Isolation and structural elucidation of novel cholestane glycosides and spirostane saponins from Polygonatum odoratum

Article abstract of DOI:10.1016/j.steroids.2013.11.013

Much attention has been paid to cholestane-type steroidal glycosides because of their importance from the perspectives of both chemical diversity and significant biological activities. A phytochemical investigation of the rhizomes of Polygonatum odoratum (Liliaceae) resulted in the isolation of three novel cholestane-type steroidal glycosides (1-3) with unique Δ^14,16-unsaturated D-ring structures as well as two novel spirostane-type steroidal saponins (4 and 5) and three known steroidal glycosides (6-8). Their structures were determined by various spectroscopic methods and chemical reactions. Steroidal saponin 7 showed significant antifungal activity against Candida albicans JCM1542 (MIC 3.1 μg/mL) and Aspergillus fumigatus JCM1738 (MIC 6.3 μg/mL).

Full text of DOI:10.1016/j.steroids.2013.11.013

Steroids 80 (2014) 7–14
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Isolation and structural elucidation of novel cholestane glycosides
and spirostane saponins from Polygonatum odoratum
Hong Bai a, , bWei Li , Huanxin Zhao , Yojiro Anzai , Haiming Li , Huanjie Guo , Fumio Kato ,
⇑
b,
⇑
a
b
a
a
b
Kazuo Koike
a
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2013
Received in revised form 14 October 2013
Accepted 9 November 2013
Available online 27 November 2013
Much attention has been paid to cholestane-type steroidal glycosides because of their importance from
the perspectives of both chemical diversity and significant biological activities. A phytochemical investi-
gation of the rhizomes of Polygonatum odoratum (Liliaceae) resulted in the isolation of three novel
14,16
cholestane-type steroidal glycosides (1–3) with unique
D
-unsaturated D-ring structures as well as
two novel spirostane-type steroidal saponins (4 and 5) and three known steroidal glycosides (6–8). Their
structures were determined by various spectroscopic methods and chemical reactions. Steroidal saponin
Keywords:
7
showed significant antifungal activity against Candida albicans JCM1542 (MIC 3.1
lg/mL) and Aspergillus
Polygonatum odoratum
Cholestane glycosides
Spirostane saponins
Antifungal activity
fumigatus JCM1738 (MIC 6.3 g/mL).
l
Ó 2013 Elsevier Inc All rights reserved.
1
. Introduction
Steroidal glycosides are an important class of natural products,
novel spirostane saponins (4 and 5), and three known compounds,
25S)-(3b,14 )-dihydroxy-spirost-5-ene-3-O-b- -glucopyranosyl-
(1 ? 2)-[b- -xylopyranosyl-(1 ? 3)]-b- -glucopyranosyl-(1 ? 4)-b-
-galacopyranoside (6) [5], 3-O-b- -glucopyranosyl-(1 ? 2)-[b-
xylopyranosyl-(1 ? 3)]-b- -glucopyranosyl-(1 ? 4)-b- -galaco-
pyranosyl-yamogenin (7) [6], and (22S)-cholest-5-ene-1b,3b,
(
a
D
D
D
including cardiac glycosides, steroidal saponins, and pregnane
glycosides, that are isolated from plants and have been reported
to show various biological activities. Recently, much attention
has been paid to steroidal glycosides with aglycones that have cho-
lestane skeletons, which are not included in the aforementioned
types, because of their importance from the perspectives of both
chemical diversity and significant biological activities. However,
the number of known compounds of this type is still quite limited
D
D
D-
D
D
16b,22-tetrol-1-O-a-L-rhamnopyranosyl-16-O-b-D-glucopyranoside
(8) [7] (Fig. 1).
2. Experimental
[
1].
Polygonatum odoratum (Mill.) Druce is a perennial herbaceous
2.1. General
plant in the Liliaceae family and is widespread in China and Eur-
asia. Its rhizomes have been used in traditional Chinese medicines
for the treatment of diabetes, hypoimmunity, and rheumatic heart
disease [2]. Previous phytochemical studies have reported that this
natural medicine contains spirostane- and furostane-type steroidal
saponins [3].
During our ongoing studies concerning the discovery of novel
steroidal glycosides from traditional Chinese medicines [4], we
performed a further phytochemical investigation of the rhizomes
of P. odoratum. Herein, we report the isolation and structural
determination of three novel cholestane glycosides (1–3), two
Melting points were determined in a SGW X-4 micro-melting
point (INESA Instrument, Shanghai, China) apparatus and were
uncorrected. The IR spectra were measured using a Nexus 670
FT-IR spectrometer (Thermo Nicolet, Madison, WI, USA) with the
KBr disk method. The specific rotations were measured on a P-
1
2200 polarimeter (JASCO, Tokyo, Japan) in a 0.5-dm cell. The
H
1
3
and C NMR spectra were measured on an ECP-500 spectrometer
(JEOL, Tokyo, Japan) using TMS as the internal reference, and the
chemical shifts are expressed in d (ppm). The ESIMS was taken
on a Trap VL mass analyzer (Agilent Technologies, Palo Alto, CA,
USA), and the HRESIMS was taken on an LTQ Orbitrap XL mass
spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Pre-
parative HPLC was performed on an Agilent 1100 apparatus (Agi-
lent Technologies, Palo Alto, CA, USA) equipped with a G1314A
⇑
Corresponding authors. Tel.: +86 531 82919971 (H. Bai), +81 47 472 1161
W. Li).
E-mail addresses: baihong@gmail.com (H. Bai), liwei@phar.toho-u.ac.jp (W. Li).
(
0
039-128X/$ - see front matter Ó 2013 Elsevier Inc All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.11.013

8
H. Bai et al. / Steroids 80 (2014) 7–14
Fig. 1. The structures of steroidal glycosides isolated from P. odoratum.
