A steroidal saponin from the seeds of Allium tuberosum

Article abstract of DOI:10.1016/S0031-9422(01)00216-3

A steroidal saponin, named tuberoside, together with seven known compounds, were isolated from the seeds of Allium tuberosum Rottl, ex Spreng. Its structure was established by spectroscopic data, hydrolysis, and comparison with spectral data of known compounds to be (2α, 3β, 5α, 25S)-2,3,27-trihydroxyspirostane 3-O-α-L-rhamnopyranoyl-(1→2)-O-[α-L-rhamnopyranoyl- (1→4)]ν-D-glucopyranoside,

Full text of DOI:10.1016/S0031-9422(01)00216-3

Phytochemistry 57 (2001) 1219–1222
www.elsevier.com/locate/phytochem
A steroidal saponin from the seeds of Allium tuberosum
a
a,
b
Zhong-Mei Zou , De-Quan Yu *, Pu-Zhu Cong
aInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
bInstitute of Medicinal Plants Developments, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100094, China
Received in revised form 25 May 1999
Abstract
A steroidal saponin, named tuberoside, together with seven known compounds, were isolated from the seeds of Allium tuberosum
Rottl. ex Spreng. Its structure was established by spectroscopic data, hydrolysis, and comparison with spectral data of known
compounds to be (2a, 3b, 5a, 2 5S )-2,3,27-trihydroxyspirostane 3-O-a-l-rhamnopyranoyl-(1!2)-O-[a-l-rhamnopyranoyl-(1!4)]-
b-d-glucopyranoside. # 2001 Published by Elsevier Science Ltd.
Keywords: Allium tuberosum; Liliaceae; Saponin; Spirostanol glycoside; Tuberoside
1
. Introduction
2. Results and discussion
Allium tuberosum Rottl. ex Spreng is widely cultivated
Commercially available seeds of Allium tuberosum
(9.5 kg) were defatted with petroleum ether followed by
extraction with hot 70% EtOH. The concentrated
extract was partitioned between CHCl and H O, then
between n-BuOH and H O. The n-BuOH soluble phase
2
was subjected to silica gel column chromatography to
give a new compound, named tuberoside (1).
and used as food in China, whose seeds have been reput-
edly used as a traditional Chinese medicine for treating
both impotence and nocturnal emissions (Jiangsu New
Medicinal College, 1979). Species of Allium genus are
famous for their production of sulfur-containing biologi-
cally active natural products. With regard to the saponin
constituents of the Allium genus, many steroidal saponins
have been reported (Zou et al., 1999). However, there has
been no exploration of the secondary metabolites of A.
tuberosum seeds. Our detailed investigation of its seeds has
led to the discovery of a new saponin (1), together with
eight known compounds, gitogenin 3-O-a-l-rhamnopyr-
anosyl-(1!2)-b-d-galactopyranoside (Mimaki et al.,
3
2
1
996), S-allylcysteine (3), l-tyrosine (4), adenosine (5),
inosine (6), allantoin (7) and mannitol (8). The known
compounds were identified by direct comparison with
authentic samples (co-TLC, IR, MS, NMR) or with
reported spectral and physical data. This paper deals
with the isolation and structural elucidation of the new
saponin (1).
Tuberoside (1) was obtained as an amorphous powder,
with the molecular formula C H O , which was
4
5
74 18
deduced from the FAB–mass spectrum showing an
M+H]+ ion at m/z 903 and the 13C NMR spectrum
Table 1). The IR spectrum of 1 showed the characteristic
absorption of hydroxyl groups at 3422 cm . The FAB–
mass spectrometry of compound 1 showed fragment ion
[
(
*
Corresponding author. Tel.: +86-10-63036781; fax: +86-10-
3017757.
E-mail address: dquanyu@public.bta.net.cn (D.-Q. Yu).
ꢀ1
6
0
031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0031-9422(01)00216-3

