25D
gel, giving 1-8. Compounds 6-8 were identified as 3β-[(O- -82.0° (MeOH). The high-resolution (HR) FABMS (positive
β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-R- mode) of 1 showed an accurate [M + Na]+ ion peak at m/z
L-arabinopyranosyl)oxy]olean-12-en-28-oic acid (6),5 3β-[(O- 1359.6653 in accordance with an empirical molecular
formula of C64H104O29, which was supported by the 13C
* To whom correspondence should be addressed. Tel: +81-426-76-4577. NMR spectrum showing a total of 64 signals and various
Fax: +81-426-76-4579. E-mail: [email protected].
†
Tokyo University of Pharmacy and Life Science. DEPT data (C × 8, CH × 33, CH2 × 14, and Me × 9). The
‡
Japanese Foundation for Cancer Research. IR spectrum of 1 showed a broad absorption band for
10.1021/np040088k CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/29/2004
1512 Journal of Natural Products, 2004, Vol. 67, No. 9 Mimaki et al.

Table 1. 1H and 13C NMR Data for the Glycosidic Moiety of 1 in Pyridine-d5 at 318 K
position 1H J (Hz) 13C position 1H J (Hz) 13C

Ara 1 4.86 d 6.0 105.1 Glc 1 4.97 d 7.8 103.0

2 4.57 dd 7.5, 6.0 75.7 2 3.88 dd 9.0, 7.8 74.1
3 4.28 dd 7.5, 4.0 74.4 3 4.21 dd 9.0, 9.0 76.3
4 4.27 br s 69.2 4 4.17 dd 9.0, 9.0 81.8
5a 4.32 m 65.5 5 3.89 br dd 9.0, 2.8 76.5
b 3.84 br d 10.3 6a 4.47 dd 11.4, 2.8 61.8
b 4.43 br d 11.4
Rha 1 6.24 br s 101.5 Glc′ 1 5.12 d 7.8 104.9
2 4.90 br d 2.9 71.9 2 4.04 dd 9.0, 7.8 74.8
3 4.69 dd 9.5, 2.9 82.0 3 4.19 dd 9.0, 9.0 78.2
4 4.44 dd 9.5, 9.5 72.7 4 3.97 dd 9.0, 9.0 71.7
5 4.63 dq 9.5, 6.1 69.8 5 4.06 ddd 9.0, 5.6, 2.1 76.7
6 1.56 d 6.1 18.4 6a 4.65 dd 10.5, 2.1 68.5
b 3.96 dd 10.5, 5.6
Rib 1 5.85 d 5.3 104.6 Rha′ 1 5.44 d 1.3 102.7
2 4.12 dd 5.3, 3.2 72.5 2 4.76 dd 3.4, 1.3 71.9
3 4.67 dd 3.2, 3.2 69.2 3 4.55 dd 9.1, 3.4 72.6
4 4.34 m 76.5 4 4.24 dd 9.1, 9.1 74.1
5a, b 4.31 m 61.6 5 4.29 dq 9.1, 6.0 69.8
6 1.60 d 6.0 18.6

hydroxyl groups at 3387 cm-1, as well as absorption due as irradiation of other nonoverlapping proton signals in a
to a carbonyl group at 1687 cm-1. The 1H NMR spectrum series of 1D TOCSY experiments. Subsequent analysis of
of 1 measured in pyridine-d5 showed signals for seven the 1H-1H COSY spectrum resulted in the sequential
tertiary methyl groups at δ 1.31, 1.30, 1.13, 1.02, 0.98, 0.97, assignments of all the proton resonances due to the six
and 0.84 (each s) and a trisubstituted olefinic proton at δ monosaccharides, including identification of most of their
5.48 (t-like, J ) 3.2 Hz), which was typical of the oleanolic multiplet patterns and coupling constants as shown in
acid skeleton. In addition, signals for six anomeric protons Table 1. The HSQC spectrum correlated the proton reso-
were observed at δ 6.24 (br s), 5.85 (d, J ) 5.3 Hz), 5.44 (d, nances with those of the corresponding one-bond coupled
J ) 1.3 Hz), 5.12 (d, J ) 7.8 Hz), 4.97 (d, J ) 7.8 Hz), and carbons and the HSQC-TOCSY spectrum associated the
4.86 (d, J ) 6.0 Hz). The two three-proton doublet signals anomeric protons with their respective skeleton carbon
at δ 1.60 (d, J ) 6.0 Hz) and 1.56 (d, J ) 6.1 Hz) indicated atoms, leading to unambiguous assignments of the carbon
the presence of two deoxyhexopyranosyl units in 1. Acid shifts (Table 1). Comparison of the carbon chemical shifts
hydrolysis of 1 with 1 M HCl in dioxane-H2O (1:1) gave thus assigned with those of the reference methyl glyco-
olean-12-en-28-oic acid (oleanolic acid), together with L- sides,23,24 taking into account the known effects of O-
arabinose, D-glucose, L-rhamnose, and D-ribose. The mono- glycosylation, indicated that 1 contained an R-L-rhamnopy-
saccharides, including their absolute configurations, were ranosyl unit (Rha′) as the terminal glycosyl moiety and an
identified by direct HPLC analysis of the hydrolysate, R-L-arabinopyranosyl unit (Ara), two β-D-glucopyranosyl
which was performed on an aminopropyl-bonded silica gel units (Glc and Glc′), a β-D-ribopyranosyl unit (Rib), and an
column using MeCN-H2O (3:1) as solvent system, with R-L-rhamnopyranosyl unit (Rha) as the substituted sugar
detection being carried out by using a combination of RI moieties. The relatively large 3JH-1,H-2 values of the
and optical rotation (OR) detectors. In the 13C NMR arabinosyl, glucosyl, and ribosyl moieties (5.3-7.8 Hz)
spectrum of 1, the C-3 and C-28 carbon signals were indicated an R anomeric orientation for the arabinosyl and
