Flavanone glycosides from Alhagi pseudalhagi

Article abstract of DOI:10.1016/S0031-9422(99)00010-2

Two new flavanone glycosides, alhagitin and alhagidin, have been isolated from the whole plant of Alhagi pseudalhagi and their structures established respectively as naringenin 5-methyl ether 4'-glucoside and hesperitin 7-galactosyl(1 → 2)[rhamnosyl(1 → 6)]glucoside by chemical and spectroscopic methods.

Full text of DOI:10.1016/S0031-9422(99)00010-2

Phytochemistry 51 (1999) 587±590
Flavanone glycosides from Alhagi pseudalhagi
V.P. Singh*, Bineeta Yadav, V.B. Pandey
Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, India
Received 1 September 1998
Abstract
Two new ¯avanone glycosides, alhagitin and alhagidin, have been isolated from the whole plant of Alhagi pseudalhagi and
their structures established respectively as naringenin 5-methyl ether 4'-glucoside and hesperitin 7-galactosyl(1 4 2)[rhamnosyl(1
4 6)]glucoside by chemical and spectroscopic methods. # 1999 Published by Elsevier Science Ltd. All rights reserved.
Keywords: Alhagi pseudalhagi; Leguminosae; Whole plant; Flavanone glycosides; Alhagitin; Alhagidin
1
3000±3400 cm (br) for a polyhydroxy system and at
1
1640 cm for a conjugated carbonyl group. On acid
1. Introduction
Alhagi pseudalhagi (Bieb.) Desv. (Leguminosae) is
distributed throughout India, where it is used as tra-
ditional herbal medicine (Kirtikar, & Basu, 1984;
Khushbaktova et al., 1992; Viramani et al., 1992).
Phenolic constituents previously reported from this
plant include: (+)-catechin, ( )-epigallocatechin, (2)-
gallocatechin and leucodelphinidin (Islambekov,
Mirzakhidov, Karimolzhanov, & Ishbaev, 1982), quer-
cetin (Khaiitbaev, Sultan, Ganiev, & Aslanov, 1993)
and rutin (Svistunova, Khalmatov, & Khazanovich,
1972). We report here the isolation of two new ¯ava-
none glycosides, alhagitin (1) and alhagidin (2), from
the whole plant of A. pseudalhagi.
hydrolysis, it gave glucose and aglycone (3) which
1
furnished a diacetate (4). The H NMR spectrum of 3
showed a methoxyl group signal (d 3.78), a typical
four peak pattern doublet for a 4'-oxygenated B-ring
(d 6.80, 7.30, J=8.5 Hz, each), two meta coupled pro-
tons merged as a broad singlet (d 5.91) and the same
ABX pattern of protons as for naringenin 5-methyl
ether (Maruyama, Hayasaka, & Sasaki, 1974). The
mass spectrum showed a molecular ion peak at m/z
286.0846 and had characteristic ion peaks (m/z 166
and m/z 120) due to retro-Diel's-Alder type fragmenta-
tion indicating the presence of a methoxyl group in
ring A. These data suggest that 3 is naringenin 5-
methyl ether and was further corroborated by the 13C
NMR data (Harborne, & Mabry, 1982) (Table 1),
which also indicated the presence of only one sugar
from the single anomeric carbon signal at d 101.4. The
attachment of a glucose unit at C-4' was apparent
from the UV spectrum of 1 which showed a batho-
chromic shift of 10 nm in the presence of sodium acet-
ate and no bathochromic shift with alkali. The struc-
ture of 1 is thus determined as naringenin 5-methyl
ether 4'-glucoside which is a new ¯avanone glycoside.
2. Results and discussion
Chromatographic resolution of the methanolic
extract of the whole plant of A. pseudalhagi yielded the
glycosides, alhagitin (1), C22H24O10 and alhagidin (2),
C34H44O20. Both compounds developed a magenta
color with Mg/HCl and exhibited UV absorption
bands typical of ¯avanones (Imperato, 1978).
Alhagidin (2) showed peaks in its IR spectrum at
1
Alhagitin (1) showed peaks in its IR spectrum at
3200±3500 cm
(br) for a polyhydroxy system, at
1
2910 cm for a methoxyl group and at 1635 cm for
1
* Corresponding author.
a conjugated carbonyl group. On acid hydrolysis, 2
0031-9422/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00010-2