1220
Z.-M. Zou et al. / Phytochemistry 57 (2001) 1219–1222
peaks at m/z 757 (M+H-146), 611 (M+H-146-146), 449
M+H-146-146-162), which suggested the sequential loss
of two deoxy hexose and one hexose moiety. The NMR
(Table 1) indicated 1a is a spirostanol derivative with
three hydroxyl groups, and the base peak at m/z 155 in
EI–MS spectrum of 1a proved a hydroxyl group to be
located at the F ring (Cong, 1987). Further analysis of
(
spectra (Tables 1 and 2) of 1 also showed the presence
of three sugars from the three anomeric protons at d
1
Table 2
1H NMR spectroscopic data of compound 1 (500 MHz, pyridine-d
5
.00 (d, J=7.3 Hz), 6.38 (s) and 5.38 (s) in the H NMR
)a
5
spectrum, and the three anomeric carbon signals at d
1
3
1
02.9, 102.2, 100.9 in the C NMR spectrum.
Position
ꢀH
J (Hz)
On acid hydrolysis, 1 afforded d-glucose, l-rham-
nose, which were identified by co-TLC with authentic
samples and aglycone (1a). The NMR spectral data
H-1a
H-1b
H-2b
H-3a
H-4a
H-4b
H-5a
H-6
2.22 dd
1.16 m
12.7, 4.7
4.07 ddd
3.88 dd
12.1, 11, 4.7
11.4, 11.0
2.0 overlap
1.39 overlap
1.01 overlap
1.20 m
Table 1
13C NMR spectroscopic data of compounds 1 and 1a (125 MHz)a
C
1
1
1a
1.08 m
0.83 m
1.48 m
45.9
73.1
85.1
33.5
44.6
28.1
32.3
34.6
54.4
36.9
21.5
45.0
H-7
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
70.6
76.4
37.4
44.9
27.8
32.1
35.6
54.3
37.6
21.2
H-8
H-9
1.39 overlap
0.62 ddd
1.48 m
11, 11, 2.5
11.8, 11.8
H-11
1.23 m
H-121.63 overlap
1.03 dd
1.00 m
2.00 overlap
1.39 overlap
4.54 m
1.39 overlap
0.80 s
H-14
H-15
0
1
240.1
39.9
H-16
H-17
H-18
H-19
H-20
H-21
H-23
H-23
H-24
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
40.6
56.4
32.2
81.280.9
63.1
16.6
15.6
42.1
15.1
109.7
31.6
24.1
39.2
64.1
64.4
100.9
41.7
56.2
32.8
0.94 s
1.95 m
1.12 d
63.0
16.2
13.6
41.7
14.4
109.2
31.9
24.5
38.2
63.1
63.4
6.9
1.74 m
1.74 m
1.80 overlap
0.96 m
2.06 m
4.13 dd
3.89 dd
3.71 dd
3.63 dd
5.00 d
H-25
H-26eq
H-26ax
H-27a
H-27b
Glc H-1
H-24.19
H-3
11.4, 5.2
11.4, 11.1
10.7, 5.1
10.7, 7.2
7.3
Glc- 1
t
4.21 t
8.9, 7.3
8.9
8.9
2
3
4
5
6
78.0
78.0
78.7
77.2
61.1
H-4
H-5
4.38 t
3.75 d
8.9
12.3, 3.6
12.3, 5.1
H-6
4.07 dd
4.24 dd
6.38 s
Rha- 1
102.2
Rha H-1
H-24.82
H-3
b
2
3
4
5
6
72
br.s
72.7a
73.9b
70.5
4.59 dd
4.34 dd
4.92 dd
1.63 d
9.3, 3.3
9.4, 9.3
9.4, 6.1
6.1
H-4
H-5
18.6
102.9
H-6
Rha- 1
Rha H-1
H-24.66
H-3
5.85 s
2
3
4
5
6
72
brs
72.7a
74.1b
69.6
4.47 dd
4.32 dd
4.85 dd
1.66 d
9.3, 3.4
9.4, 9.3
9.4, 6.3
6.3
H-4
H-5
18.6
H-6
a
a
1
1
13
1
Spectra were measured in pyridine-d (1) and chloroform-d (1a).
a and b signals may be interchangeable.
5
All assignments were confirmed by H– H COSY, C– H COSY
and DEPT spectra.
b

