Steroidal saponins from the whole plants of Agave utahensis and their cytotoxic activity

Article abstract of DOI:10.1016/j.phytochem.2009.02.013

Phytochemical investigation of the whole plants of Agave utahensis Engelm. (Agavaceae) has resulted in the isolation of 15 steroidal saponins (1-15), including five spirostanol saponins (1-5) and three furostanol saponins (11-13). Structures of compounds

Full text of DOI:10.1016/j.phytochem.2009.02.013

Phytochemistry 70 (2009) 807–815
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Steroidal saponins from the whole plants of Agave utahensis and
their cytotoxic activity
*
*
Akihito Yokosuka , Yoshihiro Mimaki
Laboratory of Medicinal Pharmacognosy, Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, 1432-1, Horinouchi, Hachiouji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation of the whole plants of Agave utahensis Engelm. (Agavaceae) has resulted in
the isolation of 15 steroidal saponins (1–15), including five spirostanol saponins (1–5) and three furost-
anol saponins (11–13). Structures of compounds 1–5 and 11–13 were determined by spectroscopic anal-
ysis and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic
activity against HL-60 human promyelocytic leukemia cells.
Received 5 November 2008
Received in revised form 4 February 2009
Available online 22 April 2009
Keywords:
Agave utahensis Engelm.
Agavaceae
Ó 2009 Elsevier Ltd. All rights reserved.
Spirostanol saponins
Furostanol saponins
Cytotoxic activity
HL-60 cells
1. Introduction
fractionated by the combined use of repeated column chromatog-
raphy on a porous–polymer polystyrene resin (Diaion HP-20), silica
Agave utahensis Engelm. (Agavaceae) is native to South and
Central America (Tsukamoto, 1989). The leaves of this plant are
roasted and eaten, and the fiber is twisted for use us ropes and other
applications (Irish and Irish, 2000). Previously, we reported the
structural characterization of four new steroidal glycosides on the
gel, and octadecylsilanized (ODS) silica gel to yield 1–15.
Compounds 6–10, 14, and 15 were identified as (25R)-5 -spiro-
stan-3b-yl O-b- -glucopyranosyl-(1?2)-O-[b- -glucopyranosyl-
(1?3)]-O-b- -glucopyranosyl-(1?4)-b- -galactopyranoside (6)
(Wu et al., 1992; Kuroda et al., 1995), (25R)-2 -hydroxy-5
spirostan-3b-yl O-b- -glucopyranosyl-(1?2)-O-[b- -glucopyrano-
syl-(1?3)]-O-b- -glucopyranosyl-(1?4)-b- -galactopyranoside (7)
(Achenbach et al., 1994), (25R)-3b-[(O-b- -glucopyranosyl-(1?2)-
O-b- -glucopyranosyl-(1?4)-b- -galactopyranosyl)oxy]-5 -spiro-
stan-12-one (8) (Xu et al., 2000), (25R)-3b-[(O-b- -glucopyranosyl-
(1?2)-O-b- -glucopyranosyl-(1?4)-b- -galactopyranosyl)oxy]-
-spirostan-12-one (9) (Mimaki et al., 1998), (25R)-
-spirostan-3b-yl O-b- -glucopyranosyl-(1?2)-O-
-galactopyranoside (10) (Yahara
et al., 1994), (25R)-26-[(b- -glucopyranosyl)oxy]-2 -hydroxy-
22 -methoxy-5 -furostan-3b-yl O-b- -glucopyranosyl-(1?2)-O-
b- -glucopyranosyl-(1?4)-b- -galactopyranoside (14) (Yahara
et al., 1994), and (25R)-26-[(b- -glucopyranosyl)oxy]-2 -hydro-
xy-22 -methoxy-5 -furostan-3b-yl O-b- -glucopyranosyl-(1?2)-
O-[b- -glucopyranosyl-(1?3)]-O-b- -glucopyranosyl-(1?4)-b-
-galactopyranoside (15) (Yahara et al., 1994), based on their phys-
ical and spectroscopic data.
a
D
D
D
D
a
D
a-
basis of (25R)-5
a
-spirostane-3b,6a-diol (chlorogenin) as the agly-
D
cone from the whole plants of A. utahensis (Yokosuka and Mimaki,
2007). Our further phytochemical analysis of the polar fraction of
the MeOH extract of this plant, in which steroidal saponins are
enriched, has resulted in the isolation of five new spirostanol sapo-
nins (1–5) and three new furostanol saponins (11–13), together
with seven known steroidal saponins (6–10, 14, and 15). This paper
reports the structural determination of the new saponins on the
basis of extensive spectroscopic analysis, including two-dimen-
sional (2D) NMR spectroscopic data and the results of hydrolytic
cleavage. The cytotoxic activity of the isolated compounds against
HL-60 human promyelocytic leukemia cells is also described.
D
D
D
D
D
a
D
D
D
2
a
-hydroxy-5
-hydroxy-5
a
a
2a
D
b-D-glucopyranosyl-(1?4)-b-D
D
a
a
D
a
D
D
D
a
a
a
D
2. Results and discussion
D
D
D
2.1. Structure elucidation
Compound 1 was isolated as an amorphous solid, and its mole-
cular formula was deduced to be C44H72O18 on the basis of the
HRESI–TOFMS (m/z: 889.4731 [M + H]+), 13C NMR, and DEPT
spectroscopic data. The 1H NMR spectrum of 1 showed two
three-proton singlet signals at d 0.81 (3H, s) and 0.72 (1H, s), indi-
cating the presence of two angular methyl groups, as well as two
The fresh whole plants of A. utahensis (2.5 kg) were extracted
with MeOH. After removal of solvent, the crude extract was
* Corresponding authors. Tel.: +81 42 676 4577; fax: +81 42 676 4579.
E-mail addresses: yokosuka@ps.toyaku.ac.jp (A. Yokosuka), mimakiy@ps.toyaku.
ac.jp (Y. Mimaki).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.02.013

808
A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815

A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
809
three-proton doublet resonances at d 1.13 (3H, d, J = 6.9 Hz), 0.69
(3H, d, J = 5.8 Hz), which were characteristic of the steroidal skele-
ton. In addition, three anomeric protons were observed at d 5.18
(1H, d, J = 7.9 Hz), 5.08 (1H, d, J = 6.8 Hz), and 4.93 (1H, d,
J = 7.7 Hz), which implied the presence of three monosaccharides.
