Steroidal glycosides from the bulbs of Bessera elegans and their cytotoxic activities

Article abstract of DOI:10.1016/j.phytochem.2013.09.023

Examination of the bulbs of Bessera elegans (Liliaceae) led to isolation of nine new and five known steroidal glycosides. The structures of the nine compounds were determined based on the results of spectroscopic analysis, including two-dimensional NMR, and hydrolysis followed by chromatographic and spectroscopic analysis. The isolated compounds and derivatives were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 normal human diploid fibroblasts. One compound, the pseudo-furostanol glycoside, induced apoptosis in HL-60 and A549 cells in a time-dependent manner and cell-cycle arrest at the G0/G1 phase in A549 cells.

Full text of DOI:10.1016/j.phytochem.2013.09.023

Phytochemistry 96 (2013) 244–256
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Steroidal glycosides from the bulbs of Bessera elegans and their cytotoxic
activities
⇑
Yukiko Matsuo , Nana Akagi, Chisato Hashimoto, Fumito Tachikawa, Yoshihiro Mimaki
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:
Received 20 May 2013
Received in revised form 6 September 2013
Available online 19 October 2013
Examination of the bulbs of Bessera elegans (Liliaceae) led to isolation of nine new and five known steroi-
dal glycosides. The structures of the nine compounds were determined based on the results of spectro-
scopic analysis, including two-dimensional NMR, and hydrolysis followed by chromatographic and
spectroscopic analysis. The isolated compounds and derivatives were evaluated for cytotoxicity against
HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 normal
human diploid fibroblasts. One compound, the pseudo-furostanol glycoside, induced apoptosis in HL-60
and A549 cells in a time-dependent manner and cell-cycle arrest at the G0/G1 phase in A549 cells.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Bessera elegans
Liliaceae
Steroidal glycoside
A549 cells
HL-60 cells
Cytotoxicity
Apoptosis
1. Introduction
human promyelocytic leukemia cells, A549 human lung adenocar-
cinoma cells, and TIG-3 normal human diploid fibroblasts.
Through our continuing search for bioactive natural products, a
number of natural products have been discovered with antitumor
activity in plants of the family Liliaceae, such as Ornithogalum saun-
dersiae and Galtonia candicans (Kuroda et al., 2000), which are not
used in traditional herbal medicine. For example, the acylated cho-
lestane glycoside, OSW-1, which was isolated from the bulbs of O.
saundersiae, is a promising lead for developing anticancer agents
(Kubo et al., 1992; Mimaki et al., 1997). This current phytochemical
study focused on Bessera elegans Schult. f., which belongs to the
family Liliaceae and is indigenous to Mexico. It produces slender
stems of 50 cm or taller, and is a popular ornamental garden plant
(Tsukamoto, 1989). However, no systematic chemical analysis has
been reported for B. elegans. Preliminary TLC analysis of the MeOH
extract of B. elegans bulbs, however, suggested that it contained
numerous steroidal glycosides. Herein is reported a detailed
chemical study of the bulbs of B. elegans, and the isolation of nine
new (1–9) and five known (10–14) steroidal glycosides. The
structures of the new steroidal glycosides were determined based
on the results of spectroscopic analysis, including two-dimensional
NMR spectroscopic data, and hydrolysis followed by chromato-
graphic and spectroscopic analysis. The isolated compounds and
their aglycones were evaluated for cytotoxicity against HL-60
2. Results and discussion
2.1. Structural elucidation
The bulbs of B. elegans (2.8 kg fr. wt) were extracted with hot MeOH.
The MeOH extract was passed through a porous-polymer polystyrene
resin (Diaion HP-20) column, and the MeOH-eluted fraction was
subjected to silica gel and octadecylsilanized (ODS) silica gel column
chromatography (CC), and to reversed-phase preparative HPLC, giving
compounds 1–14 (Fig. 1). Compounds 10–14 were identified as (25R)-
5
a
-spirostan-3b-yl O-
nosyl-(1 ? 3)]-O-b-
anosyl-(1 ? 2)]-b- -galactopyranoside (10) (Ikeda et al., 2000),
(25R)-2 -hydroxy-5 -spirostan-3b-yl O-b- -galactopyranosyl-
(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-O-b- -glucopyranosyl-
(1 ? 4)-b- -galactopyranoside (11) (Perrone et al., 2005), (25R)-26-
[(b- -glucopyranosyl)oxy]-5 -furostan-2 ,3b,22 -triol (12) (Barile
et al., 2005), (25R)-26-[(b- -glucopyranosyl)oxy]-22 -hydroxy-5
furostan-3b-ylO-b- -galactopyranosyl-(1 ? 2)-O-[b- -xylopyrano-
syl-(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-b- -galactopyranoside
(13) (Perrone et al., 2005), and (25R)-26-[(b- -glucopyranosyl)oxy]-
,22 -dihydroxy-5 -furostan-3b-yl O-b- -galactopyranosyl-
(1 ? 2)-O-[b- -xylo-pyranosyl-(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-
b- -galactopyranoside (14) (Perrone et al., 2005), respectively, by
a
-
L
-arabino-pyranosyl-(1 ? 2)-O-[b-
D-xylopyra-
D
-glucopyranosyl-(1 ? 4)-O-[ -rhamnopyr-
a-L
D
a
a
D
D
D
D
D
a
a
a
D
a
D
a-
D
D
D
D
2a
a
a
D
⇑
Corresponding author. Tel.: +81 42 676 4577; fax: +81 42 676 4579.
E-mail addresses: matsuoy@toyaku.ac.jp (Y. Matsuo), mimakiy@toyaku.ac.jp
D
D
(Y. Mimaki).
D
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.09.023

Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
245
Fig. 1. Steroidal glycosides isolated from Bessera elegans.
comparing their physical and spectroscopic data with literature
values.
Compound 1 was obtained as an amorphous solid, and its
aglycone at dC 84.3. Thus, 1 was assigned as (25R)-2
spirostan-3b-yl O-b- -galactopyranosyl-(1 ? 2)-O-[b-
-glucopyranosyl-(1 ? 4)-b-
a
-hydroxy-5
a-
D
D
-glucopyr-
anosyl-(1 ? 3)]-O-b-
D
D-
molecular formula was assigned as C51H84O24 based on data from
high-resolution electrospray ionization time-of-flight mass spec-
trometry (HR-ESI-TOF-MS; m/z 1103.5248 [M+Na]+, calcd.
1103.5250) and 13C NMR (51 carbon signals). The IR spectrum of
1 suggested the presence of hydroxy groups (3362 cmꢀ1). The 13C
NMR spectrum showed an acetal carbon signal at dC 109.1, and res-
onances for four methyl groups at dC 17.2, 16.5, 14.9, and 13.3 (Ta-
ble 1), which were characteristic of the spirostan skeleton. Its 1H
NMR spectrum showed two singlet signals for tertiary methyl
groups at dH 0.79 and 0.68, two doublet resonances for secondary
methyl groups at dH 1.12 (J = 6.8 Hz) and 0.69 (J = 7.0 Hz), and four
anomeric proton doublet signals at dH 5.50 (J = 7.8 Hz), 5.19
(J = 7.8 Hz), 5.14 (J = 8.0 Hz), and 4.94 (J = 7.8 Hz) (Tables 2 and
3). These NMR spectroscopic data suggested that 1 was a spirosta-
nol tetraglycoside. Enzymatic hydrolysis of 1 with naringinase gave
galactopyranoside.
Compound 2 (C51H84O25) showed spectroscopic features similar
to those of 1, although the molecular formula of 2 contained one
extra oxygen atom. Significant differences were also observed be-
tween the two compounds in the 1H and 13C signals for ring D
(C-13 to C-17): the C-14 carbon at dC 56.2 (CH) in 1 was replaced
by a downfield-shifted quaternary carbon at dC 87.0 (C) in 2, and
the H-14 proton resonance, which was observed at dH 0.99 (m)
in 1, disappeared. These spectroscopic data suggested that 2 was
the C-14 hydroxy derivative of 1. This was confirmed by long-range
correlations between C-14 (dC 87.0) and H-8 (dH 1.88, ddd, J = 11.5,
11.5, 3.6 Hz), H-15a (dH 2.34, dd, J = 12.6, 7.5 Hz), dH H-15b (1.84,
dd, J = 12.6, 6.0 Hz), and Me-18 (dH 1.03, s) in the HMBC spectrum
of 2. The significant downfield shifts of the H-9, H-12ax, H-16, and
H-17 protons, and the upfield shifts of the C-9, C-12, and C-17 car-
1a as the aglycone (C27H44O4), and
carbohydrate moieties. Aglycone 1a was identified as (25R)-5
spirostane-2 ,3b-diol (gitogenin) from its physical and spectro-
scopic data (Gvazava and Kikoladze, 2006). The monosaccharides
and their absolute configurations were determined by direct HPLC
analysis of the hydrolysate. The 1H–1H COSY analysis of 1 allowed
sequential assignment of the signals from H-1 to H2-6 of the mono-
saccharides. The signal multiplet patterns and coupling constants
D
-galactose and
D
-glucose as the
bons could be explained by the 1,3-diaxial interactions and
fects with the C-14 hydroxy group. Enzymatic hydrolysis of 2
with naringinase gave the aglycone 2a (C27H44O5), together with
-galactose and -glucose. The physical and spectroscopic data
were consistent with the structure of 2a (25R)-5 -spirostane-
,3b,14
-triol. The 1H and 13C NMR spectroscopic data and the
c-ef-
a
-
a
a
D
D
a
2
a
a
HMBC correlations of 2 confirmed that the tetraglycoside linked
to C-3 of the aglycone was the same as that of 1. Accordingly, 2
(Table 2) indicated the presence of two b-
D
-glucopyranosyl units
was assigned as (25R)-2
D
a
,14
-galactopyranosyl-(1 ? 2)-O-[b-
-glucopyranosyl-(1 ? 4)-b- -galactopyranoside.
The spectroscopic data for compound 3 (C50H82O24) showed it
a
-dihydroxy-5
a-spirostan-3b-yl O-b-
(Glc (I) and Glc (II)) and two b- -galactopyranosyl units (Gal (I)
D
D
-glucopyranosyl-(1 ? 3)]-O-b-
and Gal (II)). The proton signals were correlated with the carbon
resonances through one-bond coupling in the HMQC spectrum.
In the HMBC spectrum of 1, long-range correlations were observed
between the anomeric proton (H-1000) of Gal (II) at dH 5.50 and C-200
of Glc (I) at dC 80.9, between H-10000 of Glc (II) at dH 5.19 and C-300 of
Glc (I) at dC 87.4, between H-100 of Glc (I) at dH 5.14 and C-40 of Gal
(I) at dC 79.6, and between H-10 of Gal (I) at dH 4.94 and C-3 of the
D
D
was a spirostanol tetraglycoside closely related to 2, except for
one terminal monosaccharide constituent. Enzymatic hydrolysis
of 3 afforded 2a as the aglycone, and D-xylose, D-galactose, and D-
glucose were detected by HPLC analysis of the sugar fraction of
the hydrolysate. When the 1H and 13C NMR spectra of 3 were

