Four new steroidal saponins from the rhizomes of Helleborus orientalis

Article abstract of DOI:10.3987/COM-04-10319

A phytochemical analysis has been carried out on theglycoside-enriched fraction of a McOH extract of Helleborus orientalis rhizomes, resulting in the isolation of two new bisdesmosidic furostanol saponins (1 and 2) and two new furospirostanol saponins (3 and 4). The structures of 1-4 were determined on the basis of extensive spectroscopic analysis and acid hydrolysis followed by chromatographic and spectroscopic analysis. Compound (3) showed moderate cytotoxic activity against HSC-2 human squamous cell carcinoma cells with an IC₅₀ value of 16 μg/mL.

Full text of DOI:10.3987/COM-04-10319

HETEROCYCLES, Vol. 65, No. 4, 2005
775
HETEROCYCLES, Vol. 65, No. 4, 2005, pp. 775 - 785
Received, 27th December, 2004, Accepted, 7th February, 2005, Published online, 8th February, 2005
FOUR NEW STEROIDAL SAPONINS FROM THE RHIZOMES OF
HELLEBORUS ORIENTALIS
a)
b)
a),*
Kazuki Watanabe, Hiroshi Sakagami, and Yoshihiro Mimaki
a
Laboratory of Medicinal Pharmacognosy, Tokyo University of Pharmacy and
Life Science, School of Pharmacy, 1432-1, Horinouchi, Hachioji, Tokyo
192-0392, Japan
b
Department of Dental Pharmacology, Meikai University School of Dentistry,
1-1, Keyaki-dai, Sakado, Saitama 350-0283, Japan.
Abstract – A phytochemical analysis has been carried out on the
glycoside-enriched fraction of a MeOH extract of Helleborus orientalis rhizomes,
resulting in the isolation of two new bisdesmosidic furostanol saponins (1 and 2)
and two new furospirostanol saponins (3 and 4). The structures of 1-4 were
determined on the basis of extensive spectroscopic analysis and acid hydrolysis
followed by chromatographic and spectroscopic analysis. Compound (3)
showed moderate cytotoxic activity against HSC-2 human squamous cell
carcinoma cells with an IC50 value of 16 µg/mL.
INTROCUCTION
Helleborus orientalis, known by the name ‘Lenten Rose’ in Europe, is a perennial plant belonging to the
family Ranunculaceae and is indigenous to Greece and Turkey.1 Its rhizomes are known to contain
several bufadienolide glycosides and were used as a cardiotonic agent.2 Plants containing cardiac
glycosides such as Digitalis purpurea3 and Convallaria majalis4 concomitantly produce steroidal
glycosides. We have now made a phytochemical screening of the glycoside-enriched fraction prepared
from a MeOH extract of the rhizomes of H. orientalis and isolated two new bisdesmosidic furostanol
saponins (1 and 2) and two new furospirostanol saponins (3 and 4). This paper reports the structural
elucidation of the new compounds on the basis of extensive spectroscopic analysis and acid hydrolysis
followed by chromatographic and spectroscopic analysis. The cytotoxic activity of the isolated
compounds against HSC-2 human squamous cell carcinoma cells is briefly described.

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RESULTS AND DISCUSSION
A concentrated MeOH extract of H. orientalis rhizomes was passed through a porous-polymer resin
(Diaion HP-20) column and the 80% MeOH eluate fraction, in which steroidal glycosides were enriched,
was subjected to column chromatography over silica gel and octadecylsilanized (ODS) silica gel, as well
as preparative HPLC, giving to compounds (1) (8.6 mg), (2) (12.0 mg), (3) (106 mg), and (4) (6.8 mg).
