Triterpene glycosides from the whole plant of Anemone hupehensis var. japonica and their cytotoxic activity

Article abstract of DOI:10.1248/cpb.57.1425

Three new triterpene glycosides (1-3), together with eight known triterpene glycosides (4-11), were isolated from the whole plant of Anemone hupehensis var. japonica (Ranunculaceae). The structures of the new compounds were determined on the basis of spec

Full text of DOI:10.1248/cpb.57.1425

December 2009
Notes
Chem. Pharm. Bull. 57(12) 1425—1430 (2009)
1425
Triterpene Glycosides from the Whole Plant of Anemone hupehensis var.
japonica and Their Cytotoxic Activity
Akihito YOKOSUKA,*,a Tomoe SANO,a Ken HASHIMOTO,b Hiroshi SAKAGAMI,b and Yoshihiro MIMAKI*,a
a Laboratory of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1432–1
Horinouchi, Hachioji, Tokyo 192–0392, Japan: and b Department of Diagnostic and Therapeutic Sciences, School of
Dentistry, Meikai University; 1–1 Keyakidai, Sakado, Saitama 350–0283, Japan.
Received August 20, 2009; accepted September 28, 2009; published online September 29, 2009
Three new triterpene glycosides (1—3), together with eight known triterpene glycosides (4—11), were iso-
lated from the whole plant of Anemone hupehensis var. japonica (Ranunculaceae). The structures of the new
compounds were determined on the basis of spectroscopic analysis and the results of hydrolytic cleavage experi-
ments. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human leukemia cells,
HSC-2 human oral squamous carcinoma cells, HSC-4 human oral squamous carcinoma cells, and A549 human
lung adenocarcinoma cells.
Key words triterpene glycoside; Anemone hupehensis var. japonica; Ranunculaceae; cytotoxic activity
Anemone hupehensis LEM. var. japonica (THUNB.) BOWLES
The whole plant of A. hupehensis var. japonica (3.3 kg)
et STEARN is a perennial plant belonging to the family Ranun- was extracted with hot MeOH. After removal of solvent, the
culaceae and is distributed throughout Japan and China.1) Its MeOH extract was passed through a porous-polymer poly-
roots have been used in traditional Chinese medicine for the styrene resin (Diaion HP-20) column successively eluted
treatment of fever.2) Although the triterpene glycosides hupe- with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc.
hensis saponin A—G were isolated from A. hupehensis,3) The MeOH eluted fraction was repeatedly subjected to silica
there have been no reports concerning the secondary metabo- gel and octadecylsilanized (ODS) silica gel column chro-
lites of A. hupehensis var. japonica. Therefore, in this study, matography to afford compounds 1—11. Compounds 4—11
we conducted a phytochemical screening of the whole plant. were identified by their physical and spectroscopic data as
As a result, three new triterpene glycosides (1—3) and eight 3b-[(O-b-D-ribopyranosyl-(1→3)-O-a-L-rhamnopyranosyl-
known triterpene glycosides (4—11) were isolated. Here, we (1→2)-a-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid
describe the isolation and structural elucidation of the three (prosapogenin CP4, 4),4) 3b-[(O-b-D-ribopyranosyl-(1→3)-
new triterpene glycosides (1—3) on the basis of spectro- O-a-L-rhamnopyranosyl-(1→2)-b-D-xylopyranosyl)oxy]olean-
scopic analysis, including various two-dimensional (2D) 12-en-28-oic acid (huzhangoside A, 5),4) 23-hydroxy-3b-
NMR spectroscopic studies, and the results of hydrolytic [(O-b-D-ribopyranosyl-(1→3)-O-a-L-rhamnopyranosyl-
cleavage experiments. The cytotoxic activities of the isolated (1→2)-a-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid
compounds against HL-60 human leukemia cells, A549 (prosapogenin CP6, 6),4) olean-12-en-28-oic acid O-a-L-
human lung adenocarcinoma cells, HSC-2 human oral squa- rhamnopyranosyl-(1→4)-O-b-D-glucopyranosyl-(1→6)-b-D-
mous carcinoma cells, and HSC-4 human oral squamous car- glucopyranosyl ester (cussonoside B, 7),5) 3b-[(O-b-D-ribo-
cinoma cells are also reported.
pyranosyl-(1→3)-O-a -L-rhamnopyranosyl-(1→2)-a -L-
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yokosuka@toyaku.ac.jp; mimakiy@toyaku.ac.jp

1426
Vol. 57, No. 12
arabinopyranosyl)oxy]olean-12-en-28-oic acid O-a-L-rhamno- ence (HMQC) and 1H-detected heteronuclear single-quantum
pyranosyl-(1→4)-O-b -D-glucopyranosyl-(1→6)-b -D- coherence (HSQC)-TOCSY spectra correlated the proton
glucopyranosyl ester (huzhangoside B, 8),4) 3b-[(a-L-ara- resonances with those of the corresponding one-bond cou-
binopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-a- pled carbons, leading to unambiguous assignments of the
L-rhamnopyranosyl-(1→4)-O-b-D-glucopyranosyl-(1→6)-b- carbon shifts. Comparison of the carbon chemical shifts thus
D-glucopyranosyl ester (9),6) 23-hydroxy-3b-[(O-b-D-ribopy- assigned with those of reference methyl glycosides,12,13) tak-
ranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→2)-a-L-ara- ing into account the known effects of O-glycosylation, indi-
binopyranosyl)oxy]olean-12-en-28-oic acid O-a-L-rhamnopy- cated that 1 contains a C-2 substituted a-L-arabinopyranosyl
ranosyl-(1→4)-O-b-D-glucopyranosyl-(1→6)-b-D-glucopyra- moiety (Ara), two C-3 substituted a-L-rhamnopyranosyl
nosyl ester (huzhangoside D, 10),4) 23-hydroxy 3b-[(O-b- moieties (Rha, Rhaꢃ), a terminal b-D-ribopyranosyl moiety
D-ribopyranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→2)- (Rib), a C-6 substituted b-D-glucopyranosyl moiety (Glc), a
a-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-a-L- C-4 substituted b-D-glucopyranosyl moiety (Glcꢃ), and a ter-
1
rhamnopyranosyl-(1→4)-O-b-D-glucopyranosyl-(1→6)-b-D- minal b-D-glucopyranosyl moiety (Glcꢄ). In the H-detected
glucopyranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→4)-O-b- heteronuclear multiple-bond connectivity (HMBC) spectrum
D-glucopyranosyl-(1→6)-b-D-glucopyranosyl ester (hupe- of 1, long-range correlations were observed between the
hensis saponin F, 11),7) respectively.