observed at δ 88.7 and 180.2, respectively, implying that β for the glucosyls and ribosyl. The large 1JH-1,C-1 values
no sugar linkage was formed at the C-28 carboxyl group of the rhamnosyl moieties (Rha, 174 Hz; Rha′, 168 Hz)
and that a hexaglycoside was attached to the C-3 hydroxyl confirmed that the anomeric protons were equatorial (R-
group of the aglycone. The negative-ion FABMS showed pyranoid anomeric form).25 Finally, the 3JC,H correlations
an [M - H]- at m/z 1335 and prominent fragments at m/z from each anomeric proton across the glycosidic bond to
1189 [(M - H) - 146]- (cleavage of one deoxyhexose unit), the carbon of another substituted monosaccharide and the
1027 [(M - H) - 146 - 162]- (cleavage of one deoxyhexose aglycone revealed the sugar sequence. In the HMBC
unit and one hexose unit), 865 [(M - H) - 146 - 162 × spectrum, the anomeric proton signals at δ 6.24 (Rha), 5.85
2]- (cleavage of one deoxyhexose unit and two hexose (Rib), 5.44 (Rha′), 5.12 (Glc′), 4.97 (Glc), and 4.86 (Ara)
units), 733 [(M - H) - 146 - 162 × 2 - 132]- (cleavage of showed long-range correlations with C-2 of Ara at δ 75.7,
one deoxyhexose unit, two hexose units, and one pentose C-3 of Rha at δ 82.0, C-6 of Glc′ at δ 68.5, C-4 of Glc at δ
unit), and 587 [(M - H) - 146 × 2 - 162 × 2 - 132]- 81.8, C-4 of Rib at δ 76.5, and C-3 of the aglycone at δ 88.7,
(cleavage of two deoxyhexose units, two hexose units, and respectively. All of these data were consistent with the
one pentose unit). A peak at m/z 455 was assigned to the structure 3β-[(O-R-L-rhamnopyranosyl-(1f6)-O-β-D-glu-
aglycone moiety. This simple fragment ion pattern and the copyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f4)-O-β-D-ri-
results of acid hydrolysis suggested that 1 had a linear bopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-R-L-ar-
sugar sequence of rhamnosyl-glucosyl-glucosyl-ribosyl- abinopyranosyl)oxy]olean-12-en-28-oic acid, which was
rhamnosyl-arabinosyl or rhamnosyl-glucosyl-glucosyl-ara- assigned to 1.
binosyl-rhamnosyl-ribosyl in order of the terminal unit to Compound 2 was isolated as an amorphous solid with a
the inner unit attached to the aglycone. The exact sugar molecular formula of C70H114O34, as determined from data
sequence and its linkage position to the aglycone were of the positive-ion FABMS (m/z 1521 [M + Na]+), negative-
solved by detailed analysis of the 1D TOCSY and 2D NMR ion FABMS (m/z 1497 [M - H]-), 13C NMR spectrum (70
spectra. The 1H NMR subspectra of individual monosac- carbon signals), and elemental analysis. The 1H NMR
charide units were obtained by using selective irradiation spectrum of 2 showed signals for seven anomeric protons
of the easily identifiable anomeric proton signals, as well at δ 6.31 (d, J ) 8.1 Hz), 6.24 (br s), 5.85 (d, J ) 5.4 Hz),
Triterpene Saponins from Clematis chinensis Journal of Natural Products, 2004, Vol. 67, No. 9 1513

5.42 (d, J ) 1.4 Hz), 5.10 (d, J ) 7.8 Hz), 4.95 (d, J ) 7.8 Table 2. 13C NMR Data for the Aglycone Moiety of 1-4, 4a,
Hz), and 4.84 (d, J ) 6.0 Hz), along with signals for seven and 5 in Pyridine-d5
tertiary methyl groups at δ 1.28, 1.25, 1.13, 1.07, 0.91, 0.88, position 1 2 3 4 4a 5
and 0.85 (each s) and a trisubstituted olefinic proton at δ 1 38.8 38.9 38.8 38.9 39.0 38.8
5.42 (t-like, J ) 3.1 Hz), as observed for 1. Acid hydrolysis 2 26.6 26.6 26.5 26.6 28.1 26.6
of 2 yielded oleanolic acid, L-arabinose, D-glucose, L- 3 88.7 88.7 88.6 88.7 78.1 88.6
rhamnose, and D-ribose. Comparison of the 1H and 13C 4 39.5 39.6 39.5 39.5 39.4 39.5
NMR spectra of 2 with those of 1 revealed that 2 had a 5 56.0 56.0 55.9 56.0 55.9 55.9
6 18.5 18.5 18.5 18.4 18.8 18.4
terminal β-D-glucopyranosyl moiety (Glc′′) in addition to a
7 33.1 33.1 33.0 33.1 33.5 32.9
hexaglycoside group attached to C-3 of the aglycone, which 8 39.7 39.9 39.8 39.7 39.7 39.8
was identical to that of 1. Since the C-28 carbonyl carbon 9 48.0 48.0 47.9 48.1 48.2 47.9
signal of the aglycone of 2 was shifted upfield by 3.8 ppm 10 37.0 37.0 36.9 37.0 37.4 36.9
in comparison with that of 1, the additional glucosyl moiety 11 23.7 23.8 23.7 23.8 23.9 23.7
was presumed to be located at C-28 in an ester linkage 12 122.5 122.8 122.7 122.7 122.5 122.7
13 144.8 144.1 144.1 144.3 145.1 144.1
form. This was confirmed by alkaline hydrolysis of 2 with 14 42.1 42.1 42.0 42.4 42.6 42.0
6% KOH (EtOH-H2O, 1:1), giving 1, and by an HMBC 15 28.3 28.2 28.1 28.7 28.8 28.1