Compound (1) was obtained as an amorphous solid, [α]D -84.0°(MeOH). Its molecular formula was
13
determined to be C55H88O26 by the FAB-MS (m/z 1187 [M + Na]+), C-NMR spectrum, and elemental
analysis data. The glycosidic nature of 1 was suggested by strong absorption bands at 3288 and 1043
1
cm-1. The H-NMR spectrum of 1 contained signals for two tertiary methyl groups at δ 1.43 and 0.82
(each s), a secondary methyl group at δ 1.11 (d, J = 6.9 Hz), an exomethylene group at δ 5.33 and 5.04
(each br s), an olefinic proton at δ 5.58 (br d, J = 5.5 Hz), and five anomeric protons at δ 6.35 (d, J = 1.5
Hz), 6.23 (d, J = 2.5 Hz), 4.99 (d, J = 7.5 Hz), 4.91 (d, J = 7.8 Hz), and 4.69 (d, J = 7.3 Hz). Acid
hydrolysis of 1 with 0.2 M HCl in dioxane-H2O (1:1) gave a steroidal sapogenin, identified as
spirosta-5,25(27)-diene-1β,3β-diol (1a),5 together with D-apiose, L-arabinose, D-glucose, L-rhamnose,
and D-xylose. The monosaccharides, including their absolute configurations, were identified by direct
HPLC analysis of the hydrolysate, which was performed on an aminopropyl-bonded silica gel column
using MeCN-H2O (17:3) as solvent system, with detection being carried out using a combination of RI
and optical rotation (OR) detectors. The above spectral and chemical data, as well as a positive color
reaction in Ehrlich’s test, the three-proton singlet signal at δ 3.24 (3H, s), and the acetalic carbon signal at
δ 112.4 (C) indicated that 1 was the corresponding bisdesmosidic 22α-methoxyfurostanol saponin6 of 1a,
1
whose sugar moieties were composed of a total of five monosaccharides. The H-1H COSY and 2D
TOCSY experiments with 1 allowed the sequential assignments from H-1 to CH2-5, Me-6, and CH2-6 of
four monosaccharides. Their signal multiplet patterns and coupling constants enabled the identification
of an α-L-arabinopyranosyl (4C1) unit, a β-D-glucopyranosyl (4C1) unit, an α-L-rhamnopyranosyl (1C4)
unit, and a β-D-xylopyranosyl (4C1) unit (Table 1). In addition, two pairs of ABq-like signals at δ 6.23
and 4.82 (J = 2.5 Hz) and δ 4.64 and 4.28 (J = 9.2 Hz), and two-proton broad singlet signal at δ 4.15,
along with the results of acid hydrolysis, were indicative of an apiofuranosyl unit. The relatively large J
values of the anomeric protons of the arabinosyl (7.3 Hz), glucosyl (7.8 Hz), and xylosyl (7.5 Hz)
moieties indicated an α anomeric orientation for the arabinosyl and β for the glucosyl and xylosyl. For
1
the rhamnosyl moiety, the large JC,H values (170 Hz <) confirmed that the anomeric proton was
13
equatorial thus possessing an α-pyranoid anomeric form.7 The C-NMR shifts of the anomeric carbon
of the apiosyl at δ 111.6 indicated a β-orientation of the anomeric center.8 All the proton signals for the
sugar moiety thus assigned were associated with the one-bond coupled carbon signals using the HMQC

HETEROCYCLES, Vol. 65, No. 4, 2005
777
spectrum. The apiosyl, glucosyl, and xylosyl residues were considered to be the terminal units, as
shown by the absence of any glycosylation shift for their carbon resonances, while C-2 and C-3 of the
arabinosyl unit, and C-3 of the rhamnosyl unit were suggested to be substituted by comparison with those
of authentic methyl glycosides.9 In the HMBC spectrum of 1, the anomeric proton of the apiosyl at δ
6.23 showed a 3JC,H correlation with C-3 of the rhamnosyl at δ 79.8, whose anomeric proton at δ 6.35, in
turn, showed a long-range correlation with C-2 of the arabinosyl at δ 73.6. The anomeric proton of the
xylosyl at δ 4.99 was correlated to C-3 of the arabinosyl at δ 84.7. The arabinosyl moiety was thus
shown to be glycosylated at C-2 and C-3, and its anomeric proton at δ 4.69 exhibited an HMBC
correlation with C-1 of the aglycone at δ 84.1. As is usual with naturally occurring furostanol
bisdesmosides,10 a glucosyl group was revealed to be linked to the C-26 hydroxyl group of the aglycone
in 1 by an HMBC correlation of the anomeric proton of the glucosyl at δ 4.91 to C-26 of the aglycone at δ
72.0. Accordingly, the structure of 1 was formulated as 26-[(β-D-glucopyranosyl)oxy]-3β-hydroxy-
22α-methoxyfurosta-5,25(27)-diene-1β-yl O-β-D-apiofuranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-O-
[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.
1
Compound (2) (C52H84O23) was obtained as an amorphous solid, [α]D -96.0°(MeOH). The H-NMR
spectrum of 2 showed signals for four anomeric protons at δ 6.32 (d, J = 0.6 Hz), 5.09 (d, J = 7.8 Hz),
4.96 (d, J = 7.7 Hz), and 4.92 (d, J = 7.8 Hz), as well as signals for two tertiary methyl groups at δ 1.03
and 0.79 (each s), a secondary methyl group at δ 1.17 (d, J = 6.9 Hz), an exomethylene group at δ 5.36
and 5.07 (each br s), an olefinic proton at δ 5.31 (br d, J = 4.9 Hz), and a methoxy group at δ 3.26 (s).
