Steroidal glycosides from the underground parts of Yucca glauca and their cytotoxic activities

Article abstract of DOI:10.1016/j.phytochem.2014.02.002

Six steroidal glycosides and 14 known compounds were isolated from the underground parts of Yucca glauca (Agavaceae). Their structures were determined from extensive spectroscopic analysis, including analysis of two-dimensional NMR data, and from chemical

Full text of DOI:10.1016/j.phytochem.2014.02.002

Phytochemistry 101 (2014) 109–115
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Steroidal glycosides from the underground parts of Yucca glauca
and their cytotoxic activities
⇑
⇑
Akihito Yokosuka , Tomoka Suzuki, Satoru Tatsuno, Yoshihiro Mimaki
Department 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:
Six steroidal glycosides and 14 known compounds were isolated from the underground parts of Yucca gla-
uca (Agavaceae). Their structures were determined from extensive spectroscopic analysis, including anal-
ysis of two-dimensional NMR data, and from chemical transformations. The compounds were also
evaluated for cytotoxic activities against HL-60 human leukemia cells and A549 human lung adenocar-
cinoma cells. Four spirostanol glycosides and three furostanol glycosides exhibited cytotoxic activities
against both HL-60 and A549 cells. Two of the compounds induced apoptosis in HL-60 cells.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 30 November 2013
Received in revised form 20 January 2014
Available online 5 March 2014
Keywords:
Soapweed
Yucca glauca
Agavaceae
Steroidal glycosides
Spirostanol glycosides
Furostanol glycosides
HL-60 cells
A549 cells
Apoptosis
Introduction
steroidal glycosides of the underground parts of Y. glauca were
investigated. Six new steroidal glycosides (1–5 and 13) and 14
Plants of the family Agavaceae are widely distributed in tropical
and subtropical regions throughout the world. Plants of the genera
Agave, Cordyline, Dracaena, and Yucca in Agavaceae are rich sources
of steroidal sapogenins and saponins (Mahato et al., 1982; Minaki
et al., 1998; Miyakoshi et al., 2000; Yokosuka and Mimaki, 2009a).
Previous studies of Agave utahensis, Dracaena thalioides, and Cordy-
line terminalis led to isolation of a variety of steroidal glycosides,
some of which showed cytotoxic activities against cultured tumor
cells (Yokosuka and Mimaki, 2007, Yokosuka et al., 2012, 2013a).
For example, the 5b-spirostanol glycosides isolated from whole
plants of A. utahensis induced apoptotic cell death in HL-60 human
promyelocytic leukemia cells via caspase-3 activation (Yokosuka
et al., 2009b).
known compounds (6–12 and 14–20) were isolated. Reported
herein is the structural determination of the new glycosides on
the basis of extensive spectroscopic analysis, including two-
dimensional NMR spectroscopy, and chemical transformations.
Cytotoxic activities of the isolated compounds against HL-60 cells
and A549 human lung adenocarcinoma cells and the apoptotic
induction properties of 1 and 16 in HL-60 cells are also described.
Results and discussion
Structural elucidation
The genus Yucca comprises about 60 species, and Yucca glauca
Nutt. ex J. Fraser is a perennial plant native to the southwest of
the United States (Tsukamoto, 1988). Roots of the plant are known
as soapweed and are an herbal medicine used for the treatment of
inflammation, wounds, and bleeding cuts. As part of a series of
phytochemical studies on plants of the family Agavaceae, the
The underground parts of Y. glauca (2.0 kg) were extracted with
MeOH (5 l). After the solvent was removed, the crude extract was
fractionated by repeated column chromatography on porous poly-
styrene resin (Diaion HP-20), silica gel, and octadecylsilanized
(ODS) silica gel to yield compounds 1–20. Compounds 6–12 and
14–20 were identified as (25R)-5b-spirostan-3b-yl O-b-
syl-(1 ? 2)-b- -galactopyranoside (6) (Agrawal et al., 1985; Nakano
et al., 1989); (25R)-12b-hydroxy-5b-spirostan-3b-yl O-b- -glucopyran-
osyl-(1 ? 2)-b- -galactopyranoside (7) (Nakano et al., 1991); (25R)-
3b-[(O-b- -glucopyranosyl-(1 ? 2)-b- -galactopyranosyl)oxy]-5b-
spirostan-12-one (8) (Pant et al., 1986); 2b-hydroxy-5b-spirost-25
D-glucopyrano-
D
D
D
⇑
Corresponding authors. Tel.: +81 42 676 4577; fax: +81 42 676 4579.
E-mail addresses: yokosuka@toyaku.ac.jp (A. Yokosuka), mimakiy@toyaku.ac.jp
D
D
(Y. Mimaki).
http://dx.doi.org/10.1016/j.phytochem.2014.02.002
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

110
A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
(27)-en-3b-yl O-b-
D
-glucopyranosyl-(1 ? 2)-b-
D
-galactopyrano-
pyranosyl)oxy]-22
et al., 2007); and (25R)-26-[(b-
xy-5b-furostan-3b-yl O-b- -glucopyranosyl-(1 ? 4)-b-D
pyranoside (20) (Cheng et al., 2009), respectively. Compounds 6
and 12 also contain the corresponding 25S isomers by analysis of
the 1H and 13C NMR spectra (Fig. 1).