Comparison of the 1H and 13C NMR spectra of 1 with those of 10
showed considerable structural similarity and indicated that the
signals assignable to a terminal b-
D
-xylopyranosyl group were
newly observed at d 108.0, 76.5, 77.9, 70.4, and 67.4 in 1, and the
anomeric carbon resonance was associated with the d 5.08 (d,
J = 6.8 Hz) signal by the HMQC spectrum. Acid hydrolysis of 1 with
1 M HCl (dioxane–H2O, 1:1) gave (25R)-5
(1a, gitogenin) (Agrawal et al., 1985), -glucose,
-xylose. Identification of the monosaccharides, including their
a-spirostane-2a,3b-diol
D
D
-galactose, and
D
triglycoside moiety included a C-2 substituted b-
D
-glucopyranosyl
absolute configurations, was carried out by direct HPLC analysis
of the hydrolysate. In the HMBC spectrum of 1, the anomeric pro-
ton (H-1) of the xylosyl group at d 5.08 showed a long-range corre-
unit and a C-4 substituted b- -galactopyranosyl unit, as in 10. The
D
difference between 1 and 10 consisted only of resonances due to
the terminal monosaccharide constituent. Instead of the reso-
lation with C-2 of the b-
D-glucopyranosyl moiety at d 86.4, of
nances for the terminal b-D-glucopyranosyl group, five carbon
which H-1 at d 5.18 exhibited a long-range correlation with C-4

810
A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
Fig. 1. HMBC correlations of the sugar moiety of 1.
of the b-
D-galactopyranosyl moiety at d 80.4 (Fig. 1). An HMBC
The 1H–1H COSY experiment with 4 allowed the sequential assign-
correlation was also observed between H-1 of the b-
D
-galactopyr-
ments from H-1 to H2-6 of the monosaccharides, including identi-
fication of their multiplet patterns and coupling constants. The
HMQC correlated the proton resonances with those of one-bond
coupled carbons, leading to unambiguous assignments of the car-
bon shifts. The 1H and 13C NMR signals thus assigned were indica-
anosyl moiety at d 4.93 and C-3 of the aglycone moiety at d 84.8.
The b-orientations of the anomeric centers of the sugar
moieties were supported by the relatively large J values of their
anomeric protons (J = 6.8–7.9 Hz). The structure of 1 was formu-
lated as (25R)-2
a
-hydroxy-5
a
-spirostan-3b-yl O-b-
D
-xylopyrano-
tive of the presence of two terminal b-D
-glucopyranosyl units (Glc0)
syl-(1?2)-O-b- -glucopyranosyl-(1?4)-b-
D
D
-galactopyranoside. To
[dH 5.60 (d, J = 7.9 Hz); dC 104.8, 76.0, 78.2, 71.3, 78.6, 62.3], (Glc00)
[dH 5.31 (d, J = 7.9 Hz); dC 104.5, 75.3, 78.7, 71.6, 78.4, 62.6], a C-2
the best of our knowledge, the structure of the sugar moiety for
1 has not been reported previously.
and C-3 disubstituted b-
J = 7.9 Hz); dC 104.7, 81.3, 88.7, 70.7, 77.5, 63.0], and a C-4 substi-
tuted b- -galactopyranosyl unit (Gal) [dH 4.91 (d, J = 7.8 Hz); dC
103.1, 72.5, 75.5, 79.7, 75.7, 60.5] in 4. In the HMBC spectrum,
long-range correlations were observed between H-1 of Glc0 at d
5.60 and C-2 of Glc at d 81.3, H-1 of Glc00 at d 5.31 and C-3 of Glc
at d 88.7, H-1 of Glc at d 5.18 and C-4 of Gal at d 79.7, and between
H-1 of Gal at d 4.91 and C-3 of the aglycone at d 83.7. Accordingly, 4
D-glucopyranosyl unit (Glc) [dH 5.18 (d,
Compound
2 was shown to have the molecular formula
C44H70O19 on the basis of the positive-ion HRFABMS and 13C
NMR spectroscopic data. The 1H NMR spectrum of 2 showed
signals for four steroidal methyl groups at d 1.35 (d, J = 6.9 Hz),
1.07 (s), 0.75 (s), and 0.69 (d, J = 5.8 Hz) and three anomeric protons
at d 5.18 (d, J = 7.9 Hz), 5.08 (d, J = 6.3 Hz), and 4.92 (d, J = 7.7 Hz),
as in 1. The presence of a carbonyl group in 2 was established from
the IR (1707 cmꢀ1) and 13C NMR spectra (d 212.4). Acid hydrolysis
D
was formulated as (25R)-3b-[(O-b-
-glucopyranosyl-(1?3)]-O-b- -glucopyranosyl-(1?4)-b-
topyranosyl)oxy]-2 -hydroxy-5 -spirostan-12-one.
D
-glucopyranosyl-(1?2)-O-[b-
of
2
gave (25R)-2
a
,3b-dihydroxy-5
a
-spirostan-12-one (2a)
D
D
D
-galac-
(Mimaki et al., 1995),
D
-xylose, -galactose, and
D
D
-glucose. The
a
a
results of acid hydrolysis, and the 1H and 13C NMR spectroscopic
data provided evidence that the structure of the sugar moiety of
2 was the same as that of 1. Thus, 2 was determined to be
Compound 5 was deduced as C51H82O24 by the positive-ion
HRFABMS and 13C NMR data. Comparison of the 1H and 13C NMR
spectra of 5 with those of 4 showed that 5 was closely related that
of 4. However, the molecular formula of 5 was lower by one oxygen
atom than that of 4. When the 13C NMR spectrum of 5 was com-
pared with that of 4, the signals due to the C-2 oxymethine carbon,
which was observed at d 70.2 in 4, was displaced by a methylene
carbon resonance at d 29.6 in 5. Acid hydrolysis of 5 gave (25R)-
(25R)-2a-hydroxy-3b-[(O-b-
D
-xylopyranosyl-(1?2)-O-b-
D-gluco-
pyranosyl-(1?4)-b-
D-galactopyranosyl)oxy]-5
a-spirostan-12-one.
