Steroidal glycosides from the bulbs of Fritillaria meleagris and their cytotoxic activities

Article abstract of DOI:10.1016/j.steroids.2013.02.012

Steroidal glycosides (1-18), including 10 new compounds (1-10), were isolated from the bulbs of Fritillaria meleagris (Liliaceae). The structures of the new compounds were determined by two-dimensional (2D) NMR analysis, and by hydrolytic cleavage followed by spectroscopic and chromatographic analysis. The isolated compounds and their aglycones were evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells. Morphological observation and flow cytometry analysis showed that 5β-spirostanol glycoside (2) and a cholestane derivative (17a) induced apoptotic cell death in HL-60 cells through different mechanisms of action. Furthermore, the (22R)-spirosolanol glycoside (11) selectively induced apoptosis in A549 cells without affecting the caspase-3 activity level.

Full text of DOI:10.1016/j.steroids.2013.02.012

Steroids 78 (2013) 670–682
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroidal glycosides from the bulbs of Fritillaria meleagris
and their cytotoxic activities
⇑
⇑
Yukiko Matsuo , Daisuke Shinoda, Aina Nakamaru, Yoshihiro Mimaki
Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, 1432-1, Horinouchi, Hachioji, 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 3 August 2012
Received in revised form 29 January 2013
Accepted 27 February 2013
Available online 13 March 2013
Steroidal glycosides (1–18), including 10 new compounds (1–10), were isolated from the bulbs of Fritil-
laria meleagris (Liliaceae). The structures of the new compounds were determined by two-dimensional
(2D) NMR analysis, and by hydrolytic cleavage followed by spectroscopic and chromatographic analysis.
The isolated compounds and their aglycones were evaluated for cytotoxic activity against HL-60 human
promyelocytic leukemia cells and A549 human lung adenocarcinoma cells. Morphological observation
and flow cytometry analysis showed that 5b-spirostanol glycoside (2) and a cholestane derivative
(17a) induced apoptotic cell death in HL-60 cells through different mechanisms of action. Furthermore,
the (22R)-spirosolanol glycoside (11) selectively induced apoptosis in A549 cells without affecting the
caspase-3 activity level.
Keywords:
Fritillaria meleagris
Liliaceae
Steroidal glycosides
HL-60 cells
Ó 2013 Elsevier Inc. All rights reserved.
A549 cells
Apoptosis
1. Introduction
2. Experimental
Plants of the genus Fritillaria belong to the family Liliaceae, and
their bulbs contain steroidal alkaloids with various biological activ-
ities such as anticholinergic, antitussive, expectorant, and anti-
inflammatory effects [1–3]. Fritillaria meleagris L., commonly called
snake’s head fritillary, is a perennial plant distributed widely
throughout Asia and northwestern Europe. The plant grows to a
height of 30–40 cm and has flowers with a checkered pattern [4].
Only one systematic chemical characterization of F. meleagris has
been conducted; a publication in 1958 reported the putative pres-
ence of two alkaloids by paper chromatography [5]. As part of our
continuing chemical investigation of the saponin constituents of
Liliaceae plants [6], a phytochemical screen of the bulbs of F. mel-
eagris was performed. We report the discovery of a new steroidal
alkaloid (1), two new spirostanol glycosides (2, 3), five new furost-
anol glycosides (4–8), two new cholestane-type glycosides (9, 10),
together with eight known compounds (11–18). Extensive spectro-
scopic studies were conducted to determine the structures of the
new steroidal glycosides, including two-dimensional (2D) NMR,
and hydrolytic cleavage followed by spectroscopic and chromato-
graphic analysis. The cytotoxicity of the isolated compounds and
their aglycones was evaluated against HL-60 human promyelocytic
leukemia cells and A549 human lung adenocarcinoma cells.
2.1. General methods
Optical rotations were obtained using a P-1030 (Jasco, Tokyo,
Japan) automatic digital polarimeter. IR spectra were recorded
with
a FT-IR 620 spectrophotometer (Jasco). NMR spectra
(500 MHz for 1H NMR) were recorded with a DRX-500 spectrome-
ter (Bruker, Karlsruhe, Germany), using standard Bruker pulse pro-
grams. Chemical shifts are given as
d values referenced to
tetramethylsilane (TMS) as an internal standard. HRESITOFMS data
were obtained with an LCT mass spectrometer (Waters-Micromass,
Manchester, U.K.). 5 ppm error in HRESITOFMS data has achieved
the level of accuracy for formula confirmation and established
the molecular formula of isolated compound. Porous-polymer
polystyrene resin Diaion HP-20 (Mitsubishi-Chemical, Tokyo,
Japan), BW-300 silica gel (Fuji-Silysia Chemical, Aichi, Japan), and
octadecylsilanized (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, Darmstadt,
Germany) 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
composed of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010
controller (Tosoh), an RI-8010 detector (Tosoh), and a Rheodyne
injection port. A TSK gel ODS-100Z column (10 mm i.d. ꢀ 250 mm,
⇑
Corresponding authors. Tel.: +81 42 676 4577; fax: +81 42 676 4579.
E-mail addresses: matsuoy@toyaku.ac.jp (Y. Matsuo), mimakyi@toyaku.ac.jp
5
lm, Tosoh) was employed for the preparative HPLC. Purities of
all isolated compounds were confirmed by NMR, optical rotation,
(Y. Mimaki).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.02.012

Y. Matsuo et al. / Steroids 78 (2013) 670–682
671
TLC, and mass spectrometry, respectively. The following materials
and reagents were used for the cell cultures and the assay of cyto-
toxic activities: Spectra Classic microplate reader (Tecan, Salzburg,
Austria); 96-well flat-bottom plate (Iwaki Glass, Chiba, Japan);
JCRB 0085 HL-60 cells and JCRB 0076 A549 cells (Human Science
Research Resources Bank, Osaka, Japan); fetal bovine serum (FBS)
(Bio-Whittaker, Walkersville, MD, U.S.A.); 0.25% Trypsin-EDTA
solution, RPMI 1640 medium, minimum essential medium
(MEM), phosphate buffered saline (PBS) (Wako Pure Chemical
Industries, Osaka, Japan), etoposide, and 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma, St.
Louis, U.S.A.); penicillin G sodium salt and streptomycin sulfate
(Gibco, Grand Island, NY, U.S.A.). Paraformaldehyde (Wako Pure
Chemical Industries, Osaka, Japan), Triton X-100 (Sigma), propidi-
um iodide (PI) (Molecular Probes, Eugene, OK, U.S.A.), and ribonu-
clease A (RNase) (Wako Pure Chemical Industries, Osaka, Japan). All
other chemicals used were of biochemical reagent grade.
2.3.2. Acid hydrolysis of 1
A solution of 1 (10.0 mg) in 1 M HCl in dioxane/H2O (1:1;
3.0 mL) was heated at 95 °C for 1 h under an Ar atmosphere. The
reaction mixture was cooled, neturalized with aqueous 1 M NaOH
(2.0 mL), diluted with H2O (20 mL), and extracted with CHCl3
(20 mL ꢀ 3). The CHCl3 extract (7.8 mg) was purified by prepara-
tive TLC (CHCl3/MeOH, 30:1) to give tomatidenol (1a, 2.4 mg). Ste-
roid aglycones which were stable towards acid were obtained by
the above procedures. The purity of aglycone was confirmed by
NMR, optical rotation, TLC, and mass spectrometry. The aqueous
residue (2.9 mg) was directly analyzed by HPLC under the follow-
ing conditions that b-
as one peak: Capcell Pak NH2 UG80 column (4.6 mm
i.d. ꢀ 250 mm, 5 m, Shiseido, Tokyo, Japan); MeCN/H2O (85:15);
detection by refractive index (RI) and optical rotation (OR); flow
rate of 1.0 mL/min. -Glucose and -rhamnose in the aqueous res-
D-glycoside and a-D-glycoside were detected
l
D
L
idue were identified by comparing their retention times (tR [min])
and signs of optical rotation with those of authentic samples [7,8]:
L-rhamnose (7.89, negative optical rotation), and D-glucose (13.99,
2.2. Plant material
positive optical rotation).
The bulbs of F. meleagris, which were collected in India in 2007,
were obtained from Sakata-no-Tane (Kanagawa, Japan) and identi-
fied by Dr. Yutaka Sashida, professor emeritus at Tokyo University
of Pharmacy and Life Sciences. A voucher specimen was deposited
in our laboratory (voucher no. 07-004-FM, Department of Medici-
nal Pharmacognosy).
2.3.3. Compound 1a
An amorphous solid; ½a D25
ꢂ57.2 (c 0.05, MeOH) (Lit. ꢂ23.5 [9]);
ꢁ
13C NMR (125 MHz, CDCl3): d 36.7(C-1), 31.6 (C-2), 71.7 (C-3), 42.3
(C-4), 140.8 (C-5), 121.4 (C-6), 32.1 (C-7), 31.4 (C-8), 50.1 (C-9),
37.2 (C-10), 20.9 (C-11), 39.9 (C-12), 40.7 (C-13), 56.0 (C-14),
32.7 (C-15), 78.8 (C-16), 62.0 (C-17), 16.8 (C-18), 19.4 (C-19),
42.8 (C-20), 15.8 (C-21), 99.2 (C-22), 26.6 (C-23), 28.4 (C-24),
29.7 (C-25), 50.0 (C-26), 19.4 (C-27); HRESITOFMS m/z: 414.3374
[M + H]+ (calcd for C27H44NO2: 414.3372).
2.3. Extraction and isolation
The F. meleagris bulbs (fresh weight, 6.0 kg) were extracted with
hot MeOH (20 L) for 12 h. After removing the solvent, the MeOH
extract (300 g) was resuspended in MeOH/H2O (3:7) to pass
through a Diaion HP-20 column (50 mesh, 2000 g, 8.5 ꢀ 60 cm)
and then successively eluted with MeOH/H2O (3:7), MeOH, EtOH,
and EtOAc (each 9 L). CC of the MeOH-eluted fraction (80.0 g) on
silica gel (200–300 mesh, 1500 g, 8.5 ꢀ 30 cm), eluted with a step-
wise gradient mixture of CHCl3/MeOH/H2O (40:10:1; 20:10:1 and
7:4:1) and finally with MeOH alone, provided five fractions (A–
E). Fraction B was chromatographed on ODS silica gel (100–
200 mesh, 1500 g, 8.5 ꢀ 30 cm) eluted with MeCN/H2O (1:4; 1:3;
1:2 and 1:1) to afford 3 (19.1 mg) and 10 (8.7 mg). Fraction C
was separated by an ODS silica gel column (100–200 mesh,
1500 g, 8.5 ꢀ 30 cm) eluted with MeCN/H2O (1:4, 1:3, 1:2 and
1:1) to give 2 (14.6 mg), 13 (3.3 mg), 14 (2.1 mg), 15 (13.0 mg),
16 (3.9 mg), and 17 (37.0 mg). Fraction D was chromatographed
on silica gel (200–300 mesh, 1800 g, 8.5 ꢀ 35 cm) eluted with
CHCl3/MeOH/H2O (30:10:1; 20:10:1 and 7:4:1) and on ODS silica
gel (100–200 mesh, 1500 g, 8.5 ꢀ 30 cm) eluted with MeCN-H2O
(1:3; 1:2 and 1:1) to afford 1 (91.8 mg), 4 (158 mg), 5 (10.1 mg),
6 (32.0 mg), 7 (9.2 mg), 8 (39.4 mg), 9 (3.5 mg), 11 (1.4 mg), 12
(8.0 mg), and 18 (1.3 mg). Isolated compounds have been at-
tempted to crystallize but all attempts were unsuccessful. Steroidal
glycosides were described as ‘‘amorphous solids’’ and melting
point determinations should not be needed for compounds de-
scribed as ‘‘amorphous solids’’.
2.3.4. Compound 2
(25R)-5b-spirostan-3b-ylO-b-D-glucopyranosyl-(1 ? 4)-O-[a-L-
rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranoside (2): an amor-
phous solid; ½a 2D5
ꢁ
ꢂ60.4 (c 0.10, MeOH); IR mmax (film) cmꢂ1
:
3397 (OH), 2927 (CH); 1H NMR (500 MHz, C5D5N) and 13C NMR
(125 MHz, C5D5N), see Tables
1 and 2; HRESITOFMS m/z:
887.4977 [M + H]+ (calcd for C45H75O17: 887.5004).
2.3.5. Enzymatic hydrolysis of 2
Compound 2 (5.0 mg) was treated with naringinase (EC 232-96-
4, Sigma; 554 mg) in a HOAc/KOAc buffer (pH 4.3, 5.0 mL) at room
temperature for 432 h. The reaction mixture was purified by CC on
silica gel (CHCl3/MeOH/H2O; 10:1:0, 7:4:1) (200–300 mesh, 100 g,
2 ꢀ 30 cm) to give smilagenin (2a, 0.3 mg), and a sugar fraction
(2.3 mg). Acid hydrolysis of 2 with 1 M HCl have resulted in giving
only
D-glucose and L-rhamnose, whereas the labile aglycone
decomposed under acidic conditions [10]. The preparation of the
aglycone 2a has been finally completed by enzymatic hydrolysis
under the above conditions. HPLC analysis of the sugar fraction un-
der the same conditions as those for 1 showed the presence of D-
glucose (14.22, positive optical rotation) and
negative optical rotation).
L-rhamnose (7.98,
2.3.6. Compound 2a
An amorphous solid; ½a D25
ꢂ53.6 (c 0.05, MeOH) (Lit. ꢂ61 [11]);
ꢁ
2.3.1. Compound 1
13C NMR (125 MHz, C5D5N): d 30.6(C-1), 28.6 (C-2), 66.1 (C-3), 34.4
(C-4), 37.0 (C-5), 27.2 (C-6), 26.9 (C-7), 35.6 (C-8), 41.0 (C-9), 35.6
(C-10), 21.2 (C-11), 40.1 (C-12), 40.4 (C-13), 56.6 (C-14), 32.2 (C-
15), 81.3 (C-16), 63.2 (C-17), 16.6 (C-18), 24.3 (C-19), 42.0 (C-20),
15.1 (C-21), 109.3 (C-22), 31.8 (C-23), 29.3 (C-24), 30.0 (C-25),
66.9 (C-26), 17.3 (C-27); HRESITOFMS m/z: 417.3382 [M + H]+
(calcd for C27H45O3: 417.3369).
(22S,25S)-spirosol-5-en-3b-ylO -b-D-glucopyranosyl-(1 ? 4)-O-
[a-L-rhamnopyranosyl-(1 ? 2)]-b-D-glucopyranoside (1): amor-
phous solid; ½a 2D5
ꢁ
ꢂ64.8 (c 0.05, MeOH); IR mmax (film) cmꢂ1
:
3445 (OH), 2932 (CH); 1H NMR (500 MHz, C5D5N) and 13C NMR
(125 MHz, C5D5N), see Tables
1 and 2; HRESITOFMS m/z:
884.5045 [M + H]+ (calcd for C45H74O16N: 884.5008).