Compound 1 was obtained as an amorphous solid with a molec-
ular formula of C39H62O13, as determined by HRESI–TOFMS (m/z:
739.4244 [M+H]+), 13C NMR, and DEPT (Table 1). The 1H NMR spec-
trum of 1 contained signals for two tertiary methyl groups at dH
0.95 (3H, s) and 0.81 (3H, s), a secondary methyl group at dH 1.09
(3H, d, J = 6.8 Hz), an exomethylene group at dH 4.81 and 4.77 (each
1H, br s), and two anomeric protons at dH 5.28 (1H, d, J = 7.7 Hz)
and 4.89 (1H, d, J = 7.5 Hz). Acid hydrolysis of 1 with 1 M HCl
yielded a sapogenin identified as 5b-spirost-25(27)-en-3b-ol (1a)
a
-hydroxy-5b-furostan-12-one (19) (Zhang
-glucopyranosyl)oxy]-22 -hydro-
-galacto-
side (9) (Miyakoshi et al., 2000); (25R)-2b-hydroxy-5b-spirostan-
3b-yl O-b- -glucopyranosyl-(1 ? 2)-b- -galactopyranoside (10)
(Nakano et al., 1989); 5b-spirost-25(27)-en-3b-yl O-b- -glucopyr-
anosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-b- -galactopyran-
oside (11) (Miyakoshi et al., 2000); (25R)-5b-spirostan-3b-yl O-b-
-glucopyranosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-b- -gala-
ctopyranoside (12) (Miyakoshi etal., 2000; Inoueet al., 1995); (25R)-
26-[(b- -glucopyranosyl)oxy]-2b,22 -dihydroxy-5b-furostan-3b-
yl O-b- -glucopyranosyl-(1 ? 2)-b- -galactopyranoside (14)
(Peng et al., 1994); (25S)-26-[(b- -glucopyranosyl)oxy]-2b,22
dihydroxy-5b-furostan-3b-yl O-b- -glucopyranosyl-(1 ? 2)-b-
galactopyranoside (15) (Kang et al., 2006); 26-[(b-
syl)oxy]-22 -hydroxy-5b-furost-25(27)-en-3b-yl
pyranosyl-(1 ? 2)-b- -galactopyranoside (16) (Chen et al., 2007);
(25R)-26-[(b- -glucopyranosyl)oxy]-22 -hydroxy-5b-furostan-3b-
yl O-b- -glucopyranosyl-(1 ? 2)-b- -galactopyranoside (17) (Chen
et al., 2007); (25S)-26-[(b- -glucopyranosyl)oxy]-22 -hydroxy-
5b-furostan-3b-yl O-b- -glucopyranosyl-(1 ? 2)-b- -galactopy-
ranoside (18) (Nagumo et al., 1991); (25R)-3b-[(O-b-
glucopyranosyl-(1 ? 2)-b- -galactopyranosyl)oxy]-26-[(b- -gluco-
D
a
D
D
D
D
D
D
D
D
D
D
a
D
D
D
a
D
-
-
D
D
-glucopyrano-
O-b- -gluco-
a
D
D
D
a
(Miyakoshi et al., 2000), D-galactose and D-glucose. Identification
D
D
of monosaccharides, including their absolute configurations, was
carried out by direct HPLC analysis of the hydrolysate. These data
suggested that 1 was a 5b-spirostan diglycoside. The NMR spectro-
scopic data for 1 implied that its sugar moiety was composed of a
D
a
D
D
D
-
D
D
b-D
-galactopyranosyl (4C1) unit (Gal) [dH 4.89 (1H, d, J = 7.5 Hz);
Fig. 1. Structures of compounds 1–20.

A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
111
Table 1
13C NMR chemical shift assignments for the aglycone moiety of 1–5, and 13 in C5D5N.
aglycone at dC 74.8. Thus, 3 was assigned as 3b-[(O-b-
anosyl-(1 ? 2)-O-[b- -xylopyranosyl-(1 ? 3)]-b-
syl)oxy]-5b-spirost-25(27)-en-12-one.
