Isolation, chemical analysis, and study of the hypolipidemic activity of the total flavonoid extract from thermopsis altherniaflora

Full text of DOI:10.1023/A:1010410029719

Pharmaceutical Chemistry Journal
Vol. 35, No. 3, 2001
MEDICINAL PLANTS
ISOLATION, CHEMICAL ANALYSIS, AND STUDY
OF THE HYPOLIPIDEMIC ACTIVITY OF THE TOTAL
FLAVONOID EXTRACT FROM Thermopsis altherniaflora
1
1
1
1
Z. A. Khushbaktova, S. Kh. Faizieva, V. N. Syrov, M. P. Yuldashev,
1
1
É. Kh. Batirov, and A. U. Mamatkhanov
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 3, pp. 35 – 38, March, 2001.
Original article submitted April 17, 2000.
Previously we reported that the total flavonoid extract
from plants of Thermopsis altherniaflora exhibited hypolipi-
demic activity on the endogenous, triton, ethanol, and adren-
aline hyperlipidemia models [1]. In this work, we determined
the qualitative and quantitative composition of the total
flavonoid extract from this plant, studied the effect of this
preparation on the lipid metabolism characteristics in ani-
mals with expermental atherosclerosis, and established the
active component of the sum of flavonoids.
formed on Silufol UV-254 plates (Czech Republic) eluted ei-
ther in chloroform – methanol mixtures (1) 97 : 3, (2) 19 : 1,
(
(
3) 9 : 1, or (4) 4 : 1, or in a (5) 1-butanol – methanol – water
5 : 3 : 1) system.
Isolation of the total flavonoid fraction from
Thermopsis altherniaflora herbs. A charge (14.0 kg) of
crushed air-dry above-ground material was triply extracted
with water to remove carbohydrates, salts, alkaloids, organic
acids, and other hydrophilic substances. Then the material
was exhaustively extracted with an 80% aqueous ethanol so-
lution at room temperature, after which the extract volume
was reduced to 6 liters by evaporation. Then the aqueous eth-
Samples of Thermopsis altherniaflora Rgl. et Schmalh
(
above-ground parts) were collected during the blooming pe-
riod near Khumsan village of the Tashkent district of
Uzbekistan. The sum of flavonoids (called flateron) isolated
from a dense alcohol extract of this plant was separated by
column chromatography into six main flavonoid compo-
nents: formononetin, chrysoeriol, apigenin, luteolin, thermo-
psoside, and cynaroside.
anol extract was treated with chloroform (5 ´ 2 liter) on a
shaker. The resulting aqueous chloroform solution was al-
lowed to stand for one day (20 – 24 h) at room temperature,
after which the precipitate was separated by filtration,
washed with chloroform (0.5 liter), and dried at 95 – 100°C
to obtain 330.0 g of the total dry flavonoid extract (2.35% of
the initial air-dry material weight).
EXPERIMENTAL CHEMICAL PART
The IR spectra were measured on
a
UR-20
A part of the product (33.0 g) was chromatographed on a
spectrophotometer (Carl Zeiss – Jena, Germany) using sam-
ples pelletized with KBr. The UV spectra were recorded on a
Hitachi EPS-3T spectrophotometer (Japan) in ethanol solu-
tions. The mass spectra were obtained using an MX-1310 in-
column (95 ´ 5 cm) filled with silica gel (660 g) and eluted
sequentially with chloroform and chloroform – methanol
mixtures 1, 2, 3, and 4, collecting 500-ml fractions. From the
fractions eluted with system 1, we isolated 8.67 g (26.2% of
the initial product weight) of formononetin (I) and 9.4 g
(28.5%) of chrysoeriol (II). Then the column was eluted with
system 2 to obtain 1.05 g (3.2%) of apigenin (III) and with
system 3, which yielded 8.02 g (24.3%) of luteolin (IV).