Variable Wavelength Detector and a YMC-Pack RP-C18 column
(57 g) rich in steroidal glycosides. This fraction was chromato-
graphed on a silica-gel column with a gradient of CHCl –MeOH–
O to yield ten subfractions (A1–A10). Three subfractions, A6
(2.6 g), A7 (13.2 g) and A8 (5.8 g), were further separated by re-
peated silica gel, Sephadex LH-20 and ODS column chromatogra-
phy before being finally purified by preparative HPLC to afford
compound 8 (12 mg) from subfraction A6, compounds 4 (26 mg),
(
150 ꢀ 20 mm, i.d., YMC, Kyoto, Japan). Diaion HP-20 (20–60 mesh,
3
Mitsubishi Chemical, Tokyo, Japan), silica gel (200–300 mesh,
Qingdao Marine Chemical Factory, Qingdao, China), Sephadex
LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and
H
2
ODS (50
lm, YMC, Kyoto, Japan) were used for column
chromatography.
5
(3 mg), 6 (6 mg) and 7 (45 mg) from subfraction A7, and com-
pounds 1 (23 mg), 2 (23 mg) and 3 (15 mg) from subfraction A8.
2.2. Plant material
The rhizomes of P. odoratum used in this study were collected
2.3.1. Polygonatumoside A (1)
2
D
0
from Hunan province, PR China in Aug. 2008 and identified by
Dr. Wei Yingqin (Shandong Polytechnic University). A plant speci-
men (PO200808) is deposited in the herbarium of Institute of
Materia Medica, Shandong Academy of Medical Sciences.
Amorphous powder, mp 226–227 °C;
½
aꢁ
+46.0 (c 1.26,
MeOH); IR (KBr)
mmax: 3386, 2932, 1701, 1632, 1384, 1160, 1074,
ꢂ1
1
13
1036, 895 cm
(pyridine-d
m/z 1215 [M+Na] ; positive-ion HRESIMS m/z 1215.5436 (calcd
for C56 27Na, 1215.5405).
5
; H NMR (pyridine-d , 500 MHz) and C NMR
5
, 125 MHz), see Tables 1 and 2; positive-ion ESIMS
+
88
H O
2
.3. Extraction and isolation
The air-dried rhizomes of P. odoratum (10 kg) were extracted
with 70% aqueous EtOH at the boiling point for 2 h for three times.
The extract (780 g) was concentrated under reduced pressure and
2.3.2. Polygonatumoside B (2)
2
D
0
Amorphous powder, mp 219–220 °C; ½
aꢁ
ꢂ28.6 (c 1.33,
MeOH); IR (KBr)
m
max: 3417, 2933, 1701, 1635, 1364, 1159, 1074,
ꢂ1
1
13
then partitioned between n-BuOH and H
2
O. The n-BuOH extract
1038, 894 cm
(pyridine-d
m/z 1215 [M+Na] ; positive-ion HRESIMS m/z 1215.5418 (calcd
for C56 27Na, 1215.5405).
5
; H NMR (pyridine-d , 500 MHz) and C NMR
(
688 g) was subjected to Diaion HP-20 column chromatography
and eluted with gradients of H O and 20%, 70% and 95% EtOH, suc-
2
cessively. The 70% EtOH eluate was concentrated to yield a fraction
5
, 125 MHz), see Tables 1 and 2; positive-ion ESIMS
+
88
H O

H. Bai et al. / Steroids 80 (2014) 7–14
9
894 cm ; 1H NMR (pyridine-d
ꢂ1
, 500 MHz) and 13C NMR (pyri-
Table 1
5
1
3C NMR data of 1–5 (125 MHz in pyridine-d
5
).
dine-d
939 [M+Na] ; positive-ion HRESIMS m/z 939.4575 (calcd for C45-
19Na, 939.4560).
5
, 125 MHz), see Tables 1 and 2; positive-ion ESIMS m/z
+
Aglycone
1
2
3
4
5
72
H O
1
2
3
4
5
6
7
8
9
37.8
30.1
78.3
39.2
37.8
30.1
78.3
39.2
37.8
30.2
78.2
39.3
37.7
30.3
78.2
39.3
37.7
30.3
78.1
39.3
2.3.5. Polygonatumoside E (5)
20
D
Amorphous powder, mp 224–225 °C; ½
a
ꢁ
ꢂ38.1 (c 0.29,
140.5
121.5
29.4
32.1
53.7
37.6
21.2
35.4
54.1
159.8
119.4
125.3
156.5
18.2
19.4
45.1
18.4
210.4
38.1
28.4
33.4
75.1
17.0
140.6
121.4
29.4
32.1
53.6
37.6
21.3
35.7
54.2
159.9
119.4
125.7
156.3
18.0
19.4
45.7
18.3
210.5
38.3
28.5
33.6
75.0
17.3
140.5
121.5
29.4
32.1
53.8
37.6
21.3
35.4
54.1
159.8
119.4
125.4
156.6
18.3
19.4
45.2
18.5
210.4
38.1
28.4
33.5
75.1
17.1
140.5
122.3
26.7
35.6
43.6
37.4
20.4
32.0
45.1
86.4
40.0
82.0
59.8
20.1
19.3
42.6
15.2
110.1
26.6
26.3
27.6
65.1
16.3
140.6
123.1
26.8
35.5
43.6
37.4
20.4
32.1
45.6
85.8
40.8
81.3
59.5
20.4
19.3
42.2
16.8
110.9
28.0
28.0
30.8
69.9
17.3
MeOH); IR (KBr) mmax: 3417, 2933, 1631, 1384, 1158, 1071, 920,
ꢂ1
1
13
8
94 cm
dine-d
39 [M+Na] ; positive-ion HRESIMS m/z 939.4578 (calcd for C45-
19Na, 939.4560).
;
H NMR (pyridine-d
5
, 500 MHz) and C NMR (pyri-
5
, 125 MHz), see Tables 1 and 2; positive-ion ESIMS m/z
+
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
72
H O
2.3.6. Compound 6
Amorphous powder, mp 258–259 °C (lit. [5]: mp 258–260 °C);
24
D
20
D
½
a
ꢁ
ꢂ45.7 (c 0.50, MeOH) [lit. [5]: ½
aꢁ
ꢂ63.0 (c 0.15, MeOH)].