588
V.P. Singh et al. / Phytochemistry 51 (1999) 587±590
Table 1
13C NMR spectral data in `d' value of compounds 1±3
permethylate furnished 3,4-di-methylglucose, 2,3,4-tri-
methylrhamnose and 2,3,4,6-tetramethyl galactose. The
structure of 2 is thus established as hesperitin 7-galac-
tosyl(1 4 2)[rhamnosyl(1 4 6)glucoside, which is a new
¯avanone glycoside, designated alhagidin.
Carbon number
1
2
3
C-2
C-3
78.2
42.2
78.49
42.13
78.2
42.0
C-4
C-5
196.2
163.1
96.0
197.17
163.10
96.48
165.17
95.65
163.15
103.42
130.96
114.24
148.61
146.52
112.06
118.11
99.51
73.19
76.35
70.66
75.60
66.18
100.70
70.37
69.55
72.17
68.43
17.95
92.69
69.04
68.88
70.47
69.68
60.72
55.76
196.0
163.0
95.8
C-6
C-7
166.1
95.0
166.0
94.8
C-8
C-9
163.0
102.0
128.33
115.1
128.0
157.1
128.0
115.1
101.4
73.8
162.8
101.8
128.3
115.0
128.0
157.0
128.0
115.0
C-10
C-1'
C-2'
C-3'
C-4'
C-5'
C-6'
C-10
C-20
C-30
C-40
C-50
C-60
C-11
C-21
C-31
C-41
C-51
C-61
C-100
C-200
C-300
C-400
C-500
C-600
-OCH3
77.6
70.8
77.0
62.0
3. Experimental
gave galactose, glucose and rhamnose (co-PC) and an
aglycone (5). The aglycone (5) C16H14O6, showed a
methoxyl group signal (d 3.70), four meta coupled pro-
tons, one ortho coupled proton and an ABX pattern
of protons like that of 5,7,3'-trihydroxy-4'-methoxy¯a-
vanone (hesperitin) (Harborne, & Mabry, 1982). The
mass spectrum showed a molecular ion peak at m/z
302 and had characteristic ion peaks (m/z 152 and
150) due to retro-Diel's-Alder type fragmentation indi-
cating the presence of a methoxyl group in ring B.
These data suggest that 5 is hesperitin. UV spectra of
2 showed a bathochromic shift of 22 nm with alumi-
num chloride but the absence of a shift with sodium
acetate indicated the attachment of sugar residues at
the C-7 position. 1H and 13C NMR of 2 showed re-
spectively three anomeric protons (d 5.20, 2H, br s and
5.45, 1H, m) and three anomeric carbons (d 99.5,
100.6 and 92.6). FAB±MS exhibited a molecular ion
peak at m/z 811 [M++K]+ which favoured the pre-
sence of one mole each of galactose, glucose and
rhamnose. Permethylation of 2 and hydrolysis of the
Mps were uncorr. CC was carried out on silica gel
(BDH, 60-120 mesh), TLC on silica gel and PC on
Whatman No 1 paper. Solvents used for TLC were
C6H6±CHCl3 (1:4, solvent A), CHCl3±MeOH (3:1, sol-
vent B) and MeOH±H2O (10:1, solvent C) and for PC:
n-BuOH±HOAc±H2O (4:1:5, solvent D). Liebermann±
Burchard reagent was used for developing TLC plates.
PCs were developed with acetonic AgNO3±NaOH and
washed with Na2S2O3 solution. 1H NMR and 13C
NMR spectra were recorded on 300 and 100 MHz
Varian spectrometers, respectively. TMS was used as
int. standard and chemical shift values were recorded
in `d' ppm. EIMS and FAB±MS were performed on a
Kratos MS-50 instrument at 70 eV with evaporation
of sample in the ion source. Whole plants of A. pseu-
dalhagi were collected from the Varanasi District,
U.P., India and the identi®cation veri®ed by the
Department of Botany, Banaras Hindu University,
Varanasi. A herbarium specimen is kept in the
Department of Medicinal Chemistry, IMS, BHU.
The whole plant (3 kg) was dried, powdered and

