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R. Harini et al. / Journal of Pharmacy Research 2012,5(1),707-710
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Antihyperlipidemic effect of biochanin A on streptozotocin induced diabetic rats.
R. Harini, A. Sundaresan and K. V. Pugalendi*
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamilnadu, India
Received on:10-11-2011; Revised on: 15-12-2011; Accepted on:12-01-2012
ABSTRACT
Diabetes mellitus is a chronic metabolic disease with the highest rates of prevalence and mortality worldwide and hyperlipidemia has been ranked as one of
the greatest risk factors contributing to coronary heart disease. This study was undertaken to evaluate the antiatherogenic and antihyperlipidemic effect of
biochanin A on streptozotocin (STZ)-diabetic rats. Diabetes was induced in male albino Wistar rats by the administration of STZ (40 mg/kg BW) and
biochanin A was administered post orally at a dose of 10 mg/kg BW for 45 days. The result showed an increase in plasma glucose, atherogenic index and also
showed an elevation in total cholesterol (TC), triglycerides (TG), very low density lipoproteins-cholesterol (VLDL-C) and low density lipoproteincholesterol (LDL-C) and decrease in the level of plasma insulin and high density lipoprotein-cholesterol (HDL-C) in diabetic rats, whereas, diabetic rats
treated with biochanin A shifted all these parameters towards normality. The effect of biochanin A was comparable with glibenclamide, a well known
hypoglycemic drug. These findings suggest that biochanin A treatment has a therapeutic property by showing antiatherogenic and antihyperlipidemic effect
on STZ-diabetic rats.
Key words: Diabetes mellitus; streptozotocin; biochanin A; atherogenic index; lipid profile; glibenclamide
INTRODUCTION
Diabetes mellitus is a chronic metabolic disease with the highest rates of
prevalence and mortality worldwide that is caused by an absolute or relative
lack of insulin and/or reduced insulin activity, which results in hyperglycemia and abnormalities in carbohydrate, protein, and fat metabolism [1]. It can
be associated with serious complications and premature death, but people
with diabetes should take steps to control the disease and lower the risk of
complications [2]. Diabetes mellitus is a major risk factor for the development
of cardiovascular complications, and cardiovascular disease now accounts
for 80% of all diabetic mortality. Atherosclerosis, a chronic disease of the
vessel wall is associated with diabetes mellitus, which underlies the development of many acute cardiovascular disease events including myocardial infarction and stroke. During diabetes, a profound alteration in the concentration and composition of lipid occurs. Lipid-lowering therapy in diabetes is
effective in reducing the risk of vascular complications [3], in spite of the use
of many oral hypoglycemic agents such as sulphonylureas and biguanides.
All of these pharmacological modalities also show limited efficacy and certain adverse effects such as liver toxicity, lactic acidosis, diarrhea and attenuation of response after prolonged use and are also expensive for developing
countries. Comparatively very less side effects and low cost of phytochemicals
from natural resources open new avenues for the treatment of various diseases including diabetes [4]. Therefore, there is a need for phytochemicals that
have antihyperglycemic and hypolipidemic potential, which are cost-effective and also safe without side effects on long-term usage.
mild estrogenic, and hypolipidemic activity [5]. Earlier study reports that
consumption of isoflavones is associated with protection against atherosclerosis. This observation is supported by experimental studies in diverse animal models of atherosclerosis showing that dietary isoflavone can inhibit the
disease. Isoflavones are naturally occurring compounds in certain foods. Soy
isoflavones are safe to ingest in amounts upto 100 mg/day. Salam et al.,
reported that out of a natural product library comprising 200 compounds,
isoflavones regulates the fatty acid metabolism via the nuclear receptors
PPARa and PPAR? [6]. Thus the soy isoflavones could be used therapeutically with a low occurrence of unwanted side effects. Biochanin A is an Omethylated isoflavone. It is a natural organic compound in the class of
phytochemicals known as flavonoids. Biochanin A can be found in red clover
in soy, alfalfa sprouts, peanuts, chickpea (Cicer arietinum) and in other
legumes. As a flavonoid, it also exhibits various pharmacological properties
such as, anti-inflammatory, anti-carcinogenic, hypolipidemic effect.