Table 1
1H and 13C NMR chemical shift assignments for the aglycone moiety of compounds 1–10 in C5D5N.
Position
1
2
3
4
5
dH
J (Hz)
dC
dH
J (Hz)
dC
dH
J (Hz)
dC
dH
J (Hz)
dC
dH
J (Hz)
dC
1
2
ax
eq
ax
eq
0.98
1.73
1.87
2.10
3.88
2.71
2.76
–
ddd
m
m
m
m
13.9, 13.9, 3.8
37.5
1.81
1.49
1.51
1.97
4.21
1.82
1.86
2.14
1.91
1.38
0.97
1.32
1.55
1.31
–
1.21
1.36
1.10
1.69
–
1.09
2.04
1.40
4.60
1.85
0.82
1.07
1.96
1.14
–
m
br d
m
m
m
m
m
br dd
m
m
30.8
1.01
1.79
1.91
2.15
3.86
2.75
ddd
br d
m
m
m
12.4, 12.4, 3.6
12.4
37.6
1.79
1.50
1.49
1.94
4.21
1.78
1.84
2.13
1.91
1.32
0.97
1.31
1.54
1.29
–
1.21
1.35
1.11
1.75
–
1.09
2.03
1.39
4.96
1.95
0.87
1.07
2.23
1.32
–
m
m
m
m
m
m
br d
br d
m
m
m
m
m
30.8
1.85
1.50
1.53
1.98
4.25
1.85
1.91
2.17
1.98
1.41
1.10
1.31
1.48
1.31
–
m
m
m
m
m
m
m
br dd
m
m
m
m
m
31.0
11.5
30.1
26.8
32.1
26.8
26.9
3
4
W1/2 = 21.7
13.1, 12.8
13.1, 5.3
78.1
38.9
W1/2 = 21.1
6.0, 6.0
76.0
30.9
W1/2 = 22.5
78.1
39.0
W1/2 = 19.6
76.1
30.9
W1/2 = 23.9
76.2
31.0
ax
eq
br dd
br dd
m
9.8
10.6
5
6
140.8
121.8
37.1
26.7
–
5.32
140.9
121.8
37.1
26.8
11.3, 3.6
37.2
26.9
5.30
br d
4.9
br d
3.7
7
ax
eq
1.86
1.49
1.54
0.92
–
m
m
m
m
32.4
br ddd
m
m
13.4, 13.4, 2.4
26.7
1.53
1.92
1.64
0.98
–
1.49
1.60
1.50
2.12
–
2.07
2.21
1.51
4.46
–
0.96
1.09
2.27
1.22
–
m
m
m
m
32.4
26.7
26.9
8
9
10
11
31.6
50.4
37.1
21.1
35.5
40.3
35.3
21.1
32.3
50.2
37.1
20.9
35.5
40.3
35.3
21.1
35.3
40.3
35.3
21.5
m
t
7.0
m
1.45
m
m
m
m
br d
m
m
m
m
m
m
m
m
1.38
m
12
ax
eq
1.11
1.67
–
ddd
br dd
11.5, 10.5, 4.5
11.5, 3.5
40.1
40.3
32.0
40.4
1.17
1.73
–
ddd
m
11.9, 11.9, 3.2
40.1
13.8
13
14
15
46.7
56.0
33.1
40.9
56.5
32.1
45.1
54.0
31.8
41.2
56.4
43.9
54.8
34.4
1.02
2.04
1.46
4.17
1.57
0.86
1.05
1.84
1.06
–
m
m
m
m
dd
s
s
m
d
m
m
m
m
dd
s
m
m
m
dd
t
6.2
0.88
2.12
1.45
4.83
2.48
0.71
1.10
–
m
m
m
m
d
a
b
m
m
m
dd
s
s
m
d
32.4
81.2
64.0
16.7
23.8
40.6
16.4
110.6
37.2
16
17
18
19
20
21
22
23
78.7
62.2
16.8
19.8
43.0
16.2
99.4
27.1
81.2
63.1
16.6
23.8
42.0
15.0
109.2
31.8
7.2, 6.1
7.0
90.0
90.1
17.1
19.4
44.7
9.7
84.6
64.7
14.4
23.8
103.6
11.8
152.4
23.7
8.2, 8.2
7.2
8.3, 6.6
7.3, 6.4
6.8
6.8
s
s
m
d
s
s
s
br dd
d
6.9, 6.6
6.9
1.65
–
s
109.8
30.3
ax
eq
1.44
1.73
1.55
m
m
m
1.66
1.61
1.59
br dd
m
m
13.0, 12.5
1.90
2.15
1.58
br dd
br d
m
12.1, 10.4
10.4
2.01
1.95
2.02
1.65
1.91
3.92
3.61
0.97
m
m
m
m
m
dd
dd
d
2.23
2.18
1.85
1.49
1.97
3.96
3.62
1.03
m
m
m
m
m
dd
dd
d
24
29.3
29.2
28.8
28.3
31.5
25
26
1.67
2.94
2.83
0.82
m
dd
dd
d
31.4
50.6
1.56
3.51
3.59
0.69
m
dd
dd
d
30.5
66.9
1.59
3.50
3.52
0.68
m
dd
dd
d
30.4
66.7
34.2
75.2
33.5
75.0
ax
eq
10.8, 10.8
10.8, 3.8
6.5
10.5, 10.5
10.5, 3.0
5.3
10.5, 10.5
10.5, 2.9
5.3
9.3, 5.6
9.3, 6.0
6.7
9.3, 6.8
9.3, 5.5
6.6
27
19.8
17.3
17.3
17.4
17.4
Position
6
7
8
9
10
1
2
ax
eq
ax
eq
0.95
1.73
1.85
2.08
3.84
2.70
2.73
–
5.26
1.89
1.51
1.53
0.97
–
m
m
m
m
m
m
m
37.5
30.1
0.99
1.76
1.86
2.10
3.80
2.71
m
m
m
m
m
m
37.2
30.0
0.97
1.78
1.85
2.09
3.76
2.69
m
m
m
m
m
br s
37.5
30.2
1.01
1.56
1.59
2.05
3.96
1.73
2.40
2.06
–
1.99
2.31
1.63
1.09
–
br dd
br dd
m
m
m
m
br d
dd
15.6, 15.0
15.6, 4.1
36.7
29.5
1.23
1.72
1.95
2.29
3.78
1.66
2.03
2.30
–
2.89
2.46
2.35
2.20
–
ddd
br dd
br dd
m
m
dd
13.0, 13.0, 3.0
13.0, 3.0
13.2, 13.0
37.3
31.4
3
4
W1/2 = 24.5
78.1
38.9
W
1/2 = 21.2
78.2
39.0
W1/2=26.5
78.1
38.9
W1/2 = 25.4
76.7
27.0
W1/2 = 22.5
12.7, 12.2
12.2
70.1
31.9
ax
eq
13.0
12.9, 2.5
br d
m
5
6
7
140.7
121.8
32.4
–
140.8
121.8
32.1
–
140.7
121.8
32.4
56.4
209.7
46.7
56.9
211.8
42.9
br d
m
m
m
m
3.9
5.29
1.48
1.87
1.56
0.89
–
br s
m
br d
m
5.26
1.49
1.89
1.55
0.96
–
br d
m
m
m
m
3.8
ax
eq
dd
dd
m
12.7, 12.7
12.7, 4.6
dd
dd
m
12.7, 12.7
12.7, 3.9
13.3
8
9
10
32.1
50.3
37.1
31.7
50.4
37.1
32.1
50.2
37.1
37.8
53.7
40.9
40.9
46.8
40.7
m
m
ddd
12.0, 12.0, 4.0

