Isolation, chemical analysis, and study of the hepatoprotector and biligenic activity of total flavonoid preparations from Thermopsis dolichocarpa and Vexibia alopecuroides

Full text of DOI:10.1023/A:1010450709980

Pharmaceutical Chemistry Journal
Vol. 35, No. 1, 2001
MEDICINAL PLANTS
ISOLATION, CHEMICAL ANALYSIS, AND STUDY OF THE
HEPATOPROTECTOR AND BILIGENIC ACTIVITY OF TOTAL
FLAVONOID PREPARATIONS FROM Thermopsis dolichocarpa
AND Vexibia alopecuroides
V. N. Syrov,1 M. P. Yuldashev,1 M. I. Mamutova,1 Z. A. Khushbaktova,1 and É. Kh. Batirov1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 1, pp. 29 – 32, January, 2001.
Original article submitted April 10, 2000.
Some flavonoids were reported to produce a positive
pharmacotherapeutic effect in cases of hepatobiliary pathol-
ogy [1, 2]. In the course of a broad chemico-biological inves-
tigation devoted to flavonoids of the plants occurring in Cen-
tral Asia, we observed a high yield of these substances from
the plants Thermopsis dolichocarpa and Vexibia alopecuroi-
des. It was decided not only to qualitatively characterize the
isolated preparations, but to evaluate their efficacy as poten-
tial means of treating disorders in the functional state of liver
metabolism in cases of toxic damage.
The samples of Thermopsis dolichocarpa V. Nikitin
(aboveground parts) were collected during the fructification
period in the Gissar district of Tajikistan. The roots of
Vexibia alopecuroides (L.) Yakovl. were collected at the
Kibrai village (Tashkent district, Uzbekistan) at the end of
the vegetation period.
Isolation of the total flavonoid fraction from the
Thermopsis dolichocarpa herbs. A sample (2.0 kg) of
crushed air-dry aboveground material was triply extracted
with water to remove carbohydrates, alkaloids, and other hy-
drophilic substances. Then the material was extracted eight
times with an 80% aqueous ethanol solution, after which the
extract volume was reduced to 3 liter by evaporation. In or-
der to remove chlorophyll and waxy components, the etha-
nol – water extract was washed with chloroform
(5 ´ 800 ml).
EXPERIMENTAL CHEMICAL PART
The column chromatography was effected using a
100/250 mm silica gel fraction (Chemapol, Czech Republic).
TLC analyses were performed on Silufol UV-254 plates
(Kavalier, Czech Republic) eluted in various chloro-
form – methanol solvent systems: (1) 97 : 3; (2) 19 : 1;
(3) 9 : 1; (4) 17 : 3; and (5) 4 : 1.
Flavonoids from T. dolichocarpa aboverground parts
OH
HO
O
The mass spectra were obtained using an MX-1310 in-
strument with an electron-impact ionization source operated
at an electron beam energy of 50 eV. The IR spectra were
measured on a UR-20 spectrophotometer (Carl Zeiss – Jena,
Germany) using samples pelletized with KBr. The UV spec-
tra were recorded on a Hitachi EPS-3T spectrophotometer
(Japan) in ethanol solutions. The 1H NMR spectra were mea-
sured on a Tesla BS-567A spectrometer (Czech Republic)
operated at a working frequency of 100 MHz. The measure-
ments were performed using Py-d5 as the solvent; the chemi-
cal shifts (d, ppm) were determined relative to the peaks of
HMDS internal standard.
OH
OH
OH
O
I
RO
O
O
OH
OH
RO
O
II: R = H
IV: R = b-D-Glc
OH
OH
O
III: R = H
V: R = b-D-Glc
1
The purified extract was allowed to stand for one day, after
which the precipitate was separated by filtration, washed
Institute of the Chemistry of Plant Substances, Academy of Sciences of
the Republic of Uzbekistan, Tashkent, Uzbekistan.
