Steroidal glycosides from Allium cyrillii Bulbs

Article abstract of DOI:10.1007/s10600-012-0219-z

Two spirostanol saponins, one of which was a new compound, were isolated among the steroidal glycosides of Allium cyrillii Ten. Bulbs. The structures of these glycosides were established using chemical and spectral analytical methods as β-D-glycopyranosyl-(1 →2)-[β-D-xylopyranosyl-(1 →3)]-β-D-glucopyranosyl-(1 →4)-β-D-galactopyranosyl- (1→3)-(25R)-5α-spirostan-2α,3β-diol and β-D-glucopyranosyl-(1→2)-[4-O-(3-hydroxy-3-methylglutaryl) -β-D-xylopyranosyl-(1 →3)]-β-D-glucopyranosyl-(1 →4)-β-D-galactopyranosyl-(1→3)-(25R)-5α-spirostan-2α, 3β-diol.

Full text of DOI:10.1007/s10600-012-0219-z

Chemistry of Natural Compounds, Vol. 48, No. 2, May, 2012 [Russian original No. 2, March-April, 2012]
STEROIDAL GLYCOSIDES FROM Allium cyrillii BULBS
N. V. Tolkacheva,1* A. S. Shashkov, and V. Ya Chirva3
2
UDC 582.573.16:547.918
Two spirostanol saponins, one of which was a new compound, were isolated among the steroidal glycosides
of Allium cyrillii Ten. Bulbs. The structures of these glycosides were established using chemical and spectral
analytical methods as ꢀ-D-glycopyranosyl-(1ꢁ2)-[ ꢀ-D-xylopyranosyl-(1ꢁ3)]- ꢀ-D-glucopyranosyl-(1ꢁ4)-
ꢀ
-D-galactopyranosyl-(1ꢁ3)-(25R)-5ꢂ-spirostan-2ꢂ,3ꢀ-diol and ꢀ-D-glucopyranosyl-(1ꢁ2)-[4-O-(3-
hydroxy-3-methylglutaryl)- ꢀ-D-xylopyranosyl-(1ꢁ3)]- ꢀ-D-glucopyranosyl-(1ꢁ4)- ꢀ-D-galactopyranosyl-
1ꢁ3)-(25R)-5ꢂ-spirostan-2ꢂ,3ꢀ-diol.
(
Keywords: Allium cyrillii, steroidal glycosides.
Steroidal glycosides represent a broad class of natural saponins, which are interesting to researchers because of their
broad spectrum of biological activity and ecological safety [1].
Plants of the genus Allium, which are indigenous to Crimea, are promising in the search for saponin-bearing species.
Moreover, data on the steroidal glycosides of the majority of Crimean species have not been reported. Hence the study of the
chemical structure of steroidal glycosides from representatives of the Alliaceae family is timely.
Bulbs of A. cyrillii yielded chromatographically pure glycosides 1 (0.085 g) and 2 (0.154 g). The aglycon of both
compounds was identified after acid hydrolysis as gitogenin [2]. TLC and comparison with authentic sugar samples detected
glucose, xylose, and galactose in a 2:1:1 ratio. Alkaline hydrolysis of 1 and TLC using an authentic standard detected
3
-hydroxy-3-methylglutaric acid (3, HMG) as a substituent.
O
O
OH
HO
O
O
HO
O
RO
HO
O
O
O
OH
OH
O
H
HO
OH
OH
HO
HO
O
OH
1, 2
CH
C
OH
3
CH
1
: R =
C
O
CH
C
O
OH
2: R = H
2
2
The empirical formulas of 1 and 2 were determined as C H O and C H O . These were confirmed as peaks of
5
6
90 27
50 82 23
–
negative ions [M – H] in mass spectra with m/z 1193.5602 and 1049.5149 that correlated to molecular weights (MWs)
1
194.5680 and 1050.5228 Da (calculated MWs 1194.5670 and 1050.5247 Da, respectively). The 13C NMR spectrum of 1
(
(
Table 1) showed resonances of the carbohydrate part and the HMG residue as 27 C atom resonances including 4 methyl
ꢃ 13.3, 15.0, 16.6, 17.3), 10 methylene, 10 methine, and 3 quaternary (ꢃ 36.8, 40.8, 109.4) atoms. The spectrum of 2 also
1
3
contained 27 resonances for C atoms of the aglycon. The PMR and C NMR spectra indicated that 1 and 2 were spirostane
derivatives. Thus, the C chemical shift (CS) of C-22 in both compounds was 109.4 ppm. The CSs of C-5 (ꢃ 44.5), C-9
1
3
(
54.3), and C-19 (13.3) were consistent with cis-fusion of rings A and B.