Z.-M. Zou et al. / Phytochemistry 57 (2001) 1219–1222
1221
the H and 13C NMR spectra of 1a allowed its identifi-
cation as (2a, 3b, 5a, 2 5S )-spirostan-2,3,27-triol (cres-
tagenin), isolated previously from the leaves of Digitalis
canariensis (Dolgado et al., 1969; Gonzalez, et al. 1983).
1
3.3. Extration and isolation.
Air-dried and powdered seeds of A. tuberosum Rottl.
Ex Spreng (9.5 kg) were defatted by percolation with
petroleum ether followed by extraction with hot 75%
EtO. The combined EtOH extracts were filtered, evap.
1
13
The H and C NMR spectral data assignments of
1
1
compound 1 were performed via analyses of the H– H
COSY, 13C– H COSY and DEPT spectra. The presence
of two terminal a-l-rhmnopyranosyl moieties in the
molecule of 1 was demonstrated by the two sets of char-
acteristic six carbon signals at ꢀ 102.2, 72.6, 72.7, 73.9,
1
under red. press. and the residue suspended in H O and
2
partitioned with CHCl and n-BuOH, respectively.
3
The ꢁ-BuOH extract (62g) was fractionated on a
silica gel column with a gradient mixture of CHCl3–
MeOH and finally with MeOH to give five Frs. (A –A ).
1
3
70.5, 18.6; 102.9, 72.5, 72.7, 74.1, 69.6, 18.6 in the
NMR spectrum. The C-5 signals of two rhamnose units
C
1
5
Fr. A was chromatographied on silica gel, eluting with
1
at ꢀ 70.5 and 69.6, respectively, indicated their a-config-
CHCl –MeOH (9:1) to give compounds 2 (10 mg) and 5
3
1
3
urations (Seo et al., 1978). Comparison of CNMR data
of the sugar moieties with the corresponding methyl b-d-
glucopyranoside (Agrawal et al., 1985) indicated that the
C-2and C-4 of the glucosyl moiety were shifted down-
field to ꢀ 78.0 and 78.7 from 73.7 and 70.3, respectively.
The significant glycosylation shifts clearly showed the
two terminal rhamnopyranoses were linked to the C-2
and C-4 positions of the inner glucopyranose. The b-
configuration of the glucopyranose was further confirmed
by the large J1,2 couplings of its anomeric proton.
(176 mg), while Fr. A2 was repeatedly chromato-
graphied on silica gel to give compounds 6 (15 mg) and
7 (7 mg). Fr.A was subjected to silica gel with CHCl -
3
3
MeOH (20:3) to give compounds 1 (31mg) and 8 (38 mg).
Fr.A was repeatedly chromatographied on silica gel CC
4
to give compound 3 (162mg). Fr. A was subjected to
5
silica gel CC with CHCl –MeOH (5:1) to give com-
3
pound 4 (16 mg).
3.4. Tuberoside A (1)
The linkage of the sugar chain was concluded to be at
the C-3 hydroxyl position of the aglycone because, in
the 13C NMR spectrum of 1, the signal due to C-3
shifted to a lower field by 8.7 ppm, whereas the signals
due to C-2and C-4 moved to upper fields by 2. 5 and 3.9
ppm, as compared with those of 1a. Accordingly, the
structure of 1 was elucidated as (2a, 3b, 5a, 2 5S )-2,3,27-
trihydroxyspirostane 3-O-a-l-rhamnopyranoyl-(1!2)-
O-[a-l-rhamnopyranoyl-(1!4)]-b-d-glucopyranoside
ꢁ
25
C H O , amorphous powder, mp 292–293 , [a]
D
4
ꢁ
5
74 18
ꢀ1
ꢀ33 ; c 0.02, MeOH. IR (KBr) cm 3422, 2932, 1452,
+
1381, 1043, 987, 912. FABMS m/z 903 [M+H] , 757
+
+
[M+H-146] , 611 [M+H-2ꢂ146] , 449 [aglyco-
+
1
13
ne+H] , 431, 413. H and C NMR data, see Tables 1
and 2.
3.5. Acid hydrolysis of 1
(1).
Compound 1 (20 mg) was heated with 3% H SO
2
(EtOH, 1:1) at 100 C for 3 h. After cooling, the reaction
mixture was neutralized with 1 N NaOH and parti-
4
ꢁ
3
. Experimental
tioned between AcOEt and H O. The AcOEt soluble
2
3
.1. General
phase was concentrated and subjected to silica gel CC
with CHCl to give the known crestaganin 1a.
3
+
Mps were obtained on a Boetius miromelting appa-
Compound 1a: white powder, EI–MS m/z 448 (M ),
1
ratus and are uncorrected. Optical rotations were mea-
sured with Perkin-Elmer 241 polarimeter. IR spectra
were recorded on a Perkin-Elmer 683 instrument. EIMS
was obtained on a VG Zab-2f and FABMS on a VG
Zabspec mass spectrometer. The H NMR spectra (500
MHz) and 13C NMR (125 MHz) were recorded on a
155 (100). H NMR (CDCl ) ꢀ 0.97 (3H, d, J=7.8 Hz),
3
0.86 (3H, s), 0.76 (3H, s), 3.71 (dd, J=10.7, 5.1 Hz,
CH -27), 3.63 (dd, J=10.7, 7.1 Hz, CH -27), 3.89 (1H,
dd, J =11.4 11.1 Hz, CH -26), 4.13 (dd, J=11.4, 5.2
2
2
,
2
1
Hz, CH -26). 13C NMR data, see Table 1.
2
The H O soluble phase was concentrated and exam-
2
Bru
¨
ker AM-500 spectrometer and the chemical shifts
are reported in ppm using the solvent as reference.
ined by comparison with authentic samples through
TLC (CHCl –MeOH–H O, 7:3:0.1) to detect d-glucose
3
2
and l-rhamnose.
3
.2. Plant materials.
The seeds of Allium tuberosum Rottl. ex Spreng were
Acknowledgements
purchased from Beijing Tong-Ren-Tang Group, China,
and were identified by Mr. Wei-Ze Liu, Institute of
Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College, China.
The authors are grateful to Mr. Wei-Ze Liu, Institute of
Materia Medica, CAMS and PUMC, for the identifica-
tion of plant sample and to Professors Wen-Yi He and

1222
Z.-M. Zou et al. / Phytochemistry 57 (2001) 1219–1222
Man Kong, for measurements of NMR spectra. Our
thanks are also due to Mr. Li-Jun Li, Institute of Materia
Medica, CAMS and PUMC, for measurements of MS.
Jiangsu New Medicinal College, 1979. The Dictionary of Chinese
herbal Medicines, Vol. 2. Shanghai People’s Publishing Press,
Shanghai, pp. 1646.
Mimaki, Y., Kanmoto, T., Kuroto, M., Sashida, Y., Satomi, Y.,
Nishino, A., Nishino, H., 1996. Steroidal saponins from Hosta
longipes and their inhibitory activity on tumor promoter -induced
phospholipid. Phytochemistry 42, 1065–1070.
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