Spirostanol saponins from the rhizomes of Tacca chantrieri and their cytotoxic activity.

Article abstract of DOI:10.1016/S0031-9422(02)00168-1

The rhizomes of Tacca chantrieri have been analysed for steroidal saponin constituents, resulting in the isolation of four new spirostanol saponins (1-4), along with one known saponin (5); their structures were elucidated on the basis of extensive spectro

Full text of DOI:10.1016/S0031-9422(02)00168-1

Phytochemistry 61 (2002) 73–78
www.elsevier.com/locate/phytochem
Spirostanol saponins from the rhizomes of Tacca chantrieri
and their cytotoxic activity
Akihito Yokosuka, Yoshihiro Mimaki*, Yutaka Sashida
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1,
Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 24 January 2002; received in revised form 5 April 2002
Abstract
The rhizomes of Tacca chantrieri have been analysed for steroidal saponin constituents, resulting in the isolation of four new
spirostanol saponins (1–4), along with one known saponin (5); their structures were elucidated on the basis of extensive spectro-
scopic analysis, including 2D NMR, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cyto-
toxic activity against HL-60human promyelocytic leukemia cells. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Tacca chantrieri; Taccaceae; Spirostanol saponins; Cytotoxic activity; HL-60cells
1
. Introduction
Tacca chantrieri Andre
of the isolated saponins against HL-60leukemia cells is
also reported.
´
(Taccaceae) is a perennial
plant that grows in southeastern China. Its rhizomes
have been employed in traditional Chinese medicine for
the treatment of gastric ulcer, enteritis, and hepatitis
2. Results and discussion
(
Jiang Su New Medicinal College, 1977). Previously, we
A concentrated MeOH extract of T. chantrieri rhi-
zomes was passed through a porous-polymer poly-
styrene resin (Diaion HP-20) column, and the MeOH
eluate fraction, with enriched steroidal glycosides, was
subjected to CC using silica gel and octadecylsilanized
(ODS) silica gel to yield 1 (3.70g), 2 (79.0mg), 3 (9.0
mg), 4 (100 mg), and 5 (390mg). Compound 5 was
identified as (25S)-spirost-5-en-3b-yl O-a-l-rhamno-
pyranosyl-(1!2)-O-[a-l-rhamnopyranosyl-(1!3)]-b-d-
glucopyranoside (Sati and Pant, 1985).
have reported the structural determination of two new
diarylheptanoids and seven diarylheptanoid glycosides
isolated from the rhizomes of T. chantrieri, some of which
exhibited considerable cytotoxic activities against HL-
6
0human promyelocytic leukemia cells and HSC-2
human squamous carcinoma cells (Yokosuka et al., 2002).
Further phytochemical examination of T. chantrieri
rhizomes were carried out with particular attention to
the steroidal glycosides, because a literature survey
showed that some Taccaceae plants contained steroidal
compounds (Zhou et al., 1983; Abdel-Aziz et al.,
Compound 1 was isolated as an amorphous solid,
ꢀ
[a] À86.0 (CHCl –MeOH, 1:1). The positive-ion HR–
D
3
+
1
4
990a,b). As a result, four new spirostanol saponins (1–
), along with one known saponin (5), were isolated.
FABMS showed an accurate [M+Na] ion at m/z
1053.5208, corresponding to the empirical molecular
formula of C H O , which was also deduced by ana-
This paper deals with the structural elucidation of the
new saponins on the basis of extensive spectroscopic
analysis, including 2D NMR spectroscopic data, and
the results of hydrolytic cleavage. The cytotoxic activity
5
1
82 21
lysis of the 13C NMR and DEPT spectral data. The
glycosidic nature of 1 was suggested by strong IR
À1
1
absorption bands at 3400 and 1040 cm . The H NMR
spectrum of 1 (C D N) exhibited two three-proton
5
5
singlet signals at ꢀ 1.04 and 0.82, indicating the presence
of two angular methyl groups, as well as two three-pro-
ton doublet signals at ꢀ 1.15 (J=6.9 Hz) and 1.08
*
Corresponding author. Tel.: +81-426-76-4577; fax: +81-426-76-
579.
E-mail address: mimakiy@ps.toyaku.ac.jp (Y. Mimaki).