anomeric proton (H-1) of Rib at dH 5.93 and C-3 of Rha at
Compound 1 was obtained as an amorphous solid and dC 81.2, H-1 of Rha at dH 6.27 and C-2 of Ara at dC 75.3, H-
showed an accurate [MꢀNa]ꢀ ion at m/z 1521.7109 in the 1 of Ara at dH 4.82 and C-3 of the aglycone at dC 88.7, H-1
high resolution (HR)-electrospray ionization (ESI)-time of of Glcꢄ at dH 5.34 and C-3 of Rhaꢃ at dC 83.8, H-1 of Rhaꢃ at
flight (TOF)-MS, corresponding to the empirical molecular dH 5.84 and C-4 of Glcꢃ at dC 77.2, H-1 of Glcꢃ at dH 4.90
formula C70H114O34. The IR spectrum of 1 was suggestive of and C-6 of Glc at dC 69.3, and between H-1 of Glc at dH 6.22
a glycoside (3376, 1064 cmꢁ1) and indicated the presence of and C-28 of the aglycone at dC 176.5 (Fig. 1). Accordingly,
a carbonyl group (1740 cmꢁ1) in the molecule. The 1H-NMR the structure of 1 was formulated as 3b-[(O-b-D-ribopyra-
spectrum of 1 contained signals for seven quaternary methyl nosyl-(1→3)-O-a -L-rhamnopyranosyl-(1→2)-a -L-ara-
groups at d 1.28, 1.23, 1.13, 1.06, 0.89, 0.87, and 0.85 (each binopyranosyl)oxy]olean-12-en-28-oic acid O-b-D-glucopy-
s), an olefinic proton at d 5.39 (br s), and seven anomeric ranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→4)-O-b-D-glu-
protons at d 6.27 (br s), 6.22 (d, Jꢂ8.0 Hz), 5.93 (d, Jꢂ4.4 copyranosyl-(1→6)-b-D-glucopyranosyl ester.
Hz), 5.84 (br s), 5.34 (d, Jꢂ7.8 Hz), 4.90 (d, Jꢂ7.9 Hz), and
Compound 2 was found to have a molecular formula of
4.82 (d, Jꢂ6.0 Hz), and the methyl groups of two 6-deoxy- C76H124O39 as determined by HR-ESI-TOF-MS analysis (m/z
hexopyranosyl moieties at d 1.63 (d, Jꢂ6.2 Hz) and 1.52 (d, 1661.7778 [MꢀH]ꢀ). The deduced molecular weight of 2
Jꢂ6.1 Hz). Acid hydrolysis of 1 with 1 M HCl in dioxane– was higher than that of 1, with the difference corresponding
H2O (1 : 1) yielded 3b-hydroxyolean-12-en-28-oic acid (1a, to a C6H10O5 unit. The 1H-NMR spectrum of 2 contained sig-
oleanolic acid), as well as L-rhamnose, D-ribose, L-arabinose, nals for eight anomeric protons at d 6.32 (br s), 6.26 (d, Jꢂ
and D-glucose as the carbohydrate moieties. Identification of 8.1 Hz), 5.98 (d, Jꢂ4.2 Hz), 5.88 (br s), 5.38 (d, Jꢂ7.8 Hz),
the monosaccharides, including their absolute configurations, 5.14 (d, Jꢂ7.8 Hz), 4.90 (d, Jꢂ7.9 Hz), 4.72 (d, Jꢂ6.8 Hz).
was carried out by direct HPLC analysis of the hydrolysate. On the basis of the spectral properties of 2 and the results of
The C-3 oxymethine carbon and C-28 carbonyl carbon were acid hydrolysis experiments, in which 2 yielded 1a, L-rham-
observed at d 88.7 and 176.5, respectively, in the 13C-NMR nose, D-ribose, L-arabinose, and D-glucose, 2 was shown to
of 1, which suggests that 1 is a 3,28-bisdesmoside of oleano- be a triterpene glycoside closely related to 1; however, the
lic acid. In a comparison of the whole 13C-NMR spectrum
of 1 with that of 3b-[(O-b-D-ribopyranosyl-(1→3)-O-a-L-
rhamnopyranosyl-(1→2)-a-L-arabinopyranosyl)oxy]olean-
12-en-28-oic acid b-D-glucopyranosyl ester (clematichineno-
side A),8) the signals due to the aglycone moiety and the
triglycoside residue linked to C-3 of the aglycone were