Acid hydrolysis of 2 gave a steroidal sapogenin, identified as spirosta-5,25(27)-dien-3β-ol (2a),11 and
1
D-glucose and L-rhamnose. These H-NMR and chemical data, together with an acetalic carbon signal
at δ 112.4 and a positive color reaction with Ehrlich’s reagent, implied that 2 was the corresponding
1
bisdesmosidic 22α-methoxyfurostanol saponin of 2a. Analysis of the H-1H COSY, 2D TOCSY, and
HMQC spectra of 2 allowed the identification of the proton signals, including their multiplet patterns and
coupling constant, and the carbon chemical shifts of the sugar moieties of 2 as shown in Table 1. The
results indicated the presence of two terminal β-D-glucopyranosy units, an terminal α-L-rhamnopyranosyl
units, and a C-2 and C-6 disubstituted β-D-glucopyranosy unit in 2. In the HMBC spectrum of 2, the
anomeric protons at δ 6.32 (rhamnosyl) and 5.09 (terminal glucosyl) exhibited long-range correlations
with C-2 (δ 77.5) and C-6 (δ 69.8) of the inner glucosyl unit, respectively. Forthermore, the anomeric
protons of the inner glucosyl at δ 4.96 showed an HMBC correlation with C-3 of the aglycone at δ 78.5,
whereas that of the remaining glucosyl at δ 4.92 was correlated to C-26 of the aglycone at δ 72.0. Thus,
the structure of 2 was elucidated as 26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurosta-5,25(27)-diene-

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HETEROCYCLES, Vol. 65, No. 4, 2005
3β-yl O-β-D-glucopyranosyl-(1→6)-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside.
Compound (3) (C38H60O14) was suggested to be also a steroidal saponin by its spectral properties. The
1H-NMR spectrum of 3 showed signals for three tertiary methyl groups at δ 1.37, 1.20, and 0.79 (each s),
HO
O
O
HO
HO
HO1'''''
27
OMe
22
21
HO
18
O
O
O
O 19
1
HO
O
H
H
HO
1'
HO1''''
O
H
H
1''
3
HO
Me
O
O
HO
O
OH
1'''
HO
HO OH
1
HO
HO
O
HO1''''
O
HO
OMe
O
HO
H
H
O
HO
O
HO
H
H
HO1'''
O
HO
HO
O
1'
O
1''
Me
O
HO
HO
OH
2
HO
HO
O
O
HO
H
HO
O
O
HO
HO
HO1''
O
HO1'
O
26
27
HO
HO
HO
O
O
O
O
HO
11
H
H
H
HO1'
H
H
H
H
HO
HO
3
4

HETEROCYCLES, Vol. 65, No. 4, 2005
779
a secondary methyl group at δ 1.00 (d, J = 6.9 Hz), an olefinic proton at δ 5.54 (br d, J = 5.7 Hz), and two
anomeric protons at δ 4.93 (d, J = 7.7 Hz) and 4.73 (d, J = 7.4 Hz). In the 13C-NMR spectrum of 3, the
C-22 acetalic carbon signal was observed at δ 120.2, which was typical of furospirostanol type steroids.12
1
Table 1. H- and 13C-NMR spectral data of the sugar moieties of 1 and 2 in C5D5N
1
2
1H
J (Hz)
13C
1H
J (Hz)
13C
4.69 d
4.62 dd
4.08 dd
4.40 br d 3.1
4.24 *
7.3
8.7, 7.3
8.7, 3.1
100.6
73.6