4
0
031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00168-1

74
A. Yokosuka et al. / Phytochemistry 61 (2002) 73–78
(
J=7.1 Hz) assignable to secondary methyl groups of
(25S)-spirostanol tetraglycoside. Sequential assignments
of the signals from H-1 to H -6 or Me-6 of each mono-
the steroidal skeleton. In addition, signals for the
anomeric protons of four monosaccharides and methyl
groups of two deoxyhexopyranoses were observed at ꢀ
2
saccharide, including their multiplet patterns and cou-
1
pling constants, in the H NMR spectrum of 1, were
1
1
5
.81 (1H, br s), 5.76 (1H, br s), 5.24 (1H, d, J=7.8 Hz),
established by analysis of the H– H COSY and 2D
HOHAHA spectra (Table 1). Then, the HMQC spec-
trum was applied to associate the protons with the cor-
responding one-bond coupled carbon resonances.
Comparison of the carbon chemical shift thus assigned
with those of the reference methyl glycosides (Agrawal
et al., 1985; Agrawal, 1992), taking into account the
known effects of O-glycosylation, indicated that 1 con-
tained a terminal b-d-glucopyranosyl, a terminal a-l-
rhamnopyranosyl, a C-4 substituted a-l-rhamnopy-
ranosyl, and a C-2 and C-3 disubstituted b-d-glucopy-
ranosyl unit. The b-orientations of the anomeric centers
of the two glucosyl moieties were supported by the
relatively large J values of their anomeric protons
and 4.90(1H, d, J=7.8 Hz), and 1.76 (3H, d, J=6.2 Hz)
and 1.69 (3H, d, J=6.2 Hz). Acid hydrolysis of 1 with 1
M HCl in dioxane–H O (1:1) gave an aglycone identi-
2
fied as (25S)-spirost-5-en-3b-ol (yamogenin) (Agrawal et
al., 1985), and d-glucose and l-rhamnose as the carbo-
hydrate moieties. 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, with detection
being carried out using a combination of refractive
index (RI) and optical rotation (OR) detectors. The
above data, along with four anomeric carbon signals at
ꢀ
106.5, 103.1, 102.6, and 99.9, implied that 1 was a

A. Yokosuka et al. / Phytochemistry 61 (2002) 73–78
75
Table 1
1H and 13C NMR chemical shift assignments of the tetraglycoside
(25S)-spirost-5-en-3b-yl O-a-l-rhamnopyranosyl-(1!2)-
O-[O-b-d-glucopyranosyl-(1!4)-a-l-rhamnopyranosyl-
5 5
moiety of 1 in C D N
(1!3)]-b-d-glucopyranoside.
Position
1H
J (Hz)
13C
Compound 2 was shown to have the molecular for-
mula C H O on the basis of the positive-ion HR–
5
1
82 22
0
0
Glc 1
2
4.90, d
4.05, dd
4.18, dd
4.08, dd
3.78, m
4.44
7.8
99.9
78.6
86.3
69.7
78.0
FABMS (m/z 1069.5183 [M+Na] ), 13C NMR, and
+
9.3, 7.8
9.3, 9.1
9.1, 9.1
1
0
DEPT spectral data. The H NMR spectrum contained
signals for four steroid methyl groups at ꢀ 1.31 (3H, d, J
3
0
4
0
5
=7.0Hz), 1.17 (3H, d, J=6.9 Hz), 1.02 (3H, s), and
.80 (3H, s), and four anomeric protons at ꢀ 5.80(1H,
0
6
a
0
b
0
0
0
0
0
0
0
00
00
00
00
00
00
br s), 5.74 (1H, br s), 5.23 (1H, d, J=7.8 Hz), and 4.88
(1H, d, J=7.9 Hz), as in 1. Comparison of the 13C
NMR spectrum of 2 with that of 1 showed considerable
structural similarity. However, the molecular formula of
Rha 1
5.81, br s
4.73, br d
4.52, dd
4.31, dd
4.87, dq
1.76, d
102.6
72.5
72.8
73.8
69.9
18.7
103.1
72.1
72.4
84.5
68.7
18.3
106.5
76.4
78.6
71.4
78.4
62.6
0
0
0
0
0
2
3
4
5
6
3.1
3.1, 9.5
9.5, 9.5
9.5, 6.2
6.2
2
was higher by one oxygen atom than that of 1 and
