Steroidal glycosides from the bulbs of Ornithogalum thyrsoides

Article abstract of DOI:10.1021/np040108i

Phytochemical analyses have been carried out on the fresh bulbs of Ornithogalum thyrsoides with particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol saponins and seven new cholestane glycosides, together with three known steroidal compounds. The structures of the new glycosides were determined on the basis of their spectroscopic data, including 2D NMR spectroscopy, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells.

Full text of DOI:10.1021/np040108i

1
690
J . Nat. Prod. 2004, 67, 1690-1696
Ster oid a l Glycosid es fr om th e Bu lbs of Or n ith oga lu m th yr soid es
†
,†
†
‡
†
Minpei Kuroda, Yoshihiro Mimaki,* Kazutomo Ori, Hiroshi Sakagami, and Yutaka Sashida
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, J apan,
and Department of Dental Pharmacology, Meikai University School of Dentistry, 1-1 Keyaki-dai,
Sakado, Saitama 350-0283, J apan
Received April 29, 2004
Phytochemical analyses have been carried out on the fresh bulbs of Ornithogalum thyrsoides with
particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol
saponins and seven new cholestane glycosides, together with three known steroidal compounds. The
structures of the new glycosides were determined on the basis of their spectroscopic data, including 2D
NMR spectroscopy, and the results of hydrolytic cleavage. The isolated compounds were evaluated for
their cytotoxic activities against HL-60 human promyelocytic leukemia cells and HSC-2 human oral
squamous cell carcinoma cells.
Ornithogalum thyrsoides (Liliaceae) is a perennial plant
native to southwestern Cape Province. Although O. thyr-
soides has no medicinal folkloric background and is now
cultivated for ornamental purposes, a MeOH extract of the
bulbs showed potent cytotoxic activity against HL-60
human promyelocytic leukemia cells. We have reported the
structural determination of 12 cholestane glycosides iso-
lated from O. thyrsoides bulbs as the cytotoxic ingredients.1
Further phytochemical analysis of the bulb extract with
particular attention to the steroidal glycoside constituents
led to the isolation of four new spirostanol saponins (1 and
3
-5) and seven new cholestane glycosides (8-14), along
with three known steroidal compounds (2, 6, and 7). This
paper deals with the structural elucidation of the new
compounds on the basis of their spectroscopic data, includ-
ing 2D NMR spectroscopy, and the results of hydrolytic
cleavage. The cytotoxic activities of 1-14 against HL-60
human promyelocytic leukemia cells and HSC-2 human
oral squamous cell carcinoma cells are also described.
Resu lts a n d Discu ssion
A concentrated MeOH extract of O. thyrsoides bulbs was
passed through a porous-polymer polystyrene resin (Diaion
HP-20) column eluted with 30% MeOH, 50% MeOH, and
EtOH. The EtOH eluate fraction was repeatedly subjected
to column chromatography over silica gel and octadecyl-
silanized (ODS) silica gel, as well as preparative HPLC, to
give compounds 1 (71.1 mg), 2 (112 mg), 3 (33.9 mg), 4 (28.5
mg), 5 (22.1 mg), 6 (22.5 mg), 7 (50.0 mg), 8 (17.6 mg), 9
(14.3 mg), 10 (102 mg), 11 (19.7 mg), 12 (11.7 mg), 13 (26.9
mg), and 14 (14.1 mg).
Compounds 2, 6, and 7 were identified as (25R)-3â-
hydroxyspirost-5-en-1â-yl O-R-L-rhamnopyranosyl-(1f2)-
2
R-L-arabinopyranoside (2), (22S)-16â-[(â-D-glucopyranosyl)-
oxy]-3â,22-dihydroxy-5R-cholestan-1â-yl R-L-rhamnopyrano-
3
side (6), and (22S)-16â-[(6-O-acetyl-â-D-glucopyranosyl)-
oxy]-3â,22-dihydroxy-5R-cholestan-1â-yl R-L-rhamnopyra-
3
noside (7), respectively.
Compound 1 was obtained as an amorphous solid,
2
D
5
[
R] -68.0° (MeOH). Its molecular formula was deter-
+
mined as C33
54 9
H O by HRESIMS, showing an [M + H]
1
peak at m/z 595.3836. The H NMR spectrum of 1 contained
signals for four steroidal methyl groups at δ 1.10 (d, J )
*
To whom correspondence should be addressed. Tel: +81-426-76-4577.
Fax: +81-426-76-4579. E-mail: mimakiy@ps.toyaku.ac.jp.
†
Tokyo University of Pharmacy and Life Science.
Meikai University School of Dentistry.
‡
1
0.1021/np040108i CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/17/2004

Steroidal Glycosides from Ornithogalum
J ournal of Natural Products, 2004, Vol. 67, No. 10 1691
protons at δ 1.43 (s), 1.09 (d, J ) 7.0 Hz), 0.85 (s), and 0.68
(
d, J ) 5.3 Hz). Acid hydrolysis of 3 yielded a steroidal
5
sapogenin, identified as (25R)-spirost-5-ene-1â,3â-diol (3a ),
together with L-arabinose, L-rhamnose, and D-xylose. On
comparison of the entire 13C NMR spectrum of 3 with that
of 2, a set of five additional signals, corresponding to a
terminal â-D-xylopyranosyl unit, appeared at δ 106.5 (CH),
7
2
4.6 (CH), 78.3 (CH), 71.0 (CH), and 67.0 (CH ), and the
signal due to C-3 of the arabinosyl moiety was markedly
deshielded by 8.6 ppm and was observed at δ 84.6,
suggesting the C-3 hydroxyl group of the arabinosyl moiety
was the position to which the D-xylose unit was attached.
This was confirmed by an HMBC correlation from the
anomeric proton of the xylosyl unit at δ 4.98 to the
arabinose C-3′ carbon at δ 84.6. Long-range correlations
between the anomeric proton of the rhamnosyl at δ 6.34
and C-2′ of the arabinosyl at δ 74.2 and between the
anomeric proton of the arabinosyl at δ 4.73 and C-1 of the
aglycone at δ 83.8 were also noted. Accordingly, the
structure of 3 was determined to be (25R)-3â-hydrox-
yspirost-5-en-1â-yl O-R-L-rhamnopyranosyl-(1f2)-O-[â-D-
xylopyranosyl-(1f3)]-R-L-arabinopyranoside.