Finally, the column was eluted with system 4 to isolate
0.69 g (2.1%) of thermopsoside (V) and 1.58 g (4.8%) of
cynaroside (VI).
strument operated at an electron-impact ionization energy of
1
5
0 eV. The H NMR spectra were measured on a Tesla
BS-567A spectrometer (Czech Republic) operated at a work-
ing frequency of 100 MHz. The column chromatography was
effected using KSK grade silica gel. TLC analyses were per-
1
Institute of the Chemistry of Plant Substances, Academy of Sciences of
the Republic of Uzbekistan, Tashkent, Uzbekistan.
155
0
091-150X/01/3503-0155$25.00 © 2001 Plenum Publishing Corporation

1
56
Z. A. Khushbaktova et al.
Flavonoids from Thermopsis altherniaflora
dride leads to the formation of tetraacetate with m.p.
+
224 – 225°C; [M] , 454 [5].
HO
O
Thermopsoside (V). Composition, C H O ; m.p.,
2
2
22 11
OCH3
174 – 175°C; UV spectrum (l
spectrum (nmax, cm ): 3450 – 3280 (OH), 2930 (OCH3),
max, nm): 255, 268, 348; IR
–
1
O
HO
O
O
OH
I
1666 (C=O), 1614, 1504 (C=C arom.), 1120, 1045, 1025
1
(
3
C=O in glycosides); H NMR spectrum in Py-d (d, ppm):
5
.70 (s, OCH ), 3.80 – 4.54 (carbohydrate protons), 5.64 (d,
OH
3
OCH3
J 6.0 Hz, H-1²), 6.70 (bs, H-6), 6.82 (bs, H-8), 6.94 (s, H-3),
.08 (d, J 8.5 Hz, H-5¢), 7.49 (m, H-2¢, H-6¢). The acid hy-
III
RO
O
7
OH
drolysis of thermopsoside leads to chrysoeriol (II) and
D-glucose (TLC, system 5) [6].
OH
RO
O
O
OH
O
OH
Cynaroside (VI). Composition, C H O ; m.p.,
2
1
20 11
II: R = H;
V: R = b-D-Glc
240 – 242°C (with decomp); UV spectrum (lmax, nm): 256,
68, 350; IR spectrum (nmax, cm – 1): 3480 – 3260 (OH),
2
1
OH
665 (C=O), 1560, 1510 (C=C arom.), 1095, 1030 (C – O in
IV: R = H;
VI: R = b-D-Glc
1
glycosides); H NMR spectrum in Py-d5 (d, ppm):
3
6
7
.90 – 4.05 (carbohydrate protons), 5.65 (d, J 7.0 Hz, H-1²),
.65 (d, J 2.5 Hz, H-6), 6.75 (s, H-3), 6.83 (d, J 2.5 Hz, H-8),
.13 (d, J 8.0 Hz, H-5¢), 7.37 (dd, J 8.0 and 2.5 Hz, H-6¢),
Formononetin (I). Composition, C H O ; m.p.,
16
12
4
2
60 – 261°C; UV spectrum (l , nm): 239 sh, 251, 261 sh,
max
3
04; IR spectrum (nmax, cm – 1): 3150 (OH), 1641 (C=O
7.73 (d, J 2.5 Hz, H-2¢). The acid hydrolysis of cynaroside
leads to the formation of luteolin (IV) and D-glucose (TLC,
system 5). The acetylation of cynaroside with acetic anhy-
dride in the presence of pyridine leads to the formation of a
g-pyrone), 1624, 1613 – 1600 (C=C arom.), 1573, 1518,
+
1
2
458, 1031; mass spectrum, m/z (I , %): [M] 268(100),
rel
67(34), 253(22), 239(3), 225(12), 197(5), 136(5), 132(70),
1
heptaacetyl derivative with the composition C H O
1
17(20), 108(10), 89(27); H NMR spectrum in Py-d (d,
35 34 18
5
+
([M] , 742); m.p., 121 – 123°C [7, 8].
ppm): 3.57 (s, OCH ), 6.99 (d, J 9.0 Hz, H-3¢, H-5¢), 7.07
3
(
bs, H-8), 7.14 (dd, J 9.0 and 2.0 Hz, H-6), 7.72 (d, J 9.0 Hz,
EXPERIMENTAL PHARMACOLOGICAL PART
H-2¢, H-6¢), 8.10 (s, H-2), 8.40 (d, J 9.0 Hz, H-5) [2].