30
0091-150X/00/3501-0030$25.00 © 2001 Plenum Publishing Corporation

Isolation, Chemical Analysis, and Study of the Activity of Total Flavonoid Preparations
31
(+CH3ONa); 1H NMR spectrum in Py-d5 (d, ppm):
3.90 – 4.60 (glucose protons), 5.64 (d, 1H, J 6.5 Hz, H-1²),
6.70 (d, 1H, J 2.5 Hz, H-6), 6.90 (d, 1H, J 2.5 Hz, H-8), 7.15
(d, 2H, J 8.0 Hz, H-3¢, H-5¢), 7.55 (d, 2H, J 8.0 Hz, H-2¢,
H-6¢), 8.02 (s, 1H, H-2). The acid hydrolysis of glycoside V
leads to the formation of genistein III and D-glucose. The
acetylation of compound V with acetic anhydride in the pres-
ence of pyridine leads to the formation of a hexaacetyl deriv-
ative with m.p., 189 – 191°C; [M+], 684 [3, 4, 9].
with chloroform (0.4 liter), and dried at 95 – 100°C to obtain
52 g of the total dry flavonoid extract (2.6% of the initial
air-dry material weight).
Half of the product mixture (26 g) was chromatographed
on a silica gel column eluted in system 2 to isolate 3.59 g
(14.8% of the initial weight) of orobol (I) and 6.83 g (26.3%)
of luteolin (II). Then the column was eluted in system 3 to
obtain 2.91 g (11.2%) of genistein (III). Finally, the column
was eluted in system 5 to isolate 2.44 g (9.4%) of cynaroside
(IV) and 1.97 g (7.6%) of genistin (V).
Isolation of the total flavonoid fraction from the
Vexibia alopecuroides roots. A sample (3.0 kg) of crushed
air-dry roots was exhaustively (eight times) extracted with
95% aqueous ethanol, after which the extract volume was re-
duced to 1.5 liter by evaporation. Then the ethanol extract
was diluted with water (1 : 1) and successively treated by
shaking with petroleum ether, chloroform, and ethyl acetate.
The chloroform tincture yields (upon solvent removal) 100 g
of the total dry flavonoid extract. A half of this amount
(50.0 g) was chromatographed on a silica gel column eluted
in system 1 to isolate 0.75 g (1.5% of the initial weight) of
glabrol (VI), 0.95 g (1.9%) of vexibidin (VII), 0.13 g
(0.26%) of isobavachin (VIII), and 2.45 g (4.9%) of
vexibinol (IX).
Orobol (I). Composition, C15H10O6; m.p., 272 – 273°C;
UV spectrum (lmax, nm): 264, 293 (ethanol); 269, 320
(+CH3COONa);
270,
364
(+AlCl3);
272,
370
(+AlCl3 + HCl); IR spectrum (nmax, cm – 1): 3420 – 3270
(OH), 1660 (C=O g-pyrone), 1622, 1576 (C=C arom.); mass
spectrum, m/z (Irel, %): [M+] 286 (100), 268 (2), 153 (30),
134 (27), 124 (17) [3 – 5].
Luteolin (II). Composition, C15H10O6; m.p.,
328 – 331°C; UV spectrum (lmax, nm): 260, 274, 356 (etha-
nol); IR spectrum (nmax, cm – 1): 3450 – 3300 (OH), 1658
1
(C=O g-pyrone), 1612, 1584 (C=C arom.); H NMR spec-
trum in Py-d5 (d, ppm): 6.58 (d, 1H, J 2.0 Hz, H-6), 6.67 (d,
1H, J 2.0 Hz, H-8), 6.78 (s, 1H, H-3), 7.07 (d, 1H, J 8.0 Hz,
H-5¢), 7.50 (bs, 1H, H-2¢), 7.54 (dd, 1H, J 2.0 and 8.0 Hz,
H-6¢). The acetylation of compound II with acetic anhydride
in the presence of pyridine leads to the formation of
tetraacetate with m.p., 225 – 226°C; [M+], 454 [3, 4, 6].
Genistein (III). Composition, C15H10O5; m.p.,
299 – 302°C; UV spectrum (lmax, nm): 263, 329 (ethanol);
272, 323 (+CH3COONa); 271, 369 (+AlCl3); 272, 370
(+AlCl3/HCl); 275, 328 (+CH3ONa); mass spectrum, m/z
(Irel, %): [M+] 270 (100), 153 (69), 152 (36), 124 (11), 118
Flavonoids from V. alopecuroides roots
HO
O
O
OH
VI
HO
O
O
1
OH
(20); H NMR spectrum in Py-d5 (d, ppm): 6.46 (d, 1H,
J 2.0 Hz, H-6), 6.55 (d, 1H, J 2.0 Hz, H-8), 7.06 (d, 2H,
J 8.5 Hz, H-3¢, H-5¢), 7.54 (d, 2H, J 8.5 Hz, H-2¢, H-6¢), 7.92
(s, 1H, H-2) [3, 4, 7].