246
Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
Table 1
13C NMR spectroscopic (125 MHz) data for the aglycone moiety of 1–9 in pyridine-d5.
Position
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
45.5
70.4
84.3
34.1
44.6
28.0
32.1
34.5
54.3
36.8
21.3
40.0
40.7
56.2
32.0
81.0
62.9
16.5
13.3
41.9
14.9
45.8
70.4
84.6
34.2
44.6
28.2
27.4
38.5
46.9
37.1
20.8
32.3
45.2
87.0
39.8
81.9
59.7
20.3
13.3
42.0
15.3
45.7
70.3
84.3
33.9
44.4
28.0
27.2
38.4
46.8
36.9
20.7
32.2
45.1
87.0
39.6
81.8
59.6
20.2
13.1
41.9
15.2
45.9
70.5
84.8
34.2
44.7
28.2
27.4
38.5
47.0
37.1
20.9
32.3
45.2
87.0
39.9
82.0
59.8
20.3
13.3
42.1
15.3
36.6
29.6
76.7
34.1
44.3
28.5
31.7
34.4
55.3
36.3
37.9
212.8
55.5
55.8
31.4
79.6
54.2
16.0
11.8
42.5
13.8
109.2
31.6
29.1
30.4
66.9
17.2
45.6
70.4
84.4
34.3
44.8
28.1
32.2
34.5
54.4
36.9
21.3
40.1
41.1
56.3
32.1
81.1
63.9
16.4
13.4
40.7
16.7
45.9
70.4
84.7
34.2
44.7
28.2
27.4
38.5
47.0
37.1
20.9
32.1
45.5
87.0
40.0
81.8
60.3
20.4
13.3
40.7
16.7
45.6
70.4
84.5
34.2
44.7
28.1
32.4
34.3
54.3
36.9
21.6
39.8
43.7
54.6
34.4
84.5
64.6
14.4
13.4
103.6
11.8
152.3
23.7
31.5
33.5
74.9
17.3
37.3
30.0
77.0
34.4
44.7
29.0
32.6
35.0
54.4
35.9
21.4
39.9
43.7
54.8
34.4
84.5
64.6
14.4
12.4
103.6
11.8
152.3
23.7
31.5
33.5
74.9
17.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
109.1
31.7
29.2
30.5
66.8
17.2
109.5
31.9
29.3
30.6
66.8
17.3
109.5
31.8
29.2
30.4
66.7
17.2
110.0
31.7
24.1
39.2
64.0
64.4
110.6
37.2
28.4
34.3
75.2
17.4
110.9
37.2
28.4
34.3
75.3
17.5
compared with those of 2, signals belonging to a terminal b-
D
-xylo-
indicated that the carbonyl group was located at C-12 of the aglycone.
pyranosyl unit were observed instead of the resonances for the
terminal b-
-glucopyranosyl group. Furthermore, the 1H and 13C
NMR assignments of the tetraglycoside moiety of 3 were in good
agreement with those of 11. Thus, 3 was assigned as (25R)-2
14 -dihydroxy-5 -spirostan-3b-yl O-b- -galactopyranosyl-(1 ? 2)-
O-[b- -xylopyranosyl-(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-b-
galactopyranoside.
Enzymatic hydrolysis of 5 with naringinase furnished (25R)-3b-hy-
D
droxy-5
with -arabinose,
The 1H and 13C NMR spectra and the 1H-1H COSY and HMQC spectra,
showed that the pentaglycoside moietyof5wascomposedof -ara-
binopyranosyl (Ara), b- -galactopyranosyl (Gal), b- -glucopyranosyl
(Glc), -rhamnopyranosyl (Rha), and b- -xylopyranosyl units. In
a-spirostan-12-one(5a, hecogenin)(Caietal., 2001),together
L
D
-galactose, -glucose, -rhamnose, and -xylose.
D
L
D
a
,
a
a
D
a-L
D
D
D
-
D
D
a-L
D
The 1H and 13C NMR spectra of 4 (C50H82O25) and 3 suggested
that they possessed similar structures. However, the molecular for-
mula of 4 had one extra oxygen atom compared with that of 3, and
differences were observed in the NMR signals for ring F of the agly-
cone. These data implied that one more hydroxy group was present
in ring F. When the 1H NMR spectrum of 4 was compared with that
of 3, the tetraglycoside signals were consistent with those of 3,
whereas the Me-27 proton signal observed at dH 0.65 (d,
J = 4.7 Hz) in 3 was replaced with oxymethylene proton resonances
at dH 3.72 (dd, J = 10.7, 5.2 Hz) and 3.65 (dd, J = 10.7, 3.4 Hz) in 4.
An NOE correlation between H-23ax (dH 1.84) and H-25 (dH
2.06), and the proton spin-coupling constant of 10.2 Hz (H-25/H-
26ax), were indicative of a 25S configuration. Acid hydrolysis of 4
the HMBC spectrum, long-range correlations were observed between
H-10000 of Ara at dH 4.96 and C-2000 of Glc at dC 81.5, between H-100000 of Xyl
at dH 5.21 and C-3000 ofGlc at dC 87.8, between H-100of Rha at dH 6.29 and
C-20 of Gal at dC 77.3, between H-1000 of Glc at dH 5.35 and C-40 of Gal at
dC 81.4, andbetweenH-10 ofGalatdH 4.84andC-3oftheaglyconeatdC
76.7, respectively. Therefore, 5 was assigned as (25R)-3b-[(O-
binopyranosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-O-b-
glucopyranosyl-(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b-
-galactop- yranosyl)oxy]-5 -spirostan-12-one, respectively.
The acetal carbon signals at dC 110.6 in 6 and dC 110.9 in 7, and
a-L-ara-
D
D-
a
-L
D
a
the positive color reactions in the Ehrlich test suggested that 6
(C57H96O30) and 7 (C56H94O30) were 22-hydroxyfurostanol glyco-
sides. The 1H NMR spectrum of 6 showed singlets for two tertiary
methyl groups at dH 0.86 and 0.68, doublets for two secondary
methyl groups at dH 1.32 (J = 6.8 Hz) and 0.99 (J = 6.6 Hz), and
doublets for five anomeric protons at dH 5.51 (J = 7.8 Hz), 5.20
(J = 7.8 Hz), 5.15 (J = 8.0 Hz), 4.88 (J = 7.5 Hz), and 4.82
afforded aglycone 4a (C27H44O6),
lose. The physical and spectroscopic data allowed aglycone 4a to
be identified as (25S)-5 -spirostane-2 ,3b,14 ,27-tetrol. Thus, 4
was assigned as (25S)-2 ,14 ,27-trihydroxy-5 -spirostan-3b-yl
O-b- -galactopyranosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-O-
b- -glucopyranosyl-(1 ? 4)-b- -galactopyranoside.
The1Hand13CNMRspectraof5(C55H88O26)suggesteditwasaspi-
D-galactose, D-glucose, and D-xy-
a
a
a
a
a
a
D
D
(J = 7.7 Hz). Enzymatic hydrolysis of 6 with b-
the corresponding spirostanol saponin 1 and
enzymatic hydrolysis of 6 with naringinase gave the spirostanol
sapogenin 1a (gitogenin) (Gvazava and Kikoladze, 2006), -galact-
ose, and -glucose. A b- -glucopyranosyl group linkage to the C-26
hydroxy group of the aglycone was confirmed by an HMBC
correlation between the anomeric proton (H-100000) of one b-
-gluco-
D-glucosidase yielded
D
D
D-glucose, whereas
rostanol pentaglycoside, based on the signals for four methyl groups
at dH 1.35 (d, J = 6.9 Hz), 1.07 (s), 0.88 (s), and 0.69 (d, J = 5.7 Hz),
and five anomeric protons at dH 6.29 (br s), 5.35 (d, J = 7.6 Hz), 5.21
(d, J = 7.8 Hz), 4.96(d, J = 7.8 Hz), and 4.84(d, J = 7.7 Hz). The presence
of a carbonyl group was indicated by the 13C NMR signals at dC 212.8
and an IR absorption band at 1690 cmꢀ1. The HMBC correlations be-
tween C-12 (dC 212.8) and H-11ax (dH 2.41, dd, J = 13.8, 13.7 Hz), H-
11 eq (dH 2.25, dd, J = 13.8, 5.0 Hz), H-14 (dH 1.38, ddd, J = 13.2, 7.1,
5.5 Hz), H-17 (dH 2.76, dd, J = 6.8, 6.6 Hz), and Me-18 (dH 1.07, s)
D
D
D
D
pyranosyl unit at dH 4.82 (d, J = 7.7 Hz) and C-26 of the aglycone at
dC 75.2. The NOE correlations between the signals of the H-20
proton at dH 2.23 and the H2-23 protons at dH 2.06 (2H) confirmed
the C-22
a
configuration. Accordingly, 6 was assigned as (25R)-26-
,22 - dihydroxy-5 -furostan-3b-yl
[(b- -glucopyranosyl) oxy]-2
D
a
a
a