84.7
69.5
66.9
4.96 d
7.7
100.6
77.5
79.4
71.6
1
2
3
4
5
1
2
3
4
5
6
'
'
'
'
'
'
'
'
'
'
'
4.19 dd
4.23 dd
4.13 dd
9.2, 7.7
9.4, 9.2
9.4, 8.8
4.01 ddd 8.8, 5.5, 1.7 76.8
a
b
3.69 br d 11.3
4.76 dd
4.33 dd
11.6, 1.7
11.6, 5.5
69.8
a
b
6.35 d
1.5
101.4
71.7
79.8
72.5
69.5
19.0
1
2
3
4
5
6
''
''
''
''
''
''
4.95 dd
4.66 dd
4.38 dd
4.83 dq
1.68 d
3.1, 1.5
9.4, 3.1
9.4, 8.5
8.5, 6.0
6.0
6.32 d
0.6
102.0
72.5
72.8
74.1
69.4
18.6
1
2
3
4
5
6
''
''
''
''
''
''
4.78 dd
4.61 dd
4.34 dd
4.96 dq
1.77 d
3.3, 0.6
9.6, 3.3
9.6, 9.6
9.6, 6.2
6.2
6.23 d
4.82 d
-
4.64 d
4.28 d
4.15 br s
2.5
2.5
111.6
77.7
80.2
75.1
1
2
3
4
'''
'''
'''
'''
5.09 d
7.8
105.3
75.1
78.4
71.6
1
2
3
4
5
6
'''
'''
'''
'''
'''
'''
4.02 dd
4.19 dd
4.21 dd
8.4, 7.8
9.2, 8.4
9.2, 8.3
9.2
9.2
a
b
(2H)
65.5
3.91 ddd 8.3, 5.1, 2.3 78.4
5
'''
4.50 dd
4.36 dd
11.8, 2.3
11.8, 5.1
62.7
a
b
4.99 d
3.90 dd
4.11 dd
4.12 *
4.26 *
3.68 dd
7.5
8.6, 7.5
8.6, 8.1
106.4
74.6
78.3
70.9
67.1
1
2
3
4
5
''''
''''
''''
''''
''''
4.92 d
7.8
103.8
75.1
78.6
71.7
1
2
3
4
5
6
''''
''''
''''
''''
''''
''''
4.08 dd
4.26 dd
4.24 dd
8.5, 7.8
8.9, 8.5
9.1, 8.9
a
b
12.5, 11.0
3.96 ddd 8.9, 5.4, 2.4 78.4
4.91 d
7.8
103.8
75.1
78.5
71.7
4.55 dd
4.43 dd
62.8
11.9, 2.4
11.9, 5.4
1
2
3
4
5
6
a
b
'''''
'''''
'''''
'''''
'''''
'''''
4.06 dd
4.24 dd
4.23 dd
3.95 ddd 8.7, 5.5, 2.5 78.6
4.55 dd
7.8, 7.7
8.7, 7.7
8.7, 8.7
11.9, 2.5
11.9, 5.5
62.8
a
b
4.39 dd
* Multiplicities are not clear due to overlapping.
Acid hydrolysis of 3 with 2 M HCl in dioxane-H2O (1:1) gave L-arabinose and D-glucose, as well as a

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HETEROCYCLES, Vol. 65, No. 4, 2005
pseudo-aglycone, identified as spirost-5-ene-1β,3β,25-triol (3a).13 This was assumed to be produced
from the corresponding furospirostanol sapogenin of 22,25-epoxyfurost-5-ene-1β,3β-diol under acidic
conditions.14 When the 1H- and 13C-NMR signals arising from the F-ring part of 3a were compared with
those of (25R)- and (25S)-spirost-5-en-3β,25-diol,15 they were in good agreement with those of the
(25S)-isomer. Analysis of the 1H- and 13C-NMR spectra of 3 resulted in the identification of signals for
an α-L-arabinopyranosyl unit and a β-D-glucopyranosyl unit (Table 2). In the HMBC spectrum, the
anomeric proton of the arabinosyl moiety at δ 4.73 exhibited a long-range correlation with C-1 of the
aglycone at δ 83.1, whereas that of the glucosyl at δ 4.93 was correlated to C-26 of the aglycone at δ 77.4.
The steructure of 3 was characterized as (25S)-22α,25-epoxy-26-[(β-D-glucopyranosyl)oxy]-3β-hydroxy-
furost-5-en-1β-yl α-L-arabinopyranoside.
