Spirostanol steroids from the roots of Allium tuberosum

Article abstract of DOI:10.1016/j.steroids.2015.03.015

Three new spirostanol saponins named tuberosines A-C (1-3), together with three known ones tuberoside O (4), 25(S)-Schidigera-saponin D5 (5), and shatavarin IV (6) were isolated from the roots of Allium tuberosum. Their structures were established on the basis of extensive spectroscopic analyses. Whereas compounds 5 and 6 exhibited potent antibacterial activities against Bacillus subtilis (32 μg/mL) and Escherichia coli (16 μg/mL), the new saponin 2 showed only moderate antibacterial activities against these pathogens. The relationship between the antibacterial activities and the structures of these saponins are described.

Full text of DOI:10.1016/j.steroids.2015.03.015

Steroids 100 (2015) 1–4
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Spirostanol steroids from the roots of Allium tuberosum
Yun-Shan Fang a,b,1, Le Cai a,1, Ying Li a, Jia-Peng Wang a, Huai Xiao a, Zhong-Tao Ding a,
⇑
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China
b School of Chemical Science and Technology, Kunming University, Kunming 650214, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new spirostanol saponins named tuberosines A–C (1–3), together with three known ones tubero-
side O (4), 25(S)-Schidigera-saponin D5 (5), and shatavarin IV (6) were isolated from the roots of Allium
tuberosum. Their structures were established on the basis of extensive spectroscopic analyses. Whereas
Received 19 January 2015
Received in revised form 12 March 2015
Accepted 22 March 2015
compounds 5 and 6 exhibited potent antibacterial activities against Bacillus subtilis (32
lg/mL) and
Available online 30 March 2015
Escherichia coli (16 g/mL), the new saponin 2 showed only moderate antibacterial activities against
l
these pathogens. The relationship between the antibacterial activities and the structures of these
saponins are described.
Keywords:
Allium tuberosum
Spirostanol saponins
Tuberosines A–C
Antibacterial activities
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
three new steroidal saponins, tuberosines A–C (1–3) (Fig. 1),
together with three known ones tuberoside O (4) [15], 25(S)-
Plants of the genus Allium (Liliaceae), well known for their culi-
nary uses, contain abundant sulfur compounds [1] and steroidal
saponins [2–4]. Chinese chive (Allium tuberosum) grows naturally
in central and northern parts of Asia and is cultured in China,
Japan, Korea, India, Nepal, Thailand, and Philippines. It is a peren-
nial plant whose stems, leaves and inflorescence are edible and
have been used as herbal medicine for treatment of abdominal
pain, diarrhea, hematemesis, snakebite, and asthma [5]. Its seeds
have been reputedly used as a traditional Chinese medicine for
treating both impotence and nocturnal emissions [6]. There is a
popular belief in Chinese folk that A. tuberosum roots are effective
in resisting gastric ulcer and treating dyspepsia [7]. Previous
research revealed that A. tuberosum seeds contain amounts of ster-
oidal saponins [8–11]. The steroidal saponins are naturally occur-
ring glycosides that possess properties such as froth formation,
hemolytic activity, toxicity to fish and complex formation with
cholesterin [12]. During recent years, steroidal glycosides have
attracted a growing interest owing to the wide range of their bio-
logical actions on living organisms, including antidiabetic [13],
antitumor [14], antitussive actions, and as platelet aggregation
inhibitors [15]. To find more bioactive steroidal saponins, we
carried out a detailed phytochemical investigation on the roots of
A. tuberosum, whose secondary metabolites has never been
explored previously. Our experiments leaded to the discovery of
Schidigera-saponin D5 (5) [16], and shatavarin IV (6) [17]. The
antibacterial activities of the isolated compounds have also been
investigated. This paper deals with the isolation, structural elucida-
tion, and antibacterial activities of the steroidal saponins from A.
tuberosum roots.