Y. Matsuo et al. / Steroids 78 (2013) 670–682
673
2.3.7. Compound 3
(25R)-17
a-hydroxyspirost-5-en-3b-ylO-a-L-rhamnopyranosyl-
(1 ? 2)-b-
D
-xylopyranoside (3): an amorphous solid; ½a D25
ꢂ79.7 (c
ꢁ
0.08, MeOH); IR m
max (film) cmꢂ1: 3365 (OH), 2930 and 2871 (CH);
1H NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N), see Ta-
bles 1 and 2; HRESITOFMS m/z: 731.3961 [M + Na]+ (calcd for C38-
H60O12Na: 731.3982).
2.3.8. Acid hydrolysis of 3
A solution of 3 (8.0 mg) in 0.5 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 cooled, and then neturalized with an Amber-
lite IRA-96SB column (Organo, Tokyo, Japan) (16–50 mesh, 100 g,
2.0 ꢀ 30 cm). The mixture was then eluted through a Diaion HP-
20 column (MeOH/H2O, 2:3; MeOH; EtOH/Me2CO, 1:1) (50 mesh,
50 g, 2.0 ꢀ 15 cm). The fraction eluted with EtOH/Me2CO (1:1)
(5.3 mg) was purified by CC on silica gel (CHCl3/MeOH, 15:1)
(200–300 mesh, 50 g, 2 ꢀ 18 cm) to give pennogenin (3a, 1.9 mg).
HPLC analysis of the fraction eluted with MeOH/H2O (2:3)
(2.7 mg) under the same conditions as those used for 1 showed
the presence of
L-rhamnose (7.70, negative optical rotation) and
D-xylose (9.13, positive optical rotation).
2.3.9. Compound 3a
An amorphous solid; ½a D25
ꢂ87.3 (c 0.08, MeOH) (Lit. ꢂ74.1
ꢁ
[12]); 13C NMR (125 MHz, C5D5N): d 37.8(C-1), 32.4 (C-2), 71.3
(C-3), 43.5 (C-4), 141.9 (C-5), 121.0 (C-6), 32.1 (C-7), 32.4 (C-8),
50.3 (C-9), 37.0 (C-10), 21.0 (C-11), 32.6 (C-12), 44.8 (C-13), 53.1
(C-14), 31.8 (C-15), 90.0 (C-16), 90.1 (C-17), 17.2 (C-18), 19.6 (C-
19), 45.2 (C-20), 9.7 (C-21), 109.8 (C-22), 32.1 (C-23), 28.8 (C-24),
30.2 (C-25), 66.7 (C-26), 17.3 (C-27); HRESITOFMS m/z: 431.3162
[M + H]+ (calcd for C27H43O4: 431.3161).
2.3.10. Compound 4
(25R)-26-[(b-
3b-ylO-b-
(1 ? 2)]-b-
D-glucopyranosyl)oxy]-22a-hydroxy-5b-furostan-
D
-glucopyranosyl-(1 ? 4)-O-[a-L-rhamnopyranosyl-
D
-glucopyranoside (4): an amorphous solid; ½a D25
ꢂ43.7
ꢁ
(c 0.26, MeOH); IR m
max (film) cmꢂ1: 3396 (OH), 2929 (CH); 1H NMR
(500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N), see Tables 1
and 2; HRESITOFMS m/z: 1089.5491 [M + Na]+ (calcd for C51H86O23-
Na: 1089.5458).
2.3.11. Acid hydrolysis of 4
A solution of 4 (20.0 mg) was subjected to the acid hydrolysis
described for 3 to give 2a (1.9 mg) and a sugar fraction (5.6 mg).
HPLC analysis of the sugar fraction under the same conditions as
those used for 1 showed the presence of
D-glucose (13.91, positive
optical rotation) and -rhamnose (7.98, negative optical rotation).
L
2.3.12. Enzymatic hydrolysis of 4
Compound 4 (15.0 mg) was treated with b-D-glucosidase (EC
232-589-7, Sigma) (25.0 mg) in HOAc/NaOAc buffer (pH 5.0,
3.0 mL) at room temperature for 23 h. The reaction mixture was
purified by CC on silica gel (CHCl3/MeOH/H2O, 30:10:1; 7:4:1)
(200–300 mesh, 100 g, 2 ꢀ 20 cm) to yield 2 (10.4 mg) and
D-glu-
cose (13.97, positive optical rotation).
2.3.13. Compound 5
(25R)-26-[(b-D-glucopyranosyl)oxy]-5b-furost-20(22)-en-3b-yl
O-b-
b-
D-glucopyranosyl-(1 ? 4)-O-[a-L-rhamnopyranosyl-(1 ? 2)]-
D
-glucopyranoside (5); an amorphous solid; ½a D25
ꢁ
ꢂ31.7 (c 0.29,
MeOH); IR mmax (film) cmꢂ1: 3445 (OH), 2928 (CH); 1H NMR
(500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N), see Tables 1
and 2; HRESITOFMS m/z: 1049.5518 [M + H]+ (calcd for
C51H85O22: 1049.5533).

Table 2
1H and 13C NMR chemical shift assignments for the sugar moieties of compounds 1–10 in C5D5N.
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
Glc (I) 10
4.95
d
6.8
100.0 Glc (I) 10
4.80
d
7.3
101.9 Xyl 10
4.86
d
7.0
101.2 Glc (I) 10
4.78
d
7.8
101.9 Glc (I) 10
4.82
d
7.5
102.0
76.0
76.5
82.3
76.1
62.1
20
30
40
50
60
4.22 dd 8.8, 6.8
4.23 dd 9.1, 8.8
4.21 dd 9.1, 8.8
77.3
77.7
82.0
76.2
61.9
20
30
40
50
60
4.25 dd 8.4, 7.3
4.23 dd 8.8, 8.4
4.20 dd 8.8, 8.8
76.6
78.0
82.2
76.2
62.0
20
30
40
50
4.19 dd 7.8, 7.0 77.9
4.15 dd 8.0, 7.8 79.6
4.13
4.28 br d 10.6
3.61 dd 10.6, 9.3
20
30
40
50
60
4.20 dd 8.8, 7.8
4.21 dd 8.8, 8.8
4.19 dd 9.2, 8.8
76.1
76.5
82.1
76.0
20
30
40
50
60
4.28 dd 8.2, 7.5
4.21 dd 9.0, 8.2
4.22 dd 9.0, 9.0
3.83
4.52
4.47
6.38 br s
4.72 br s
4.56 dd 9.3, 2.7
4.33 dd 9.3, 9.3
4.81
1.74
5.13
4.06 dd 8.6, 7.9
4.26 dd 8.8, 8.6
4.29 dd 8.8, 8.8
4.74
4.45 br d 13.2
4.31
4.85
m
71.4
67.0
3.85
m
3.81
m
a
b
3.79
m
m
m
m
a
b
4.52 br d 11.0
4.46 br d 11.0
6.25 br s
4.74 br d 1.8
4.58 dd 9.3, 3.2
4.34 dd 9.3, 9.3
4.94
1.76
5.13
a
b
4.50 br d 12.2
4.44 br d 12.2
6.34 br s
4.71 br s
4.53 dd 9.3, 3.0
4.31 dd 9.5, 9.3
4.76
1.72
5.11
4.05 dd 8.5, 7.9
4.22 dd 9.3, 8.5
4.24 dd 9.3, 9.3
3.95
4.44 br d 12.2
4.30 br d 12.2
a
b
4.48 dd 11.1, 2.9 61.9
4.42 br d 11.1
6.33 br s
4.68 br s
4.50 dd 8.8, 3.1
4.30 dd 9.0, 8.8
4.75
1.71
5.10
a
b
Rha
100
101.8 Rha
72.4
72.8
74.1
69.5
100
200
300
400
500
600
101.3 Rha 100
6.35 br s
4.80 br s
4.61 br d 9.3
4.34 dd 9.3, 9.3 74.1
4.95
1.76
102.2 Rha
72.5
72.8
100
101.3 Rha
100
200
300
400
500
600
101.3
72.4
72.7
74.1
69.4
18.8
105.2
75.0
78.1
71.2
78.3
62.1
200
300
400
500
600
72.3
72.6
74.0
69.4
18.7
105.1
74.9
78.3
71.2
78.4
62.0
200
300
400
500
600
200
72.2
72.6
73.9
69.3
18.7
300
400
m
d
d
m
d
d
m
d
69.5
18.6
500
m
d
d
m
d
d
6.2
7.9
18.6
6.1
7.9
5.8
600
6.1
7.8
6.3
7.9
Glc (II) 1000
105.2 Glc (II) 1000
Glc (II) 1000
105.1 Glc (II) 1000
2000
3000
4000
5000
6000
4.06 dd 8.4, 7.9
4.22 dd 9.1, 8.4
4.27 dd 9.1, 9.1
74.9
78.3
71.2
78.5
2000
3000
4000
5000
6000
2000
3000
4000
5000
6000
4.02 dd 8.3, 7.8
4.20 dd 9.1, 8.3
4.21 dd 9.1, 9.1
74.8
78.2
71.1
78.3
2000
3000
4000
5000
6000
3.98
m
m
3.94
m
m
a
b
4.46 dd 11.0, 2.1 62.0
4.33 dd 11.0, 4.8
a
b
a
b
4.41 dd 12.5, 2.4
4.29 br d 12.5
a
b
m
Glc (III) 10000
4.78
d
7.8
104.8 Glc (III) 10000
d
7.8
104.9
75.2
78.6
71.7
78.5
20000
30000
40000
50000
60000
4.00 dd 8.0, 7.8
4.20 dd 8.8, 8.0
4.18 dd 9.2, 8.8
3.90
4.51
4.35
75.1
78.5
71.6
78.4
62.7
20000
30000
40000
50000
60000
4.05 dd 8.6, 7.8
4.24 dd 8.8, 8.6
4.25 dd 8.8, 8.8
3.95 m
4.57 dd 11.8, 2.7 62.9
4.41 dd 11.8, 5.0
m
m
m
a
b
a
b
6
7
8
9
10
Position
dH
J (Hz) dC
Position
dH
J (Hz) dC
Position
dH
J (Hz) dC
Position
dH
J (Hz)
dC
Position
dH
J
dC
(Hz)
Glc (I)
10
4.93
4.22
d
7.7 100.0 Xyl
10
20
4.83
4.19
d
m
7.7
100.8 Xyl
10
4.80
4.17
d
m
7.6
100.8 Glc (I)
77.3
10
20
5.06
d
7.7
102.2 Glc 10
4.96
4.06 dd 8.8,
7.6
4.24 dd 9.0,
8.8
4.29 dd 9.0,
9.0
d
7.6
106.4
75.4
20
30
40
50
60
dd 8.0,
7.7
dd 8.8,
8.8
dd 8.8,
8.8
m
77.3
77.7
82.0
76.1
61.9
77.3
77.4
79.3
64.3
20
30
40
50
4.07 dd 8.6, 7.7 75.2
4.25 dd 8.8, 8.6 78.6
4.15 dd 9.0, 8.8 71.7
20
30
40
50
60
4.23
4.22
3.85
4.51
4.44
6.23
4.73
4.57
4.33
30
40
50
4.23
4.24
m
m
4.19
4.18
m
m
77.4
79.3
64.3
30
40
50
60
78.5
71.5
78.4
a
4.38 brd 10.0
a
4.36 br 11.6
4.10 ddd 9.0, 5.3, 77.3
1.6
4.88 dd 11.4, 1.6 70.3
4.30 m
d
a
dd 11.8,
b
3.63 dd 10.0,
9.2
b
3.60 dd 11.6,
9.3
a
a
4.51 br 10.7 62.4
3.4
d
b
dd 11.8,
b
4.34 dd 11.4, 5.3
b
4.35 brd 10.7
5.5
Rha
100
200
300
400
br
s
br
s
101.8 Rha 100
6.29 br
102.2 Rha 100
6.25 br
102.1 Glc (II)
72.4
100
200
300
400
5.13
d
7.8
105.5
s
s
72.4
72.7
74.1
200
300
400
4.77 br
72.4
72.7
74.1
200
300
400
4.75 br 3.0
4.07 dd 8.6, 7.8 75.4
4.25 dd 8.8, 8.6 78.6
s
d
dd 9.3,
3.3
dd 9.3,
9.3
4.57 br 9.4
4.56 dd 9.4,
3.0
4.33 dd 9.4,
9.4
72.7
d
4.35 dd 9.4,
9.4
74.1
4.27 dd 8.8,8.8
71.9

