Life Sciences 79 (2006) 1578 1584

www.elsevier.com/locate/lifescie

Effects of soy protein and genistein on blood glucose, antioxidant enzyme

activities, and lipid profile in streptozotocin-induced diabetic rats
Jeong-Sook Lee
Department of Food and Nutrition, Kosin University, Busan, 606-701, South Korea
Received 18 May 2006; accepted 19 June 2006

Abstract
In the current study, the effect of soy protein and genistein, one of the main isoflavones in soybeans, on blood glucose, lipid profile, and
antioxidant enzyme activities in streptozotocin (STZ)-induced diabetic rats was investigated. Male SpragueDawley rats were divided into
nondiabetic control, STZ, STZ-genistein supplemented group (STZ-G; 600 mg/kg diet), and STZ-isolated soy protein supplemented group (STZISP; 200 g/kg diet). Diabetes was induced by a single injection of STZ (50 mg/kg BW) freshly dissolved in 0.1 mol/L citrate buffer (pH 4.5) into
the intraperitonium. Diabetes was confirmed by measuring the fasting blood glucose concentration 48-h post-injection. The rats with blood
glucose level above 350 mg/dL were considered to be diabetic. Genistein and ISP were supplemented in the diet for 3 weeks.
The supplementation of genistein and ISP increased the plasma insulin level but decreased the HbAIC level of the STZ-induced diabetic rats.
The supplementation of genistein and ISP increased the glucokinase level of the STZ-induced diabetic rats. A significant reduction in glucose-6phosphatase was observed in the groups treated with genistein and ISP in comparison with the diabetic control group. Hepatic superoxide
dismutase, catalase, and glutathione peroxidase activities of the STZ-induced diabetic rats were significantly decreased in comparison with the
control rats. Administering genistein and ISP to the STZ-induced diabetic rats significantly increased those enzyme activities. The concentration of
thiobarbituric acid reactive substances in the STZ-induced diabetic rats was significantly elevated, while the genistein and ISP supplement
decreased it to the control concentration. Genistein and ISP supplements seem to be beneficial for correcting the hyperglycemia and preventing
diabetic complications.
2006 Elsevier Inc. All rights reserved.
Keywords: Soy protein; Genistein; Streptozotocin-induced diabetic rats; Antioxidant enzymes

Introduction
Diabetes mellitus is a major endocrine disorder and growing
health problem in most countries (Gavard et al., 1993; Anderson
et al., 2001). Recently, it was suggested that formation of free
radicals is involved in the pathogenesis of diabetes and the
development of diabetic complications because prolonged
exposure to hyperglycemia increases the generation of free
radicals and reduces capacities of the antioxidant defense system
(Sanders et al., 2001). In spite of the presentation of many
hypoglycemic agents, diabetes and its related complications are
still a major medical problem.

Tel.: +82 51 990 2328; fax: +82 51 403 3760.

E-mail address: jslee@Kosin.ac.kr.
0024-3205/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.06.030

The interest in the potential health effects of soy and soy

isoflavones is growing as epidemiological studies have associated
with a diet rich in isoflavones with a lower risk of certain diseases
(Anderson et al., 1995; Potter, 1998; Hermansen et al., 2001). Soy
intake has been linked to the improved blood lipid levels in
humans and animals and decreased arterial fatty streaks in animals, therefore reducing the risk of developing atherosclerosis
(Adams et al., 2002; Lichtenstein, 2001; Iqbal et al., 2002).
Recently, isoflavones as an important bio-active component of
soy also have been investigated (Adams et al., 2002; Clarkson,
2002; Nestel, 2002).
Considerable research efforts have focused on isoflavones as
the main hypolipidemic agent in soy because of their antioxidative and mild estrogenic activity (Anthony, 2000; Polkowski and Mazurek, 2000; Wilson et al., 2002). Some studies
have shown that removal of the isoflavone-containing fraction of

J.-S. Lee / Life Sciences 79 (2006) 15781584

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soy protein results in the loss of soy's beneficial effect on blood

lipids (Anthony et al., 1998; Crouse et al., 1999). It remains a
possibility that soy has a positive and direct effect on the management of diabetes by some yet-unrecognized mechanism.
However, the interaction between soy protein and isoflavone and
diabetic complications is little known. In this study, the possible
anti-diabetic effects of soy protein and genistein, one of the main
isoflavones in soybeans, in streptozotocin-induced diabetic rats
have been evaluated.

The food consumption and weight gain were measured

daily and weekly, respectively. At the end of the experimental
period (3 weeks), the rats were anesthetized with ether following a 16-h fast. Blood samples were taken from the abdominal aorta using heparin-coated syringes for plasma and
regular syringes for serum. Plasma and serum were obtained
by centrifuging the blood at 3000 rpm for 15 min at 4 C. The
livers were removed and rinsed with physiological saline. All
samples were stored at 70 C until analyzed.