correlation from the anomeric proton of Glc′′ at δ 6.31 to 16 23.6 23.4 23.3 26.9 27.4 23.2
C-28 of the aglycone at δ 176.4. Accordingly, the structure 17 46.6 47.0 46.9 47.4 47.2 46.9
of 2 was determined as 3β-[(O-R-L-rhamnopyranosyl-(1f6)- 18 41.9 41.7 41.6 41.7 42.1 41.5
19 46.4 46.2 46.1 41.3 41.7 46.1
O-β-D-glucopyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f4)-O-
20 30.9 30.7 30.6 35.6 35.9 30.6
β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-R- 21 34.2 34.0 33.9 73.4 73.8 33.9
L-arabinopyranosyl)oxy]olean-12-en-28-oic acid β-D-gluco- 22 33.2 32.5 32.4 39.6 40.4 32.4
pyranosyl ester. 23 28.1 28.1 28.0 28.1 28.8 28.0
Compound 3 was analyzed for C58H94O25 by combined 24 17.1 17.1 17.0 17.1 16.6 17.0
25 15.5 15.6 15.5 15.7 15.7 15.6
positive-ion FABMS (m/z 1213 [M + Na]+), 13C NMR with 26 17.3 17.4 17.3 17.5 17.6 17.4
DEPT data, and elemental analysis. The 1H NMR spectrum 27 26.1 26.1 26.0 25.6 25.7 26.0
of 3 showed five anomeric proton signals at δ 6.28 (d, J ) 28 180.2 176.4 176.4 176.5 180.3 176.5
8.1 Hz), 6.19 (br s), 5.83 (d, J ) 5.5 Hz), 4.98 (1H, d, J ) 29 33.2 33.1 33.0 28.3 28.5 33.0
7.8 Hz), and 4.81 (d, J ) 6.0 Hz), along with seven tertiary 30 23.7 23.6 23.5 24.9 25.1 23.6
methyl and one olefinic proton signals characteristic of
oleanolic acid. The 13C NMR spectroscopic features were at δ 2.39 (dd, J ) 14.4, 2.7 Hz, H-22a) and 2.34 (dd, J )
also suggestive of oleanolic acid 3,28-bisdesmoside. Acid 14.4, 2.7 Hz, H-22b) in the 1H-1H COSY spectrum and
hydrolysis of 3 with 1 M HCl furnished oleanolic acid, HMBC correlations with C-17 (δ 47.2), C-19 (δ 41.7), C-20
L-arabinose, D-glucose, L-rhamnose, and D-ribose, whereas (δ 35.9), C-29 (δ 28.5), and C-30 (δ 25.1), indicating the
alkaline treatment of 3 with 6% KOH (EtOH-H2O, 1:1) presence of a hydroxyl group at C-21. NOE correlations
yielded a known oleanolic acid tetraglycoside (9),4,10 which from H-21 to both Me-29 and Me-30 confirmed the C-21R
was identical to the compound produced by alkaline treat- configuration. The downfield shift of the H-19 axial proton
ment of 8. These facts indicated that the tetraglycoside [δ 2.62 (dd, J ) 13.9, 13.9 Hz)] and upfield shift of the C-19
linked to C-3 of the aglycone of 3 was the same as that of carbon (δ 41.7) were consistent with the presence of the
8. When the 13C NMR spectrum of 3 was compared with C-21R-axial hydroxyl group. The structure of 4a was
that of 9, a set of additional six signals corresponding to a determined to be 21R-hydroxyoleanolic acid. Although 4a
terminal β-D-glucosyl group appeared, and an HMBC has been reported from Olax dissitiflora,26 Amaracus
correlation was observed between the signals of the ano- dictamnus,27 Origanum compactum,28 and Mentha cit-
meric proton of the glucosyl group (δ 6.28) and the C-28 rata,29 no spectroscopic data for intact 4a are available.
carbonyl carbon of the aglycone (δ 176.4). Accordingly, the The IR, 1H NMR, and 13C NMR data for 4a are reported
structure of 3 was assigned as 3β-[(O-β-D-glucopyranosyl- in Table 2 and the Experimental Section. As for the sugar
(1f4)-O-β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl- sequences of 4, the tetraglycoside and the triglycoside,
(1f2)-R-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-β- which were attached to C-3 and C-28 of the aglycone,
D-glucopyranosyl ester. respectively, were suggested to be identical with those of
Compound 4 had the molecular formula C70H114O35 on 8. This was confirmed by analysis of the HMBC spectrum
the basis of the positive-ion FABMS (m/z 1537 [M + Na]+), of 4, resulting in the detection of the 3JC,H correlations from
negative-ion FABMS (m/z 1513 [M - H]-), 13C NMR each anomeric proton across the glycosidic bond to the
spectrum, and elemental analysis. The 1H NMR spectrum carbon of another substituted monosaccharide or the ag-
contained seven three-proton singlet signals between δ 1.34 lycone. The structure of 4 was thus defined as 3β-[(O-β-D-
and 0.87, an olefinic proton signal at δ 5.48 (t-like, J ) 3.1 glucopyranosyl-(1f4)-O-β-D-ribopyranosyl-(1f3)-O-R-L-
Hz), and seven anomeric proton signals. Comparison of the rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl)oxy]-21R-
1H and 13C NMR spectra of 4 with those of 8 showed their hydroxyolean-12-en-28-oic acid O-R-L-rhamnopyranosyl-
considerable structural similarity. Acid hydrolysis of 4 with (1f4)-O-β-D-glucopyranosyl-(1f6)-β-D-glucopyranosyl ester.