+
Chrysoeriol (II). Composition, C H O ; M , 300;
16
12
6
The activity of flavonoids (flateron) was studied on chin-
chilla rabbits weighing 2.5 – 3.0 kg with experimental ath-
erosclerosis induced by peroral introduction of cholesterol
with cotton oil in a daily dose of 0.3 g/kg over 120 days
[9, 10]. There were three series of experiments (each in a
group of 8 – 10 animals), in which the test rabbits received
m.p., 335 – 337°C (with decomp); UV spectrum (lmax, nm):
2
2
53, 270, 350; IR spectrum (nmax, cm – 1): 3540 – 3290 (OH),
1
930 (OCH ), 1665 (C=O), 1615, 1584 (C=C arom.); H
3
NMR spectrum in Py-d (d, ppm): 3.70 (s, OCH ), 6.60 (d, J
5
3
2
8
.0 Hz, H-6), 6.70 (d, J 2.0 Hz, H-8), 6.82 (s, H-3), 7.12 (d, J
.0 Hz, H-5¢), 7.47 (bs, H-2¢), 7.52 (dd, J 2.0 and 8.0 Hz,
(i) cholesterol, (ii) cholesterol and flateron (50 mg/kg, p.o.),
H-6¢) [3].
Apigenin (III). Composition, C H O ; M , 270; m.p.,
and (iii) cholesterol and the reference drug miscleron
(clofibrate) (250 mg/kg; Chinoin Company, Hungary). The
atherosclerotic damage of the aorta in the experimental ani-
mals was determined by direct planimetry and expressed by
the damage index calculated as described in [11].
+
16
10
5
3
45 – 346°C; UV spectrum (lmax, nm): 270, 298 sh, 338; IR
spectrum (nmax, cm – 1): 3520 – 3300 (OH), 1660 (C=O),
1
1
612, 1583 (C=C arom.); H NMR spectrum in Py-d (d,
5
The hyperlipidemia model was induced by
intraperitoneal injection of triton WR-1339 in a dose of
ppm): 6.62 (d, J 2.0 Hz, H-6), 6.71 (d, J 2.0 Hz, H-8), 6.80 (s,
H-3), 7.05 (d, J 9.0 Hz, H-3¢, H-5¢), 7.83 (d, J 9.0 Hz, H-2¢,
225 mg/kg [12]. The total flavonoid preparations were intro-
H-6¢), 13.65 (bs, 5-OH) [4].
duced perorally in a dose of 50 mg/kg (2 h before and 16 h
after triton WR-1339 injection)
The effects of individual flavonoids entering into the
flateron composition on the cholesterol level was studied in
vitro using subendothelial foam cells of the aorta intima of ill
patients, isolated by autopsy 2 – 3 h after death. The cells
Luteolin (IV). Composition, C H O ; m.p.,
15
10
6
3
28 – 331°C; UV spectrum (lmax, nm): 260, 274 sh, 356
ethanol); IR spectrum (nmax, cm – 1): 3450 – 3300 (OH),
658 (C=O), 1612, 1584 (C=C arom.); mass spectrum, m/z
(
1
+
(
I , %): [M] 286, 258, 229, 213, 153, 149, 137, 134, 129,
rel
1
1
6
7
8
09, 107, 91, 81, 69; H NMR spectrum in Py-d (d, ppm):
were isolated by incubating the intima for 3 h in an 0.1% col-
5
.58 (d, J 2.0 Hz, H-6), 6.67 (d, J 2.0 Hz, H-8), 6.79 (s, H-3),
.07 (d, J 8.0 Hz, H-5¢), 7.50 (bs, H-2¢), 7.55 (dd, J 2.0 and
.0 Hz, H-6¢). The acetylation of luteolin with acetic anhy-
lagenase solution. The cholesterol content was determined
by extracting lipids from the cells with an n-hex-
ane – isopropanol mixture [13]. These experiments were car-

Isolation, Chemical Analysis, and Study of the Hypolipidemic Activity
157
TABLE 1. Effects of Flateron and Clofibrate on the Lipid Metabolism Characteristics (mmole/liter) in Blood Serum of Rabbits with Experi-
mental Atherosclerosis
a-Choles-
terol/total
cholesterol
Cholesterol
atherogenicity terol/phospho-
coefficient
1.31
Total choles-
