Steroidal and pregnane glycosides from the rhizomes of Tacca chantrieri

Article abstract of DOI:10.1021/np020094l

The rhizomes of Tacca chantrieri have been analyzed as part of a systematic study on saponin constituents of medicinal plants. This has resulted in the isolation of three new bisdesmosidic furostanol saponins (1-3), two new bisdesmosidic pseudofurostanol saponins (4, 5), and two new pregnane glycosides (6, 7). Their structures were determined on the basis of extensive spectroscopic studies and a few chemical transformations.

Full text of DOI:10.1021/np020094l

J . Nat. Prod. 2002, 65, 1293-1298
1293
Ster oid a l a n d P r egn a n e Glycosid es fr om th e Rh izom es of Ta cca ch a n tr ier i
Akihito Yokosuka, Yoshihiro Mimaki,* and Yutaka Sashida
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1, Horinouchi, Hachioji, Tokyo 192-0392, J apan
Received March 8, 2002
The rhizomes of Tacca chantrieri have been analyzed as part of a systematic study on saponin constituents
of medicinal plants. This has resulted in the isolation of three new bisdesmosidic furostanol saponins
(1-3), two new bisdesmosidic pseudofurostanol saponins (4, 5), and two new pregnane glycosides (6, 7).
Their structures were determined on the basis of extensive spectroscopic studies and a few chemical
transformations.
In a continuation of our study on saponin constituents
of medicinal plants, we have examined the saponin-
enriched fraction prepared from the MeOH extract of the
rhizomes of Tacca chantrieri Andre´ (Taccaceae). This plant
is indigenous to areas in southeast China and has been
used in traditional Chinese medicine for the treatment of
gastric ulcer, enteritis, and hepatitis.1 Previously, we
reported the structural characterization of two new dia-
rylheptanoids and seven diarylheptanoid glucosides iso-
lated from T. chantrieri rhizomes and their cytotoxic
activities against HL-60 leukemia cells, HSC-2 cells, and
HGF.2 Further phytochemical analysis has been carried out
on the rhizomes, with particular attention to the steroidal
glycoside constituents, and has resulted in the isolation of
three new bisdesmosidic furostanol saponins (1-3), two
new bisdesmosidic pseudofurostanol saponins (4, 5), and
two new pregnane glycosides (6, 7), among which 1 is the
main secondary metabolite with a yield of 0.14% based on
dry weight of the plant material. This paper reports the
structural determination of the new glycosides.
Resu lts a n d Discu ssion
The fractions enriched with steroidal glycosides, pre-
pared by passing the MeOH extract of T. chantrieri
rhizomes through a porous-polymer polystyrene resin
(Diaion HP-20) column eluted with MeOH-H2O mixtures
followed by MeOH, were subjected to multiple chromato-
graphic steps over silica gel and octadecylsilanized (ODS)
silica gel, giving compounds 1 (0.14%), 2 (0.0027%), 3
(0.00090%), 4 (0.0018%), 5 (0.00029%), 6 (0.00082%), and
7 (0.00041%).
Compound 1 was obtained as an amorphous solid. The
negative-ion FABMS of 1 showed an [M - H]- ion at m/z
1223, compatible with the molecular formula C58H96O27
,
which was confirmed by data from the 13C NMR spectrum,
with a total of 58 carbon signals, and the results of
1
elemental analysis. The H NMR spectrum showed signals
indicated that 1 had two deoxy sugars. When 1 was
submitted to acid hydrolysis with 1 M HCl in dioxane-
H2O (1:1), it was hydrolyzed to yield a sapogenin identified
as (25S)-spirost-5-en-3â-ol.3 The monosaccharides of the
acidic hydrolysate of 1 were identified as D-glucose and
L-rhamnose on the basis of the HPLC analysis, which was
performed on an aminopropyl-bonded silica gel column,
with detection being carried out by using a combination of
refractive index (RI) and optical rotation (OR) detectors.
The above 1H NMR and chemical data, together with an
acetalic carbon signal at δ 112.7 in the 13C NMR spectrum3
and a positive coloration with Ehrlich’s reagent,4,5 indicated
for four steroid methyl groups at δ 1.17 (3H, d, J ) 6.9
Hz), 1.05 (3H, d, J ) 6.7 Hz), 1.04 (3H, s), and 0.82 (3H,
s), an olefinic proton at δ 5.33 (1H, br d, J ) 4.6 Hz), and
a methoxyl group at δ 3.26 (3H, s), as well as signals for
five anomeric protons at δ 5.81 (1H, br s), 5.75 (1H, br s),
5.24 (1H, d, J ) 7.8 Hz), 4.89 (1H, d, J ) 7.8 Hz), and 4.85
(1H, d, J ) 7.7 Hz). The two methyl carbon signals at δ
18.6 and 18.3 and their corresponding proton signals at δ
1.76 (3H, d, J ) 6.2 Hz) and 1.69 (3H, d, J ) 6.2 Hz)
* To whom correspondence should be addressed. Tel: +81-426-76-4577.
Fax: +81-426-76-4579. E-mail: mimakiy@ps.toyaku.ac.jp.
10.1021/np020094l CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/15/2002

1294 J ournal of Natural Products, 2002, Vol. 65, No. 9
Yokosuka et al.