The eventual objective of the present study is to evaluate the antiatherogenic
and antihyperlipidemic effect of biochanin A on STZ- induced diabetic rats.
HO
O
OH
O
OCH 3
Most recent studies on the treatment of type-2 diabetes have focused on the
Fig.1 Biochanin A
potential use of plant constituents with hypoglycemic and hypolipidemic
effects. As such, there has been a growing interest in flavonoids, which are MATERIALS AND METHODS
widely distributed in plants and ingested by humans, due to their antioxidative,
Animals
Male albino (9 week-old) rats of Wistar strain with a body weight ranging
*Corresponding author.
from 180 to 200 g, were procured from Central Animal Hourse, Department
Dr. K.V. Pugalendi
of Experimental Medicine, Rajah Muthiah Medical College and Hospital,
Professor and Head
Annamalai University, and were maintained in an air conditioned room (25 ±
Department of Biochemistry and Biotechnology
1ºC) with a 12 h light/12 h dark cycle. Feed and water approved by the
Annamalai University
Institutional Animals Ethics Committee of Rajah Muthiah Medical College
Tamilnadu,India
and Hospital Reg No. 160/1999/CPCSEA, Proposal number: 738, Annamalai
University, Annamalainagar.
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R. Harini et al. / Journal of Pharmacy Research 2012,5(1),707-710
Chemicals
Streptozotocin (STZ) and biochanin A were purchased from Sigma-Aldrich
(St. Louis, Missouri, USA). All other chemicals used in this study were of
analytical grade obtained from E. Merck and HIMEDIA, Mumbai, India.
Experimental induction of diabetes
After an overnight fast, the animals were rendered diabetic by a single intraperitoneal injection of STZ (40 mg/kg BW) in freshly prepared citrate buffer
(0.1 M pH 4.5). The STZ-injected animals were given 20 % glucose solution
for 24 h to prevent initial drug-induced hypoglycemic mortality. The STZinjected animals exhibited hyperglycemia within a few days. Diabetes in
STZ rats was confirmed by measuring the blood glucose (by glucose oxidase
method) 96 h after injection with STZ. The animals with blood glucose above
230 mg/dL were considered diabetic and used for the experiments.
Experimental design
The animals were randomly divided into five groups of six animals each as
given below. Biochanin A and glibenclamide were dissolved in 0.5% DMSO
and administered orally once in a day in the morning for 45 days.
Group I : Control (0.5% DMSO)
Group II : Control + biochanin A (10 mg/kg BW/day)
Group III : Diabetic control (0.5% DMSO)
Group IV : Diabetic + biochanin A (10 mg/kg BW/day)
Group V : Diabetic + glibenclamide (600 µg/kg BW/day).
After 45 days, the animals were anaesthetized using ketamine (24 mg/kg/
body weight, intramuscular injection), and sacrificed by cervical dislocation.
Between 8:00 am and 9:00 am blood was collected in tubes with a mixture of
potassium oxalate and sodium fluoride (1:3) to get plasma for various assays.
Table 1. Effect of Biochanin A on and plasma glucose and insulin in
STZ-diabetic rats
Groups
0 day
Control
Control + Biochanin A
(10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A
(10 mg/kg BW)
Diabetic + glibenclamide
(600 µg/kg BW)
Plasma glucose (mg/dL)
45th day
Change (%)
(+) 2.29
(-) 3.17
14.98 ± 1.11a
15.08 ± 1.15a
242.02 ± 9.32 285.72 ± 11.33b
241.15 ± 9.21 121.24 ± 9.81c
(+) 15.08
(-) 49.72
7.55 ± 0.39b
13.34 ± 0.65c
238.45 ± 9.34 98.54 ± 6.81d
(-) 58.67
14.86 ± 1.23a,c
90.85 ± 4.61
88.45 ± 4.59
92.76 ± 4.56a
85.64 ± 5.86a
Insulin
(µU/mL)
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
Tissues (liver and kidney) were collected and stored at 4 ºC for the measurement of various parameters.
Biochemical estimations
Glucose was estimated by the method of Trinder using a reagent kit (Trinder.