The spectroscopic features of 3 (C44H68O19) were quite similar
to those of 2, and the 1H and 13C NMR spectra allowed for the iden-
tification of the triglycoside moiety attached to C-3 of the aglycone
as being same as that of 2. However, the molecular formula of 3
was less than by two hydrogen atoms, and the IR (1673 cm-1),
3b-hydroxy-5a-spirostan-12-one (hecogenin) (Mimaki et al.,
1995), -galactose, and
D
D-glucose. Analysis of the HMBC spectrum
UV [kmax 236.4 nm (log
e
4.07)], and 13C NMR [d 204.2 (C@O),
of 5 indicated that the tetraglycoside attached to C-3 of the agly-
cone is the same as that of 4. Thus, 5 was formulated as (25R)-
170.4 (C), 120.1 (CH)] indicated the presence of a conjugated car-
bonyl group in 3. In the HMBC spectrum, long-range correlations
were observed between the olefinic proton at d 5.95 (s, H-11)
and C-8 (d 36.1)/C-10 (d 40.5)/C-13 (d 51.3), and between the car-
bonyl carbon at d 204.2 and H-17 (d 2.63)/Me-18 (d 1.01)/H-14 (d
1.72), which are consistent with the 9-en-12-one structure. Acid
3b-[(O-b-
O-b- -glucopyranosyl-(1?4)-b-
rostan-12-one.
D
-glucopyranosyl-(1?2)-O-[b-
D
-glucopyranosyl-(1?3)]-
-spi-
D
D
-galactopyranosyl)-oxy]-5a
Compound 11 was obtained as an amorphous solid with a
molecular formula of C51H86O24. The 1H NMR spectrum of 11
showed signals for four anomeric protons at d 5.17 (1H, d,
J = 7.9 Hz), 5.07 (1H, d, J = 7.2 Hz), 4.93 (1H, d, J = 7.7 Hz), and
4.85 (1H, d, J = 7.8 Hz), as well as signals for four steroid methyl
groups at d 1.18 (d, J = 6.8 Hz), 1.00 (d, J = 6.7 Hz), 0.78 (s), and
0.72 (s), and a methoxy group at d 3.26 (3H, s). From the 1H NMR
spectroscopic data, an acetic carbon signal at d 112.6 in the 13C
NMR spectrum, and a positive color reaction in Ehrlich’s test
(Kiyosawa and Hutoh, 1968; Nohara et al., 1975) indicated 11 to
be a 22-methoxyfurostanol saponin with four monosaccharides.
hydrolysis of 3 gave (25R)-2
one (3a) (Mimaki et al., 1995),
a
D
,3b-dihydroxy-5
-xylose, -galactose, and
-hydroxy-3b-[(O-b-
-glucopyranosyl-(1?4)-b- -galactopyr-
-spirost-9-en-12-one.
a
-spirost-9-en-12-
D
D-glucose.
Accordingly, 3 was formulated as (25R)-2
xylopyranosyl-(1?2)-O-b-
anosyl)oxy]-5
a
D-
D
D
a
Compound 4 was analyzed for C51H82O25 by combined positive-
ion HRFABMS and 13C NMR data. The 1H NMR spectrum of 4
showed signals for four anomeric protons at d 5.60 (d, J = 7.9 Hz),
5.31 (d, J = 7.9 Hz), 5.18 (d, J = 7.9 Hz), and 4.91 (d, J = 7.8 Hz), as
well as signals for four steroid methyl groups at d 1.36 (d,
J = 6.9 Hz), 1.07 (s), 0.74 (s), and 0.70 (d, J = 5.8 Hz). Analysis of
the 13C 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. Acid hydrolysis of 4 with 1 M
Enzymatic hydrolysis of 11 with b-D-glucosidase gave 1 and D-glu-
cose. In addition, a long-range correlation as observed between H-1
of Glc0000 at d 4.85 and C-26 of the aglycone at d 75.2. Thus, 11 was
demonstrated to be the corresponding 22-methoxyfurostanol
saponin of 1 with a glucosyl unit at C-26. A NOE correlation be-
tween the methoxy proton at d 3.26 and the H-16 proton signal
at d 4.45 was consistent with the C-22 d configuration. Thus, 11
HCl (dioxane–H2O, 1:1) resulted in the production of 2a,
D-galact-
ose, and -glucose. Thus, 4 was suggested to be a 2a tetraglycoside.
D

A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
811
Table 1
1H and 13C NMR chemical shift assignments for the sugar moieties of compounds 1–6 in C5D5Na.
1
2
3
4
1H
13C
1H
13C
1H
13C
1H
13C
Gal
1
2
3
4
5
6a
6b
4.93 d (7.7)
103.8
72.6
75.5
80.4
75.7
60.3
Gal
1
2
3
4
5
6a
6b
4.92 d (7.7)
103.8
72.6
75.5
80.4
75.7
60.3
Gal
1
2
3
4
5
6a
6b
4.93 d (7.7)
103.7
72.7
75.5
80.4
75.7
60.3
Gal
1
2
3
4
5
6a
6b
4.91 d (7.8)
103.1
72.5
75.5
79.7
75.7
60.5
4.48 dd (9.9, 7.7)
4.15 dd (9.9, 2.7)
4.57 br d (2.7)
4.08 m
4.75 dd (9.7, 9.7)
4.23 dd (9.7, 5.0)
4.48 dd (9.6, 7.7)
4.15 dd (9.6, 3.0)
4.57 br d (3.0)
4.07 m
4.74 dd (9.8, 9.8)
4.22 dd (9.8, 5.1)
4.47 dd (9.1, 7.7)