1
13
H
NMR (pyridine-d
5
,
500 MHz) and
5
C NMR (pyridine-d ,
1
25 MHz) data agree well with the values reported for (25S)-
)-dihydroxy-spirost-5-ene-3-O-b- -glucopyranosyl-
1 ? 2)-[b- -xylopyranosyl-(1 ? 3)]-b- -glucopyranosyl-(1 ? 4)-
-galacopyranoside.
(3b,14
a
D
(
D
D
b-
D
2.3.7. Compound 7
Amorphous powder, mp 277–278 °C (lit. [5]: mp 278–279 °C; lit
29
D
20
D
[
6]: mp 280–283 °C); ½
a
ꢁ
ꢂ74.6 (c 0.50, C
, 500 MHz) and
, 125 MHz) data agree well with the values re-
-glucopyranosyl-(1 ? 2)-[b- -xylopyranosyl-
-glucopyranosyl-(1 ? 4)-b- -galacopyranosyl-
H
5 5
N) (lit. [6]: ½
a
ꢁ
1
13
ꢂ63.0 (c 1, C
5
H
5
N)). H NMR (pyridine-d
5
C
Sugar
b-
D-gal
b-
D-gal
b-
D
-gal
b-
D-gal
b-D-gal
NMR (pyridine-d
5
1
2
3
4
5
6
102.8
73.1
75.5
79.8
75.1
60.5
102.8
73.2
75.6
79.8
75.1
60.6
102.8
73.3
75.6
81.0
75.1
60.5
102.7
73.3
75.6
81.0
75.1
60.5
102.6
73.3
75.6
81.0
75.1
60.5
ported for 3-O-b-
1 ? 3)]-b-
yamogenin.
D
D
(
D
D
0
0
0
0
0
0
2.3.8. Compound 8
Amorphous powder, mp 245–246 °C; ½
b-
D-glc
b-
D-glc
b-
D-glc
b-
D-glc
b-D-glc
2
7
1
2
3
4
5
6
105.1
81.3
86.8
70.5
77.6
63.0
105.1
81.3
86.8
70.5
77.6
63.0
105.2
86.2
78.5
71.9
78.2
63.2
105.2
86.1
78.5
71.9
78.2
63.2
105.2
86.1
78.5
71.9
78.2
63.2
a
ꢁ
ꢂ24.1 (c 0.50, MeOH)
D
2
D
7
1
(
5
lit. [7]: ½
00 MHz) and C NMR (pyridine-d
with the values reported for (22S)-cholest-5-ene-1b,3b,16b,22-te-
trol-1-O- -rhamnopyranosyl-16-O-b- -glucopyranoside.
aꢁ
ꢂ36.0 (c 0.29, MeOH)). H NMR (pyridine-d
5
,
13
5
, 125 MHz) data agree well
a
-
L
D
0
00
00
00
00
b-
D-glc
b-
D-glc
b-
D-glc
b-
D-glc
b-D-glc
1
2
3
4
5
6
104.8
76.2
77.7
71.0
78.7
62.5
104.8
76.2
77.7
71.1
78.7
62.5
107.0
76.7
77.7
70.3
79.0
61.6
107.0
76.8
77.7
70.4
79.0
61.6
106.9
76.8
77.7
70.4
79.0
61.6
2
.4. Acid hydrolysis of compounds 1–5
Compounds 1 (8 mg), 2 (8 mg), 3 (1 mg), 4 (1 mg) and 5 (1 mg)
were separately dissolved in 1 M HCl (dioxane–H O, 1:1, 25 mL)
and then heated at 100 °C for 2 h. After the dioxane was removed,
the solutions were extracted with EtOAc (10 mL ꢀ 3).
2
b-
D-xyl
b-D-xyl
1
2
3
4
5
104.9
75.3
78.6
70.7
67.3
104.9
75.3
78.6
70.7
67.3
2 3
The aqueous layers were neutralized with Ag CO and filtered,
and the filtrates were concentrated under reduced pressure to
yield the sugar fractions. The sugar fractions were dissolved in
H
(
4
2
O (4 mL), and S-(ꢂ)-
12 mg) in EtOH (2 mL) was added. The solution was stirred at
0 °C for 4 h, after which glacial acetic acid (0.4 mL) was added be-
3
a-methylbenzylamine (14 lL) and NaBH CN
000
000
000
b-
D-glc
b-
D-glc
b-D-glc
1
2
3
4
5
6
105.0
75.2
78.6
71.7
78.5
62.9
105.0
75.2
78.6
71.7
78.5
62.9
105.0
75.2
78.6
71.8
78.5
62.9
fore evaporating to dryness. The resulting solids were acetylated
with acetic anhydride (0.6 mL) in pyridine (0.6 mL) for 24 h at
room temperature. After evaporation, H
the residue, and the solutions were filtered through a Cleanert
18-SPE cartridge (Agela, Tianjin, China) and washed with H
and 20% and 50% CH CN (each 15 mL), successively. The 50% CH3-
CN eluate was analyzed, and the 1-[(S)-N-acetyl- -methylbenzyla-
2
O (2 mL) was added to
C
2
O
2
.3.3. Polygonatumoside C (3)
Amorphous powder, mp 222–223 °C;
MeOH); IR (KBr) max: 3405, 2933, 1702, 1639, 1384, 1161, 1073,
2
D
0
3
½
a
ꢁ
+32.2 (c 0.47,
a
m
mino]-1-deoxy-alditol acetate derivatives of the monosaccharides
were identified by HPLC analysis with the derivatives of standard
sugars prepared under the same conditions. The HPLC conditions
were as follows: column, Agilent SB-C18 (4.6 ꢀ 250 mm, Agilent
ꢂ1
1
13
8
96 cm
dine-d
083 [M+Na] ; positive-ion HRESIMS m/z 1083.5003 (calcd for C51-
23Na, 1083.4983).