RO
VIII
HO
O
Cynaroside (IV). Composition, C21H20O11; m.p.,
240 – 242°C; UV spectrum (lmax, nm): 256, 268, 350 (etha-
nol); IR spectrum (nmax, cm – 1): 3480 – 3300 (OH), 1665
(C=O g-pyrone), 1560, 1510 (C=C arom.), 1095, 1030 (C–O
OH
OH
O
VII: R = CH3
IX: R = H
1
HO
bonds in glycosides); H NMR spectrum in Py-d5 (d, ppm):
3.90 – 4.05 (glucose protons), 5.66 (d, 1H, J 7.0 Hz, H-1¢),
6.67 (d, 1H, J 2.5 Hz, H-6), 6.77 (s, 1H, H-3), 6.84 (d, 1H, J
2.5 Hz, H-8), 7.13 (d, 1H, J 8.0 Hz, H-5¢), 7.38 (dd, 1H, J 2.5
and 8.0 Hz, H-6¢), 7.74 (d, 1H, J 2.5 Hz, H-2¢). The acid hy-
drolysis of glycoside IV leads to the formation of luteolin II
and D-glucose. The acetylation of compound IV with acetic
anhydride in the presence of pyridine leads to the formation
of a heptaacetyl derivative with the composition C35H34O18
([M+], 742); m.p., 121 – 123°C [3, 4, 8].
HO
OH
OH
O
X
HO CH2
O
O
O
O
H
OH
HO
Genistin (V). Composition, C21H20O10; m.p., 253 –
255°C; UV spectrum (lmax, nm): 263, 329 (ethanol); 262,
330 (+CH3COONa); 273, 373 (+AlCl3); 272, 355
O
O
H
OH
XI

32
V. N. Syrov et al.
Then the column was eluted in system 3 to obtain 0.11 g
(0.22%) of ammothamnidin (X). Finally, the column was
3CH3–C=), 2.27 (m, H-3²), 3.08 (m, H-1², H-2²), 4.62, 4.72
(2bs, 2H-9²), 5.10 (m, H-4²), 6.35 – 6.70 (m, H-3, H-5,
H-5¢), 7.62 (d, 8.0 Hz, H-6), 7.87 (d, J 9.0 Hz, H-6¢), 8.02 (d,
eluted in system 4 to isolate 0.97 g (1.94%) of trifolirhizin (XI).
Glabrol (VI). Composition, C25H25O4; m.p.,
136 – 137°C; UV spectrum (lmax, nm): 288, 312 (ethanol);
256, 284, 323, 330 (+CH3COONa); 294, 329, 334
(+CH3ONa); 287, 313 (+AlCl3); IR spectrum (nmax, cm – 1):
3390 (OH), 1662 (C=O g-pyrone), 1602, 1588, 1516 (C=C);
mass spectrum, m/z (Irel, %): [M+] 392 (93), 377 (5), 349
(70), 337 (18), 205 (36), 204 (30), 189 (16), 189 (20), 149
(100), 133 (40). The acetylation of compound VI with acetic
anhydride in the presence of pyridine leads to the formation
of a diacetyl derivative with m.p., 106 – 107°C [10, 11].
Vexibidin (VII). Composition, C26H30O6; m.p.,
156 – 157°C; UV spectrum (lmax, nm): 293, 340 (ethanol);
294, 341 (+CH3COONa); 244, 332 (+CH3ONa); 311, 376
(+AlCl3); mass spectrum, m/z (Irel, %): [M+] 438 (8), 315
(76), 285 (2.9), 219 (18), 203 (2), 191 (9), 177 (8), 166 (14),
165 (100), 151 (22), 150 (10), 137 (9A), 135 (6), 123 (14).
The acetylation of compound VII with acetic anhydride in
the presence of pyridine leads to the formation of a triacetyl
derivative with m.p., 77 – 78°C. The methylation of com-
pound VII with diazomethane yields a dimethyl ester of
compound VII; mass spectrum, m/z (Irel, %): [M+] 466
[3, 12].