Compound 4 was assigned the molecular formula
+
C
38
H
60
O
13 using HRESIMS (m/z 747.3935 [M + Na] ). The
1
H NMR spectrum showed signals for four steroid methyl
groups at δ 1.44 (s), 1.12 (d, J ) 7.0 Hz), 1.08 (d, J ) 6.5
Hz), and 0.86 (s) and two anomeric protons at δ 6.34 (br s)
and 4.73 (d, J ) 7.3 Hz). Acid hydrolysis of 4 with 1 M
HCl gave L-arabinose and L-rhamnose, together with
several unidentified artifactual sapogenols; no genuine
1
13
aglycone could be obtained. Comparison of the H and
C
NMR spectra of 4 with those of 2 revealed that the
structures of the ring A-E portion and diglycoside moiety
attached at C-1 of the aglycone were identical to those of
2
. However, significant differences were recognized in the
signals from the ring F portion (C-22-C-27) between 2 and
4
4
, and an additional oxymethine proton was observed at δ
.01 in 4, which was shifted downfield by 1.19 ppm on
complete acetylation of 4. These data indicated that 4 was
a steroidal glycoside closely related to 2 with a hydroxyl
1
1
group at ring F. The H- H COSY and 2D TOCSY spectra
were inspected to assign the locus of the hydroxyl group,
with the methyl doublet due to Me-27 at δ 1.08 (J ) 6.5
Hz) being used as the starting point of the analysis. The
subsequent spin-coupling correlations led us to propose the
F ring as -CH
2
-CH(-OH)-CH(-Me)-CH -O-, giving
2
evidence for the presence of a C-24 hydroxyl group. NOE
correlations from H-26ax (δ 3.59) to H-16 (δ 4.54) and Me-
2
7 and from H-26eq (δ 3.69) to H-25 (δ 1.82) in the
6
.9 Hz), 1.03 (s), 0.85 (s), and 0.70 (d, J ) 5.3 Hz) and one
anomeric proton at δ 5.05 (d, J ) 7.7 Hz). Acid hydrolysis
O (1:1) resulted in the
PHNOESY spectrum and spin-coupling constants between
H
3
3
2
-23 and H-24 ( J H-23eq,H-24 ) 4.7 Hz, J H-23ax,H-24 ) 12.2
of 1 with 1 M HCl in dioxane-H
2
3
Hz) and between H-25 and H
2
-26 ( J H-25,H-26eq ) 4.8 Hz,
production of an aglycone, identified as (25R)-5R-spiro-
3
4
J H-25,H-26ax ) 11.3 Hz) were indicative of the 22R, 24S,
stane-1â,3â-diol (1a ), and D-glucose as the carbohydrate
and 25S configurations. Thus, the structure of 4 was
defined as (24S,25S)-3â,24-dihydroxyspirost-5-en-1â-yl O-R-
L-rhamnospyranosyl-(1f2)-R-L-arabinopyranoside.
component. The signal at δ 3.98 (dd, J ) 11.5, 4.2 Hz)
showed long-range correlations with C-9 (δ 54.8), C-10 (δ
4
was assigned to H-1. In the HMQC spectrum, the signal
assigned to H-1 was correlated to the oxygen-bearing
carbon signal at δ 81.2 (C-1), with which the anomeric
proton of the glucosyl moiety had an HMBC correlation.
Thus, the structure of 1 was elucidated as (25R)-3â-
hydroxy-5R-spirostan-1â-yl â-D-glucopyranoside.
0.4), and C-19 (δ 8.2) in the HMBC spectrum of 1 and
Compound 5 was deduced as C43
H
68
O
17 from its HRESI-
+
1
MS (m/z 879.4302 [M + Na] ). The prominent H NMR
signals arising from the aglycone moiety of 5 at δ 5.58 (br
d, J ) 5.7 Hz, H-6), 4.01 (ddd, J ) 12.1, 10.3, 4.7 Hz, H-24),
3.88 (m, W1/2 ) 20.0 Hz, H-3), 3.83 (dd, J ) 12.0, 3.9 Hz,
H-1), 1.42 (s, Me-19), 1.12 (d, J ) 6.9 Hz, Me-21), 1.08 (d,
J ) 6.5 Hz, Me-27), and 0.85 (s, Me-19) were superimpos-
able on those of 4, whereas the signals due to the anomeric
protons at δ 6.34 (br s), 4.98 (d, J ) 7.5 Hz), and 4.74 (d,
J ) 7.3 Hz) were coincident with those of 3. Acid hydrolysis
of 5 with 1 M HCl gave L-arabinose, L-rhamnose, and
Compound 3 had the molecular formula C43
H
68
O
16 on the
+
basis of HRESIMS, exhibiting an [M + Na] peak at m/z
1
8
63.4404. The H NMR spectrum of 3 showed three
anomeric protons at δ 6.34 (br s), 4.98 (d, J ) 7.5 Hz), and
.73 (d, J ) 7.3 Hz), along with four steroidal methyl
4

1
692 J ournal of Natural Products, 2004, Vol. 67, No. 10
Kuroda et al.
D-xylose. Comparison of the 13C NMR spectrum of 5 with
those of 3 and 4 and the results of acid hydrolysis, as well
as the above H NMR data, allowed the structural assign-
ment of 5 as (24S,25S)-3â,24-dihydroxyspirost-5-en-1â-yl
O-R-L-rhamnopyranosyl-(1f2)-O-[â-D-xylopyranosyl-(1f3)]-
R-L-arabinopyranoside.
comparison with those of 9 implied that 10 differed from 9
in the presence of an aromatic acid ester group. The
aromatic acid was suggested to be 3,4,5-trimethoxybenzoic
1
-
1
acid by the IR [1716 cm (CdO); 1590, 1504, and 1459
-
1
1
cm (aromatic ring)], UV [λmax 266 nm (log ꢀ 4.01)], H
NMR [δ 7.71 (2H, s), 3.97 (3H, s), and 3.85 (3H × 2, s)],
and 13C NMR [δ 126.2 (C), 107.9 (CH × 2), 153.8 (C × 2),
Compound 8 was isolated as an amorphous solid with
1
43.1 (C), 166.3 (CdO), 60.7 (OMe), and 56.2 (OMe × 2)]
the molecular formula
C
46
H
78
O
18, as determined by
+
spectral data. Alkaline hydrolysis of 10 with 4% KOH in
EtOH yield 9 and 3,4,5-trimethoxybenzoic acid. Therefore,
HRESIMS data exhibiting an [M + Na] peak at m/z
1
9
41.5048. The H NMR spectrum of 8 showed signals for
1
0 was a 3,4,5-trimethoxybenzoyl ester of 9. The anomeric
five typical steroidal methyl groups at δ 1.14 (d, J ) 7.0
Hz), 0.98 (s), 0.97 (s), 0.95 (d, J ) 6.2 Hz), and 0.93 (d, J )
proton of one glucosyl moiety at δ 4.75 (d, J ) 7.8 Hz)
showed a long-range correlation with C-16 of the aglycone
at δ 82.9, whereas that of another glucosyl unit at δ 4.97
6
.2 Hz) and suggested that the aglycone of 8 was the same
as that of 7. The presence of a 6-O-acetyl-â-D-glucopyra-
nosyl moiety attached to C-16 of the aglycone and an R-L-
rhamnopyraosyl attached to C-1 in 8 as in 7 was also shown
(
d, J ) 7.8 Hz) gave an HMBC correlation with C-1 of the
aglycone at δ 82.8. The H -6′ protons due to the glucosyl
2
group attached to C-1 of the aglycone at δ 5.15 and 5.08
were correlated to the carbonyl carbon of the 3,4,5-
trimethoxybenzoyl moiety at δ 166.3. Thus, 10 was defined
as (22S)-16â-[(â-D-glucopyranosyl)oxy]-3â,22-dihydroxy-5R-
cholestan-1â-yl 6-O-(3,4,5-trimethoxybenzoyl)-â-D-glucopy-
ranoside.
1
13
by analysis of the H and C NMR spectra of 8. However,
the molecular formula of 8 was higher by C than that
5
8 4
H O
of 7, and three anomeric proton signals were observed at
δ 5.92 (br s), 5.78 (d, J ) 3.6 Hz), and 4.60 (d, J ) 7.8 Hz).
