Flavonoid glycosides from Astragalus galegiformis leaves

Article abstract of DOI:10.1007/s10600-006-0251-y

New flavonoid oligosides, the structures of which were established by chemical transformations and UV, IR, PMR, and ¹³C NMR spectra, were isolated from Astragalus galegiformis leaves.

Full text of DOI:10.1007/s10600-006-0251-y

Chemistry of Natural Compounds, Vol. 42, No. 6, 2006
FLAVONOID GLYCOSIDES FROM Astragalus galegiformis LEAVES
1
1
2
M. D. Alaniya, N. Sh. Kavtaradze, C. Bassarello,
UDC 547.972
1
2
1
A. V. Skhirtladze, C. Pizza, and I. Kutateladze
Newflavonoid oligosides, the structures of which were established by chemical transformations and UV, IR,
PMR, and 13C NMR spectra, were isolated from Astragalus galegiformis leaves.
Key words: Astragalus galegiformis, flavonol, quercetin, isorhamnetin, flagaloside.
We reported earlier on the isolation from Astragalus galegiformis L. (Fabaceae L.) flowers of flavonoid glycosides that
were derivatives of isorhamnetin, astragalegoside and isoastragalegoside, which possess diuretic and hyponitrogenous activity
[1, 2].
Herein we examine the structure of flavonoid glycosides isolated from leaves of A. galegiformis. They are difficult to
separate by chromatography (R 0.34 ± 0.05) and isolate in pure states. This may be due to either H-bonds between the
f
compounds or gums in the Astragalus species.
We were able to isolate six compounds by using classical separation methods for flavonoids and their modifications.
Two compounds turned out to be known flavonoids that were previously isolated from other Astragalus species, astragalin (1)
and kaempferol (2) [3].
The other compounds were new. We called them flagalosides (2-5). They all gave a positive qualitative reaction
-1
characteristicofflavonoidglycosides[4]. Their IRspectra contained absorption bandsfor hydroxyls(3200-3400cm ), γ-pyrone
-1
-1
carbonyl (1665-1660 cm ), and aromatic rings (1620, 1575 cm ) [5].
UV spectra of3 and 4 in ethanol with added ionizing and complexing reagents produced absorption bands that revealed
the absence of substituents on C , C , C , and C ; for 2 and 5, the presence of substituents on C and C , and C and C ,
7
5
4
3′
7
3
3
3′
respectively.
Acid hydrolysis of the glycosides produced the aglycons quercetin (from 2-4) and isorhamnetin (from 5). The yields
of the aglycons indicated that 2 was a tetraoside; 3, a pentaoside; 4, a trioside; and 5, a bioside. The carbohydrate part of 2
contained D-glucose, D-galactose, L-arabinose, and L-rhamnosebondedtoC andC ofquercetin; of3, D-glucose, D-galactose,
3
7
L-arabinose, D-xylose, and L-rhamnose also bonded to C of quercetin.
3
In view of the fact that 2 and 3 were isolated in minor amounts, it was not possible to establish their complete chemical
structures.
The PMR spectrum of 4 recorded in CD OD (Table 1) contained a strong singlet at 12.8 ppm, which is characteristic
3
of a 5-OH group. The presence of this hydroxyl was confirmed by the UV spectrum recorded in ethanol with added AlCl , in
3
which the absorption band at 401 nm underwent a bathochromic shift of 46 nm [6]. The size of bathochromic shifts produced
by adding AcONa + H BO and MeONa is indicative of a dihydroxy group in the 3′,4′-positions and a free 4′-OH group [6].
3
3
Signals of aromatic protons are observable at weak field in the PMR spectra at 7.65, 6.90, and 6.92 (ring B) and 6.45 and
5
.25 ppm (ring A). The chemical shifts and SSCC of protons in ring B indicated that they were located on C , C , and C
2 5′ 6′
′
(
Table 1).
The 13C NMR spectra led to an analogous conclusion, where signals for secondaryC atoms at C-5, C-7, C-4′, and C-3′
were clearly observed. The chemical shift of C-3 was indicative of its glycosylation compared with C-3 of the aglycon. This
C atom appeared at 135.6 ppm (136.73 ppm [7]).
1
) I. G. Kutateladze Institute of Pharmaceutical Chemistry, 0159, Tbilisi, Georgia, fax (995) 32 25 00 23, e-mail:
merialania@yahoo.com; 2) Department of Pharmaceutical Sciences, University of Solerno, Solerno, Italy. Translated from
Khimiya Prirodnykh Soedinenii, No. 6, pp. 555-558, November-December, 2006. Original article submitted August 14, 2006.
©
0
009-3130/06/4206-0681 2006 Springer Science+Business Media, Inc.
681