Material and methods

Glucose tolerance test

Animals and diets

After 18 days of treatment, a fasting blood sample was taken

from all the groups of rats. Four more blood samples were
collected at 30-, 60-, 90-, and 120-min intervals after administration of glucose at a concentration of 2 g/kg of body weight
(Joy and Kuttan, 1999). All the blood samples were collected
with potassium oxalate and sodium fluoride solution for the
estimation of glucose.

At the beginning of the experiment, Male SpragueDawley

rats weighing between 80 and 90 g were purchased from Daehan
Laboratory Animal Research Center (Daegu, Korea). The animals were all individually housed in stainless steel cages in an
air-conditioned room with controlled temperature (2022 C)
and automatic lighting (alternation 12-h periods of light and
dark) and fed an AIN-93 (Reeves et al., 1993) standard
laboratory diet for 21 days after arrival. The animals were divided into two groups: a nondiabetic control and a diabetic
group. Diabetes was induced by a single injection of STZ
(50 mg/kg BW; Sigma, USA) freshly dissolved in a 0.1 mol/L
citrate buffer (pH 4.5) into the intraperitonium. The control rats
were only injected with the citrate buffer. Diabetes was confirmed in the STZ-treated rats by measuring the fasting blood
glucose concentration 48-h post-injection. The rats with blood
glucose level above 350 mg/dL were considered to be diabetic
and were used in the experiment. The diabetic rats were
randomly divided into three sub-groups, diabetic controls (STZ),
diabetic rats given genistein (STZ-G; 600 mg/kg diet), and
diabetic rats given isolated soy protein (STZ-ISP; 200 g/kg diet).
In this study, 32 rats were used (eight control and 24 diabetic).
The composition of the experimental diet, as shown in Table 1,
was based on the AIN-93 standard laboratory diet. The rats were
given free access to food and distilled water, and all animals were
observed daily for any clinical signs of disease.
Table 1
Composition of control and experimental diet (g/100 g diet)
Control

STZ-G

STZ-ISP

Caseina
Isolated soy proteinb
Cornstarch
Sucrose
Corn oil
Cellulose
Mineral mixturec
Vitamin mixtured
Choline bitartrate
DL-methione
Genistein

20.0

55.0
10.0
5.0
5.0
3.5
1.0
0.2
0.3

20.0

54.94
10.0
5.0
5.0
3.5
1.0
0.2
0.3
0.06

20.0
55.0
10.0
5.0
5.0
3.5
1.0
0.2
0.3

b
c
d

The livers were homogenized in 20 parts (w/v) of a 0.25 mol/L

sucrose solution using a tissue homogenizer with a Teflon pestle
at 4 C. The homogenate was centrifuged at 600 g for 10 min to
discard any cell debris, then the supernatant was further centrifuged at 10,000 g for 20 min to remove the mitochondria pellet.
Finally, the supernatant was further ultracentrifuged at 105,000 g
for 60 min to obtain the cytosol supernatant. The amounts of
protein in the mitochondrial and cytosolic fractions were
measured using the method of Lowry et al. (1951) with bovine
serum albumin as the standard.
Plasma insulin and glycosylated hemoglobin levels
Plasma insulin was determined by using a rat insulin
radioimmunoassay kit (Linco Research Inc., St. Charles, USA)
in a gamma counter (Peckard, USA) based on the method of
Andersen et al. (1993). The glycosylated hemoglobin (HbAIC)
was determined using a commercial kit (Roche Co., Basel,
Switzland) based on the method of Goldstein et al. (1986).
Glucokinase and glucose-6-phosphatase activities

Component

Tissue preparations

Casein (ICN Biomedicals, Costa Mesa, USA).

Soy protein isolates (Protein Technologies International, St. Louis, USA).
AIN-93 mineral mixture.
AIN-93 vitamin mixture.

The glucokinase activity was determined using the method of

Davidson and Arion (1987). The activity of glucose-6-phosphatase was measured using the method of Alegre et al. (1988).
Serum and hepatic lipids
The serum total cholesterol level was determined using a
commercial kit (Sigma, USA) based on a modification of the
cholesterol oxidase method of Allain et al. (1974). The HDLfractions were separated using a Sigma kit based on the heparinmanganese precipitation procedure (Warnick and Albers, 1978),
and the HDL-cholesterol concentration was determined using the
same enzymatic method. The serum triglyceride concentration