4.14 dd (9.1, 3.1)
4.57 br d (3.1)
4.07 m
4.74 dd (9.7, 9.7)
4.23 dd (9.7, 5.0)
4.54 dd (9.0, 7.8)
4.12 dd (9.0, 3.3)
4.28 br d (3.3)
4.04 m
4.63 dd (10.5, 9.0)
4.33 m
Glc
Xyl
1
2
3
4
5
6a
6b
5.18 d (7.9)
105.2
86.4
78.0
72.2
78.0
63.3
Glc
Xyl
1
2
3
4
5
6a
6b
5.18 d (7.9)
105.2
86.4
78.0
72.2
78.0
63.3
Glc
Xyl
1
2
3
4
5
6a
6b
5.18 d (7.8)
105.2
86.4
78.0
72.2
78.0
63.2
Glc
Glc
Glc
1
2
3
4
5
6a
6b
5.17 d (7.9)
4.35 dd (8.9, 7.9)
4.19 dd (8.9, 8.9)
3.82 dd (8.9, 8.9)
3.83 m
104.7
81.3
88.7
70.7
77.5
63.0
4.13 dd (8.5, 7.9)
4.27 dd (8.5, 8.5)
3.95 dd (8.5, 8.5)
4.00 m
4.63 br d (9.9)
4.10 m
4.14 dd (8.6, 7.9)
4.28 dd (8.6, 8.6)
3.95 dd (8.6, 8.6)
3.99 m
4.63 br d (10.6)
4.10 m
4.13 dd (8.4, 7.8)
4.27 dd (8.4, 8.4)
3.96 dd (8.4, 8.4)
3.99 m
4.63 br d (10.6)
4.11 m
4.48 m
4.02 m
1
2
3
4
5a
5b
5.08 d (6.8)
4.02 m
4.01 m
3.95 m
4.48 m
108.0
76.5
77.9
70.4
67.4
1
2
3
4
5a
5b
5.08 d (6.3)
4.02 m
4.01 m
3.95 m
4.48 m
108.0
76.5
77.9
70.2
67.4
1
2
3
4
5a
5b
5.07 d (6.1)
4.02 m
4.01 m
3.94 m
4.47 m
108.0
76.5
77.9
70.4
67.4
71.6
1
2
3
4
5
6a
6b
5.06 d (7.9)
104.8
76.0
78.2
71.3
78.6
62.3
4.05 dd (8.9, 7.9)
4.17 dd (8.9, 8.9)
4.12 dd (8.9, 8.9)
3.89 m
4.54 dd
4.22 dd (12.1, 5.1)
3.67 dd (11.0, 11.0)
3.67 dd (10.8, 10.8)
3.66 dd (11.0, 11.0)
1
2
3
4
5
6a
6b
5.31 d (7.9)
104.5
75.3
78.7
71.6
78.4
62.6
4.04 dd (8.8, 7.9)
4.20 dd (8.8, 8.8)
4.15 dd (8.8, 8.8)
4.04 m
4.54 m
4.27 dd (11.8, 6.1)
5
11
12
13
1H
13C
1H
13C
1H
13C
1H
13C
Gal
1
2
3
4
5
6a
6b
4.85 d (7.7)
102.4
73.2
75.6
80.2
75.3
60.6
Gal
1
2
3
4
5
6a
6b
4.93 d (7.7)
103.8
72.6
75.4
80.4
75.7
60.3
Gal
1
2
3
4
5
6a
6b
4.94 d (7.7)
103.6
72.6
75.4
80.3
75.7
60.3
Gal
1
2
3
4
5
6a
6b
4.91 d (7.7)
102.4
73.3
75.6
81.0
75.2
60.5
4.42 dd (9.5, 7.7)
4.10 dd (9.5, 3.1)
4.58 br d (3.1)
4.00 m
4.68 dd (10.1, 9.4)
4.20 m
4.48 dd (9.1, 7.7)
4.15 dd (9.1, 3.4)
4.57 br d (3.4)
4.09 m
4.75 dd (9.7, 9.7)
4.24 m
4.48 dd (9.4, 7.7)
4.15 dd (9.4, 3.5)
4.57 br d (3.5)
4.08 m
4.75 dd (9.6, 9.6)
4.24 m
4.50 dd (9.4, 7.7)
4.12 dd (9.4, 4.2)
4.59 br d (4.2)
4.11 m
4.78 dd (10.1, 9.8)
4.26 dd (9.9, 4.5)
Glc
Glc
Glc
a
1
2
3
4
5
6a
6b
5.14 d (7.9)
4.38 dd (8.9, 7.9)
4.20 dd (8.9, 8.9)
3.83 dd (8.9, 8.9)
3.84 m
105.0
81.4
88.4
70.8
77.5
63.0
Glc
Xyl
Glc
1
2
3
4
5
6a
6b
5.17 d (7.9)
105.2
86.4
77.9
72.2
78.0
63.2
Glc
Xyl
Glc
1
2
3
4
5
6a
6b
5.18 d (7.8)
105.1
86.4
77.9
72.2
78.0
63.2
Glc
Glc
Glc
1
2
3
4
5
6a
6b
5.15 d (7.8)
4.15 dd (8.8, 7.8)
4.27 m
3.98 m
3.97 m
105.2
86.1
78.4
71.8
78.2
63.2
4.13 dd (8.6, 7.9)
4.27 dd (8.6, 8.6)
3.95 dd (8.6, 8.6)
3.97 m
4.63 br d (9.6)
4.10 m
4.14 dd (8.8, 7.8)
4.28 dd (8.8, 8.8)
3.94 dd (8.8, 8.8)
3.97 m
4.64 br d (9.7)
4.10 dd (10.6, 7.6)
4.48 m
4.01 m
4.63 br d (10.3)
4.12 m
1
2
3
4
5
6a
6b
5.58 d (7.8)
104.9
76.1
77.9
70.9
78.6
62.3
1
2
3
4
5a
5b
5.07 d (7.2)
4.02 m
4.01 m
3.96 m
4.48 dd (11.1, 6.3)
3.67 dd (11.1, 11.1)
108.0
76.5
77.9
70.4
67.4
1
2
3
4
5a
5b
5.07 d (7.2)
4.02 m
4.01 m
3.94 m
4.46 m
108.0
76.5
77.9
70.4
67.4
1
2
3
4
5
6a
6b
5.22 d (7.5)
4.06 m
4.12 m
4.22 m
3.80 m
106.9
76.7
77.6
70.3
78.9
61.6
4.06 dd (9.2, 7.8)
4.14 dd (9.2, 9.2)
4.24 dd (8.9, 8.9)
3.85 m
4.52 br d (12.9)
4.37 m
3.65 dd (11.0, 11.0)
4.59 m
4.37 dd (12.4, 3.5)
1
2
3
4
5
6a
6b
5.30 d (7.9)
104.5
75.3
78.6
71.6
78.6
62.3
1
2
3
4
5
6a
6b
4.85 d (7.8)
105.0
75.2
78.6
71.8
78.5
62.9
1
2
3
4
5
6a
6b
4.85 d (7.8)
105.0
75.2
78.6
71.7
78.5
62.8
1
2
3
4
5
6a
6b
4.85 d (7.8)
4.04 dd (8.7, 7.8)
4.24 m
4.24 m
3.96 m
105.0
75.2
78.6
71.7
78.5
62.9
4.05 dd (8.7, 7.9)
4.20 dd (8.7, 8.7)
4.14 dd (8.7, 8.7)
4.03 m
4.55 dd (12.0, 2.2)
4.26 dd (12.0, 6.3)
4.04 dd (8.2, 7.8)
4.26 dd (8.2, 8.2)
4.23 dd (8.2, 8.2)
3.98 m
4.57 dd (11.7, 2.3)
4.40 dd (11.7, 5.4)
4.04 dd (8.6, 7.8)
4.24 dd (8.6, 8.6)
4.23 dd (8.6, 8.6)
3.98 m
4.58 dd (11.7, 2.4)
4.40 dd (11.7, 5.4)
4.57 dd (11.7, 2.4)
4.40 dd (11.7, 5.4)
Values in parentheses are coupling constants in Hz.
was assigned as (25R)-26-[(b-
22 -methoxy-5 -furostan-3b-yl O-b-
-glucopyranosyl-(1?4)-b- -galactopyranoside.