500
600
4.92
1.75
m
d
69.4
18.7
500
600
4.89
1.76
m
d
69.6
18.7
500
600
4.88
1.75
m
d
69.5
18.6
500
600
3.96
4.53 br
d
4.40 br
d
m
78.5
62.8
6.1
7.8
6.2
7.8
6.2
7.8
a
11.3
11.3
b
Glc (II)
1000
2000
3000
4000
5.12
4.04
4.22
4.26
d
105.2 Glc
(I)
74.9
1000
2000
3000
4000
5.01
d
104.0 Glc
(I)
74.6
1000
2000
3000
4000
4.98
d
103.9
dd 8.3,
7.8
dd 8.8,
8.3
dd 8.8,
8.8
m
br 11.0
d
brd 11.0
4.00 dd 8.4,
3.98 dd 8.4,
74.6 Glc (III)
78.4
1000
2000
3000
4.78
d
7.8
104.9
7.8
4.23 dd 8.8,
8.4
4.24 dd 8.8,
8.8
3.98
4.53 br 12.7
7.8
4.18 dd 8.8,
8.4
4.20 dd 8.8,
8.8
3.96
4.50 br 11.5
d
78.2
71.2
78.4
71.5
4.00 dd 8.6, 7.8 75.1
4.19 dd 8.8, 8.6 78.6
4.28 dd 8.8, 8.8 71.8
71.4
5000
6000
3.97
4.45
78.4
62.1
5000
6000
m
78.2
62.4
5000
6000
m
78.2
62.3
4000
5000
a
a
a
4.03
m
78.5
d
b
4.32
b
4.34 br 12.7
d
b
4.31 brd 11.5
6000
a
4.63 br
d
11.0
63.0
b
4.43 dd 11.0, 5.6
Glc (III)
10000
20000
30000
40000
4.81
4.02
4.22
4.21
d
7.7
104.9 Glc
(II)
1000
’
4.82
d
7.7
104.9 Glc
(II)
75.2
10000
20000
30000
40000
4.81
d
7.6
104.9
75.1
78.6
71.7
dd 8.1,7.7 75.2
20000
30000
40000
4.03 dd 8.2,
4.01 dd 8.2,
7.7
4.22 dd 8.8,
8.2
4.25 dd 8.8,
8.8
3.96
4.56 br 12.5,
4.5
7.6
4.22 dd 8.8,
8.2
4.21 dd 8.8,
8.8
3.94
4.53 br 11.8
dd 8.8,
78.6
71.7
78.6
71.7
8.1
dd 8.8,
8.8
50000
60000
3.95
4.54
m
78.4
62.8
50000
60000
m
78.3
62.8
50000
60000
m
78.5
62.8
a
br 11.8
d
a
a
d
d
b
4.37
br 11.8
d
b
4.38 br 12.5
d
b
4.36 br 11.8
d