observed at almost the same positions for each of the com-
pounds. However, differences were recognized in the
tetraglycoside moiety attached to C-28 of the aglycone. The
exact structures of the sugar moieties and their linkage
positions to the aglycone were solved by detailed analysis of
the one-dimensional (1D) totally correlated spectroscopy
1
(TOCSY) and 2D NMR spectra. The H-NMR subspectra of
individual monosaccharide units were obtained by using se-
lective irradiation of easily identifiable anomeric proton sig-
nals, as well as non-overlapping proton signals in a series of
1D TOCSY experiments.9—11) Subsequent analysis of the
1H–1H shift correlation spectroscopy (COSY) spectrum re-
sulted in the sequential assignment of all the proton reso-
nances due to the seven glycosyl units, including identifica-
tion of their multiplet patterns and coupling constants (Table
1
1). The H-detected heteronuclear multiple-quantum coher- Fig. 1. HMBC Correlations of the Glycoside Moieties of 1

December 2009
1427
Table 1. 1H- and 13C-NMR Chemical Shift Assignments of the Sugar Moieties for Compounds 1—3a)
1
2
3
Position
1H
J (Hz)
6.0
8.7, 6.0
8.7, 4.7
13C
Position
1H
J (Hz)
13C
Position
1H
J (Hz)
13C
Ara 1ꢃ
4.82 d
105.2
75.3
74.7
69.4
65.7
Ara 1ꢃ
4.72 d
6.8
8.2, 6.8
8.2
105.3
75.8
75.0
80.4
65.5
Ara 1ꢃ
5.06 d
6.6
8.1, 6.6
8.1, 4.4
105.0
75.2
75.4
69.8
66.4
2ꢃ
3ꢃ
4ꢃ
5ꢃ
4.57 dd
4.24 dd
4.21 m
4.29 br d 11.0
3.80 br d 11.0
6.27 br s
4.89 br d
4.72 dd
4.42 dd
4.61 dq
1.52 d
2ꢃ
3ꢃ
4ꢃ
5ꢃ
4.51 dd
4.24 br d
4.23 m
4.38 br d
3.75 br d
6.32 br s
4.93 br d
4.75 dd
4.46 dd
4.66 dq
1.58 d
2ꢃ
3ꢃ
4ꢃ
5ꢃ
4.59 dd
4.02 dd
4.12 m
4.25 dd
3.67 br d 10.9
6.36 br s
a
b
a
b
10.7
10.7
a
b
9.7, 2.9
Rha 1ꢄ
2ꢄ
101.4
72.0
81.2
72.8
69.8
18.4
104.6
72.8
70.2
68.9
65.3
Rha 1ꢄ
2ꢄ
101.4
72.0
81.1
72.9
69.9
18.6
104.8
72.8
70.3
68.8
65.3
Rha 1ꢄ
2ꢄ
101.3
72.0
81.2
72.9
69.8
18.4
104.7
72.8
70.3
68.9
65.3
3.7
2.8
4.90 br d 2.9
3ꢄ
4ꢄ
5ꢄ
6ꢄ
9.7, 3.7
9.7, 9.7
9.7, 6.1
6.1
3ꢄ
4ꢄ
5ꢄ
6ꢄ
9.6, 2.8
9.6, 9.6
9.6, 6.1
6.1
3ꢄ
4ꢄ
5ꢄ
6ꢄ
4.77 dd
4.43 dd
4.71 dq
1.55 d
9.6, 2.9
9.6, 9.6
9.6, 6.2
6.2
Rib 1ꢅ
2ꢅ
5.93 d
4.4
Rib 1ꢅ
2ꢅ
5.98 d
4.2
Rib 1ꢅ
2ꢅ
5.96 d
4.4
4.29 m
4.51 m
4.17 m
4.34 br d
4.16 br d
6.22 d
4.09 dd
4.19 dd
4.32 dd
4.06 m
4.63 br d 11.3
4.28 br d 11.3
4.90 d
3.89 dd
4.10 dd
4.40 dd
3.53 m
4.33 m
4.51 m
4.18 m
4.31 m
4.51 m
4.16 m
4.31 br d 10.8
4.14 br d 10.8
3ꢅ
4ꢅ
5ꢅ
3ꢅ
4ꢅ
3ꢅ
4ꢅ
5ꢅ
a
b
8.5
8.5
8.0
8.9, 8.0
8.9, 8.9
8.9, 8.5
5ꢅ (2H) 4.18 m
a
b
Glc 1ꢄꢄ
2ꢄꢄ
95.6
73.8
78.7
70.8
77.9
69.3
Glc 1ꢄꢄ
5.14 d
7.8
106.8
75.5
78.8
71.3
78.8
62.6
Glc 1ꢄꢄ
2ꢄꢄ
6.25 d
8.1
95.6
73.9
78.7
70.8
77.9
69.3
2ꢄꢄ
3ꢄꢄ
4ꢄꢄ
5ꢄꢄ
6ꢄꢄ
4.03 dd
4.17 dd
4.24 dd
3.89 m
4.49 br d
4.37 br d
6.26 d
4.10 dd
4.19 dd
4.33 dd
4.08 m
8.6, 7.8
8.6, 8.6
8.6, 8.6
4.08 dd
4.20 dd
4.32 dd
4.02 m
4.65 br d 11.1
4.30 br d 11.1
9.2, 8.1
9.2, 9.2
9.2, 8.5
3ꢄꢄ
4ꢄꢄ
5ꢄꢄ
6ꢄꢄ
3ꢄꢄ
4ꢄꢄ
5ꢄꢄ
6ꢄꢄ
a
b
a
b
11.5
11.5
8.1
8.7, 8.1
8.7, 8.7
8.7, 8.7
a
b
Glcꢃ 1ꢄꢅ
2ꢄꢅ
7.9
105.0
75.3
76.4
77.2
77.0
61.2
Glcꢃ 1ꢄꢅ
2ꢄꢅ
95.7
73.9
78.6
70.9
77.9
69.3
Glcꢃ 1ꢄꢅ
2ꢄꢅ
4.93 d
7.9
104.7
75.4
76.4
77.2
77.1
61.2
8.7, 7.9
9.4, 8.7
9.4, 9.4
3.91 dd
4.13 dd
4.43 dd