D
-glucopyranosyl)oxy]-2
a
-hydroxy-
shown by the IR (1671 cmꢀ1), UV [kmax 236.6 nm (log
e
3.93)],
a
a
D-xylopyranosyl-(1?2)-O-b-
and 13C NMR [d: 204.2 (C@O), 170.4 (C), 120.0 (CH)] spectra. Enzy-
D
D
matic hydrolysis of 12 with b-
Thus, 12 was established as (25R)-26-[(b-
-hydroxy-22 -methoxy-3-[(O-b- -xylopyranosyl-(1?2)-O-b-
-galactopyranosyl)oxy]-5 -furost-9-en-
D
-glucosidase gave 3 and
D-glucose.
Compound 12 (C51H82O25) was shown to be a 22-methoxyfu-
D
-glucopyranosyl)oxy]-
rostanol saponin by Ehrlich’s test, and 1H NMR [d 3.27 (3H, s)]
and 13C NMR [d 112.9 (C-22), 47.4 (Me)] spectra. The presence of
a conjugated carbonyl group in the aglycone moiety of 12 was
2
a
a
D
D-
glucopyranosyl-(1?4)-b-
D
a
12-one.

812
A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
Compound 13 (C52H88O24) was also a 22-methoxyfurostanol
saponin. Treatment of 13 with b- -glucosidase gave a known
spirostanol saponin identified as (25R)-5 -spirostan-3b-yl O-b-
glucopyranosyl-(1?2)-O-b- -glucopyranosyl-(1?4)-b-
pyranoside (13a) (Murakami et al., 1981) and
Accordingly, 13 was formulated as (25R)-26-[(b-
syl)oxy]-22 -methoxy-5 -furostan-3b-yl O-b- -glucopyranosyl-
(1?2)-O-b- -glucopyranosyl-(1?4)-b- -galactopyranoside.
PX-8010 controller (Tosoh), a RI-8010 (Tosoh) and a Shodex OR-2
D
(Showa-Denko, Tokyo, Japan) detector, and a Rheodyne injection
port. HL-60 cells were obtained from the Human Science Research
Resources Bank (JCRB 0085, Osaka, Japan). The following reagents
were obtained from the indicated companies: RPMI 1640 medium
a
D-
D
D
-galacto-
-glucose.
-glucopyrano-
D
D
and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
a
D
a
D
bromide (MTT) (Sigma, St. Louis, MO, USA); fetal bovine serum
(FBS) (Bio-Whittaker, Walkersville, MO, USA); penicillin G sodium
salt and streptomycin sulfate (Gibco, Grand Island, NY, USA). All
other chemicals used were of biochemical reagent grade.
D
2.2. Cytotoxic activity
The isolated compounds were evaluated for their cytotoxic
activity against HL-60 cells (Table 3). Compounds 1, 6, 7, and 10
showed moderate cytotoxicity, relative to the control, etoposide,
3.2. Plant material
A. utahensis was purchased from a garden center in Japan Cactus
Planning Co. (Fukushima, Japan) in 2000 and identified by Dr. Yu-
taka Sashida, emeritus professor of Medicinal Pharmacognosy at
Tokyo University of Pharmacy and Life Sciences, on the basis of
its morphological properties. A voucher specimen has been depos-
ited in our laboratory (voucher No. AU-2000-001, Laboratory of
Medicinal Pharmacognosy).
with respective IC50 values of 12.3, 9.4, 5.5, and 11.3
contrast, compounds 2, 4, 5, 8, and 9 did not exhibit apparent
cytotoxicity even at a sample concentration of 20 g/ml. The above
lg/ml. In
l
results indicated that the C-12 carbonyl group significantly reduces
the cytotoxic activity in these spirostanol saponins. The furostanol
saponins (11–15) were not cytotoxic to HL-60 cells at the sample
concentration of 20 lg/ml.
3.3. Extraction and isolation
2.3. Conclusions
The plant material (2.5 kg) was extracted for 3 h with hot MeOH
twice (each 6 l). The combined MeOH extract (12 l) was concen-
trated under reduced pressure, and the viscous concentrate
(190 g) was passed through a Diaion HP-20 column, successively
eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc (each
4 l). The 50% MeOH eluate portion was subjected to silica gel CC
eluted with CHCl3–MeOH–H2O (40:10:1) to give four fractions
(Ia–IVa). Fraction IIIa was applied to an ODS silica gel eluted with
MeOH–H2O (1:1; 7:5) and then silica gel CC with CHCl3–MeOH–
H2O (50:20:1) to yield 12 (27 mg), 13 (58 mg), and 14 (38 mg).
The MeOH eluate portion was subjected silica gel CC1 eluted with
a stepwise gradient mixture of CHCl3–MeOH (9:1; 4:1; 3:1; 2:1;
1:1), and finally with MeOH alone, to give five fractions (Ib–Vb).
A
D
number of steroidal saponins with the triglycoside of
-glucosyl-(1?2)-[b- -glucosyl-(1?3)]-b- -glucosyl-(1?4)-b-
-galactoside and b- -glucosyl-(1?2)-b- -glucosyl-(1?4)-b-D-
b-
D
D
D
D
D
galactoside have been isolated from the Liliaceae, Agavaceae, and
Solanaceae plants such as Allium chinense (Kuroda et al., 1995),
Allium amperoprasum (Mimaki et al., 1999), Chlorophytum malay-
ence (Li et al., 1990), Chlorophytum comosum (Mimaki et al.,
1996), Hosta longips (Mimaki et al., 1995), and Hosta sieboldii
(Mimaki et al., 1998) (Liliaceae), Agave sisalana (Ding et al.,
1993), Agave uthahensis (Yokosuka and Mimaki, 2007), and
Polianthes tuberosa (Jin et al., 2004) (Agavaceae), and Solanum
nigrum (Ikeda et al., 2000), Solanum dulcamara (Lee et al., 1994),
and Capsicum annuum (Yahara et al., 1994) (Solanaceae).