676
Y. Matsuo et al. / Steroids 78 (2013) 670–682
2.3.14. Acetylation of 5
2.3.21. Compound 7a
A mixture of 5 (2.0 mg) and Ac2O (1.0 mL) in pyridine (1.0 mL)
was stirred at room temperature for 24 h. After the excess Ac2O
was decomposed by H2O (10 mL), the reaction mixture was ex-
tracted with EtOAc (10 mL ꢀ 3). The EtOAc extract was purified
by preparative TLC (hexane/Me2CO, 1:1) to yield 5b (1.3 mg).
An amorphous solid; ½a D25
ꢂ135.9 (c 0.05, MeOH) (Lit. ꢂ123
ꢁ
[13]); 13C NMR (125 MHz, C5D5N): d 37.8(C-1), 31.7 (C-2), 71.3
(C-3), 42.0 (C-4), 142.0 (C-5), 121.0 (C-6), 32.1 (C-7), 31.8 (C-8),
50.4 (C-9), 37.0 (C-10), 21.2 (C-11), 39.9 (C-12), 40.5 (C-13), 56.7
(C-14), 32.3 (C-15), 81.1 (C-16), 62.9 (C-17), 16.4 (C-18), 19.6 (C-
19), 40.5 (C-20), 15.0 (C-21), 109.3 (C-22), 31.7 (C-23), 29.2 (C-
24), 30.2 (C-25), 66.9 (C-26), 17.3 (C-27); HRESITOFMS m/z:
415.3221 [M + H]+ (calcd for C27H43O3: 415.3212).
2.3.15. Preparation of 5b from 4
A mixture of 4 (10.0 mg) and Ac2O (2.0 mL) in pyridine (2.0 mL)
was stirred at 110 °C for 3 h. After the excess Ac2O was decom-
posed by H2O (10 mL), the reaction mixture was extracted with
EtOAc (10 mL ꢀ 3). The EtOAc extract was purified by preparative
TLC (hexane/Me2CO, 1:1) to yield 5b (1.0 mg).
2.3.22. Compound 8
(25R)-26-[(b-
5-en-3b-yl O-b-
syl-(1 ? 2)]-b-
D-glucopyranosyl)oxy]-17a,22a-dihydroxyfurost-
D
-glucopyranosyl-(1 ? 4)-O-[a-L-rhamnopyrano-
D
-xylopyranoside (8); an amorphous solid; ½a D25
ꢁ
ꢂ62.3 (c 0.25, MeOH); IR mmax (film) cmꢂ1: 3445 (OH), 2934
(CH); 1H NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N),
see Tables 1 and 2; HRESITOFMS m/z: 1073.5187 [M + Na]+ (calcd
for C50H82O23Na: 1073.5145).
2.3.16. Compound 5b
An amorphous solid; ½a D25
ꢂ12.8 (c 0.05, MeOH); IR mmax (film)
ꢁ
cmꢂ1: 2926 (OH), 1749 (C@O); 1H NMR (500 MHz, C5D5N): d 3.71
(1H, m, W1/2 = 28.5 Hz, H-3), 3.49 (1H, dd, J = 9.6, 5.7 Hz, H-26a),
2.21, 2.20, 2.16, 2.12, 2.06 ꢀ 2, 2.04 ꢀ 2, 2.03 ꢀ 2, 2.02, 2.00 ꢀ 2
(each 3H, s, Ac ꢀ 13), 1.68 (3H, s, Me-21), 1.47 (3H, d, J = 6.2 Hz,
Rha-6), 1.07 (3H, s, Me-18 or Me-19), 0.95 (3H, d, J = 6.6 Hz, Me-
27), 0.79 (3H, s, Me-18 or Me-19); HRESITOFMS m/z: 1617.6672
[M + Na]+ (calcd for C77H110O35Na: 1617.6725).
2.3.23. Enzymatic hydrolysis of 8
A solution of 7 (5.0 mg) was hydrolyzed with naringinase
(18.0 mg) under the same conditions described for 2 to yield 3a
(1.5 mg) and a sugar fraction (1.7 mg). HPLC analysis of the sugar
fraction under the same conditions as those used for 1 showed
the presence of
D-glucose (13.95, positive optical rotation),
L
-rham-
nose (7.62, negative optical rotation), and
optical rotation).
D-xylose (9.16, positive
2.3.17. Compound 6
(25R)-26-[(b-
5-en-3b-yl O-b-
syl-(1 ? 2)]-b-
D-glucopyranosyl)oxy]-17a,22a-dihydroxyfurost-
D
-glucopyranosyl-(1 ? 4)-O-[a-L-rhamnopyrano-
2.3.24. Compound 9
D
-glucopyranoside (6); an amorphous solid; ½a D25
ꢁ
(25R)-3b-[(O-b-
D
-glucopyranosyl-(1 ? 6)-b-
D
-glucopyrano-
-cholestane-6,22-dione
max (film)
ꢂ57.1 (c 0.25, MeOH); IR mmax (film) cmꢂ1: 3446 (OH), 2949
(CH); 1H NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N),
see Tables 1 and 2; HRESITOFMS m/z: 1103.5288 [M + Na]+ (calcd
for C51H84O24Na: 1103.5250).
syl)oxy]-26-[(b-D-glucopyranosyl)oxy]-5a
(9); an amorphous solid; ½a D25
ꢁ
ꢂ43.4 (c 0.18, MeOH); IR
m
cmꢂ1: 3364 (OH), 2932 and 2872 (CH), 1707 and 1648 (C@O); 1H
NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N), see Tables
1 and 2; HRESITOFMS m/z: 941.4745 [M + Na]+ (calcd for C45H74-
O19Na: 941.4722).
2.3.18. Enzymatic hydrolysis of 6
A solution of 6 (5.0 mg) was hydrolyzed with naringinase
(16.5 mg) under the same conditions described for 2 to yield 3a
(0.4 mg) and a sugar fraction (2.7 mg). HPLC analysis of the sugar
fraction under the same conditions as those used for 1 showed
2.3.25. Enzymatic hydrolysis of 9
A solution of 9 (3.0 mg) was hydrolyzed with naringinase
(20.0 mg) under the conditions described for 2 to yield 17a
(0.8 mg) and a sugar fraction (1.4 mg). HPLC analysis of the sugar
fraction under the same conditions as those used for 1 showed
the presence of
rhamnose (7.81, negative optical rotation).
A solution of 6 (2.0 mg) with b- -glucosidase (8.0 mg) was sub-
D-glucose (14.02, positive optical rotation) and L-
D
the presence of D-glucose (14.01, positive optical rotation).
jected to the enzymatic hydrolysis described for 4 to yield 14
(1.0 mg) and a sugar fraction (0.5 mg). HPLC analysis of the sugar
fraction under the same conditions as those used for 1 showed
2.3.26. Compound 17a
An amorphous solid; ½a D25
ꢂ34.5 (c 0.03, MeOH) (Lit. ꢂ16.3
ꢁ
the presence of
D
-glucose (14.04, positive optical rotation).
[14]); IR mmax (film) cmꢂ1: 2940 (CH), 1705 (C@O); 13C NMR
(125 MHz, C5D5N): d 37.0 (C-1), 31.8 (C-2), 70.0 (C-3), 27.7 (C-4),
56.9 (C-5), 210.2 (C-6), 46.8 (C-7), 37.9 (C-8), 53.8 (C-9), 40.9 (C-
10), 21.7 (C-11), 39.6 (C-12), 43.2 (C-13), 56.1 (C-14), 24.4 (C-15),
27.7 (C-16), 52.4 (C-17), 12.3 (C-18), 13.3 (C-19), 49.4 (C-20),
16.7 (C-21), 214.0 (C-22), 39.9 (C-23), 31.3 (C-24), 36.2 (C-25),
67.4 (C-26), 17.2 (C-27); HRESITOFMS m/z: 433.3327 [M + H]+
(calcd for C27H45O4: 433.3318).
2.3.19. Compound 7
(25R)-26-[(b- -glucopyranosyl)oxy]-22
3b-yl O-b-
(1 ? 2)]-b-
D
a-hydroxyfurost-5-en-
D
-glucopyranosyl-(1 ? 4)-O-[a-L-rhamnopyranosyl-
D
-xylopyranoside (7); an amorphous solid; ½a D25
ꢂ35.7
ꢁ
(c 0.42, MeOH); IR mmax (film) cmꢂ1: 3445 (OH), 2931 (CH); 1H
NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N), see Tables
1 and 2; HRESITOFMS m/z: 1057.5205 [M + Na]+ (calcd for C50H82-
O22Na: 1057.5195).
2.3.27. Compound 10
(20R,22R)-22-[(b-D-glucopyranosyl)oxy]-3b,14a,20-trihydoxy-
5
a
-cholestan-6-one (10): an amorphous solid; ½a D25
ꢂ6.3 (c 0.03,
ꢁ
2.3.20. Enzymatic hydrolysis of 7
MeOH); IR mmax (film) cmꢂ1: 3420 (OH), 2950 and 2869 (CH),
A solution of 7 (5.0 mg) was hydrolyzed with naringinase
(100 mg) under the same conditions described for 2 to yield dios-
genin (7a, 0.4 mg) and a sugar fraction (1.7 mg). HPLC analysis of
the sugar fraction under the same conditions as those used for 1
1698 (C@O); 1H-NMR (500 MHz, C5D5N) and 13C-NMR (125 MHz,
C5D5N), see Tables
1 and 2; HRESITOFMS m/z: 635.3788
[M + Na]+ (calcd for C33H56O10Na: 635.3771).
showed the presence of
-rhamnose (7.56, negative optical rotation), and
positive optical rotation).
D
-glucose (14.12, positive optical rotation),
2.3.28. Enzymatic hydrolysis of 10
A solution of 10 (5.0 mg) was hydrolyzed with naringinase
(30.0 mg) under the conditions described for 2 to yield tenuifoliol
L
D-xylose (9.39,

Y. Matsuo et al. / Steroids 78 (2013) 670–682
677
(10a, 2.8 mg) and a sugar fraction (1.7 mg). HPLC analysis of the su-
gar fraction under the same conditions as those used for 1 showed
the presence of -glucose (13.89, positive optical rotation).
2.3.31. Compound 12
An amorphous solid; ½a D25
ꢁ
ꢂ36.5 (c 0.36, MeOH) (Lit. ꢂ36.5
D
[14]); 13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
722.4495 [M + H]+ (calcd for C39H64 NO11: 722.4479).
2.3.29. Compound 10a
An amorphous solid; ½a D25
ꢁ
6.22 (c 0.14, MeOH) (Lit. 16.8 [15]); IR
2.3.32. Compound 13
mmax (film) cmꢂ1: 3446 (OH), 2926 (CH), 1693 (C@O); 13C NMR
(125 MHz, C5D5N): d 37.3(C-1), 31.4 (C-2), 70.1 (C-3), 31.9 (C-4),
56.9 (C-5), 211.8 (C-6), 42.9 (C-7), 41.0 (C-8), 46.8 (C-9), 40.8 (C-
10), 21.3 (C-11), 33.0 (C-12), 48.9 (C-13), 83.9 (C-14), 32.4 (C-15),
21.6 (C-16), 50.2 (C-17), 17.8 (C-18), 13.0 (C-19), 76.8 (C-20),
21.2 (C-21), 76.9 (C-22), 30.3 (C-23), 37.2 (C-24), 28.2 (C-25),
22.4 (C-26), 23.2 (C-27); HRESITOFMS m/z: 451.3428 [M + H]+
(calcd for C27H47O5: 451.3424).
An amorphous solid; ½a D25
ꢂ93.8 (c 0.11, MeOH) (Lit. ꢂ136.0
ꢁ
[18]); 13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
739.4292 [M + H]+ (calcd for C39H63O13: 739.4269).
2.3.33. Compound 14
An amorphous solid; ½a D25
ꢁ
ꢂ62.4 (c 0.11, MeOH); 1H-NMR
(500 MHz, C5D5N): d 5.28 (1H, br d, J = 4.9 Hz, H-6), 3.84 (1H, m,
W1/2 = 27.5 Hz, H-3), 3.51 (2H, br d, J = 9.8 Hz, H-26), 1.23 (3H, d,
J = 7.2 Hz, Me-21), 1.09 (3H, s, Me-19), 0.97 (3H, s, Me-18), 0.69
(3H, d, J = 5.8 Hz, Me-27), 6.26 (1H, br s, H-100 of Rha), 5.13 (1H,
d, J = 7.9 Hz, H-1000 of Glc (II)), 4.97 (1H, d, J = 7.7 Hz, H-10 of Glc
(I)), 4.75 (1H, br s, H-200 of Rha), 4.58 (1H, dd, J = 9.2, 3.0 Hz, H-300
of Rha), 4.06 (dd, J = 8.5, 7.9 Hz, H-2000 of Glc (II)), 1.76 (3H, d,
2.3.30. Compound 11
An amorphous solid; ½a D25
ꢂ104.3 (c 0.04, MeOH) (Lit. ꢂ86.5
ꢁ
[16]); 13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
722.4482 [M + H]+ (calcd for C39H64 NO11: 722.4479).
Table 3
13C-NMR (125 MHz, C5D5N) spectral assignments for 11–18.
Position
11
37.5
12
37.5
13
37.6
14
37.5
15
37.5
16
36.7
17
36.7
18
37.7
1
2
30.2
78.3
39.0
140.9
121.8
32.4
31.6
50.3
37.2
21.2
40.1
40.6
56.7
32.6
78.8
63.5
16.5
19.4
41.6
15.7
98.4
34.7
31.1
31.6
48.1
19.8
30.2
78.3
39.0
140.9
121.8
32.4
31.6
50.4
37.2
21.1
40.1
40.7
56.0
33.1
78.7
62.2
16.8
19.4
43.0
16.2
99.4
27.1
29.3
31.4
50.6
19.8
30.2
78.2
39.0
140.8
121.8
32.4
31.6
50.2
37.2
20.9
31.8
45.1
53.0
32.1
90.0
90.1
17.1
19.4
44.8
9.7
30.1
78.1
38.9
140.8
121.8
32.4
31.8
50.2
37.2
21.0
32.0
44.8
53.0
32.1
90.1
90.2
17.1
19.4
45.1
9.7
30.1
78.1
38.9
140.8
121.8
32.2
31.7
50.3
37.1
21.1
39.8
40.5
56.7
31.4
81.4
62.9
16.3
19.4
42.0
15.0
111.8
32.3
29.3
35.5
103.1
16.7
55.6
39.4
76.2
26.5
56.5
209.4
46.7
37.3
53.7
41.0
21.5
39.6
40.9
56.3
31.7
80.8
62.8
16.4
13.1
41.9
14.9
109.2
31.8
29.2
30.6
66.9
17.3
29.4
76.7
27.0
56.3
209.8
46.6
37.7
53.7
40.8
21.5
39.5
43.1
55.9
24.2
27.6
52.3
12.2
13.0
49.3
16.6
213.9
39.6
27.8
33.4
74.9
17.3
30.4
78.0
39.1
140.9
121.8
32.3
32.1
50.3
37.2
21.0
32.0
45.1
53.0
32.5
90.5
90.8
17.2
19.4
43.6
10.5
111.4
36.9
28.0
34.3
75.2
17.5
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
109.8
30.0
28.8
30.4
66.7
17.3
109.8
30.0
28.8
30.4
66.7
17.3
Position
Glc (I)
11
12
Position
Glc (I)
13
14
15
16
Position
17
Position
Glc (I)
18
100.4
77.8
79.7
71.8
77.8
62.7
102.1
72.6
72.9
74.2
69.5
18.7
100.4
77.9
79.6
71.8
77.9
62.7
102.1
72.6
72.8
74.2
69.5
18.7
100.3
77.8
79.6
71.8
77.9
62.7
102.1
72.6
72.8
74.2
69.5
18.7
100.0
77.3
77.7
82.0
76.2
61.9
101.8
72.5
72.8
74.2
69.5
18.7
105.2
75.0
78.3
71.2
78.5
62.1
100.0
77.3
77.7
82.0
76.2
61.9
101.8
72.4
72.8
74.1
69.5
18.6
105.2
75.0
78.3
71.2
78.5
62.1
100.0
78.2
79.5
72.0
78.4
62.8
102.2
72.5
72.8
74.1
69.5
18.7
Glc (I)
102.1
75.3
78.6
71.7
78.5
62.8
104.9
75.1
78.6
71.8
78.5
63.0
101.3
77.9
79.6
71.8
77.9
62.8
102.1
72.6
72.8
74.2
69.5
18.7
105.0
75.2
78.6
71.4
78.5
62.8
Rha
Rha
Glc (II)
Rha
Glc (II)
Glc (II)