1 M HCl resulted in the production of an aglycone (4a), Compound 5 was the most polar triterpene saponin, and
together with L-arabinose, D-glucose, L-rhamnose, and its molecular formula was derived as C82H134O43 from the
D-ribose. The spectroscopic properties of 4a were closely positive-ion FABMS (m/z 1829 [M + Na]+), negative-ion
related to those of oleanolic acid; however, the molecular FABMS (m/z 1805 [M - H]-), 13C NMR, and elemental
formula of 4a (C30H48O4) was higher by one oxygen atom analysis. The 1H NMR spectrum of 5 displayed signals for
than that of oleanolic acid, implying the presence of one nine anomeric protons at δ 6.22 (br s), 6.20 (d, J ) 8.1 Hz),
more hydroxyl group in addition to the C-3β hydroxyl 5.83 (br s), 5.81 (d, J ) 5.5 Hz), 5.40 (br s), 5.11 (d, J ) 7.8
group. The hydroxymethine proton at δ 3.81 (dd, J ) 2.7, Hz), 4.99 (d, J ) 7.8 Hz), 4.95 (d, J ) 7.8 Hz), and 4.83 (d,
2.7 Hz) showed spin-couplings with the methylene protons J ) 5.9 Hz), as well as signals for seven tertiary methyl
1514 Journal of Natural Products, 2004, Vol. 67, No. 9 Mimaki et al.

groups and an olefinic proton due to the oleanolic acid Plant Material. The plant material defined as the roots of
moiety. The C-3 and C-28 bisdesmosidic nature of 5 was C. chinensis was obtained from a wholesale firm in Uchida-
shown by the 13C NMR shifts of C-3 (δ 88.6) and C-28 (δ Wakanyaku, Tokyo, Japan, and authenticated by one of the
176.5). Acid hydrolysis of 5 with 1 M HCl furnished authors (Y.S.). A small amount of the sample is preserved in
our laboratory (00-CC-UW-07).
oleanolic acid, L-arabinose, D-glucose, L-rhamnose, and
Extraction and Isolation. The plant material (5.0 kg) was
D-ribose, whereas alkaline hydrolysis with 6% KOH (EtOH-
extracted with hot MeOH (3L × 3). The MeOH extract was
H2O, 1:1) yielded 1. The triglycoside attached to C-28 was concentrated under reduced pressure, and the viscous concen-
presumed to be the same as that of 4 and 8, confirmative trate (432 g) was passed through a Diaion HP-20 column,
evidence for which was obtained by HMBC correlations successively eluting with 30% MeOH, 60% MeOH, 80% MeOH,
from the anomeric protons assigned for the triglycoside MeOH, EtOH, and EtOAc. The MeOH eluate fraction (39 g)
moiety to their respective linkage carbons. The structure exhibited a potent cytotoxic activity against HL-60 cells (98.6%
of 5 was elucidated as 3β-[(O-R-L-rhamnopyranosyl-(1f6)- cell growth inhibition at a sample concentration of 10 µg/mL;
O-β-D-glucopyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f4)-O- IC50 1.4 µg/mL). Column chromatography of the MeOH eluate
β-D-ribopyranosyl-(1f3)-O-R-L-rhamnopyranosyl-(1f2)-R- portion on silica gel and elution with a stepwise gradient
mixture of CHCl3-MeOH (4:1; 2:1; 1:1) and finally with MeOH
L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-R-L-rham-
gave five fractions I-V. Fraction I was subjected to a silica
nopyranosyl-(1f4)-O-β-D-glucopyranosyl-(1f6)-β-D-glucopy- gel column eluting with CHCl3-MeOH-H2O (40:10:1) to give
ranosyl ester. 6 (19.8 mg). Fraction II was purified by silica gel column
Compounds 1-8 were evaluated for their cytotoxic chromatography eluting with CHCl3-MeOH-H2O (7:4:1) and
activity against HL-60 human promyelocytic leukemia cells ODS silica gel column chromatography with MeOH-H2O (4:
using an MTT assay method.30 The monodesmosidic sa- 1) to yield 3 (14.2 mg). Fraction III was separated by column
ponins (1 and 6) showed cytotoxic activity against HL-60 chromatography on silica gel eluting with CHCl3-MeOH-H2O
cells with the IC50 values of 2.8 and 2.3 µM, respectively. (7:4:1) and ODS silica gel with MeOH-H2O (8:5) to afford 1
Although the main saponin constituent 5 of C. chinensis (435 mg). Fraction IV was subjected to a silica gel column
eluting with CHCl3-MeOH-H2O (7:4:1) and an ODS silica gel
roots did not exhibit any apparent cytotoxicity even at a column with MeOH-H2O (8:5) and MeCN-H2O (5:8) to obtain
concentration of 20 µM, it can be converted to the cytotoxic 2 (21.1 mg), 4 (119 mg), 7 (32.1 mg), and 8 (627 mg). Fraction
saponin 1 by cleavage of the C-28 triglycosyl ester linkage V was chromatographed on silica gel eluting with CHCl3-
and is concluded to have a cytotoxic potentiality. Compound MeOH-H2O (7:4:1) and ODS silica gel with MeOH-H2O (8:
1 exhibited no significant differential cellular sensitivities 5) to furnish 5 (5.41 g).