Acid hydrolysis of 8 with 0.2 M HCl in dioxane-H O (1:1)
2
gave an aglycone (8a ), identified as (22S)-5R-cholestane-
â,3â,16â,22-tetraol (8a ),3 and D-apiose, D-glucose, and
L-rhamnose as the carbohydrate moieties. In the H NMR
Compound 11 analyzed for C49
75.4915 [M + Na] ). Comparison of the H and C NMR
H
76
O
18 by HRESIMS (m/z
1
+
1
13
9
1
spectra of 11 with those of 10 showed their considerable
structural similarity. The differences consisted only in the
signals of the methyl groups assignable to Me-26 and Me-
spectrum of 8, the signals at δ 5.78 and 4.76 (J ) 3.6 Hz),
4
.73 and 4.31 (J ) 9.2 Hz), and 4.15 and 4.12 (J ) 11.3
Hz) were assignable to the protons of the apiofuranosyl
residue, and the quaternary carbon at δ 80.1 was typical
of C-3 of apiofuranose. The â-furanoid anomeric form of
the apiofuranosyl residue was indicated by the deshielded
2
7. The two doublets for Me-26 and Me-27 observed in the
1
H NMR spectrum of 10 were displaced by two three-proton
singlets at δ 1.74 and 1.66 in that of 11, suggesting that
1
1 was a C-24/C-25 dehydro derivative of 10. This was
6
anomeric carbon signal at δ 111.0. The apiosyl group was
confirmed by catalytic hydrogenation of 11 over PtO
2
,
involved in a glycosidic linkage at C-4 of the glucosyl group,
because the signal due to C-4 of the glucosyl was remakedly
deshielded by 8.1 ppm, while the signals due to C-3 and
C-5 were shielded by 2.1 and 1.8 ppm, respectively, when
comparing the 13C NMR spectrum of 8 with that of 7. This
was confirmed by the observation of a long-range correla-
tion from the anomeric proton of the apiosyl group at δ
resulting in the formation of 9. The structure of 11 was
formulated as (22S)-16â-[(â-D-glucopyranosyl)oxy]-3â,22-
dihydroxy-5R-cholest-24-en-1â-yl 6-O-(3,4,5-trimethoxyben-
zoyl)-â-D-glucopyranoside.
Compound 12 had the molecular formula C39
H
66
O
12 on
1
+
the basis of HRESIMS (m/z 727.4636 [M + H] ). The H
NMR spectrum of 12 exhibited signals for five steroidal
methyl groups at δ 1.28 (s), 1.17 (d, J ) 6.9 Hz), 1.00 (s),
and 0.86 (3H × 2, d, J ) 6.5 Hz), an olefinic proton at δ
5
.78 to C-4′′ of the glucosyl moiety at δ 80.0 in the HMBC
spectrum. The anomeric proton of the substituted glucosyl
group at δ 4.60 showed an HMBC correlation to C-16 of
the aglycone at δ 83.0, whereas that of the rhamnosyl at δ
5
.63 (br d, J ) 5.6 Hz), and two anomeric protons at δ 5.66
and 5.27 (each br s). Acid hydrolysis of 12 gave an aglycone,
5
.92 to C-1 of the aglycone appeared at δ 81.8. Long-range
7
identified as (22S)-cholest-5-ene-1â,3â,16â,22-tetraol (12a),
correlations from H -6′′ of the glucosyl at δ 4.75 and 4.73
2
13
and L-rhamnose. The C NMR spectrum of 12 showed the
to the carbonyl carbon of the acetyl group at δ 170.5 were
also observed. The data led to the full structure of 8 as
presence of two terminal R-L-rhamnopyranosyl units in 12,
and HMBC correlations were observed from δ 5.66 to δ 82.1
(22S)-16â-[(O-â-D-apiofuranosyl-(1f4)-6-O-acetyl-â-D-glu-
(C-1 of aglycone) and from δ 5.27 to δ 81.3 (C-16 of
copyranosyl)oxy]-3â,22-dihydroxy-5R-cholestan-16â-yl R-L-
rhamnopyranoside.
aglycone), allowing the assignment of the structure of 12
as (22S)-16â-[(R-L-rhamnopyranosyl)oxy]-3â,22-dihydroxy-
cholest-5-en-1â-yl R-L-rhamnopyranoside.
Compound 9 analyzed for C39
83.4490 [M + Na] ). The spectroscopic data of 9 showed
68
H O14 by HRESIMS (m/z
+
7
+
Compound 13 showed an accurate [M + Na] ion at m/z
that 9 was a cholestane glycoside closely related to 6. The
7
66 12
49.4474, corresponding to the molecular formula C39H O .
1
13
H and C NMR spectra of 9 exhibited signals of two â-D-
1
13
The H and C NMR spectroscopic data of 13 indicated
that 13 contained two R-L-rhamnopyranosyl units, whose
anomeric protons were observed at δ 5.61 (br s) and 5.25
glucopyranosyl residues, along with signals for five steroid
methyl groups at δ 1.13 (d, J ) 7.0 Hz), 1.02 (s), 0.96 (s),
0
.93 (d, J ) 6.2 Hz), and 0.92 (d, J ) 6.2 Hz). Acid
(br s), and that the aglycone of 13 was the same as that of
hydrolysis of 9 yielded 8a and D-glucose. In the HMBC
spectrum of 9, the anomeric proton signals of the two
glucosyl groups at δ 5.06 and 4.75 showed long-range
correlations with C-1 (δ 81.0) and C-16 (δ 82.7) of the
aglycone, respectively. The structure of 9 was shown to be
1
1. The linkage positions of two rhamnosyl units were
revealed to be at C-1 and C-16 of the aglycone by the
observation of long-range correlations from δ 5.61 to δ 81.7
(C-1 of the aglycone) and from δ 5.25 to δ 81.9 (C-16 of the
aglycone). The structure of 13 was elucidated as (22S)-16â-
[(R-L-rhamnopyranosyl)oxy]-3â,22-dihydroxycholest-24-en-
1â-yl R-L-rhamnopyranoside.
(
22S)-16â-[(â-D-glucopyranosyl)oxy]-3â,22-dihydroxy-5R-
cholestan-1â-yl â-D-glucopyranoside.
Compound 10 exhibited the molecular formula C49
H
78
O
18
+
Compound 14 was a cholestane glycoside with the
on the basis of HRESIMS (m/z 977.5065 [M + Na] ).