678
Y. Matsuo et al. / Steroids 78 (2013) 670–682
J = 6.3 Hz, H-600 of Rha); HRESITOFMS m/z: 923.4651 [MH + Na]+
(calcd for C45H72O18Na: 923.4616).
2.3.34. Compound 15
An amorphous solid; ½a D25
ꢂ60.1 (c 0.59, MeOH) (Lit. ꢂ95.9
ꢁ
[19]); 13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
937.4803 [M + Na]+ (calcd for C46H74O18Na:937.4773).
2.3.35. Compound 16
An amorphous solid; ½a D25
ꢂ96.9 (c 0.15, MeOH) IR mmax (film)
ꢁ
cmꢂ1: 3410 (OH), 2926 (CH), 1708 (C@O); 1H-NMR (500 MHz,
C5D5N): d 4.01 (1H, m, W1/2 = 18.5 Hz, H-3), 3.58 (1H, dd, J = 10.7,
3.7 Hz, H-26a), 3.49 (1H, dd, J = 10.7, 10.7 Hz, H-26b), 2.38 (1H,
dd, J = 12.9, 4.4 Hz, H-7 eq), 1.13 (3H, d, J = 6.9 Hz, Me-21), 1.13
(3H, d, J = 6.9 Hz, Me-21), 1.03 (1H, ddd, J = 13.6, 13.6, 3.8 Hz, H-
1), 0.78 (3H, s, Me-18), 0.77 (3H, s, Me-19), 0.69 (3H, d, J = 5.9 Hz,
Me-27), 6.34 (1H, br s, H-100 of Rha), 4.65 (1H, dd, J = 9.2, 3.0 Hz,
H-300 of Rha), 1.78 (3H, d, J = 6.2 Hz, H-600 of Rha); HRESITOFMS
m/z: 761.4092 [M + Na]+ (calcd for C39H62O13Na: 761.4088).
2.3.36. Compound 17
An amorphous solid; ½a D25
ꢂ40.3 (c 0.10, MeOH) (Lit. ꢂ42.4
ꢁ
[20]); IR mmax (film) cmꢂ1: 3420 (OH), 2939 (CH), 1699 (C@O);
13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
779.4130 [M + Na]+ (calcd for C39H64O14Na: 779.4194).
2.3.37. Compound 18
An amorphous solid; ½a D25
ꢂ109.7 (c 0.08, MeOH) (Lit. ꢂ85.2
ꢁ
[18]); 13C NMR (125 MHz, C5D5N), see Table 3; HRESITOFMS m/z:
871.4699 [M + H–OH–OCH3]+ (calcd for C44H71O17: 871.4691).
2.4. Cell culture assays
The growth of HL-60 and A549 cells was measured with an MTT
assay according to a previously reported method [21].
2.4.1. Assay for caspases-3, -8, and -9 activation
Fig. 1. Steroidal glycosides isolated from Fritillaria meleagris.
The activities of caspases-3, -8, and -9 in HL-60 cells were mea-
sured by using a commercially available kit (Apopcyto Caspases-3,
-8, and -9 Colorimetric Assay Kit, Medical and Biological Laborato-
ries, Aichi, Japan) as previously described [21]. A549 cells (2 ꢀ 106)
were treated with each compound for 12 and for 24 h, in separate
experiments, and the cells were centrifuged and collected. The cell
3. Results and discussion
3.1. Structural elucidation
lysate (50
lL, equivalent to 200
lg of protein) was mixed with por-
The bulbs of F. meleagris (6.0 kg of fresh weight) were extracted
with hot MeOH. The MeOH extract (300 g) was passed through a
Diaion HP-20 column, and the MeOH-eluted fraction (80.0 g), in
which steroidal glycosides were enriched, was subjected to CC
using silica gel and ODS silica gel, and to reversed-phase prepara-
tive HPLC, giving compounds 1–18 (Fig. 1). Compounds 11–18
tions of reaction buffer (50
l
L) containing the substrates for casp-
ases-3, -8, and -9 (DEVD-p-nitroanilide (pNA), IETD-pNA, and
LEHD-pNA). After incubation for 5 h at 37 °C, the absorbance at
405 nm of the liberated pNA chromophore was measured using a
microplate reader. The activities of caspases-3, -8, and -9 were
evaluated in triplicate.
were identified as (22R,25R)- spirosol-5-en-3b-yl O-
pyranosyl-(1 ? 2)-b- -glucopyranoside (11) [16], (22S,25S)-
spirosol-5-en-3b-yl O- -rhamnopyranosyl-(1 ? 2)-b- -glucopy-
ranoside (12) [22,23], (25R)-17 -hydroxy-spirost-5-en-3b-yl O-
-rhamnopyranosyl-(1 ? 2)-b- -glucopyranoside (13) [24], (25R)-
17 -hydroxyspirost-5-en-3b-yl O-b- -glucopyranosyl-(1 ? 4)-O-
-rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside (14) [17],
(25R,26R)-26-methoxyspirost-5-en-3b-yl O-b- -glucopyranosyl-
(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside
(15) [25], (25R)-3b-[(O- -rhamnopyranosyl-(1 ? 2)-b- -gluco-
a-L-rhamno-
D
a-
L
D
2.4.2. Cell cycle analysis by flow cytometry
HL-60 cells (3 ꢀ 106) were treated with each compound for 12
and for 24 h, in separate experiments, washed with PBS, and fixed
with 1% paraformaldehyde at 0 °C for 10 min and 70% EtOH at
ꢂ20 °C overnight. The cells were treated with 0.25% Triton X-100
and were stained with PI for 20 min. Analysis of the cell cycle dis-
tribution was performed using a flow cytometer (FACSCanto II, BD
Biosciences, USA).
a
D
a-
L
a
D
[
a
-
L
D
D
a
-
L
D
a
-L
D