D
-glucopyr-
D
D-galactopyrano-
Position
1
2
3
4
5
13
40.4
Compound 4 had a molecular formula of C44H70O18 as deter-
mined by HRESI–TOFMS data (m/z: 909.4509 [M+Na]+). Its 13C
NMR spectrum was very similar to 3, with the exception of the
aglycone F-ring (C-22–C-27) signals. Comparing the 1H and 13C
NMR spectra of 4 with those of 3 showed that the resonances from
the C-25(27)-exomethylene group in 3 were replaced by those
from a Me-CH group at dH 0.70 (3H, d, J = 5.6 Hz) and 1.57 (1H,
m), and dC 17.3 (Me) and 30.5 (CH) in 4. Acid hydrolysis of 4 gave
(25R)-3b-hydroxy-5b-spirostan-12-one (4a) (Nakano et al., 1991),
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30.7
26.6
75.2
30.7
36.7
26.6
26.8
35.4
40.1
35.1
21.0
40.1
40.8
56.3
32.0
81.5
63.2
16.4
23.9
41.7
14.9
30.6
26.5
75.2
30.7
36.5
26.8
26.4
34.7
41.9
35.7
37.7
30.3
26.5
74.8
30.5
36.0
26.7
26.4
34.7
41.9
35.7
37.7
213.1
55.6
56.0
31.4
80.1
54.3
16.0
23.1
42.5
13.8
109.5
33.2
28.9
144.2
65.0
108.8
30.3
26.5
74.8
30.5
36.1
26.8
26.4
34.7
41.9
35.7
37.7
30.3
26.5
74.8
30.5
36.0
26.8
26.4
34.7
41.9
35.7
37.7
213.0
55.6
56.0
31.4
80.1
53.9
16.0
23.1
42.9
13.8
111.9
41.8
70.5
39.9
65.4
13.6
67.2
81.7
31.7
36.4
26.2
26.7
35.4
41.3
37.0
21.2
40.2
41.1
56.2
32.2
81.2
63.8
16.6
23.8
40.6
16.3
110.3
37.9
28.3
147.2
72.0
110.7
D
-galactose,
as (25R)-3b-[(O-b-
syl-(1 ? 3)]-b- -galactopyranosyl)oxy]-5b-spirostan-12-one.
D
-glucose, and
D
-xylose. Accordingly, 4 was assigned
213.0
55.6
56.0
31.4
80.1
54.3
16.0
23.1
42.5
13.9
109.5
33.2
28.9
144.2
65.1
108.8
213.1
55.6
56.0
31.5
79.8
54.3
16.0
23.1
42.6
13.9
109.3
31.8
29.2
30.5
66.9
17.3
D
-glucopyranosyl-(1 ? 2)-O-[b-
D
-xylopyrano-
D
Compound 5 had a molecular formula of C44H70O19, as deter-
mined by HRESI–TOFMS (m/z: 925.4352 [M+Na]+). The molecular
formula was higher than that of 4 by one oxygen atom. Acid hydro-
lysis of 5 gave
D-galactose, D-glucose, and D-xylose, and the aglycone
decomposed under acidic conditions. Comparison of the 1H and 13
C
NMR assignments of 5 with those of 4 established that portions A–E
(C-1–C-21) of the aglycone and the sugar moiety attached to C-3 of
the aglycone were the same as those of 4. However, differences were
observed in the signals from the F-ring (C-22–C-27). In the 1H-1H
COSY spectrum of 5, an oxymethine proton at dH 4.03 exhibited
spin-coupling correlations with methylene protons at dH 2.33 and
2.02 (H2-23), and methine protons dH 1.85 (H-25). The methine
proton was coupled with oxymethylene protons at dH 3.71 and
3.60 (H2-26) and methyl protons at dH 1.10 (Me-27). This showed
the presence of a hydroxy group at C-24. The 24R and 25S configura-
tions were revealed by ROE correlations from H-24 at dH 4.03 to
H-26ax at dH 3.60 and Me-27 at dH 1.10 observed in the ROESY
spectrum of 5 (Fig. 2). Thus, 5 was assigned as (24R,25S)-3b-
109.4
28.8
33.1
144.3
65.0
108.7
dC 102.2, 81.2, 75.1, 69.7, 76.5, and 62.0] and a b-D-glucopyranosyl
(4C1) unit (Glc) [dH 5.28 (1H, d, J = 7.7 Hz); dC 105.7, 76.7, 77.9, 71.6,
78.3, and 62.7] (Agrawal et al., 1985). The absence of a glycosyla-
tion shift in the carbon resonances suggested that the glucosyl res-
idue was a terminal unit (Table 2). In the HMBC spectrum of 1, the
anomeric proton (H-1) of Glc at dH 5.28 showed a long-range cor-
relation with C-2 of Gal at dC 81.2, and H-1 of Gal at dH 4.89 showed
a correlation with C-3 of the aglycone at dC 75.2. Thus, 1 was
[(O-b-
-galactopyranosyl)oxy]-24-hydroxy-5b-spirostan-12-one.
Compound 13 had a molecular formula of C45H74O20 as deter-
D-glucopyranosyl-(1 ? 2)-O-[b-D-xylopyranosyl-(1 ? 3)]-b-
D
assigned as 5b-spirost-25(27)-en-3b-yl O-b-
(1 ? 2)-b- -galactopyranoside.