Ta ble 1. 1H NMR Chemical Shift Assignments for the
Glycoside Moieties of Compound 1 in C5D5N
position
1′
1H
J (Hz)
7.8
Glc
4.89, d
2′
3′
4′
5′
6′a
b
1′′
2′′
3′′
4.05, dd
4.17, dd
4.08, dd
3.78, ddd
4.44, dd
4.36, dd
5.81, br s
4.73, br d
4.51, dd
4.31, dd
4.87, dq
1.76, d
8.7, 7.8
9.1, 8.7
9.1, 9.1
9.1, 4.5, 2.3
11.8, 2.3
11.8, 4.5
Rha
Rha
Glc
3.2
9.3, 3.2
9.7, 9.3
9.7, 6.2
6.2
4′′
5′′
6′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′a
b
1′′′′′′
2′′′′′′
3′′′′′′
4′′′′′′
5′′′′′′
6′′′′′′a
b
5.75, br s
4.80, br d
4.57, dd
4.43, dd
4.83, dq
1.69, d
3.3
9.1, 3.3
9.1, 9.1
9.1, 6.2
6.2
5.24, d
7.8
4.09, dd
4.21, dd
4.27, dd
3.78, ddd
4.41, dd
4.34, dd
4.85, d
4.05, dd
4.25, dd
4.22, dd
3.97, ddd
4.57, dd
4.39, dd
8.8, 7.8
9.0, 8.8
9.4, 9.0
9.4, 4.9, 2.3
11.5, 2.3
11.5, 4.9
7.7
8.7, 7.7
9.1, 8.7
9.1, 9.1
9.1, 5.3, 2.5
11.8, 2.5
11.8, 5.3
Glc
glucosyl) and δC 74.9 (C-26 of aglycon) implied that one
glucose unit was attached at C-26 of the aglycon, which is
a structural feature most frequently encountered in the
plant furostanol saponins.3 Consequently, a tetraglycoside
was assumed to be located at C-3 of the aglycon. Further
HMBC correlations from δ 5.81 (H-1 of terminal rhamno-
syl) to δ 78.6 (C-2 of 2,3-disubstituted glucosyl), δ 5.24 (H-1
of terminal glucosyl) to δ 84.4 (C-4 of substituted rham-
nosyl), δ 5.75 (H-1 of substituted rhamnosyl) to δ 86.2 (C-3
of 2,3-disubstituted glucosyl), and δ 4.89 (H-1 of 2,3-
disubstituted glucosyl) to δ 77.8 (C-3 of aglycon) confirmed
the tetraglycoside sequence as rhamnosyl-(1f2)-[glucosyl-
(1f4)-rhamnosyl-(1f3)]-glucosyl, which was attached at
C-3 of the aglycon. An NOE correlation from the methoxyl
proton signal at δ 3.26 to the H-16 proton signal at δ 4.45
was consistent with the C-22R configuration. Thus, 1 was
determined to be (25S)-26-[(â-D-glucopyranosyl)oxy]-22R-
methoxyfurost-5-en-3â-yl O-R-L-rhamnopyranosyl-(1f2)-O-
[O-â-D-glucopyranosyl-(1f4)-R-L-rhamnopyranosyl-(1f3)]-
â-D-glucopyranoside.
1 to be a saponin based upon (25S)-22-methoxyfurost-5-
ene-3â,26-diol6 with up to five monosaccharides. Sequential
assignments of the signals from H-1 to H2-6 or Me-6 of each
monosaccharide, including their multiplet patterns and
coupling constants, in the 1H NMR spectrum of 1, were
established by analysis of the 1H-1H COSY and 2D
HOHAHA spectra (Table 1). Then, the HMQC spectrum
was applied to associate the protons with the corresponding
one-bond coupled carbon resonances. Comparison of the
carbon chemical shift thus assigned with those of the
reference methyl glycosides,3,7 taking into account the
known effects of O-glycosylation, indicated that 1 contained
two terminal â-D-glucopyranosyl units, a terminal R-L-
rhamnopyranosyl unit, a C-4 substituted R-L-rhamnopy-
ranosyl unit, and a C-2 and C-3 disubstituted â-D-
glucopyranosyl unit. The â-orientations of the anomeric
centers of the three glucosyl moieties were supported by
the relatively large J values of their anomeric protons (J
) 7.7-7.8 Hz). For the rhamnosyl moieties, the large 1J C,H
values (166.1 and 172.1 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, thus possessing an R-pyr-
anoid anomeric form.8 In the HMBC spectrum, a correla-
tion peak between the signals at δΗ 4.85 (H-1 of terminal
Compound 2 was shown to have the molecular formula
C
60H98O28 on the basis of the negative-ion FABMS (m/z
1265 [M - H]-), 13C NMR spectrum (60 signals), and
elemental analysis. The H and 13C NMR spectra of 2 were
1
quite similar to those of 1. In addition, the presence of an
acetyl group in 2 was shown by the IR (1730 cm-1), 1H
NMR [δ 2.01 (3H, s)], and 13C NMR [δ 170.8 (CdO) and
20.7 (Me)] spectra. Alkaline treatment of 2 with 3% NaOMe
in MeOH furnished 1, indicating that 2 was a monoacetate
of 1. In the HMBC spectrum, correlation peaks were
observed from the H2-6 protons due to the 2,3-disubstituted
inner glucosyl moiety at δ 4.86 and 4.72 to the acetyl
carbonyl carbon. The structure of 2 was assigned as (25S)-
26-[(â-D-glucopyranosyl)oxy]-22R-methoxyfurost-5-en-3â-
yl O-R-L-rhamnopyranosyl-(1f2)-O-[O-â-D-glucopyranosyl-

Steroidal and Pregnane Glycosides from Tacca
J ournal of Natural Products, 2002, Vol. 65, No. 9 1295
(1f4)-R-L-r h a m n opyr a n osyl-(1f3)]-6-O-a cet yl-â-D-
glucopyranoside.
of 6 revealed the presence of four methyl groups on the
aglycon and five monosaccharides and were essentially
analogous to those of 4. However, 6 was different from 4
in the lack of the signals assignable to the tetrasubstituted
olefinic group forming the bond between C-20 and C-22 and
in the presence of a ketone carbonyl carbon signal at δ
205.5 and an ester carbonyl carbon signal at δ 173.3. The
HMBC spectrum exhibited correlations of the ketone
carbonyl carbon with H-17 at δ 2.50 (1H, d, J ) 7.6 Hz)
and Me-21 at δ 2.14 (3H, s) and of the ester carbonyl carbon
with H-16 at δ 5.67 (1H, m) and H2-23 at δ 2.44 and 2.36,
indicating that C-20 and C-22 of 6 were the carbonyl
groups, instead of the olefinic group in 4. The structure of
6, including the absolute configuration at C-25, was
confirmed by the following chemical conversion. When the
C-20 and C-22 bond of 4a was oxidatively cleaved by
treating it with CrO3 in AcOH at room temperature for 2
h, the resultant product was completely consistent with the
peracetyl derivative of 6 (6a ). Accordingly, 6 was charac-
terized as 16â-[[(4S)-5-(â-D-glucopyranosyloxy)-4-methyl-
1-oxopentyl]oxy]-3â-[(O-R-L-rhamnopyranosyl-(1f2)-O-[O-
â-D-glucopyranosyl-(1f4)-R-L-rhamnopyranosyl-(1f3)]-â-
D-glucopyranosyl)oxy]pregn-5-en-20-one.