0
0
differences were recognized in the carbon signals from
the ring F portion (C-22–C-27). The 1H– H COSY
spectrum was carefully inspected to assign the structure
of the ring F residue, with the three-proton doublet sig-
nal at ꢀ 1.31 (J=7.0Hz) attributable to Me-27, being
used as the starting point of analysis. As a result, the
Rha 1
5.76, br s
4.80, br d
4.58, dd
4.44, dd
4.83, dq
1.69, d
1
2
3
4
5
6
3.1
0
0
0
0
9.2, 3.1
9.3, 9.2
9.3, 6.2
6.2
0
0
0
0
0
0
000
000
000
000
000
000
Glc 1
5.24, d
7.8
structural fragment of ring F, –C(23)H –C(24)H(–O–)–
2
2
3
4
5
6
4.10, dd
4.21, dd
4.28, dd
3.78, ddd
4.41, dd
4.36, dd
8.9, 7.8
8.9, 8.9
9.3, 8.9
9.3, 6.7, 2.5
12.2, 2.5
12.2, 6.7
C(25)H(–C(26)H –O–)–Me(27), was revealed and the location
2
of an oxygen atom at C-24 was evident. On treatment of 2
with Ac O in pyridine, eleven acetyl groups were intro-
2
a
duced to the tetraglycoside residue and one acetyl to the
1
b
aglycone moiety (2a). When the H NMR spectrum of 2a
was compared to that of 2, the H-24 oxymethine proton
was shifted downfield by 0.88 ppm and was observed at
ꢀ 5.52, which was consistent with the presence of a C-24
hydroxyl group in 2. The spin-coupling constants of H-24,
(
J=7.8 Hz). For the rhamnosyl moieties, the large 1JC,H
values (168.1 and 166.9 Hz) and three-bond coupled
strong HMBC correlations from the anomeric proton to
the C-3 and C-5 carbons (the dihedral angles between
H-1 and C-3, and between H-1 and C-5 about 180 ),
indicated that each anomeric proton was equatorial
3J
3J
=11.6 Hz, 3J
=5.1 Hz, and
=5.1 Hz, gave evidence for the 24S and 25R
HÀ24,HÀ23ax
HÀ24,HÀ25
HÀ24,HÀ23eq
ꢀ
configurations. The structure of 2 was formulated as
(24S,25R)-24-hydroxyspirost-5-en-3b-yl O-a-l-rhamno-
pyranosyl-(1!2)-O- [O-b-d-glucopyranosyl-(1!4)-a-l-
rhamnopyranosyl-(1!3)]-b-d-glucopyranoside.
thus possessing an a-pyranoid anomeric form (Jia et al.,
1
998). In the HMBC spectrum, correlation peaks were
observed from ꢀ 5.81 (H-1 of terminal rhamnosyl) to ꢀ
8.6 (C-2 of 2,3-disubstituted glucosyl), ꢀ 5.24 (H-1 of
7
Compound 3 was was found to be C H O by
4
5
72 17
terminal glucosyl) to ꢀ 84.5 (C-4 of substituted rham-
nosyl), d 5.76 (H-1 of substituted rhamnosyl) to d 86.3
combined positive-ion HR–FABMS (m/z 907.4692
[M+Na] ) and 13C NMR spectroscopic analyses,
together with DEPT data. The 1H NMR spectrum
showed signals for three anomeric protons at ꢀ 6.37
(1H, br s), 5.25 (1H, d, J=7.8 Hz), and 4.92 (1H, d,
+
(
C-3 of 2,3-disubstituted glucosyl), and d 4.90(H-1 of
,3-disubstituted glucosyl) to d 77.8 (C-3 of aglycone)
Fig. 1). Thus, the structure of 1 was determined to be
2
(
Fig. 1. HMBC correlations of the sugar moiety of 1.

76
A. Yokosuka et al. / Phytochemistry 61 (2002) 73–78
J=7.8 Hz), together with those for four steroid methyl
groups at ꢀ 1.15 (3H, d, J=6.9 Hz), 1.08 (3H, d, J=7.1
and dithioerythritol, 3:1; Tokyo-Kasei, Tokyo, Japan).
1
NMR Bruker AM-500 (500 MHz for H NMR, Karls-
1
3
Hz), 0.87 (3H, s), and 0.82 (3H, s). Analysis of the
C
NMR signals arising from the sugar moiety with the aid of
ruhe, Germany). CC: Diaion HP-20(Mitsubishi-Kasei,
Tokyo, Japan); silica gel (Fuji-Silysia Chemical, Aichi,
Japan); ODS silica gel (Nacalai Tesque, Kyoto, Japan).