D-glucopyranosyl-
mined by HRESI–TOFMS (m/z: 957.4672 [M+H]+). Its 1H NMR spec-
trum showed signals for three steroid methyl groups at dH 1.30 (d,
J = 6.9 Hz), 0.94 (s), and 0.83 (s), an exomethylene group at dH 5.33
and 5.04 (each 1H, br s), as well as resonances for three anomeric
protons at dH 5.28 (1H, d, J = 7.7 Hz), 4.98 (1H, d, J = 7.6 Hz), and
4.89 (1H, d, J = 7.7 Hz). The 1H NMR data, the acetalic carbon signal
at dC 110.3 in the 13C NMR spectrum, and a positive color reaction
in Ehrlich’s test, indicated that 13 was a 22-hydroxyfurostanol
saponin with three monosaccharides (Kiyosawa and Hutoh,
D
Compound 2 was obtained as an amorphous solid with a molec-
ular formula C39H60O14 as determined by HRESI–TOFMS (m/z:
753.4061 [M+H]+) data. Comparison of the 13C NMR spectrum of 2
indicated that it possessed a diglycoside moiety identical to that of
1, but that the aglycone structure was slightly different. The IR
(1705 cmÀ1) and 13C NMR (dC 213.0) spectra indicated that the agly-
cone contained a carbonyl group. The HMBC correlations between C-
12 at dC 213.0 and H2-11 at dH 2.36 (dd, J = 13.6, 13.6 Hz), and dH 2.19
(dd, J = 13.6, 4.8 Hz), H-17 at dH 2.82 (dd, J = 8.2, 7.1 Hz), and Me-18
at dH 1.09 (s) showed that the carbonyl was located at C-12. Acid
hydrolysis of 2 gave 3b-hydroxy-5b-spirost-25(27)-en-12-one (2a)
1968; Nohara et al., 1975). Enzymatic hydrolysis of 13 with b-
D-
glucosidase gave 9 and -glucose. The HMBC spectrum of 13
D
showed a long-range correlation between H-1 of Glc (II) at dH
4.98 and C-26 of the aglycone at dC 72.0, showing that a glucosyl
group was linked to the C-26 hydroxy group of the aglycone, which
is typical of naturally occurring furostanol glycosides (Table 2).
(Miyakoshi et al., 2000), together with
Thus, 2 was assigned as 3b-[(O-b- -glucopyranosyl-(1 ? 2)-b-
galactopyranosyl)oxy]-5b-spirost-25(27)-en-12-one.
D-galactose and D-glucose.
D
D-
Compound 3 had a molecular formula C44H68O18 as determined
by HRESI–TOFMS (m/z: 907.4250 [M+Na]+). Its molecular formula
was larger than that of 2 by C5H8O4, and the 1H NMR spectrum
showed signals for three anomeric protons at dH 5.57 (1H, d,
J = 7.9 Hz), 5.23 (1H, d, J = 7.7 Hz), and 4.88 (1H, d, J = 7.7 Hz). Acid
Accordingly, 13 was assigned as 26-[(b-
2b,22f-dihydroxy-5b-furostan-25(27)-en-3b-yl O-b-
osyl-(1 ? 2)-b- -galactopyranoside.
D
-glucopyranosyl)oxy]-
D-glucopyran-
D
Cytotoxic activity
hydrolysis of 3 yielded 2a,
When the 13C NMR spectrum of 3 was compared with that of 2,
a set of five additional signals corresponding to a terminal b-
xylopyranosyl moiety (Xyl, see Table 1) was observed. The signals
assigned to C-3 of the glucosyl moiety linked to C-3 of the aglycone
and its neighboring carbons varied, whereas all other resonances
were mainly unaffected. In the HMBC spectrum of 3, long-range
correlations were observed between H-1 of Glc at dH 5.57 and
C-2 of Gal at dC 77.6; between H-1 of Xyl at dH 5.23 and C-3 of
Gal at dC 84.2; and between H-1 of Gal at dH 4.88 and C-3 of the
D-galactose, D-glucose, and D-xylose.
The isolated compounds (1–20) were evaluated for their cyto-
toxic activities against HL-60 cells using a modified MTT assay
(Table 3). The 5b-spirostanol glycosides (1, 6, and 9–12) and
5b-furostanol glycosides (13–18 and 20) showed cytotoxic activ-
D
-
ity against HL-60 cells with IC50 values from 2.5 to 17.8 lM.
These compounds were also subjected to a cytotoxic screening
test using A549 cells. Compounds 1, 6, 11, 12, and 16–18 exhib-
ited cytotoxic activity against HL-60 and A549 cells. Compounds
9, 10, and 13–15, which were the 2b-hydroxy derivatives of 1, 6,

112
A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
Table 2
1H and 13C NMR chemical shift assignments for the sugar moieties of 1–5, and 13 in C5D5N.a
1
2
3
1H
13C
1H
13C
1H
13C
Gal
1
2
3
4
4.89 d (7.5)
102.2
81.2
75.1
69.7
76.5
62.0
105.7
76.7
77.9
71.6
78.3
62.7
Gal
1
2
3
4