1
3
TABLE 1. PMR and C NMR Spectra of Flavonoid Glycosides from Astragalus galegiformis Leaves
Compound
C atom
4
5
δC
δH
δC
δH
2
3
4
5
6
7
8
9
158.10
135.60
178.00
162.10
99.20
158.20
134.25
179.10
163.10
99.70
6.26, d, 2.22
6.45, d, 2.08
6.20, d, 2.2
165.00
94.30
165.80
94.61
6.45, d, 2.02
157.60
105.10
122.10
117.20
145.00
149.50
115.90
122.10
158.30
105.95
123.45
114.52
150.55
146.89
116.05
123.38
56.92
10
1
2
3
4
5
6
′
′
′
′
′
′
7.92, d, 2.10
7.95, d, 2.20
6.90, d, 8.0
7.65
6.93, d, 8.43
7.64, dd, 2.2; 8.43
3.81, s
OCH3
1
2
3
4
5
6
″
″
″
″
″
″
105.70, d, 8.30
72.70
5.09, d, 8.30
3.86, dd, 8.3; 9.4
3.60, dd, 9.40; 3.8
3.83
104.06
83.27
75.65
70.36
66.35
4.80, d, 7.50
4.36, d, 7.30
3.41, t, 9.0
75.00
69.90
75.20
67.60
3.78, td, 9.0; 5.0
3.65, dd, 10.0; 9.0
3.90, dd, 10.0; 5.0
3.71
3.54; 3.75
1
2
3
4
5
6
′″
′″
′″
′″
′″
′″
101.60
71.40
82.30
72.50
69.10
4.56, d, 2.1
3.78, dd, 3.8; 2.1
3.57, dd, 9.6; 3.8
3.47, dd, 9.60
3.60
17.60
1.19
1
2
3
4
5
″″
″″
″″
″″
″″
106.30
75.00
77.20
70.70
66.60
4.36, d, 7.30
3.25, dd, 9.0; 7.3
3.33, t, 9.0
3.49, td, 9.5
3.14, dd, 9.0; 9.0
106.10
75.02
77.20
70.70
66.70
4.71, d, 7.0
3.20
3.32
3.45
3.14, 3.80
3.82, dd, 10.5; 5.0
The glycoside was readily hydrolyzed by aqueous H SO to afford an aglycon with mp 303-305°C that was identified
2
4
as quercetin [8]. The aglycon yield was 38%, from which the trioside nature of this flavonoid was deduced. This conclusion
was confirmed by the PMR spectrum, in which signals of three anomeric protons were observed at 5.09 (d, J = 8.3 Hz), 4.56
(d, J = 2.1 Hz), and 4.36 ppm (d, J = 7.3 Hz). The methyl of L-rhamnose appeared at 1.19 ppm.
PC of the hydrolysate using System 4 detected D-galactose, L-rhamnose, and D-xylose. The bonding between sugar
units was determined using 13C NMR spectra (Table 1) and chemical transformations of 1. Stepwise hydrolysis of 4 formed
D-xylose and a less polar glycoside, enzymatic decomposition of which produced robinobiose [9] and quercetin. It was
established that D-galactose was directly bonded to the aglycon through a 3→1 bond. L-Rhamnose was bonded to it through
a 6→1 bond. The terminal unit was D-xylose bonded to L-rhamnose through a 3→1 bond with the pyranose form of the rings
[
10, 11]. Alkaline hydrolysis of flagaloside C did not change the structure of the glycoside.
Judging from the results, glycoside 4 was characterized as quercetin-3-O-β-D-galactopyranosyl-(6→1)-O-α-L-
rhamnopyranosyl-(3→1)-O-β-D-xylopyranoside (flagaloside C).
6
82