molecular formula C39
H
64
O
12 (HRESIMS m/z 747.4266
1
13
+
Analysis of the H and C NMR spectra of 10 and
[M + Na] ), and the spectroscopic data implied that it was

Steroidal Glycosides from Ornithogalum
J ournal of Natural Products, 2004, Vol. 67, No. 10 1693
Ta ble 1. 13C NMR Spectroscopic Data for 1, 3-5, and 8-14 in C5D5N
carbon
1
3
4
5
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
9
81.2
37.7
67.6
39.7
42.9
28.9
32.4
36.4
54.8
40.4
23.7
40.7
41.5
56.7
32.4
81.1
63.2
16.9
8.2
42.1
14.9
109.2
31.8
29.3
30.6
66.8
17.3
100.6
75.6
78.6
72.3
78.4
63.6
83.8
37.5
68.2
43.9
139.5
124.7
32.4
33.1
50.3
42.9
24.1
40.3
40.2
56.8
32.0
81.1
63.0
16.7
15.0
41.9
15.0
109.2
31.8
29.3
30.6
66.8
17.3
100.5
74.2
84.6
69.7
67.1
83.5
37.4
68.2
43.9
139.6
124.7
32.4
33.1
50.4
42.9
24.0
40.3
40.2
56.8
32.0
81.5
62.6
16.7
15.0
42.2
15.0
111.8
41.9
70.6
40.0
65.3
13.6
100.3
75.1
75.9
70.1
67.3
83.8
37.5
68.2
43.8
139.5
124.7
32.4
33.1
50.3
42.9
24.0
40.3
40.2
56.8
31.9
81.5
62.6
16.7
14.9
42.2
15.0
111.8
41.8
70.6
40.0
65.3
13.6
100.5
74.2
84.5
69.6
67.1
81.8
37.4
67.6
39.6
42.9
29.0
32.0
36.5
55.1
41.6
24.5
40.9
42.3
55.2
37.2
83.0
58.1
13.9
8.1
36.0
12.6
73.2
33.7
36.7
28.9
23.1
23.0
98.5
72.8
73.2
73.8
71.0
18.7
106.6
75.1
76.4
80.0
73.2
64.0
111.0
77.4
80.1
64.7
75.2
81.0
37.5
67.7
39.7
42.9
28.6
32.1
36.4
54.8
41.4
23.8
40.7
42.2
55.0
37.3
82.7
58.2
14.0
8.2
36.0
12.5
73.2
33.8
36.7
28.9
23.1
23.0
100.4
75.6
78.7
71.7
78.4
63.5
107.0
75.7
78.7
71.7
78.2
63.5
82.8
38.0
67.8
39.8
43.1
28.9
32.1
36.5
54.9
41.3
23.7
40.6
42.3
54.9
37.3
82.9
58.2
14.1
8.3
35.9
12.5
73.3
33.7
36.7
28.9
23.1
23.0
101.5
75.3
78.6
72.3
74.9
66.0
107.0
75.7
78.8
71.7
78.2
62.9
82.7
38.0
67.8
39.8
43.1
28.9
32.1
36.5
54.9
41.3
23.7
40.6
42.3
54.9
37.2
82.9
58.2
14.1
8.3
35.5
12.4
72.9
34.7
126.2
131.3
26.0
18.2
101.5
75.3
78.6
72.2
74.9
66.0
107.0
75.7
78.8
71.7
78.2
62.9
82.1
35.8
68.1
43.8
139.3
125.0
31.5
33.5
50.8
42.9
24.9
40.5
42.1
55.1
35.9
81.3
57.9
13.6
14.6
36.1
12.0
72.5
34.5
36.8
28.7
22.9
22.8
97.7
73.0
72.9
73.7
71.0
18.7
104.9
72.7
73.2
74.0
70.7
18.4
81.7
37.2
67.6
39.6
42.9
29.0
32.0
36.6
55.0
41.6
24.6
40.9
42.2
55.1
35.9
81.9
58.0
13.7
8.2
35.3
11.8
72.0
35.4
123.1
132.3
26.0
18.1
98.4
73.1
72.8
73.8
71.0
18.8
104.9
72.6
73.2
74.0
70.9
18.4
82.0
35.9
68.1
43.8
139.2
125.0
31.5
33.5
50.8
42.8
24.9
40.6
42.1
55.1
35.8
81.3
57.9
13.6
14.6
35.3
11.9
72.1
35.4
125.0
132.3
26.0
18.1
97.7
73.0
72.9
73.7
70.9
18.7
104.9
72.7
73.2
74.0
70.7
18.4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
′
′
′
′
′
′
′′
′′
′′
′′
′′
′′
′′′
′′′
′′′
′′′
′′′
′′
′′′
′′′
′′′
′′′
′′′
′′′
101.8
72.6
72.6
74.1
69.5
19.2
106.5
74.6
78.3
71.0
67.0
101.7
72.6
72.7
74.2
69.4
19.0
101.8
72.5
72.5
74.1
69.5
19.2
106.5
74.6
78.3
71.0
67.0
126.2
107.9
153.8
143.1
153.8
107.9
166.3
60.7
126.2
107.9
153.8
143.1
153.8
107.9
166.3
60.6
OMe
OMe
Ac
56.2 × 2
56.2 × 2
170.5
2
0.8
1
closely related to that of 12. The H NMR spectrum of 14
showed two three-proton singlets at δ 1.71 and 1.68
attributable to methyl groups on a double bond, and
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured using a J ASCO DIP-360 (Tokyo, J apan)
automatic digital polarimeter. IR spectra were recorded on a
J ASCO FT-IR 620 spectrophotometer and UV spectra on a
J ASCO V-520 instrument. NMR spectra were recorded on a
catalytic hydrogenation of 14 over PtO
2
yielded 12. Thus,
4 was defined as (22S)-16â-[(R-L-rhamnopyranosyl)oxy]-
â,22-dihydroxycholesta-5,24-dien-1â-yl R-L-rhamnopyra-
1
3
noside.
The isolated compounds (1-14) were evaluated for their
cytotoxic activities against HL-60 luekemia cells and HSC-2
cells, using a modified MTT assay method. Compounds
-4, 7, 10, and 14 showed moderate cytotoxic activity
against HL-60 cells with IC50 values ranging from 1.6 to
.3 µg/mL, when that of etoposide used as positive control
1
Bruker DRX-500 spectrometer (500 MHz for H NMR, Karlsru-
he, Germany) using standard Bruker pulse programs. Chemi-
cal shifts are given as δ-values with reference to tetrameth-
ylsilane (TMS) as internal standard. ESIMS data were obtained
on a Micromass LCT mass spectrometer (Manchester, UK).
Diaion HP-20 (Mitsubishi-Chemical, Tokyo, J apan), silica gel
1
5
(Fuji-Silysia Chemical, Aichi, J apan), and ODS silica gel
was 0.23 µg/mL. As for cytotoxicty against HSC-2 cells,
which were resistant to etoposide (24.4 µg/mL), 1-3 were
also cytotoxic, with IC50 values ranging from 19.9 to 34.6
µg/mL.
(Nacalai Tesque, Kyoto, J apan) were used for column chro-
matography. TLC was carried out on precoated Kieselgel 60
F
254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254
S (0.25 mm thick, Merck) plates, and spots were visualized

1
694 J ournal of Natural Products, 2004, Vol. 67, No. 10
Kuroda et al.
Ta ble 2. Cytotoxic Activities of 1-14 and Etoposide against
preparative HPLC using MeCN-H
2
O (2:5) to yield 6 (22.5 mg)
HL-60 and HSC-2 Cells
and 8 (17.6 mg). Fraction III was separated by silica gel column
chromatography eluted with CHCl -MeOH-H O (40:10:1)
and ODS silica gel column chromatography with MeCN-H
1:2) to afford four subfractions (IIIa, IIIb, IIIc, and IIId).
Fraction IIIb was subjected to a silica gel column eluted with
CHCl -MeOH-H O (60:10:1) and an ODS silica gel column
with MeCN-H O (1:2) to give 3 (33.9 mg). Fraction IIId was
separated by a silica gel column eluted with CHCl -MeOH-
O (40:10:1; 30:10:1) and an ODS silica gel column with
MeCN-H O (2:3; 2:5) to give 5 and 9 with a few impurities.
Final purification of 5 and 9 was established by preparative
3
2
IC50 (µg/mL)
2
O
compound
HL-60
HSC-2
(
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1.6
2.2
1.9
34.6
19.9
21.2
3
2
2
5.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.4
3
>10
>10
5.3
>10
>10
5.0
>10
>10
>10
5.1
H
2
2
HPLC using MeCN-H
(14.3 mg).
2
O (2:3; 2:5) to yield 5 (22.1 mg) and 9
0
1
2
3
4
25
Com p ou n d 1: amorphous solid; [R]D -68.0° (c 0.10,
MeOH); IR (film) νmax 3363 (OH), 2950, 2926, and 2875 (CH),
-
1
1
1
7
5 5
073, 1055, 1025 cm ; H NMR (C D N) δ 5.05 (1H, d, J )
.7 Hz, H-1′), 4.59 (1H, dd, J ) 11.5, 3.3 Hz, H-6′a), 4.54 (1H,
m, H-16), 4.36 (1H, dd, J ) 11.5, 5.8 Hz, H-6′b), 4.24 (1H, dd,
etoposide
0.23
J ) 8.9, 8.9 Hz, H-3′), 4.15 (1H, dd, J ) 8.9, 8.9 Hz, H-4′),
4
.03 (1H, dd, J ) 8.9, 7.7 Hz, H-2′), 3.98 (1H, dd, J ) 11.5, 4.2
by spraying with 10% H
2
SO
4
followed by heating. HPLC was
Hz, H-1), 3.90 (1H, br m, W1 ) 22.3 Hz, H-3), 3.88 (1H, ddd,
/2
performed by using a system comprised of a CCPM pump
Tosoh, Tokyo, J apan), a CCP PX-8010 controller (Tosoh), an
J ) 8.9, 5.8, 3.3 Hz, H-5′), 3.58 (1H, dd, J ) 10.6, 2.9 Hz,
H-26eq), 3.51 (1H, dd, J ) 10.6, 10.6 Hz, H-26ax), 1.10 (3H,
d, J ) 6.9 Hz, Me-21), 1.03 (3H, s, Me-19), 0.85 (3H, s, Me-
(
RI-8010 detector (Tosoh) or a Shodex OR-2 detector (Showa-
Denko, Tokyo, J apan), and a Rheodyne injection port. A
Capcell Pak C18 UG120 column (10 mm i.d. × 250 mm, 5 µm,
Shiseido, Tokyo, J apan) was employed for preparative HPLC.