4.88 d (7.7)
4.66 dd (8.6, 7.7)
4.26 dd (8.6, 3.1)
4.56 br s
4.01 m
4.42 m
5.29 d (7.7)
4.08 d (9.1, 7.7)
4.21 dd (9.1, 9.1)
4.31 dd (9.1, 9.1)
3.85 m
102.4
81.7
75.2
69.8
76.6
62.1
106.1
76.9
78.0
71.7
78.4
62.8
Gal
1
2
3
4
4.88 d (7.7)
101.9
77.6
84.2
69.8
76.4
62.0
104.4
76.4
78.5
72.6
77.9
63.4
4.66 dd (8.5, 7.7)
4.25 dd (8.5, 2.7)
4.54 br s
4.11 m
4.39 m
4.82 dd (9.0, 7.7)
4.28 dd (9.0, 3.1)
4.77 br d (3.1)
4.04 m
4.39 m
5.57 d (7.9)
4.02 dd (9.0, 7.9)
4.23 dd (9.0, 9.0)
4.16 dd (9.0, 9.0)
3.78 m
4.47 dd (11.4, 3.3)
4.34 dd (11.4, 5.4)
5.23 d (7.7)
3.95 dd (8.6, 7.7)
4.10 dd (8.6, 8.6)
4.13 m
5
5
5
6 (2H)
1
2
3
4
5
6a
b
6 (2H)
1
2
3
4
5
6a
b
6 (2H)
1
2
3
4
5
6a
b
1
2
3
4
5a
b
Glc
5.28 d (7.7)
Glc
Glc
Xyl
4.06 dd (8.9, 7.7)
4.18 dd (8.9, 8.9)
4.26 dd (8.9, 8.9)
3.83 m
4.49 dd (11.8, 2.7)
4.42 dd (11.8, 4.5)
4.51 dd (11.6, 2.5)
4.47 m
106.2
75.1
78.5
71.0
67.1
4.19 m
3.60 dd (10.3, 10.3)
4
5
13
1H
13C
1H
13C
1H
13C
Gal
1
2
3
4
4.88 d (7.7)
4.82 dd (9.4, 7.7)
4.28 dd (9.4, 3.1)
4.78 br d (3.1)
4.03 m
101.9
77.7
84.2
69.8
76.4
62.1
Gal
1
2
3
4
4.88 d (7.7)
4.82 dd (9.5, 7.7)
4.28 dd (9.5, 3.1)
4.77 br d (3.1)
4.03 m
101.9
77.7
84.2
69.8
76.4
62.1
Gal
1
2
3
4
5
6a
b
4.89 d (7.7)
4.73 dd (9.3, 7.7)
4.28 dd (9.3, 3.1)
4.51 br s
4.11 m
4.46 m
103.2
81.5
75.1
69.7
76.9
62.0
5
5
6 (2H)
4.39 m
6 (2H)
4.39 m
4.38 m
Glc
Xyl
1
2
3
4
5
6a
b
1
2
3
4
5a
b
5.57 d (7.9)
104.4
76.4
78.5
72.7
77.8
63.4
Glc
Xyl
1
2
3
4
5
6a
b
1
2
3
4
5a
b
5.57 d (7.9)
104.4
76.4
78.5
72.7
77.8
63.4
Glc (I)
1
2
3
4
5
6a
b
1
2
3
4
5
6a
b
5.28 d (7.7)
4.09 d (9.0, 7.7)
4.20 dd (9.0, 9.0)
4.29 dd (9.0, 9.0)
3.85 m
106.0
76.9
77.9
71.6
78.5
62.7
4.02 dd (8.9, 7.9)
4.23 dd (8.9, 8.9)
4.16 dd (8.9, 8.9)
3.77 m
4.46 dd (11.4, 3.2)
4.34 dd (11.4, 5.3)
5.23 d (7.7)
3.95 dd (8.6, 7.7)
4.10 dd (8.6, 8.6)
4.13 m
4.02 dd (8.9, 7.9)
4.23 dd (8.9, 8.9)
4.16 dd (8.9, 8.9)
3.77 m
4.46 dd (11.4, 3.2)
4.34 dd (11.4, 5.3)
5.23 d (7.7)
3.95 dd (8.5, 7.7)
4.10 dd (8.5, 8.5)
4.13 m
4.52 m
4.43 m
106.2
75.1
78.5
71.0
67.1
106.2
75.1
78.5
71.0
67.2
Glc (II)
4.98 d (7.6)
4.06 d (8.9, 7.6)
4.25 dd (8.9, 8.9)
4.23 dd (8.9, 8.9)
3.93 m
103.8
75.1
78.5
71.6
78.4
62.7
4.19 m
3.60 dd (10.5, 10.5)
4.19 m
3.60 dd (10.5, 10.5)
4.54 dd (11.4, 2.0)
4.36 m
a
Values in parentheses are coupling constants in Hz.
HL-60 cells stained with DAPI suggested that HL-60 cell death
caused by 1, 6, 11, 12, and 16–18 was mediated partially through
induction of apoptosis, because nuclear chromatin condensation
and cell shrinkage were observed (Fig. 3). Caspases are cysteine
proteases and play a crucial role in the apoptotic signaling path-
ways. Caspace-3 has been shown to be the executing enzyme for
apoptosis. Compound 1 is a new 5b-spirostanol glycoside with
cytotoxicity against HL-60 cells, and 16 is the corresponding 5b-
furostanol glycoside of 1. When HL-60 cells were treated with 1
Fig. 2. Important ROE correlations in 5.
(20
pase-3 was markedly activated (Figs. 4 and 5). However, 16
(20 g/ml) failed to induce apoptotic cell death in HL-60 cells after
6 h, and required 16 h for caspase-3 activation and induction of
apoptosis in HL-60 cells.
lg/ml) for 6 h, the cell viability was reduced to 5.4% and cas-
and 16–18, were only cytotoxic to HL-60 cells. The 5b-spirosta-
nol glycosides with the C-12 carbonyl group (2–5 and 8) and
C-12 hydroxy group (7) were not cytotoxic to the HL-60 and
A549 cells.
l
Conclusion
Apoptosis induction activity
The underground parts of Y. glauca yielded 20 steroidal gly-
cosides, including five new 5b-spirostanol glycosides (1–5) and
a new 5b-furostanol glycoside (13). 5b-Steroidal glycosides are
Compounds 1, 6, 11, 12, and 16–18 showed cytotoxicity against
HL-60 and A549 cells. Morphological observations of the cultured

A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
113
Table 3
Cytotoxic activities of the isolated compounds 1–20, cisplatin, and etoposide against
HL-60 and A549 cells.