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J.-S. Lee / Life Sciences 79 (2006) 15781584

was measured enzymatically using a kit from Sigma Chemical

Co., a modification of the lipase-glycerol phosphate oxidase
method (McGrowan et al., 1983). The hepatic lipids were
extracted using the procedure of Folch et al. (1957). The dried
lipid residues were dissolved in 1 mL ethanol for cholesterol and
triglycerides assays. Triton X-100 and sodium cholate solutions
(in distilled H2O) were added to 200 L of the dissolved lipid
solution to produce final concentrations of 5 g/L and 3 mmol/L,
respectively. The hepatic cholesterol and the triglyceride were
analyzed with the same enzymatic kit used in the plasma analysis.
Antioxidant enzyme activities and TBARS concentration
The hepatic superoxide dismutase (SOD) activity was determined using Marklund and Marklund's method (Marklund
and Marklund, 1974). The hepatic catalase (CAT) activity was
measured using Abei's method (Abei, 1984). The activity of
hepatic glutathione peroxidase (GSH-Px) was measured using
Paglia and Valentine's method (Paglia and Valentine, 1967). The
hepatic thiobarbituric acid reactive substances (TBARS) were
monitored according to the method of Tarladgis et al. (1964).

Fig. 2. Plasma insulin level in experimental groups (n = 8). The insulin values
(mean SD) are expressed as ng/mL. The means sharing a common letter are not
significantly different ( p < 0.05).

multiple range test. Statistical significance was considered at

p < 0.05.

Serum aminotransferase activity and plasma urea level

Results
The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined using a commercial kit (Eiken Co., Tokyo, Japan) based on the method of Reitman
and Frankel (1957). Plasma concentration of urea was determined
by the methods of Patton and Crouch (1977).
Statistical analyses
All data are presented as the mean SE. The data were evaluated by a one-way ANOVA using the SPSS program, and the
differences between the means assessed using Duncan's

Fig. 1. Oral glucose tolerance test in experimental groups (n = 8). The blood
glucose levels (mean SD) are expressed as mg/dL. The means sharing a
common letter are not significantly different ( p < 0.05).

Blood glucose, plasma insulin and glycosylated hemoglobin

levels
Fig. 1 shows the blood glucose levels of control and
experimental groups of rats after oral administration of glucose.
The blood glucose level in the control rats rose to a peak value
30 min after glucose load and decreased to near normal levels at
120 min. In diabetic control rats, the peak increase in blood
glucose concentration was observed after 30 min and remained
high over the 90 min. Genistein and ISP treated diabetic rats
showed significant decreases in blood glucose level at 30 and
120 min compared with diabetic rats.

Fig. 3. Glacosylated hemoglobin level in experimental groups (n = 8). The glycosylated hemoglobin values (mean SD) are expressed as mg/g of hemoglobin.
The means sharing a common letter are not significantly different ( p < 0.05).

J.-S. Lee / Life Sciences 79 (2006) 15781584

Table 2
Effect of genistein and isolated soy protein on hepatic glucokinase and glucose6-phosphatase activities in diabetic rats
Control
1

STZ

STZ-G

Control
b

Glucokinase
0.261 0.023 0.079 0.009 0.103 0.012 0.133 0.012
Glucose-60.165 0.011d 0.457 0.014a 0.396 0.022b 0.302 0.013c
2
phosphatase

Values are mean SD of 8 rats from each group.

Means in the same row not sharing a common superscript are significantly
different ( p < 0.05) between groups.
1
Glucokinase: units/h/mg of protein.
2
Glucose-6-phosphatase: units/min/mg of protein.
a,b,c

Plasma insulin levels were lower in the STZ-induced diabetic

rats compared with the control rats (Fig. 2). The supplementation
of genistein and ISP increased the plasma insulin level of the STZinduced diabetic rats. The effect was more pronounced in the ISP
supplemented rats than in the genistein-supplemented rats.
HbAIC levels were higher in the STZ-induced diabetic rats
compared to the control rats (Fig. 3). The supplementation of
genistein and ISP decreased the HbAIC level of the STZinduced diabetic rats. The effects were more potent in the STZISP group than in the STZ-G group.
Glucokinase and glucose-6-phosphatase activities
Table 2 shows the effect of genistein and ISP on glucokinase
and glucose-6-phosphatase activities of normal and STZ diabetic
rats. The glucokinase activity was lower in the STZ-induced
diabetic rats compared with the control rats. The supplementation
of genistein and ISP increased the glucokinase level of the STZinduced diabetic rats. The glucose-6-phosphatase level was increased in diabetic rats. A significant reduction in glucose-6phosphatase was observed in the groups treated with genistein and
ISP compared with the diabetic control group. The effects were
more potent in the STZ-ISP group than in the STZ-G group.
Serum and hepatic lipids
The total cholesterol and triglyceride concentrations in the
serum and liver were significantly higher in the STZ-induced
Table 3
Effect of genistein and isolated soy protein on serum and hepatic lipids in
diabetic rats
Control