Compound 3 analyzed as C64H106O32 by combined nega-
tive-ion FABMS (m/z 1385 [M - H]-), 13C NMR (64
signals), and elemental analysis. The deduced molecular
1
formula was higher by C6H10O5 than that of 1, and the H
NMR spectrum showed signals for six anomeric protons
at δ 5.80 (1H, br s), 5.74 (1H, br s), 5.23 (1H, d, J ) 7.8
Hz), 5.11 (1H, d, J ) 7.9 Hz), 4.88 (1H, d, J ) 7.9 Hz), and
4.77 (1H, d, J ) 7.8 Hz), along with signals for four steroid
methyl protons at δ 1.18 (3H, d, J ) 6.8 Hz), 1.04 (3H, d,
J ) 6.7 Hz), 1.03 (3H, s), and 0.83 (3H, s). Acid hydrolysis
of 3 with 1 M HCl resulted in the production of (25S)-
spirost-5-en-3â-ol, D-glucose, and L-rhamnose. On compari-
son of the whole 13C NMR spectrum of 3 with that of 1, a
set of six signals corresponding to a terminal â-D-glucopy-
ranosyl moiety were observed at δ 105.4 (CH), 75.1 (CH),
78.4 (CH), 71.5 (CH), 78.4 (CH), and 62.7 (CH2), and the
signals due to C-6 of the glucosyl moiety attached at C-26
of the aglycon and its neighboring carbons varied, while
all other signals remained almost unaffected. The HMBC
spectrum showed a long-range correlation peak from the
anomeric proton of the terminal glucosyl group at δ 5.11
to C-6 of the inner glucosyl unit at δ 70.1, whose anomeric
proton at δ 4.77, in turn, exhibited a correlation with C-26
of the aglycon at δ 74.8. The structure of 3 was thus
formulated as (25S)-26-[(O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranosyl)oxy]-22R-methoxyfurost-5-en-3â-yl O-R-L-
rhamnopyranosyl-(1f2)-O-[O-â-D-glucopyranosyl-(1f4)-R-
L-rhamnopyranosyl-(1f3)]-â-D-glucopyranoside.
Compound 4 was deduced as C57H92O26 by the positive-
ion FABMS (m/z 1215 [M + Na]+), 13C NMR (57 signals),
and elemental analysis and was suggested to be a furo-
stanol saponin closely related to 1 from its spectral data.
It differed from 1 in the presence of one more olefinic
functionality [δC 152.4 (C) and 103.5 (C)] in addition to the
5(6)-ene group. Furthermore, the Me-21 methyl doublet
signal observed at δ 1.17 (J ) 6.9 Hz) in the 1H NMR
spectrum of 1 was absent from 4, but was replaced by a
methyl singlet at δ 1.63. These data were suggestive of 4
being the corresponding ∆20(22)-furostanol saponin of 1. This
was confirmed by the fact that the peracetate (4a ) of 4
agreed with the product obtained by treatment of 1 with
Ac2O in pyridine at 110 °C for 2.5 h, during which
dehydration at C-20 and C-22, as well as the introduction
of an acetyl group to all the hydroxyl groups of the sugar
moiety, occurred to 1. The structure of 4 was established
as (25S)-26-[(â-D-glucopyranosyl)oxy]furosta-5,20(22)-dien-
3â-yl O-R-L-rhamnopyranosyl-(1f2)-O-[O-â-D-glucopyra-
nosyl-(1f4)-R-L-rhamnopyranosyl-(1f3)]-â-D-glucopyrano-
side.
Compound 7 had a molecular formula of C45H70O20
,
established by analysis of the positive-ion FABMS (m/z 953
[M + Na]+) and 13C NMR spectrum (45 signals) and by
1
elemental analysis. The H NMR spectrum of 7 displayed
two three-proton singlet signals at δ 1.04 and 0.93, indicat-
ing the presence of two angular methyl groups, and a
methyl singlet at δ 2.25 attached to a deshielding moiety,
as well as four anomeric proton signals at δ 5.80 (1H, br
s), 5.74 (1H, br s), 5.24 (1H, d, J ) 7.8 Hz), and 4.88 (1H,
d, J ) 7.8 Hz). The existence of an R,â-unsaturated
carbonyl group was verified by the IR (1660 cm-1), UV
[239.8 nm (log ꢀ 3.84)], and 13C NMR [δ 196.3 (CdO), 155.2
(C), and 144.7 (CH)] spectra. These spectral data and
comparison with those of previously reported compounds
allowed identification of the aglycon of 7 as 3â-hydroxypre-
1
gna-5,16-dien-20-one.9 The H and 13C NMR shifts of the
tetraglycoside moiety linked to C-3 of the pregnane were
superimposable on those of 1, 3, 4, and 6. Furthermore,
alkaline treatment of 6 afforded 7 and 16-methoxy-3â-[(O-
R-L-rhamnopyranosyl-(1f2)-O-[O-â-D-glucopyranosyl-(1f4)-
R-L-rhamnopyranosyl-(1f3)]-â-D-glucopyranosyl)oxy]pregn-
5-en-20-one (8). All of these data were consistent with the
structure 3â-[(O-R-L-rhamnopyranosyl-(1f2)-O-[O-â-D-glu-
copyranosyl-(1f4)-R-L-rhamnopyranosyl-(1f3)]-â-D-glu-
copyranosyl)oxy]pregna-5,16-dien-20-one, which was as-
signed to 7.