1
1
the H– H COSY, HMQC, and HMBC spectra revealed
that 3 was a spirostanol triglycoside structurally related to
TLC: precoated Kieselgel 60F
(0.25 mm thick,
54
2
1
with the terminal rhamnosyl group attached at the inner
Merck, Darmstadt, Germany) and RP-18 F254S (0.25
mm thick, Merck). HPLC: a Tosoh HPLC system
(pump, CCPM; controller, CCP controller PX-8010)
equipped with an RI detector (RI-8010, Tosoh, Tokyo,
Japan) and an OR detecter (Shodex OR-2, Showa-
Denko, Tokyo, Japan). The following reagents were
obtained from the indicated companies: RPMI 1640
medium (Gibco, Grand Island, NY, USA); FBS (Bio-
Whittaker, Walkersville, MD, USA); MTT (Sigma, St.
Louis, MO, USA); penicillin and streptomycin (Meiji-
Seika, Tokyo, Japan). All other chemicals used were of
biochemical reagent grade.
glucosyl moiety being absent from 1. This was con-
firmed by the result of acid hydrolysis of 3, giving
yamogenin, d-glucose and l-rhamnose, and by HMBC
correlations from ꢀ 5.25 (H-1 of terminal glucosyl) to ꢀ
8
5.7 (C-4 of substituted rhamnosyl), ꢀ 6.37 (H-1 of sub-
stituted rhamnosyl) to ꢀ 82.4 (C-3 of inner glucosyl),
and from ꢀ 4.92 (H-1 of inner glucosyl) to ꢀ 78.1 (C-3 of
aglycone). The structure of 3 was determined to be
(
25S)-spirost-5-en-3b-yl O-b-d-glucopyranosyl-(1!4)-
O-a-l-rhamnopyranosyl-(1!3)-b-d-glucopyranoside.
Compound 4 was deduced as C H O from its
72 17
4
5
positive-ion HR–FABMS (m/z 885.4810[M+H] +) and
1
3
C NMR spectroscopic analysis together with DEPT
3.2. Plant material
1
data. The H NMR spectrum of 4 showed four steroid
methyl signals at ꢀ 1.32 (3H, d, J=7.0Hz), 1.18 (3H, d,
J=7.0Hz), 1. 03 (3H, s), and 0.81 (3H, s), and three
anomeric proton signals at ꢀ 5.87 (1H, br s), 5.76 (1H, br
s), and 4.91 (1H, d, J=7.8 Hz). Analysis of the C NMR
spectrum of 4 and comparison with that of 2 implied that
the aglycone of 4 was identical to that of 2, but differed
from 2 in terms of the glycoside structure. Two terminal
a-l-rhamnopyranosyl units and a 2,3-branched b-d-
glucopyranosyl unit were found to be included in the
The rhizomes of T. chantrieri were collected at Yun-
nan Province, People’s Republic of China in October
1996, and identified by one of the authors, Professor Y.
Sashida. A voucher specimen has been deposited in the
laboratory of Y. Sashida (voucher No. TC-96–003.,
Laboratory of Medicinal Plant Science).
1
3
3.3. Extraction and isolation
1
1
structure of 4 by analysis of the H– H COSY, HMQC,
and HMBC spectra. Accordingly, the triglycoside moiety
of 4 was shown to be the same as that of 5 and the
structure of 4 was assigned as (24S,25R)-24-hydroxy-
spirost-5-en-3b-yl O-a-l-rhamnopyranosyl-(1!2)-O-[a-
l-rhamnopyranosyl-(1!3)]-b-d-glucopyranoside.
The plant material (7.3 kg) was extracted with hot
MeOH (3 lÂ2). The MeOH extract was concentrated
under reduced pressure, and the viscous concentrate
(115 g) was passed through a Diaion HP-20column
eluting with 30% aq MeOH, 50% aq MeOH, MeOH,
EtOH, and EtOAc. Preliminary TLC analysis of these
fractions suggested that the MeOH eluate portion con-
tained several steroidal glycosides, on which further
fractionation was carried out. CC of the MeOH eluate
portion on silica gel and elution with a stepwise gradient
The isolated compounds were evaluated for their cyto-
toxic activities against HL-60leukemia cells. Compounds
1
and 5 showed considerable cytotoxicity with respective
IC50 values of 1.8 and 2.1 mM, whereas etoposide used as
positive control gave an IC50 of 0.37 mM. Compounds 2
and 4, the corresponding C-24 hydroxy derivatives of 1
and 5, and 3, which is structurally related to 1 with a
terminal rhamnosyl group linked to C-2 of the inner glu-
cosyl residue absent from 1, did not show any cell growth
inhibitory activity at the sample concentration of 10 mg/
ml, suggesting that the structures of both the aglycone
and sugar moieties contribute to the cytotoxicity.
mixture of CHCl –MeOH (9:1; 4:1; 3:1; 2:1; 1:1), and
3
finally with MeOH alone, gave five fractions (I–V).