1969) [7]. The insulin in the rat plasma was measured by the method of Burgi
et al. (1988) [8]. Total lipids were extracted from the liver and kidney tissues
according to the method of Folch et al., [9], Total cholesterol was estimated
by the method of Allain et al., [10]. HDL-C was estimated by the method of
Izzo et al.,[11]. Atherogenic index was calculated by the method of Malspina
et al.,[12]. VLDL-C and LDL-C were calculated by the method of Friedewald
et al.,[13]. Triglycerides were estimated by the method of McGowan et al.,[14]
. Free fatty acid content was estimated by the method of Falholt et al.,[15],
Phospholipid was estimated by the method of Silversmit and Davis [16].
Statistical analysis
Values are given as means ± S.D. for six rats in each group. Data were
analyzed by one-way analysis of variance followed by Duncan’s Multiple
Range Test (DMRT) using SPSS version 10 (SPSS, Chicago, IL). The limit of
statistical significance was set at P = 0.05.
RESULTS
Table 1 shows the effect of oral administration of biochanin A on the level of
plasma glucose and insulin in control and STZ-diabetic rats. Diabetic rats
showed increased plasma glucose and decreased insulin level. Administration of biochanin A to diabetic rats significantly decreased the plasma glucose and increased the insulin level.
Table 2 shows that the TC, TG, VLDL-C, LDL-C and atherogenic index
increased and HDL-C decreased in diabetic rats, and treatment with biochanin
A shifted all these parameters toward normal levels.
Table 3 shows the effect of biochanin A on free fatty acids and phospholipids in control and STZ-induced diabetic rats. Diabetic rats showed increased
level of FFA and PL, and treatment with biochanin A significantly decreased
the levels of FFA and PL.
Tables 4, 5 and 6 show the effect of biochanin A on total cholesterol, triglycerides, free fatty acids and phospholipids in the liver, kidney and heart on
control and STZ-diabetic rats. Diabetic rats showed increased TC, TG, FFA
and PL levels and treatment with biochanin A brought these changes to
normal levels.
Table 2. Effect of Biochanin A on total cholesterol, triglycerides, high density lipoprotein-C, very low density lipoprotein-C, low density
lipoprotein-C and atherogenic index in the plasma of STZ-diabetic rats
Groups
TC
(mg/dL)
TG
(mg/dL)
HDL-C
(mg/dL)
VLDL-C
(mg/dL)
LDL-C
(mg/dL)
Atherogenic
Index
(TC/HDL-C)
Control
Control + Biochanin
A(10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A
(10 mg/kg BW)
Diabetic + glibenclamide
(600 µg/kg BW)
79.27±3.46
74.39 ± 2.76 a
60.17 ± 2.75a
58.53 ± 2.62a
49.17 ± 2.35a
47.24 ± 2.62a
12.03 ± 0.55a
11.70 ± 0.52a
18.06 ± 0.55a
15.44 ± 0.38a
1.61 ± 0.01 a
1.57 ± 0.02 a
155.07 ± 11.08b
92.42 ± 4.88 c
159.27 ± 10.43b
79.46 ± 4.56c
24.89 ± 1.29b
41.82 ± 2.73c
31.56 ± 2.07b
15.89 ± 0.91c
98.38 ± 7.75b
34.70 ± 1.23c
6.22 ± 0.12 b
2.22 ± 0.04 c
84.28 ± 3.73 d
70.24 ± 3.55d
44.19 ± 3.46c,a
14.04 ± 0.70d
26.05 ± 0.43d
1.90 ± 0.06 d
a,d
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
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Table 3. Effect of Biochanin A on free fatty acids and phospholipids in