Compounds 1-7 are new steroidal glycosides. The known
naturally occurring 22,26-hydroxyfurostanol saponins ex-
clusively exist in the form of glycoside, bearing a monosac-
charide at C-26.3 The monosaccharide in the furostanol
saponins reported thus far is limited to be â-D-glucopyra-
nose except for one furostanol saponin from Dracaena
afromontana, which has an R-L-rhamnopyranosyl group at
C-26.10 Compound 3 is distinctive in carrying a diglucosyl
group, O-glucosyl-(1f6)-glucosyl, in place of a monoglucosyl
unit at C-26. A few compounds related to 6 have been
isolated;11-13 however, the C-25 configuration of them is
not clearly presented in all the reports. In this investiga-
tion, we unequivocally determined the C-25 configuration
of 6 as S by a chemical correlation method. Compound 6
could be defined as a pregnane glycoside rather than a
furostanol saponin.11-13 Compounds 1-7 did not show
apparent cytotoxic activity against HL-60 cells at a sample
concentration of 10 µg/mL, although some furostanol sa-
Compound 5 (C59H94O27) was obtained as an amorphous
1
solid. Its H and 13C NMR spectral properties were in good
agreement with those of 4, except for the presence of the
signals for one acetyl group [δΗ 2.01 (3H, s); δC 170.8
(CdO) and 20.7 (Me)]. Alkaline treatment of 5 with 3%
NaOMe in MeOH furnished 4, and the HMBC spectrum
showed correlation peaks between the signals of the H2-6
protons due to the 2,3-disubstituted inner glucosyl moiety
at δ 4.85 and 4.72 and the acetyl carbonyl carbon. The
structure of 5 was shown to be (25S)-26-[(â-D-gluco-
pyranosyl)oxy]-22R-methoxyfurosta-5,20(22)-dien-3â-yl O-R-
L-rhamnopyranosyl-(1f2)-O-[O-â-D-glucopyranosyl-(1f4)-
R-L-rhamnopyranosyl-(1f3)]-6-O-acetyl-â-D-glucopyrano-
side.
Compound 6 was deduced as C57H92O28 from its positive-
ion FABMS (m/z 1247 [M + Na]+), 13C NMR (57 signals),
1
and elemental analysis data. The H and 13C NMR spectra

1296 J ournal of Natural Products, 2002, Vol. 65, No. 9
Yokosuka et al.
Ta ble 2. 13C NMR Chemical Shift Assignments of Compounds
1-7 in C5D5N
ponins and pregnane glycosides have been reported to be
cytotoxic.11-14
position
1
2
3
4
5
6
7
Exp er im en ta l Section
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
1′
2′
3′
4′
5′
37.5
30.0
77.8
38.6
37.5
30.1
78.4
38.7
37.4
30.0
77.8
38.6
37.5
30.0
77.7
38.6
37.6
30.1
78.4
38.7
37.4
30.0
77.8
38.6
37.3
30.0
77.8
38.7
Gen er a l Exp er im en ta l P r oced u r es. The instruments
and experimental conditions, as well as the plant material
used, were the same as described in the previous paper.2
Extr a ction a n d Isola tion . The plant material (dry wt, 7.3
kg) was extracted with hot MeOH (3 L × 2). The MeOH extract
(630 g) was passed through a Diaion HP-20 (2.2 kg, Mitsubishi-
Kasei, Tokyo, J apan) column, eluting with 30% MeOH, 50%
MeOH, MeOH, EtOH, and EtOAc (4 L of each). The 50%
MeOH eluate portion (70 g) was chromatographed on a silica
gel (1.2 kg, 200-400 mesh, Fuji-Silysia Chemical, Aichi,
J apan) column, eluting with a stepwise gradient mixture of
CHCl3-MeOH (9:1, 4:1, 3:1, 2:1, and 1:1; 4 L of each) and
finally with MeOH (4 L). The CHCl3-MeOH (2:1) eluate
portion (15 g) was subjected to column chromatography on
silica gel (400 g), eluting with CHCl3-MeOH-H2O (20:10:1,
2 L) to give 5 (21 mg, 0.00029%). The CHCl3-MeOH (1:1) and
MeOH eluate portions were combined (22 g), which was then
chromatographed on ODS silica gel (500 g, 75 µm, Nacalai
Tesque, Kyoto, J apan) with MeOH-H2O (2:1, 2 L) and on ODS
silica gel (400 g) with MeCN-H2O (2:5, 1.5 L; 1:2, 2 L) to give
4 (130 mg, 0.0018%). The MeOH eluate portion (115 g) was
chromatographed on silica gel (1.2 kg), eluting with a stepwise
gradient mixture of CHCl3-MeOH (9:1, 4:1, 3:1, 2:1, and 1:1;
4 L of each) and finally with MeOH (4 L). The CHCl3-MeOH
(1:1) and MeOH eluate portions were combined (40 g), which
was further subjected to column chromatography on silica gel
(800 g) eluting with CHCl3-MeOH-H2O (30:10:1; 20:10:1, 3
L of each) and on ODS silica gel (500 g) with MeOH-H2O (2:
1, 2 L; 4:1, 3 L) and MeCN-H2O (1:2, 1.5 L) to give 1 (10 g,
0.14%), 2 (200 mg, 0.0027%), 3 (66 mg, 0.00090%), 6 (60 mg,
0.00082%), and 7 (30 mg, 0.00041%).