b-, Pre-b-lipo-
Experiment Total cholesterol a-Cholesterol
Triglycerides
Phospholipids
proteins
lipids
1.098
Intact animals
1.56 ± 0.61 0.675 ± 0.033 0.685 ± 0.15
1.55 ± 0.15
1.42 ± 0.20
0.43
Control (athero-
sclerosis)
23.05 ± 1.35 0.641 ± 0.011
< 0.001 > 0.5
10.27 ± 0.27 0.718 ± 0.015
< 0.001 < 0.001
13.84 ± 0.36 0.704 ± 0.011
< 0.001 < 0.001
6.49 ± 0.19
< 0.001
12.02 ± 0.57
< 0.001
4.34 ± 0.71
< 0.001
0.028
34.9
13.3
18.6
5.311
3.591
4.943
p
1
Flateron
2.93 ± 0.45
< 0.001
5.46 ± 0.41
< 0.001
2.86 ± 0.39
> 0.05
0.069
0.051
p
2
Clofibrate
2.84 ± 0.44
< 0.001
5.42 ± 0.28
< 0.001
2.8 ± 0.33
> 0.05
p
2
Note: p and p values indicate reliable differences from data for the intact and control groups, respectively.
1
2
sion of Prof. A. N. Orekhov). The total cholesterol in the
blood serum and aorta cells was determined according to
TABLE 2. Effects of Flateron and Clofibrate on the Lipid Content
in Aorta of Rabbits with Experimental Atherosclerosis
[14], and the concentrations of triglycerides, b- and
Cholesterol,
mg/g
Triglycer-
ides, mg/g
Experiment
p
p
pre-b-lipoproteins, and a-cholesterol were determined ac-
cording to [15 – 17].
Intact animals
1.24 ± 0.76
24.4 ± 1.35
1.31 ± 0.34
The effects of individual flavonoid components of
Control (athero-
sclerosis)
flateron (taken in a concentration of 2 ´ 10 – 5 M) on the
malonic dialdehyde (MDA) accumulation were studied un-
der the conditions of iron-induced ascorbate-dependent lipid
peroxidation (LPO) in rat liver homogenate. The LPO reac-
tion was induced by 10 mM FeSO4 in the presence of
< 0.001 9.25 ± 0.52 < 0.001
Atherosclero-
sis + flateron
13.34 ± 1.36 < 0.001
5.1 ± 0.30 < 0.001
Atherosclero-
sis + clofibrate 16.86 ± 0.79
< 0.001 6.88 ± 0.56 < 0.001
Note: p values indicate reliable differences from data for the control
group.
200 mM of ascorbate in a medium containing 145 mM KCl
and 25 mM Tris-HCl (pH 7.4). The accumulation of LPO
products was determnined using a color reaction with
thiobarbituric acid [18].
ried out at the Cardiological Research Center of the Ministry
of Public Health of the Russian Federation (under supervi-
The experimental data were statistically processed using
the Student t-criterion.
TABLE 3. Hypocholesterinemic and Antioxidant Effects of Flateron and Individual Flavonoid Components on Foam
Cells of Aorta Intima
Foam aorta cells
Triton hyperlipidemia
Cholesterol, Triglycerides,
Antioxidant effect
Experiment
Cholesterol, mg/mg protein
MDA accumulation,
mM
mM
nmole/mg protein
0.3 mg/ml
0.03 mg/ml
Intact animals
–
–
0.71 ± 0.06
6.6 ± 0.21
0.44 ± 0.03
6.3 ± 0.56
20.19 ± 1.4
Control (atherosclerosis)
Flateron
124 ± 9
46 ± 10*
97 ± 7
123 ± 7
47 ± 8*
98 ± 8
4.31 ± 0.36*
4.95 ± 0.8*
4.47 ± 0.34*
4.50 ± 0.32*
4.36 ± 0.30*
4.41 ± 0.38*
3.81 ± 0.49*
3.94 ± 0.97*
4.57 ± 0.20*
3.55 ± 0.25*
13.27 ± 0.87*
7.32 ± 1.15*
18.07 ± 0.37
20.01 ± 0.46
12.43 ± 1.45*
Luteolin
Chrysoeriol
Formononetin
Cynaroside
90 ± 5*
52 ± 10*
59 ± 8*
40 ± 3*
49 ± 9*
43 ± 7*
1
1
1
1
1
Apigenin
–
–
–
–
–
Thermopsoside
87 ± 5*
99 ± 7
5.03 ± 0.23
5.58 ± 6.42
21.66 ± 0.940
*
p < 0.05 relative to the control group; 1 apigenin was not studied.