Table 2
1H NMR spectroscopic (500 MHz) data for the aglycone moiety of 1–9 in pyridine-d5.
1
2
3
4
5
Position
dH
J (Hz)
Position
dH
J (Hz)
Position
dH
J (Hz)
Position
dH
J (Hz)
Position
dH
J (Hz)
1
ax
eq
1.17 dd
12.7, 10.4
1
ax
eq
1.20 dd
12.1, 12.1
1
ax
eq
1.18 dd
12.0, 12.0
1
ax
eq
2.20 overlapping
1
2
ax
0.76 ddd
12.0, 11.1,
3.8
12.0
2.18 dd
12.7, 4.5
2.24 dd
3.98 ddd
12.1, 4.6
12.1, 12.1,
3.7
W1/2
= 21.8
12.4, 12.4,
12.2
12.4, 4.5
2.21 dd
3.96 overlapping
12.0, 4.4
2.25 dd
12.7, 4.6
eq
ax
1.36 br d
1.76 dddd 12.9, 11.1,
11.1, 3.4
2
3
4
3.96
m
2
3
4
2
3
4
2
3
4
3.97
m
3.84
m
W1/2
3.81
m
3.77
m
W1/2
3.82
m
W1/2
= 21.3
12.8, 12.5,
12.3
12.8, 3.5
eq
2.00 br d
11.1
= 25.4
12.0, 11.9,
11.6
11.6, 4.4,
4.3
= 24.0
12.7, 12.5,
12.2
12.2, 3.6,
3.4
ax
eq
1.49 ddd
1.85 ddd
ax
eq
1.51 ddd
1.82 dd
ax
eq
1.49 ddd
1.80 ddd
ax
eq
1.52 ddd
1.83 dd
3
4
3.88
m
W1/2 = 20.9
ax
eq
1.78 ddd
1.90 br d
12.9, 11.1,
11.1
12.9
5
6
1.03
m
5
6
1.03 overlapping
1.12 dddd 12.6, 12.6,
12.1, 3.5
5
6
0.99 overlapping
1.09 dddd
5
6
1.05 overlapping
1.12 dddd
ax
1.04 overlapping
ax
ax
12.6, 12.2,
11.7, 3.0
11.7
ax
12.8, 12.8,
12.1, 3.4
12.8
5
0.86
m
eq
ax
1.16 br d
0.76 dddd
12.1
eq
ax
1.21 br d
1.63
12.1
eq
ax
1.17 br d
eq
ax
1.29 br d
6
7
(2H) 1.16
m
m
7
12.1, 11.8,
11.4, 3.8
11.4
11.8, 11.4,
10.8, 3.8
11.4, 10.8,
3.2
7
m
7
1.62
m
7
1.63
m
ax
eq
0.78
eq
1.38 br d
1.37 dddd
eq
1.55 br d
1.88 ddd
12.9
11.5, 11.5,
3.6
12.8, 11.5,
3.5
eq
1.53 br d
1.84 ddd
12.7
12.6, 12.3,
3.5
12.4, 12.3,
3.5
eq
1.55 br d
1.91 ddd
12.8
12.3, 11.4,
3.6
12.9, 12.3,
3.7
1.63 br d
1.65 dddd 12.5, 12.0,
12.0, 3.3
12.0
8
8
8
8
8
9
0.57 ddd
9
1.64 ddd
–
9
1.62 ddd
9
1.65 ddd
9
0.92 ddd
12.5, 10.5,
2.7
10
–
10
10
–
10
–
10
–
11 ax
1.19 dddd
12.7, 11.4,
10.4, 2.8
11 ax
1.36 dddd 12.8, 12.8,
11.4, 2.4
1.56 overlapping
11 ax
1.32 dddd
12.4, 12.4,
11.2, 3.4
11 ax
1.36 dddd
12.9, 12.9,
12.7, 3.6
11 ax
2.41 dd
13.8, 13.7
13.8, 5.0
eq
12 ax
1.42 overlapping
1.06 overlapping
eq
12 ax
eq
12 ax
1.55 overlapping
2.12 ddd
eq
12 ax
1.56 overlapping
2.15 ddd
eq
12
2.25 dd
–
2.14 ddd
13.1, 12.8,
4.4
12.4, 11.2,
3.9
12.9, 12.3,
4.0
eq
13
1.65 br d
–
11.6
eq
13
1.38 br d
–
13.1
eq
13
1.35 br d
–
11.2
eq
13
1.40 br d
–
12.9
13
14
–
1.38 ddd
13.2, 7.1,
5.5
14
0.99
m
14
–
14
–
14
–
15
a
2.10 ddd
1.61 ddd
12.5, 7.1,
7.0
12.5, 6.3,
6.2
15
16
a
2.00 dd
1.44 dd
12.0, 5.7
12.0, 8.2
15
16
a
2.34 dd
12.6, 7.5
15
16
a
2.35 dd
12.5, 7.5
15
16
a
2.33 dd
12.6, 7.4
b
b
b
1.84 dd
5.07 ddd
12.6, 6.0
7.5, 7.2,
6.0
b
1.83 dd
5.06 ddd
12.5, 5.9
7.5, 7.2,
5.9
b
1.86 dd
5.10 ddd
12.6, 6.0
7.5, 7.4,
6.0
16
17
4.99
m
4.54
m
2.76 dd
6.8, 6.6
6.9
17
18
19
20
21
22
1.78 dd
0.79
0.68
1.94 dq
1.12
–
7.8, 7.0
17
18
19
20
21
22
2.77 dd
1.03
0.77
2.07 dq
1.16
–
7.3, 7.2
17
18
19
20
21
22
2.74 dd
1.01
0.74
2.04 dq
1.14
–
7.2, 7.1
17
18
19
20
21
22
2.79 dd
1.05
0.78
2.10 dd
1.19
–
7.5, 7.3
18
19
20
21
22
1.07
0.88
1.92
1.35
–
s
s
m
d
s
s
s
s
s
s
s
s
6.8, 6.7
6.8
7.3, 6.9
6.9
6.8, 6.4
6.8
7.0, 6.8
7.0
d
d
d
d
23 (2H) 1.69
24 (2H) 1.56
25
26 ax
eq
27
m
m
m
23 (2H) 1.67
24 (2H) 1.56
25
26 ax
eq
27
m
m
m
23 (2H) 1.70
24 (2H) 1.57
25
26 ax
eq
27
m
m
m
23 (2H) 1.68
24 (2H) 1.55
25
26 ax
eq
27
m
m
m
23 (2H) 1.84
24 (2H) 1.82
25
26 ax
eq
27
m
m
m
1.57
1.59
3.50 dd
3.58 dd
1.58
3.48 dd
3.54 br d
0.67
1.55
3.44 br d
3.51 dd
2.06
3.48 dd
3.59 br d
11.8, 10.6
11.8
5.7
10.6, 10.4
10.6, 2.4
7.0
10.4, 10.4
10.4
5.1
10.3
10.3, 10.2
4.7
4.13 dd
3.90 dd
3.72 dd
3.65 dd
5.60 br s
11.3, 10.2
11.3, 3.9
10.7, 5.2
10.7, 3.4
0.69
d
0.69
d
d
0.65
d
a
b
2-OH
5.69 br s
2-OH
5.67 br s
2-OH
5.69 br s
2-OH
(continued on next page)

Table 2 (continued)
6
7
8
9
Position
dH
J (Hz)
Position
dH
J (Hz)
Position
1
dH
J (Hz)
Position
dH
J (Hz)
1
ax
eq
1.13
2.20
3.98
3.85
1.52
1.82
1.01
1.03
1.11
0.74
1.40
1.38
0.58
–
1.24
1.45
1.05
1.66
–
1.00
2.07
1.47
4.96
1.93
0.86
0.68
2.23
1.32
–
dd
dd
12.1, 11.5
12.1, 4.8
overlapping
1
ax
eq
1.21
2.25
3.98
3.78
1.52
1.83
1.06
1.09
1.15
1.63
1.55
1.87
1.68
–
dd
dd
12.6, 11.8
12.6, 4.4
overlapping
ax
eq
1.18
2.20
3.97
3.87
1.54
1.88
1.04
1.05
1.16
0.80
1.49
1.31
0.58
–
1.24
1.43
1.06
1.65
–
0.78
2.08
1.42
4.78
2.42
0.68
0.70
–
dd
dd
12.6, 11.8
12.6, 5.0
overlapping
1
2
ax
eq
ax
eq
0.82
1.59
1.78
2.06
3.93
1.67
1.92
0.92
1.17
1.53
0.81
1.38
0.52
–
1.24
1.42
1.10
1.70
–
0.82
2.10
1.45
4.78
2.44
0.71
0.89
–
dd
11.4, 10.8
ddd
dddd
dd
m
br d
11.4, 2.8, 2.6
11.4, 11.4, 11.2, 3.2
11.4, 3.2
W1/2 = 20.2
12.1
2
3
4
2
3
4
2
3
4
m
W1/2 = 23.8
12.1, 11.9, 11.8
11.8
m
W1/2 = 22.2
12.5, 12.2, 12.0
12.5
11.4, 10.6
11.6, 11.4, 10.6
11.6, 4.5
12.0, 11.6, 11.4, 3.4
12.0
11.6, 11.5, 3.5
11.5, 11.3, 3.4
m
W1/2 = 26.3
12.3, 12.2, 11.6
12.3, 3.8, 3.2
ax
eq
ddd
br d
m
dddd
dd
dddd
br d
m
ax
eq
ddd
br d
dd
ddd
dd
dddd
br d
ddd
ddd
ax
eq
ddd
ddd
m
3
4
ax
eq
5
6
5
6
5
6
overlapping
ax
eq
ax
eq
12.5, 11.8, 11.5, 3.1
11.5, 2.4
11.8, 10.9, 10.3, 2.7
11.8
ax
eq
ax
eq
ax
eq
ax
eq
overlapping
5
6
7
m
m
ddd
br d
br d
dddd
br d
dddd
ddd
11.8
(2H)
ax
7
7
7
12.7, 12.7, 11.2, 3.3
12.7
11.8, 11.2, 10.4, 3.5
11.8, 10.4, 3.0
12.9, 3.0, 3.0
12.9
overlapping
eq
8
9
8
9
8
9
10
11
8
9
ddd
11.8, 11.5, 3.2
ddd
11.2, 11.1, 3.5
12.8, 12.6, 6.8, 4.0
10
11
10
11
10
11
ax
eq
ax
eq
m
ax
eq
ax
eq
1.40
1.57
2.17
1.43
–
overlapping
ax
eq
ax
eq
dddd
br d
12.6, 12.6, 11.8, 3.5
12.6
overlapping
ax
eq
ax
eq
dddd
br d
ddd
br d
11.8
12.6, 11.8, 3.1
12.6
br d
ddd
dd
13.0
12.5, 12.3, 4.4
12.5, 2.4
overlapping
12
12
12
12
ddd
br d
12.8, 12.3, 4.2
12.3
br d
12.6
13
14
15
13
14
15
13
14
15
13
14
15
dd
dd
dd
m
dd
s
12.5, 6.6
12.5, 6.6
12.5, 7.3
–
dd
dd
dd
m
d
12.7, 5.9
12.9, 6.9
12.9, 5.5
ddd
ddd
ddd
m
d
s
10.4, 6.6, 3.2
12.7, 12.1, 6.6
12.7, 12.6, 5.6
a
a
2.31
1.84
5.41
2.88
1.08
0.78
2.36
1.35
–
2.09
2.06
1.95
3.95
3.65
1.25
dd
12.1, 6.6
overlapping
a
b
a
b
b
b
16
17
18
19
20
21
22
23
24
16
17
18
19
20
21
22
23
24
25
26
dd
dd
s
7.2, 6.6
7.2, 6.8
16
17
18
19
20
21
22
23
24
16
17
18
19
20
21
22
23
24
7.4, 6.8
10.1
10.1
s
s
s
s
s
br d
d
6.8
6.8
br d
d
6.8
6.8
1.62
–
s
1.64
–
s
(2H)
a
2.06
2.04
1.68
1.86
3.65
3.83
0.99
5.62
m
(2H)
(2H)
m
m
m
(2H)
a
b
2.23
1.47
1.82
1.96
3.62
3.91
1.03
5.65
m
m
m
m
dd
dd
d
(2H)
a
2.23
1.48
1.84
1.96
3.62
3.95
1.03
m
overlapping
overlapping
m
b
m
m
dd
b
25
26
a
overlapping
25
26
25
26
m
a
12.2, 10.8
overlapping
b
dd
d
9.5, 6.0
6.1
a
b
11.8, 10.4
11.8, 5.7
6.6
a
overlapping
overlapping
b
27
b
27
2-OH
d
br s
6.6
27
2-OH
27
d
6.7
2-OH
5.60
br s
br s

Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
249
O-b-
O-b-
D
-galactopyranosyl-(1 ? 2)-O-[b-
-glucopyranosyl-(1 ? 4)-b- -galactopyranoside.
-glucosidase gave the corre-
-glucose, whereas acid hydro-
lysis of 7 gave spirostanol sapogenin 2a, -galactose, -glucose, and
D
-glucopyranosyl-(1 ? 3)]-
and did not affect cell growth of the TIG-3 normal cells. Although
previously reported pseudo-furostanol glycosides have not shown
cytotoxic activity (Liu et al., 2003; Yokosuka et al., 2002), pseudo-
furostanol glycoside 9 exhibited significant tumor-selective cyto-
toxicity. Comparing cytotoxic activities of 6 and 13, those of 1
and 2, and those of 7 and 13 suggested that the presence of a
D
D
Enzymatic hydrolysis of 7 with b-
sponding spirostanol saponin 3 and
D
D
D
D
D
-xylose. The HMBC correlation of 7 between the anomeric proton
(H-100000) of one b-
D-glucopyranosyl group at dH 4.83 (d, J = 7.8 Hz)
C-2a
hydroxy group did not affect cytotoxic activity, whereas
position reduced the
and C-26 of the aglycone at dC 75.3 confirmed that a b-
D-glucopyr-
introducing a hydroxy group to the C-14a
anosyl unit was attached at C-26. NOE correlations between the
signals of the H-20 proton at dH 2.36 and the H2-23 protons at dH
cytotoxicity. Preliminary analysis showed that the C-6 hydroxy
group or C-6 O-glucosyl group significantly reduced cytotoxic
activity in the spirostanol glycosides (Lu et al., 2011; Yokosuka
et al., 2009b). In the present study, these results also suggested
that introduction of a hydroxy group to the steroidal aglycone re-
duced cytotoxicity. Compounds 1 and 11, as well as 6 and 14, are
2.09 (2H) indicated a C-22
a
configuration. Thus, 7 was identified
-glucopyranosyl)oxy]-2 ,14 ,22 -trihydroxy-
O-b- -galactopyranosyl-(1 ? 2)-O-[b- -xylo-
-glucopyranosyl-(1 ? 4)-b- -galactopy-
as
(25R)-26-[(b-
-furostan-3b-yl
D
a
a
a
5a
D
D
pyranosyl-(1 ? 3)]-O-b-
D
D
ranoside.
related; the terminal b-
D-glucopyranosyl residue of 1 and 6 was re-
The spectroscopic data for 8 (C56H92O28) showed that it had a
structure similar to 14. However, the 13C NMR signals at dC 152.3
(C) and 103.6 (C) indicated the presence of an olefinic moiety in
8. Furthermore, the Me-21 methyl doublet observed at dH 1.32
(J = 6.8 Hz) in the 1H NMR spectrum of 14 was absent from that
of 8, and was replaced by a methyl singlet at dH 1.62. The H-17 pro-
ton resonance appeared at dH 2.42 as a doublet with a coupling
constant of 10.1 Hz. These spectroscopic properties and the HMBC
correlations of C-20 (dC 103.6) and C-22 (dC 152.3) with H-17 (dH
2.42, d, J = 10.1 Hz), Me-21 (dH 1.62, s), and H2-23 (dH 2.23, m) sug-
placed by a b- -xylopyranosyl group in 11 and 14. No difference in
D
cytotoxicity was observed between 1 and 11, and between 6 and
14. On the other hand, the aglycones, 1a, 2a, 4a, 5a, and 9a, did
not show cytotoxicity against any cells.
Fig. 2 shows the time course of the antiproliferative effects of
the tumor-selective compounds 9 in HL-60 cells and A549 cells
at 3.0, 10, and 20 lg/ml. Interestingly, pseudo-furostanol glycoside
9 appeared to inhibit HL-60 and A549 cell growth in a time-depen-
dent manner. Morphological observation of HL-60 and A549 cells
stained with 40,6-diamidino-2-phenylindole (DAPI) suggested that
9 and 14 induced apoptosis in both HL-60 and A549 cells; the cells
displayed nuclear chromatin condensation and apoptotic bodies
(data not shown). Furthermore, an increase of caspase-3 activity
in HL-60 cells and A549 cells was evident after 16 h of treatment
with 9 and 14 (Fig. 3). Finally, the cell cycle distribution of HL-60
cells and A549 cells treated with 9 and 14 was analyzed by flow
cytometry (Table 5). Compounds 9 and 14 induced a sub-G1 pop-
ulation of 51.9 3.4% and 55.9 2.1% in HL-60 cells and
6.0 0.3% and 20.6 3.3% in A549 cells, respectively. This suggests
that 9 and 14 may induce apoptotic cell death through caspase-3
gested that 8 was the corresponding
coside of 14. This was supported by enzymatic hydrolysis of 8 with
b- -glucosidase giving spirostanol glycoside 11 and -glucose,
whereas enzymatic hydrolysis using naringinase gave spirostanol
sapogenin 1a (Gvazava and Kikoladze, 2006), -galactose,
-glucose, and -xylose. Thus, was assigned as (25R)-26-
[(b- -glucopyranosyl)oxy]-2 -hydroxy-5 -furost-20(22)-en-3b-yl
O-b- -galactopyranosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-O-
b- -glucopyranosyl-(1 ? 4)-b- -galactopyranoside.
Compound 9 (C61H100O30) was also expected to be a D20(22)
D
20(22)-pseudo-furostanol gly-
D
D
D
D
D
8
D
a
a
D
D
D
D
-
pseudo-furostanol glycoside, based on the characteristic 1H and
13C NMR signals at dH 2.44 (d, J = 10.1 Hz, H-17) and 1.64 (s, Me-
21), and at dC 152.3 (C) and 103.6 (C), and the HMBC correlations
of C-20 (dC 103.6) and C-22 (dC 152.3) with H-17 (dH 2.44, d,
J = 10.1 Hz), Me-21 (dH 1.64, s), and H2-23 (dH 2.23, m). Enzymatic
activity. Additionally,
9
induced
a
G0/G1 population of
84.7 0.2% in A549 cells. This result implies that 9 also induce
cell-cycle arrest at the G0/G1 phase in A549 cells.
3. Concluding remarks
hydrolysis of 9 with b-
10 and -glucose, whereas acid hydrolysis of 9 gave the spirostanol
sapogenin (25R)-5 -spirostan-3b-ol (9a, tigogenin) (Gvazava and
Kikoladze, 2010), -arabinose, -galactose, -glucose, -rhamnose,
and -xylose. Therefore, 9 was the corresponding
20(22)-pseudo-
furostanol glycoside of 10: (25R)-26-[(b- -glucopyranosyl)oxy]
-5 -furost-20(22)-en-3b-yl O- -arabinopyranosyl-(1 ? 2)-O-[b-
-xylopyranosyl-(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-O-[
-galactopyranoside.
D-glucosidase gave the spirostanol glycoside
D
In conclusion, nine new and five known steroidal glycosides
were isolated from the bulbs of B. elegans and evaluated for their
cytotoxic activities against HL-60 and A549 tumor cells, and TIG-
3 normal cells. Compounds 9 and 14 increased caspase-3 activity
and induced cell accumulation at the sub-G1 phase of cell cycle
in HL-60 cells and A549 cells, respectively. These results indicated
that 9 and 14 had potent apoptotic activity in HL-60 cells and A549
cells. Previous studies of steroidal glycosides found that pseudo-
furostanol glycoside did not show cytotoxic activity (Liu et al.,
2003; Yokosuka et al., 2002). Thus, it is notable that pseudo-furost-
anol glycoside 9 appeared to inhibit tumor cells growth in a time-
dependent manner and induce apoptosis in HL-60 and A549 cells.
Compound 9 also induced cell-cycle arrest at the G0/G1 phase in
A549 cells.
a
L
D
D
L
D
D
D
a
a-L
D
D
a-L-
rhamnopyranosyl-(1 ? 2)]-b-
D
2.2. Cytotoxic activity
It has been reported that steroidal glycosides have potent anti-
cancer activity. Relationships between chemical structure and
cytotoxicity of steroidal glycosides have been studied (Yokosuka
et al., 2009a). The aglycone moiety and the sugar moiety play an
important role in the cytotoxicity.
Isolated compounds 1–14 were evaluated for their cytotoxic
activities against HL-60 and A549 tumor cells, and TIG-3 normal
cells. Etoposide and cisplatin were used as positive controls, and
4. Experimental
4.1. General
had IC50 values of 0.3 0.01 and 4.1 0.23
lM against HL-60 cells,
and 1.2 0.03 and 2.3 0.04 M against A549 cells, respectively.
Compounds 1, 5, 6, 9–11, 13, and 14 showed cytotoxic activity
against both HL-60 and A549 cells with IC50 values ranging from
l
Optical rotations were measured using an automatic digital
polarimeter (P-1030, Jasco, Tokyo, Japan). IR spectra were recorded
on a spectrophotometer (FT-IR 620, Jasco). NMR spectra were
recorded at 500 MHz for 1H NMR using standard Bruker pulse pro-
grams at 300 K (DRX-500, Bruker, Karlsruhe, Germany). Chemical
0.5 0.01 to 8.1 0.28
lM, respectively (Table 4). Compounds 6,
9, 11, 13, and 14 were selectively cytotoxic to the tumor cells