Fraction II was applied to a silica gel column which was
eluted with CHCl –MeOH–H O (50:10:1) and ODS
3
2
silica gel with MeOH–H O (4:1; 2:1) to give 3 (9.0mg),
2
4 (100 mg), and 5 (390mg). Fraction IV was subjected
to CC on silica gel eluting with CHCl –MeOH–H O
3
2
(40:10:1; 30:10:1; 20:10:1) and ODS silica gel with
MeOH–H O (4:1; 2:1) to yield 1 (3.70g) and 2 (79.0
mg).
2
3
. Experimental
3.4. Compound 1
3
.1. General
2
5
ꢀ
An amorphous solid. ½ꢁ ꢁ À86.0 (CHCl –MeOH,
D
0.10). HR–FABMS (positive mode) m/z:
3
FABMS: Finnigan MAT TSQ-700 (San Jose, CA,
USA) (matrix: Magic Bullet, a mixture of dithiothreitol
1:1;
1053.5208 (C H O Na requires
c
[M+Na]+
5
1
82 21

A. Yokosuka et al. / Phytochemistry 61 (2002) 73–78
77
À1
00
00
00
000
1
1
(
(
053.5246); IR n
(KBr) cm : 3400 (OH), 2930 (CH),
00
73.7 (C-4 ), 69.9 (C-5 ), 18.6 (C-6 ), 103.1 (C-1 ), 72.0
(C-2 ), 72.3 (C-3 ), 84.3 (C-4 ), 68.7 (C-5 ), 18.2 (C-
max
1
000 000 000 000
000 0000 0000 0000
6 ), 106.4 (C-1 ), 76.4 (C-2 ), 78.5 (C-3 ), 71.4 (C-
040; H NMR (C D N): ꢀ 5.81 (1H, br s, H-1 ), 5.76
5
5
000
1H, br s, H-1 ), 5.33 (1H, br d, J=5.0Hz, H-6), 5.24
0
0000
0000
0000
0000
1H, d, J=7.8 Hz, H-1 ), 4.90(1H, d, J=7.8 Hz, H-1 ),
00
4 ), 78.4 (C-5 ), 62.5 (C-6 ).
1
.76 (3H, d, J=6.2 Hz, Me-6 ), 1.69 (3H, d, J=6.2 Hz,
000
Me-6 ), 1.15 (3H, d, J=6.9 Hz, Me-21), 1.08 (3H, d,
J=7.1 Hz, Me-27), 1.04 (3H, s, Me-19), 0.82 (3H, s,
3.7. Acetylation of 2
Me-18); 13C NMR (pyridine-d ): ꢀ 37.5 (C-1), 30.0 (C-
Compound 2 (10mg) was treated with Ac O (1 ml)
2
5
2
(
3
(
2
2
), 77.8 (C-3), 38.6 (C-4), 140.8 (C-5), 121.8 (C-6), 32.3
C-7), 31.7 (C-8), 50.3 (C-9), 37.1 (C-10), 21.1 (C-11),
9.8 (C-12), 40.4 (C-13), 56.6 (C-14), 32.2 (C-15), 81.2
C-16), 62.7 (C-17), 16.3 (C-18), 19.4 (C-19), 42.4 (C-
0), 14.9 (C-21), 109.7 (C-22), 26.4 (C-23), 26.2 (C-24),
7.5 (C-25), 65.1 (C-26), 16.3 (C-27). Signals for the
and pyridine (1 ml) at room temperature for 12 h. After
addition of H O (2 ml) into the reaction mixture fol-
2
lowed by evaporation to dryness, it was chromato-
graphed on silica gel eluting with hexane–Me CO (2:1)
to give the corresponding dodecaacetate (2a, 10mg).
2
sugar moiety: Table 1.
3.8. Compound 2a
2
D
5
ꢀ
3
.5. Acid hydrolysis of 1
An amorphous solid. ½ꢁ ꢁ À76.0 (CHCl ; c 0.10).