1
58
Z. A. Khushbaktova et al.
2. S. S. Yusupova, É. Kh. Batirov, F. Kiyamitdinova, and
RESULTS AND DISCUSSION
V. M. Malikov, Khim. Prir. Soedin., No. 5, 639 – 640 (1986).
It was established that the considerably increased blood
content of cholesterol, triglycerides, phospholipids, and
b-lipoproteins observed in animals with experimental athero-
sclerosis was significantly reduced by flateron administration
3. N. Sh. Kattaev and G. K. Nikonov, Khim. Prir. Soedin., No. 2,
140 – 144 (1975).
4. P. Khozhambergenova and K. F. Blinova, Khim. Prir. Soedin.,
No. 4, 566 – 567 (1980).
(
Table 1). The hypocholesterinemic affect of flateron ex-
5. A. Sakushima, H. Hisoda, Y. Ogihara, and S. Nichibe, Chem.
ceeded that of clofibrate.
Pharm. Bull., 28(4), 1219 – 1223 (1980).
Analogous results were obtained for the content of lipids
in rabbits with experimental atherosclerosis (Table 2). The
aorta intima in most rabbits of the control group (receiving
only cholesterol) was covered by atherosclerotic plaques of
various sizes and shapes. The degree of damage varied from
6. N. Sh. Kattaev and G. K. Nikonov, Khim. Prir. Soedin., No. 1,
115 – 117 (1973).
7
8
. Sh. V. Abdullaev, A. Sattikulov, É. Kh. Batirov, et al., Khim.
Prir. Soedin., No. 1, 104 – 106 (1983).
. T. J. Mabry, K. R. Markham, and M. B. Thomas, The System-
1
5 to 85%. In four rabbits of the group of eight treated with
atic Identiaication of Flavanoids, Springer-Verlag, Berlin
flateron, the aorta intima exhibited no visible damage and the
area damaged by sclerotic plaques in the remaining four ani-
mals was 5 – 28%. The average index of atherosclerotic
aorta damage in this group was 3.3 times smaller than in the
control group. In the reference group treated with clofibrate,
only one rabbit had an aorta without visible damage, one ani-
mal exhibited separate plaques (4%), and the atherosclerotic
damage in the other rabbits varied from 12 to 35%. The aver-
age index of the atherosclerotic aorta damage in the refer-
ence group was smaller than that in the control group by a
factor of 2.2.
(
1970).
9
. N. N. Anichkov, On the Initial Stage of Arterial Atherosclerosis
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0. É. P. Tsyganov, USSR Inventor¢s Certificate, No. 1508269,
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1
1. G. G. Avtandilov, Classification and Planimetric Evaluation of
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(
12. P. F. Schurr, J. R. Schulz, and T. M. Parkinson, Lipids, 7(1),
68 – 72 (1972).
As seen from the data presented in Table 3, most of the
individual flavonoids entering into flateron produce a
hypocholesterinemic effect both in vitro (in foam cells of the
human aorta intima) and in vivo (triton hyperlipidemia model
in rabbits). In addition, some of these flavonoids (luteolin
and cynaroside) exhibited antioxidant properties.
13. A. N. Orekhov, V. V. Tertov, and S. A. Kudryashov, Atheroscle-
rosis, No. 60, 101 – 104 (1986).
1
4. L. L. Abell, B. B. Levy, and F. E. Kendoll, J. Biol. Chem., 195,
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3
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(
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