1
) Nikitskii Botanical Garden, National Science Center, Ukraine, Nikita, Yalta, 98648, e-mail: tolkacheva_n@mail.ru;
2
1
) N. D. Zelinskii Institute of Organic Chemistry, Russian Academy of Sciences, Russia, Leninskii Prosp., 47, Moscow,
19991; 3) Tavrida National V. I. Vernadskii University, Ukraine, Prosp.Akad. Vernadskogo, 4, Simferopol. 95007. Translated
from Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2012, pp. 243–246. Original article submitted September 29, 2011.
272
0009-3130/12/4802-0272 ©2012 Springer Science+Business Media, Inc.

TABLE 1. Chemical Shifts (Py-d , 0 = TMS, ꢃ, ppm) of 13C Atoms of 1 and 2
5
C atom
1
2
C atom
1
2
1
2
3
4
5
6
7
8
9
45.3
70.4
84.0
33.7
44.5
28.0
32.0
34.5
54.3
36.8
21.4
40.0
40.8
56.3
32.2
81.2
62.9
16.6
13.3
42.0
15.0
109.4
31.7
29.2
30.5
66.9
17.3
45.4
70.4
84.0
33.8
44.5
28.1
32.1
34.6
54.3
36.9
21.4
40.1
40.8
56.3
32.2
81.2
62.9
16.6
13.4
42.0
15.0
109.4
31.8
29.2
30.6
66.9
17.3
HMG 1*
179.6
47.4
70.4
47.3
171.5
28.2
102.9
72.4
75.2
79.4
75.6
60.9
104.2
80.8
86.7
70.0
77.2
62.5
104.2
75.6
77.6
71.1
78.2
62.4
104.4
74.6
74.3
2*
3*
4*
5*
6*
Gal-1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
103.0
72.5
75.3
79.4
75.7
60.8
104.3
80.9
87.1
70.1
77.4
62.6
104.4
75.7
77.9
71.2
78.3
62.5
104.8
74.9
78.3
70.6
67.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
6ꢅ
Glc-1ꢅꢅ
2ꢅꢅ
3ꢅꢅ
4
5
6
ꢅꢅ
ꢅꢅ
ꢅꢅ
Glc-1ꢅꢅꢅ
2
3
4
5
6
ꢅꢅꢅ
ꢅꢅꢅ
ꢅꢅꢅ
ꢅꢅꢅ
ꢅꢅꢅ
Xyl-1ꢅꢅꢅꢅ
2ꢅꢅꢅꢅ
3ꢅꢅꢅꢅ
4ꢅꢅꢅꢅ
5ꢅꢅꢅꢅ
7
6
2.8
3.2
The CSs of the CH -27 doublet (ꢃ 0.71–0.72) and the H-26 methylene protons (3.50, 1H, H-26a; 3.61, 1H, H-26b)
3
indicated unambiguously that CH -27 had an equatorial orientation (Table 2). Thus, the aglycon of 1 and 2 was a
3
(
25R)-spirostane. The 25R-configuration was confirmed by the CSs of C-20, C-21, C-23, C-24, C-25, and C-27 and a comparison
with the literature [3, 4]. The 5ꢂ-configuration was determined based on correlations among resonances for protons at ꢃ 0.70
CH -19) and C atoms at ꢃ 36.8 (C-10), 54.3 (C-9), 44.5 (C-5), and 45.3 (C-1) in the 2D HMBC spectrum. Resonances of four
(
3
methyls at ꢃ 0.81 (3H, s, Me-18), 0.70 (3H, s, Me-19), 0.71 (0.72) (3H, d, J = 7.0 Hz, Me-27), and 1.14 (1.15) (3H, d,
J = 7.0 Hz, Me-21) were clearly resolved in the strong-field region of PMR spectra of both glycosides.
The location of the hydroxyl on C-2 of the aglycon was confirmed by the weak-field position of its resonance compared
with literature data for the non-hydroxylated aglycons [5] and taking into account the significant positive ꢂ-effects of
hydroxylation and the small and usually negative ꢀ-effects. The 2ꢂ,3ꢀ-configuration was determined based on an analysis of
spin–spin coupling constants of the H-2 and H-3 protons with neighboring protons.