À1 1
max
3
IR n
(KBr) cm : 2970(CH), 1750(C=O);
00
NMR (C D N): ꢀ 5.54 (1H, br s, H-1 ), 5.52 (1H, br s,
5 5
H
A solution of 1 (10mg) in 1 M HCl (dioxane–
ꢀ
000
H O,1:1; 2 ml) was heated at 95 C for 2 h under an Ar
2
atmosphere. After cooling, the reaction mixture was
neutralized by passage through an Amberlite IRA-
H-1 ), 5.52 (1H, m, H-24), 5.44 (1H, br d, J=4.5 Hz,
0
000
H-6), 5.33 (1H, d, J=7.9 Hz, H-1 ), 4.93 (1H, d, J=7.8
0
Hz, H-1 ), 2.20, 2.18, 2.16, 2.15, 2.14, 2.07Â2, 2.05, 2.04,
93ZU (Organo, Tokyo, Japan) column and subjected to
silica gel chromatography using a gradient mixture of
2.03Â2, 2.01 (each 3H, s, AcÂ12), 1.61 (3H, d, J=6.1
0
0
000
Hz, Me-6 ), 1.47 (3H, d, J=6.2 Hz, Me-6 ), 1.16 (3H,
d, J=6.9 Hz, Me-21), 1.10(3H, s, Me-19), 1.10(3H, d,
J=7.0Hz, Me-27), 0. 85 (3H, s, Me-18).
CHCl –MeOH (19:1; 1:1) to give (25S)-spirost-5-en-3b-
3
ol (3.0mg) as the aglycone and a sugar fraction (2.7
mg). HPLC analysis of the sugar fraction under the
following conditions showed the presence of d-glucose
3.9. Compound 3
and l-rhamnose. Column: Kaseisorb LC NH -60–5
2
2
D
5
ꢀ
UG80(4.6 mm i.d. Â250mm, 5 mm, Tokyo-Kasei,
An amorphous solid. ½ꢁ ꢁ À86.0 (CHCl –MeOH,
3
Tokyo, Japan); solvent: MeCN–H O (3:1); flow rate:
2
1:1; c 0.10). HR–FABMS (positive mode) m/z: 907.4692
[M+Na]+ (C H O Na requires 907.4667); IR n
(KBr) cm : 3200 (OH), 2930 (CH), 1070; H NMR
000
(C D N): ꢀ 6.37 (1H, br s, H-1 ), 5.30(1H, br d, J=5.1
5 5
0
.8 ml/min. Rt (min): 8.10( l-rhamnose, negative
45
72 17
max
À1
1
optical rotation); 12.60( d-glucose, positive optical
rotation).
0
000
Hz, H-6), 5.25 (1H, d, J=7.8 Hz, H-1 ), 4.92 (1H, d,
000
0
3
.6. Compound 2
An amorphous solid. ½ꢁ ꢁ À108.0 (CHCl –MeOH,
J=7.8 Hz, H-1 ), 1.77 (3H, d, J=6.2 Hz, Me-6 ), 1.15
3H, d, J=6.9 Hz, Me-21), 1.08 (3H, d, J=7.1 Hz, Me-
(
2
5
ꢀ
27), 0.87 (3H, s, Me-19), 0.82 (3H, s, Me-18); 13C NMR
(C D N): ꢀ 37.4 (C-1), 30.1 (C-2), 78.1 (C-3), 39.1 (C-4),
D
:1; c 0.10). HR–FABMS (positive mode) m/z: 1069.5138
3
1
[
5
5
M+Na]+ (C H O Na requires 1069.5195); IR n
140.8 (C-5), 121.8 (C-6), 32.2 (C-7), 31.6 (C-8), 50.2 (C-9),
37.0(C-1 0) , 21.1 (C-11), 39.8 (C-12), 4 0. 4 (C-13), 56.6 (C-
14), 32.1 (C-15), 81.1 (C-16), 62.7 (C-17), 16.3 (C-18), 19.3
(C-19), 42.4 (C-20), 14.8 (C-21), 109.7 (C-22), 26.4 (C-23),
26.2 (C-24), 27.5 (C-25), 65.1 (C-26), 16.3 (C-27), 102.2 (C-
5
1
82 22
max
À1
1
(
(
5
KBr) cm : 3400 (OH), 2930 (CH), 1040; H NMR
C D N): ꢀ 5.80(1H, br s, H-1 ), 5.74 (1H, br s, H-1 ),
00
000
5
5
.32 (1H, br d, J=4.8 Hz, H-6), 5.23 (1H, d, J=7.8 Hz,
0
0000
H-1 ), 4.88 (1H, d, J=7.9 Hz, H-1 ), 4.64 (1H, ddd,
J=11.6, 5.1, 5.1 Hz, H-24), 1.75 (3H, d, J=6.2 Hz, Me-
6
0
0
0
0
0
1 ), 75.9 (C-2 ), 82.4 (C-3 ), 69.5 (C-4 ), 78.3 (C-5 ), 62.4 (C-
6 ), 102.1 (C-1 ), 72.6 (C-2 ), 72.0(C-3 ), 85.7 (C-4 ),
00
000
0 000 000 000 000
), 1.69 (3H, d, J=6.2 Hz, Me-6 ), 1.31 (3H, d, J=7.0
000 000 0000 0000
68.0(C-5 ), 18.6 (C-6 ), 106.8 (C-1 ), 76.6 (C-2 ), 78.7
Hz, Me-27), 1.17 (3H, d, J=6.9 Hz, Me-21), 1.02 (3H, s,
Me-19), 0.80 (3H, s, Me-18); 13C NMR (C D N): ꢀ 37.4
0000
0000
0000
0000
(C-3 ), 71.3 (C-4 ), 78.5 (C-5 ), 62.5 (C-6 ).