Compounds 1–3, 11, and 12 are rare type of steroidal saponins
with the sugar moiety of b-
D-xylosyl-(1?2)-b-D-glucosyl-(1?4)-
Table 2
13C NMR chemical shift assignments for the aglycone moiety of compounds 1–5 and
11–13 in C5D5N.
b- -galactoside at C-3 of the aglycone. Steroidal saponin with the
D
same sugar moiety as 1–3, 11, and 12 has been isolated only from
Solanum lyratum (Solanaceae) (Lee et al., 1997).
Position
1
2
3
4
5
11
45.6
12
43.5
13
37.2
1
2
3
4
5
6
7
8
9
45.7
70.4
84.8
34.1
44.7
28.1
32.2
34.6
54.3
36.9
21.4
40.0
40.7
56.3
32.1
81.1
63.0
16.6
13.4
42.0
15.0
45.1
70.4
84.5
34.0
44.5
27.8
31.4
33.7
55.7
37.3
38.0
43.5
70.3
84.0
33.7
42.5
27.1
32.5
36.1
170.4
40.5
120.1
204.2
51.3
52.6
31.8
80.2
54.5
15.2
19.3
42.9
13.8
109.4
31.8
29.2
30.5
67.0
17.3
45.0
70.2
83.7
33.9
44.3
27.8
31.4
33.7
55.7
37.2
38.0
36.6
29.6
77.1
34.6
44.4
28.5
31.4
34.3
55.5
36.2
37.9
70.4
84.8
34.1
44.7
28.1
32.2
34.5
54.3
36.8
21.3
39.8
41.1
56.2
32.1
81.3
64.3
16.5
13.4
40.5
16.3
112.6
30.8
28.2
34.2
75.2
17.1
47.2
70.2
83.9
33.7
42.5
27.1
32.4
36.1
170.4
40.5
120.0
204.2
51.7
52.5
31.6
80.3
55.6
15.2
19.3
41.2
14.7
112.9
30.5
28.1
34.1
75.2
17.1
47.4
30.8
77.4
34.8
44.7
28.9
32.4
35.2
54.4
35.8
21.2
40.0
41.1
56.3
32.1
81.3
64.3
16.5
12.3
40.5
16.3
112.6
30.0
28.2
34.2
75.2
17.1
47.2
3. Experimental
3.1. General
Optical rotations were measured using a JASCO DIP-360 (Tokyo,
Japan) automatic digital polarimeter. IR spectra were recorded on a
JASCO FT–IR 620 spectrophotometer. UV spectra were recorded on
a JASCO V-520 spectrophotometer. NMR spectra were recorded on
a Bruker DRX-500 (500 MHz for 1H NMR, Karlsruhe, Germany)
spectrophotometer using standard Bruker pulse programs. Chemi-
cal shifts are given as d values with reference to tetramethylsilane
(TMS) as an internal standard. FABMS was recorded on a Finnigan
MAT TSQ-700 (San Jose, CA, USA). Elemental analysis was carried
out using an Elementar Vario EL elemental analyzer (Hanau, Ger-
many). Diaion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), silica
gel (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai
Tesque, Kyoto, Japan) were used for column chromatography (CC).
TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm thick,
Merck, Darmstadt, Germany) and RP18 F254 S plates (0.25 mm thick,
Merck), and spots were visualized by spraying the plates with 10%
H2SO4 solution, followed by heating. HPLC was performed by using
a system composed of a CCPM pump (Tosoh, Tokyo, Japan), a CCP
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
212.4
55.3
55.3
31.8
79.7
54.3
16.1
12.8
42.6
13.9
109.3
31.5
29.2
30.5
66.9
17.3
212.5
55.3
55.7
31.8
79.7
54.3
16.1
12.8
42.6
13.9
109.3
31.5
29.2
30.5
66.9
17.3
212.8
55.3
55.9
31.7
79.7
54.3
16.0
11.7
42.6
13.9
109.3
31.8
29.2
30.5
66.9
17.3
109.2
31.8
29.2
30.6
66.9
17.3

A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
813
Table 3
9.9, 5.4 Hz, H-3), 1.35 (3H, d, J = 6.9 Hz, Me-21), 1.07 (3H, s, Me-
18), 0.75 (3H, s, Me-19), 0.69 (3H, d, J = 5.8 Hz, Me-27); For 1H
NMR spectroscopic data of the sugar moiety, see Table 1; for
13C NMR (125 MHz, C5D5N) spectroscopic data, see Tables 1 and
2; HRFABMS (positive mode) m/z: 925.4409 [M + Na]+ (calculated
for C44H70O19Na, 925.4409).
Cytotoxic activity of the isolated compounds 1–15 on HL-60 leukemia cells.
Compound
IC50
(
l
g/ml)a
Compound
IC50 (lg/ml)
1
2
3
4
5
6
7
8
12.3
9
10
11
12
13
14
15
–
11.3
–
–
–
b
–
–
–
–
9.4
5.5
–
3.7. Acid hydrolysis of 2
–
–
A solution of 2 (9.8 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give 2a (4.5 mg) and a sugar fraction (2.8 mg). HPLC
analysis of the sugar fraction under the same conditions as in the
Etoposide
0.20
a
Data represent mean of three independent experiments.
Means IC50 > 20 lg/ml.
b
case of 1 showed the presence of
glucose. tR (min): 10.46 ( -xylose, positive optical rotation), 14.66
-galactose, positive optical rotation), 15.86 ( -glucose, positive
D-xylose, D-galactose, and D-
D
(D
D
Fraction IIb was applied to a silica gel column eluted with CHCl3–
MeOH–H2O (60:10:1; 50:10:1) and then to ODS silica gel with
MeOH–H2O (8:3; 4:1) and CH3CN–H2O (5:7) to give 1 (28 mg), 2
(15 mg), 3 (15 mg), 8 (26 mg), 9 (16 mg), and 10 (9.0 mg). Fraction
IVb was subjected to silica gel CC eluted with CHCl3–MeOH–H2O
(20:10:1; 7:4:1) and then to ODS silica gel CC with MeOH–H2O
(2:1; 4:1) and CH3CN–H2O (1:2; 1:3) to yield 4 (15 mg), 5
(7.0 mg), 6 (10 mg), 7 (32 mg), 11 (49 mg), and 15 (15 mg).
optical rotation).
