Steroidal Glycosides from the Flowers of Allium leucanthum

Article abstract of DOI:10.1007/s10600-015-1444-z

Furostanol and spirostanol glycosides 1 and 2 were isolated from the flowers of Allium leucanthum, a Caucasian endemic species that grows in Georgia. The structures were established on the base of chemical evidence and spectral analyses ( ¹ H, ¹³ C NMR, ¹ H- ¹ H COSY, ¹ H- ¹³ C COSY, HMBC, and HR-MS) data. Compound 1 (leucofuranoside A) was reported for the first time and was identified as 26-O-β-Dglucopyranosyl-(25R)-5α-furostane-3β,6β-diol-3-O-β-D-glucopyranosyl-(1→2)-O-β-D-xylopyranosyl-(1→3)-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside. Compound 2 was identified as (25R)-5α-spirostane-3β,6β-diol-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and described for the first time in the genus Allium.

Full text of DOI:10.1007/s10600-015-1444-z

DOI 10.1007/s10600-015-1444-z
Chemistry of Natural Compounds, Vol. 51, No. 5, September, 2015
STEROIDAL GLYCOSIDES FROM THE FLOWERS OF Allium leucanthum
1*
1
2
Lasha Mskhiladze, David Chincharadze, Vakhtang Mshvildadze,
2
3
4
4
Andre Pichette, Michel Frederich, Evelyne Ollivier, and Riad Elias
Furostanol and spirostanol glycosides 1 and 2 were isolated from the flowers of Allium leucanthum,
a Caucasian endemic species that grows in Georgia. The structures were established on the base of chemical
1
13
1
1
1
13
evidence and spectral analyses ( H, C NMR, H– H COSY, H– C COSY, HMBC, and HR-MS) data.
Compound 1 (leucofuranoside A) was reported for the first time and was identified as 26-O-ꢀ-D-
glucopyranosyl-(25R)-5ꢁ-furostane-3ꢀ,6ꢀ-diol-3-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-O-ꢀ-D-xylopyranosyl-
(1ꢂ3)-O-ꢀ-D-glucopyranosyl-(1ꢂ4)-ꢀ-D-galactopyranoside. Compound2was identified as (25R)-5ꢁ-spirostane-
3ꢀ,6ꢀ-diol-3-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-ꢀ-D-glucopyranosyl-(1ꢂ4)-ꢀ-D-galactopyranoside and
described for the first time in the genus Allium.
Keywords: Allium leucathum, Alliaceae, spirostanol glycoside, furostanol glycoside, leucofuranoside A.
Saponins are an important family of glycosylated secondary metabolites that are widely distributed in the plant
kingdom [1]. Besides food industrial and pharmaceutical applications, saponins are known to be the main constituents of
several herbal drugs and folk medicines used worldwide [2]. Their biological activities are linked to the structure of both the
aglycone and the sugar moieties. The incredibly wide chemical diversity of saponins has led to a sustained and renewed
interest in these compounds.
The genus Allium includes up to 800 species of the worldꢃs flora.Among them, some 70 grow in the Caucasian region
and 35 species are described in Georgia [3]. Allium leucanthum K. Koch (Alliaceae), called “whiteflower onion” in Georgia,
is a Caucasian endemic species and, along with other Allium species, is widely used in Georgian traditional medicine as an
antiseptic and antibacterial remedy. Various secondary metabolites have been identified in the genus Allium [4, 5]. Among
them, steroidal saponins have been investigated for their antibacterial, antileishmanial, antifungal [6–9], cytotoxic [10, 11],
and antioxidant activities [12]. Furthermore, steroidal saponins isolated from different species of Allium have shown significant
cytotoxic activity [13–16].
This paper describes the isolation of spirostanol and furostanol glycosides from the flowers of Allium leucanthum
1
13
that grow in Georgia and the structural determination of two these compounds by H, C nuclear magnetic resonance (NMR),
1
1
1
13
H– H correlation spectroscopy (COSY), H– C COSY, heteronuclear multiple bond correlation (HMBC), and high-resolution
mass spectroscopy (HR-MS), and the results of hydrolytic cleavage.
Dried and powdered flowers of Allium leucanthum (500 g) were extracted twice with hot MeOH–H O (8:2, v/v).