;
H NMR (pyridine-d
5
, 500 MHz) and C NMR (pyri-
5
, 125 MHz), see Tables 1 and 2; positive-ion ESIMS m/z
+
1
80
H O
3
Technologies, Palo Alto, CA, USA); solvent, 40% CH CN; flow rate,
0
.8 mL/min; and detection, DAD 230 nm. The identification of the
monosaccharides present in the sugar fraction was carried out by
a comparison of the retention times t (min) of their derivatives
with those of authentic samples: 21.80 (derivative of -glucose),
2
.3.4. Polygonatumoside D (4)
2
D
0
Amorphous powder, mp 241–242 °C; ½
aꢁ
ꢂ57.8 (c 1.61,
R
MeOH); IR (KBr)
m
max: 3386, 2934, 1631, 1371, 1157, 1070, 921,
D

1
0
H. Bai et al. / Steroids 80 (2014) 7–14
Table 2
1
5
H NMR data of 1–5 (500 MHz in pyridine-d ).
Aglycone
1
2
3
4
5
a
a
a
a
a
a
1.77a
1
1
2
2
3
4
4
6
7
7
8
9
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
a
b
a
b
1.71 (m)
0.95a
2.10 (m)
1.75 (m)
3.90a
2.68 (m)
2.44 (t, 13.0)
5.40 (br s)
2.17 (m)
1.70
0.96
2.10
1.70
3.86
2.67
1.71 (m)
0.97 (m)
2.10 (m)
1.75 (m)
3.90
2.68 (m)
2.47 (t,11.4)
5.41 (br s)
2.17 (m)
1.74 (m)
1.01 (m)
2.10 (m)
1.03 (m)
2.11 (m)
a
a
1.74
1.72
a
3.88 (m)
3.87 (m)
2.69 (m)
a
b
2.70 (dd, 13.7,4.8)
a
a
2.44 (t, 11.2)
5.39 (br s)
2.15 (m)
2.46
2.46
5.38 (br d, 4.8)
5.38 (d, 4.8)
a
a
a
b
2.50
2.46
1.83
a
a
1.86
a
2.32 (m)
0.86 (m)
1.49 (m)
1.92 (m)
0.77 (m)
5.89 (br s)
2.31 (m)
0.88 (m)
1.52 (m)
2.32 (m)
0.86 (m)
1.49 (m)
1.92 (m)
0.77 (m)
5.89 (br s)
2.04
2.00 (td, 11.2, 5.3)
1.81 (m)
1.80 (td, 11.3, 3.8)
a
a
1
1.54
1.53
a
2a
2b
5a
5b
6
7
8
9
0
2.07
2.25 (m)
1.45 (m)
2.26 (m)
1.46 (m)
0.91 (m)
5.91 (d, 1.8)
2.31 (dd, 12.7, 7.8)
1.88 (dd, 12.7, 6.0)
5.07 (ddd, 7.8, 6.8, 6.0)
2.77 (dd, 7.8, 6.8)
1.07 (s)
2.34 (dd, 12.5, 7.2)
1.94 (dd, 12.5, 6.2)
a
6.17 (d,1.8)
6.23 (d, 1.8)
6.17 (d, 1.6)
4.88
2.54 (dd, 8.0, 6.4)
1.21 (s)
1.10 (s)
0.96 (s)
3.52 (q, 6.9)
1.34 (d, 6.9)
2.62 (m)
2.56 (m)
1.96 (m)
1.56 (m)
1.03 (s)
0.96 (s)
1.10 (s)
0.96 (s)
0.98 (s)
0.96 (s)
a
a
3.52 (q, 6.9)
1.36 (d, 6.9)
3.52 (q, 6.9)
1.34 (d, 6.9)
2.62 (m)
2.56 (m)
1.96 (m)
1.56 (m)
1.90 (m)
2.05
2.46
1
1.20 (d, 7.1)
1.95 (td, 13.5, 4.8)
1.50 (m)
1.07 (d, 7.1)
1.70 (m)
a
3a
3b
4a
4b
5
6a
6b
7
2.67
2.54 (m)
1.97 (m)
1.55 (m)
1.88 (m)
2.17 (tt, 13.5, 4.8)
1.38 (m)
1.53a
a
1.90 (m)
1.55
1.63 (m)
3.95a
3.97
a
3.95
a
4.05 (dd, 10.7, 1.8)
3.44 (d, 10.7)
1.08 (d, 5.9)
3.75 (t, 10.7)
3.74 (d, 10.7)
0.69 (d, 6.4)
3.47 (dd, 9.4, 6.1)
0.92 (d, 6.8)
3.48 (dd, 9.4, 6.2)
0.94 (d, 6.9)
3.47 (dd, 9.4, 6.0)
0.92 (d, 6.6)
Sugar
b-
D-gal
b-
D-gal
b-
D-gal
b-D
-gal
b-D-gal
1
2
3
4
5
4.89 (d, 7.6)
4.87 (d, 7.5)
4.91 (d, 7.8)
4.88 (d, 7.8)
4.88 (d, 7.6)
4.39a
4.39
4.11
a
4.50 (dd, 9.4, 7.8)
4.49 (dd, 8.6, 7.8)
4.49 (dd, 9.5, 7.6)
4.11a
a
4.10
a
4.10
a
4.10
a
a
4.60 (br d, 3.0)
4.59 (br d, 3.0)
4.57
4.57 (br d, 2.3)
4.57 (br d, 2.7)
3.95a
3.95
a
3.97
a
3.97
a
3.97
a
6
6
a
b
4.67 (m)
4.17a
4.65 (m)
4.17
4.76 (m)
4.18
4.74 (m)
4.18 (m)
4.74 (m)
4.18 (m)
a
a
0
0
0
0
0
b-
D-glc
b-
D-glc
b-
D-glc
b-D
-glc
b-D-glc
1
2
3
4
5
6
6
5.19 (d, 7.8)
5.18 (d, 8.1)
5.14 (d, 7.8)
5.14 (d,7.8)
5.14 (d,7.8)
4.39a
4.39
4.12
a
4.15 (dd, 8.7, 7.8)
4.28 (dd, 8.7, 8.4)
4.15 (dd, 8.4, 7.8)
4.27 (dd, 8.4, 8.2)
4.15 (dd, 8.7, 7.8)
4.27 (dd, 8.7, 8.2)
4.12a
a
a
a
a
3.82 (t, 9.1)
3.81 (t, 9.0)
3.97
3.97
3.97
3.97
a
a
a
a
a
3.85
3.85
4.50
4.02
3.97
3.97
a
a
b
4.50a
4.02a
4.63 (br d, 10.8)
4.62 (br d, 10.3)
4.62 (br d, 10.5)
a
a
a
a
4.09
4.09
4.09
00
00
00
00
00
b-
D-glc
b-
D-glc
b-
D-glc
b-D
-glc
b-D-glc
1
2
3
4
5
6
6
5.57 (d, 7.5)
5.57 (d, 7.6)
5.23 (d, 7.8)
4.06
5.23 (d, 7.5)
5.23 (d,7.6)
4.06a
4.06
4.08
4.20
3.87
a
a
4.06 (dd, 8.5, 7.5)
4.12 (dd, 8.9, 8.5)
4.22 (dd, 8.9, 8.1)
3.80 (m)
4.58 (br d, 11.4)
4.37 (br d, 11.4)
4.06 (dd, 9.4, 7.6)
4.12 (dd, 9.4, 9.1)
4.22 (dd, 9.1, 8.7)
3.80 (m)
4.58 (br d, 11.7)
4.37 (br d, 11.7)
a
a
4.08
4.12 (t, 8.7)
4.20a
a
4.22
a
a
a
3.87
3.80 (m)
a
a
a
b
4.38a
4.55a