J 16 Hz, H-a), 8.70 (d, J 16 Hz, H-b). The acetylation of
compound X with acetic anhydride in the presence of
pyridine leads to the formation of a tetraacetyl derivative
with the composition C33H36O9 [14].
Trifolirhizin (XI). Composition, C22H24O10; m.p.,
140 – 142°C; UV spectrum (lmax, nm): 279, 286, 312 (etha-
nol); IR spectrum (nmax, cm – 1): 3370 – 3220 (OH), 1625,
1580 (C=C arom.), 1076, 1045, 1020 (C–O), 936 (OCH2O);
1H NMR spectrum in Py-d5 (d, ppm): 3.06 – 3.85 (m, H-6),
3.86 – 4.53 (sugar residue protons, H-6a), 5.44 (d, J 7.0 Hz,
H-1¢), 5.55 (d, 1H, J 6.0 Hz, H-11a), 5.85 (bs, OCH2O), 6.58
(s, H-10), 6.78 (s, H-7), 7.06 (m, H-2, H-4), 7.42 (d, J 9.0 Hz,
H-1). The acetylation of compound XI with acetic anhydride
in the presence of pyridine leads to the formation of a
tetraacetyl derivative with the composition C30H30O14 ([M+],
614). The acid hydrolysis of compound XI leads to the for-
mation of inermin and D-glucose.
EXPERIMENTAL BIOLOGICAL PART
The experiments were performed on white mongrel male
rats weighing 150 – 180 g (6 – 8 animals in each group). The
total flavonoid extract from T. dolichocarpa and V.
alopecuroides (preparations I and II, respectively) were in-
troduced to the test animals in a preliminarily selected single
effective daily dose (50 mg/kg, p.o.) over a period of five
days. The model toxic liver damage was induced by
Isobavachin (VIII). Composition, C20H20O4; m.p.,
203 – 204°C; UV spectrum (lmax, nm): 288, 312 (ethanol);
289, 338 (+CH3COONa); 290, 349 (+CH3ONa); 290, 311
(+AlCl3); IR spectrum (nmax, cm – 1): 3280 (OH), 1645
1
(C=O), 1585, 1520 (C=C); H NMR spectrum in Py-d5 (d,
ppm): 1.59 (bs, =C–CH3), 1.72 (bs, =C–CH3), 2.80 – 3.05
(m, H-3), 3.54 (d, J 7.0 Hz, Ar–CH2–), 5.16 – 5.68 (m, H-2,
=CH–CH2–), 6.81 (d, J 8.6 Hz, H-6), 7.10 (d, J 9.0 Hz, H-3¢,
H-5¢), 7.43 (d, J 9.0 Hz, H-2¢, H-6¢), 7.89 (d, J 8.6 Hz, H-5)
[10, 13].
paracetamol in a dose of 2.5 g/kg introduced in the form of a
25% aqueous suspension during the first two days via a spe-
cial intragastric tube [16]. After termination of the treatment
with flavonoid preparations I and II, the animals were decap-
itated under light ether narcosis. The efficacy of the treat-
ment was evaluated by its effect upon the activity of alanine
(AlAT) and aspartate (AsAT) aminotransferases [17] and al-
kaline phosphatase (AlkP) in the blood serum [18] and by the
content of malonic dialdehyde (MDA) [19] and glycogen
[20] in liver homogenates.
Vexibinol (IX). Composition, C25H28O6; m.p.,
173 – 175°C; UV spectrum (lmax, nm): 292, 339 (ethanol);
mass spectrum, m/z (Irel, %): [M+] 424 (14), 409 (3), 408 (3),
407 (5), 406 (18), 391 (7), 389 (3), 363 (7), 338 (3.9), 337
(10), 302 (17), 301 (75), 284 (23), 283 (100), 219 (13), 166
(5), 165 (37), 139 (5), 137 (4), 136 (6), 124 (6.8), 123 (9),
109 (13), 107 (6). The acetylation of compound VII with
acetic anhydride in the presence of pyridine leads to the for-
mation of a tetraacetyl derivative with m.p., 70 – 72°C. The
methylation of compound IX with an ether solution of diazo-
methane yields a trimethyl ester of compound IX, which is
identical to the aforementioned dimethyl ester of vexibidin
[12].