2
After removal of solvent, the extract was suspended in water and then extracted with n-BuOH. The n-BuOH extract was
chromatographed over Diaion HP-20, using MeOH–H O as the eluent in gradient conditions and EtOAc. The spirostanol
2
fraction was collected in MeOH–H O (7:3, v/v), and the furostanol fraction in MeOH–H O (5:5, v/v). The spirostanol and
2
2
furostanol fractions were subjected to column chromatography (CC) on silica gel to give compounds 1 and 2.
1) Department of Pharmacognosy and Botany, Faculty of Pharmacy, Tbilisi State Medical University, 33, Vazha
PshavelaAve., 0177, Tbilisi, Georgia, e-mail: lashamskhiladze@gmail.com; 2) Laboratoire LASEVE, Departement des Sciences
Fondamentales, Universite du Quebec a Chicoutimi, 555 Boul. Universite, Chicoutimi, Quebec, Canada, G7H2B1; 3) Laboratoire
de Pharmacognosie, Departement de Pharmacie, Centre Interfacultaire de Recherche du Medicament-CIRM, University of
Liege, CHU-B36, B-4000 Liege, Belgium; 4) Laboratoire de Pharmacognosie-Ethnopharmacologie, UMR-MD3, AMU Faculte
de Pharmacie, 27 Boul. Jean Moulin, CS30064, 13385 Marseille, cedex 5, France. Published in Khimiya Prirodnykh Soedinenii,
No. 5, September–October, 2015, pp. 772–775. Original article submitted February 7, 2014.
900
0009-3130/15/5105-0900 02015 Springer Science+Business Media New York

1
13
TABLE 1. H (400 MHz) and C (100 MHz) NMR Data of Compound 1 (pyridine-d –D O, 10:1, ꢅ, ppm)
5
2
C atom
C atom
ꢅC (DEPT)
ꢅH
ꢅC (DEPT)
ꢅH
Aglycone
Glc-A
1
2
3
4
5
6
Glc-B
1
2
3
4
5
6
Xyl
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Gal
1
38.8 (CH2)
29.9 (CH2)
78.4 (CH)
32.4 (CH2)
47.8 (CH)
70.8 (CH)
40.1 (CH2)
30.4 (CH)
54.5 (CH)
35.9 (C)
1.53, 0.89
2.13, 1.77
4.08
2.27 2.13
1.07
4.00
1.76, 1.14
2.17
104.4 (CH)
80.8 (CH)
86.9 (CH)
69.9 (CH)
77.1 (CH)
62.4 (CH2)
5.15
4.32
4.12
3.77
3.85
4.50, 4.06
104.2 (CH)
75.6 (CH)
77.4 (CH)
70.8 (CH)
78.2 (CH)
62.0 (CH2)
5.58
4.06
4.21
4.20
3.96
0.63
–
1.42
20.9 (CH2)
40.4 (CH2)
41.2 (C)
2.06, 1.14
–
1.11
2.10, 1.50
4.99
4.50, 4.39
56.1 (CH)
32.3 (CH2)
81.2 (CH)
63.4 (CH)
16.7 (Me)
15.9 (Me)
40.4 (CH)
16.2 (Me)
110.9 (C)
36.7 (CH2)
28.1 (CH2)
34.1 (CH)
75.3 (CH2)
17.3 (Me)
1
2
3
4
104.6 (CH)
74.7 (CH)
77.9 (CH)
70.4 (CH)
66.9 (CH2)
5.19
3.95
4.13
2.01
0.89
1.21
2.25
1.36
–
2.07
4.13
5
4.26, 3.71
Glc-C
1
2
3
4
5
6
104.4 (CH)
74.8 (CH)
77.9 (CH)
71.3 (CH)
78.0 (CH)
62.4 (CH2)
4.81
4.03
4.29
4.15
3.97
2.04, 1.65
1.96
3.98, 3.67
1.03
4.52, 4.31
102.1 (CH)
72.7 (CH)
75.1 (CH)
79.7 (CH)
75.1 (CH)
60.7 (CH2)
5.01