Compounds
IC50
(
l
M)a
HL-60
A549
1
2
2.5 0.47
>20
7.3 0.63
–
b
3
>20
–
4
>20
–
5
>20
–
6
7
3.1 0.35
>20
8.4 0.34
–
8
>20
–
9
10
11
12
13
14
15
16
5.0 0.09
11.3 1.42
4.9 0.43
4.2 0.37
13.3 0.09
17.8 2.47
9.2 1.21
4.4 0.10
3.7 0.55
3.3 0.15
>20
>20
>20
6.0 0.65
5.9 0.43
>20
>20
>20
11.9 0.70
7.0 0.29
9.3 2.07
–
Fig. 4. Caspase-3 activity in the lysates of cells treated with 1, 16, or etoposide HL-
60 cells were incubated at 37 °C for 6 h or 16 h with 20 lg/ml of 1 or 16, or 10 lg/ml
of etoposide (E). The data represent the mean S.E.M. of three experiments. Results
17
18
19
with p < 0.05 were considered significantly different from the control group, and are
⁄
marked with
.
20
Cisplatin
Etoposide
14.3 0.07
1.7 0.06
0.39 0.08
>20
2.1 1.10
–
a
Data are represent the mean value S.E.M. of three experiments performed in
triplicate.
b
Not determined.
Fig. 5. Cell viability of HL-60 cells treated with 1, 16, or etoposide. Cell viability was
determined by an MTT assay after incubation with 20 g/ml of 1, 16, or 10 g/ml of
l
l
etoposide (E) at 6, 16, and 24 h. The data represent the mean S.E.M. of three
experiments.
Experimental
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. NMR spectra were recorded
Fig. 3. Morphology of representative fields of HL-60 cells stained with DAPI after
treatment with 1, 16, or etoposide for 16 h to evaluate fragmented and condensed
nuclear chromatins.
on
a Bruker DRX-500 spectrometer (Karlsruhe, Germany) at
500 MHz for 1H NMR, using standard Bruker pulse programs. Chem-
ical shifts are given as d values with reference to tetramethylsilane
(TMS) as an internal standard. HRESI–TOFMS was recorded on a
Waters-Micromass LCT mass spectrometer (Manchester, UK). Dia-
ion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), silica gel (Fuji-Sily-
sia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto,
Japan) were used for column chromatography (CC). TLC was carried
out on precoated silica gel 60 F254 (0.25 mm thick, Merck, Darms-
tadt, Germany) and RP18 F254S (0.25 mm thick, Merck) plates, and
spots were visualized by spraying the plates with 10% H2SO4 aque-
ous solution, followed by heating. HPLC was performed on a system
composed of a DP-8020 (Tosoh, Tokyo, Japan) pump, a RI-8021
a small group of naturally occurring steroidal glycosides com-
pared to 5a- and 5(6)-ene steroidal glycosides, and are known
to occur only in a limited number of higher plants belonging
to the families Agavaceae and Liliaceae (Yokosuka et al.,
2009b; Higano et al., 2007). Seven compounds (1, 6, 11, 12,
and 16–18) showed cytotoxic activity against both HL-60 and
A549 cells. Compound 1 induced apoptotic cell death in HL-60
cells and caspase-3 activation after 6 h. In contrast, 16 required
16 h for caspase-3 activation and induction of apoptosis in
HL-60 cells.

114
A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
(Tosoh) or a Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector,
and a Rheodyne injection port. A Capcell pak C18 AQ column
IRA-96SB (Organo, Tokyo, Japan) column and was applied to silica
gel column eluted with CHCl3–MeOH (9:1) followed by MeOH to
yield 1a (2.8 mg) and a sugar fraction (3.0 mg). The sugar fraction
was passed through a Sep-Pak C18 cartridge (Waters, Milford, MA,
USA), and was then analyzed by HPLC under the following condi-
tions: column, Capcell Pak NH2 UG80 (4.6 mm i.d. Â 250 mm,
(10 mm i.d. Â 250 mm, ODS 5
lm, Shiseido, Tokyo, Japan) was used
for preparative HPLC. The following reagents were used: RPMI 1640
medium, MEM and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) (Sigma–Aldrich, St. Louis, MO, USA);
fetal bovine serum (FBS) (Bio-Whittaker, Walkersville, MO, USA);
penicillin G sodium salt and streptomycin sulfate (Meiji-Seika,
Tokyo, Japan). All other chemicals used were of biochemical reagent
grade.
5
l
m, Shiseido); solvent, MeCN–H2O (17:3); flow rate, 0.8 ml/
min; detection, RI and OR. Identification of -galactose and -glu-
D
D
cose present in the sugar fraction was carried out by comparison
of their retention times and optical rotations with those of authen-
tic samples. tR (min): 13.88 (
D-galactose, positive optical rotation),
Plant material
14.84 ( -glucose, positive optical rotation).