Y. Matsuo et al. / Steroids 78 (2013) 670–682
679
pyranosyl)oxy]-5
copyranosyl)oxy]-26-[(b-
6,22-dione (17) [20], and (25R)-26-[(b-
17 ,22 -dihydroxyfurost-5-en-3b-yl O-
(1 ? 2)-b- -glucopyranoside (18) [24], respectively, by compari-
a
-spirostan-6-one (16) [26], (25R)-3b-[(b-
-glucopyranosyl)oxy]-5 -cholestane-
-glucopyranosyl)oxy]-
-rhamnopyranosyl-
D
-glu-
The 1H NMR spectrum of 3 (C38H60O12) displayed signals for
four steroidal methyl groups at dH 1.22 (d, J = 7.0 Hz), 1.09 (s),
0.96 (s), and 0.68 (d, J = 5.3 Hz), and two anomeric protons at dH
6.35 (br s) and 4.86 (d, J = 7.0 Hz), which suggested that it was a
steroidal diglycoside closely related to 13. Acid hydrolysis of 3 with
D
a
D
a
a
a-L
D
son of their physical and spectroscopic data with literature values.
0.5 M HCl in dioxane/H2O (1:1) furnished (25R)-17
a
-hydroxyspi-
Compound 1 was obtained as an amorphous solid, ½a D25
ꢁ
ꢂ64.8 in
rost-5-en-3b-ol (pennogenin, 3a), [27]
L
-rhamnose, and -xylose.
D
MeOH, and showed a positive color reaction in the Dragendorff test
on TLC, which indicates the presence of an alkaloid. The molecular
formula of 1 was assigned as C45H74O16N based on the HRESI-
TOFMS (m/z 884.5045 [M + H]+, calcd. 884.5008), and the 13C
NMR data (45 carbon signals). The IR spectrum of 1 suggested
the presence of hydroxy groups (3445 cmꢂ1). The 1H NMR spec-
trum showed two singlet signals for tertiary methyl groups at dH
1.05 and 0.86 (each s), and three doublet signals for secondary
methyl groups at dH 1.76 (d, J = 6.2 Hz), 1.06 (d, J = 7.2 Hz), and
0.82 (d, J = 6.5 Hz). The signal at dH 1.76 was assigned to the methyl
group of 6-deoxyhexose. The above spectral properties, together
with a quaternary carbon signal at dC 99.4 (C) and a pair of olefinic
carbon signals at dC 140.8 (C) and 121.8 (CH) in the 13C NMR spec-
trum, and a broad doublet olefinic proton signal at dH 5.30 (br d,
J = 4.9 Hz) in the 1H NMR spectrum suggested that the aglycone
of 1 was a spirosol-5-ene derivative. Furthermore, the 1H and 13C
NMR spectra of 1, contained signals for three anomeric protons
at dH 6.25 (br s), 5.13 (d, J = 7.9 Hz), and 4.95 (d, J = 6.8 Hz), and
the corresponding carbon signals at dC 105.2 (CH), 101.8 (CH),
and 100.0 (CH). Acid hydrolysis of 1 with 1 M HCl in dioxane/
H2O (1:1) gave (22S,25S)-spirosol-5-en-3b-ol (tomatidenol, 1a)
The results of acid hydrolysis of 3 and the comparison of the 1H
and 13C NMR spectra with those of 13 implied that the inner mono-
saccharide constituent of 3 was different from that of 13. Instead of
the signals for a 2-substituted glucopyranosyl moiety, five signals
were observed, which could be assigned to a 2-substituted b-D-
xylopyranosyl residue (Xyl) [dH-1 4.86 (d, J = 7.0 Hz); dC 101.2
(CH), 77.9 (CH), 79.6 (CH), 71.4 (CH), and 67.0 (CH2)]. In the HMBC
spectrum of 3, the H-100 proton of Rha at dH 6.35 showed a long-
range correlation with the C-20 of Xyl at dC 77.9, of which H-10 at
dH 4.86 exhibited a correlation with the C-3 of the aglycone at dC
78.1. Accordingly, 3 was assigned as (25R)-17
a-hydroxyspirost-5-
en-3b-yl O- -rhamnopyranosyl-(1 ? 2)-b- -xylopyranoside.
a
-L
D
The HRESITOFMS data (m/z 1089.5491 [M + Na]+, calcd.
1089.5458) showed 4 had a molecular formula of C51H86O23. The
1H NMR spectrum of 4 showed signals for two tertiary methyl
groups at dH 1.07 and 0.87 (each s), two secondary methyl groups
at dH 1.32 (d, J = 6.8 Hz), and 0.97 (d, J = 6.7 Hz), and for four ano-
meric protons at dH 6.33 (br s), 5.10 (d, J = 7.8 Hz), and 4.78 (d,
2H, J = 7.8 Hz). In addition, an acetalic carbon signal at d 110.6
and a positive color reaction in Ehrlich’s test, suggested that 4
was a furostanol glycoside with four monosaccharides. Enzymatic
[9] as the aglycone, and
D
-glucose and
L
-rhamnose as the carbohy-
hydrolysis of 4 with b-
whereas acid hydrolysis of 4 with 0.5 M HCl in dioxane/H2O (1:1)
gave the corresponding spirostanol sapogenin 2a, -glucose, and
D-glucosidase yielded 2 and D-glucose,
drate moieties. The NOE correlations between Me-21 (dH 1.06) and
H2-23 (dH 1.73 and 1.44), and H-23 eq (dH 1.73) and H-16 (dH 4.17)/
H-17 (dH 1.57) in the NOESY spectrum of 1 were consistent with
the 22S configuration of the aglycone. The monosaccharides and
their absolute configurations were identified by direct HPLC analy-
sis of the hydrolysate. The 1H–1H COSY and the HMQC spectra of 1
suggested that the triglycoside attached to the C-3 hydroxy group,
D
L
-rhamnose. The HMBC spectrum of 4 showed a long range corre-
lation between H-10000 of Glc (III) at dH 4.78 (d, J = 7.8 Hz) and C-26
of the aglycone at dC 75.2, which is typical of naturally occurring
furostanol glycosides. The absolute configuration of the C-22 hy-
droxy group of 4 was established as C-22a based on the NOE cor-
which was composed of a C-2 and C-4 disubstituted b-
anosyl unit (Glc (I)), a terminal -rhamnopyranosyl unit (Rha),
and a terminal b- -glucopyranosyl unit (Glc (II)), was the same
D
-glucopyr-
relations between the signals of the H-20 proton at d 2.23 and the
H2-23 protons at dH 2.01 and 1.95. The 13C NMR signals of C-22 and
its neighboring carbons of 4 were similar to those of a reported
a-L
D
as that of the known compounds 14 and 15. This was determined
by HMBC correlations between the H-100 proton of Rha at dH 6.25
and C-20 of Glc (I) at dC 77.3; the H-1000 proton of Glc (II) at dH
5.13 and C-40 of Glc (I) at dC 82.0; and between the H-10
proton of Glc (I) at dH 4.95 and C-3 of the aglycone at dC 78.1. Thus,
22
assignment of the absolute configuration [28]. Accordingly, the
structure of was assigned as (25R)-26-[(b- -glucopyrano-
-hydroxy-5b-furostan-3b-yl O-b- -glucopyranosyl-
-rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside.
a-hydroxyl furostanol glycoside, which also supported the
4
D
syl)oxy]-22
a
D
(1 ? 4)-O-[
a-
L
D
1 was assigned as (22S,25S)-spirosol-5-en-3b-ylO-b-
osyl-(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside.
The HRESITOFMS data (m/z 887.4977 [M + H]+, calcd.
887.5004) showed that 2 had a molecular formula of C45H74O17
D
-glucopyran-
The spectroscopic data for 5 (C51H84O22) suggested it was a
furostanol glycoside, with a similar structure to 4. However, the
molecular formula of 5 was H2O smaller than that of 4 and the
13C NMR spectrum of 5 indicated the presence of an olefinic func-
tionality [dC 152.4 (C) and 103.6 (C)]. Furthermore, the Me-21 dou-
blet signal observed at dH 1.32 (d, J = 6.8 Hz) and the H-17 signal at
dH 1.95 (dd, J = 7.3, 6.4 Hz) in the 1H NMR spectrum of 4 were re-
placed by a deshielded methyl singlet signal at dH 1.65 (Me-21)
and a doublet signal at dH 2.48 (H-17, d, J = 6.8 Hz), respectively,
in the spectrum of 5. These data suggested that 5 was the
a-L
D
.
The 1H NMR spectrum of 2 showed signals for four steroidal
methyl groups at dH 1.14 (d, J = 6.9 Hz), 1.07 (s), 0.82 (s), and
0.69 (d, J = 5.3 Hz), and for three anomeric protons at dH 6.34
(br s), 5.11 (d, J = 7.9 Hz), and 4.80 (d, J = 7.3 Hz). The 13C NMR
spectrum of 2 showed signals which were assigned to an acetalic
carbon (dC 109.2), four methyl groups (dC 23.8, 17.3, 16.6, and
15.0), and three anomeric carbons (dC 105.1, 101.9, and 101.3).
These NMR data suggested that 2 was a spirostanol glycoside.
Enzymatic hydrolysis of 2 with naringinase gave (25R)-5b-spiro-
D
20(22)-pseudo-furostanol glycoside of 4. This was confirmed by
the fact that the tridecaacetate of 5 (5b) was the same as the prod-
uct obtained by treating 4 with Ac2O in pyridine at 110 °C for 3 h,
during which the dehydration occurred between C-20 and C-22,
and all the hydroxy groups were acetylated. The structure of 5
stan-3b-ol (smilagenin, 2a) [27], D-glucose, and L-rhamnose. The
NOE correlations between H-5 (dH 2.14) and Me-19 (dH 1.07),
and Me-19 and H-8 (dH 1.55) in the NOESY spectrum of 2 pro-
vided evidence for the steroidal aglycone A/B cis (5b) ring func-
tion. Analysis of the 1H NMR, 13C NMR, HMQC, and HMBC
spectra of 2 indicated that the triglycoside linked to C-3 of the
aglycone was the same as that of 1. Based on the above data, 2
was assigned as (25R)-26-[(b-
20(22)-en-3b-yl O-b- -glucopyranosyl-(1 ? 4)-O-[
anosyl-(1 ? 2)]-b- -glucopyranoside.
The HRESITOFMS data (m/z 1103.5288 [M + Na]+, calcd.
1103.5250) showed that the molecular formula of was
51H84O24, and its spectroscopic data showed that it was the 26-
(b- -glucopyranosyl)oxy-22 -hydroxyfurostanol glycoside of 14.
D-glucopyranosyl)oxy]-5b-furost-
D
a-L-rhamnopyr-
D
6
was assigned as (25R)-5b-spirostan-3b-ylO-b-
D-glucopyranosyl-
C
(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside.
a-
L
D
D
a