3.56 m
8.5, 7.9
9.3, 8.5
9.3, 9.3
3ꢄꢅ
4ꢄꢅ
5ꢄꢅ
3ꢄꢅ
4ꢄꢅ
5ꢄꢅ
3ꢄꢅ
4ꢄꢅ
5ꢄꢅ
6ꢄꢅ a 4.14 br d
2.5
6ꢄꢅ a 4.64 br d
11.1
6ꢄꢅ a 4.15 br d 11.9
b
4.00 br d 12.5
b
4.29 br d
4.90 d
3.90 dd
4.11 dd
4.43 dd
3.57 m
11.1
7.9
8.9, 8.5
9.0, 8.9
9.0, 9.0
b
4.03 br d 11.9
5.87 br s
4.90 br d 2.6
Rhaꢃ1ꢄꢅꢃ
2ꢄꢅꢃ
5.84 br s
4.90 br d
4.63 dd
4.50 dd
5.03 dq
1.63 d
102.4
71.9
83.8
73.0
70.0
18.4
Glcꢄ 1ꢄꢅꢃ
2ꢄꢅꢃ
105.1
75.4
76.5
77.3
77.1
61.2
Rhaꢃ1ꢄꢅꢃ
2ꢄꢅꢃ
102.4
71.9
83.9
73.0
70.0
18.4
2.8
3ꢄꢅꢃ
4ꢄꢅꢃ
5ꢄꢅꢃ
6ꢄꢅꢃ
9.3, 2.8
9.3, 9.3
9.3, 6.2
6.2
3ꢄꢅꢃ
4ꢄꢅꢃ
5ꢄꢅꢃ
3ꢄꢅꢃ
4ꢄꢅꢃ
5ꢄꢅꢃ
6ꢄꢅꢃ
4.65 dd
4.43 dd
5.04 dq
1.66 d
9.3, 2.6
9.3, 9.3
9.3, 6.2
6.2
6ꢄꢅꢃ a 4.16 br d
10.5
10.5
b
4.03 br d
5.88 br s
4.88 br d
4.67 dd
4.50 dd
5.06 dq
1.67 d
Glcꢄ 1ꢄꢅꢃꢃ 5.34 d
2ꢄꢅꢃꢃ 4.07 dd
3ꢄꢅꢃꢃ 4.19 dd
4ꢄꢅꢃꢃ 4.26 dd
5ꢄꢅꢃꢃ 3.83 m
7.8
106.7
76.0
78.4
71.3
78.4
62.3
Rhaꢃ1ꢄꢅꢃꢃ
2ꢄꢅꢃꢃ
102.5
72.0
84.0
73.0
70.0
18.5
Glcꢄ 1ꢄꢅꢃꢃ
2ꢄꢅꢃꢃ
5.37 d
7.8
106.8
76.0
78.4
71.3
78.4
62.3
8.8, 7.8
8.8, 8.8
8.8, 8.5
3.0
4.09 dd
4.21 dd
4.26 dd
3.85 m
8.8, 7.8
8.8, 8.8
8.8, 8.8
3ꢄꢅꢃꢃ
4ꢄꢅꢃꢃ
5ꢄꢅꢃꢃ
6ꢄꢅꢃꢃ
9.1, 3.0
9.6, 9.1
9.6, 6.1
6.1
3ꢄꢅꢃꢃ
4ꢄꢅꢃꢃ
5ꢄꢅꢃꢃ
6ꢄꢅꢃꢃ a 4.40 br d 11.2
4.34 br d 11.2
6ꢄꢅꢃꢃ a 4.41 br d 11.8
4.35 br d 11.8
b
b
Glcꢅ1ꢄꢅꢃꢄ
2ꢄꢅꢃꢄ
5.38 d
7.8
106.8
76.1
78.4
71.3
78.4
62.4
4.07 dd
4.20 dd
4.28 dd
3.86 m
8.8, 7.8
8.8, 8.8
8.8, 8.8
3ꢄꢅꢃꢄ
4ꢄꢅꢃꢄ
5ꢄꢅꢃꢄ
6ꢄꢅꢃꢄa 4.41 br d
4.34 br d
11.6
11.6
b
a) Spectra were measured in C5D5N.
sugar chain attached at C-3 of the aglycone moiety was made nosyl moiety is the glycosylated position at which the addi-
up of four monosaccharides and differed from that of 1 by tional D-glucosyl unit is linked. In the HMBC spectrum of 2,
the presence of an additional glucosyl unit. In a comparison long-range correlations were observed between H-1 of Rib at
of the 13C-NMR spectrum of 2 with that of 1, a set of six ad- d 5.98 and C-3 of Rha at d 81.1, H-1 of Rha at d 6.32 and C-
ditional signals corresponding to a terminal b-D-glucopyra- 2 of Ara at d 75.8, H-1 of Glc at d 5.14 and C-4 of Ara at d
nosyl unit (Glc) [dH-1 5.14 (1H, d, Jꢂ7.8 Hz); dC 106.8, 75.5, 80.4, H-1 of Ara at d 4.72 and C-3 of the aglycone at d 88.7,
78.8, 71.3, 78.8, and 62.6] appeared, and the signal due to C- H-1 of Glcꢅ at d 5.38 and C-3 of Rhaꢃ at d 84.0, H-1 of Rhaꢃ
4 of the arabinosyl moiety shifted downfield by 11.0 ppm to at d 5.88 and C-4 of Glcꢄ at d 77.3, H-1 of Glcꢄ at d 4.90
d 80.4, suggesting that the C-4 hydroxyl group of the arabi- and C-6 of Glcꢃ at d 69.3, and between H-1 of Glcꢃ at d 6.26

1428
Vol. 57, No. 12
Table 2. Cytotoxic Activities of Compounds 1—11 against Four Human Cancer Cell Linesa,b)
IC50 (mM)
Compound
HL-60
A549
HSC-2
HSC-4
4
5
6
3.1ꢇ0.15
2.3ꢇ0.09
5.7ꢇ0.58
ꢈ10
2.9ꢇ0.20
1.5ꢇ0.14
9.6ꢇ0.03
—
—
6.4ꢇ0.43
ꢈ20
1.7ꢇ0.03
5.9ꢇ0.16
5.7ꢇ0.46
16.3ꢇ0.06
—
13.0ꢇ0.14
11.7ꢇ0.20
ꢈ20
—
—
ꢈ20
ꢈ20
c)
Oleanolic acid
Hederagenin
Cisplatind)
ꢈ10
—
1.6ꢇ0.03
0.38ꢇ0.02
0.07ꢇ0.01
16.9ꢇ0.60
ꢈ20
0.25ꢇ0.03
Etoposided)
Doxorubicind)
0.88ꢇ0.04
a) Data represent the meanꢇS.E.M. of three independent experiments. b) Compounds 1—3 and 7—11 were not cytotoxic to HL-60 cells (IC50ꢈ10 mM). c) Not deter-
mined. d) Positive control substances.