4.38
4.37
a
a
4.55
4.58
b-
D-xyl
b-D-xyl
1
2
3
4
5
5
5.24 (d, 7.8)
5.23 (d, 7.8)
a
a
3.95
3.95
4.06a
4.06
4.09
a
a
a
4.09
a
a
b
4.22a
4.22
3.67 (t, 10.5)
3.67 (t, 10.3)
000
000
000
b-
D-glc
b-
D-glc
b-D-glc
1
2
3
4
5
6
6
4.81 (d, 7.6)
4.80 (d, 7.8)
4.81 (d, 7.8)
3.98a
3.98
4.23
4.21
3.93
a
3.98
a
a
a
a
4.23
4.23
4.21a
3.93a
a
4.21
a
a
a
3.93
a
a
a
a
b
4.55
4.55
4.55
4.38a
4.38
a
4.38
a
a
Overlapped.

H. Bai et al. / Steroids 80 (2014) 7–14
11
2
1
0.31 (derivative of
9.78 (derivative of
L
-glucose), 18.48 (derivative of
-galactose), 16.28 (derivative of
-xylose). The sugars in compounds 1–5
-glucose, -galactose and -xylose.
D
-galactose),
spectroscopic data also revealed the presence of five sugar units,
whose b-anomeric configurations were determined by the large
coupling constant values of their corresponding anomeric protons
(7.5–7.8 Hz). Further detailed analyses of the DQFCOSY, TOCSY,
HMQC, HMBC and NOESY spectroscopic data established the pres-
L
D-xylose)
and 15.69 (derivative of
were determined to be
L
D
D
D
The EtOAc extracts obtained from compounds 1 and 2 were
purified by semi-preparative HPLC with gradient elution of 80–
ence of a b-
D
-glucopyranosyl-(1 ? 2)-[b-
-glucopyranosyl-(1 ? 4)-b- -galactopyranosyl moiety and
-glucopyranosyl moiety. The C27-steroidal aglycone structure
D-xylopyranosyl (1 ? 3)]-
1
(
00% MeOH, respectively, and afforded the same compound 9
0.7 mg from 1 and 0.6 mg from 2).
25R)-3ß-Hydroxy-spirost-5,14-diene (9): Positive-ion ESIMS m/z
b-
b-
D
D
a
D
(
was inferred from the presence of a cholestane skeleton, as shown
by two typical methyl singlets at d 1.10 (s, H -18) and 0.96 (s, H
19) and two methyl doublets at d 1.34 (d, J = 6.9 Hz, H -21) and
-27) in the H NMR spectrum. In addition,
+
1
4
12.9 [M+H] . H NMR (500 MHz, pyridine-d
5
): 1.90 (1H, m, H-1a),
3
3
-
1
.14 (1H, overlapped, H-1b), 2.10 (1H, m, H-2a), 1.79 (1H, over-
3
1
lapped, H-2b), 3.84 (1H, m, H-3), 2.61 (2H, m, H-4), 5.40 (1H, br d,
J = 5.0 Hz, H-6), 2.18 (1H, m, H-7a), 2.00 (1H, m, H-7b), 2.20 (1H,
m, H-8), 1.05 (1H, overlapped, H-9), 1.53 (2H, m, H-11), 1.65 (1H,
overlapped, H-12a), 1.05 (1H, overlapped, H-12b), 5.44 (1H, br s,
H-15), 5.21 (1H, br d, J = 7.5 Hz, H-16), 2.52 (1H, dd, J = 10.0,
0.92 (d, J = 6.8 Hz, H
3
three trisubstituted C@C bonds were found, and their positions
were determined to be C-5(6), C-14(15) and C-16(17) by the HMBC
correlations from d
2.32 (H-8) to d
and 156.5 (C-17), d
(H-15) to d 32.1 (C-8) (Fig. 2). In addition, the carbonyl carbon
at d 210.4 was determined to be C-22 by the HMBC correlations
from d 1.34 (H -21) to 210.4 (C-22) and d 1.96, 1.56 (H -24) to
210.4 (C-22). The relative configurations in the cholestane A–D
ring system were typically confirmed by the 1,3-diaxial NOE corre-
lations of H-1 /H-3, H-4b/H -19, H-8/H -19 and H-8/H -18. Fur-
H
1.71 (H-1) and 0.96 (H
121.5 (C-6), d 1.10 (H -18) to d
1.34 (H -21) to d 156.5 (C-17), and d
3
-19) to d
C
140.5 (C-5),
159.8 (C-14)
5.89
d
H
C
H
3
C
7
.5 Hz, H-17), 1.13 (3H, s, H-18), 1.05 (3H, s, H-19), 1.90 (1H, over-
H
3
C
H
lapped, H-20), 1.15 (3H, d, J = 7.0 Hz, H-21), 1.65 (2H, overlapped,
H-23), 1.79 (1H, overlapped, H-24a), 1.65 (1H, overlapped, H-
C
2
0
3
6
4b), 1.65 (1H, overlapped, H-25), 3.64 (2H, overlapped, H-26),
H
3
H
2
.73 (3H, d, J = 6.5 Hz, H-27). 13C NMR (125 MHz, pyridine-d
):
d
C
5
7.6 (C-1), 32.6 (C-2), 71.3 (C-3), 43.4 (C-4), 141.3 (C-5), 120.8 (C-
), 31.9 (C-7), 31.3 (C-8), 50.5 (C-9), 37.4 (C-10), 21.3 (C-11), 33.6
a
3
3
3
000
(
(
C-12), 46.9 (C-13), 159.8 (C-14), 121.8 (C-15), 85.4 (C-16), 60.4
C-17), 28.4 (C-18), 19.3 (C-19), 44.4 (C-20), 13.8 (C-21), 106.4 (C-
thermore, the HMBC correlations of Gal-H-1/C-3 and Glc -H-1/C-
26 established the bisdesmoside structure. However, the absolute
configuration of C-25 remained unsolved.