140.8 140.8 140.7 140.7 140.8 140.9 141.2
121.8 121.8 121.8 121.8 121.8 121.6 121.6
32.2
31.6
50.2
37.1
21.0
39.7
40.8
56.5
32.3
81.3
64.1
16.3
19.3
40.4
16.2
32.2
31.6
50.3
37.1
21.0
39.7
40.8
56.5
32.3
81.3
64.1
16.3
19.3
40.4
16.2
32.2
31.6
50.2
37.1
21.0
39.7
40.8
56.5
32.3
81.3
64.2
16.4
19.3
32.4
31.4
50.2
37.1
21.2
39.6
43.4
54.9
34.5
84.5
64.5
14.1
19.4
32.4
31.4
50.3
37.1
21.2
39.6
43.4
54.9
34.5
84.5
64.5
14.1
19.4
31.9
31.0
50.4
37.0
20.6
38.1
42.3
54.0
35.5
74.7 144.7
66.6 155.2
13.7
19.4
31.8
30.3
50.7
37.1
20.9
35.1
46.3
56.4
32.3
15.9
19.2
40.5 103.5 103.5 205.5 196.3
16.2
11.8
11.8
30.5
27.1
112.7 112.7 112.7 152.4 152.4 173.3
30.9
28.1
34.4
74.9
17.5
47.3
30.9
28.1
34.4
74.9
17.5
47.3
31.0
28.1
34.3
74.8
17.5
47.4
99.8
78.6
86.2
69.7
78.0
62.1
31.4
23.6
33.7
75.2
17.1
31.4
23.6
33.7
75.2
17.1
32.2
29.0
33.5
74.7
16.9
99.8 100.1
99.8 100.1
99.8
78.6
86.3
69.7
78.1
62.2
99.8
78.6
86.2
69.7
78.0
62.2
78.6
86.2
69.7
78.0
62.2
78.6
85.6
69.9
74.7
64.1
78.6
86.2
69.7
78.1
62.1
78.6
85.6
69.9
74.7
64.2
6′
1′′
2′′
3′′
4′′
5′′
6′′
25
102.6 102.7 102.6 102.6 102.7 102.6 102.6
Com p ou n d 1: amorphous solid; [R]D -82.0° (c 0.10,
72.5
72.8
73.8
69.9
18.6
72.4
72.7
73.7
70.0
18.6
72.3
72.7
73.7
69.9
18.6
72.5
72.8
73.8
69.9
18.7
72.4
72.7
73.8
70.0
18.6
72.5
72.8
73.8
69.9
18.7
72.5
72.8
73.8
69.9
18.6
CHCl3-MeOH, 1:1); IR (KBr) νmax 3400 (OH), 2930 (CH), 1040
cm-1; 1H NMR (C5D5N) δ 5.81 (1H, br s, H-1′′), 5.75 (1H, br s,
H-1′′′), 5.33 (1H, br d, J ) 4.6 Hz, H-6), 5.24 (1H, d, J ) 7.8
Hz, H-1′′′′), 4.89 (1H, d, J ) 7.8 Hz, H-1′), 4.85 (1H, d, J ) 7.7
Hz, H-1′′′′′), 4.45 (1H, m, H-16), 3.91 (1H, m, W1/2 ) 26.3 Hz,
H-3), 3.26 (3H, s, OMe), 1.76 (3H, d, J ) 6.2 Hz, Me-6′′), 1.69
(3H, d, J ) 6.2 Hz, Me-6′′′), 1.17 (3H, d, J ) 6.9 Hz, Me-21),
1.05 (3H, d, J ) 6.7 Hz, Me-27), 1.04 (3H, s, Me-19), 0.82 (3H,
s, Me-18); 13C NMR, see Table 2; FABMS (negative mode) m/z
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′
1′′′′′′
2′′′′′′
3′′′′′′
4′′′′′′
5′′′′′′
6′′′′′′
Ac
103.1 103.2 103.1 103.1 103.2 103.1 103.1
72.0
72.3
84.4
68.7
18.3
72.0
72.4
84.4
68.7
18.2
72.0
72.4
84.3
68.7
18.2
72.0
72.4
84.5
68.7
18.3
72.0
72.4
84.5
68.7
18.2
72.1
72.4
84.4
68.7
18.3
72.0
72.4
84.4
68.7
18.3
1223 [M - H]-; anal. C 55.60%, H 8.14%, calcd for C58H96O27
3/2H2O, C 55.62%, H 7.97%.
‚
106.5 106.5 106.4 106.5 106.5 106.5 106.5
Acid Hyd r olysis of 1. A solution of 1 (10 mg) in 1 M HCl
(dioxane-H2O, 1:1, 2 mL) was heated at 95 °C for 2 h under
an Ar atmosphere. After cooling, the reaction mixture was
neutralized by passage through an Amberlite IRA-93ZU
(Organo, Tokyo, J apan) column and chromatographed on silica
gel (15 g) eluting with CHCl3-MeOH (9:1 to 1:1, 100 mL of
each) to give an aglycon [(25S)-spirost-5-en-3â-ol] (3.0 mg) and
a sugar fraction (2.7 mg). The sugar fraction was dissolved in
H2O (1 mL) and passed through a Sep-pak C18 cartridge
(Waters, Milford, MA), which was then analyzed by HPLC
under the following conditions: column, Kaseisorb LC NH2-
60-5 UG80 (4.6 mm i.d. × 250 mm, 5 µm, Tokyo-Kasei, Tokyo,
J apan); solvent, MeCN-H2O (3:1); flow rate, 0.8 mL/min;
detection, RI and OR. Identification of D-glucose and L-
rhamnose present in the sugar fraction was carried out by
comparing their retention times and optical rotations with
those of authentic samples: tR (min) 8.1 (L-rhamnose, negative
optical rotation); 12.6 (D-glucose, positive optical rotation).
76.4
78.5
71.4
78.4
62.5
76.4
78.6
71.4
78.4
62.5
76.4
78.5
71.4
78.4
62.5
76.4
78.6
71.4
78.4
62.5
76.4
78.6
71.4
78.4
62.5
76.4
78.5
71.4
78.4
62.6
76.4
78.6
71.4
78.4
62.6
105.0 105.1 104.8 105.2 105.1 105.0
75.2
78.6
71.7
78.5
62.9
75.2
78.6
71.7
78.5
62.9
75.0
78.5
71.6
77.2
70.1
105.4
75.1
78.4
71.5
78.4
62.7
75.2
78.6
71.7
78.5
62.8
75.2
78.6
71.7
78.4
62.8
75.2
78.6
71.7
78.6
62.8
170.8
20.7
170.8
20.7
25
Com p ou n d 2: amorphous solid; [R]D -106.0° (c 0.10,
CHCl3-MeOH, 1:1); IR (KBr) νmax 3400 (OH), 2930 (CH), 1730
1
(CdO), 1040 cm-1; H NMR (C5D5N) δ 5.82 (1H, br s, H-1′′),
21), 1.05 (3H, d, J ) 6.7 Hz, Me-27), 1.02 (3H, s, Me-19), 0.81
(3H, s, Me-18); 13C NMR, see Table 1; FABMS (negative mode)
m/z 1265 [M - H]-; anal. C 55.87%, H 8.20%, calcd for
5.76 (1H, br s, H-1′′′), 5.32 (1H, br d, J ) 4.7 Hz, H-6), 5.28
(1H, d, J ) 7.8 Hz, H-1′′′′), 4.87 (1H, d, J ) 7.6 Hz, H-1′), 4.86
and 4.72 (each 1H, m, H2-6′), 4.85 (1H, d, J ) 7.8 Hz, H-1′′′′′),
4.46 (1H, m, H-16), 3.89 (1H, m, W1/2 ) 24.6 Hz, H-3), 3.26
(3H, s, OMe), 2.01 (3H, s, Ac), 1.75 (3H, d, J ) 6.2 Hz, Me-6′′),
1.67 (3H, d, J ) 6.2 Hz, Me-6′′′), 1.17 (3H, d, J ) 6.9 Hz, Me-
C
60H98O28‚H2O, C 56.06%, H 7.86%.