Experimental
and C-28 at the aglycone at d 176.5. Thus, the structure of 2
Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan)
automatic digital polarimeter. IR spectra were recorded on a JASCO FT-IR
620 spectrophotometer. NMR spectra were recorded on a Bruker DRX-500
was determined to be 3b-[(O-b-D-ribopyranosyl-(1→3)-O-a-
L-rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-
(500 MHz for 1H-NMR, Karlsruhe, Germany) and a Bruker DRX-600
(600 MHz for 1H-NMR) spectrometer using standard Bruker pulse pro-
grams. 1D-TOCSY and HSQC-TOCSY spectra were recorded at the mixing
time of 120 ms. Chemical shifts are given as d values in reference to tetra-
methylsilane (TMS) as an internal standard. ESI-TOF-MS spectra were
recorded on a Waters-Micromass LCT mass spectrometer (Manchester,
U.K.). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica gel (Fuji
Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto,
Japan) were used for column chromatography. TLC was carried out on Silica
gel 60 F254 (thickness: 0.25 mm, Merck, Darmstadt, Germany) and RP18
F254S plates (thickness: 0.25 mm, Merck), and spots were visualized by
spraying the plates with 10% H2SO4 solution, followed by heating. HPLC
was performed by using a system composed of a CCPM pump (Tosoh,
Tokyo, Japan), a PX-8010 controller (Tosoh), an RI-8010 (Tosoh) and a
Shodex OR-2 (Showa Denko, Tokyo, Japan) detector, and a Rheodyne injec-
tion port. The following reagents were obtained from the indicated compa-
nies: RPMI 1640, minimum essential medium (MEM), and 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-
L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-b-D-glu-
copyranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→4)-O-b-D-
glucopyranosyl-(1→6)-b-D-glucopyranosyl ester.
Compound 3 was shown to have a molecular formula
C70H114O35 on the basis of HR-ESI-TOF-MS analysis (m/z:
1
1515.7216 [MꢀH]ꢀ). Analysis of the H- and 13C-NMR
spectra of 3 and comparison with that of 1 indicated that the
triglycoside structure attached to C-3 of the aglycone and the
tetraglycoside attached to C-28 are identical to those of 1;
however, 3 slightly differs from 1 in terms of the aglycone
moiety. The molecular weight of 3 was higher than that of 1,
with the difference corresponding to one oxygen atom. In the
13C-NMR spectrum, the signal due to the C-23 methyl group,
which was observed at d 28.2 in 1, appeared as a signal due
to a hydroxymethyl carbon at d 64.0. Furthermore, ABq sig-
nals at d 4.30 and 3.93 (Jꢂ10.3 Hz) were associated with the Aldrich, St. Louis, MO, U.S.A.); fetal bovine serum (FBS) (BioWhittaker,
hydroxymethyl carbon signal by analysis of the HMQC spec-
trum. Acid hydrolysis of 3 yielded 3b,23-dihydroxyolean-12-
en-28-oic acid (hederagenin, 3a), L-rhamnose, D-ribose, L-
Walkersville, MO, U.S.A.); Dulbecco’s modified Eagle’s medium (DMEM),
penicillin G sodium salt, and streptomycin sulfate (Gibco, Grand Island, NY,
U.S.A.). All other chemicals used were of biochemical reagent grade.
Plant Material The young plants of Anemone hupehensis var. japonica
arabinose, and D-glucose. On the basis of these data, the
structure of 3 was found to be 3b-[(O-b-D-ribopyranosyl-
(1→3)-O-a-L-rhamnopyranosyl-(1→2)-a-L-arabinopyra-
nosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-b-D-glu-
copyranosyl-(1→3)-O-a-L-rhamnopyranosyl-(1→4)-O-b-D-
glucopyranosyl-(1→6)-b-D-glucopyranosyl ester.
The isolated compounds (1—11) were evaluated for their
cytotoxic activity against HL-60 cells (Table 2). The triter-
pene monodesmosides (4—6) showed moderate cytotoxic ac-
tivity against HL-60 cells with IC50 values of 3.1, 2.3, and
5.7 mM, respectively. Oleanolic acid (the aglycone of 4 and
5), hederagenin (the aglycone of 6), and bisdesmosides with
the sugar units at both the C-3 hydroxy group and C-28 car-
boxy group (1—3 and 7—11) did not exhibit cytotoxicity
even at a sample concentration of 10 mM. Compounds 4—6
were purchased from Sakata Seed Co., Ltd. (Kanagawa, Japan) in June
2002. The plants were cultivated in the botanical garden of Tokyo University
of Pharmacy and Life Sciences, and identified by Dr. Yutaka Sashida, emeri-
tus professor of medicinal pharmacognosy at Tokyo University of Pharmacy
and Life Sciences. A voucher specimen has been deposited in our laboratory
(voucher No. AH-2003-001, Laboratory of Medicinal Pharmacognosy).