2. Results and discussion
Compound 1 was obtained as an amorphous powder, with
the molecular formula C33H54O9, which was deduced from the
HRESIMS data showing a [M-H]ꢀ ion at m/z 593.3693. The IR spec-
trum of 1 showed the characteristic absorption of hydroxy groups
at 3428 cmꢀ1. Of the 33 carbons, 27 were assigned to the aglycon
part and 6 to the oligosaccharide moiety. The 1H NMR spectrum
of the aglycon part of 1 showed two angular methyl signals at dH
0.75 and 0.97 (each 3H, s), two secondary methyl signal at dH
0.95 (3H, d, J = 6.6 Hz) and 1.05 (3H, d, J = 7.2 Hz) (Table 1). The
13C NMR spectrum of 1 showed two signals at lower field than
100 ppm (Table 1); the signal at dC 103.8 was due to the anomeric
carbon, and the signal at dC 111.1 was assignable to the C-22 car-
bon of the spirostan skeleton [18]. The above data were consistent
with a (25S)-spirostanol monosaccharide. The chemical shift and
coupling constant of H-27 (dH 1.05 d, J = 7.2 Hz) in 1 further con-
firmed the b-orientation of methyl-27 [19]. Comparison of the sig-
nals from the aglycon moiety in the 13C NMR spectra with those
from markogenin [(25S)-5b-spirostane-2b,3b-diol] [20] showed
that the aglycone moiety of 1 was markogenin and the sugar was
attached to its C-3 position, which was further supported by the
⇑
Corresponding author. Fax: +86 0871 65033910.
E-mail address: ztding@ynu.edu.cn (Z.-T. Ding).
1
These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.steroids.2015.03.015
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

2
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
27
The anomeric proton coupling constant of 7.8 Hz indicated
21
20
the b-configuration of the glucopyranosyl [21], whose absolute
configuration was confirmed to be D on the basis of the acid
hydrolysis of 1 with 1 M HCl and the GC analysis of their
trimethylsilyl L-cysteine derivatives. Therefore, the structure of
1 was determined as (25S)-5b-spirostan-2b,3b-diol 3-O-b-D-glu-
26
25
O
22
18
12
11
23
24
17
R3
1
13
14
O
19
10
OH
6'
16
R1
O
2
8
9
6
15
3
5'
O
5
7
1'
copyranoside and named tuberosine A.
R5O
4
4'
R2
2'
Compound 2,
a white amorphous powder, exhibited the
3'
pseudomolecular ion peak at m/z 609.3641 [M-H]ꢀ in the
HO
OR4
1
R1=OH, R2=R3=R4=R5=H
2 R1=R3=OH, R2=R4=R5=H
3 R1=OH, R2=R3=R4=H, R5=α-L-Rha
HRESIMS data, corresponding to the molecular formula
C
33H54O10. A detailed comparison of the 1H, 13C NMR chemical shift
(Table 1) of 1 and 2, revealed that 2 was also a spirostanol steroidal
saponin with a b-glucopyranosyl group at C-3. The main difference
between them was that compound 2 possessed only one angular
methyl group which was one less than 1. Meanwhile, compound
2 exhibited the signals of an additional oxygenated CH2 group
(dH 3.40 d, J = 11.6 Hz; 3.87 d, J = 11.6 Hz; dC 65.2 t) in its NMR
spectra. Combining with its molecular formula, there might be a
hydroxy group substituted at C-18 or C-19 in compound 2. The
absence of the angular methyl group at about 24 ppm indicated
that the hydroxy group should be located at C-19, which was fur-
ther supported by the HMBC correlations from H-19 to C-1 (dC 33.3
t), C-5 (dC 41.9 d), C-9 (dC 29.2 d), and C-10 (dC 41.9 s) (Fig. 2). On
4
R1=R2=OH, R3=R4=R5=H
5 R1=R2=R3=R5=H, R4=β-D-Glc
6
R1=R2=R3=H, R4=β-D-Glc, R5=α-L-Rha
Fig. 1. Structures of compounds 1–6.
HMBC correlations of anomeric proton H-10 (dH 4.27 d, J = 7.8 Hz)
with C-3 (dC 80.3) and H-3 (dH 3.99 m) with anomeric carbon C-
10 (dC 103.8) (Fig. 2). The sugar was assigned as glucopyranosyl
moiety on the basis of a set of characteristic six carbon signals at
dC 103.8, 78.9, 78.8, 75.6, 72.5, 63.5 in the 13C NMR spectrum.
Table 1
acid hydrolysis, 2 afforded
analysis of its trimethylsilyl
2 was established as (25S)-5b-spirostan-2b,3b,19-triol 3-O-b-D-
D
-glucose, which was identified by GC
-cysteine derivative. Thus, compound
NMR spectral data of 1 (1H: 600 MHz; 13C: 150 MHz) and 2 (1H: 400 MHz; 13C:
L
100 MHz) in CD3OD.
Position
1
1
2
glucopyranoside and named tuberosine B.