4.44
4.20
4.61
4.09
2
3
4
5
6
4.63, 4.28
Glc B
OH
OH
OH
27
21
OH 23
O
O
HO
20
O
18
H
HO
O
HO
25
22
OH
Glc C
11
9
26
OH
17
O
HO
HO
19
O
13
O
HO
H
1
H
O
OH
OH
O
15
10
5
O
H
H
3
Xyl
7
OH
Glc A
O
H
HO
OH
OH
1
Gal
24
Compound 1 was obtained as an amorphous solid: [ꢁ] –45.3ꢄ (c 0.35, MeOH). High-resolution (HR)-electrospray
D
+
ionization (ESI)-time of flight (TOF)-MS of 1 showed an [M + Na] peak at m/z 1253.5773 corresponding to the empirical
1
molecular formula C H O . The H NMR spectrum of 1 (Table 1) showed characteristic proton signals due to two tertiary
56 94 29
methyls at ꢅ 0.89 (3H, s) and 1.21 (3H, s), two secondary methyls at ꢅ 1.03 (3H, d, J = 6.5 Hz) and 1.36 (3H, d, J = 6.8 Hz), and
five anomeric protons at ꢅ 5.01 (1H, d, J = 7.6 Hz), 5.15 (1H, d, J = 7.8 Hz), 5.58 (1H, d, J = 7.9 Hz), 5.19 (1H, d, J = 7.8 Hz),
13
and 4.81 (1H, d, J = 7.8 Hz). The C NMR spectrum showed 56 peaks: 29 for the sugar moieties, including five anomeric
1
carbons at ꢅ 102.1, 104.4, 104.2, 104.6, and 104.4, and 27 for the aglycone part (Table 1). The H NMR data, an acetylic
13
carbon signal at ꢅ 110.9 in the C NMR spectrum, and a positive color reaction in Ehrlichꢃs test indicated 1 to be a
C
901

furostanol saponin. Enzymatic hydrolysis of 1 produced a spirostanol saponin, identified by comparison with reference samples
as (25R)-5ꢁ-spirostane-3ꢀ,6ꢀ-diol-3-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-[ꢀ-D-xylopyranosyl-(1ꢂ3)]-ꢀ-D-glucopyranosyl-(1ꢂ4)-
ꢀ-D-galactopyranoside [4] and glucose. Chiral GC-FID analyses of the monosaccharides obtained after acid hydrolysis of 1
confirmed the D conformation of galactose, glucose, and xylose. As is usual with naturally occurring furostanol glycosides,
one glucosyl group was shown to be linked to the C-26 hydroxyl group of the aglycone by an HMBC correlation of the
anomeric proton at ꢅ 4.81 (3H, d, J = 7.8 Hz) with C-26 of the aglycone at ꢅ 75.3. Thus, 1 was identified as 26-O-ꢀ-D-
C
glucopyranosyl-(25R)-5ꢁ-furostane-3ꢀ,-6ꢀ-diol-3-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-O-ꢀ-D-xylopyranosyl-(1ꢂ3)-O-ꢀ-D-
glucopyranosyl-(1ꢂ4)-ꢀ-D-galactopyranoside and was named leucofuroside A.
24
Compound 2 was isolated as an amorphous solid: [ꢁ] –34.0ꢄ (c 0.10, MeOH). HR-ESI-TOF-MS of 2 showed a
D
+
pseudomolecular ion peak at m/z 941.4716 [M + Na] , corresponding to the empirical molecular formula C H O , which
was also deduced by analysis of its C NMR and distortionless enhancement by polarization transfer (DEPT) spectral data.