V.P. Singh et al. / Phytochemistry 51 (1999) 587±590
589
MeOH
max
repeatedly extracted with MeOH by cold percolation
at 258. The MeOH extract a€orded a green semi-solid
(60 g) which was chromatographed over a silica gel
column eluting with solvents of increasing polarity.
The eluants from C6H6±CHCl3 (1:1), (1:2), CHCl3,
CHCl3±MeOH (1:1) and (1:2) furnished, respectively,
apigenin (30 mg), naringenin (35 mg), hesperidin (28
mg), alhagitin (1) (25 mg) and alhagidin (2) (36 mg).
[C34H44O20+K]+ (FAB±MS); UV l
270 (log e
4.19), 3.12 (log e 3.78); +AlCl3 292 (log e 4.15), 352
(log e 3.00) nm; IR nmax (KBr, cm 1): 3200±3540 (-
OH), 2910 (-OCH3), 1635, 1605 (>C=0), 1515, 1355,
1
1275, 1200, 1130, 1090; H NMR (DMSO-d6, d): 1.10
(3H, d, J=5 Hz, rhamnosyl CH3), 2.78 (1H, dd, J=2
Hz and 10.5 Hz, H-3), 3.15 (1H, m, H-3), 3.75 (3H, s,
-OCH3), 3.20±4.50 (complex, glucosyl, rhamnosyl and
galactosyl protons), 5.20 (2H, br, s, one glucosyl and
one rhamnosyl anomeric protons), 5.45 (2H, m, H-2
and one galactosyl anomeric proton), 6.15 (1H, d,
J=2.0 Hz, H-6), 6.17 (1H, d, J=2 Hz, H-8), 6.80 (3H,
m, H-2', H-5' and H-6'), 9.15 (1H, br, s, 3'-OH), 12.0
(1H, br, s, 5-OH). 13C NMR (DMSO-d6, d): see Table
1.
3.1. Alhagitin (1)
Compound 1 crystallized from MeOH as light yel-
low granules, Rf 0.19 (solvent A), 0.45 (solvent B);
molecular ion peak at m/z 449 as a cationized cluster
ion [C22H24O10+H]+ (FAB±MS); IR nmax (KBr,
cm 1) 3000±3400, 1640, 1460, 1310, 1250, 1175, 1160,
1060; UVMmaexOH: 287 (log e 4.26), 325 sh (log e 3.16)
nm; 13C NMR (DMSO-d6): see Table 1.
3.4. Hydrolysis of alhagidin (2)
Compound 2 (30 mg) was dissolved in MeOH (12
ml) and H2O (2 ml) and re¯uxed with H2SO4 (1 ml)
for 6 h. The reaction mixture was worked up in the
usual manner and extracted with CHCl3. The CHCl3
extract yielded hesperitin (5) as cream colored gran-
3.2. Hydrolysis of alhagitin (1)
Compound 1 (28 mg) was dissolved in MeOH (10
ml) and H2O (1 ml) and re¯uxed with H2SO4 (1 ml)
for 5 h. The reaction mixture was poured into H2O,
the MeOH removed and the mixture extracted with
CHCl3. The CHCl3 extract yielded naringenin 5-methyl
ules, mp 132±368; C16H14O6 (M+, 302); UV l
MeOH
max
275 (log e 4.00), 303 (log e 3.12) nm; 1H NMR
(DMSO-d6, d ): 2.77 (2H, m, H-3), 3.70 (3H, s, -
OCH3), 5.50 (1H, m, H-2), 6.19 (1H, d, J=2 Hz, H-6),
6.24 (1H, d, J=2 Hz, H-8), 6.89 (3H, m, H-2', H-5'
and H-6'), 9.00 (1H, br, s, 7-OH, exchangeable with
D2O), 11.90 (1H, br, s, 5-OH, exchangeable with D2O.
MS: m/z 302 (M+), 274, 273, 179, 152, 150, 124, 120,
98, 97. The aqueous hydrolysate showed three spots
on PC (solvent D) which corresponded to galactose,
glucose and rhamnose (co-PC with authentic samples).
ether (3) (17 mg) as colorless needles, mp 257±598,
C16H14O5 (M+ 286.0846, HRMS); UV l
285, 322
MeOH
max
sh nm: 1H NMR (CCl4, d): 2.70 (2H, m, H-3), 3.78