when 1 was evaluated in the Japanese Foundation for Compound 1: amorphous solid; [R]D25 -82.0° (c 0.25,
Cancer Research 39 cell line assay.31 However, BSY-1 MeOH); IR (film) νmax 3387 (OH), 2941 (C-H), 1687 (CdO),
breast cancer cells, U251 and SF-295 CNS cancer cells, and 1057 cm-1; 1H NMR (pyridine-d5) δ 5.48 (1H, t-like, J ) 3.2
PC-3 prostate cancer cells were relatively sensitive to 1 Hz), 3.29 (1H, overlapping, H-18), 3.28 (1H, dd, J ) 11.1, 4.1
Hz, H-3), 1.31 (3H, s, Me-27), 1.30 (3H, s, Me-23), 1.13 (3H, s,
with respective LC50 values of 5.9, 6.3, 6.0, and 6.0 µM,
Me-24), 1.02 (3H, s, Me-30), 0.98 (3H, s, Me-26), 0.97 (3H, s,
and NCI-H460 lung cells, OVCAR-3 and OVCAR-8 ovarian Me-29), 0.84 (3H, s, Me-25), signals for the sugar moiety, see
cells, and stomach MKN28 cells were relatively resistant, Table 2; 13C NMR, see Tables 1 and 2; HRFABMS (positive
with respective LC50 values of 40, 47, 71, and 30 µM. mode) m/z 1359.6653 [M + Na]+ (calcd for C64H104O29Na,
1359.6561); FABMS (negative mode) m/z 1335 [M - H]-, 1189
Experimental Section [(M - H) - deoxyhexosyl]-, 1027 [(M - H) - deoxyhexosyl -
General Experimental Procedures. Optical rotations hexosyl]-, 865 [(M - H) - deoxyhexosyl - hexosyl × 2]-, 733
[(M - H) - deoxyhexosyl - hexosyl × 2 - pentosyl]-, 587 [(M
were measured by using a JASCO DIP-360 (Tokyo, Japan)
- H) - deoxyhexosyl × 2 - hexosyl × 2 - pentosyl]-, 455.
automatic digital polarimeter. IR spectra were recorded on a
Acid Hydrolysis of 1. A solution of 1 (45 mg) in 1 M HCl
JASCO FT-IR 620 spectrophotometer. NMR spectra were (dioxane-H2O, 1:1, 4 mL) was heated at 90 °C for 1 h under
recorded on a Bruker DRX-500 spectrometer (500 MHz for 1H an Ar atmosphere. After cooling, the mixture was neutralized
NMR, Karlsruhe, Germany) using standard Bruker pulse by passage through an Amberlite IRA-93ZU (Organo, Tokyo,
programs. Chemical shifts are given as δ values with reference Japan) column and chromatographed on silica gel eluting with
to tetramethylsilane (TMS) as internal standard. MS were CHCl3-MeOH (99:1; 1:1) to give oleanolic acid (11.4 mg) and
recorded on a Finnigan MAT TSQ-700 (San Jose, CA) mass a sugar fraction (7.1 mg). The sugar fraction was passed
spectrometer, using a dithiothreitol and dithioerythritol (3:1) through a Sep-Pak C18 cartridge (Waters, Milford, MA) and
matrix. Elemental analyses were carried out using an Elemen- a Toyopak IC-SP M cartridge (Tosoh), which was then ana-
tal Vario EL (Hanau, Germany) elemental analyzer. Silica gel lyzed by HPLC under the following conditions: column,
(Fuji-Silysia Chemical, Aichi, Japan), ODS silica gel (Nacalai Kaseisorb NH2-60-5 LC (4.6 mm i.d. × 250 mm, 5 µm); solvent,
Tesque, Kyoto, Japan), and Diaion HP-20 (Mitsubishi-Chemi- MeCN-H2O (3:1); flow rate, 0.6 mL/min; detection, RI and OR.
cal, Tokyo, Japan) were used for column chromatography. TLC The identification of L-arabinose, D-glucose, L-rhamnose, and
was carried out on precoated Kieselgel 60 F254 (0.25 mm, D-ribose present in the sugar fraction was carried out by
Merck, Darmstadt, Germany) and RP-18 F254S (0.25 mm, comparison of their retention times and polarities with those
Merck) plates, and spots were visualized by spraying the plates of authentic samples; tR (min) 10.49 (L-rhamnose, negative
with 10% H2SO4 solution, followed by heating. HPLC was polarity), 10.90 (D-ribose, negative polarity), 13.26 (L-arabinose,
performed by using a system comprised of a CCPM pump positive polarity), 16.04 (D-glucose, positive polarity).
Compound 2: amorphous solid; [R]D25 -70.0° (c 0.25,
(Tosoh, Tokyo, Japan), a CCP PX-8010 controller (Tosoh), an
MeOH); IR (film) νmax 3387 (OH), 2940 (C-H), 1746 (CdO),
RI-8010 detector (Tosoh), a Shodex OR-2 detector (Showa-
1067 cm-1; 1H NMR (pyridine-d5) δ 6.31 (1H, d, J ) 8.1 Hz,
Denko, Tokyo, Japan), and a Rheodyne injection port with a H-1 of Glc′′), 6.24 (1H, br s, H-1 of Rha), 5.85 (1H, d, J ) 5.4
20 µL sample loop. A Kaseisorb NH2-60-5 LC column (4.6 mm Hz, H-1 of Rib), 5.42 (1H, d, J ) 1.4 Hz, H-1 of Rha′), 5.42
i.d. × 250 mm, 5 µm, Tokyo-Kasei, Tokyo, Japan) was (1H, t-like, J ) 3.1 Hz, H-12), 5.10 (1H, d, J ) 7.8 Hz, H-1 of