OH
OH
OCH3
OH
HO
O
O
HO
O
O
O
O
OH
CH
OH
O
2
O
HO
O
HO
O
OH
O
HO
OH
CH
O
3
OH
OH
O
OH
O
HO
OH
OH
4
5
HO
OH
UV spectra of glycoside 5 (356, 255 nm) were characteristic of flavonol derivatives. Its IR spectrum had absorption
-1
bands due to hydroxyls (3560-3200 cm ), methoxyl (2980), γ-pyrone carbonyl (1650), aromatic rings (1635, 1587), and
carbohydrate C–O vibrations (1100, 1078, 1050).
The glycoside was hydrolyzed by H SO (2%) to form D-xylose and the aglycon (62%), indicating it was a biose. The
2
4
aglycon melted at 289-300°C. Fusion with base gave phloroglucinol and vanillic acid [2]. The UV spectrum of the aglycon
produced by adding CH COONa + H BO was consistent with the absence of a C-3′,4′ dihydroxy group. Melting-point
3
3
3
depression was not observed upon mixing with an authentic sample of isorhamnetin. Therefore, we characterized it as
isorhamnetin [9].
Glycoside 5 was unaffected by base. This meant that the sugar was bonded to the aglycon at C-3. This conclusion was
consistent with the PMR spectrum, in which signals for a 5-OH were observed at 12.05 ppm; five signals of aromatic H of rings
A and B, at 7.65-6.25 ppm; and two signals of anomeric carbohydrate protons, at 4.80 and 4.71 ppm. The methoxyls of the
aglycon were observed as a singlet at 3.18 ppm (Table 1).
The chemical shifts of the carbohydrate atoms (Table 1) indicated a 2→1 bond between the sugars and the
β-configuration and pyranose form for the ring for xylose units bonded as a biose to C-3 of isorhamnetin [8, 10]. Thus, this
glycoside was characterized as isorhamnetin-3-O-β-D-xylopyranosyl-(2→1)-O-β-D-xylopyranoside (flagaloside D).
EXPERIMENTAL
General Comments. Plants were collected near the resort Bakuriani (Georgia) during full flowering in 1999. Raw
material was extracted with ethanol (80°). Chromatography was performed on Filtrak FN-11 paper using n-butanol:acetic
acid:water (4:1:2, 1), acetic acid (15%, 2), ethylacetate:benzene:acetic acid (74.5:23.5:2, 3), and n-butanol:
pyridine:benzene:water (5:3:1:3, 4). Column chromatography used polyamide [12]. Molecular weights were determined as
before [13].
NMR spectra were recorded in CD OD on a Bruker DRX-600 instrument; UV spectra, on a Lomo SF-16
3
spectrophotometer; IR spectra, on a U-20 instrument as KBr disks.
Isolation of Flavonoids. Ground raw material (1 kg, air-dried) was extracted three times with ethanol (80°) in a 1:10
ratio. The extract was condensed to an aqueous residue and purified with CHCl . The purified aqueous extract was extracted
3
exhaustively with ethylacetate. The ethylacetate was evaporated to afford a dry solid (3.8 g, fraction 1). After the ethylacetate
was removed from the aqueous extract, it was fractionated over a polyamide column (d, 1.5; h, 50 cm) with elution successively
by water and ethanol (45 and 90%). The alcohol (45%) fractions contained almost all flavonoids (7.2 g, fraction 2).
Fractions (3 g each) were separated over a polyamide column (d, 2.5; h, 90). Compounds of fraction 1 were eluted by
CHCl and CHCl :alcohol of increasing alcohol concentration. Compounds of fraction 2 were eluted by ethylacetate and
3
3
ethylacetate:alcohol (1:4). This isolated compounds 1-6.
Astragalin, mp 195-197°C, [α] -10° (c 0.1, ethanol); UV spectrum (EtOH, λ , nm): 375, 268; +AlCl : 410, 275;
D
max
3
+
CH ONa: 420, 290; +CH COONa: 385, 275.
3
3
683