Spectral data were compared with the literature [6] and confirmed the structure of the aglycon as (25R)-5ꢂ-spirostan-
2
1
ꢂ,3ꢀ-diol. It was also noticed that C-3 was substituted (weak-field shift from 76.5 ppm to 84.0). The 13C NMR spectrum of
exhibited six additional resonances for two methylenes (ꢃ 47.3 and 47.4), a methyl (28.2), a carboxylic group (179.6), an
ester (171.5), and a tertiary C atom bound to the hydroxyl (70.4 ppm). The CSs of the methylene protons of this substituent
were ꢃ 2.75, 2.93, 2.84, and 3.07 ppm; of the methyl protons, 1.57 ppm. A comparison of these values with the literature [7]
confirmed that glycoside 1 contained HMG. The acid was bonded at O-4 of a xylose unit as indicated by the weak-field CS of
H-4ꢄꢄ (ꢃ 5.17) compared with that of 2 (4.12), which did not contain an HMG residue (Table 2). Also, ꢂ-effects (for
C-4ꢄꢄ) and ꢀ-effects (for C-3ꢄꢄ and C-5ꢄꢄ) of the 4-O-acylation of the xylose unit were characteristic in the 13C NMR spectrum
(
Table 1).
273

TABLE 2. Observed Correlations in HSQC and HMBC Spectra of the Glycosides
ꢃ
Í
HSQC
Í
ꢃ HSQC
Atom
HMBC
Atom
HMBC
1
2
1
2
1
1
2
a
b
2.18 dd
1.19 m
3.96 m
3.89 m
1.87 q
1.47 m
1.02 m
1.18 m
1.03 m
1.51 m
0.78 m
1.40 m
0.58 td
1.44 m
1.22 m
1.64 m
1.03 m
1.05 m
2.04 m
1.41 m
4.57 m
1.82 t
2.18 dd
1.18 m
3.98 m
3.91 m
1.88 q
1.50 m
1.03 m
1.17 m
1.06 m
1.53 m
0.79 m
1.42 m
0.58 td
1.47 m
1.22 m
1.65 m
1.05 m
1.03 m
2.06 m
1.42 m
4.57 m
1.82 t
C-2, 10
C-2, 10, 19
C-3
C-2
C-2, 3, 6, 10
C-3, 5
C-1, 6, 19
C-10
C-7
C-5, 6, 8
Gal-1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅa
6ꢅe
Glc-1ꢅꢅ
2ꢅꢅ
3ꢅꢅ
4ꢅꢅ
5ꢅꢅ
6ꢅꢅa
6ꢅꢅe
Glc-1ꢅꢅꢅ
2ꢅꢅꢅ
3ꢅꢅꢅ
4ꢅꢅꢅ
5ꢅꢅꢅ
6ꢅꢅꢅa
6ꢅꢅꢅe
Xyl-1ꢅꢅꢅꢅ
2ꢅꢅꢅꢅ
3ꢅꢅꢅꢅ
4ꢅꢅꢅꢅ
5ꢅꢅꢅꢅa
5ꢅꢅꢅꢅe
HMG
2à*
2b*
4a*
4b*
6*
4.92 d
4.49 m
4.16 m
4.57 m
4.08 m
4.55 m
4.27 m
5.14 d
4.27 t
4.93 d
4.52 m
4.17 m
4.58 m
4.10 m
4.56 m
4.27 m
5.16 d
4.30 t
C-3
C-1ꢅ, 3ꢅ
C-2ꢅ, 5ꢅ
C-2ꢅ, 3ꢅ, 1ꢅꢅ
C-1ꢅ, 3ꢅ, 6ꢅ
C-5ꢅ
3
4
a
4
b
5
C-4ꢅ
6a
6b
7a
7b
C-4ꢅ, 5ꢅꢅ
C-3ꢅꢅ, 1ꢅꢅꢅ
C-1ꢅꢅꢅꢅ, 1ꢅꢅ, 2ꢅꢅ
C-3ꢅꢅ, 5ꢅꢅ, 6ꢅꢅ
C-4ꢅꢅ
4.14 t
3.76 t
4.09 t
3.76 t
8
C-14
C-19
C-8
3.82 m
4.46 m
4.03 m
5.58 d
4.05 m
4.21 m
4.08 m
3.94 m
4.50 m
4.39 dd
5.23 d
3.92 t
4.25 m
5.17 m
3.50 t
3.82 m
4.47 m
4.04 m
5.59 d
4.04 m