The H NMR spectrum of 2 showed characteristic proton signals due to two tertiary methyls at ꢅ 0.82 (3H, s) and 1.05 (3H, s),
45 74 19
13
1
two secondary methyls at ꢅ 0.79 (3H, d, J = 6.4 Hz) and 0.95 (3H, d, J =7.0 Hz), and three anomeric protons at ꢅ 4.39 (1H, d,
13
J = 7.2 Hz), 4.53 (1H, d, J = 7.2 Hz), and 4.66 (1H, d, J = 7.8 Hz). The C NMR spectrum showed 45 peaks: 18 for the sugar
moieties, including three anomeric carbons at ꢅ 102.5, 104.8, and 106.2, and 27 for the aglycone part. Acid hydrolysis of 2
with 1 M HCl in dioxane–H O (1:1, v/v) produced steroidal sapogenin, glucose, and galactose. Chiral gas chromatography
2
flame ionization detector (GC-FID) analyses of the monosaccharides confirmed their D-configuration. The physical and spectral
data allowed the identification of the sapogenin as (25R)-5ꢁ-spirostane-3ꢀ,-6ꢀ-diol (ꢀ-chlorogenin) [17], suggesting 2 to be a
1
1
ꢀ-chlorogenin triglycoside. H– H shift COSY allowed the sequential assignments from H-1 to H -6 of each monosaccharide,
2
including identification of their multiple patterns and coupling constants. These sugar linkages were confirmed by long-range
1
13
correlations on the HMBC spectrum. The H and C NMR signals indicated the presence of a terminal ꢀ-D-glucopyranosyl
unit (Glc B) [ꢅ 4.66 (d, J = 7.8 Hz); ꢅ 106.2, 76.3, 77.5, 70.7, 78.7, 61.9], a C-2 substituted ꢀ-D-glucopyranosyl unit
H
C
(Glc A) [ꢅ 4.53 (d, J = 7.2 Hz); ꢅ 104.8, 84.9, 78.1, 71.7, 77.8, 63.2], and a C-4 substituted ꢀ-D-galactopyranosyl unit (Gal)
H
C
[ꢅ 4.39 (d, J = 7.2 Hz); ꢅ 102.5, 73.2, 75.1, 80.5, 75.6, 60.8]. In the HMBC spectrum, correlations were observed from
H
C
ꢅ 4.66 (H-1 of Glc B) to ꢅ 84.9 (C-2 of Glc A), ꢅ 4.53 (H-1 of Glc A) to ꢅ 80.5 (C-4 of Gal), and ꢅ 4.39 (H-1 of Gal) to 79.8
(C-3 of the aglycone). Thus, compound 2 was identified as (25R)-5ꢁ-spirostane-3ꢀ,6ꢀ-diol-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-
O-ꢀ-D-glucopyranosyl-(1ꢂ4)-ꢀ-D-galactopyranoside, recently isolated and characterized by Akihito et al. as compound 2
[18]. The mentioned compound was described for the first time in the genus Allium.
EXPERIMENTAL
1
13
General. Spectra were recorded on a Bruker DRX-500 and a Bruker Avance 400 instrument. H and C NMR
chemical shifts in ppm were referenced with the residual solvent (CD OD) signals (ꢅ 3.31 and ꢅ 49.0) or with TMS as
3
H
C
internal standard (for pyridine-d –H O). High-resolution electrospray ionization mass spectrometry was conducted in the
5
2
positive mode on an Applied Biosystems/MDS Sciex QSTAR XL QqTOF MS system. The flowers were dried by microwave
irradiation oven Pr KS-22E, 850 W, 2450 MHz. GC-FID analysis was performed on an Agilent 7890Aseries equipped with an
InertCap Chiramix column (30 m ꢆ 0.25 mm ꢆ 0.25 ꢇm). The temperature program was 120ꢄC for 1 min, followed by a rise of
25
4ꢄC/min to 180ꢄC, which was maintained for 120 min. Optical rotation [ꢁ] was measured on an Autopol IV polarimeter. For
D
CC, silica gel 60 (40–63 ꢇm, Merck) and Diaion HP20 resin (Mitsubishi) were used. Thin-layer chromatography (TLC)
analysis of saponins was performed on silica gel 60 F254 plates (Merck) and eluted with CH Cl –MeOH–H O (26:14:3 v/v/v).
2
2
2
Spots were detected by spraying the plates with vanillin-sulfuric acid (in EtOH) reagent, followed by heating at 110ꢄC
(spirostanols were colored yellow, furostanols dark green, and they gave a positive color reaction in Ehrlichꢃs test).
Plant Material. The flowers of Allium leucanthum K. Koch were collected in the Dmanisi region of Georgia (June 2005)
and identified by Prof. Jumber Kuchukhidze. A voucher specimen is kept in the Department of Pharmacognosy and Botany,
Faculty of Pharmacy, Tbilisi State Medical University, Tbilisi, Georgia (flowers No. AL 0605).