The following reagents were obtained from the indicated
companies: RPMI 1640 medium and Dulbecco’s modified
Eagle’s medium (DMEM) (Gibco, Grand Island, NY); fetal
bovine serum (FBS) (Bio-Whittaker, Walkersville MD or J RH
Biosciences, Lenexa, KS); 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-2H-tetrazolium bromide (MTT) (Sigma, St. Louis, MO);
penicillin and streptomycin (Sigma or Meiji-Seika, Tokyo,
J apan). All other chemicals used were of biochemical reagent
grade.
P la n t Ma ter ia l. The bulbs of O. thyrsoides were purchased
from a nursery in Heiwaen, Nara, J apan. The bulbs were
cultivated, and the flowered plant was identified by one of the
authors (Y.S.). A voucher specimen of the plant has been
deposited in our laboratory (99-4-OT).
Extr a ction a n d Isola tion . The plant material (fresh
weight, 15.4 kg) was extracted with hot MeOH (twice). The
MeOH extract was concentrated under reduced pressure, and
the viscous concentrate (702 g) was passed through a Diaion
HP-20 column, successively eluted with 30% MeOH, 50%
MeOH, and EtOH. Column chromatography of the EtOH
eluate portion on silica gel and elution with a stepwise gradient
18), 0.70 (3H, d, J ) 5.3 Hz, Me-27); HRESIMS m/z 595.3836
+
[
M + H] (calcd for C33
H
55
O
9
, 595.3846).
Acid Hyd r olysis of 1. A solution of 1 (20.0 mg) in 1 M HCl
O, 1:1, 5 mL) was heated at 95 °C for 1 h under
an Ar atmosphere. After cooling, the reaction mixture was
neutralized by passage through an Amberlite IRA-93ZU
(
dioxane-H
2
(Organo, Tokyo, J apan) column and chromatographed on
Diaion HP-20 eluted with H O-MeOH (3:2) followed by Me -
2
2
CO-EtOH (1:1) to give an aglycone fraction and a sugar
fraction (5.0 mg). The aglycone fraction was chromatographed
on silica gel eluted with CHCl -MeOH (19:1) to give 1a (12.1
3
mg). The sugar fraction was passed through a Sep-Pak C18
cartridge (Waters, Milford, MA) and a Toyopak IC-SP-M
cartridge (Tosoh), which was then analyzed by HPLC under
the following conditions: column, a Capcell Pak C18 UG80 (4.6
mm i.d. × 250 mm, 5 µm, Shiseido); solvent, MeCN-H
2
O
(17:3); flow rate, 0.9 mL/min; detection, RI and OR. Identifica-
tion of D-glucose present in the sugar fraction was carried out
by the comparison of its retention time and polarity with that
of an authentic sample; t
R
(min) 17.57 (D-glucose, positive
polarity).
2
5
Com p ou n d 3: amorphous solid; [R]D -54.0° (c 0.10,
MeOH); IR (film) νmax 3377 (OH), 2950, 2928 and 2905 (CH),
-
1 1
1
(
4
090, 1050 cm ; H NMR (C
1H, br d, J ) 5.7 Hz, H-6), 4.98 (1H, d, J ) 7.5 Hz, H-1′′′),
.73 (1H, d, J ) 7.3 Hz, H-1′), 4.52 (1H, q-like, J ) 7.4 Hz,
5 5
D N) δ 6.34 (1H, br s, H-1′′), 5.58
mixture of CHCl
finally with MeOH alone gave four fractions (I, II, III, and IV).
Fraction II was subjected to a silica gel column eluted with
3
2
-MeOH-H O (90:10:0; 40:10:1; 20:10:1) and
H-16), 3.89 (1H, m, W1/2 ) 21.2 Hz, H-3), 3.82 (1H, dd, J )
1
3
2.0, 3.9 Hz, H-1), 3.56 (1H, dd, J ) 10.5, 2.6 Hz, H-26eq),
.48 (1H, dd, J ) 10.5, 10.5 Hz, H-26ax), 1.74 (3H, d, J ) 6.2
CHCl
and IIb). Fraction IIa was chromatographed on silica gel eluted
with CHCl -MeOH-H O (80:10:1; 70:10:1) and ODS silica gel
with MeOH-H O (4:1) to yield 1 (71.1 mg) and 2 (112 mg).
Fraction IIb was subjected to an ODS silica gel column eluted
with MeCN-H O (1:2) and a silica gel column with CHCl
MeOH-H O (60:10:1) and was divided into four further
fractions (IIb-1, IIb-2, IIb-3, and IIb-4). Fraction IIb-1 was
subjected to a silica gel column eluted with CHCl -MeOH-
O (60:10:1) and preparative HPLC using MeCN-H O (2:3)
to give 4 (28.5 mg). Fraction IIb-2 was chromatographed on
silica gel eluted with CHCl -MeOH-H O (80:10:1) and ODS
silica gel with MeCN-H O (2:5) to give a mixture of 7 and 14,
which was then separated by preparative HPLC using MeCN-
O (1:2) to yield 7 (50.0 mg) and 14 (14.1 mg). Fraction IIb-3
was subjected to a silica gel column eluted with CHCl
3
-MeOH-H
2
O (60:10:1) to collect two subfractions (IIa
Hz, Me-6′′), 1.43 (3H, s, Me-19), 1.09 (3H, d, J ) 7.0 Hz, Me-
3
2
2
1), 0.85 (3H, s, Me-18), 0.68 (3H, d, J ) 5.3 Hz, Me-27);
2
+
HRESIMS m/z 863.4404 [M + Na] (calcd for C43
63.4405).
Acid Hyd r olysis of 3. A solution of 3 (25.0 mg) was
68
H O16Na,
8
2
3
-
2
subjected to acid hydrolysis as described for 1 to give an
aglycone fraction and a sugar fraction (11.2 mg). The aglycone
fraction was chromatographed on silica gel eluted with CHCl -
MeOH (19:1) to give 3a (10.1 mg). HPLC analysis of the sugar
fraction under the same conditions as for 1 showed the
3
H
2
2
3
3
2
presence of L-arabinose, L-rhamnose, and D-xylose; t
.71 (L-rhamnose, negative polarity); 10.43 (L-arabinose, posi-
tive polarity); 11.04 (D-xylose, positive polarity).