Alk a lin e Meth a n olysis of 2. Compound 2 (12.0 mg) was
treated with 3% NaOMe in MeOH (5 mL) at room temperature
for 1 h. The reaction mixture was neutralized by passage

Steroidal and Pregnane Glycosides from Tacca
J ournal of Natural Products, 2002, Vol. 65, No. 9 1297
through an Amberlite IR-120B (Organo, Tokyo, J apan) column
and purified by silica gel (15 g) column chromatography eluting
with CHCl3-MeOH-H2O (20:10:1, 100 mL) to give 1 (9.0 mg).
5.75 (1H, br s, H-1′′′), 5.67 (1H, m, H-16), 5.32 (1H, br d, J )
4.5 Hz, H-6), 5.24 (1H, d, J ) 7.8 Hz, H-1′′′′), 4.90 (1H, d, J )
7.7 Hz, H-1′), 4.80 (1H, d, J ) 7.8 Hz, H-1′′′′′), 3.91 (1H, m,
W1/2 ) 29.4 Hz, H-3), 2.50 (1H, d, J ) 7.6 Hz, H-17), 2.44 and
2.36 (each 1H, m, H2-23), 2.14 (3H, s, Me-21), 1.75 (3H, d, J )
6.2 Hz, Me-6′′), 1.69 (3H, d, J ) 6.1 Hz, Me-6′′′), 1.22 (3H, s,
Me-18), 1.05 (3H, s, Me-19), 0.92 (3H, d, J ) 6.7 Hz, Me-27);
13C NMR, see Table 1; FABMS (positive mode) m/z 1247 [M +
Na]+; anal. C 53.18%, H 7.73%, calcd for C57H92O28‚3H2O, C
53.51%, H 7.72%.
Acetyla tion of 6. Compound 6 (10 mg) was acetylated with
Ac2O (2 mL) in pyridine (2 mL) at room temperature for 12 h.
The reaction mixture was chromatographed on silica gel (20
g) eluting with hexane-Me2CO (1:1, 200 mL) to give peracetate
6a (10 mg).
25
Com p ou n d 3: amorphous solid; [R]D -54.0° (c 0.10,
CHCl3-MeOH, 1:1); IR (film) νmax 3388 (OH), 2934 (CH), 1047
cm-1; 1H NMR (C5D5N) δ 5.80 (1H, br s, H-1′′), 5.74 (1H, br s,
H-1′′′), 5.32 (1H, br d, J ) 4.0 Hz, H-6), 5.23 (1H, d, J ) 7.8
Hz, H-1′′′′), 5.11 (1H, d, J ) 7.9 Hz, H-1′′′′′′), 4.88 (1H, d, J )
7.9 Hz, H-1′), 4.77 (1H, d, J ) 7.8 Hz, H-1′′′′′), 4.45 (1H, m,
H-16), 3.90 (1H, m, W1/2 ) 24.3 Hz, H-3), 3.28 (3H, s, OMe),
1.75 (3H, d, J ) 6.2 Hz, Me-6′′), 1.69 (3H, d, J ) 6.2 Hz, Me-
6′′′), 1.18 (3H, d, J ) 6.8 Hz, Me-21), 1.04 (3H, d, J ) 6.7 Hz,
Me-27), 1.03 (3H, s, Me-19), 0.83 (3H, s, Me-18); 13C NMR, see
Table 1; FABMS (negative mode) m/z 1385 [M - H]-; anal. C
50.27%, H 7.72%, calcd for C64H106O32‚8H2O, C 50.19%, H
8.03%.
P r ep a r a tion of 6a (P er a ceta te of 6) fr om 4a (P er a c-
eta te of 4). The CrO3 (160 mg) solution in AcOH-H2O (19:1,
10 mL) was added to 4a (1.5 g) dissolved in AcOH-H2O (19:1,
20 mL), and it was stirred at room temperature for 2 h. After
the excess CrO3 was decomposed by MeOH (20 mL), the
reaction mixture was diluted with H2O (100 mL) and extracted
with Et2O (40 mL × 4). The Et2O extract was chromatographed
on silica gel (50 g) eluting with hexane-Me2O (1:1, 500 mL)
to give 6a (200 mg).
Acid Hyd r olysis of 3. A solution of 3 (10 mg) in 1 M HCl
(dioxane-H2O, 1:1, 2 mL) was subjected to acid hydrolysis as
described for 1 to give an aglycon [(25S)-spirost-5-en-3â-ol] (2.0
mg) and a sugar fraction (5.0 mg). HPLC analysis of the sugar
fraction under the same conditions as for 1 showed the
presence of D-glucose and L-rhamnose; tR (min) 8.1 (L-rham-
nose, negative optical rotation); 12.6 (D-glucose, positive optical
rotation).