Table 3
1H and 13C NMR spectroscopic (500 and 125 MHz) data for the sugar moieties of 1–9 in pyridine-d5.
1
2
3
4
5
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Gal (I) 10
4.94
d
7.8
103.1 Gal (I)
72.3
75.6
79.6
75.5
10
20
30
40
50
60
4.91
d
7.7
103.1 Gal (I) 10
4.89
d
7.7
102.9 Gal (I) 10
4.93
d
7.5
103.2 Gal 10
4.84
d
7.7
100.0
77.3
76.1
81.4
75.1
60.3
20
30
40
50
60
4.52 dd 8.2, 7.8
4.10 br d 8.2
4.55 br s
4.46 dd 8.6, 7.7
4.08 dd 8.6, 2.7
4.54 br d 2.7
4.03
4.60
72.5
75.6
79.6
75.6
60.4
20
30
40
50
60
4.47 dd 9.6, 7.7
4.08 dd 9.6, 2.1
4.52 br d 2.1
72.4
75.4
79.3
75.6
60.5
20
30
40
50
60
4.48 dd 9.6, 7.5
4.11 dd 9.6, 2.9
4.57 br d 2.9
72.6
75.7
79.4
75.7
60.4
20
30
40
50
60
4.39 dd 8.7, 7.7
4.15 dd 9.1, 3.2
4.50 br d 3.2
3.97 m
4.67 br d 11.4
4.03
m
m
m
4.03
m
4.05
m
a
b
4.59 dd 10.0, 9.3 60.4
4.17 overlapping
a
b
a
b
4.56 dd 10.9, 7.9
4.17 dd 10.9, 5.1
a
b
4.63 dd 11.7, 8.9
4.16 dd 11.7, 5.9
a
b
4.15 dd 9.1, 3.4
4.21 dd 11.4, 6.1
Glc (I) 100
5.14
d
8.0
105.1 Glc (I)
80.9
87.4
70.7
77.4
100
200
300
400
500
600
5.14 7.9
d
105.1 Glc
81.0
87.5
70.8
77.5
100
200
300
400
500
600
5.16
d
7.9
105.0 Glc
80.9
85.9
70.3
77.4
100
200
300
400
500
600
5.19
d
7.9
105.3 Rha 100
6.29 br s
101.6
72.3
72.6
73.9
69.5
18.4
200
300
400
500
600
4.45 dd 8.6, 8.0
4.16 dd 8.6, 8.5
3.78 dd 8.5, 8.4
3.81
4.46 br d 12.4
3.97 br d 12.4
4.45 dd 8.7, 7.9
4.16 dd 8.7, 8.7
3.78 dd 8.7, 8.7
4.43 dd 8.6, 7.9
4.07 dd 9.0, 8.6
3.72 dd 9.2, 9.0
3.81
4.46 br d 12.6
4.52 dd 8.8, 7.9
4.14 dd 9.8, 8.8
3.79 dd 9.4, 9.0
3.85
4.50 br d 12.7
4.04 br d 12.7
81.1
85.4
70.4
77.3
62.7
200
300
400
500
4.79 br d 1.6
4.53 dd 9.1, 3.2
4.31 dd 9.4, 9.1
m
3.83
m
m
m
4.89
600 1.72
m
d
a
b
62.9
a
b
4.45 overlapping
3.97 br d 12.2
63.0
a
b
62.8
a
b
6.2
3.99 dd 12.6, 9.3
Glc 1000
2000
5.35
d
7.6
105.7
81.5
87.8
70.3
77.6
62.8
Gal (II) 1000
5.50
d
7.8
105.3 Gal (II)
73.5
74.4
70.4
77.0
1000
2000
3000
4000
5000
6000
5.49
d
7.8
105.3 Gal (II) 1000
5.48
d
7.7
105.1 Gal (II) 1000
5.50
d
7.8
105.3
73.7
74.3
70.4
77.3
62.7
4.23 dd 8.6, 7.6
4.06 dd 8.7, 8.6
3.82 dd 8.7, 7.6
3.84 m
4.51 br d 12.7
4.03 br d 12.7
2000
3000
4000
5000
6000
4.56 dd 8.2, 7.8
3.98 dd 8.2, 2.1
4.31 br d 2.1
4.58 dd 8.6, 7.8
3.98 dd 8.6, 3.1
4.29 br s
73.6
74.4
70.5
77.1
2000
3000
4000
5000
6000
4.56 dd 9.1, 7.7
3.98 br d 9.1
4.29 br s
73.5
74.2
70.2
77.1
62.3
2000
3000
4000
5000
6000
4.61 dd 9.5, 7.8
3.96 dd 9.5, 2.8
4.27 br d 2.8
3000
4000
5000
4.03
m
4.03
m
4.05
m
4.06
m
6000
a
b
a
b
4.67 dd 11.4, 6.4 62.5
4.39 dd 11.4, 4.3
a
b
4.67 dd 11.4, 5.7 62.6
4.38 dd 11.4, 5.3
a
b
4.69 dd 11.2, 6.5
4.35 dd 11.2, 4.4
a
b
4.70 dd 11.4, 6.5
4.39 dd 11.4, 4.5
Ara 10000
20000
4.96
d
7.8
105.4
73.2
74.6
69.6
67.2
Glc (II) 10000
5.19
d
7.8
104.4 Glc (II) 10000
75.1 20000
5.19
d
7.8
104.5 Xyl
75.2
78.5
71.5
78.5
10000
20000
30000
40000
50000
5.08
d
7.6
104.7 Xyl
74.9
78.3
70.6
67.1
10000
20000
30000
40000
50000
5.12
d
7.5
104.8
75.0
78.5
70.7
67.3
4.42 dd 8.3, 7.8
4.01 dd 8.3, 3.4
4.17 m
4.20 br d 11.2
3.66 br d 11.2
20000
30000
40000
50000
60000
4.02 dd 8.6, 7.8
4.13 dd 8.6, 7.7
4.12 dd 8.6, 7.7
3.96
4.53 br d 11.5
4.02 dd 8.9, 7.8
4.13 dd 9.1, 8.9
4.12 dd 9.1, 8.9
3.98
4.52 br d 11.8
3.91 dd 8.4, 7.6
3.99 dd 8.4, 9.3
4.07
4.17 dd 10.9, 5.1
3.57 dd 10.9, 10.7
3.94 dd 8.4, 7.5
3.98 dd 8.9, 8.4
4.08
4.20 dd 11.4, 5.3
3.60 dd 11.4, 10.7
30000
78.5 30000
40000
50000
71.5 40000
m
m
a
b
m
78.5 50000
m
a
b
a
b
a
62.2 60000
a
62.3
b
4.24 dd 11.5, 6.0
b
4.25 dd 11.8, 5.7
Xyl 100000
200000
5.21
d
7.8
104.9
75.0
78.6
70.6
67.2
3.95 dd 8.6, 7.8
4.08 dd 8.7, 8.6
4.12 m
4.21 dd 11.2, 5.3
3.65 dd 11.2, 10.7
300000
400000
500000
a
b

Table 3 (continued)
6
7
8
9
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Position
dH
J (Hz)
dC
Gal (I)
10
20
30
40
50
60
4.88
4.48 dd
4.07 dd
4.52 br d 2.8
4.04
4.63 dd
4.16 dd
d
7.5
8.9, 7.5
8.9, 2.8
103.2 Gal (I)
72.4
75.7
79.6
75.7
10
20
30
40
50
60
4.91
4.48 dd
4.10 dd
4.58 br d 2.6
4.03
4.61 dd
4.17 dd
d
7.6
8.7, 7.6
8.7, 2.6
103.1 Gal (I)
72.5
75.6
79.5
75.7
10
20
30
40
50
60
4.97
4.50 dd
4.13 dd
4.58 br d 2.7
4.08
4.62
d
8.1
8.9, 8.1
8.9, 2.7
103.2 Gal
72.4
75.8
79.4
75.6
10
20
30
40
50
60
4.88
4.41 dd
4.14 dd
4.51 br d 3.3
3.95
4.74 dd
4.21 dd
d
7.7
8.7, 7.7
8.7, 3.3
100.1
77.4
76.2
81.5
75.1
60.4
m
m
m
m
m
a
b
11.0, 7.4
11.0, 6.1
60.4
a
b
11.5, 8.7
11.5, 4.2
60.5
a
b
60.4
a
b
11.4, 2.6
11.4, 6.0
4.17 dd
12.2, 5.4
Glc (I)
10
5.15
d
8.0
105.2 Glc (I)
81.1
87.5
70.8
77.6
100
200
300
400
500
600
5.18
d
7.9
105.3 Glc (I)
81.0
86.0
70.5
77.6
100
200
300
400
500
600
5.20
d
7.9
105.3 Rha
81.1
85.8
70.5
77.6
100
200
300
400
500
600
6.27 br s
4.79 br d 2.1
4.55 dd
4.31 dd
101.7
72.4
72.7
74.0
69.6
18.5
200
300
400
500
600
4.46 dd
4.19 dd
3.81 dd
8.8, 8.0
8.9, 8.8
8.9, 8.8
4.50 dd
4.13 dd
3.78 dd
3.83
4.55 br d 12.5
4.01 dd 12.5, 8.2
8.6, 7.9
9.0, 8.6
9.3, 9.0
4.52 dd
4.14 dd
3.78 dd
3.86
4.51 br d 12
4.05 dd 12.0, 8.6
8.9, 7.9
9.1, 8.9
9.3, 9.1
9.5, 2.1
9.5, 9.4
3.83
m
m
m
4.91
1.73
m
d
a
b
4.41 br d 11.3
3.96 br d 11.3
63.1
a
b
63.0
a
b
63.0
6.2
Glc (I)
1000
2000
3000
4000
5000
6000
5.35
d
7.6
105.9
81.6
87.9
70.4
77.7
62.9
Gal (II)
1000
2000
3000
4000
5000
6000
5.51
4.60 dd
3.99 dd
4.32 br d 2.9
4.05
4.70 dd
4.40 dd
d
7.8
8.9, 7.8
8.9, 2.9
105.4 Gal (II) 1000
5.50
4.59 dd
3.97 br d 9.3
4.28 br s
4.04
d
7.8
9.3, 7.8
105.3 Gal (II) 1000
5.51
4.60 dd
3.98 dd
4.29 br d 1.9
4.10
4.70 dd
4.41 br d 11.5
d
7.8
8.7, 7.8
8.7, 1.9
105.3
73.7
74.3
70.5
77.3
62.6
4.25 dd
4.08 dd
3.82 dd
8.6, 7.6
8.6, 8.5
8.6, 8.6
73.6
74.3
70.4
77.1
62.8
2000
3000
4000
5000
6000
73.6
74.3
70.4
77.2
62.6
2000
3000
4000
5000
6000
3.86
m
m
m
m
a
b
4.56 br d 12.8
4.03 br d 12.8
a
b
11.6, 6.9
11.6, 5.3
a
b
4.68 dd
4.39 dd
11.2, 7.3
11.2, 4.8
a
b
11.5, 6.6
Ara
10000
20000
30000
40000
50000
4.98
4.43 dd
3.99 dd
d
7.8
8.1, 7.8
8.1, 2.8
105.5
73.3
74.7
69.7
67.3
Glc (II)
10000
20000
30000
40000
50000
60000
5.20
d
7.8
104.5 Xyl
75.3
78.5
71.7
78.5
10000
20000
30000
40000
50000
5.12
3.93 dd
3.99 dd
4.08
4.19 dd
3.60 dd
d
7.6
8.7, 7.6
8.7, 8.5
104.8 Xyl
75.0
78.5
70.7
67.3
10000
20000
30000
40000
50000
5.12
3.93 dd
3.96 dd
4.09
4.19 dd
3.60 dd
d
7.4
8.3, 7.4
8.6, 8.3
104.8
75.2
78.5
70.7
67.3
4.03 dd
4.13 dd
4.12 dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
4.17
m
m
m
a
b
4.21 br d 12.4
3.67 br d 12.4
3.96
m
a
b
10.9, 5.3
10.9, 10.7
a
b
11.0, 5.0
11.0, 9.0
a
b
4.53 br d 11.1
4.25 br d 11.1
62.3
Xyl
100000
200000
300000
400000
500000
5.22
3.94 dd
4.08 dd
4.13
4.21 dd
3.64 dd
d
7.8
8.7, 7.8
8.8, 8.7
104.9
75.1
78.8
70.7
67.3
Glc (III) 100000
4.82
d
7.7
105.0 Glc (II) 100000
4.83
d
7.8
105.0 Glc (II) 100000
4.85
d
7.5
104.9
75.2
78.6
71.7
78.5
62.9
200000
300000
400000
500000
600000
4.01 dd
4.25 dd
4.23 dd
8.3, 7.7
8.3, 8.3
8.3, 8.3
75.2
78.6
71.7
78.6
62.8
200000
300000
400000
500000
600000
4.01 dd
4.23 dd
4.21 dd
8.7, 7.8
8.7, 8.7
9.2, 8.7
75.2
78.5
71.7
78.6
62.8
200000
300000
400000
500000
600000
4.07 dd
4.27 dd
4.25 dd
8.6, 7.5
8.6, 7.7
8.2, 7.7
m
a
b
10.7, 5.3
10.7, 10.7
3.94
m
3.94
m
3.96
m
a
b
4.55 br d 11.0
4.37 br d 11.0
a
b
4.57 br d 12.5
4.36 br d 12.5
a
b
4.57 br d 11.0
4.38 br d 11.0
Glc (II) 1000000
4.85
d
7.8
105.0
75.2
78.6
71.7
78.5
62.9
2000000
3000000
4000000
5000000
6000000
3.96 dd
4.26 dd
4.23 dd
3.98
4.59 br d 12.2
4.40 dd 12.2, 5.1
8.3, 7.8
8.6, 8.3
8.6, 7.3
m
a
b