5
5
(
C-1), 30.0 (C-2), 77.8 (C-3), 38.6 (C-4), 140.7 (C-5),
1
1
3
21.8 (C-6), 32.2 (C-7), 31.6 (C-8), 50.2 (C-9), 37.1 (C-
0), 21.0 (C-11), 39.8 (C-12), 40.4 (C-13), 56.6 (C-14),
2.1 (C-15), 81.5 (C-16), 62.3 (C-17), 16.2 (C-18), 19.3
3.10. Acid hydrolysis of 3
Compound 3 (4.7 mg) was subjected to acid hydrolysis
as described for 1 to give (25S)-spirost-5-en-3b-ol (1.3 mg)
and a sugar fraction (1.6 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.
(
C-19), 42.5 (C-20), 14.8 (C-21), 111.4 (C-22), 35.9 (C-
2
9
5
3), 66.4 (C-24), 35.8 (C-25), 64.5 (C-26), 9.7 (C-27),
0
0
0
0
9.8 (C-1 ), 78.6 (C-2 ), 86.2 (C-3 ), 69.7 (C-4 ), 78.0(C-
00
0
0
00
00
), 62.1 (C-6 ), 102.6 (C-1 ), 72.4 (C-2 ), 72.7 (C-3 ),

78
A. Yokosuka et al. / Phytochemistry 61 (2002) 73–78
3
.11. Compound 4
An amorphous solid. ½ꢁ ꢁ À112.0 (CHCl –MeOH,
Briefly, after termination of the cell culture, 10 ml of 5
mg/ml MTT in phosphate buffered saline was added to
2
5
ꢀ
every well and the plate was further reincubated in 5%
ꢀ
D
:1; c 0.10). HR–FABMS (positive mode) m/z: 885.4810
3
1
CO /air for 4 h at 37 C. The plate was then centrifuged
2
+
[M+H] (C H O requires 885.4848); IR ꢂ (KBr)
max
cm : 3400 (OH), 2930 (CH), 1040; H NMR (C D N):
ꢀ
br d, J=5.0Hz, H-6), 4.91 (1H, d, J=7.8 Hz, H-1 ),
4
at 1500 g for 5 min to precipitate cells and MTT for-
mazan. An aliquot of 150 ml of the supernatant was
removed from every well, and 175 ml of DMSO was
added to dissolve the MTT formazan crystals. The plate
was mixed on a microshaker for 10min, and then read
on a microplate reader (Immuno-Mini NJ-2300, Inter
Med, Tokyo, Japan) at 550nm. A dose–response curve
was plotted for 1 and 5, which showed more than 50%
of cell growth inhibition at the sample concentration of
10 mg/ml, and the concentration giving 50% inhibition
(IC ) was calculated.