the plasma of STZ-diabetic rats
Groups
FFA
(mg/dL)
PL
(mg/dL)
Control
Control + Biochanin A (10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A (10 mg/kg BW)
Diabetic + glibenclamide (600 µg/kg BW)
60.57 ± 5.23a
58.12 ± 5.14a
113.14 ± 10.26b
70.21 ± 6.84c
63.45 ± 5.26a
81.54 ± 7.54a
80.29 ± 6.23a
149.68 ± 11.15b
101.28 ± 9.47c
99.84 ± 7.72d
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
Table 4. Effect of Biochanin A on total cholesterol, triglycerides, free
fatty acids and phospholipids in the liver of streptozotocin-diabetic
rats
Groups
(mg/g wet tissue)
TG
FFA
TC
Control
Control + Biochanin A
(10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A
(10 mg/kg BW)
Diabetic + glibenclamide
(600 µg/kg BW)
a
a
PL
a
4.28 ± 0.31
3.74 ± 0.16a
3.52 ± 0.30
3.49 ± 0.32ad
7.92 ± 0.45 22.12 ± 1.58a
7.78 ± 0.59a 22.74 ± 1.82a
6.47 ± 0.49b
4.92 ± 0.38c
7.82 ± 0.47b
4.88 ± 0.47c
13.54 ± 0.81b 47.27 ± 4.16b
9.44 ± 0.83c 29.52 ± 1.70c
4.52 ± 0.33a
4.42 ± 0.43d
8.92 ± 0.70ad
27.73 ± 2.19c
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
Table 5. Effect of Biochanin A on total cholesterol, triglycerides, free
fatty acids and phospholipids in the kidney of STZ-diabetic rats
Groups
( mg/g wet tissue )
TG
FFA
TC
Control
Control + Biochanin A
(10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A
(10 mg/kg BW)
Diabetic + glibenclamide
a
a
PL
a
4.83 ± 0.27
4.72 ± 0.34a
3.79 ± 0.38
3.54 ± 0.27a
4.53 ± 0.38
4.44 ± 0.32a
17.12 ± 1.49a
17.27 ± 1.39a
7.24 ± 0.49b
5.73 ± 0.29a
7.24 ± 0.52b
4.64 ± 0.43c
10.78 ± 0.58b
6.16 ± 0.25c
34.32 ± 2.93b
22.34 ± 1.97c
4.24 ± 0.41c
4.24 ± 0.25c
5.42 ± 0.40d
21.23 ± 1.72c
(600 µg/kg BW)
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
Table 6. Effect of Biochanin A on total cholesterol, triglycerides, free
fatty acids and phospholipids in the heart of STZ-diabetic rats
Groups
TC
Control
Control + Biochanin A
(10 mg/kg BW)
Diabetic control
Diabetic + Biochanin A
(10 mg/kg BW)
Diabetic + glibenclamide
(600µg/kg BW)
(mg/g wet tissue)
TG
FFA
PL
2.67 ± 0.19a,d 4.29 ± 0.25a
2.48 ± 0.18a 4.57 ± 0.29a,d
5.42 ± 0.35a
5.27 ± 0.32a
12.12 ± 1.12a
12.74 ± 0.82a
5.34 ± 0.29b 7.22 ± 0.40b
2.91 ± 0.13c 4.73 ± 0.39c
15.14 ± 1.43b
8.45 ± 0.23c
25.37 ± 1.87b
18.32 ± 1.24c
2.79 ± 0.13d 4.12 ± 0.32d
6.11 ± 0.32ad
14.53 ± 1.10e
Values are given as means ± S.D for six rats in each group.
Values not sharing a common superscript differ significantly at P< 0.05 (DMRT).
DISCUSSION
Streptozotocin is well known for its selective pancreatic islet ß-cell cytotoxicity and has been extensively used to induce diabetes mellitus in animals [17].
In the present study, we observed an increase in the level of plasma glucose
and decrease in the level of insulin in STZ-diabetic rats. The diabetic rats
treated with biochanin A decreases the plasma glucose and increases the
insulin level which might be due to elevated secretion of insulin from the
existing ß-cells, which, in turn, increases the utilization of glucose by the
tissue. However the standard drug glibenclamide showed a better reduction
of blood glucose that biochanin A.