R
(min)
2
8
H
2
2
3
3
-
Com p ou n d 4: amorphous solid; [R]D -72.0° (c 0.10,
MeOH); IR (film) νmax 3386 (OH), 2951, 2927 and 2906 (CH),
MeOH-H
2
O (80:10:1) and an ODS silica gel column with
O (2:5) to give 10 (102 mg) and 13 (26.9 mg) in pure
-
1
1
MeCN-H
2
5 5
1084, 1054, 1037 cm ; H NMR (C D N) δ 6.34 (1H, br s,
form and a mixture of 11 and 12. The mixture was separated
H-1′′), 5.58 (1H, br d, J ) 5.7 Hz, H-6), 4.73 (1H, d, J ) 7.3
Hz, H-1′), 4.54 (1H, q-like, J ) 7.4 Hz, H-16), 4.01 (1H, ddd, J
) 12.2, 10.5, 4.7 Hz, H-24), 3.87 (1H, m, W1/2 ) 19.5 Hz, H-3),
3.84 (1H, dd, J ) 12.1, 3.9 Hz, H-1), 3.69 (1H, dd, J ) 11.3,
by preparative HPLC using MeCN-H
19.7 mg) and 12 (11.7 mg). Fraction IIb-4 was subjected to
an ODS silica gel column eluted with MeCN-H O (2:5) and
2
O (1:2) to furnish 11
(
2

Steroidal Glycosides from Ornithogalum
J ournal of Natural Products, 2004, Vol. 67, No. 10 1695
rhamnose; t (min) 8.01 (D-apiose, positive polarity), 8.69 (L-
4
.8 Hz, H-26eq), 3.59 (1H, dd, J ) 11.3, 11.3 Hz, H-26ax), 2.31
1H, dd, J ) 12.7, 4.7 Hz, H-23eq), 1.98 (1H, dd, J ) 12.7,
2.2 Hz, H-23ax), 1.82 (1H, m, H-25), 1.75 (3H, d, J ) 6.1 Hz,
Me-6′′), 1.44 (3H, s, Me-19), 1.12 (3H, d, J ) 7.0 Hz, Me-21),
.08 (3H, d, J ) 6.5 Hz, Me-27), 0.86 (3H, s, Me-18); HRESIMS
R
(
1
rhamnose, negative polarity), 17.55 (D-glucose, positive polar-
ity).
2
4
Com p ou n d 9: amorphous solid; [R]D -20.0° (c 0.10,
MeOH); IR (film) νmax 3357 (OH), 2926 and 2870 (CH), 1074
1
+
m/z 747.3935 [M + Na] (calcd for C38
H
60
O
13Na, 747.3932).
-1 1
5 5
cm ; H NMR (C D N) δ 5.06 (1H, d, J ) 7.6 Hz, H-1′), 4.75
Acid Hyd r olysis of 4. A solution of 4 (5.0 mg) was
subjected to acid hydrolysis as described for 1 to give a mixture
of several decomposed sapogenols and a sugar fraction (1.5
mg). HPLC analysis of the sugar fraction under the same
conditions as for 1 showed the presence of L-arabinose and
L-rhamnose.
(1H, d, J ) 7.8 Hz, H-1′′), 4.51 (1H, m, H-16), 4.30 (1H, br d,
J ) 8.9 Hz, H-22), 3.98 (1H, dd, J ) 11.5, 4.2 Hz, H-1), 3.93
(1H, m, W1/2 ) 23.9 Hz, H-3), 1.13 (3H, d, J ) 7.0 Hz, Me-21),
1.02 (3H, s, Me-19), 0.96 (3H, s, Me-18), 0.93 (3H, d, J ) 6.2
Hz, Me-26), 0.92 (3H, d, J ) 6.2 Hz, Me-27); HRESIMS m/z
+
783.4490 [M + Na] (calcd for C39
68
H O14Na, 783.4507).
Acetyla tion of 4. Compound 4 (8.5 mg) was treated with
Acid Hyd r olysis of 9. A solution of 9 (5.0 mg) was
subjected to acid hydrolysis as described for 1 to give an
aglycone fraction and a sugar fraction (1.1 mg). The aglycone
Ac
methylamino)pyridine (8.0 mg) as catalyst at room tempera-
ture for 12 h. After addition of H O (3 mL) into the reaction
mixture followed by evaporation to dryness, it was chromato-
graphed on silica gel eluted with hexane-Me CO (5:2) to give
the corresponding heptaacetate (4a , 8.3 mg).
Com p ou n d 4a : amorphous solid; IR (film) νmax 2952 (CH),
2
O (2 mL) and pyridine (1 mL) in the presence of 4-(di-
fraction was chromatographed on silica gel eluted with CHCl -
3
2
MeOH (19:1) to give 8a (1.8 mg). HPLC analysis of the sugar
fraction under the same conditions as for 1 showed the
presence of D-glucose.
2
2
2
Com p ou n d 10: amorphous solid; [R]D +2.0° (c 0.10,
MeOH); IR (film) νmax 3386 (OH), 2935 and 2876 (CH), 1716
739 (CdO), 1038 m-1; H NMR (C
1
1
5
1
5 5
D N) δ 5.63 (1H, br d, J )
-
1
(
CdO), 1590, 1504 and 1459 (aromatic ring), 1076, 1045 cm ;
.6 Hz, H-6), 5.60 (1H, br s, H-1′′), 5.20 (1H, ddd, J ) 10.9,
0.9, 4.8 Hz, H-24), 4.87 (1H, m, W1/2 ) 19.8 Hz, H-3), 4.63
1
UV λmax (MeOH) 266 nm (log ꢀ 4.01); H NMR (C
(
5
5
D
5
N) δ 7.71
2H, s, H-2′′′ and H-6′′′), 5.15 (1H, dd, J ) 11.4, 7.3 Hz, H-6′a),
(
1H, d, J ) 7.6 Hz, H-1′), 4.51 (1H, t-like, J ) 7.3 Hz, H-16),
.08 (1H, d, J ) 11.4, 2.1 Hz, H-6′b), 4.97 (1H, d, J ) 7.8 Hz,
3
5
2
(
)
.68 (1H, dd, J ) 12.0, 4.0 Hz, H-1), 3.63 (1H, dd, J ) 11.3,
.0 Hz, H-26eq), 3.55 (1H, dd, J ) 11.3, 11.3 Hz, H-26ax), 2.20,
.18, 2.15, 2.09, 2.08, 2.03, and 2.02 (each 3H, s, Ac × 7), 1.48
H-1′), 4.75 (1H, d, J ) 7.8 Hz, H-1′′), 4.52 (1H, m, H-16), 4.28
1H, m, H-22), 3.97 (3H, s, OMe), 3.89 (1H, m, H-3), 3.85 (3H
2, s, OMe), 3.82 (1H, dd, J ) 11.6, 4.0 Hz, H-1), 1.07 (3H, d,
(
×
3H, d, J ) 6.2 Hz, Me-6′′), 1.29 (3H, s, Me-19), 1.13 (3H, d, J
J ) 7.0 Hz, Me-21), 1.00 (3H, s, Me-19), 0.97 (3H, s, Me-18),
6.9 Hz, Me-21), 0.89 (3H, s, Me-18), 0.80 (3H, d, J ) 6.5 Hz,
0
.93 (3H × 2, d, J ) 6.2 Hz, Me-26 and Me-27); HRESIMS
Me-27).
+
m/z 977.5065 [M + Na] (calcd for C49
78
H O18Na, 977.5086).
2
D
6
Com p ou n d 5: amorphous solid; [R] -48.0° (c 0.10,
Alk a lin e Hyd r olysis of 10. Compound 10 (20.0 mg) was
treated with 4% KOH in EtOH (7 mL) at room temperature
for 1 h. The reaction mixture was neutralized by passage
through an Amberlite IR-120B (Organo) column and chro-
MeOH); IR (film) νmax 3386 (OH), 2950, 2928 and 2905 (CH),
-
1 1
1
5 5
047 cm ; H NMR (C D N) δ 6.34 (1H, br s, H-1′′), 5.58 (1H,
br d, J ) 5.7 Hz, H-6), 4.98 (1H, d, J ) 7.5 Hz, H-1′′′), 4.74
1H, d, J ) 7.3 Hz, H-1′), 4.54 (1H, q-like, J ) 7.4 Hz, H-16),
.01 (1H, ddd, J ) 12.1, 10.3, 4.7 Hz, H-24), 3.88 (1H, m, W1/2
20.0 Hz, H-3), 3.83 (1H, dd, J ) 12.0, 3.9 Hz, H-1), 3.71
1H, dd, J ) 11.3, 3.3 Hz, H-26eq), 3.59 (1H, dd, J ) 11.3,
1.3 Hz, H-26ax), 2.30 (1H, dd, J ) 12.7, 4.7 Hz, H-23eq), 1.97
(
4
)
(
1
(
(
)
matographed on silica gel eluted with CHCl
0:1) to give 9 (11.5 mg) and 3,4,5-trimethoxybenzoic acid (1.1
mg).