1
Table 2. H- and 13C-NMR spectral data of the sugar moieties of 3 and 4 in C5D5N
3
4
1H
J (Hz)
13C
1H
J (Hz)
13C
1
2
3
4
1
2
3
4
5
6
4.73 d
4.38 dd
4.12 dd
4.26 br d 3.4
4.29 dd
7.4
9.4, 7.4
9.4, 3.4
102.2
72.6
74.7
69.6
67.5
4.93 d
7.8
105.3
75.3
78.3
71.5
'
'
'
'
'
'
'
'
'
'
4.02 dd
4.26 dd
4.27 dd
8.9, 7.8
8.9, 8.6
9.4, 8.6
5 a
'
11.3, 2.1
3.92 ddd 9.4, 5.0, 2.4 78.4
b
3.71 br d 11.3
4.51 dd
4.40 dd
11.9, 2.4
11.9, 5.0
62.6
a
b
1
''
2
''
3
''
4
''
4.93 d
7.7
105.3
75.3
78.3
71.5
4.02 dd
4.25 dd
4.26 dd
8.9, 7.7
9.4, 8.9
9.4, 8.6
5
''
6 a
''
3.91 ddd 8.6, 5.2, 2.4 78.4
4.51 dd
4.39 dd
11.9, 2.4
11.9, 5.2
62.5
b
Compound (4) was isolated as an amorphous solid with the molecular formula C33H52O11. Analysis of
the 1H- and 13C-NMR spectra of 4 and comparison with those of 3 implied that 4 was closely related to 3.
However, the signals due to the α-L-arabinopyranosyl group linked to C-1 of the aglycone could not be
observed in the 1H- and 13C-NMR spectra of 4, and the C-1 carbon signal of the aglycone moiety of 4 was
shifted upfield by 7.3 ppm in comparison with that of 3. Furthermore, 4 differed from 3 in the presence
of one more hydroxymethine group (δH 4.24; δC 66.0), which was shown to be located at C-11 by proton
spin-coupling correlations of the signal at δ 4.24 with H-9 at δ 1.47 and H2-12 at δ 2.42 and 1.55. The
large proton coupling constants between H-9 and H-11 (9.9 Hz), and between H-11 and H-12 axial (10.8

HETEROCYCLES, Vol. 65, No. 4, 2005
781
Hz) indicated that the C-11 hydroxy group was an α-equatorial-oriented configuration. The above data
led to the structure of 4 as (25S)-22α,25-epoxy-3β,11α-dihydroxyfurost-5-en-26-yl β-D-glucopyranoside.
Compounds (1-4) were evaluated for their cytotoxic activity against HSC-2 human squamous cell
carcinoma cells. Only 3 showed cytotoxic activity with an IC50 value of 16 µg/mL, when etoposide used
as a positive control, to which HSC-2 cells are relatively resistant, had an IC50 value of 24 µg/mL.
EXPERIMENTAL
Optical rotations were measured using a JASCO DIP-360 (Tokyo, Japan) automatic digital polarimeter.
IR spectra were recorded on a JASCO FT-IR 620 spectrophotometer. NMR spectra were recorded on a
1
Bruker DRX-500 spectrometer (500 MHz for H NMR, Karlsruhe, Germany) using standard Bruker
pulse programs. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as
internal standard. MS spectra were recorded on a Finnigan MAT TSQ-700 (San Jose, CA) or a
Micromass LCT (Manchester, UK) mass spectrometer. Elemental analysis was carried out using an
Elemental Vario EL (Hanau, Germany) elemental analyzer. Silica gel (Fuji-Silysia Chemical, Aichi,
Japan), ODS silica gel (Nacalai Tesque, Kyoto, Japan), and Diaion HP-20 (Mitsubishi-Chemical, Tokyo,
Japan) were used for column chromatography. TLC was carried out on precoated Kieselgel 60 F254
(0.25 mm thick, Merck, Darmstadt, Germany) and RP-18 F254S (0.25 mm thick, Merck) plates, and spots
were visualized by spraying the plates with 10% H2SO4 solution, followed by heating. HPLC was
performed using a system comprised of 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, Japan), and a
Rheodyne injection port. A Capcell Pak C18 UG120 column (10 mm i.d. x 250 mm, 5 µm, Shiseido,
Tokyo, Japan) was employed for preparative HPLC. The following reagents were obtained from the
indicated companies: Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA);
fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA); penicillin and streptomycin sulfate
(Meiji-Seica, Tokyo, Japan); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
(Sigma, St. Louis, MO, USA). All other chemicals used were of biochemical reagent grade.
Plant material
Helleborus orientalis was purchased from a nursery in Heiwaen, Japan, in November, 1999, and was
identified by an emeritus professor of Tokyo University of Pharmacy and Life Science, Dr. Y. Sashida.
A voucher specimen has been deposited in our laboratory (voucher No. 99-11-7-HO, Laboratory of
Medicinal Pharmacognosy).