D
The plant material, defined as the underground parts of Y. glau-
ca Nutt. ex J. Fraser, was obtained from a wholesale firm in Rich-
ters, Ontario, Canada in 1999. A voucher specimen has been
deposited in our laboratory (voucher No. YG-99-001, Department
of Medicinal Pharmacognosy).
Compound 2
3b-[(O-b-
D
-glucopyranosyl-(1 ? 2)-b-D-galactopyranosyl)oxy]-
25
5b-spirost-25(27)-en-12-one; amorphous solid; [
0.10; MeOH); IR
a]
À1.6 (c
D
m
max (film) cmÀ1: 3363 (OH), 2925 (CH), 1705
(C@O), 1077, 1041; 1H NMR (500 MHz, C5D5N): d 4.82 and 4.79
(each 1H, br s, H2-27), 4.55 (1H, m, H-16), 4.46 and 4.04 (each
1H, d, J = 12.3 Hz, H2-26), 4.25 (1H, br s, H-3), 2.82 (dd, J = 8.2,
7.1 Hz, H-17), 2.36 (dd, J = 13.6, 13.6 Hz, H-11a), 2.19 (dd, J = 13.6,
4.8 Hz, H-11b), 1.32 (3H, d, J = 6.9 Hz, Me-21), 1.09 (3H, s,
Me-18), 0.99 (3H, s, Me-19): For 1H NMR spectroscopic data of
the sugar moiety, see Table 1; for 13C NMR (125 MHz, C5D5N) spec-
troscopic data, see Table 2; HRESI–TOFMS m/z: 753.4061 [M+H]+
(calculated for C39H61O14, 753.4061).
Extraction and isolation
The plant material (dry weight 2.0 kg) was extracted with hot
MeOH (2 Â 5 l). The combined MeOH extract was concentrated un-
der reduced pressure, and the viscous concentrate (435 g) was
passed through a Diaion HP-20 column (90 mm i.d. Â 300 mm),
successively eluted with MeOH–H2O (3:7, 5:5), MeOH, EtOH, and
EtOAc (5 l each). Silica gel CC (75 mm i.d. Â 220 mm) of the MeOH
eluted fraction (50 g), and elution with a stepwise gradient mixture
of CHCl3–MeOH–H2O (9:1:0, 40:10:1, 20:10:1, 1:1:0), and finally
with MeOH alone, gave 12 fractions (I–XII). Fraction VI was applied
to an ODS silica gel column eluted with MeCN–H2O (1:2, 2:3, 1:1)
to give 1 (43.5 mg), 6 (315 mg), and 8 (152 mg), respectively. Frac-
tion VIII was separated by ODS silica gel CC eluted with MeOH–
H2O (6:4; 2:1; 7:3; 8:2) to give 2 (10.8 mg), 3 (11.0 mg), 4
(265 mg), 7 (379 mg), 9 (158 mg), 10 (94.7 mg), 11 (6.1 mg), and
12 (9.6 mg). Fraction IX was separated by ODS silica gel CC eluted
with MeCN–H2O (1:4, 1:3, 1:2) to give nine subfractions (IXa–IXi).
Fraction IXb was purified on a silica gel column eluted with CHCl3–
MeOH–H2O (30:10:1, 20:10:1) to give 19 (59 mg). Fraction IXc was
purified on a silica gel column eluted with CHCl3–MeOH–H2O
(20:10:1) to afford 5 (10.0 mg). Fraction IXd was purified by pre-
parative HPLC using MeCN–H2O (1:4) to yield 16 (13.5 mg), 17
(315 mg), and 18 (180 mg). Fraction IXg was purified on a silica
gel column eluted with CHCl3–MeOH–H2O (30:10:1) to afford 20
(9.0 mg). Fraction XI was purified on an ODS silica gel column
eluted with MeCN–H2O (1:4, 2:7, 2:5, 2:3) and by preparative HPLC
using MeCN–H2O (1:4) to yield 13 (22.0 mg), 14 (21.5 mg), and 15
(19.0 mg).
Compound 3
3b-[(O-b-D-glucopyranosyl-(1 ? 2)-O-[b-D-xylopyranosyl-(1 ? 3)]-
b-
solid; [a]D À6.8 (c 0.10; MeOH); IR (film) mmax 3376 (OH), 2925
(CH), 1706 (C@O), 1040 cmÀ1 1H NMR (500 MHz, C5D5N) d 4.82
D-galactopyranosyl)oxy]-5b-spirost-25(27)-en-12-one; amorphous
25
;
and 4.78 (each 1H, br s, H2-27), 4.55 (1H, m, H-16), 4.46 and 4.04
(each 1H, d, J = 12.3 Hz, H2-26), 4.24 (1H, br s, H-3), 1.32 (3H, d,
J = 7.0 Hz, Me-21), 1.08 (3H, s, Me-18), 0.96 (3H, s, Me-19), sig-
nals for the sugar moiety, see Table 1; 13C NMR, see Table 2;
HRESI–TOFMS m/z 907.4250 [M+Na]+ (calcd for C44H68
O18Na, 907.4303).