Compound 3, a white amorphous powder, gives a molecular for-
mula of C39H64O13 by HRESIMS (negative ion mode) at m/z
739.4271 [M-H]ꢀ. Its spectral features and physicochemical
properties suggested 3 to be a spirostanol saponin. Compound 3
containing 39 carbons, including 27 of the aglycon part and 12 of
the oligosaccharide moiety. Comparison of the NMR data of 3 with
those of 1 (Table 2) revealed that they shared the same skeleton
and same glycosidic position at C-3. The molecular weight of 3
was 146 mass units greater than that of 1 indicating that 3 had
an additional deoxyhexose group, which was identified as a rham-
nose by its NMR data (dH 4.80 brs, 1.23 d, J = 6.0 Hz; dC 102.9 d, 73.7
d, 72.4 d, 72.2 d, 70.6 d, 17.8 d) [19]. The HMBC correlation
between H-100 (dH 4.80 brs) and C-40 (dC 79.6) (Fig. 2) proved the
(1 ? 4) linkage between glucose and rhamnose. The evaluation
of chemical shifts and spin–spin couplings of two anomeric
protons allowed the identification of one b-glucopyranose and
dC
dH (J in Hz)
dC
dH (J in Hz)
40.8
1.65 m
1.41 m
3.58 m
3.99 m
1.77 m
1.61 m
1.30 m
1.98 m
1.39 m
1.31 m
1.24 m
1.57 m
1.77 m
–
1.49 m
1.31 m
1.72 m
1.17 m
–
1.17 m
1.92 m
1.20 m
4.35 m
1.71 m
0.75 s
0.97 s
33.3
1.80 m
1.53 m
3.63 m
4.02 m
1.94 m
1.21 m
1.38 m
1.81 m
1.24 m
1.43 m
1.05 m
1.59 m
2.14 m
–
1.55 m
1.34 m
1.72 m
1.14 m
–
1.14 m
1.86 m
1.66 m
4.36 m
1.71 m
0.74 s
2
3
4
68.8
80.3
32.4
68.0
78.5
32.4
5
6
43.3
27.6
41.9
26.4
7
27.8
27.2
8
9
10
11
37.6
37.9
38.6
23.0
36.5
29.2
41.9
22.0
12
42.1
41.4
one
glucose was determined as
described above. Therefore, the structure of 3 was determined as
(25S)-5b-spirostan-2b,3b-diol 3-O- -rhamnopyranoyl-(1 ? 4)-
O-b- -glucopyranoside and named tuberosine C.
a-rhamnopyranose [2]. The absolute configuration of
13
14
15
42.6
58.3
33.5
41.6
57.8
30.9
D
and that of rhamnose as , as
L
a-L
16
17
18
19
83.3
64.5
17.8
24.9
82.4
63.4
16.9
65.2
D
It’s worthy to note that all the three new spirostanol steroidal
saponins (1–3) possess an unusual b-hydroxy substitution at C-2,
which is normally a-orientation in this kind of compounds.
The antimicrobial activities of the isolated compounds against
Escherichia coli, Bacillus subtilis have also been investigated
(Table 3). The results showed that 5 and 6 exhibited potent antibac-
3.87 d (11.6)
3.40 d (11.6)
1.82 m
0.95 d (7.2)
–
20
21
22
23
44.3
15.6
111.1
28.5
1.81 m
0.95 d (6.6)
–
1.42 m
1.04 m
1.87 m
1.39 m
1.63 m
3.88 dd (10.8, 2.4)
3.23 (9.6, 7.8)
1.05 d (7.2)
4.27 d (7.8)
3.20 m
3.23 m
3.23 m
3.31 m
3.81 m
43.4
14.7
111.2
26.9
Ha1.90 m; Hb1.34 m
terial activities against B. subtilis (32
(positive control, kanamycin: 2 g/mL). New saponin 2 showed
moderate antibacterial activities against B. subtilis (64 g/mL) and
E. coli (64 g/mL). Compounds 1, 3, and 4 showed no or only very
weak growth inhibition against above two microbes.