3.8. Compound 3
(25R)-2a-Hydroxy-3b-[(O-b-
D
-xylopyranosyl-(1?2)-O-b-
-spirost-9-en-
ꢁ ꢀ30.0 (c 0.10; MeOH); IR mmax
D-glu-
copyranosyl-(1?4)-b-D-galactopyranosyl)oxy]-5a
12-one (3); amorphous solid; ½a D23
(film) cmꢀ1: 3375 (OH), 2928 (CH), 1673 (C@O), 1074, 1048; UV
kmax: 236.4 nm (MeOH, loge
4.07); 1H NMR (500 MHz, C5D5N): d
3.4. Compound 1
5.95 (1H, s, H-11), 4.53 (1H, m, H-16), 4.05 (1H, m, H-2), 3.86
(1H, ddd, J = 10.5, 9.3, 5.3 Hz, H-3), 2.63 (1H, dd, J = 8.1, 7.7 Hz, H-
17), 1.72 (1H, m, H-14), 1.40 (3H, d, J = 6.8 Hz, Me-21), 1.01 (3H,
s, Me-18), 0.91 (3H, s, Me-19), 0.70 (3H, d, J = 5.2 Hz, Me-27); For
1H NMR spectroscopic data of the sugar moiety, see Table 1; for
13C NMR (125 MHz, C5D5N) spectroscopic data, see Tables 1 and
2; HRESI–TOFMS m/z: 901.4417 [M + H]+ (calculated for
C44H68O19, 901.4433).
(25R)-2b-Hydroxy-5
a-spirostan-3b-yl
O-b-
D
-xylopyranosyl-
(1?2)-O-b-D-glucopyranosyl-(1?4)-b-D-galactopyranoside
(1);
amorphous solid; ½a D23
ꢀ46.0 (c 0.10; MeOH); IR mmax (film)
ꢁ
cmꢀ1: 3357 (OH), 2928 (CH), 1050; 1H NMR (500 MHz, C5D5N): d
4.55 (1H, m, H-16), 3.98 (1H, m, H-2), 3.88 (1H, ddd, J = 10.8, 9.1,
4.9 Hz, H-3), 1.13 (3H, d, J = 6.9 Hz, Me-21), 0.81 (3H, s, Me-18),
0.72 (3H, s, Me-19), 0.69 (3H, d, J = 5.8 Hz, Me-27); For 1H NMR
spectroscopic data of the sugar moiety, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI–
3.9. Acid hydrolysis of 3
TOFMS m/z: 889.4731 [M + H]+ (calculated for C44H73O18
889.4797).
,
A solution of 3 (10 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give 3a (4.7 mg) and a sugar fraction (2.3 mg). HPLC
analysis of the sugar fraction under the same conditions as in the
3.5. Acid Hydrolysis of 1
case of 1 showed the presence of
glucose. tR (min): 10.43 ( -xylose, positive optical rotation), 14.63
-galactose, positive optical rotation), 15.86 ( -glucose, positive
D-xylose, D-galactose, and D-
D
A solution of 1 (9.8 mg) in 1 M HCl (dioxane–H2O, 1:1; 2 ml)
was heated at 95 °C for 2 h under Ar atmosphere. After cooling,
the reaction mixture was neutralized by passage through an
Amberlite IRA-93ZU (Organo, Tokyo, Japan) column and then sub-
jected to silica gel CC1 eluted with CHCl3–MeOH (9:1) to yield 1a
(3.7 mg) and a sugar fraction (4.5 mg). The sugar fraction was
passed through a Sep-Pak C18 cartridge (Waters, Milford, MA,
USA) and a Toyopak IC-SP M cartridge (Tosoh), which was then
analyzed by HPLC under the following conditions: column, Capcell
Pak NH2 UG80 (4.6 mm i.d. x 250 mm, 5
pan); solvent, MeCN–H2O (17:3); flow rate, 0.9 ml/min; detection,
RI and OR. Identification of -xylose, -galactose, and -glucose
present in the sugar fraction was carried out by comparison of their
retention times and optical rotations with those of authentic
(D
D
optical rotation).
3.10. Compound 4
(25R)-3b-[(O-b-
D
-Glucopyranosyl-(1?2)-O-[b-
D-glucopyrano-
syl-(1?3)]-O-b-
D
-glucopyranosyl-(1?4)-b- -galactopyranosyl)-
D
oxy]-2 -hydroxy-5a-spirostan-12-one (4); amorphous solid;
a
½
a 2D5
ꢁ
ꢀ20.0 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3364 (OH),
2926 (CH), 1706 (C@O), 1074; 1H NMR (500 MHz, C5D5N): d
4.48 (1H, m, H-16), 3.92 (1H, ddd, J = 11.4, 9.0, 4.6 Hz, H-2), 3.86
(1H, m, H-3), 1.36 (3H, d, J = 6.9 Hz, Me-21), 1.07 (3H, s, Me-18),
0.74 (3H, s, Me-19), 0.70 (3H, d, J = 5.8 Hz, Me-27); For 1H NMR
spectroscopic data of the sugar moiety, see Table 1; for 13C NMR
lm, Shiseido, Tokyo, Ja-
D
D
D
samples. tR (min): 9.70 (
D
-xylose, positive optical rotation), 13.70
(125 MHz, C5D5N) spectroscopic data, see Tables
1 and 2;
HRFABMS (positive mode) m/z: 1117.5019 [M + Na]+ (calculated
(D
-galactose, positive optical rotation), 15.11 (D
-glucose, positive
optical rotation).
3.6. Compound 2
(25R)-3b-[(O-b-
for C51H82O25Na, 1117.5043).
3.11. Acid hydrolysis of 4
D
-Glucopyranosyl-(1?2)-O-[b-
D-glucopyrano-
A solution of 4 (5.0 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give 2a (2.5 mg) and a sugar fraction (1.5 mg). HPLC
analysis of the sugar fraction under the same conditions as in the
syl-(1?3)]-O-b-
D
-glucopyranosyl-(1?4)-b- -galactopyranosyl)-
D
oxy]-2 -hydroxy-5a-spirostan-12-one (2); amorphous solid;
a
½
a 2D4
ꢁ
ꢀ8.0 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3375 (OH), 2927
case of 1 showed the presence of
(min): 17.43 ( -galactose, positive optical rotation), 19.27 (
cose, positive optical rotation).