4.19 m
4.09 m
3.96 m
4.53 m
4.41 dd
5.21 d
3.94 t
9
11a
11b
12a
12b
C-5ꢅꢅ
C-2ꢅꢅ
C-1ꢅꢅ, 3ꢅꢅꢅ
C-4ꢅꢅꢅ
C-3ꢅꢅꢅ, 6ꢅꢅꢅ
C-2ꢅꢅꢅ
C-4ꢅꢅꢅ
C-9, 11, 14
C-13
C-13, 18
C-13, 17
C-14, 16
C-13
C-12, 16, 18, 21
C-12, 13, 14, 17
C-1, 5, 9, 10
C-13, 17, 21, 22
C-17, 20, 22
C-25
1
4
1
1
5a
5b
1
1
1
1
2
2
6
7
8
9
0
1
C-3ꢅꢅ
0.81 s
0.70 s
0.81 s
0.70 s
C-1ꢅꢅꢅꢅ, 3ꢅꢅꢅꢅ
C-1ꢅꢅꢅꢅ, 5ꢅꢅꢅꢅ
C-3ꢅꢅꢅꢅ
4.10 m
4.12 m
3.68 t
1.96 m
1.14 d
1.69 m
1.71 m
1.61 m
1.58 m
1.60 m
3.61 dd
3.50 t
1.97 m
1.15 d
1.69 m
1.73 m
1.61 m
1.57 m
1.60 m
3.61 dd
3.51 t
C-1ꢅꢅꢅꢅ, 3ꢅꢅꢅꢅ
C-4ꢅꢅꢅꢅ
2
2
2
2
3a
3b
4a
4b
4.20 m
4.22 m
C-22
C-25
C-23
HMG
2.93 d
2.76 d
3.07 d
2.85 d
1.57 s
C-1*, 4*, 6*
C-1*, 4*, 6*
C-1*, 3*, 6*
C-1*, 3*, 6*
C-2*, 3*, 4*
2
5
2
2
6à
6b
C-22, 24
C-22, 24, 25
C-24, 25, 26
2
7
0.71 d
0.72 d
Four resonances for anomeric protons of the monosaccharides at ꢃ 5.58 (5.59) (1H, d, J = 7.5 Hz), 5.23 (5.21) (1H, d,
J = 7.5 Hz), 5.14 (5.16) (1H, d, J = 7.5 Hz), and 4.92 (4.93) (1H, d, J = 7.5 Hz) appeared at weak field in the PMR spectra of
and 2. Thus, the carbohydrate chain in both glycosides consisted of four sugar units. The spin–spin coupling constants
1
J1,2 were indicative of the ꢀ-configurations of the glycoside bonds in all monosaccharide residues.
The CSs of all four monosaccharide protons were found using a combination of 2D TOCSY and COSY spectra. The
1
3
CSs of the resonances of the corresponding C atoms were assigned unambiguously using 2D HSQC spectra (Table 2). These
data indicated that one ꢀ-D-galactopyranose, one ꢀ-D-xylopyranose, and two ꢀ-D-glucopyranose residues were present.
The correlation between Gal H-1ꢅ and C-3 of the aglycon (Table 2) defined the site of attachment of the carbohydrate
chain to the aglycon. A CS of ꢃ 79.4 was observed for Gal C-4ꢅ. This characterized the site of attachment of the next
monosaccharide unit. The HMBC spectrum showed structurally informative cross-peaks between resonances Xyl H-1ꢄꢄ
(
ꢃ 5.23) and Glc C-3ꢅꢅ (86.7); Glc H-1ꢅꢅꢅ (5.58) and Glc C-2ꢅꢅ (80.8); and Glc H-1ꢅꢅ (5.14) and Gal C-4ꢅ (79.4) for 1 in addition
to Xyl H-1ꢄꢄ (5.21) and Glc C-3ꢅꢅ (87.1); Glc H-1ꢅꢅꢅ (5.59) and Glc C-2ꢅꢅ (80.9); and Glc H-1ꢅꢅ (5.16) and Gal (C-4ꢅ)
79.4) for 2.