252
Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
Table 4
Diaion HP-20 (50 mesh, Mitsubishi-Chemical, Tokyo, Japan), BW-
300 silica gel (200–300 mesh, Fuji Silysia Chemical, Kasugai,
Japan), and ODS silica gel COSMOSIL 75C18-OPN (75 lM, Nacalai
Cytotoxic activities of 1–14, 1a, 2a, 4a, 5a, and 9a against HL-60, A549 and TIG-3 cells.
Compounds
IC50
(
l
M)a
Tesque, Kyoto, Japan) were used for CC. TLC was carried out on pre-
coated silica gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Ger-
many) and RP18 F254S plates (0.25 mm thick, Merck), and the
spots were visualized by spraying the plates with 10% H2SO4 in
H2O and then heating. HPLC was performed with a system com-
posed of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010 con-
troller (Tosoh), an RI-8010 detector (Tosoh), and a Rheodyne
injection port (Rohnert Park, CA, USA). A TSK gel ODS-100Z column
HL-60
A549
TIG-3
1
1a
2
2a
3
4
4a
5
5a
6
7
8
9
9a
10
11
5.4 0.2
>10
>10
>10
>10
>10
>10
8.1 0.3
>10
6.2 0.04
>10
>10
3.8 0.1
>10
3.1 0.1
4.1 0.2
>10
6.1 0.1
4.2 0.04
0.3 0.01
1.2 0.03
4.1 0.1
not determined
>10
>10
>10
>10
>10
3.6 0.1
>10
3.8 0.1
>10
3.7 0.2
1.6 0.1
>10
1.1 0.01
1.5 0.04
>10
0.5 0.01
1.3 0.02
4.1 0.2
2.3 0.04
1.8 0.01
>10
>10
>10
>10
>10
>10
1.7 0.01
8.1 0.5
>10
>10
2.0 0.01
>10
>10
1.1 0.01
>10
>10
>10
>10
11.2 1.4
2.0 0.3
(10 mm i.d. ꢁ 250 mm, 5
lm, Tosoh) was employed for preparative
HPLC. Purities of all isolated compounds were confirmed by NMR,
optical rotation, TLC, and mass spectrometry, respectively. The fol-
lowing materials and reagents were used for the cell cultures and
the cytotoxicity assays: a microplate reader (Spectra Classic, Tecan,
Salzburg, Austria); a 96-well flat-bottom plate (Iwaki Glass, Funab-
ashi, Japan); JCRB 0085 HL-60 cells, JCRB 0076 A549 cells, and JCRB
0506 TIG-3 cells (Human Science Research Resources Bank, Osaka,
Japan); fetal bovine serum (FBS; Bio-Whittaker, Walkersville, MD,
USA); 0.25% trypsin–EDTA solution, RPMI-1640 medium, mini-
mum essential medium (MEM), etoposide, Triton X-100, and 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT; Sigma, St. Louis, MO, USA); penicillin G sodium salt and
streptomycin sulfate (Gibco, Grand Island, NY, USA); paraformal-
12
13
14
Etoposide
Cisplatin
a
Data are represented the mean value S.E.M. of three experiments performed
in triplicate.
dehyde and ribonuclease
A (RNase; Wako Pure Chemical
shifts are given as d values relative to tetramethylsilane (TMS) used
as an internal standard. HR-ESI-TOF-MS data were recorded on a
LCT mass spectrometer (Waters-Micromass, Manchester, UK).
Industries, Osaka, Japan); and propidium iodide (PI; Molecular
Probes, Eugene, OK, USA). All other chemicals used were of bio-
chemical reagent grade.
(B)
(A)
100
140
120
100
80
80
60
3
60
40
3
10
40
10
20
20
20
20
0
0
0h 3h 6h 16h 24h 48h 72h
0h 3h 6h 16h 24h 48h 72h
Fig. 2. Cell viability of tumor-selective compounds 9 in HL-60 cells and A549 cells. Cell viability was measured for 0, 3, 16, 24, 48, 72 h at 3.0, 10, and 20 lg/ml using trypan
blue staining in HL-60 cells (A), and using the MTT assay in A549 cells (B). Each value is the mean S.E. of three experiments.
(A)
(B)
1.2
1
0.25
0.2
6h
6h
16h
16h
0.8
0.6
0.4
0.2
0
0.15
0.1
0.05
0
C
E
9
14
C
E
9
14
Fig. 3. Caspase-3 activity in 9 and 14 or etoposide-treated HL-60 cell lysates (A) and A549 cell lysates (B). Both cells were incubated at 37 °C for 6 and 16 h, respectively, with
20 g/ml of the compound or etoposide. Each value is the mean S.E. of three experiments.
l

Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
253
Table 5
Effects of 9 and 14 on cell cycle distribution of HL-60a and 9, 11 and 14 on cell cycle distribution of A549a for 24 h.
%Sub G0/G1
%G0/G1
%S
%G2–M
HL-60
A549
Control
9 (48 h)
14
7.4 0.3
51.9 3.5
55.9 2.1
44.7 0.8
49.1 0.4
25.3 1.3
23.0 1.5
24.5 3.7
21.6 0.2
8.7 0.8
12.3 0.5
19.1 1.5
20.5 0.7
14.6 2.2
6.3 1.2
9.1 0.9
Etoposide
Control
9 (48 h)
14
1.5 0.2
6.0 0.3
20.6 3.3
14.2 0.01
71.6 6.7
84.7 0.2
62.6 2.3
48.3 0.2
6.0 0.2
0.2 0.01
4.8 0.3
10.1 2.5
9.0 0.4
12.9 1.1
18.7 1.6
Etoposide
18.2 1.8
a
The cell cycle distribution was determined by flow cytometry. Date are represented the mean value S.E.M. of three experiments performed in triplicate.
4.2. Plant material
4.5. Enzymatic hydrolysis of 1
B. elegans bulbs were obtained from the Sakata Seed Corpora-
tion (Yokohama, Japan) in 2007 and were identified by Dr. Yutaka
Sashida, Professor Emeritus of Tokyo University of Pharmacy and
Life Sciences. A voucher specimen is retained in our laboratory
(07-006-Be).
Compound 1 (4.5 mg) was treated with naringinase (EC 232-96-
4, Sigma; 90.0 mg) in a HOAc/KOAc buffer (pH 4.3, 3.0 ml) at room
temp. for 49 h. The reaction mixture was purified by silica gel CC
(100 g, 2 cm i.d. ꢁ 30 cm) eluted with CHCl3–MeOH–H2O (7:4:1)
to give gitogenin (1a, 2.7 mg). The sugar fraction (0.9 mg) was ana-
lyzed by HPLC under the following conditions: Capcell Pak NH2
UG80 column (4.6 mm i.d. ꢁ 250 mm, 5
pan); mobile phase of MeCN–H2O (85:15); detection by refractive
index and optical rotation, and at a flow rate of 1.0 ml/min. -Gal-
actose and -glucose were identified by comparing their retention
times (tR) and optical rotation with those of authentic samples:
lm, Shiseido, Tokyo, Ja-
4.3. Extraction and isolation
D
D
Bulbs of B. elegans (2.8 kg fresh weight) were extracted with hot
MeOH (each 8 l ꢁ 3 times) for 6 h. After removing the solvent, the
MeOH extract (200 g) was passed through a Diaion HP-20 column
(2000 g, 8.5 cm i.d. ꢁ 60 cm) and successively eluted with MeOH–
H2O (3:7, 1:1), MeOH, EtOH, and EtOAc (9 l of each eluent). CC of
D
-
galactose (13.40 min, positive optical rotation), and
(15.85 min, positive optical rotation).
D-glucose
the MeOH-eluted fraction (15.1 g; cytotoxicity IC50 5.7 lg/ml) on
4.6. Compound 2
silica gel (2000 g, 8.5 cm i.d. ꢁ 60 cm), eluted with a stepwise gra-
dient mixture of CHCl3–MeOH–H2O (80:10:1, 70:20:1, 20:10:1,
7:4:1) and finally with MeOH, provided eight fractions (A–H). Frac-
tion C was separated on an ODS silica gel column (1500 g, 8.5 cm
i.d. ꢁ 30 cm) eluted with MeCN–H2O (1:2, 1:1) and MeOH–H2O
(2:1, 1:1) to give 12 (10.9 mg). Fraction D was subjected to silica
gel CC (1500 g, 8.5 cm i.d. ꢁ 30 cm) eluted with CHCl3–MeOH–
H2O (50:10:1, 20:10:1, 7:4:1) and on ODS silica gel (1000 g,
6.0 cm i.d. ꢁ 30 cm) eluted with MeOH–H2O (2:1) and MeCN–
H2O (2:5) to afford 15 (2.1 mg). Fraction E was applied to a silica
gel CC (1500 g, 8.5 cm i.d. ꢁ 30 cm) eluted with CHCl3–MeOH–
H2O (30:10:1, 7:4:1) and on ODS silica gel (1000 g, 6.0 cm
i.d. ꢁ 30 cm) eluted with MeCN–H2O (1:3, 1:2, 1:1) to afford 11
(63.1 mg). Fraction F was subjected to silica gel CC (1800 g,
8.5 cm i.d. ꢁ 35 cm) eluted with hexane–EtOAc (2:1, 3:1) and
CHCl3–MeOH–H2O (30:10:1, 7:4:1) and on ODS silica gel (1000 g,
6.0 cm i.d. ꢁ 30 cm) eluted with MeOH–H2O (1:2, 2:3, 1:2, 1:1) to
give 1 (38.7 mg), 3 (81.7 mg), 5 (14.5 mg), and 10 (95.6 mg). Frac-
tion G was purified using silica gel (1500 g, 8.5 cm i.d. ꢁ 30 cm)
eluted with CHCl3–MeOH–H2O (30:10:1, 7:4:1) and on ODS silica
gel (1000 g, 6.0 cm i.d. ꢁ 30 cm) eluted with MeOH–H2O (1:4)
(25R)-2
anosyl-(1 ? 2)-O-[b-
a
,14
a
-Dihydroxy-5
a
-spirostan-3b-yl O-b-
D
-galactopyr-
D
-glucopyranosyl-(1 ? 3)]-O-b-
D-glucopyran-
25
osyl-(1 ? 4)-b-D-galactopyranoside (2); amorphous solid; [a]
D
ꢀ39.3 (c 0.73, MeOH); IR mmax (film) cmꢀ1: 3388 (OH), 2927
(CH); for 1H and 13C NMR spectroscopic data, see Tables 1–3;
HR-ESI-TOF-MS m/z: 1119.5201 [M+Na]+ (calcd. for C51H84NaO25
,
1119.5199).
4.7. Enzymatic hydrolysis of 2
Compound 2 (8.5 mg) was treated with naringinase (120.0 mg)
in HOAc/KOAc buffer (pH 4.3, 3.0 ml) at room temp. for 140 h. The
reaction mixture was purified by CC on silica gel (90 g, 2 cm
i.d. ꢁ 18 cm) eluted with CHCl3–MeOH–H2O (7:4:1) to yield 2a
(1.6 mg) and a sugar fraction. HPLC analysis of the sugar fraction
under the same conditions as those for 1 showed the presence of
D
-galactose (13.14 min, positive optical rotation) and D-glucose
(15.88 min, positive optical rotation).
and separated by HPLC using MeCN–H2O (1:3) to give
2
4.8. Compound 2a
(29.0 mg), 4 (11.6 mg), 6 (10.8 mg), 7 (11.6 mg), 8 (13.2 mg), 9
(33.6 mg), 13 (13.4 mg), and 14 (33.3 mg), respectively.
(25R)-5a-Spirostane-2a,3b,14a-triol (2a); amorphous solid;
D
[a]
ꢀ11.6 (c 0.13, MeOH); IR mmax (film) cmꢀ1: 3330 (OH),
25
2932 (CH); 1H NMR (500 MHz, C5D5N): dH 1.12 (3H, d, J = 7.9 Hz,
Me-21), 0.98 (3H, s, Me-18), 0.74 (3H, s, Me-19), 0.73 (3H, d,
J = 6.3 Hz, Me-27);13C NMR (125 MHz, C5D5N): dC 108.9 (C-22),
87.1 (C-14), 79.2 (C-16), 73.0 (C-3), 70.5 (C-2), 67.0 (C-26), 61.9
(C-17), 49.2 (C-9), 44.9 (C-1), 44.7 (C-13), 44.3 (C-5), 43.3 (C-20),
39.5 (C-15), 38.8 (C-8), 35.6 (C-10), 33.5 (C-4), 31.9 (C-12), 31.4
(C-23), 30.4 (C-25), 29.3 (C-24), 28.8 (C-6), 28.2 (C-7), 22.5 (C-
11), 20.1 (C-18), 17.1 (C-27), 14.2 (C-21), 13.9 (C-19); HR-ESI-
TOF-MS (m/z: 431.3183 [M+H]+–H2O, calcd. for C27H43O4:
431.3161).
4.4. Compound 1
(25R)-2
syl-(1 ? 2)-O-[b-
syl-(1 ? 4)-b-
a
-Hydroxy-5
a
-spirostan-3b-yl O-b-
D
-galactopyrano-
-glucopyrano-
D
-glucopyranosyl-(1 ? 3)]-O-b-
D
25
D
-galactopyranoside (1); amorphous solid; [a]
D
ꢀ46.9 (c 0.12, MeOH); IR mmax (film) cmꢀ1: 3362 (OH), 2925
(CH); for 1H and 13C NMR spectroscopic data, see Tables 1–3;
HR-ESI-TOF-MS m/z: 1103.5248 [M+Na]+ (calcd. for C51H84NaO24
:
1103.5250).