D
-glucose and
D
-galactose. tR
(CH), 1707 (C@O), 1072, 1041; 1H NMR (500 MHz, C5D5N): d
4.49 (1H, m, H-16), 3.94 (1H, m, H-2), 3.85 (1H, ddd, J = 11.1,
D
D-glu-

814
A. Yokosuka, Y. Mimaki / Phytochemistry 70 (2009) 807–815
3.12. Compound 5
(25R)-3b-[(O-b-
3.17. Enzymatic hydrolysis of 12
Compound 12 (11 mg) was subjected to enzymatic hydrolysis
D-Glucopyranosyl-(1?2)-O-[b-D-glucopyranosyl-
(1?3)]-O-b-
D
-glucopyranosyl-(1?4)-b-
D
-galactopyranosyl)oxy]-5
ꢁ
a
-
as described for 11 to yield 5 (8.0 mg) and
D-glucose (1.0 mg).
spirostan-12-one (5); amorphous solid; ½a D25
ꢀ22.0 (c 0.10; MeOH);
IR mmax (film) cmꢀ1: 3376 (OH), 2927 (CH), 1707 (C@O), 1071; 1H
NMR (500 MHz, C5D5N): d 4.48 (1H, m, H-16), 3.87 (1H, m, H-3),
1.35 (3H, d, J = 6.9 Hz, Me-21), 1.08 (3H, s, Me-18), 0.69 (3H, d,
J = 5.8 Hz, Me-27), 0.65 (3H, s, Me-19); For 1H NMR spectroscopic
data of the sugar moiety, see Table 1; for 13C NMR (125 MHz,
C5D5N) spectroscopic data, see Tables 1 and 2; HRFABMS (positive
mode) m/z: 1101.5096 [M + Na]+ (calculated for C51H82O24Na,
1101.5094).
3.18. Compound 13
(25R)-26-[(b-
D
-Glucopyranosyl)oxy]-22
a
-methoxy-5a-furostan-
3b-yl O-b-D-glucopyranosyl-(1?2)-O-b-D-glucopyranosyl-(1?4)-b-
D
-galactopyranoside (13); amorphous solid; ½a D24
ꢀ36.0 (c 0.10;
ꢁ
MeOH); IR mmax (film) cmꢀ1: 3376 (OH), 2929 (CH), 1070; 1H NMR
(500 MHz, C5D5N): d 4.46 (1H, m, H-16), 3.95 (1H, m, H-3), 3.27
(3H, s, OMe), 1.19 (3H, d, J = 6.8 Hz, Me-21), 1.00 (3H, d, J = 6.7 Hz,
Me-27), 0.80 (3H, s, Me-18), 0.65 (3H, s, Me-19); For 1H NMR spec-
troscopic data of the sugar moiety, see Table 1; for 13C NMR
(125 MHz, C5D5N) spectroscopic data, see Tables 1 and 2; HRESI–
3.13. Acid hydrolysis of 5
TOFMS m/z: 1065.5441 [M ꢀ OMe]+ (calculated for C51H85O23
,
A solution of 5 (5.0 mg) was subjected to acid hydrolysis as de-
scribed for 1 to give hecogenin (1.2 mg) and a sugar fraction
(2.5 mg). HPLC analysis of the sugar fraction under the same con-
1065.5482); FABMS (positive mode) m/z: 1119 [M + Na]+ (Found:
C, 54.78; H, 8.26. C52H88O24ꢂ5/2H2O requires, C, 54.68; H, 8.21%).
ditions as in the case of 1 showed the presence of
and -glucose. tR (min): 17.39 ( -galactose, positive optical rota-
tion), 19.21 ( -glucose, positive optical rotation).
D-galactose
3.19. Enzymatic hydrolysis of 13
D
D
D
Compound 13 (8.6 mg) was subjected to enzymatic hydrolysis
as described for 11 to yield 13a (3.0 mg) and D-glucose (1.0 mg).
3.14. Compound 11
3.20. HL-60 cell culture assay
(25R)-26-[(b-
-furostan-3b-yl O-b-
syl-(1?4)-b- -galactopyranoside (11); amorphous solid;
D
-Glucopyranosyl)oxy]-2
a
-hydroxy-22
a-methoxy-
5a
D-xylopyranosyl-(1?2)-O-b-
D-glucopyrano-
The cell growth was measured with an MTT reduction assay as
described in a previous paper (Yokosuka et al., 2002). Briefly, HL-60
cells were maintained in RPMI 1640 medium containing heat-inac-
D
½ ꢁ
a 2D8
ꢀ12.0 (c 0.10; MeOH); IR mmax (film) cmꢀ1: 3375 (OH), 2928 (CH),
1070; 1H NMR (500 MHz, C5D5N): d 4.45 (1H, m, H-16), 3.97 (1H,
m, H-2), 3.88 (1H, m, H-3), 3.26 (3H, s, OMe), 1.18 (3H, d,
J = 6.8 Hz, Me-21), 1.00 (3H, d, J = 6.7 Hz, Me-27), 0.78 (3H, s, Me-
18), 0.72 (3H, s, Me-19); For 1H NMR spectroscopic data of the sugar
moiety, see Table 1; for 13C NMR (125 MHz, C5D5N) spectroscopic
data, see Tables 1 and 2; HRESI–TOFMS m/z: 1051.5308 [M ꢀ OMe]+
(calculated for C50H83O23, 1051.5325); FABMS (positive mode) m/z:
1105 [M + Na]+ (Found: C, 53.67; H, 8.05. C51H86O24 ꢂ 3H2O requires,
C, 53.86; H, 8.15%).
tivated 10% (v/v) FBS supplemented with
penicillin G sodium salt, and 100
L
-glutamine, 100 unit/ml
l
g/ml streptomycin sulfate. The
cells (4 ꢃ 104 cells/ml) were continuously treated with each com-
pounds for 72 h, and cell growth was measured with an MTT
reduction assay procedure. A dose-response curve was plotted
for 1, 6, 7, and 10, and the concentration giving 50% inhibition
(IC50) was calculated.
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