Extraction and Isolation The plant material (dry weight, 3.3 kg) was
extracted with hot MeOH (26 l). The MeOH extract was concentrated under
reduced pressure, and the viscous concentrate (180 g) was passed through a
Diaion HP-20 column (85 mm inner diameter (i.d.).ꢆ200 mm) that was suc-
cessively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc
(each 7 l). Column chromatography of the MeOH eluate portion (50 g) on
silica gel (70 mm i.d.ꢆ330 mm) and elution with a stepwise gradient mix-
ture of CHCl3–MeOH–H2O (9 : 1 : 0; 30 : 10 : 1; 20 : 10 : 1; 7 : 4 : 1), and fi-
nally with MeOH alone, yielded eight fractions (A—H). Fraction D was
chromatographed on an ODS silica gel column (60 mm i.d.ꢆ240 mm) eluted
with MeOH–H2O (4 : 1) to give 14 subfractions (D-1—D-14). Fraction D-7
was suspended in MeOH and the insoluble solid was filtrated to give 6
also exhibited cytotoxic activities against A459 and HSC-2 (1.00 g). Fraction D-11 was chromatographed on silica gel (25 mm
solid tumor cells greater than or equal to those of cisplatin,
which was used as a positive control. A459 and HSC-2 cells
were not sensitive to etoposide. Compounds 4 and 5 were
i.d.ꢆ200 mm) eluted with CHCl3–MeOH–H2O (50 : 10 : 1) to give 4 (15.1
mg). Fraction D-12 was chromatographed on silica gel (35 mm i.d.ꢆ200
mm) eluted with CHCl3–MeOH–H2O (70 : 10 : 1) to give 5 (137 mg). Frac-
tion E was chromatographed on an ODS silica gel column (40 mm
moderately cytotoxic to HSC-4 cells, which were resistant to
both cisplatin and etoposide. Compound 6 is the C-23 hy-
droxy derivative of 4, and it was presumed that the hydroxy
group in 6 diminishes its cytotoxicity against HSC-4 cells.
i.d.ꢆ220 mm) eluted with MeOH–H2O (7 : 3) to give seven subfractions (E-
1—E-7). Fraction E-3 was subjected to column chromatography on silica gel
(35 mm i.d.ꢆ200 mm) eluted with CHCl3–MeOH–H2O (30 : 10 : 1; 20 : 10 :
1) and on ODS silica gel (25 mm i.d.ꢆ240 mm) eluted with MeOH–H2O
(2 : 1) to give 9 (6.2 mg). Fraction E-4 was subjected to column chromatog-
raphy on silica gel (35 mm i.d.ꢆ200 mm) eluted with CHCl3–MeOH–H2O

December 2009
1429
Compound 3: Amorphous solid. [a]D25 ꢁ29.3° (cꢂ0.10, MeOH). HR-ESI-
TOF-MS m/z: 1515.7216 [MꢀH]ꢀ (Calcd for C70H115O35: 1515.7219). IR
nmax (film) cmꢁ1: 3376 (OH), 2936 (CH), 1732 (CꢂO), 1260 and 1061 (C–
(30 : 10 : 1) and on ODS silica gel (25 mm i.d.ꢆ250 mm) eluted with
MeOH–H2O (7 : 3) to give 7 (17.6 mg). Fraction F was separated by an ODS
silica gel column (40 mm i.d.ꢆ240 mm) eluted with MeOH–H2O (7 : 3) to
give 8 (371 mg). Fraction G was subjected to silica gel column chromatogra-
phy (60 mm i.d.ꢆ250 mm) eluted with CHCl3–MeOH–H2O (20 : 10 : 1) and
ODS silica gel column chromatography (40 mm i.d.ꢆ240 mm) eluted with
MeOH–H2O (7 : 3) to give 10 (527 mg). Fraction H was chromatographed on
an ODS silica gel column (60 mm i.d.ꢆ300 mm) eluted with MeOH–H2O
(7 : 3) to give 12 subfractions (H-1—H-12). Fraction H-3 was subjected to
column chromatography on ODS silica gel (40 mm i.d.ꢆ220 mm) eluted
with MeOH–H2O (3 : 2) and on silica gel (25 mm i.d.ꢆ270 mm) eluted with
CHCl3–MeOH–H2O (7 : 4 : 1) to give 11 (37.8 mg). Fraction H-4 was chro-
matographed on silica gel (40 mm i.d.ꢆ250 mm) eluted with CHCl3–
MeOH–H2O (20 : 10 : 1) and on ODS silica gel (25 mm i.d.ꢆ240 mm) eluted
with MeOH–H2O (3 : 2) to give 3 (35.3 mg). Fraction H-6 was subjected to
column chromatography on silica gel (35 mm i.d.ꢆ230 mm) eluted with
CHCl3–MeOH–H2O (20 : 10 : 1) and on ODS silica gel (25 mm i.d.ꢆ230
mm) eluted with MeOH–H2O (2 : 1) to give 2 (6.7 mg). Fraction H-9 was
chromatographed on silica gel (20 mm i.d.ꢆ220 mm) eluted with CHCl3–
MeOH–H2O (20 : 10 : 1) to give 1 (45.3 mg).
1
O). H-NMR (500 MHz, C5D5N) d: 5.41 (1H, br s, H-12), 4.29 (1H, m, H-
3), 4.30 (1H, d, Jꢂ10.3 Hz, H-23a), 3.93 (1H, d, Jꢂ10.3 Hz, H-23b), 3.17
(1H, dd, Jꢂ13.4, 3.3 Hz, H-18), 1.19 (3H, s, Me-27), 1.14 (3H, s, Me-24),
1.11 (3H, s, Me-26), 0.97 (3H, s, Me-25), 0.88 (3Hꢆ2, s, Me-29 and Me-
30); for signals of sugar moieties, see Table 1. 13C-NMR (125 MHz, C5D5N)
d: 39.1 (C-1), 26.4 (C-2), 81.1 (C-3), 43.6 (C-4), 47.7 (C-5), 18.1 (C-6),
32.8 (C-7), 39.9 (C-8), 48.2 (C-9), 36.9 (C-10), 23.8 (C-11), 123.0 (C-12),
144.1 (C-13), 42.1 (C-14), 28.3 (C-15), 23.3 (C-16), 47.0 (C-17), 41.6 (C-
18), 46.2 (C-19), 30.7 (C-20), 34.0 (C-21), 32.5 (C-22), 64.0 (C-23), 14.1
(C-24), 16.2 (C-25), 17.5 (C-26), 26.0 (C-27), 176.5 (C-28), 33.1 (C-29),
23.7 (C-30); for signals of sugar moieties, see Table 1.