2
2), 30.2 (C-23), 29.3 (C-24), 30.9 (C-25), 67.3 (C-26), 17.4 (C-27).
Polygonatumoside B (2) was isolated in the same fraction as
compound 1 and their retention times for preparative HPLC were
2.5. Antimicrobial activity
very similar. They have the same molecular formula, C56
88 27
H O ,
which was determined by positive-ion HRESIMS. These two com-
Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and
1
13
pounds showed a high similarity in their H and C NMR spectro-
scopic data, although they exhibited some slight differences
Micrococcus luteus ATCC 9341), Gram-negative bacteria (Salmonella
enterica serovar Typhimurium ATCC 14028 and Escherichia coli
ATCC 25922), yeast (Candida albicans JCM 1542) and fungus (Asper-
gillus fumigatus JCM 1738) were used as indicator microbes for
antimicrobial evaluation. The antimicrobial activity of isolated gly-
cosides was assayed by the micro-broth dilution method using
Mueller–Hinton broth (Becton, Dickinson and Company, USA) for
bacteria and potato dextrose broth (Becton, Dickinson and Com-
pany, USA) for yeast and fungus according to previous reports
2
D
0
(
+
Tables 1 and 2). However, their optical rotation values {½
46.0 for 1 and ꢂ28.6 for 2} differed greatly. All of these data sug-
gested that compound 2 is a C-25 diastereomer of compound 1.
a
ꢁ
It is always difficult to assign the C-25 absolute configuration in
cholestane-type compounds. In this study, several approaches
were utilized to solve this problem. An empirical rule by calculat-
ing the chemical shift difference between H
for 25S, and dab 60.48 ppm for 25R, when in pyridine-d
2
-26 (dab P0.57 ppm
) [11]
5
[
8,9]. The lowest antibiotic concentration that prevented the
growth of a given test organism was determined as the minimal
inhibitory concentration (MIC).
was applied to determine the C-25 absolute configuration of cho-
lestane glycosides [12,13], however, compounds 1 and 2 did not
follow this rule. Furthermore, on acid hydrolysis of compounds 1
and 2, only the same compound (25R)-3b-hydroxy-spirost-5,14-
diene (9) could have been isolated as the major product, possibly
due to the equilibrium of C-25 epimerization [14] (Fig. 3). Thus,
3
. Results and discussion
A 70% EtOH extract from the rhizomes of P. odoratum was par-
titioned between n-BuOH and H O. The n-BuOH fraction was sep-
2
comparison of the chemical shifts of H
utilized to assign the 25R/25S-configuration because for most
furostane saponins, the resonance from H -27 in the 25R configu-
ration appears at a higher field than that in the 25S configuration
[15]. Using this empirical observation, compound 1 was deter-
mined to possess the 25R configuration, and compound 2 possesses
the 25S configuration. Thus, polygonatumoside A (1) was deter-
3
-27 between 1 and 2 was
arated by Diaion HP-20, silica gel, ODS, and Sephadex LH-20
column chromatography, as well as preparative HPLC, to afford
four cholestane-type steroidal glycosides (1–3, 8) and four spiros-
tane-type steroidal saponins (4–7). The structures of known com-
pounds (6–8) were identified by detailed NMR analysis and
comparison with literature data. The structures of the new com-
pounds were determined by various spectroscopic analyses and
chemical reactions.
3
mined to be (25R)-26-O-b-
5,14,16-trien-22-one-3-O-b-
pyranosyl (1 ? 3)]-b- -glucopyranosyl-(1 ? 4)-b-
oside, and the structure of polygonatumoside B (2) is (25S)-26-O-
b- -glucopyranosyl-3b-hydroxy-cholest-5,14,16-trien-22-one-3-O
-b- -glucopyranosyl-(1 ? 2)-[b- -xylopyranosyl (1 ? 3)]-b- -glu-
copyranosyl-(1 ? 4)-b- -galactopyranoside.
D
-glucopyranosyl-3b-hydroxy-cholest-
-glucopyranosyl-(1 ? 2)-[b- -xylo-
-galactopyran-
D
D
D
D
Polygonatumoside A (1) was isolated as an amorphous powder.
Its molecular formula was determined to be C56
H
88
O
27 by positive-
D
ion HRESIMS analysis. The 13C NMR spectroscopic data revealed 56
carbon resonances, of which 29 were assigned to the sugar por-
tions and the remaining 27 were assigned to the C27-steroidal agly-
cone. Upon acid hydrolysis, the component sugar composition was
D
D
D
D
Polygonatumoside C (3) was obtained as an amorphous powder.
+
HRESIMS analysis (m/z 1083.5003 [M+Na] ) indicated a molecular
determined to consist of
ratio of 3:1:1 by HPLC analysis after conversion of monosaccha-
rides to their corresponding 1-[(S)-N-acetyl- -methylbenzylami-
no]-1-deoxy-alditol acetate derivatives [10]. The H and C NMR
D
-glucose,
D
-xylose and
D
-galactose in a
formula of C51
pound 3 with those of 1 suggested that 3 had the same aglycone as
that of 1 but different sugar moieties. The presence of three b-
glucopyranosyl moieties and one b- -galactopyranosyl in 3 was
80
H O23. Careful comparison of the NMR data of com-
a
D-
1
13
D

1
2
H. Bai et al. / Steroids 80 (2014) 7–14
Fig. 2. Key DQFCOSY and HMBC correlations of compounds 1 and 4.