680
Y. Matsuo et al. / Steroids 78 (2013) 670–682
Enzymatic hydrolysis of 6 with b-
cose, whereas enzymatic hydrolysis of 6 using naringinase gave 3a
as the corresponding spirostanol sapogenin, -glucose, and -rham-
nose. The HMBC correlations of 6 confirmed that the triglycoside
linked to C-3 of the aglycone was the same as that of 14, and that
D
-glucosidase gave 14 and
D
-glu-
The HRESITOFMS data showed that 10 had a molecular formula
of C33H56O10. The IR spectrum of 10 showed a prominent carbonyl
absorption at 1698 cmꢂ1 and the 1H NMR spectrum exhibited sin-
glet signals for three tertiary methyl groups at dH 1.52, 1.35, and
0.81 (each s), two secondary methyl groups at dH 0.83 (d,
J = 6.3 Hz), and 0.82 (d, J = 6.8 Hz), and an anomeric proton at dH
4.96 (d, J = 7.6 Hz). These spectral features suggested that 10 was
also a cholestane glycoside with a carbonyl group. Enzymatic
hydrolysis of 10 with naringinase gave (20R,22R)-3b,20,22-trihy-
D
L
one b-
ture of 6 was assigned as (25R)-26-[(b-
17 ,22 -dihydroxyfurost-5-en-3b-yl-b- -glucopyranosyl-(1 ? 4)-
O-[ -rhamnopyranosyl-(1 ? 2)]-b- -glucopyranoside.
Compound 7 (C50H82O22) appeared to be a furostanol saponin
D
-glucopyranosyl unit was attached to C-26. Thus, the struc-
D-glucopyranosyl)oxy]-
a
a
D
a
-L
D
doxy-5
a
-cholestan-6-one (tenuifoliol, 10a) [15,29] and
D-glucose.
related to 6. The 1H NMR spectrum displayed signals for two ter-
tiary methyl groups at dH 1.05 and 0.90 (each s), two secondary
methyl groups at dH 1.34 (d, J = 6.9 Hz) and 1.00 (d, J = 6.6 Hz),
and for four anomeric protons at dH 6.29 (br s), 5.01 (d,
J = 7.8 Hz), 4.83 (d, J = 7.7 Hz), and 4.82 (d, J = 7.7 Hz). Enzymatic
hydrolysis of 7 with naringinase gave (25R)-spirost-5-en-3b-ol
In the HMBC spectrum of 10, H-10 of the b-
D-glucopyranosyl unit
at dH 4.96 (d, J = 7.6 Hz) showed a long-range correlation with C-
22 of the cholestane aglycone at dC 90.0. The structure of 10 was
identified as (20R,22R)-22-[(b-D-glucopyranosyl)oxy]-3b,14a,20-
trihydoxy-5 -cholestan-6-one.
a
A variety of steroidal glycosides (1–17) were isolated from the
bulbs of F. meleagris, and they were classified as steroidal alkaloid
glycosides of (1, 11, and 12), spirostanol derivatives (2, 3, and 13–
16), furostanol derivatives (4–7, 8, and 18), pseudo-furostanol
derivatives (5), and cholestane derivatives (9 and 17). Compounds
1–10 are new naturally occurring steroidal glycosides, and 3, 7, and
8 are rare types of steroidal glycosides which contain an inner b-D-
xylopyranosyl moiety directly attached to the C-3 hydroxy group
of the aglycone.
(diosgenin, 7a) [27],
1H–1H COSY and HMQC correlations indicated that 7 contains a
C-2 and C-4 disubstituted b- -xylopyranosyl moiety [dH-1 4.83
(1H, d, J = 7.7 Hz); dC 100.8, 77.3, 77.4, 79.3, 64.3 (C-10 to C-60)], a
terminal -rhamnopyranosyl moiety [dH-1 6.29 (1H, br s); dC
102.2, 72.4, 72.7, 74.1, 69.6, 18.7 (C-100 to C-600)], and two terminal
b- -glucopyranosyl moieties [dH-1 5.01 (1H, d, J = 7.8 Hz); dC 104.0,
D-glucose, L-rhamnose, and D-xylose. The
D
a
-L
D
74.6, 78.4, 71.5, 78.2, 62.4 (C-1000 to C-6000) (Glc (I)); dH-1 4.82 (1H, d,
J = 7.7 Hz); dC 104.9, 75.2, 78.6, 71.7, 78.3, 62.8 (C-10000 to C-60000) (Glc
(II))]. In the HMBC spectrum, long-range correlations were ob-
served between H-100 of Rha at dH 6.29 and C-20 of Xyl at dC 77.3;
H-1000 of Glc (I) at dH 5.01 and C-40 of Xyl at dC 79.3; H-10 of Xyl
at dH 4.83 and C-3 of the aglycone at dC 78.2; and between H-10000
of Glc (II) at dH 4.82 and C-26 of the aglycone at dC 75.3. The NOE
correlations between the H-20 proton at dH 2.24 and the H2-23
3.2. Cytotoxic activity
The isolated compounds 1–18 were evaluated for their cyto-
toxic activities against HL-60 and A549 cells using a modified
MTT assay method (Table 4). Compounds 4 and 13–15 exhibited
cytotoxic activities against both HL-60 and A549 cells with IC50
protons at dH 2.05 (2H) were consistent with the C-22
tion. Based on these data, the structure of 7 was assigned as (25R)-
26-[(b- -glucopyranosyl)oxy]-22 -hydroxyfurost-5-en-3b-yl O-b-
-glucopyranosyl-(1 ? 4)-O-[ -rhamnopyranosyl-(1 ? 2)]-b-
xylopyranoside.
a configura-
values ranging from 3.8 0.25
lM to 6.8 0.25 lM. Etoposide
and cisplatin were used as the positive controls, and had IC50 val-
ues of 0.3 0.01 lM and 4.8 0.15 lM against HL-60 cells, and
D
a
D
a
-L
D-
Compound 8 (C50H82O23) showed spectral features similar to
those of 7. However, the molecular formula of 8 was one oxygen
atom in excess of 7 and significant differences were observed in
the signals for ring D (C-13 to C-17). When the 1H and 13C NMR
spectra of 8 were compared with those of 7, the signal for the C-
17 carbon at dC 63.9 (CH) was displaced by the downfield-shifted
quaternary carbon at dC 90.8 (C). Enzymatic hydrolysis of 8 with
Table 4
Cytotoxic activities of 1–18, 1a, 2a, 3a, 7a, 10a, and 17a against HL-60 and A549 cells.
Compounds
IC50
(
l
M)a
HL-60
A549
1
1a
2
2a
3
3a
4
5
6
7
7a
8
9
10
10a
11
12
13
14
15
16
17
17a
18
Etoposide
Cisplatin
5.0 0.16
>10
5.7 0.10
>10
>10
>10
3.8 0.25
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.8 0.25
7.6 0.24
4.5 0.05
>10
>10
>10
>10
>10
>10
7.9 0.16
>10
6.5 0.22
4.4 0.09
5.2 0.02
>10
naringinase gave 3a,
data indicated a hydroxy group was located at C-17
lowed the structure of 8 to be assigned as (25R)-26-[(b-
anosyl)oxy]-17 ,22 -dihydroxyfurost-5-en-3b-yl O-b-
-rhamnopyranosyl-(1 ? 2)]-b-
D
-glucose,
L
-rhamnose, and
D
-xylose. These
in 8 and al-
-glucopyr-
-gluco-
a
D
a
a
D
pyranosyl-(1 ? 4)-O-[
a
-
L
D-
xylopyranoside.
The molecular formula of 9 (C45H74O19) was C6H10O5 in excess
of 17, which corresponded to a hexosyl unit. The 1H and 13C
NMR data for 9 were analogous to those of 17; the cholestane skel-
>10
>10
>10
>10
eton had two carbonyl groups and two b-
ties [Glc (I) and Glc (II)]. However, unlike 17, 9 had signals which
could be assigned to one more b- -glucopyranosyl residue (Glc
(III)). Enzymatic hydrolysis of 9 with naringinase gave (25R)-
3b,26-dihydroxy-5 -cholestane-6,22-dione (17a) [14] and -glu-
D-glucopyranosyl moie-
>10
D
4.4 0.92
6.1 0.04
6.8 0.05
4.7 0.09
>10
>10
7.8 0.09
>10
a
D
cose. In the HMBC spectrum, long-range correlations were ob-
served between H-100 of Glc (II) at dH 5.13 and C-60 of Glc (I) at dC
70.3; H-10 of Glc (I) at dH 5.06 and C-3 of the aglycone at dC 76.7;
and between H-1000 of Glc (III) at dH 4.78 and C-26 of the aglycone
at dC 75.0. The anomeric configuration of the Glc (III) unit was as-
signed as b from the relatively large J value of the anomeric proton
>10
>10
>10
4.8 0.15
2.4 0.06
0.3 0.01
1.1 0.01
a
Data are represented the mean value S.E.M. of three experiments performed
in triplicate.
(7.8 Hz). Thus, the structure of 9 was (25R)-3b-[(O-b-
osyl-(1 ? 6)-b- -glucopyranosyl)oxy]-26-[(b- -glucopyranosyl)
oxy]-5 -cholestane-6,22-dione.
D-glucopyran-
D
D
a

Y. Matsuo et al. / Steroids 78 (2013) 670–682
681
A
B
2, 24 h
2, 12 h
P2: sub-G1
P3: G0/G1
P4: S
P5: G2/M
control
17a,12 h
17a, 24 h
PI
Fig. 2. (A) Caspases-3, -8, and -9 activities in 2, 17a, or etoposide-treated HL-60 cell lysates. HL-60 cells were incubated with 20
l
g/mL of 2, 17a, or etoposide at 37 °C for 17 h.
Each value represents the mean standard error of triplicate measurements. (B) The cell populations in HL-60 cells stained with propidium iodide (PI) determined by flow
cytometry (FACS). HL-60 cells were incubated with 20 g/mL of 2, 17a or etoposide for 12 and 24 h, respectively. The experiments were performed in triplicate.
l
1.1 0.01
l
M and 2.4 0.06
l
M against A549 cells, respectively.
and 17a was evaluated. Although no significant activation of casp-
ases-8 and -9 were observed, caspase-3 was activated when the
HL-60 cells were treated with 2 and 17a at a sample concentration
The (22S)-spirosol glycosides, (1 and 12) only showed cytotoxic
activities against HL-60 cells with respective IC50 values of
5.0 0.16 and 4.4 0.92
(11) was selectively cytotoxic to A549 cells with an IC50 value of
7.9 0.16 M. Interestingly, the absolute configuration of C-22
contributed to the selective cytotoxicity of the spirosol glycosides.
The comparison of the cytotoxic activities of 6, 13, and 14 with
l
M, whereas the (22R)-spirosol glycoside
of 20 lg/mL for 17 h (Fig. 2). Furthermore, the cell cycle distribu-
tion of HL-60 cells treated with 2 and 17a for 12 and 24 h was ana-
lyzed using flow cytometry (Table 5). The sub-G1 population of HL-
60 cells, which was quantified with the apoptosis index, was
4.4 0.46% in the vehicle control. When HL-60 cells were cultured
l
those of 3, 7, and 8, showed that replacing the b-
unit at C-3 of the aglycone with the b- -xylopyranosyl unit dimin-
ished the cytotoxic activities of 6, 13, and 14. This showed that the
b- -glucopyranosyl moiety at C-3 in the cytotoxic glycosides plays
an important role in their activity. Morphological observation of
the cultured tumor cells stained with DAPI suggested that 2 and
17a induced apoptosis in HL-60 cells, and 11 induced apoptosis
in 549 cells (data not shown). The activation of caspase by 1, 11,
D
-glucopyranosyl
with 2 (20 lg/mL) for 12 and 24 h, the sub-G1 population in-
D
creased to 36.0 0.60% and 88.1 0.95%, respectively. This implied
that the inhibition of growth by 2 was mediated by the time-
dependent induction of apoptosis, rather than cell cycle arrest in
HL-60 cells. In contrast, the sub-G1 peak appeared after HL-60 cells
were treated with 17a continuously for 24 h, and the G2/M phase
cell population also increased to 27.7 0.05%. This showed that
17a simultaneously arrested HL-60 cell proliferation in the G2/M
D

682
Y. Matsuo et al. / Steroids 78 (2013) 670–682
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Effects of 2 and 17a on cell cycle distribution of HL-60.a
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% sub G0/G1
% G0/G1
% S
% G2-M
Control
2, 12 h
2, 24 h
17a, 12 h
17a, 24 h
Etoposide
4.4 0.46
36.0 0.60
88.1 0.95
6.0 1.10
17.9 1.30
42.9 4.20
51.5 0.73
32.3 5.82
1.6 0.45
46.3 2.30
22.7 1.60
33.9 1.14
26.4 0.87
13.6 0.83
6.7 0.85
24.2 4.00
18.3 1.42
18.5 3.23
16.4 1.77
9.7 0.99
1.1 0.01
19.9 3.60
27.7 0.05
4.2 1.08
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a
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Phytochemistry
% sub G0/G1
% G0/G1
% S
% G2-M
I
Control
11, 24 h
Etoposide
0.63 0.38
19.4 1.83
15.6 2.20
67.2 2.77
63.7 2.23
58.3 1.05
14.3 0.65
10.7 0.43
14.7 0.70
14.4 1.30
4.8 0.45
10.8 1.35
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