Lipids play an important role in the pathogenesis of diabetes mellitus. Hyperlipidemia is a recognized consequence of diabetes mellitus demonstrated
by the elevated levels of tissue cholesterol, phospholipids and free fatty
acids [18,19]. Lowering the plasma lipid levels through dietary or drug therapy
appears to be associated with a decrease in the risk of vascular disease. STZdiabetic rats showed increase in the plasma cholesterol and triglyceride concentrations [20]. Further, the abnormal high concentration of serum lipids in
the diabetic subjects is mainly due to increase in the mobilization of free fatty
acids from fat depots. The marked hyperlipidemia that characterizes the
diabetic state may be regarded as a consequence of uninhibited actions of
lipolytic hormones on fat depots [21]. Insulin is required for the inhibition of
hormone sensitive lipase and on the other hand, glucagons and other hormones enhance lipolysis. Under normal circumstances, insulin activates the
enzyme lipoprotein lipase, which hydrolyses triglycerides [22]. However, in
diabetic state lipoprotein lipase is not activated due to insulin deficiency
resulting in hypertriglyceridemia. Diabetic rats treated with biochanin A and
glibenclamide significantly decreased TC and TG towards normal level which
could be due to an increase in insulin secretion which, in turn, inhibits hormone sensitive lipase and increases the utilization of glucose thereby decreasing the mobilization of free fatty acids from fat depots. Tauseef siddiqui
reported that two isoflavones, biochanin A and formononetin isolated from
gram Cicer arietinum, have been shown to possess hypolipidemic properties for Triton WR-1339 induced hyperlipidemia in male albino rats, when
administered as a crude extract or as individual compounds [23]. Isoflavones
have mechanism of action, that potentially involving in the regulation of
fatty acid metabolism via the nuclear receptor PPARa and PPAR?. It indicates that Biochanin A is more useful in the treatment of diabetes as it has
hypolipidemic effect. Moreover, its hypolipidemic effect could represent a
protective mechanism against the development of atherosclerosis, which is
usually associated with diabetes.
The concentration of LDL-C is one of the most important predictors of
atherosclerosis and coronary heart disease [24], and reduction in its level
reduces the morbidity and mortality of patients with CHD. Normally circulating LDL-C undergoes reuptake in the liver via specific receptors and gets
cleared from the circulation [25]. This increased LDL concentration in the
plasma of diabetic rats might be due to the defect in LDL-C receptor either
through failure in its production (or) function. HDL-C is protective by
reversing cholesterol transport, inhibiting the oxidation of LDL-C and by
neutralizing the atherogenic effects of oxidized LDL-C. A greater increase of
LDL-C and VLDL-C may also cause a greater decrease of HDL-C as there is
a reciprocal relationship between the concentration of VLDL-C and HDL-C.
Decreased HDL-C may also be due to diminished lecithin cholesterol acyl
transferase activity. In our study, the diabetic rats treated with biochanin A
showed an elevation in HDL-C and reduction in LDL-C and VLDL-C. Thus,
biochanin A could alleviate the risk of cardiovascular diseases.
Isoflavones prevent the formation of atherosclerosis by scavenging the reactive oxygen species and thereby preventing oxidative damage. Moreover it
inhibits the oxidation of low density lipoprotein, which plays a central role
in the formation of atherogenesis [26]. In our study atherogenic index was
increased in diabetic rats, and treatment with biochanin A showed decrease
which might be due to consumption of the isoflavone, biochanin A which is
associated with the protection against atherosclerosis.
Phospholipids are vital components of biomembrane and play an important
role in the transport of triglygerides [27]. In STZ-diabetic rats, the elevated
level of phospholipids may be due to the elevated levels of FFA and TC
Journal of Pharmacy Research Vol.5 Issue 1.January 2012
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R. Harini et al. / Journal of Pharmacy Research 2012,5(1),707-710
which can promote the synthesis of phospholipids.[28] Diabetic rats treated
with biochanin A showed a decrease in the FFA which in turn decreased the
phospholipids level. Thus, our findings demonstrate that biochanin A is
having good antihyperglycemic and antihyperlipidemic effects, which is evidenced by the decreased levels of glucose, TC, TG, LDL-C, VLDL-C, FFA
and PL, and elevated level of insulin, HDL-C in diabetic rats.
In conclusion, our study shows that biochanin A is having antihyperglycemic,
antihyperlipidemic and antiatherogenic effect on STZ induced diabetic rats.
The effect of biochanin A is comparable with standard drug, glibenclamide.
Though the dosage of biochanin A is higher than the standard drug, due to the
nontoxic nature of biochanin A, a study on the combined therapy of biochanin
A and glibenclamide would be beneficial. Further study on the metabolism
and pharmacokinetics of biochanin A is warranted.
ACKNOWLEDGMENT
The financial assistance to Ms. R. Harini as Senior Research Fellowship
from the Indian Council of Medical Research, New Delhi is gratefully acknowledged.
11.
12.
13.
14.
15.
16.
17.
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Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.5 Issue 1.January 2012
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