Extraction and Isolation. Dried and powdered flowers of Allium leucanthum (500 g) were extracted twice with hot
MeOH–H O (8:2, v/v, 5 L). After evaporation of the solvent, the residue (79 g) was suspended in water and the saponins were
2
extracted with n-BuOH. The n-BuOH extract (32 g) was chromatographed over Diaion HP-20, using MeOH–H O as eluent in
2
gradient conditions (0ꢂ100%) and EtOAc. The spirostanol fraction (14.5 g) was collected in MeOH–H O (7:3, v/v), and the
2
furostanol fraction (5.9 g) in MeOH–H O (5:5, v/v). The furostanol saponins were subjected to CC on silica gel and eluted
2
902

with CH Cl –MeOH–H O (40:12:2, v/v/v) to give compound 1 (32 mg). The spirostanol saponins were subjected to CC on
2
2
2
silica gel and eluted with CH Cl –MeOH–H O (45:14:2, v/v/v) to give compound 2 (25 mg).
2
2
2
24
+
Compound 1. Amorphous solid, [ꢁ] –45.3ꢄ (c 0.35, MeOH). HR-ESI-TOS-MS [m/z 1230.588, [M + H] , calcd for
D
1
13
C H O Na, 1253.577]. H NMR (400 MHz) and C NMR (100 MHz) spectroscopic data are shown in Table 1.
56 94 29
Enzymatic Hydrolysis. A solution of 1 (10 mg) was treated with ꢀ-D-glycosidase (EC, Sigma, USA) (10 mg) in
AcOH–AcONa buffer (pH 5; 10 mL) at room temperature for 12 h. The reaction mixture was chromatographed on Diaion HP-20
eluted with Me CO–EtOH (3:2, v/v) and on silica gel eluted with CH Cl –MeOH–H O (45:14:2, v/v/v) to give (25R)-5ꢁ-
2
2
2
2
spirostane-3ꢀ,-6ꢀ-diol-3-O-ꢀ-D-glucopyranosyl-(1ꢂ2)-O-ꢀ-D-xylopyranosyl-(1ꢂ3)-O-ꢀ-D-glucopyranosyl-(1ꢂ4)-ꢀ-D-
galactopyranoside (6 mg) and a sugar fraction (2 mg). The sugar fraction was analyzed on silica gel TLC by comparison with
standard sugars in a CH Cl –MeOH–H O (50:25:5, v/v/v) solvent system and further developed using an orthophosphoric
2
2
2
acid solution of 5% naphthoresorsinol in EtOH, followed by heating at 110ꢄC to give glucose.
Acid Hydrolysis. A solution of 1 (10 mg) in 1 M HCl (dioxane–H O, 1:1, v/v; 3 mL) was heated at 100ꢄC for 2 h
2
under an Ar atmosphere. The reaction mixture was neutralized with N,N-dioctylmethylamine (10% in CHCl ) and was
3
chromatographed on silica gel eluted with CHCl –MeOH (9:1, v/v) to give ꢀ-chlorogenin (3.3 mg) and a sugar fraction
3
(5.8 mg). The sugar fraction was analyzed by silica gel TLC and comparison with standard sugars in a CH Cl –MeOH–H O
2
2
2
(50:25:5, v/v/v) solvent system; the monosaccharides were identified by TLC in a CH Cl –CH OH–H O (50:25:5, v/v/v)
2
2
3
2
solvent system and further developed using an orthophosphoric acid solution of 5% naphthoresorsinol in EtOH, followed by
heating at 110ꢄC to give galactose, glucose, and xylose.
Determination of Stereochemistry of Monosaccharide. The aqueous phase (after acid hydrolysis) was evaporated
and dried, and the residue containing monosaccharides was acetylated with acetic anhydride (1 mL) in pyridine (1 mL) for 24 h.
The monosaccharide acetates were extracted with EtOAc, and the organic fraction was treated with water, followed by a
saturated solution of NaHCO . The sample obtained was analyzed by GC-FID on a chiral column for comparison with authentic
3
samples of monosaccharide acetates. The retention indices of monosaccharide acetates were compared with those of authentic
samples. D-Galactose, D-glucose, and D-xylose were identified for compound 1.
ACKNOWLEDGMENT
The authors are grateful for the support of the French Embassy in Georgia. We thank Prof. Jumber Kuchukhidze for
identification of Allium leucanthum K. Koch, and Gilbert Boudon for his technical support. This research was partially supported
by the program Shota Rustaveli National Science Foundation “Research Grants for Young Scientists 2013” (Grant Agreement
Nr. YS/1/8-404/13).
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