3
2
-MeOH-H O (40:
1
2
4
Com p ou n d 11: amorphous solid; [R]D +10.0° (c 0.10,
MeOH); IR (film) νmax 3377 (OH), 2959, 2927 and 2857 (CH),
1H, dd, J ) 12.7, 12.1 Hz, H-23ax), 1.82 (1H, m, H-25), 1.74
1
715 (CdO), 1591, 1505 and 1457 (aromatic ring), 1077, 1044
3H, d, J ) 6.2 Hz, Me-6′′), 1.42 (3H, s, Me-19), 1.12 (3H, d, J
-
1
1
5 5
cm ; UV λmax (MeOH) 266 nm (log ꢀ 3.99); H NMR (C D N)
6.9 Hz, Me-21), 1.08 (3H, d, J ) 6.5 Hz, Me-27), 0.85 (3H, s,
+
δ 7.58 (2H, s, H-2′′′ and H-6′′′), 5.86 (1H, br t, J ) 8.5 Hz,
H-24), 5.15 (1H, dd, J ) 11.4, 6.9 Hz, H-6′a), 5.08 (1H, d, J )
Me-18); HRESIMS m/z 879.4302 [M + Na] (calcd for C43
68 17
H O -
Na, 879.4354).
1
1.4, 2.1 Hz, H-6′b), 4.97 (1H, d, J ) 7.7 Hz, H-1′), 4.75 (1H,
Acid Hyd r olysis of 5. A solution of 5 (6.0 mg) was
subjected to acid hydrolysis as described for 1 to a give a
mixture of several decomposed sapogenols and a sugar fraction
1.5 mg). HPLC analysis of the sugar fraction under the same
conditions as for 1 showed the presence of L-arabinose,
d, J ) 7.8 Hz, H-1′′), 4.54 (1H, m, H-16), 4.13 (1H, m, H-22),
3
3
.97 (3H, s, OMe), 3.89 (1H, m, H-3), 3.84 (3H × 2, s, OMe),
.83 (1H, dd, J ) 11.5, 3.9 Hz, H-1), 1.74 (3H, s, Me-26), 1.66
(
(
3H, s, Me-27), 1.10 (3H, d, J ) 7.0 Hz, Me-21), 0.99 (3H, s,
Me-19), 0.95 (3H, s, Me-18); HRESIMS m/z 975.4915 [M +
Na] (calcd for C49
L-rhamnose, and D-xylose.
+
76
H O18Na, 975.4929).
2
D
6
Com p ou n d 8: amorphous solid; [R] -18.0° (c 0.10,
Ca ta lytic Hyd r ogen a tion of 11. A mixture of 11 (5.5 mg)
MeOH); IR (film) νmax 3376 (OH), 2929 and 2874 (CH), 1731
and PtO (6.0 mg) was stirred in an H2 atmosphere at room
2
-
1
1
(
CdO), 1036 cm ; H NMR (C
5
D
5
N) δ 5.92 (1H, br s, H-1′),
temperature for 12 h. The reaction mixture, after removal of
the catalyst by filtration, was subjected to a silica gel column
5
4
9
.78 (1H, d, J ) 3.6 Hz, H-1′′′), 4.76 (1H, d, J ) 3.6 Hz, H-2′′′),
.75 (1H, dd, J ) 9.6, 2.1 Hz, H-6′′a), 4.73 (1H, dd, J ) 9.6,
.2 Hz, H-6′′b), 4.73 (1H, d, J ) 9.2 Hz, H-5′′′a), 4.60 (1H, d,
J ) 7.8 Hz, H-1′′), 4.50 (1H, m, H-16), 4.31 (1H, d, J ) 9.2 Hz,
H-5′′′b), 4.25 (1H, br d, J ) 8.6 Hz, H-22), 4.15 (1H, d, J )
1.3 Hz, H-4′′′a), 4.12 (1H, d, J ) 11.3 Hz, H-4′′′b), 3.88 (1H,
m, H-3), 3.72 (1H, dd, J ) 11.4, 4.2 Hz, H-1), 2.05 (3H, s, Ac),
eluted with CHCl
3
-MeOH-H
2
O (40:10:1) to give 9 (1.8 mg).
24
Com p ou n d 12: amorphous solid; [R]D -50.0° (c 0.10,
MeOH); IR (film) νmax 3376 (OH), 2933 and 2878 (CH), 1071,
-1
1
1
048 cm ; H NMR (C
5 5
D N) δ 5.66 (1H, br s, H-1′), 5.63 (1H,
1
br d, J ) 5.6 Hz, H-6), 5.27 (1H, br s, H-1′′), 4.39 (1H, m, H-16),
4
.01 (1H, br d, J ) 9.2 Hz, H-22), 3.89 (1H, m, W1/2 ) 21.5 Hz,
1
2
6
.68 (3H, d, J ) 6.0 Hz, Me-6′), 1.14 (3H, d, J ) 7.0 Hz, Me-
1), 0.98 (3H, s, Me-19), 0.97 (3H, s, Me-18), 0.95 (3H, d, J )
H-3), 3.82 (1H, dd, J ) 11.5, 3.6 Hz, H-1), 1.70 (3H, d, J ) 4.8
Hz, Me-6′′), 1.67 (3H, d, J ) 6.0 Hz, Me-6′), 1.28 (3H, s, Me-
.2 Hz, Me-26), 0.93 (3H, d, J ) 6.2 Hz, Me-27); HRESIMS
1
9), 1.17 (3H, d, J ) 6.9 Hz, Me-21), 1.00 (3H, s, Me-18), 0.86
+
m/z 941.5048 [M + Na] (calcd for C46
78
H O18Na, 941.5086).
(
3H × 2, d, J ) 6.5 Hz, Me-26 and Me-27); HRESIMS m/z
+
Acid Hyd r olysis of 8. A solution of 8 (6.0 mg) in 0.2 M
O, 1:1, 2 mL) was heated at 95 °C for 1 h
727.4636 [M + H] (calcd for C39
H
67
O
12, 727.4633).
HCl (dioxiane-H
2
Acid Hyd r olysis of 12. A solution of 12 (5.0 mg) was
subjected to acid hydrolysis as described for 1 to give an
aglycone fraction and a sugar fraction (1.1 mg). The aglycone
under an Ar atmosphere, and the hydrolysate was treated as
described for 1 to give an aglycone fraction and a sugar fraction
(
1.9 mg). The aglycone fraction was chromatographed on silica
fraction was chromatographed on silica gel eluted with CHCl -
3
MeOH (19:1) to give 12a (1.8 mg). HPLC analysis of the sugar
fraction under the same conditions as for 1 showed the
presence of L-rhamnose.
gel eluted with CHCl
analysis of the sugar fraction under the same conditions as
for 1 showed the presence of D-apiose, D-glucose, and L-
3
-MeOH (19:1) to give 8a (1.2 mg). HPLC

1
696 J ournal of Natural Products, 2004, Vol. 67, No. 10
Kuroda et al.