25
25
Com p ou n d 4: amorphous solid; [R]D -60.0° (c 0.10,
Com p ou n d 6a : amorphous solid; [R]D -50.0° (c 0.10,
1
CHCl3-MeOH, 1:1); IR (film) νmax 3389 (OH), 2932 (CH), 1041
cm-1; 1H NMR (C5D5N) δ 5.83 (1H, br s, H-1′′), 5.77 (1H, br s,
H-1′′′), 5.32 (1H, br d, J ) 4.3 Hz, H-6), 5.25 (1H, d, J ) 7.8
Hz, H-1′′′′), 4.91 (1H, d, J ) 7.8 Hz, H-1′), 4.84 (1H, d, J ) 7.8
Hz, H-1′′′′′), 4.81 (1H, m, H-16), 3.91 (1H, m, W1/2 ) 27.0 Hz,
H-3), 1.76 (3H, d, J ) 6.2 Hz, Me-6′′), 1.69 (3H, d, J ) 6.1 Hz,
Me-6′′′), 1.63 (3H, s, Me-21), 1.03 (3H, d, J ) 6.6 Hz, Me-27),
1.06 (3H, s, Me-19), 0.71 (3H, s, Me-18); 13C NMR, see Table
1; FABMS (positive mode) m/z 1215 [M + Na]+; anal. C
54.99%, H 7.67%, calcd for C57H92O26‚3H2O, C 54.88%, H
7.92%.
CHCl3); IR (KBr) νmax 2940 (CH), 1750 (CdO), 1030 cm-1; H
NMR (CDCl3) δ 5.48 (1H, ddd, J ) 7.8, 7.7, 4.5 Hz, H-16), 5.35
(1H, br d, J ) 5.2 Hz, H-6), 2.38 (1H, d, J ) 7.6 Hz, H-17),
2.15 (3H, s, Me-21), 2.09-1.93 (Ac × 15), 1.22 (3H, d, J ) 6.1
Hz, Me-6′′′), 1.18 (3H, d, J ) 6.1 Hz, Me-6′′), 1.02 (3H, s, Me-
19), 1.00 (3H, s, Me-18), 0.87 (3H, d, J ) 6.7 Hz, Me-27).
25
Com p ou n d 7: amorphous solid; [R]D -44.0° (c 0.10,
CHCl3-MeOH, 1:1); UV (MeOH) λmax 239.8 nm (log ꢀ 3.84);
IR (film) νmax 3417 (OH), 2932 (CH), 1660 (CdO), 1028 cm-1
;
1H NMR (C5D5N) δ 6.62 (1H, dd, J ) 3.1, 2.0 Hz, H-16), 5.80
(1H, br s, H-1′′), 5.74 (1H, br s, H-1′′′), 5.34 (1H, br d, J ) 5.0
Hz, H-6), 5.24 (1H, d, J ) 7.8 Hz, H-1′′′′), 4.88 (1H, d, J ) 7.8
Hz, H-1′), 3.89 (1H, m, W1/2 ) 22.5 Hz, H-3), 2.25 (3H, s, Me-
21), 1.75 (3H, d, J ) 6.2 Hz, Me-6′′), 1.68 (3H, d, J ) 6.2 Hz,
Me-6′′′), 1.04 (3H, s, Me-19), 0.93 (3H, s, Me-18); 13C NMR,
see Table 1; FABMS (positive mode) m/z 953 [M + Na]+; anal.
Acetyla tion of 4. Compound 4 (10 mg) was acetylated with
Ac2O (2 mL) in pyridine (2 mL) at room temperature for 12 h.
The reaction mixture was chromatographed on silica gel (20
g) eluting with hexane-Me2CO (1:1, 200 mL) to give peracetate
4a (9 mg).
P r ep a r a tion of 4a (P er a ceta te of 4) fr om 1. Compound
1 (2.5 g) was treated with a mixture of Ac2O (15 mL) and
pyridine (15 mL) at 110 °C for 2.5 h. After addition of H2O (20
mL) into the reaction mixture followed by evaporation to
dryness, it was chromatographed on silica gel (95 g) eluting
with hexane-Me2CO (1:1, 800 mL) to give 4a (2.0 g).
C 52.13%, H 7.71%, calcd for C
7.95%.
45H70O20‚6H2O, C 52.01%, H
P r ep a r a tion of 7 fr om 6. Compound 6 (40 mg) was treated
with 7% NaOMe in MeOH (5 mL) at room temperature for
1.5 h. The reaction mixture was neutralized by passage
through an Amberlite IR-120B column and purified by ODS
silica gel (15 g) column chromatography eluting with MeOH-
H2O (8:5, 300 mL) to give 7 (4.0 mg) and 8 (5.0 mg).
25
Com p ou n d 4a : amorphous solid; [R]D -52.0° (c 0.10,
1
CHCl3); IR (KBr) νmax 2950 (CH), 1750 (CdO), 1035 cm-1; H
25
NMR (CDCl3) δ 5.37 (1H, br d, J ) 4.8 Hz, H-6), 2.16-1.94
(Ac × 15), 1.56 (3H, s, Me-21), 1.23 (3H, d, J ) 6.1 Hz, Me-
6′′′), 1.18 (3H, d, J ) 6.2 Hz, Me-6′′), 1.00 (3H, s, Me-19), 0.89
(3H, d, J ) 6.7 Hz, Me-27), 0.66 (3H, s, Me-18).