lg/mL) and E. coli (16 lg/mL)
24
27.8
26.6
1.93 m
1.34 m
1.67 m
3.87 dd (10.2, 2.4)
3.24 dd (10.2, 7.2)
1.04 d (6.8)
4.28 d (8.0)
3.24 m
3.35 m
3.26 m
3.26 m
3.81 m
l
l
25
26
29.4
66.9
28.3
66.1
l
27
10
20
30
40
50
60
17.2
103.8
75.6
78.8
72.5
78.9
63.5
16.4
102.4
74.6
77.6
71.6
77.8
62.6
Those saponins having a saccharide moiety at C-3 without any
oxygen functionalities at C-2 (5 and 6) exhibited potent antibacter-
ial activities, indicating 2-OH was negative to antibacterial activi-
ties. Compound 2, possessing a 2-OH, exhibited moderate
antibacterial activities, probably because of the existence of 19-
OH. 4 showed no antibacterial activities indicating 5-OH has no
positive effect on antibacterial activities.
3.60 m
3.63 m

Y.-S. Fang et al. / Steroids 100 (2015) 1–4
3
O
O
HO
O
O
OH
OH
HO
O
HO
O
O
O
HO
HO
HO
HO
2
1
OH
OH
27
25
21
17
26
23
O
20
18
12
9
6"
22
24
HO
11
5"
19
13
O
4"
OH
16
1
14
3"
HO
2
6'
5'
HO
O
1' '
O
15
10
8
7
O
3
2"
HO
5
O
4
6
4'
1'
2'
3'
HO
OH
3
Fig. 2. Key 1H–1H COSY (
) and HMBC (
) correlations of compounds 1–3.
Table 2
NMR spectral data of 3 in CD3OD (1H: 600 MHz; 13C: 150 MHz).
Table 3
Antibacterial activities of compounds 1–6 (MIC:
lg/mL).
Position
1
dC
dH (J in Hz)
Position
21
dC
dH (J in Hz)
Compounds
B. subtilis
E. coil
40.0
1.66 m
1.41 m
3.58 m
3.99 brs
14.7
0.95 d (7.2)
1
2
3
4
5
6
256
64
256
256
16
256
64
256
128
32
2
3
67.9
79.4
22
23
111.1
27.7
–
1.42 m
1.04 m
1.87 m
1.39 m
1.64 m
3.88 dd (10.2, 2.4)
3.24 dd (10.2, 7.2)
1.05 d (7.2)
4
31.5
1.77 m
1.61 m
1.31 m
1.98 m
1.39 m
1.31 m
1.24 m
1.58 m
1.77 m
–
1.52 m
1.31 m
1.72 m
1.18 m
–
24
26.9
16
2
16
2
Kanamycin
5
6
42.5
26.7
25
26
29.4
66.1
3.2. Plant material
7
26.9
27
16.4
10
20
30
40
102.6
74.9
76.7
79.6
4.29 d (7.8)
3.24 m
3.43 m
The roots of A. tuberosum were collected from Guandu District,
Kunming City in Yunnan Province of China, in June 2011, and iden-
tified by professor Shu-Gang Lu from School of life Sciences,
Yunnan University. The voucher specimen (2011-JCG-1) has been
deposited in the school of Chemical Science and Technology of
Yunnan University.
8
9
10
11
36.8
37.0
37.8
22.1
3.48 m
12
13
41.3
41.7
50
60
76.8
61.9
3.31 m
3.76 m
3.61 m
4.80 brs
3.60 m
3.3. Extraction and isolation
14
15
57.4
32.6
1.17 m
1.92 m
1.20 m
4.35 m
1.71 m
0.75 s
100
200
102.9
72.2
Air-dried and powdered roots (14.0 kg) of A. tuberosum were
extracted three times with methanol at 60 °C. The extracts were
combined, and evaporated under reduced pressure. The crude resi-
due (1.4 kg) was suspended in H2O, and partitioned with petro-
leum ether, EtOAc, and 1-butanol successively. The 1-butanol
fraction (200 g) was subjected to silica gel column eluted gradually
with CHCl3–MeOH (200:1–1:1) to yield seven fractions (FrA1–A7).
FrA4 (8.3 g) was further subjected to silica gel column chro-
matography using a EtOAc–MeOH gradient (20:1–2:1) to yield
eight fractions (FrA4-1–FrA4-8). Fr4-4 (0.4 g) was separated by
repeated column chromatography of silica gel, Sephadex LH-20
and yielded compounds 1 (7 mg), 2 (48 mg), and 4 (41 mg), respec-
tively. Fr4-5 (0.3 g) was passed through a Sephadex LH-20 column
and further purified by silica gel chromatography with CHCl3–
MeOH (7:1) as the eluent to yield 3 (105 mg) and 5 (75 mg).