employed for HPLC analysis. The following reagents were Glc′), 4.95 (1H, d, J ) 7.8 Hz, H-1 of Glc), 4.84 (1H, d, J ) 6.0
obtained from the indicated companies: RPMI 1640 medium Hz, H-1 of Ara), 3.27 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 3.18 (1H,
(Gibco, Gland Island, NY); FBS (Bio-Whittaker, Walkersville, dd, J ) 13.6, 3.7 Hz, H-18), 1.58 (3H, d, J ) 5.8 Hz, Me-6 of
MD); MTT (Sigma, St. Louis, MO); penicillin and streptomycin Rha′), 1.53 (3H, d, J ) 6.1 Hz, Me-6 of Rha), 1.28 (3H, s, Me-
(Meiji-Seika, Tokyo, Japan). All other chemicals used were of 23), 1.25 (3H, s, Me-27), 1.13 (3H, s, Me-24), 1.07 (3H, s, Me-
biochemical reagent grade. 26), 0.91 (3H, s, Me-29), 0.88 (3H, s, Me-30), 0.85 (3H, s, Me-
Triterpene Saponins from Clematis chinensis Journal of Natural Products, 2004, Vol. 67, No. 9 1515

Table 3. 13C NMR Data for the Glycosidic Moieties of 2-5 in Compound 3: amorphous solid; [R]D25 -38.0° (c 0.10,
Pyridine-d5 MeOH); IR (film) νmax 3376 (OH), 2941 (C-H), 1745 (CdO),
position 2 3 4 5 1070 cm-1; 1H NMR (pyridine-d5) δ 6.28 (1H, d, J ) 8.1 Hz,
H-1 of Glc′′), 6.19 (1H, br s, H-1 of Rha), 5.83 (1H, d, J ) 5.5
Ara 1 105.2 105.1 105.2 105.1 Hz, H-1 of Rib), 5.40 (1H, t-like, J ) 3.1 Hz, H-12), 4.98 (1H,
2 75.4 75.4 75.3 75.3 d, J ) 7.8 Hz, H-1 of Glc), 4.81 (1H, d, J ) 6.0 Hz, H-1 of Ara),
3 74.6 74.4 74.6 74.5 3.24 (1H, dd, J ) 11.7, 4.1 Hz, H-3), 3.16 (1H, dd, J ) 13.6,
4 69.3 69.2 69.3 69.2 3.8 Hz, H-18), 1.51 (3H, d, J ) 6.2 Hz, Me-6 of Rha), 1.24 (3H
5 65.6 65.5 65.6 65.5
× 2, s, Me-23 and Me-27), 1.09 (3H, s, Me-24), 1.05 (3H, s,
Rha 1 101.4 101.3 101.3 101.3
Me-26), 0.89 (3H, s, Me-29), 0.86 (3H, s, Me-30), 0.82 (3H, s,
2 71.9 71.8 71.9 71.8
3 82.0 81.8 81.9 81.8 Me-25); 13C NMR, see Tables 1 and 3; FABMS (positive mode)
4 72.7 72.6 72.7 72.6 m/z 1213 [M + Na]+, 1051 [(M + Na) - hexosyl]+; anal. C
5 69.8 69.7 69.7 69.7 56.80%, H 8.32%, calcd for C58H94O25‚2H2O, C 56.76%, H
6 18.4 18.4 18.4 18.4 8.05%.
Rib 1 104.7 104.4 104.6 104.4 Acid Hydrolysis of 3. Compound 3 (5 mg) was subjected
2 72.5 72.3 72.4 72.3 to acid hydrolysis as described for 1 to give oleanolic acid (2.3
3 69.4 69.4 69.5 69.4 mg) and a sugar fraction (1.7 mg). HPLC analysis of the sugar
4 76.6 76.2 76.3 76.3 fraction under the same conditions as in the case of 1 showed
5 61.6 61.7 61.7 61.6 the presence of L-arabinose, D-glucose, L-rhamnose, and D-
Glc 1 103.1 103.3 103.4 103.0 ribose.
2 74.0 74.6 74.7 74.0 Alkaline Hydrolysis of 3 and 8. Compound 3 (5 mg) was
3 76.3 78.1 78.2 76.2 treated with 6% KOH in EtOH-H2O (1:1, 3 mL) at 95 °C for
4 81.9 71.3 71.4 81.7 1 h under an Ar atmosphere. The mixture was neutralized by
5 76.5 78.5 78.5 76.4 passage through an Amberlite IR-120B column and then
6 61.8 62.3 62.4 61.6 chromatographed over silica gel eluting with CHCl3-MeOH-
Glc′ 1 104.9 104.8
H2O (40:10:1) to yield 9 (2.7 mg). Compound 8 (80 mg) was
2 74.8 74.8
3 78.2 78.1
subjected to alkaline hydrolysis as described above to give 9
4 71.7 71.7 (42.7 mg).
5 76.7 76.6 Compound 4: amorphous solid; [R]D25 -108.0° (c 0.25,
6 68.6 68.5 MeOH); IR (film) νmax 3387 (OH), 2937 (C-H), 1733 (CdO),
Rha′ 1 102.7 102.6 1065 cm-1; 1H NMR (pyridine-d5) δ 6.25 (1H, d, J ) 8.1 Hz,
2 71.9 71.7 H-1 of Glc′′), 6.23 (1H, br s, H-1 of Rha), 5.82 (1H, d, J ) 1.5
3 72.6 72.6 Hz, Rha′′), 5.81 (1H, d, J ) 5.8 Hz, H-1 of Rib), 5.48 (1H, t-like,
4 74.1 73.9 J ) 3.1 Hz, H-12), 5.00 (1H, d, J ) 7.8 Hz, H-1 of Glc), 4.97
5 69.8 69.7 (1H, d, J ) 7.9 Hz, H-1 of Glc′′′), 4.83 (1H, d, J ) 6.0 Hz, H-1
6 18.6 18.5 of Ara), 3.68 (1H, br s, H-21), 3.36 (1H, dd, J ) 14.1, 2.6 Hz,
Glc′′ 1 95.7 95.7 95.7 95.5 H-18), 3.27 (1H, dd, J ) 11.4, 3.8 Hz, H-3), 1.68 (3H, d, J )
2 74.1 74.0 73.8 73.7 6.2 Hz, Me-6 of Rha′′), 1.52 (3H, d, J ) 6.1 Hz, Me-6 of Rha),
3 78.9 78.7 78.6 78.5 1.34 (3H, s, Me-27), 1.26 (3H, s, Me-23), 1.18 (3H, s, Me-29),
4 71.1 71.0 70.7 70.6 1.12 (3H, s, Me-24), 1.08 (3H, s, Me-26), 1.00 (3H, s, Me-30),
5 79.3 79.2 78.0 77.9 0.87 (3H, s, Me-25); 13C NMR, see Tables 1 and 3; FABMS
6 62.2 62.1 69.2 69.0 (positive mode) m/z 1537 [M + Na]+; FABMS (negative mode)
Glc′′′ 1 104.8 104.6
m/z 1513 [M - H]-; anal. C 53.65%, H 7.72%, calcd for
2 75.2 75.2
3 76.4 76.4
C70H114O35‚3H2O, C 53.52%, H 7.71%.