(3H, s, -OCH3), 5.40 (1H, m, H-2), 6.12 (1H, d, J=2
Hz, H-6), 6.32 (1H, d, J=2 Hz, H-8), 6.80 (2H, d,
J=8 Hz, H-2' and H-3'), 7.30 (2H, d, J=8 Hz, H-5'
and H-6'), 9.10 (1H, br s, 4'-OH, exchangeable with
D2O), 10.00 1H, br, s, 7-OH, exchangeable with D2O).
13C NMR (DMSO-d6, d): see Table 1. HRMS: m/z
286.0846 (M+, 100%), 286(30), 258(58), 179(10),
166(75), 138(65), 135(14), 134(16), 120(22), 119(26),
108(62), 98(24), 73(22), 57(22). On acetylation with
Ac2O/triethylamine, 3 furnished naringenin 5-methyl
ether 7, 4'-diacetate (4) as colorless needless, mp 171±
738; IR nmax (KBr cm 1): 1743, 1682; 1H NMR
(CDCl3, d): 2.25 (6H, s, 2 Â OAc), 2.72 (2H, m, H-3),
3.70 (3H, s, -OCH3), 5.42 (1H, m, H-2), 6.31 (1H, d,
J=2 Hz, H-6), 6.44 (1H, d, J=2 Hz, H-8), 7.10 (2H,
d, J=8 Hz, H-2' and H-3'), 7.44 (2H, d, J=8 Hz, H-
5' and H-6'). Found: C, 64.66, H, 5.28% calcd. for
C20H18O7: C, 64.86, H. 4.90%. The hydrolysate
showed a single spot on PC (solvent D) which corre-
sponded to glucose (co-PC with authentic sample).
3.5. Methylation of alhagidin (2)
Compound 2 was methylated using DMSO accord-
ing to the Hakomori method (Hakomori, 1964).
Alhagidin (2) (25 mg) was treated with NaH (70 mg)
and MeI (1.5 ml) in DMSO (8 ml) in N2. The reaction
mixture was diluted with H2O and extracted with
CHCl3 in the usual way. The methylated product on
puri®cation by prep. TLC gave a semi solid mass, Rf
0.20 (solvent B), which showed no hydroxyl absorption
in its IR. The permethylated product was re¯uxed with
6% HCl in MeOH for 1 h. The MeOH was removed
from the reaction mixture which was then extracted
with CHCl3. The CHCl3 extract could not be puri®ed
because of excess amount of coloring matter and the
small amount of aglycone. The aqueous hydrolysate
was concentrated and co-PC with authentic methylated
sugars indicated the presence of 3,4-di-methylglucose:
3.3. Alhagidin (2)
Compound 2 crystallized from MeOH as bu€
colored granules, Rf 0.34 (solvent C); molecular ion
2,3,4-tri-methylrhamnose
galactose (solvent D).
and
2,3,4,6-tetra-methyl
peak at m/z 811 as
a
cationized cluster ion

590
V.P. Singh et al. / Phytochemistry 51 (1999) 587±590
Khaiitbaev, Kh. Kh., Sultan, A., Ganiev, S. S., & Aslanov, Kh. A.
(1993). Khim. Prir. Soedin., 5, 664.
Acknowledgements
Khushbaktova, Z. A., Syrov, V. N., Kuliev, Z., Bashirova, N. S.,
Shadieva, Z. Kh., Gorodetskaya, Ye. A., & Medvedev, O. S.
(1992). Eksp. Klin. Farmakol. (Russ), 55, 16.
The authors are grateful to Professor Dr. G.
Rucker, Pharmazeutisches Institut, Bonn for authentic
samples and for various spectral analyses.
Kirtikar, K. R., & Basu, B. D. (1984). Indian Medicinal Plants, Vol 1
(p. 742). Delhi: Periodical Expert Book Agency.
Maruyama, M., Hayasaka, K., & Sasaki, S. (1974). Phytochemistry,
13, 286.
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