Thus, 1 was isolated from A. cyrillii and was ꢀ-D-glucopyranosyl-(1ꢁ2)-[4-O-(3-hydroxy-3-methylglutaryl)-ꢀ-D-
xylopyranosyl-(1ꢁ3)]-ꢀ-D-glucopyranosyl-(1ꢁ4)-ꢀ-D-galactopyranosyl-(1ꢁ3)-(25R)-5ꢂ-spirostan-2ꢂ,3ꢀ-diol whereas 2
(
274

had the structure ꢀ-D-glucopyranosyl-(1ꢁ2)-[ꢀ-D-xylopyranosyl-(1ꢁ3)]-ꢀ-D-glucopyranosyl-(1ꢁ4)-ꢀ-D-galactopyranosyl-
(
1ꢁ3)-(25R)-5ꢂ-spirostan-2ꢂ,3ꢀ-diol.
Glycoside 2 was isolated previously from Digitalis purpurea L. [8] whereas 1 was an unreported new compound.
EXPERIMENTAL
NMR spectra were taken from Py-d solutions (0.6 mL) with TMS standard on a Varian AS 400 instrument (400 MHz
5
for H; 100, for 13C). Mass spectra (negative-ion mode) with electrospray ionization were obtained in a micrOTOF II instrument
1
(
Bruker Daltonics). Samples (~10 ng/ꢆL) were dissolved in a H O:i-PrOH:Et N mixture (5:5:0.003) and injected at flow rate
2 3
3
ꢆL/min. The potential at the capillary entrance was 3.2 kV; drying-gas temperature, 180°C.
Analytical HPLC was performed on anAgilent 1100 chromatograph equipped with anAgilent light-scattering detector
and a Zorbax SB-C18 column (150 ꢇ 2.1 mm, 3.5 ꢆm) using gradient elution with aqueous CH CN (15%) with added CF COOH
3
3
(
TFA, 0.05%) up to MeOH (100%) in 20 min at mobile-phase flow rate 0.25 mL/min and column temperature thermostatted
at 35°C. The injected sample volume was 2 ꢆL. The N pressure in the light-scattering detector was 3.5 atm; the vaporizer
2
temperature, 100°C.
Preparative column chromatography was carried out using a column packed with C18 sorbent (100 ꢇ 20 mm, 40–
6
3 ꢆm, Aldrich) with elution successively by aqueous MeOH (20%) with added TFA (0.05%) up to MeOH (100%).
Two saponins isolated from A. cyrillii bulbs collected on the northeast slope of Paragilmen mountain in 2010 were
studied. Freshly collected and ground bulbs (1148 g) were extracted (3ꢇ) with EtOH (70%). The extraction was performed
with heating of the material on a water bath with a reflux condenser for 3 h. The resulting extract was evaporated in a rotary
evaporator at 50°C to a watery residue from which total steroidal glycosides were extracted by n-BuOH. The BuOH extract
was evaporated to dryness. TLC was performed using CHCl :MeOH:H O (65:35:7) and CHCl :MeOH (7:3). We used
3
2
3
Sorbfil AF-V plates with layer thickness 110 ꢆm for the chromatography. Chromatograms were sprayed with Erlich [9] and
Sannie [10] reagents and then heated to 120°C.
Acid Hydrolysis. Pure glycoside (10 mg) was heated at 110°C in H SO solution (1%, 5 mL) in EtOH for 2 h,
2
4
cooled, and diluted with H O. The aglycon was extracted by CHCl . The CHCl extracts were washed with H O until neutral
2
3
3
2
and concentrated in vacuo to a dry solid. The aglycon was identified using TLC and benzene:EtOH (9:1).
The aqueous layers were neutralized with anion exchanger until neutral and evaporated. The sugars were identified
using BuOH:Me CO:H O (4:5:1).
2
2
Alkaline Hydrolysis. The glycoside (10 mg) was heated in NaOH solution (10%, 10 mL) for 2 h at 50°C. The
hydrolysate was neutralized by cation exchanger, washed with MeOH, and evaporated. TLC analysis was performed using
EtOH:NH OH:H O (1:1:2) with detection by bromocresol solution (0.05%).
4
2
ACKNOWLEDGMENT
We thank Dr. Yu. A. Knirel (IOC, RAS, Moscow) for review of the article and Cand. A. N. Kondakov (IOC, RAS,
Moscow) for recording mass spectra. Access to the micrOTOF II mass spectrometer was provided by OOO Bruker (Moscow).
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