Com p ou n d 8: amorphous solid; [R]D -46.0° (c 0.10,
1
MeOH); IR (film) νmax 3388 (OH), 2933 (CH), 1048 cm-1; H
NMR (C5D5N) δ 5.82 (1H, br s, H-1′′), 5.76 (1H, br s, H-1′′′),
5.31 (1H, br d, J ) 4.6 Hz, H-6), 5.24 (1H, d, J ) 7.8 Hz, H-1′′′′),
4.90 (1H, d, J ) 7.7 Hz, H-1′), 4.53 (1H, m, H-16), 3.90 (1H,
m, W1/2 ) 23.8 Hz, H-3), 3.24 (3H, s, OMe), 2.67 (1H, d, J )
6.4 Hz, H-17), 2.17 (3H, s, Me-21), 1.76 (3H, d, J ) 6.2 Hz,
Me-6′′), 1.69 (3H, d, J ) 6.1 Hz, Me-6′′′), 1.02 (3H, s, Me-19),
0.64 (3H, s, Me-18); 13C NMR (C5D5N) δ 37.4 (C-1), 30.0 (C-2),
77.8 (C-3), 38.7 (C-4), 140.8 (C-5), 121.7 (C-6), 31.9 (C-7), 31.6
(C-8), 50.1 (C-9), 37.0 (C-10), 20.9 (C-11), 38.6 (C-12), 44.4 (C-
13), 54.6 (C-14), 32.1 (C-15), 81.8 (C-16), 71.5 (C-17), 14.5 (C-
18), 19.3 (C-19), 207.5 (C-20), 31.5 (C-21), 57.0 (OMe), 99.9
(C-1′), 37.4 (C-2′), 37.4 (C-3′), 37.4 (C-4′), 37.4 (C-5′), 37.4 (C-
6′), 102.6 (C-1′′), 72.5 (C-2′′), 72.8 (C-3′′), 73.8 (C-4′′), 69.9 (C-
5′′), 18.7 (C-6′′), 103.2 (C-1′′′), 72.1 (C-2′′′), 72.4 (C-3′′′), 71.4
(C-4′′′), 68.7 (C-5′′′), 18.3 (C-6′′′), 106.6 (C-1′′′′), 76.5 (C-2′′′′),
78.6 (C-3′′′′), 71.4 (C-4′′′′), 78.4 (C-5′′′′), 62.6 (C-6′′′′).
25
Com p ou n d 5: amorphous solid; [R]D -42.0° (c 0.10,
CHCl3-MeOH, 1:1); IR (film) νmax 3389 (OH), 2933 (CH), 1739
1
(CdO), 1044 cm-1; H NMR (C5D5N) δ 5.82 (1H, br s, H-1′′),
5.76 (1H, br s, H-1′′′), 5.32 (1H, br d, J ) 4.5 Hz, H-6), 5.23
(1H, d, J ) 7.8 Hz, H-1′′′′), 4.87 (1H, d, J ) 7.8 Hz, H-1′), 4.85
and 4.72 (each 1H, m, H2-6′), 4.83 (1H, d, J ) 7.7 Hz, H-1′′′′′),
4.82 (1H, m, H-16), 3.90 (1H, m, W1/2 ) 20.7 Hz, H-3), 2.01
(3H, s, Ac), 1.75 (3H, d, J ) 6.1 Hz, Me-6′′), 1.67 (3H, d, J )
6.2 Hz, Me-6′′′), 1.63 (3H, s, Me-21), 1.05 (3H, s, Me-19), 1.04
(3H, d, J ) 6.8 Hz, Me-27), 0.72 (3H, s, Me-18); 13C NMR, see
Table 1; FABMS (positive mode) m/z 1257 [M + Na]+ ; HR-
MALDI-TOFMS m/z 1257.5891 [M + Na]+ (calcd for C59H94O27-
Na, 1257.5880).
Alk a lin e Meth a n olysis of 5. Compound 5 (8.0 mg) was
treated with 3% NaOMe in MeOH (5 mL) at room temperature
for 1 h. The reaction mixture was neutralized by passage
through an Amberlite IR-120B column and purified by silica
gel (15 g) column chromatography eluting with CHCl3-
MeOH-H2O (20:10:1, 100 mL) to give 4 (6.0 mg).
Ack n ow led gm en t. We are grateful to Mr. Akira Higuchi,
Medicinal Research Laboratories, Taisho Pharmaceutical Co.,
Ltd., for the measurement of the HR-MALDI-TOFMS.
25
Refer en ces a n d Notes
Com p ou n d 6: amorphous solid; [R]D -22.0° (c 0.10,
CHCl3-MeOH, 1:1); IR (film) νmax 3389 (OH), 2933 (CH), 1712
(1) Dictionary of Chinese Medicinal Materials; Shanghai Scientific and
1
(CdO), 1044 cm-1; H NMR (C5D5N) δ 5.81 (1H, br s, H-1′′),
Technological Press: Shanghai, 1977; Vol. 1, p 524.

1298 J ournal of Natural Products, 2002, Vol. 65, No. 9
Yokosuka et al.
(2) Yokosuka, A.; Mimaki, Y.; Sakagami, H.; Sashida, Y. J . Nat. Prod.
2002, 65, 283-289.
(8) J ia, Z.; Koike, K.; Nikaido, T. J . Nat. Prod. 1998, 61, 1368-1373.
(9) Szendi, Z.; Forgo´, P.; Sweet, F. Steroids 1995, 60, 442-446.
(10) Reddy, K. S.; Shekhani, M. S.; Berry, D. E.; Lynn, D. G.; Hecht, S.
M. J . Chem. Soc., Perkin Trans. 1 1984, 987-992.
(11) Dong, M.; Feng, X. Z.; Wang, B. X.; Wu, L. J .; Ikejima, T. Tetrahedron
2001, 57, 501-506.
(3) Agrawal, P. K.; J ain, D. C.; Gupta, R. K.; Thakur, R. S. Phytochemistry
1985, 24, 2479-2496.
(4) Kiyosawa, S.; Hutoh, M.; Komori, T.; Nohara, T.; Hosokawa, I.;
Kawasaki, T. Chem. Pharm. Bull. 1968, 16, 1162-1164.
(5) Nohara, T.; Miyahara, K.; Kawasaki, T. Chem. Pharm. Bull. 1975,
23, 872-885.
(6) The furostanol saponins (1-3) were obtained as a mixture of C-22
hydroxyl and C-22 methoxyl forms. The C-22 hydroxyl form present
in the mixture was completely converted to the C-22 methoxyl form
by treatment with hot MeOH, and the structural elucidation of 1-3
was carried out with the C-22 methoxyl form.
(12) Dong, M.; Feng, X. Z.; Wu, L. J .; Wang, B. X.; Ikejima, T. Planta Med.
2001, 67, 853-857.
(13) Tran, Q. L.; Tezuka, Y.; Banskota, A. H.; Tran, Q. K.; Saiki, I.; Kadota,
S. J . Nat. Prod. 2001, 64, 1127-1132.
(14) Mimaki, Y.; Yokosuka, A.; Kuroda, M.; Sashida, Y. Biol. Pharm. Bull.
2001, 24, 1286-1289.
(7) Agrawal, P. K. Phytochemistry 1992, 31, 3307-3330.
NP020094L