Compound 4
(25R)-3b-[(O-b-
syl-(1 ? 3)]-b- -galactopyranosyl)oxy]-5b-spirostan-12-one; amor-
phous solid; [a]D À8.9 (c 0.10; MeOH); IR (film) mmax 3376 (OH),
2927 (CH), 1706 (C@O), 1074 cmÀ1 1H NMR (500 MHz, C5D5N) d
4.55 (1H, m, H-16), 4.25 (1H, br s, H-3), 3.59 (1H, m, H-26a), 3.49
(1H, dd, J = 10.6, 10.6 Hz, H-26b), 1.57 (1H, m, H-25), 1.36 (3H, d,
J = 6.9 Hz, Me-21), 1.08 (3H, s, Me-18), 0.96 (3H, s, Me-19), 0.70
(3H, d, J = 5.6 Hz, Me-27), signals for the sugar moiety, see Table 1;
13C NMR, see Table 2; HRESI–TOFMS m/z 909.4509 [M+Na]+ (calcd
for C44H70O18Na, 909.4460).
D-glucopyranosyl-(1 ? 2)-O-[b-D-xylopyrano-
D
25
;
Compound 1
5b-Spirost-25(27)-en-3b-yl O-b-
galactopyranoside; amorphous solid; [
IR (film) mmax 3347 (OH), 2925 (CH), 1041 cmÀ1
D-glucopyranosyl-(1 ? 2)-b-D-
25
a
]
D
À4.4 (c 0.10; MeOH);
;
1H NMR
(500 MHz, C5D5N) d 4.81 and 4.77 (each 1H, br s, H2-27), 4.61
(1H, m, H-16), 4.46 and 4.04 (each 1H, d, J = 12.3 Hz, H2-26), 4.32
(1H, br s, H-3), 1.09 (3H, d, J = 6.8 Hz, Me-21), 0.95 (3H, s, Me-19),
0.81 (3H, s, Me-18), signals for the sugar moiety, see Table 1; 13C
NMR, see Table 2; HRESI–TOFMS m/z: 739.4244 [M+H]+ (calcd for
C39H63O13, 739.4269).
Compound 5
(24R,25S)-3b-[(O-b-
anosyl-(1 ? 3)]-b- -galactopyranosyl)oxy]-24-hydroxy-5b-spiro-
stan-12-one: amorphous solid; [a]D À13.6 (c 0.10; MeOH); IR
D-glucopyranosyl-(1 ? 2)-O-[b-D-xylopyr-
D
25
(film) mmax 3377 (OH), 2927 (CH), 1704 (C@O), 1073, 1037 cmÀ1
;
1H NMR (500 MHz, C5D5N) d 4.56 (1H, m, H-16), 4.25 (1H, br s,
H-3), 4.03 (1H, m, H-24), 3.71 (1H, dd, J = 11.1, 4.7 Hz, H-26a),
3.60 (1H, dd, J = 11.1, 11.1 Hz, H-26b), 2.33 (1H, dd, J = 12.6,
4.6 Hz, H-23a), 2.02 (1H, dd, J = 12.6, 12.6 Hz, H-23b), 1.85 (1H,
m, H-25), 1.39 (3H, d, J = 6.9 Hz, Me-21), 1.10 (3H, d, J = 6.5 Hz,
Me-27), 1.07 (3H, s, Me-18), 0.96 (3H, s, Me-19), signals for the
Acid hydrolysis of 1
A solution of 1 (10.2 mg) in 1 M HCl (dioxane-H2O, 1:1; 2 ml)
was heated at 95 °C for 2 h under Ar. After cooling, the reaction
mixture was neutralized by passage through an Amberlite

A. Yokosuka et al. / Phytochemistry 101 (2014) 109–115
115
sugar moiety, see Table 1; 13C NMR, see Table 2; HRESI–TOFMS m/z
The cell lysate (50
with of reaction buffer (2 Â 50
l
l, equivalent to 200
l
g protein) was mixed
925.4352 [M+Na]+ (calcd for C44H70O19Na, 925.4409).
ll) containing the substrates for
caspase-3 [DEVD-pNA (p-nitroanilide)]. After incubation for 2 h at
37 °C, the absorbance at 405 nm of the liberated chromophore
pNA was measured using a microplate reader. The activity of cas-
pase-3 was evaluated in triplicate.
Acid hydrolysis of 2–5
Compounds 2 (6.3 mg), 3 (8.0 mg), 4 (9.7 mg), and 5 (8.3 mg)
were independently subjected to the acid hydrolysis described
for 1 to give the aglycones (2a: 3.5 mg and 2.9 mg from 2 and 3,
respectively, and 4a: 3.0 mg from 4) and sugar fractions (2:
2.7 mg, 3: 1.0 mg, 4: 3.0 mg, and 5: 3.0 mg). HPLC analysis of the
sugar fractions under the same conditions used for 1 showed the
Statistical analysis
For statistical analysis, one-way analysis of variance (ANOVA)
followed by Dunnett’s test was performed. A probability (p) value
of less than 0.05 was considered to represent a statistically signif-
icant difference.
presence of
tR (min): 9.39 (
ose, positive optical rotation), 14.84 (
rotation).
D
-xylose,
D
-galactose, and
D
-glucose in those of 2–5.
-galact-
D-glucose, positive optical
D
-xylose, positive optical rotation), 13.88 (
D
References
Compound 13
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