Ammothamnidin (X). Composition, C25H28O5; m.p.,
112 – 114°C; UV spectrum (lmax, nm): 231, 261, 321, 390
(ethanol); 398 (+CH3COONa); 395 (+CH3COONa/H3BO3);
439 (+CH3ONa); 392 (+AlCl3); IR spectrum (nmax, cm – 1):
3430 – 3145 (OH), 1628 (C=O), 1615, 1557, 1522 (C=C);
1H NMR spectrum in Py-d5 (d, ppm): 1.43, 1.47, 1.78 (3s,
For evaluating the biligenic activity of preparations I and
II, a part of the animals from each group (a total of not less
than six) were narcotized with barbamyl (100 mg/kg, i.p.)
and bile samples were taken every hour over a 4-h period of
time using a catheter inserted into the common bile duct. The
samples were analyzed for the concentration of bile acids
[21], cholesterol [22], and bilirubin [23], after which their to-
tal amounts were determined. In addition, the test animals in
all experimental series were characterized by the
cholate – cholesterol coefficient. The experimental data were
statistically processed in terms of the Student t-criterion.

Isolation, Chemical Analysis, and Study of the Activity of Total Flavonoid Preparations
33
TABLE 1. Effect of the Total Flavonoid Extracts from Thermopsis dolichocarpa (Preparation I) and Vexibia alopecuroides (Preparation II) on
Some Parameters of Liver Metabolism and Functioning in Rats with Paracetamol Hepatitis Model
Hepatitis
(paracetamol)
Paracetamol +
preparation I
Paracetamol +
preparation II
Characteristic
Intact rats
AlAT***
AsAT***
0.94 ± 0.08
1.34 ± 0.18
216.4 ± 24.8
0.496 ± 0.018
2088 ± 96
2.84 ± 0.46*
2.06 ± 0.22*
308.6 ± 16.2*
0.854 ± 0.046*
834 ± 56*
1.16 ± 0.12**
1.38 ± 0.06**
222.4 ± 12.4**
0.532 ± 0.020**
1706 ± 64*,**
0.98 ± 0.10**
1.32 ± 0.06**
214.6 ± 10.8**
0.512 ± 0.032**
1806 ± 94**
AlkP, U/liter
MDA, mmole/(mg protein)
Glycogen, mg%
Bile secretion rate, mg/(min 100 g)
5.12 ± 0.14
4.44 ± 0.12
4.38 ± 0.10
4.20 ± 0.10
1088 ± 84
3.64 ± 0.18*
3.50 ± 0.16*
3.30 ± 0.14*
3.18 ± 0.16*
817.2 ± 28*
582.8 ± 32.6*
15.4 ± 1.4
5.16 ± 0.20**
5.80 ± 0.24*,**
4.50 ± 0.18**
4.30 ± 0.16**
1182 ± 88**
5.50 ± 0.38**
5.10 ± 0.20*,**
4.80 ± 0.24**
4.76 ± 0.14*,**
1209 ± 96**
1st hour
2nd hour
3rd hour
4th hour
Total bile, mg/(100 g 4 h)
Bile acids, mg%
876.4 ± 40.4
18.8 ± 2.2
882.4 ± 42.6**
998.6 ± 54.2**
Cholesterol, mg%
18.6 ± 2.6
20.4 ± 2.8
Bilirubin, mg%
22.2 ± 3.0
17.8 ± 1.4
26.8 ± 2.4**
28.3 ± 3.0**
4.763 ± 0.362*
0.125 ± 0.006*
0.145 ± 0.010*
35.5 ± 2.2*
10.429 ± 0.690**
0.219 ± 0.014**
0.317 ± 0.026*,**
47.4 ± 4.8**
12.073 ± 0.720*,**
0.246 ± 0.018**
0.342 ± 0.028*,**
48.9 ± 5.2**
Total bile acids, mg/(100 g 4 h)
Total cholesterol, mg/(100 g 4 h)
Total bilirubin, mg/(100 g 4 h)
Cholate – cholesterol coefficient
9.535 ± 0.650
0.204 ± 0.012
0.242 ± 0.018
46.6 ± 4.2
*
p < 0.05 relative to intact control
p < 0.05 relative to untreated control
Aminotransferase activity in mmole PVA per ml serum for 1 h incubation.
**
***
RESULTS AND DISCUSSION
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