2
D
6
2
Com p ou n d 13: amorphous solid; [R] -36.0° (c 0.10,
and the plate was further incubated in 5% CO /air for 4 h at
37 °C. The plate was then centrifuged at 1500g for 5 min to
precipitate cells and formazan. An aliquot of 150 µL of the
supernatant was removed from every well, and 175 µL of
DMSO was added to dissolve the MTT formazan crystals. The
plate was mixed on a microshaker for 10 min and then read
on a microplate reader (Spectra Classic, Tecan, Salzburg,
Austria) at 550 nm. Each assay was done in triplicate, and
cytotoxicity was expressed as IC50 value, which reduced the
viable cell number by 50%.
MeOH); IR (film) νmax 3376 (OH), 2968, 2926 and 2856 (CH),
-
1 1
1
052 cm ; H NMR (C
br t, J ) 7.1 Hz, H-24), 5.25 (1H, br s, H-1′′), 4.38 (1H, m,
H-16), 4.11 (1H, br t, J ) 6.8 Hz, H-22), 3.89 (1H, m, W1/2
2.3 Hz, H-3), 3.73 (1H, dd, J ) 11.4, 4.1 Hz, H-1), 1.70 (3H,
5 5
D N) δ 5.61 (1H, br s, H-1′), 5.55 (1H,
)
2
s, Me-26), 1.69 (3H, d, J ) 5.8 Hz, Me-6′), 1.67 (3H, s, Me-27),
1
2
7
.66 (3H, d, J ) 6.0 Hz, Me-6′′), 1.20 (3H, d, J ) 6.9 Hz, Me-
1), 1.03 (3H, s, Me-19), 0.95 (3H, s, Me-18); HRESIMS m/z
49.4474 [M + Na]+ (calcd for C39
H
O
12Na, 749.4452).
Acid Hyd r olysis of 13. A solution of 13 (8.0 mg) was
subjected to acid hydrolysis as described for 1 to a give a
mixture of several decomposed sapogenols and a sugar fraction
66
HSC-2 Cell Cu ltu r e Assa y. HSC-2 cells were maintained
as monolayer cultures at 37 °C in DMEM supplemented with
2
10% heat-inactivated FBS in a humidified 5% CO atmosphere.
3
(
2.5 mg). HPLC analysis of the sugar fraction under the same
Cells were trypsinized, inoculated at 6 × 10 per each 96-
microwell plate (Falcon, flat bottom, treated polystyrene,
Becton Dickinson, San J ose, CA), and incubated for 24 h. After
washing once with PBS, they were treated for 24 h without or
with test compounds. They were washed once with PBS and
incubated for 4 h with 0.2 mg/mL MTT in DMEM supple-
mented with 10% FBS. After the medium was removed, the
cells were lysed with 0.1 mL of DMSO and the relative viable
cell number was determined by measuring the absorbance at
conditions as for 1 showed the presence of L-rhamnose.
3
D
0
Com p ou n d 14: amorphous solid; [R] -30.0° (c 0.10,
MeOH); IR (film) νmax 3322 (OH), 2922, 2881 and 2854 (CH),
-1 1
1
(
5
5 5
066, 1043 cm ; H NMR (C D N) δ 5.65 (1H, br s, H-1′), 5.62
1H, br d, J ) 6.2 Hz, H-6), 5.55 (1H, br t, J ) 7.1 Hz, H-24),
.25 (1H, d, J ) 0.7 Hz, H-1′′), 4.38 (1H, m, H-16), 4.11 (1H,
br t, J ) 6.7 Hz, H-22), 3.89 (1H, m, W1/2 ) 23.5 Hz, H-3), 3.81
(
1H, dd, J ) 11.5, 3.6 Hz, H-1), 1.71 (3H, s, Me-26), 1.69 (3H,
5
(
40 nm of the cell lysate, using Labsystems Multiskan
Biochromatic, Helsinki, Finland) connected to a Star dot
d, J ) 6.3 Hz, Me-6′), 1.68 (3H, s, Me-27), 1.66 (3H, d, J ) 6.1
Hz, Me-6′′), 1.30 (3H, s, Me-19), 1.19 (3H, d, J ) 6.9 Hz, Me-
9
,10
+
matrix printer J L-10.
2
(
1), 0.98 (3H, s, Me-18); HRESIMS m/z 747.4266 [M + Na]
calcd for C39
Ca ta lytic Hyd r ogen a tion of 14. A mixture of 14 (4.8 mg)
and PtO (5.0 mg) was stirred in an H atmosphere at room
64
H O12Na, 747.4295).
Ack n ow led gm en t. We are grateful to Dr. Y. Shida, Tokyo
University of Pharmacy and Life Science, for the measure-
ments of the mass spectra.
2
2
temperature for 12 h. The reaction mixture, after removal of
the catalyst by filtration, was subjected to an ODS silica gel
column eluted with MeCN-H O (1:1) to give 12 (1.8 mg).
2
HL-60 Cell Cu ltu r e Assa y. HL-60 cells, which were
obtained from Human Science Research Resources Bank
J CRB 0085, Osaka, J apan), were maintained in the RPMI
640 medium containing heat-inactivated 10% FBS supple-
Refer en ces a n d Notes
(
1) Kuroda, M.; Mimaki, Y.; Yokosuka, A.; Hasegawa, F.; Sashida, Y. J .
Nat. Prod. 2002, 65, 1417-1423.
(2) Ono, M.; Shoyama, K.; Nohara, T. Shoyakugaku Zasshi 1988, 42,
135-142.
(
1
(
3) Kubo, S.; Mimaki, Y.; Sashida, Y.; Nikaido, T.; Ohmoto, T. Bull. Chem.
Soc. J pn. 1992, 65, 1120-1124.
mented with L-glutamine, 100 units/mL penicillin, and 100 µg/
mL streptomycin. The leukemia cells were washed and resus-
(
4) Inoue, T.; Mimaki, Y.; Sashida, Y.; Nikaido, T.; Ohmoto, T. Phy-
tochemistry 1995, 39, 1103-1110.
4
(5) Mimaki, Y.; Kuroda, M.; Kameyama, A.; Yokosuka, A.; Sashida, Y.;
pended in the above medium to 4 × 10 cells/mL, and 196 µL
Phytochemistry 1998, 48, 485-493.
of this cell suspension was placed in each well of a 96-well
flat-bottom plate (Iwaki Glass, Chiba, J apan). The cells were
(
6) Kitagawa, I.; Sakagami, M.; Hashiuchi, F.; Zhou, J . L.; Yoshikawa,
M.; Ren, J . Chem. Pharm. Bull. 1989, 37, 551-553.
(7) Takaashi, Y.; Mimaki, Y.; Kameyama, A.; Kuroda, M.; Sashida, Y.;
incubated in 5% CO
µL of EtOH-H O (1:1) solution containing the sample was
added to give the final concentrations of 0.1-20 µg/mL; 4 µL
O (1:1) was added into control wells. The cells
2
air for 24 h at 37 °C. After incubation, 4
Nikaido, T.; Koike, K.; Ohmoto, T. Chem. Pharm. Bull. 1995, 43,
2
1
180-1185.
(
8) Sargent, J . M.; Taylor, C. G. Br. J . Cancer 1989, 60, 206-210.
of EtOH-H
2
(9) Furuya, S.; Takayama, F.; Mimaki, Y.; Sashida, Y.; Satoh, K.;
Sakagami, H. Anticancer Res. 2000, 20, 4189-4194.
were further incubated for 72 h in the presence of each agent,
and then cell growth was evaluated by an MTT assay
procedure. At the end of incubation, 10 µL of 5 mg/mL MTT
in phosphate-buffered saline (PBS) was added to every well
(
10) Furuya, S.; Takayama, F.; Mimaki, Y.; Sashida, Y.; Satoh, K.;
Sakagami, H. Anticancer Res. 2001, 21, 959-964.
8
NP040108I