Fig. 3. Acid hydrolysis of compounds 1 and 2.
suggested by the observation of four anomeric proton doublets at d
fusion was indicated by the HMBC correlations of H
C-4, H-6/C-8 and H-6/C-10. The position of the 14 -hydroxy moi-
ety was determined by comparing the downfield-shifted reso-
nances of H-9, H-11 and H-17 with those of the 14 -H
compound [16], as well as the HMBC correlations from H -18/C-
14 and H -12/C-14. The 25S configuration in 4 and 25R configura-
tion in 5 were inferred from the differences in the chemical shifts
of the H -23, H -24 and H -26 geminal protons, which were
>0.35 ppm in compound 4 and <0.20 ppm in compound 5, as well
as the fact that the H -27 resonance appears 0.39 ppm upfield in
compound 5 compared to that of 4 [17]. The component sugars
in compounds 4 and 5 were determined to be a b- -galactopyran-
osyl unit and two b- -glucopyranosyl units through detailed NMR
3
-19/C-5, H-6/
1
4
.91, 5.14, 5.23 and 4.81 in the H NMR spectrum and by the acid
a
hydrolysis results. The structures of the sugar moieties and the
location of the glycosidic bonds were determined by further
detailed analyses of 2D NMR spectroscopic data, in particular, the
a
3
0
0
0
0
HMBC correlations of Glc -H-1/Glc -C-2, Glc -H-1/Gal-C-4, Gal-
2
000
H-1/C-3 and Glc -H-1/C-26 indicated the bisdesmoside structure
of b- -glucopyranosyl-(1 ? 2)-b- -glucopyranosyl-(1 ? 4)-b-
-galactopyranosyl moiety connected at C-3 and a b- -glucopyran-
osyl moiety connected at C-26. Thus, the structure of polygona-
tumoside (3) was determined to be (25R)-26-O-b-
glucopyranosyl-3b-hydroxy-cholest-5,14,16-trien-22-one-3-O-b-
a
D
D
2
2
2
D
D
3
C
D-
D-
D
glucopyranosyl-(1 ? 2)-b-D-glucopyranosyl-(1 ? 4)-b-D-galactop-
D
yranoside.
spectroscopic analyses and acid hydrolysis. The connection and the
sequence of the sugar chain at C-3 were determined through the
same HMBC correlations as in 3 (Fig. 2). Thus, the structure of
Polygonatumoside D (4) and polygonatumoside E (5) were iso-
lated as amorphous powders with the same molecular formula,
C
45
H
72
O
19. They are spirostane-type steroidal saponins; this con-
polygonatumoside D (4) is (25S)-(3b,14
en-3-O-b- -glucopyranosyl-(1 ? 2)-b- -glucopyranosyl-(1 ? 4)-b-
-galactopyranoside, and the structure of polygonatumoside E (5)
was determined to be (25R)-(3b,14)-dihydroxy-spirost-5-en-3-O-
b- -glucopyranosyl-(1 ? 2)-b- -glucopyranosyl-(1 ? 4)-b- -gala-
ctopyranoside.
a)-dihydroxy-spirost-5-
clusion was suggested by the characteristic proton resonances for
two tertiary angular methyl and two secondary methyl groups in
the H NMR spectra and by the quaternary carbon at ca. d 110 in
the C NMR spectra (Tables 1 and 2). Compounds 4 and 5 are a
pair of C-25 diastereomers, as shown by their superimposable
D
D
D
1
1
3
D
D
D
NMR spectra, but differences were observed in the E and F ring res-
Although our knowledge of cholestane-type steroidal glycosides
is still quite limited, this class of compounds has been recognized
5
onances. In the aglycone, the presence of a
D
-unsaturated A/B ring

H. Bai et al. / Steroids 80 (2014) 7–14
13
Fig. 4. Possible biosynthetic pathway for compounds 1–8.
as the third class of C27-steroidal glycosides [1]. The biosynthesis of
4. Conclusions
cholestane-type glycosides has been proposed to involve indepen-
dent pathways from furostane- and spirostane-type steroidal sap-
onins, but in this study, the cholestane glycosides (1–3) and
spirostane saponins (4–7) are proposed to share a common biosyn-
thetic precursor, 16,26-dihydroxy-22-keto-cholesterol (Fig. 4). The
high sugar-sequence similarity between the cholestane glycosides
The phytochemical investigation of P. odoratum resulted in the
isolation of four cholestane-type steroidal glycosides (1–3, 8) and
four spirostane-type steroidal saponins (4–7). Polygonatumosides
1
4,16
A–C (1–3) have an unique
D
-unsaturated D-ring structure,
which extends the biodiversity of plant steroidal glycosides. From
the biosynthetic perspective, the cholestane-type steroidal glyco-
sides (1–3) are steroidal saponin-like glycosides, which is evident
from their close biosynthetic relationship with and high sugar-se-
quence similarity to the steroidal saponins obtained in this study.
However, further investigation is required to identify the biosyn-
thetic enzymes and intermediates involved in C27-steroidal glyco-
side biosynthesis. Furthermore, spirostane-type steroidal saponin
7 showed significant antifungal activity against C. albicans JCM
1542 and A. fumigatus JCM 1738.
(1–3) and spirostane saponins (4–7) also indicated their close bio-
synthetic relationship, compared to another cholestane-type gly-
coside (8). The occurrence of different biosynthetic reactions at
the 16-hydroxy group appears to be an important diverging point
for their biosynthetic pathways. This conclusion is also supported
by other phytochemical investigations that have used similar met-
abolic profiling [12,18].