4 78.1 78.1 Acid Hydrolysis of 4. Compound 4 (57.2 mg) was subjected
5 77.0 77.0 to acid hydrolysis as described for 1 to give 4a (15.6 mg) and
6 61.2 61.1 a sugar fraction (25.9 mg). HPLC analysis of the sugar fraction
Rha′′ 1 102.6 102.6 under the same conditions as in the case of 1 showed the
2 72.5 72.4 presence of L-arabinose, D-glucose, L-rhamnose, and D-ribose.
3 72.7 72.5 Compound 4a: amorphous solid; [R]D25 -46.4° (c 0.25,
4 73.9 73.9 MeOH); IR (film) νmax 3434 (OH), 2928 and 2871 (C-H), 1685
5 70.2 70.2 (CdO) cm-1; 1H NMR (pyridine-d5) δ 5.60 (1H, t-like, J ) 3.2
6 18.4 18.4 Hz, H-12), 3.81 (1H, dd, J ) 2.7, 2.7 Hz, H-21), 3.53 (1H, dd,
J ) 13.9, 3.2 Hz, H-18), 3.47 (1H, dd, J ) 10.6, 5.4 Hz, H-3),
2.62 (1H, dd, J ) 13.9, 13.9 Hz, H-19ax), 2.39 (1H, dd, J )
25); 13C NMR, see Tables 1 and 3; FABMS (positive mode) m/z 14.4, 2.7 Hz, H-22a), 2.34 (1H, dd, J ) 14.4, 2.7 Hz, H-22b),
1521 [M + Na]+; FABMS (negative mode) m/z 1497 [M - H]-, 1.40 (3H, s, Me-27), 1.30 (1H, dd, J ) 13.9, 3.2 Hz, H-19eq),
1335 [(M - H) - hexosyl]-, 1189 [(M - H) - hexosyl - 1.27 (3H, s, Me-29), 1.25 (3H, s, Me-23), 1.16 (3H, s, Me-30),
deoxyhexosyl]-, 1027 [(M - H) - hexosyl × 2 - deoxyhexosyl]-, 1.08 (3H, s, Me-26), 1.04 (3H, s, Me-24), 0.94 (3H, s, Me-25);
865 [(M - H) - hexosyl × 3 - deoxyhexosyl]-, 733 [(M - H) 13C NMR, see Table 1; FABMS (positive mode) m/z 473 [M +

- hexosyl × 3 - deoxyhexosyl - pentosyl]-; anal. C 53.05%, H]+; anal. C 74.64%, H 10.10%, calcd for C30H48O4‚1/2H2O, C
H 7.81%, calcd for C70H114O34‚5H2O, C 52.84%, H 7.86%. 74.80%, H 10.25%.
Acid Hydrolysis of 2. Compound 2 (6 mg) was subjected Compound 5: amorphous solid; [R]D25 -94.0° (c 0.25,
to acid hydrolysis as described for 1 to give oleanolic acid (3.1 MeOH); IR (film) νmax 3387 (OH), 2939 (C-H), 1733 (CdO),
mg) and a sugar fraction (2.4 mg). HPLC analysis of the sugar 1063 cm-1; 1H NMR (pyridine-d5) δ 6.22 (1H, br s, H-1 of Rha),
fraction under the same conditions as in the case of 1 showed 6.20 (1H, d, J ) 8.1 Hz, H-1 of Glc′′), 5.83 (1H, br s, H-1 of
the presence of L-arabinose, D-glucose, L-rhamnose, and D- Rha′′), 5.81 (1H, d, J ) 5.5 Hz, H-1 of Rib), 5.40 (1H, br s, H-1
of Rha′), 5.39 (1H, overlapping, H-12), 5.11 (1H, d, J ) 7.8
ribose.
Hz, H-1 of Glc′), 4.99 (1H, d, J ) 7.8 Hz, H-1 of Glc′′′), 4.95
Alkaline Hydrolysis of 2. Compound 2 (5 mg) was treated (1H, d, J ) 7.8 Hz, H-1 of Glc), 4.83 (1H, d, J ) 5.9 Hz, H-1 of
with 6% KOH in EtOH-H2O (1:1, 4 mL) at 95 °C for 1 h under Ara), 3.27 (1H, dd, J ) 11.0, 3.7 Hz, H-3), 3.15 (1H, dd, J )
an Ar atmosphere. The mixture was neutralized by passage 13.5, 3.7 Hz, H-18), 1.68 (3H, d, J ) 6.1 Hz, Me-6 of Rha′′),
through an Amberlite IR-120B column (Organo, Tokyo, Japan) 1.57 (3H, d, J ) 5.5 Hz, Me-6 of Rha′), 1.53 (3H, d, J ) 5.9
and then chromatographed over silica gel eluting with CHCl3- Hz, Me-6 of Rha), 1.27 (3H, s, Me-23), 1.23 (3H, s, Me-27), 1.12
MeOH-H2O (7:4:1) to yield 1 (2.8 mg). (3H, s, Me-24), 1.05 (3H, s, Me-26), 0.89 (3H, s, Me-29), 0.88
1516 Journal of Natural Products, 2004, Vol. 67, No. 9 Mimaki et al.

(3H, s, Me-30), 0.85 (3H, s, Me-25); 13C NMR, see Tables 1 References and Notes
and 3; FABMS (positive mode) m/z 1829 [M + Na]+, 1683 [(M (1) Xu, R.; Zhao, W.; Xu, J.; Shao, B.; Qin, G. Adv. Exp. Med. Biol. 1996,
+ Na) - deoxyhexosyl]+, 1359 [(M + Na) - deoxyhexosyl - 404, 371-382.