Acid Hydrolysis of Astragalin. Glycoside (20 mg) and H SO (2%) gave an aglycon with mp 270-272°C, C H O
15 10 5
2
4
that was identical to kaempferol [9] and D-glucose.
A mixed sample of 1 and astragalin did not show melting-point depression. It was identified as astragalin [9, 14].
Flagaloside A (2); pale white crystals; MW 921.5 [13]; soluble in aqueous alcohol; insoluble in water, alcohol,
ethylacetate; mp 188-192°C; [α] -161.7° (c 0.271, ethanol:DMF, 1:10). UV spectrum (EtOH, λ , nm): 360, 260; +AlCl :
D
max
3
-1
4
3
05, 270; AlCl + HCl: 390, 265; +CH COONa: 369, 269; +CH COONa + H BO : 375, 265; IR spectrum (KBr, ν , cm ):
3 3 3 3 3 max
400 (OH), 1650-1660 (γ-pyrone C=O).
Acid Hydrolysis of Flagaloside A. Glycoside (50 mg) was hydrolyzed by H SO (5 mL, 2%) on a water bath. The
2
4
resulting precipitate was separated, washed with water, dried, and recrystallized from aqueous ethanol to isolate the aglycon
15.2 mg), mp 303-305°C, which was identified as quercetin [14]. The filtrate was neutralized with AB-17 ion exchanger and
(
evaporated. PC of the solid using system 4 detected D-glucose, D-galactose, L-arabinose, and L-rhamnose.
Flagaloside B (3), pale white crystals, MW 1036.5 [13], mp 202-205°C; [α] +55.0° (c 0.68, ethanol:DMF, 1:10).
D
UV spectrum (EtOH, λmax, nm): 360, 260; +Zr(NO ) : 400, 260; +Zr(NO ) + citric acid: 390, 255; +CH COONa: 375, 265;
3 3
3 3
3
-1
+
CH COONa + H BO : 378, 265. IR spectrum (KBr, ν , cm ): 3200-3400 (OH), 1660-1600 (γ-pyrone C=O), 1550 (C=C).
3 3 3 max
Acid Hydrolysis of Flagaloside B. Glycoside (50 mg) was hydrolyzed as for 2 to afford the aglycon (13.2 mg),
mp 301-303°C, which was identified as quercetin [8, 9]. PC of the carbohydrate part using system 4 identified D-glucose,
D-galactose, D-xylose, L-arabinose, and L-rhamnose.
Flagaloside C (4); white crystals; mp 188-193°C; soluble in aqueous alcohol, water; insoluble in concentrated alcohol,
acetone; MW 726.03 [13], C H O . UV spectrum (EtOH, λ , nm): 350, 270; +AlCl : 401, 350, 300sh, 267; +AlCl +
3
2
38 19
max
3
3
HCl: 400, 350, 295, 270; +CH COONa: 370, 360, 300sh, 270; +CH COONa + H BO : 370, 270; CH ONa: 400, 320, 270.
3
3
3
3
3
-1
IR spectrum (KBr, νmax, cm ): 3200-3400 (OH), 1670 (γ-pyrone C=O), 1520, 1470 (C=C).
Acid Hydrolysis of Flagaloside C. Glycoside (20 mg) was hydrolyzed by H SO (2%) as for 1 to afford the aglycon
2
4
(
7.52 mg), mp 300-302°C, which was identified as quercetin [9]. The carbohydrate part was neutralized with AB-17 anion
exchanger. PC detected in it D-galactose, D-xylose, and L-rhamnose.
Enzymatic Hydrolysis of the Product of Partial Hydrolysis of 3 (I). Glycoside (75 mg) was hydrolyzed by aqueous
H SO (5%) on a water bath. The production of the intermediate product was monitored at 15-min intervals. The maximum
2
4
amount of intermediate product had formed after 30 min. The hydrolysis was interrupted by diluting the reaction mixture with
water and extracting with ethylacetate. The aqueous part was neutralized byAB-17 ion exchanger. PC detected in it D-xylose.
The ethylacetate extract was washed with water until the rinsings were neutral and then evaporated. The solid was hydrolyzed
by rhamnodiastase (37°C, 5 h). After the appropriate work up, PC of the hydrolysate using system 4 detected robinobiose by
comparison with an authentic sample [9, 12].
Flagaloside D (5), white crystals, MW 564.5 [13], C H O , soluble in aqueous alcohol. UV spectrum (EtOH, λmax,
26 28 14
nm): 356, 255; +AlCl : 402, 268; +AlCl + HCl: 396, 268; +CH COONa: 378, 325, 275; +CH COONa + H BO : 360, 255;
3
3
3
3
3
3
-1
+
CH ONa: 415, 275. IR spectrum (KBr, ν , cm ): 3350 (OH), 2980 (OCH ), 1650, 1620 (γ-pyrone C=O), 1580, 1410
3 max 3
(C=C).
Table 1 gives the PMR and 13C NMR spectra.
Acid Hydrolysis of 5. Glycoside (10 mg) was hydrolyzed byH SO (2%) for 2 h to afford the aglycon (65%), mp 299-
2
4
3
3
03°C. UV spectrum (EtOH, λmax, nm): 355, 308, 255; +AlCl : 405, 360sh, 267; +AlCl + HCl: 400, 356, 265; +CH COONa:
3 3 3
-1
77, 324, 274; +CH COONa + H BO : 358, 300sh, 258; +CH ONa: 422, 300sh, 268. IR spectrum (KBr, ν , cm ): 3400
3
3
3
3
max
(
OH), 2980 (OCH ), 1650, 1620 (γ-pyrone C=O), 1580, 1470 (C=C). An authentic sample of isorhamnetin and 5 gave a single
3
spot on PC. It was characterized as isorhamnetin [8, 9].
-1
Kaempferol (6), mp 270-272°C. UV spectrum (EtOH, λmax, nm): 270, 350. IR spectrum (KBr, νmax, cm ): 3200-
400 (OH), 1650 (γ-pyrone C=O).
3
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