FrA4-7 was separated by repeated silica column chromatography
and Sephadex LH-20 to yield 6 (86 mg).
16
17
18
19
20
82.4
63.6
16.9
24.0
43.4
300
400
500
600
72.4
73.7
70.6
17.8
3.80 m
3.37 m
3.93 m
1.23 d (6.0)
0.97 s
1.82 m
3. Experimental section
3.1. General experimental procedures
Melting points were determined on a XRC-1 Melting Point
Apparatus and uncorrected. Optical rotations were measured with
a
Jasco P-1020 digital polarimeter. A Nicolet Magna-IR 550
spectrometer was used for scanning IR spectroscopy with KBr pel-
lets. NMR spectra were acquired with either a Bruker AM-400
spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR)
or a Bruker DRX-600 (600 MHz for 1H NMR and 150 MHz for 13C
NMR) spectrometer. ESI-MS analyses were recorded with Agilent
G3250AA and Auto Spec Premier P776 spectrometer. Silica gel
(200–300 mesh or 300–400 mesh) was used for column chro-
matography. Sephadex LH-20 was purchased from Amersham
Biosciences.
Tuberosine A (1): white amorphous powder, m.p. 162–164 °C,
20
[
a
]
D
ꢀ60.0 (c 1.0, CH3OH); 1H and 13C NMR data: see Table 1; IR
(KBr) v: 3428, 2933, 2353, 1678, 1531, 1082 cmꢀ1; HRESIMS m/z:
593.3693 [M-H]ꢀ (calcd for C33H53O9 [M-H]ꢀ, 593.3690).
Tuberosine B (2): white amorphous powder, m.p. 180–182 °C,
20
[a
]
ꢀ56.1 (c 1.0, CH3OH); 1H and 13C NMR data: see Table 1; IR
D

4
Y.-S. Fang et al. / Steroids 100 (2015) 1–4
(KBr) v: 3427, 2931, 2353, 1633, 1454, 1044 cmꢀ1; HRESIMS m/z:
Appendix A. Supplementary data
609.3641 [M-H]ꢀ (calcd for C33H53O10 [M-H]ꢀ, 609.3639).
Tuberosine C (3): white amorphous powder, m.p. 204–206 °C,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2015.03.
015.
20
[a
]
ꢀ77.9 (c 1.0, CH3OH); 1H and 13C NMR data: see Table 1; IR
D
(KBr) v: 3428, 2933, 2353, 1678, 1531, 1081 cmꢀ1; HRESIMS m/z:
739.4271 [M-H]ꢀ (calcd for C39H63O13 [M-H]ꢀ, 739.4269).
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3.4. Acid hydrolysis and sugar analysis
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Saponin (4 mg) was dissolved in 1 M HCl (dioxane/H2O, 1:1,
2 mL) and stirred at 90 °C for 2 h. After cooling, the reaction mix-
ture was neutralized with silver carbonate, and the solvent was
evaporated. Then, the reaction mixture was extracted with CHCl3,
and the aqueous layer was evaporated to give a mixture of
monosaccharides. The residue was further dissolved in anhydrous
pyridine (0.2 mL) followed by the addition of L-cysteine methyl
ester hydrochloride (dry pyridine, 0.06 M, 0.2 mL). After heating
at 60 °C for 2 h, and trimethylsilyl thiazolidine (0.2 mL) was added.
Next, the mixture was heated at 60 °C for another 2 h and parti-
tioned with n-hexane and water. The organic layer was analyzed
by gas chromatography (GC) under the following conditions:
capillary column, HP-5 (30 m ꢁ 0.32 mm ꢁ 0.25
lm, Dikma); FID
detector at 300 °C; injection temperature, 280 °C; column
temperature, 210 °C, maintained for 20 min; carrier gas, N2. The
standard sugars were subjected to the same reaction and GC
conditions. The retention times of thiazolidine derivatives
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3.5. Antibacterial activities
Antibacterial activity assays were performed in sterilized 96-
well microplates using a microdilution method described pre-
viously [22]. The 18-h-old bacterial cultures from B. subtilis
(ATCC 6633), and E. coli (ATCC 25922) were added to LB broth med-
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Acknowledgements
This project was financially supported by a Natural Science
Foundation of China (No. 81460648) and
a grant from the
Program for Changjiang Scholars and Innovative Research Team
in University (No. IRT13095).