Extraction and isolation
The plant material (fresh weight, 2.7 kg) was extracted with MeOH (20 L) at 75 °C for 3 h twice. The
MeOH extract was concentrated under reduced pressure, and the viscous concentrate (300 g) was passed

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HETEROCYCLES, Vol. 65, No. 4, 2005
through a Diaion HP-20 column (30% MeOH, 80% MeOH, MeOH, EtOH, and EtOAc). The 80%
MeOH eluate portion (90 g) was chromatographed on silica gel, eluted with CHCl3-MeOH gradients (9:1,
4:1, 2:1) and finally MeOH, to give subfractions I-VIII. Fraction V was subjected to silica gel column
chromatography, eluted with CHCl3-MeOH-H2O (40:10:1; 30:10:1; 20:10:1), ODS silica gel with
MeCN-H2O (1:2; 1:4) and MeOH-H2O (4:5), and finally to preparative HPLC using MeCN-H2O (1:3) to
furnish 2 (12.0 mg), 3 (106 mg), and 4 (6.8 mg). Fraction VII was subjected to silica gel column
chromatography, eluted with CHCl3-MeOH-H2O (70:40:9), ODS silica gel with MeCN-H2O (1:3; 1:5),
and to preparative HPLC using MeCN-H2O (1:3) to give 1 (8.6 mg).
Compound (1): amorphous solid; [α]D -84.0°(c = 0.10, MeOH); IR (film) νmax 3288 (OH), 2948 and
2888 (CH), 1043 cm-1; 1H-NMR (C5D5N) δ 5.58 (1H, br d, J = 5.5 Hz, H-6), 5.33 and 5.04 (each 1H, br s,
H2-27), 4.60 and 4.35 (each 1H, d, J = 12.7 Hz, H2-26), 4.41 (1H, m, H-16), 3.87 (1H, m, H-3), 3.79 (1H,
dd, J = 12.0, 3.9 Hz, H-1), 3.24 (3H, s, OMe), 1.43 (3H, s, Me-19), 1.11 (3H, d, J = 6.9 Hz, Me-21), 0.82
(3H, s, Me-18), signals for the sugar moieties, see Table 1; 13C-NMR (C5D5N) δ 84.1 (C-1), 37.5 (C-2),
68.3 (C-3), 43.8 (C-4), 139.5 (C-5), 124.7 (C-6), 31.9 (C-7), 33.0 (C-8), 50.3 (C-9), 42.9 (C-10), 24.0
(C-11), 40.2 (C-12), 40.5 (C-13), 56.7 (C-14), 32.3 (C-15), 81.4 (C-16), 64.1 (C-17), 16.6 (C-18), 15.1
(C-19), 40.4 (C-20), 16.1 (C-21), 112.4 (C-22), 31.6 (C-23), 28.0 (C-24), 146.8 (C-25), 72.0 (C-26),
111.0 (C-27), 47.3 (OMe), signals for the sugar moieties, see Table 1; FAB-MS (positive-mode) m/z 1187
[M + Na]+; Anal. Calcd for C55H88O26・3H2O: C; 54.18, H; 7.77. Found: C; 54.07, H; 8.04.
Acid Hydrolysis of 1
A solution of 1 (5.8 mg) in 0.2 M HCl (dioxane-H2O, 1:1, 3 mL) was heated at 95 °C for 30 min under an
Ar atmosphere. After cooling, the reaction mixture was neutralized by passage through an Amberlite
IRA-93ZU (Organo, Tokyo, Japan) column and then passed through a Sep-Pak C18 cartridge (Waters,
Milford, MA, USA), eluted with H2O followed by MeOH. The MeOH eluate portion was
chromatographed on silica gel, eluted with CHCl3-MeOH (9:1), to give 1a (1.9 mg). The H2O eluate
portion was passed through a Toyopak IC-SP M cartridge (Tosoh, Tokyo, Japan), which was then
analyzed by HPLC under the following conditions: column, Capcell Pak NH2 UG80 (4.6 mm i.d. x 250
mm, 5 µm, Shiseido); solvent, MeCN-H2O (17:3); flow rate, 0.9 mL/min; detection, RI and OR.
Identification of D-apiose, L-arabinose, D-glucose, L-rhamnose, and D-xylose present in the sugar fraction
was carried out by comparison of their retention times and optical rotations with those of authentic
samples. tR (min): 7.32 (D-apiose, positive optical rotation), 7.91 (L-rhamnose, negative optical rotation),
9.58 (L-arabinose, positive optical rotation), 9.96 (D-xylose, positive optical rotation), 15.30 (D-glucose,
positive optical rotation).