254
Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
4.9. Compound 3
4.14. Compound 5
(25R)-2
anosyl-(1 ? 2)-O-[b-
a
,14
a
-Dihydroxy-5
a
-spirostan-3b-yl O-b-
D
-galactopyr-
(25R)-3b-[(O-
-(1 ? 3)]-O-b- -glucopyranosyl -(1 ? 4)-O-[
(1 ? 2)]-b-
phous solid; [
a
-
L
-Arabinopyranosyl-(1 ? 2)-O-[b-
D-xylopyranosyl
D
-xylopyranosyl-(1 ? 3)]-O-b-
D-glucopyrano-
D
a-L-rhamnopyranosyl-
25
syl-(1 ? 4)-b-
D-galactopyranoside (3); amorphous solid; [
a]
D-galactopyranosyl)oxy]-5a-spirostan-12-one (5); amor-
D
ꢀ29.8 (c 0.11, MeOH); IR mmax (film) cmꢀ1: 3366 (OH), 2926
a
]
ꢀ23.8 (c 0.08, MeOH); IR mmax (film) cmꢀ1
:
25
D
(CH); for 1H and 13C NMR spectroscopic data, see Tables 1–3;
3433 (OH), 2925 (CH), and 1690 (C@O); for 1H and 13C NMR spectro-
scopic data, see Tables 1–3; HR-ESI-TOF-MS m/z: 1187.5461
[M+Na]+ (calcd. for C55H88NaO26, 1187.5462).
HR-ESI-TOF-MS m/z: 1089.5093 [M+Na]+ (calcd. for C50H82NaO24
,
1089.5094).
4.15. Enzymatic hydrolysis of 5
4.10. Enzymatic hydrolysis of 3
Compound 5 (5.0 mg) was treated with naringinase (90.0 mg) in
HOAc/KOAc buffer (pH 4.3, 3.0 ml) at room temp. for 49 h. The
reaction mixture was purified by silica gel CC (100 g, 2 cm
i.d. ꢁ 20 cm) eluted with CHCl3–MeOH–H2O (7:4:1) to yield hecog-
enin (5a, 3.1 mg) and a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as those for 1 showed the pres-
Compound
3
(10.5 mg) was treated with naringinase
(120.0 mg) in HOAc/KOAc buffer (pH 4.3, 3.0 ml) at room temp.
for 140 h. The reaction mixture was purified by silica gel CC
(90 g, 2 cm i.d. ꢁ 18 cm) eluted with CHCl3–MeOH–H2O (7:4:1) to
yield 2a (4.8 mg) and a sugar fraction. HPLC analysis of the sugar
fraction under the same conditions as those for 1 showed the pres-
ence of
ose (13.37 min, positive optical rotation),
positive optical rotation), -rhamnose (7.35 min, negative optical
rotation), and -xylose (9.88 min, positive optical rotation).
L-arabinose (9.30 min, positive optical rotation),
D-galact-
ence of
D-galactose (13.24 min, positive optical rotation),
D
-glucose
D-glucose (15.43 min,
(15.25 min, positive optical rotation), and
tive optical rotation).
D-xylose (9.42 min, posi-
L
D
4.16. Compound 6
4.11. Compound 4
(25R)-26-[(b-
D
-Glucopyranosyl)oxy]-2
-galactopyranosyl-(1 ? 2)-O-[b-
-galactopyranoside (6);
a
,22
a
-dihydroxy-5
a-furo-
(25S)-2
pyranosyl-(1 ? 2)-O-[b-
anosyl-(1 ? 4)-b-
a
,14
a
,27-Trihydroxy-5
a
-spirostan-3b-yl O-b-
D-galacto-
stan-3b-yl O-b-
D
D-glucopyranosyl-
D-xylopyranosyl-(1 ? 3)]-O-b-
D
-glucopyr-
(1 ? 3)]-O-b-D-glucopyranosyl-(1 ? 4)-b-
D
25
D-galactopyranoside (4); amorphous solid; [a]
25
D
amorphous solid; [a D
]
ꢀ29.3 (c 0.35, MeOH); IR mmax (film)
ꢀ39.7 (c 0.29, MeOH); IR mmax (film) cmꢀ1: 3365 (OH), 2925 (CH);
for 1H and 13C NMR spectroscopic data, see Tables 1–3; HR-ESI-
TOF-MS m/z: 1105.5055 [M+Na]+ (calcd. C50H82NaO25: 1105.5043).
cmꢀ1: 3394 (OH), 2923 (CH); for 1H and 13C NMR spectroscopic data,
see Tables 1–3; HR-ESI-TOF-MS m/z: 1283.5886 [M+Na]+ (calcd. for
C
57H96NaO30, 1283.5884).
4.17. Enzymatic hydrolysis of 6
Compound (3.0 mg) was treated with b-D-glucosidase
4.12. Acid hydrolysis of 4
6
A solution of 4 (5.0 mg) in 1 M HCl in dioxane–H2O (1:1; 2.0 ml)
was heated at 95 °C for 2 h under an Ar atmosphere. The reaction
mixture was neturalized by passing it through an Amberlite col-
umn (IRA-96SB, Organo, Tokyo, Japan; 16–50 mesh, 100 g, 2.0 cm
i.d. ꢁ 30 cm). The mixture was then eluted through a Diaion HP-
20 column (50 g, 2.0 cm i.d. ꢁ 15 cm) with MeOH–H2O (2:3)
MeOH, and EtOH–Me2CO (1:1). The fraction eluted with EtOH–
Me2CO (1:1) (3.0 mg) was purified by silica gel CC (50 g, 2.0 cm
i.d. ꢁ 17 cm) eluted with CHCl3–MeOH (10:1) to give 4a,
(1.0 mg). HPLC analysis of the fraction eluted with MeOH–H2O
(2:3) (1.3 mg) under the same conditions as those used for 1
(10.0 mg) in HOAc/NaOAc buffer (pH 5.0, 3.0 ml) at room temp.
for 27 h. The reaction mixture was purified by silica gel CC
(100 g, 2 cm i.d. ꢁ 25 cm) eluted with CHCl3–MeOH–H2O (7:4:1)
to yield 1 (2.0 mg) and a sugar fraction (0.9 mg). HPLC analysis of
the sugar fraction under the same conditions as those for 1 showed
the presence of D-glucose (15.36 min, positive optical rotation).
A solution of 6 (4.0 mg) was hydrolyzed with naringinase
(90.0 mg) under the same conditions described for 1 to yield 1a
(4.2 mg) and a sugar fraction. HPLC analysis of the sugar fraction
under the same conditions as those used for 1 showed the presence
of D-galactose (13.49 min, positive optical rotation) and D-glucose
(16.05 min, positive optical rotation).
showed the presence of
D-galactose (13.91 min, positive optical
rotation), -glucose (15.26 min, positive optical rotation), and D-
D
xylose (9.45 min, positive optical rotation).
4.18. Compound 7
(25R)-26-[(b-
-furostan-3b-yl
pyranosyl-(1 ? 3)]-O-b-
ranoside (7); amorphous solid; [
max (film) cmꢀ1: 3398 (OH), 2925 (CH); for 1H and 13C NMR spec-
D
-Glucopyranosyl)oxy]-2
O-b- -galactopyranosyl-(1 ? 2)-O-[b-
-glucopyranosyl-(1 ? 4)-b- -galactopy-
ꢀ26.3 (c 0.40, MeOH); IR
a
,14
a
,22
a
-trihydroxy-
4.13. Compound 4a
5
a
D
D-xylo-
D
D
(25S)-5
a
-Spirostane-2
a,3b,14a,27-tetrol (4a); amorphous so-
25
1.2 (c 0.39, MeOH); IR mmax (film) cmꢀ1: 3349 (OH),
25
a]
D
lid; [a]
D
2925 (CH); 1H NMR (500 MHz, C5D5N): dH 3.58 (1H, dd, J = 11.2,
7.5 Hz, H-27a), 3.54 (1H, dd, J = 11.2, 3.6 Hz, H-27b), 0.98 (3H, s,
Me-18), 0.97 (3H, d, J = 7.0 Hz, Me-21), 0.74 (3H, s, Me-19); 13C
NMR (125 MHz, C5D5N): dC 109.6 (C-22), 87.1 (C-14), 79.3 (C-16),
73.0 (C-3), 70.7 (C-2), 65.1 (C-27), 63.4 (C-26), 61.9 (C-17), 49.2
(C-9), 46.1 (C-1), 45.1 (C-13), 44.3 (C-5), 43.4 (C-20), 39.5 (C-15),
38.8 (C-25), 38.3 (C-8), 37.9 (C-10), 33.5 (C-4), 32.2 (C-12), 30.9
(C-23), 28.2 (C-6), 27.5 (C-7), 23.2 (C-24), 22.5 (C-11), 20.1 (C-
18), 14.1 (C-21), 13.9 (C-19); HR-ESI-TOF-MS (m/z: 447.3125
[M+H]+–H2O, calcd. for C27H43O5: 447.3110).
m
troscopic data, see Tables 1–3; HR-ESI-TOF-MS m/z: 1269.5787
[M+Na]+ (calcd. for C56H94NaO30, 1269.5728).
4.19. Enzymatic hydrolysis of 7
Compound
7 (3.0 mg) was treated with b-D-glucosidase
(10.0 mg) in HOAc/NaOAc buffer (pH 5.0, 3.0 ml) at room temp.
for 27 h. The reaction mixture was purified by silica gel CC
(100 g, 2 cm i.d. ꢁ 25 cm) eluted with CHCl3–MeOH–H2O (7:4:1)

Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
255
to yield 3 (2.2 mg) and
rotation).
D-glucose (15.35 min, positive optical
positive optical rotation),
rotation),
-xylose (9.90 min, positive optical rotation).
D
-glucose (15.24 min, positive optical
L-rhamnose (7.84 min, negative optical rotation), and
D
4.20. Acid hydrolysis of 7
4.26. Cell culture assays and cytotoxicity assays
A solution of 7 (5.0 mg) was subjected to the acid hydrolysis de-
scribed for 4 to give 2a (1.1 mg) and a sugar fraction (0.7 mg). HPLC
analysis of the sugar fraction under the same conditions as those
HL-60 cells were maintained in an RPMI-1640 medium, and
A549 and TIG-3 cells were maintained in MEM containing
heat-inactivated 10% (v/v) FBS supplemented with L-glutamine,
used for 1 showed the presence of
optical rotation), -glucose (15.99 min, positive optical rotation),
and -xylose (9.87 min, positive optical rotation).
D-galactose (13.55 min, positive
D
penicillin G sodium salt (100 units/ml), and streptomycin sulfate
D
(100
l
g/ml). The HL-60 (4 ꢁ 104 cells/ml), A549 (1 ꢁ 104 cells/
ml), and TIG-3 (2 ꢁ 104 cells/ml) cells were continuously treated
with each compound for 72 h, and cell growth was measured with
an MTT reduction assay as previously described (Matsuo and
4.21. Compound 8
(25R)-26-[(b-
(22)-en-3b-yl O-b-
syl-(1 ? 3)]-O-b- -glucopyranosyl-(1 ? 4)-b-
(8); amorphous solid; [
D
-Glucopyranosyl)oxy]-2
-galactopyranosyl-(1 ? 2)-O-[b-
-galactopyranoside
a
-hydroxy-5
a-furost-20
Mimaki, 2010). Briefly, after terminating the cell culture, 10 ll of
D
D
-xylopyrano
5 mg/ml of MTT in PBS was added to every well, and the plate
was reincubated in 5% CO2/air for 4 h at 37 °C. The plate was then
centrifuged at 1500 g for 5 min to precipitate the cells and MTT for-
D
D
25
a
]
ꢀ22.7 (c 0.66, MeOH); IR mmax (film)
D
cmꢀ1: 3374 (OH), 2925 (CH); for 1H and 13C NMR spectroscopic
data, see Tables 1–3; HR-ESI-TOF-MS m/z: 1235.5616 [M+Na]+
(calcd. for C56H92NaO28, 1235.5673).
mazan. An aliquot (150
ll) of the supernatant was removed from
each well, and DMSO (175
ll) was added to dissolve the MTT for-
mazan crystals. The plate was mixed on a microshaker for 10 min,
and then read on a microplate reader at 550 nm. The concentra-
tion, up to 10 lM, resulting in a 50% inhibition (IC50 value) was
4.22. Enzymatic hydrolysis of 8
calculated from the dose–response curve.
Compound
8 (3.0 mg) was treated with b-D-glucosidase
The time course of the antiproliferative effects of compounds
were measured by trypan blue assay (HL-60 cells) and MTT assay
(A549 cells), respectively. The HL-60 cells viability was measured
as a percentage of the total cell number that remained unstained.
MTT assay was conducted for A549 cells in the same way as de-
scribed above. Data are represented the mean value S.E.M. of
three experiments performed in triplicate. Purities of all isolated
compounds were confirmed by NMR spectra, optical rotation, TLC
analysis, and mass spectrometry, respectively. The purity of the
isolated compounds was confirmed to be more than 98% on the ba-
sis of their 1H and 13C NMR spectra, in which no signals arising
from impurities could be observed. The 1H and 13C NMR spectra
are available in the Supplementary information.
(10.0 mg) in HOAc/NaOAc buffer (pH 5.0, 3.0 ml) at room temp.
for 25 h. The reaction mixture was purified by silica gel CC
(100 g, 2 cm i.d. ꢁ 25 cm) eluted with CHCl3–MeOH–H2O (7:4:1)
to yield 11 (2.2 mg) and a sugar fraction (4.8 mg). HPLC analysis
of the sugar fraction under the same conditions as those for 1
showed the presence of
rotation).
D-glucose (15.34 min, positive optical
A solution of 8 (6.5 mg) was hydrolyzed with naringinase
(90.0 mg) under the same conditions described for 1 to yield 1a
(3.0 mg) and a sugar fraction. HPLC analysis of the sugar fraction
under the same conditions as those used for 1 showed the presence
of
D
-galactose (13.13 min, positive optical rotation), and
D
-glucose
(15.69 min, positive optical rotation) and
tive optical rotation).
D-xylose (9.84 min, posi-
4.27. Assay for caspase-3 activation
4.23. Compound 9
Caspase-3 activity was measured using a commercially avail-
able kit (Apopcyto Caspase-3 Colorimetric Assay Kit, MBL, Nagoya,
Japan) as previously described (Matsuo and Mimaki, 2010). Briefly,
HL-60 cells (2 ꢁ 106 cells/ml) and A549 cells (6 ꢁ 105 cells/ml)
were treated with a compound for 6 and 16 h, respectively, and
(25R)-26-[(b-
-arabinopyranosyl-(1 ? 2)-O-[b-
-glucopyranosyl-(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b-
D
-Glucopyranosyl)oxy]-5
a-furost-20(22)-en-3b-yl
O-
a
-
L
D
-xylopyranosyl-(1 ? 3)]-O-
b-
D
a-L
25
D
-galactopyranoside (9); amorphous solid; [
a
]
ꢀ26.7 (c 1.28,
D
MeOH); IR mmax (film) cmꢀ1: 3372 (OH), 2926 (CH); for 1H and
13C NMR spectroscopic data, see Tables 1–3; HR-ESI-TOF-MS m/z:
1335.6180 [M+Na]+ (calcd. for C61H100NaO30, 1335.6197).
the cells were centrifuged and collected. The cell lysate (50
equivalent to 200 g of protein) was mixed with reaction buffer
(2 ꢁ 50 l) containing the substrates for caspase-3 (DEVD-pNA).
ll,
l
l
After incubation for 2 h (HL-60 cells) or 24 h (A549 cells) at
37 °C, the absorbance at 405 nm of the liberated p-nitroanilide
chromophore was measured using a microplate reader. The activ-
ity of caspase-3 was measured in triplicate.
4.24. Enzymatic hydrolysis of 9
Compound
9 (3.0 mg) was treated with b-D-glucosidase
(10.0 mg) in HOAc/NaOAc buffer (pH 5.0, 3.0 ml) at room temp.
for 20 h. The reaction mixture was purified by silica gel CC
(100 g, 2 cm i.d. ꢁ 25 cm) eluted with CHCl3–MeOH–H2O (7:4:1)
4.28. Cell cycle analysis by flow cytometry
to yield 10 (2.9 mg) and
D-glucose (15.89 min, positive optical
rotation).
Cell cycle distribution was performed using a flow cytometer
(FACSCanto II, BD Biosciences, Franklin Lakes, NJ). HL-60 cells
(3 ꢁ 106 cells/ml) and A549 cells (6 ꢁ 105 cells/ml) were treated
with each compound for 24 or 48 h, in separate experiments,
washed with PBS, and fixed with 1% paraformaldehyde at 0 °C for
10 min and 70% EtOH at ꢀ20 °C overnight. The cells were treated
4.25. Acid hydrolysis of 9
A solution of 9 (10.0 mg) was subjected to the acid hydrolysis
described for 4 to give tigogenin (9a, 1.0 mg) and a sugar fraction
(0.9 mg). HPLC analysis of the sugar fraction under the same con-
with 0.25% Triton X-100 and were stained with PI (5 lg/ml) and
ditions as those used for 1 showed the presence of
L
-arabinose
RNase A (1 mg/ml) for 20 min. The DNA content of tumor cells
(9.21 min, positive optical rotation), -galactose (13.87 min,
D
was analyzed and percentage of cells in each phase was calculated.

256
Y. Matsuo et al. / Phytochemistry 96 (2013) 244–256
Kuroda, M., Mimaki, Y., Sashida, Y., Yamori, T., Tsuruo, T., 2000. Galtonioside A, a
Appendix A. Supplementary data
novel cytotoxic cholestane glycoside from Galtonia candicans. Tetrahedron Lett.
41, 251–255.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
09.023.
Liu, H.W., Wang, S.L., Cai, B.Q., Ge, X., Yang, X.J., Kobayashi, H., Yao, X.S., 2003. New
furostanol glycosides from the rhizomes of Dioscorea futschauensis R. Kunth. J.
Asian Nat. Prod. Res. 5, 241–247.
Lu, Y., Luo, J., Kong, L., 2011. Steroidal alkaloid saponins and steroidal saponins from
Solanum surattense. Phytochemistry 72, 668–673.
Matsuo, Y., Mimaki, Y., 2010. Lignans from Santalum album and their cytotoxic
activities. Chem. Pharm. Bull. 58, 587–590.
Mimaki, Y., Kuroda, M., Kameyama, A., Sashida, Y., Hirano, T., Oka, K., 1997.
Cholestane glycosides with potent cytostatic activities on various tumor cells
from Ornithogalum saundersiae bulbs. Bioorg. Med. Chem. Lett. 7, 633–636.
Yokosuka, A., Jitsuno, M., Yui, S., Yamazaki, M., Mimaki, Y., 2009a. Steroidal
glycosides from Agave utahensis and their cytotoxic activity. J. Nat. Prod. 72,
1399–1404.
Yokosuka, A., Sano, T., Hashimoto, K., Sakagami, H., Mimaki, Y., 2009b. Steroidal
glycosides from Furcraea foetida and their cytotoxic activity. Chem. Pharm. Bull.
57, 1161–1166.
Perrone, A., Plaza, A., Bloise, E., Nigro, P., Hamed, A.I., Belisario, M.A., Pizza, C.,
Piacente, S., 2005. Cytotoxic furostanol saponins and a megastigmane glucoside
from Tribulus parvispinus. J. Nat. Prod. 68, 1549–1553.
Tsukamoto, S., 1989. The Grand Dictionary of Horticulture, vol. 4. Shogakukan,
Tokyo, pp. 339–340.
References
Barile, E., Capasso, R., Izzo, A.A., Lanzotti, V., Sajjadi, S.E., Zolfaghari, B., 2005.
Structure–activity relationships for saponins from Allium hirtifolium and Allium
elburzense and their antispasmodic activity. Planta Med. 71, 1010–1018.
Cai, L., Wu, Y., Zhang, J., Pei, F., Xu, Y., Xie, S., Xu, D., 2001. Steroidal saponins from
Tribulus terrestris. Planta Med. 67, 196–198.
Gvazava, L.N., Kikoladze, V.S., 2006. Steroidal glycosides from Digitalis ciliate leaves.
Chem. Nat. Compd. 42, 562–566.
Gvazava, L.N., Kikoladze, V.S., 2010. Furostane-type steroidal saponin from Digitalis
ciliate. Chem. Nat. Compd. 46, 246–249.
Ikeda, T., Tsumagari, H., Nohara, T., 2000. Steroidal oligoglycosides from Solanum
nigrum. Chem. Pharm. Bull. 48, 1062–1064.
Kubo, S., Mimaki, Y., Terao, M., Sashida, Y., Nikaido, T., Ohmoto, T., 1992. Acylated
cholestane glycosides from the bulbs of Ornithogalum saundersiae.
Phytochemistry 31, 3969–3973.
Yokosuka, A., Mimaki, Y., Sashida, Y., 2002. Steroidal and pregnane glycosides from
the rhizomes of Tacca chantrieri. J. Nat. Prod. 65, 1293–1298.