Acid Hydrolysis of 3 A solution of 3 (12.0 mg) was subjected to acid
hydrolysis using the above-mentioned procedure for the hydrolysis of 1 to
afford an aglycone fraction (3.3 mg) and a sugar fraction (7.4 mg). The agly-
cone fraction was chromatographed on silica gel eluted with hexane–Me2CO
(4 : 1) to give 3a (hederagenin, 1.7 mg). HPLC analysis of the sugar fraction
under the same conditions as in the analysis of 1 showed the presence of L-
rhamnose, D-ribose, D-arabinose, and D-glucose. tR (min): 7.6 (L-rhamnose,
negative optical rotation), 8.2 (D-ribose, negative optical rotation), 9.0 (D-
arabinose, positive optical rotation), 15.6 (D-glucose, positive optical rota-
tion).
Compound 1: Amorphous solid. [a]D25 ꢁ33.6° (cꢂ0.10, MeOH). HR-ESI-
TOF-MS m/z: 1521.7109 [MꢀNa]ꢀ (Calcd for C70H114O34Na: 1521.7089).
IR nmax (film) cmꢁ1: 3376 (OH), 2925 (CH), 1740 (CꢂO), 1260 and 1064
1
(C–O). H-NMR (500 MHz, C5D5N) d: 5.39 (1H, br s, H-12), 3.26 (1H, dd,
Cell Culture and Assay for Cytotoxic Activity against HL-60 Cells
HL-60 cells, which were obtained from the Human Science Research Re-
sources Bank (JCRB 0085, Osaka, Japan) were maintained in RPMI 1640
medium containing 10% heat-inactivated FBS and antibiotics (100 units/ml
penicillin sodium salt and 100 mg/ml streptomycin sulfate) in a 5% CO2 hu-
midified incubator at 37 °C. The cells were washed and resuspended in the
medium at 4ꢆ104 cells/ml, and 196 ml of this cell suspension was seeded
into 96-well flat bottom plates (Iwaki Glass, Chiba, Japan). The cells were
incubated in 5% CO2/air for 24 h at 37 °C. After incubation, 4 ml of
EtOH–H2O (1 : 1) solution containing the sample was added to obtain final
concentrations of 0.01—20 mM, and 4 ml of EtOH–H2O (1 : 1) was added to
the control wells. The cells were further incubated for 72 h in the presence of
each agent, and then cell growth was evaluated using a modified MTT re-
duction assay.14) At the end of the incubation period, 10 ml of 5 mg/ml MTT
in phosphate buffered saline (PBS) was added to each well, and the plate
was further incubated in 5% CO2/air for 4 h at 37 °C. Then, the plate was
centrifuged at 1500 g for 5 min to precipitate MTT formazan. An aliquot of
150 ml of the supernatant was removed from each well, and 175 ml of di-
methylsulfoxide (DMSO) was added to dissolve the MTT formazan crystals.
The plate was mixed on a microplate mixer for 10 min and then read on a
microplate reader (Spectra Classic, Tecan, Salzburg, Austria) at 550 nm.
Each assay was performed in triplicate and cytotoxicity was expressed as the
IC50 value, which reduces the number of viable cells by 50%.
Cell Culture and Assay for Cytotoxic Activity against A549, HSC-2,
and HSC-4 Cells A549 (JCRB 0076) cells were incubated at 37 °C in
MEM supplemented with 10% heat-inactivated FBS in a humidified 5%
CO2 atmosphere. HSC-2 and HSC-4 cells were maintained as monolayer
cultures at 37 °C in DMEM supplemented with 10% heat-inactivated FBS in
a humidified 5% CO2 atmosphere. Cells were trypsinized and resuspended
in the medium at 1ꢆ105 cells/ml, and 100 ml of this cell suspension was
seeded into 96-well flat bottom plates and incubated for 24 h. After washing
once with PBS, the cells were treated for 24 h in the presence of the test
compounds. The cells were washed once with PBS and incubated for 4 h
with 0.2 mg/ml MTT in MEM (A549 cells) or DMEM (HSC-2 and HSC-4
cells), supplemented with 10% heat-inactivated FBS. After the medium was
removed, the MTT formazan crystals were dissolved with 100 ml of DMSO.
The plate was mixed on a microplate mixer for 10 min and then read on a
microplate reader at 550 nm. Each assay was performed in triplicate and
cytotoxicity was expressed as the IC50 value, which reduces the number of
viable cells by 50%.
Jꢂ11.5, 3.8 Hz, H-3), 3.17 (1H, dd, Jꢂ10.1, 3.3 Hz, H-18), 1.28 (3H, s, Me-
23), 1.23 (3H, s, Me-27), 1.13 (3H, s, Me-24), 1.06 (3H, s, Me-26), 0.89
(3H, s, Me-29), 0.87 (3H, s, Me-30), 0.85 (3H, s, Me-25); for signals of
sugar moieties, see Table 1. 13C-NMR (125 MHz, C5D5N) d: 38.9 (C-1),
26.6 (C-2), 88.7 (C-3), 39.6 (C-4), 56.0 (C-5), 18.5 (C-6), 33.1 (C-7), 39.9
(C-8), 48.0 (C-9), 37.0 (C-10), 23.8 (C-11), 122.8 (C-12), 144.1 (C-13), 42.1
(C-14), 28.3 (C-15), 23.3 (C-16), 47.0 (C-17), 41.7 (C-18), 46.2 (C-19), 30.7
(C-20), 34.0 (C-21), 32.5 (C-22), 28.2 (C-23), 17.1 (C-24), 15.6 (C-25), 17.4
(C-26), 26.1 (C-27), 176.5 (C-28), 33.1 (C-29), 23.7 (C-30); for signals for
the sugar moieties, see Table 1.