Compound (2): amorphous solid; [α]D -96.0°(c = 0.10, MeOH); IR (film) νmax 3388 (OH), 2933 and
2851 (CH), 1041 cm-1; 1H-NMR (C5D5N) δ 5.36 and 5.07 (each 1H, br s, H2-27), 5.31 (1H, br d, J = 4.9

HETEROCYCLES, Vol. 65, No. 4, 2005
783
Hz, H-6), 4.63 and 4.37 (each 1H, d, J = 11.0 Hz, H2-26), 4.44 (1H, m H-16), 3.96 (1H, m, H-3), 3.26
(3H, s, OMe), 1.17 (3H, d, J = 6.9 Hz, Me-21), 1.03 (3H, s, Me-19), 0.79 (3H, s, Me-18), signals for the
13
sugar moieties, see Table 1; C-NMR (C5D5N) δ 37.5 (C-1), 30.3 (C-2), 78.5 (C-3), 39.1 (C-4), 140.9
(C-5), 121.6 (C-6), 32.2 (C-7), 31.6 (C-8), 50.2 (C-9), 37.1 (C-10), 21.0 (C-11), 39.7 (C-12), 40.7 (C-13),
56.5 (C-14), 32.1 (C-15), 81.4 (C-16), 64.0 (C-17), 16.2 (C-18), 19.4 (C-19), 40.3 (C-20), 16.2 (C-21),
112.4 (C-22), 29.9 (C-23), 28.1 (C-24), 146.8 (C-25), 72.0 (C-26), 111.1 (C-27), 47.3 (OMe), signals for
the sugar moieties, see Table 1; HR-ESI-MS (positive-mode) m/z 1045.5214 [M + H - MeOH]+ (Calcd
1045.5220 for C51H81O22).
Acid hydrolysis of 2
Compound (2) (5.4 mg) was subjected to acid hydrolysis as described for 1 to give 2a (1.2 mg) and a
sugar fraction (1.7 mg). HPLC analysis of the sugar fraction under the same conditions as in the case of
that of 1 showed the presence of D-glucose and L-rhamnose. tR (min): 7.82 (L-rhamnose, negative
optical rotation), 14.86 (D-glucose, positive optical rotation).
Compound (3): amorphous solid; [α]D -68.0°(c = 0.10, MeOH); IR (film) νmax 3363 (OH), 2890 and
2875 (CH), 1024 cm-1; 1H-NMR (C5D5N) δ 5.54 (1H, br d, J = 5.7 Hz, H-6), 4.67 (1H, q-like, J = 7.2 Hz,
H-16), 4.14 and 3.87 (each 1H, d, J = 10.0 Hz, H2-26), 3.89 (1H, m, H-3), 3.84 (1H, dd, J = 11.7, 4.0 Hz,
H-1), 1.37 (3H, s, Me-27), 1.20 (3H, s, Me-19), 1.00 (3H, d, J = 6.9 Hz, Me-21), 0.79 (3H, s, Me-18),
signals for the sugar moieties, see Table 2; 13C-NMR (C5D5N) δ 83.1 (C-1), 37.5 (C-2), 68.0 (C-3), 43.7
(C-4), 139.4 (C-5), 124.6 (C-6), 31.8 (C-7), 32.9 (C-8), 50.1 (C-9), 42.8 (C-10), 23.7 (C-11), 40.2 (C-12),
40.2 (C-13), 56.5 (C-14), 32.3 (C-15), 80.9 (C-16), 62.5 (C-17), 16.5 (C-18), 14.7 (C-19), 38.4 (C-20),
15.1 (C-21), 120.2 (C-22), 33.0 (C-23), 33.8 (C-24), 83.8 (C-25), 77.4 (C-26), 24.3 (C-27), signals for the
sugar moieties, see Table 2; FAB-MS (positive-mode) m/z 741 [M + H]+; HR-ESI-TOF-MS
(positive-mode) m/z 741.4066 [M + H]+ (Calcd 741.4061 for C38H61O14); Anal. Calcd for C38H60O14: C;
61.60, H; 8.16. Found: C; 61.48, H; 8.20.
Acid hydrolysis of 3
Compound (3) (11.2 mg) was subjected to acid hydrolysis as described for 1 to give 3a (8.6 mg) and a
sugar fraction (2.9 mg). HPLC analysis of the sugar fraction under the same conditions as in the case of
that of 1 showed the presence of L-arabinose and D-glucose. tR (min): 9.03 (L-arabinose, positive optical
rotation), 14.98 (D-glucose, positive optical rotation).