All isolated compounds were evaluated for their antimicrobial
activity against S. aureus ATCC 25923, M. luteus ATCC 9341, S. ent-
erica serovar Typhimurium ATCC 14028, E. coli ATCC 25922, C. albi-
cans JCM 1542 and A. fumigatus JCM 1738. Compound 7 showed
significant antifungal activity against C. albicans JCM 1542 (MIC
Acknowledgments
3
.1
the other seven compounds had very low activity against these
yeast and fungus strains (MICs P100 g/mL). Moreover, all iso-
lated glycosides showed no activity against the bacterial strains
using in this antimicrobial test at concentration of 100 g/mL.
l
g/mL) and A. fumigatus JCM 1738 (MIC 6.3
l
g/mL). However,
The study partially supported by Natural Science Foundation of
Shandong Province (ZR2009BQ019), the Key Laboratory for Rare
and Uncommon Diseases of Shandong Province, P. R. China, and
Grant-in-Aid for Young Scientists (B) (No. 23790025) from the Min-
istry of Education, Culture, Sports, Science and Technology of Japan.
l
l
Comparison of the structures of 7 and 6, suggested that 14-hydrox-
ylation greatly decreased the activity. In comparison of 7 with sev-
eral structurally related antifungal spirostane saponins (MICs
Appendix A. Supplementary data
6
10
l
g/mL) [19–21], it suggested that the C-5(6) double bond,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.
11.013.
the C-25 absolute configuration and the sugar structures did not
significantly influence the activity.

1
4
H. Bai et al. / Steroids 80 (2014) 7–14
[
[
[
12] Ono M, Kakiuchi T, Ebisawa H, Shiono Y, Nakamura T, Kai T, et al. Steroidal
glycosides from the fruits of Solanum viarum. Chem Pharm Bull 2009;57:
32–5.
References
6
[
[
[
1] Challinor VL, De Voss JJ. Open-chain steroidal glycosides, a diverse class of
plant saponins. Nat Prod Rep 2013;30:429–54.
2] Yu. C. A Dictionary of the Traditional Chinese Medicine. Beijing: Chinese
Medical Science Press; 1993.
3] Guo H, Zhao H, Bai H. Spectroscopic characteristics of steroidal saponins and
homoisoflavonoids from Polygonatum odoratum. Acta Chin Med Pharmacol
13] Hai L, Chou GX, Tao W, Long GY, Chun WS, Hong WC, et al. Steroidal
sapogenins and glycosides from the rhizomes of Dioscorea bulbifera. J Nat Prod
2009;72:1964–8.
14] Murakami T, Akinobu K, Matsuda H, Yoshikawa M. Medicinal foodstuffs. Part
XVII: fenugreek seed. (3): Structures of new furostanoltype steroid saponins,
trigoneosides Xa, Xb, XIb, XIIa, XIIb and XIIIa, from the seeds of Egyptian
Trigonella foenum-graecum L. Chem Pharm Bull 2000;48:994–1000.
15] Agrawal PK. Assigning stereodiversity of the 27-Me group of furostane-type
steroidal saponins via NMR chemical shifts. Steroids 2005;70:715–24.
16] Yoshikawa M, Xu F, Morikawa T, Pongpiriyadacha Y, Nakamura S, Asao Y, et al.
Medicinal flowers. XII. New spirostane-type steroid saponins with
antidiabetogenic activity from Borassus flabellifer. Chem Pharm Bull 2007;55:
2
012;40:41–6.
[
[
[
4] Bai H, Li W, Asada Y, Satou T, Wang Y, Koike K. Twelve pregnane glycosides
from Cynanchum atratum. Steroids 2009;74:198–207.
5] Son KH, Do JC, Kang SS. Steroidal saponins from the rhizomes of Polygonatum
sibiricum. J Nat Prod 1990;53:333–9.
6] Sugiyama M, Nakano K, Tomimatsu T. Five steroidal components from the
rhizomes of Polygonatum odoratum var. pluriflorum. Chem Pharm Bull
[
[
308–16.
1
984;32:1365–72.
[
17] Agrawal PK. 25R/25S stereochemistry of spirostane-type steroidal sapogenins
and steroidal saponins via chemical shift of geminal protons of ring-F Magn.
Reson Chem 2003;41:965–8.
18] Matsuo Y, Watanabe K, Mimaki Y. New steroidal glycosides from rhizomes of
Clintonia udensis. Biosci Biotechnol Biochem 2008;72:1714–21.
19] Favel A, Kemertelidze E, Benidze M, Fallague K, Regli P. Antifungal activity of
steroidal glycosides from Yucca gloriosa L. Phytother Res 2005;19:158–61.
20] Yang CR, Zhang Y, Jacob MR, Khan SI, Zhang YJ, Li XC. Antifungal activity of C-
[
[
7] Mimaki Y, Kuroda M, Fukasawa T, Sashida Y. Steroidal glycosides from the
bulbs of Allium jesdianum. J Nat Prod 1999;62:194–7.
8] Tsuruoka T, Bekh-Ochir D, Kato F, Sanduin S, Shataryn A, Ayurzana A, et al. The
essential oil of Mongolian Nepeta sibirica: a single component and its biological
activities. J Essent Oil Res 2012;24:555–9.
[
[
[
[
[
9] Sakai A, Mitsumori A, Furukawa M, Kinoshita K, Anzai Y, Kato F. Production of a
hybrid 16-membered macrolide antibiotic by genetic engineering of
Micromonospora sp. TPMA0041. J Ind Microbiol Biotechnol 2012;39:1693–701.
27 steroidal saponins. Antimicrob Agents Chemother 2006;50:1710–4.
[
[
10] Oshima R, Yamauchi Y, Kumanotani J. Resolution of the enantiomers of
aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-
methylbenzylamino)-1-deoxyalditol acetates. Carbohydr Res 1982;107:169–76.
11] Agrawal PK. Dependence of H NMR chemical shifts of geminal protons of
21] Zhang Y, Li H-Z, Zhang YJ, Jacob MR, Khan SI, Li XC, et al. Atropurosides A–G,
a-
new steroidal saponins from Smilacina atropurpurea. Steroids 2006;71:712–9.
1
glycosyloxy methylene (H2–26) on the orientation of the 27-methyl group of
furostane-type steroidal saponins. Magn Reson Chem 2004;42:990–3.