Acid Hydrolysis of 1 A solution of 1 (12.0 mg) in 1 M HCl (dioxane–
H2O, 1 : 1, 3 ml) was heated at 95 °C for 1.5 h under an Ar atmosphere. After
cooling, the reaction mixture was neutralized by passage through an Amber-
lite IRA-96SB (Organo, Tokyo, Japan) column and chromatographed on Di-
aion HP-20 eluted with MeOH–H2O (2 : 3) followed by EtOH–Me2CO
(1 : 1), to yield an aglycone fraction (3.7 mg) and a sugar fraction (8.6 mg).
The aglycone fraction was chromatographed on silica gel eluted with
hexane–Me2CO (4 : 1) to give 1a (oleanolic acid, 2.3 mg). The sugar fraction
was analyzed by HPLC under the following conditions: column, Capcell Pak
NH2 UG80 (4.6 mm i.d.ꢆ250 mm, 5 mm, Shiseido, Tokyo, Japan); solvent,
MeCN–H2O (17 : 3); flow rate, 0.9 ml/min; detection, refractive index (RI)
and optical rotation (OR). Identification of L-rhamnose, D-ribose, D-arabi-
nose, and D-glucose present in the sugar fraction was carried out by compar-
ison of their retention times (tR) and optical rotations with those of authentic
samples. tR (min): 7.6 (L-rhamnose, negative optical rotation), 8.2 (D-ribose,
negative optical rotation), 9.0 (D-arabinose, positive optical rotation), 15.6
(D-glucose, positive optical rotation).
Compound 2: Amorphous solid. [a]D25 ꢁ20.2° (cꢂ0.10, MeOH). HR-ESI-
TOF-MS m/z: 1661.7778 [MꢀH]ꢀ (Calcd for C76H125O39: 1661.7798). IR
nmax (film) cmꢁ1: 3376 (OH), 2925 (CH), 1739 (CꢂO), 1260, 1092 and
1027 (C–O). 1H-NMR (500 MHz, C5D5N) d: 5.42 (1H, br s, H-12), 3.25
(1H, dd, Jꢂ11.5, 3.8 Hz, H-3), 3.19 (1H, dd, Jꢂ13.4, 3.4 Hz, H-18), 1.31
(3H, s, Me-23), 1.26 (3H, s, Me-27), 1.18 (3H, s, Me-24), 1.09 (3H, s, Me-
26), 0.92 (3H, s, Me-29), 0.90 (3H, s, Me-30), 0.88 (3H, s, Me-25); for sig-
nals of sugar moieties, see Table 1. 13C-NMR (125 MHz, C5D5N) d: 39.0 (C-
1), 26.7 (C-2), 88.7 (C-3), 39.6 (C-4), 56.1 (C-5), 18.5 (C-6), 33.2 (C-7),
39.9 (C-8), 48.1 (C-9), 37.0 (C-10), 23.7 (C-11), 123.1 (C-12), 144.1 (C-13),
42.2 (C-14), 28.2 (C-15), 23.4 (C-16), 47.0 (C-17), 41.7 (C-18), 46.3 (C-19),
30.8 (C-20), 34.0 (C-21), 32.5 (C-22), 28.2 (C-23), 17.2 (C-24), 15.7 (C-25),
17.5 (C-26), 26.1 (C-27), 176.5 (C-28), 33.2 (C-29), 23.7 (C-30); for signals
of sugar moieties, see Table 1.
References and Notes
1) “The Grand Dictionary of Horticulture,” Vol. 1, ed. by Tsukamoto Y.,
Shogakukan, Tokyo, 1989, pp. 109—110.
2) Jiangsu New Medical College, “The Dictionary of Chinese Medicinal
Materials,” Vol. 1, Shanghai Scientific and Technological Press,
Shanghai, 1977, pp. 640—641.
3) Wang M. K., Chen Y. Z., Wu F. E., Huaxue Xuebao, 52, 609—612
(1994).
Acid Hydrolysis of 2 A solution of 2 (6.0 mg) was subjected to acid hy-
drolysis using the above-mentioned procedure for the hydrolysis of 1 to af-
ford an aglycone fraction (1.5 mg) and a sugar fraction (4.5 mg). The agly-
cone fraction was chromatographed on silica gel eluted with hexane–Me2CO
(4 : 1) to give 1a (0.8 mg). HPLC analysis of the sugar fraction under the
same conditions as in the analysis of 1 showed the presence of L-rhamnose,
D-ribose, D-arabinose, and D-glucose. tR (min): 7.7 (L-rhamnose, negative op-
tical rotation), 8.3 (D-ribose, negative optical rotation), 9.7 (D-arabinose, pos-
itive optical rotation), 17.2 (D-glucose, positive optical rotation).
4) Mizutani K., Ohtani K., Wei J. X., Kasai R., Tanaka O., Planta Med.,

1430
Vol. 57, No. 12
50, 327—331 (1984).
10) Yokosuka A., Mimaki Y., Chem. Pharm. Bull., 52, 1396—1398
(2004).
11) Yokosuka A., Kawakami S., Haraguchi M., Mimaki Y., Tetrahedron,
64, 1474—1481 (2008).
5) Dubois M. A., Ilyas M., Wagner H., Planta Med., 52, 80—83 (1986).
6) Dovgii I. I., Grishkovets V. I., Kachala V. V., Shashkov A. S., Chem.
Nat. Compd., 42, 182—185 (2006).
7) Wang M., Wu F., Chen Y., Phytochemistry, 44, 333—335 (1997).
8) Shao B., Qin G., Xu R., Wu H., Ma K., Phytochemistry, 38, 1473—
1479 (1995).
9) Yokosuka A., Mimaki Y., Sakuma C., Sashida Y., Steroids, 70, 257—
265 (2005).
12) Agrawal P. K., Jain D. C., Gupta R. K., Thakur R. S., Phytochemistry,
24, 2479—2496 (1985).
13) Agrawal P. K., Phytochemistry, 31, 3307—3330 (1992).
14) Sargent J. M., Taylor C. G., Br. J. Cancer, 60, 206—210 (1989).