Compound (4): amorphous solid; [α]D -114.0°(c = 0.10, MeOH); IR (film) νmax 3347 (OH), 2921 and
2853 (CH), 1076, 1030 cm-1; 1H-NMR (C5D5N) δ 5.60 (1H, br d, J = 5.7 Hz, H-6), 4.70 (1H, q-like, J =
7.5 Hz, H-16), 4.24 (1H, ddd, J = 10.8, 9.9, 5.4 Hz, H-11), 4.16 and 3.87 (each 1H, d, J = 9.9 Hz, H2-26),
3.93 (1H, m, H-3), 3.88 (1H, dd, J = 11.6, 5.4 Hz, H-1), 2.42 (1H, dd, J = 12.1, 5.4 Hz, H-12eq), 1.55 (1H,
dd, J = 12.1, 10.8 Hz, H-12ax), 1.47 (1H, dd, J = 9.9, 9.7 Hz, H-9), 1.37 (3H, s, Me-27), 1.39 (3H, s,

784
HETEROCYCLES, Vol. 65, No. 4, 2005
Me-19), 1.02 (3H, d, J = 6.9 Hz, Me-21), 0.85 (3H, s, Me-18), signals for the sugar moiety, see Table 2;
13C-NMR (C5D5N) δ 75.8 (C-1), 42.5 (C-2), 67.9 (C-3), 44.1 (C-4), 140.3 (C-5), 125.3 (C-6), 32.9 (C-7),
33.1 (C-8), 57.9 (C-9), 46.0 (C-10), 66.0 (C-11), 50.2 (C-12), 40.5 (C-13), 55.2 (C-14), 32.3 (C-15), 81.0
(C-16), 62.1 (C-17), 16.8 (C-18), 13.7 (C-19), 38.5 (C-20), 15.0 (C-21), 120.1 (C-22), 33.0 (C-23), 33.8
(C-24), 83.8 (C-25), 77.4 (C-26), 24.3 (C-27), signals for the sugar moiety, see Table 2;
HR-ESI-TOF-MS (positive-mode) m/z 625.3607 [M + H]+ (Calcd 625.3588 for C33H53O11).
Cell culture and assay for cytotoxic activity
HSC-2 cells were maintained as monolayer cultures at 37 °C in DMEM supplemented with 10%
heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin sulfate in a humidified 5%
CO2 atmosphere. Cells were trypsinized and inoculated at 6 x 103 per each 96-microwell plate (Falcon,
flat bottom, treated polystyrene, Becton Dickinson, San Jose, CA, USA), and incubated for 24 h. After
washing once with PBS, they were treated for 24 h without or with test compounds. The cells were
washed once with PBS and incubated for 4 h with 0.2 mg/mL MTT in DMEM medium supplemented
with 10% FBS. After the medium was removed, the cells were lysed with 0.1 mL DMSO and the
relative viable cell number was determined by measuring the absorbance at 540 nm of the cell lysate,
using Labsystems MultiskanR (Biochromatic, Helsinki, Finland) connected to a Star/DOT Matrix printer
JL-10. The IC50 value, which reduces the viable cell number by 50%, was determined from the
dose-response curve.
REFERENCES AND NOTES
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2. W. Wissner and H. Kating, Planta Med., 1974, 26, 228.
3. T. Kawasaki, I. Nishioka, T. Yamauchi, K. Miyahara, and M. Embutsu, Chem. Pharm. Bull., 1965,
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4. M. Kimura, M. Tohma, I. Yoshizawa, and A. Fujino, Chem. Pharm. Bull., 1968, 16, 2191.
5. Y. Mimaki, M. Kuroda, A. Kameyama, A. Yokosuka, and Y. Sashida, Chem. Pharm. Bull., 1998, 46,
298.
6. The furostanol saponins (1 and 2) were obtained as a mixture of C-22 hydroxy and C-22 methoxy
forms. The C-22 hydroxy form present in the mixture was completely converted to the C-22
methoxy form by treatment with hot MeOH, and the structural elucidation of 1 and 2 was carried out
with the C-22 methoxy form. The C-22 configuration was shown to be α by an NOE correlation
between the C-22 methoxy protons and H-16 proton.
7. Z. Jia, K. Koike, and T. Nikaido, J. Nat. Prod., 1998, 61, 1368.

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8. I. Kitagawa, M. Sakagami, F. Hashiuchi, J. L. Zhou, M. Yoshikawa, and J. Ren, Chem. Pharm. Bull.,
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