4
5
73 17
À1
1
5
5
00
000
5.87 (1H, br s, H-1 ), 5.76 (1H, br s, H-1 ), 5.32 (1H,
0
.64 (1H, ddd, J=11.6, 5.0, 5.0 Hz, H-24), 1.76 (3H, d,
00
000
J=6.2 Hz, Me-6 ), 1.65 (3H, d, J=6.2 Hz, Me-6 ), 1.32
3H, d, J=7.0Hz, Me-27), 1.18 (3H, d, J=7.0Hz, Me-
1), 1.03 (3H, s, Me-19), 0.81 (3H, s, Me-18); 13C NMR
C D N): ꢀ 37.5 (C-1), 30.0 (C-2), 77.8 (C-3), 38.7 (C-4),
(
2
(
1
9
5
5
5
40.7 (C-5), 121.8 (C-6), 32.3 (C-7), 31.7 (C-8), 50.2 (C-
), 37.1 (C-10), 21.1 (C-11), 39.8 (C-12), 40.4 (C-13),
6.6 (C-14), 32.1 (C-15), 81.5 (C-16), 62.4 (C-17), 16.3
5
0
(
C-18), 19.4 (C-19), 42.5 (C-20), 14.8 (C-21), 111.4 (C-
References
2
9
4
7
2), 36.0(C-23), 66.5 (C-24), 35.9 (C-25), 64.5 (C-26),
0
0
0
.7 (C-27), 99.9 (C-1 ), 78.3 (C-2 ), 87.4 (C-3 ), 69.9 (C-
0
Abdel-Aziz, A.M.E., Brain, K.R., Blunden, G., Crabb, T., Bashir,
A.K., 1990a. Steroidal sapogenins from Tacca leontopetaloides.
Planta Medica 56, 218–221.
0
0
00
00
), 78.1 (C-5 ), 62.3 (C-6 ), 102.6 (C-1 ), 72.5 (C-2 ),
00
00
00
00
2.8 (C-3 ), 73.8 (C-4 ), 69.9 (C-5 ), 18.7 (C-6 ), 103.9
000
000
000
000
(
5
C-1 ), 72.6 (C-2 ), 72.5 (C-3 ), 73.6 (C-4 ), 70.6 (C-
000
Abdel-Aziz, A., Brain, K., Shatalebi, M.A., Blunden, G., Patel, A.,
Crabb, T.A., Bashir, A.K., 1990b. A B-ring contracted spirostane
from Tacca leontopetaloides. Phytochemistry 29, 2623–2627.
Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation
of oligosaccharides and glycosides. Phytochemistry 31, 3307–3330.
Agrawal, P.K., Jain, D.C., Gupta, R.K., Thakur, R.S., 1985. Carbon-
13 NMR spectroscopy of steroidal sapogenins and steroidal sapo-
nins. Phytochemistry 24, 2479–2496.
000
), 18.4 (C-6 ).
3
.12. Cell culture assay
HL-60cells (JCRB 08 5, Human Science Research
Resources Bank, Osaka, Japan) were maintained in the
RPMI 1640medium containing 1 %0 FBS supple-
mented with l-glutamine, 100 units/ml penicillin, and
Jia, Z., Koike, K., Nikaido, T., 1998. Major triterpenoid saponins from
Saponaria officinalis. Journal of Natural Products 61, 1368–1373.
Jiang Su New Medical College, 1977. The Dictionary of Chinese
Herbal Medicines, Vol. 1. Shanghai Scientific and Technological
Press, Shanghai (p. 524).
1
00 mg/ml streptomycin. The leukemia cells were
4
washed and resuspended in the above medium to 4Â10
cells/ml, and 196 ml of this cell suspension was placed in
each well of a 96-well flat-bottom plate (Iwaki Glass,
Chiba, Japan). The cells were incubated in 5% CO /air
2
for 24 h at 37 C. After incubation, 4 ml of EtOH–H O
Sargent, J.M., Taylor, C.G., 1989. Appraisal of the MTT assay as a
rapid test of chemosensitivity in acute myeloid leukemia. British
Journal of Cancer 60, 206–210.
Sati, O.P., Pant, G., 1985. Spirostanol glycosides from Asparagus plu-
mosus.. Phytochemistry 24, 123–126.
ꢀ
2
(
1:1) solution containing the sample was added to give
Yokosuka, Y., Mimaki, Y., Sakagami, H., Sashida, Y., 2002. New
diarylheptanoids and diarylheptanoid glycosides from the rhizomes
of Tacca chantrieri and their cytotoxic activity. Journal of Natural
Products 65, 283–289.
the final concentrations of 0.1–10 mg/ml and 4 ml of
EtOH-H O (1:1) was added into control wells. The cells
were further incubated for 72 h in the presence of each
agent, and then cell growth was evaluated using a mod-
ified MTT reduction assay (Sargent and Taylor, 1989).
2
Zhou, J., Chen, C., Liu, R., Yang, C., 1983. Studies on the chemical
´
constituents of Tacca chantrieri Andre. Zhiwu Xuebao 25, 568–
573.