hexosyl × 2]+; FABMS (negative mode) m/z 1805 [M - H]-, (2) Chiu, H. F.; Lin, C. C.; Yang, C. C.; Yang, F. Am. J. Chin. Med. 1988,
1335 [(M - H) - deoxyhexosyl - hexosyl × 2]-; anal. C 16, 127-137.
(3) Ho, C. S.; Wong, Y. H.; Chiu, K. W. Am. J. Chin. Med. 1989, 17, 189-
52.94%, H 7.71%, calcd for C82H134O43‚3H2O, C 52.90%, H 202.
7.58%. (4) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1979, 27, 2388-2393.
Acid Hydrolysis of 5. Compound 5 (60.2 mg) was subjected (5) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1980, 28, 2827-2830.
to acid hydrolysis as described for 1 to give oleanolic acid (13.6 (6) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1980, 28, 3555-3560.
(7) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1982, 30, 859-865.
mg) and a sugar fraction (21.6 mg). HPLC analysis of the sugar (8) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1982, 30, 3340-3346.
fraction under the same conditions as in the case of 1 showed (9) Shao, B.; Qin, G.; Xu, R.; Wu, H.; Ma, K. Phytochemistry 1995, 38,
the presence of L-arabinose, D-glucose, L-rhamnose, and D- 1473-1479.
ribose. (10) Shao, B.; Qin, G.; Xu, R.; Wu, H.; Ma, K. Phytochemistry 1996, 42,
Alkaline Hydrolysis of 5. Compound 5 (20.3 mg) was 821-825.
(11) Mimaki, Y.; Kuroda, M.; Asano, T.; Sashida, Y. J. Nat. Prod. 1999,
treated with 6% KOH in EtOH-H2O (1:1, 4 mL) at 95 °C for 62, 1279-1283.
1 h under an Ar atmosphere. The mixture was neutralized by (12) Kuroda, M.; Mimaki, Y.; Sashida, Y.; Kitahara, M.; Yamazaki, M.;
passage through an Amberlite IR-120B column and then Yui, S. Bioorg. Med. Chem. Lett. 2001, 11, 371-374.
chromatographed over silica gel eluting with CHCl3-MeOH- (13) Mimaki, Y.; Fukushima, M.; Yokosuka, A.; Sashida, Y.; Furuya, S.;
Sakagami, H. Phytochemistry 2001, 57, 773-779.
H2O (7:4:1) to yield 1 (4.7 mg). (14) Kuroda, M.; Mimaki, Y.; Harada, H.; Sakagami, H.; Sashida. Y. Nat.
Cell Culture Assay. HL-60 cells, obtained from Human Med. (Tokyo) 2001, 55, 134-138.
Science Research Resources Bank (JCRB 0085, Osaka, Japan), (15) Yui, S.; Ubukata, K.; Hodono, K.; Kitahara, M.; Mimaki, Y.; Kuroda,
were maintained in RPMI 1640 medium containing heat- M.; Sashida, Y.; Yamazaki, M. Int. Immunopharmacol. 2001, 1, 1989-
2000.
inactivated 10% FBS supplemented with L-glutamine, 100 (16) Mimaki, Y.; Yokosuka, A.; Kuroda, M.; Hamanaka, M.; Sakuma, C.;
units/mL penicillin, and 100 µg/mL streptomycin. The leuke- Sashida, Y. J. Nat. Prod. 2001, 64, 1226-1229.
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to 4 × 104 cells/mL, and 196 µL of this cell suspension was Bull. 2002, 50, 121-125.
(18) Mimaki, Y.; Harada, H.; Sakuma, C.; Haraguchi, M.; Yui, S.; Kudo,
placed in each well of a 96-well flat-bottom plate (Iwaki Glass, T.; Yamazaki, M.; Sashida, Y. Bioorg. Med. Chem. Lett. 2003, 13,
Chiba, Japan). The cells were incubated in 5% CO2/air for 24 623-627.
h at 37 °C. After incubation, 4 µL of EtOH-H2O (1:1) solution (19) Watanabe, K.; Mimaki, Y.; Sakuma, C.; Sashida, Y. J. Nat. Prod.
containing the sample was added to give the final concentra- 2003, 66, 879-882.
tions of 0.1-50 µg/mL; 4 µL of EtOH-H2O (1:1) was added (20) Mimaki, Y.; Kuroda, M.; Yokosuka, A.; Harada, H.; Fukushima, M.;
Sashida, Y. Chem. Pharm. Bull. 2003, 51, 960-965.
into control wells. The cells were further incubated for 72 h (21) Yui, S.; Kudo, T.; Hodono, K.; Mimaki, Y.; Kuroda, M.; Sashida, Y.;
in the presence of each agent, and then cell growth was Yamazaki, M. Mediators Inflamm. 2003, 12, 157-166.
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Acknowledgment. We are grateful to Dr. Y. Shida and K.; Mita, I.; Edatsugi, H.; Matsuba, Y.; Takezawa, K.; Nakanishi, O.;
Mr. H. Fukaya, Tokyo University of Pharmacy and Life Kohno, H.; Nakajima, Y.; Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer
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Science, for the measurements of the mass spectra and
elemental analysis. NP040088K