STZ

STZ-G

STZ-ISP

Serum
Total cholesterol
2.40 0.16b 3.62 0.20a 2.61 0.09b 2.55 0.08b
(mmol/L)
HDL-cholesterol
1.22 0.05a 0.42 0.05c 0.65 0.06b 0.63 0.06b
(mmol/L)
Triglyceride (mmol/L) 1.11 0.09b 2.03 0.16a 1.16 0.09b 1.15 0.13b
Liver
Cholesterol (mmol/g) 0.13 0.02b 0.23 0.04a 0.16 0.03b 0.15 0.03b
Triglyceride (mmol/g) 0.24 0.02b 0.45 0.05a 0.28 0.03b 0.26 0.03b
Values are mean SD of 8 rats from each group.
Means in the same row not sharing a common superscript are significantly
different ( p < 0.05) between groups.

a,b,c

Table 4
Effect of genistein and isolated soy protein on hepatic SOD, CAT, GSH-Px
activities and TBARS concentration in diabetic rats

STZ-ISP
c

1581

SOD
CAT2
GSH-Px3
TBARS4

STZ
a

STZ-G
c

12.79 0.75
18.60 1.13a
3.01 0.46a
15.12 0.14b

STZ-ISP
b

6.50 0.54
11.06 0.91d
1.23 0.46c
29.53 2.12a

11.00 1.12b
14.30 1.19b
2.27 0.77b
17.91 1.23b

10.52 1.06
12.41 1.13c
2.16 0.46b
19.24 1.06b

Values are mean SD of 8 rats from each group.

Means in the same row not sharing a common superscript are significantly
different ( p < 0.05) between groups.
1
Superoxide dismutase: units/mg protein.
2
Catalase: deceased H2O2 nmol/min/mg protein.
3
Glutathione peroxidase: oxidized NADPH nmol/min/mg protein.
4
Thiobarbituric acid reactive substances: nmol/g.
a,b,c

diabetic rats than in the control rats (Table 3). The supplementation of genistein and ISP suppressed the increase in the total
cholesterol and triglyceride levels in the serum and liver of the
diabetic rats. The genistein and ISP supplementation decreased in
the serum and hepatic triglyceride levels significantly to almost
the control concentration. The HDL-cholesterol concentration
was also significantly lowered by the induction of diabetes;
however it was higher in the genistein and ISP supplemented
groups compared to the other diabetic group.
Antioxidant enzyme activities and TBARS concentration
The activities of SOD, CAT, GSH-Px and hepatic TBARS
concentration are given in Table 4. SOD and GSH-Px activities
in the liver of the STZ-induced diabetic rats were significantly
decreased compared to the control rats. Administering genistein
and ISP to the STZ-induced diabetic rats significantly increased
those enzyme activities. CAT activity of the rats treated with STZ
was significantly decreased, while genistein and ISP supplement
to the STZ-induced diabetic rats appeared to increase its activity.
The effect was more pronounced in the ISP supplemented group
than in the genistein-supplemented group.
The concentration of TBARS in the STZ-induced diabetic
rats was significantly elevated, the genistein and ISP supplement
decreased it significantly to almost the control concentration.
Serum aminotransferase activity and plasma urea level
The activities of serum AST and ALT and plasma urea level
of control and experimental animals are given in Table 5.
Supplement with the genistein and ISP along with STZ showed
Table 5
Effect of genistein and isolated soy protein on serum ALT and AST activities and
plasma urea level in diabetic rats
Control
ALT (unit/mL)
AST (unit/mL)
Urea (mg/dL)

STZ
c

35.55 1.62
70.60 2.13c
32.62 1.52c

STZ-G
a

63.88 1.90
101.66 3.06a
61.60 2.52a

STZ-ISP
b

42.09 1.06
82.19 1.56b
43.14 1.36b

43.19 1.06b
80.34 1.69b
41.06 2.02b

Values are mean SD of 8 rats from each group.

Means in the same row not sharing a common superscript are significantly
different ( p < 0.05) between groups.

a,b,c

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J.-S. Lee / Life Sciences 79 (2006) 15781584

significantly reduced levels of AST and ALT as compared with

those of the unsupplemented STZ-treated rats. The STZ-induced
diabetic group resulted in a significant increase in plasma urea
concentration compared to the control group; Supplementation
of genistein and ISP decreased the level of urea as compared
with that of the STZ-treated group.
Discussion
Diabetes arises from destruction of the -islet cells of the
pancreas, due to degranulation or reduction of insulin secretion
(Ramkumar et al., 2004). The elevation in plasma insulin in the
genistein and ISP-treated STZ-diabetic rats could be due to the
insulinotropic substances present in the fractions, which induce
the intact functional -cells of the Langerhans islet to produce
insulin, or the protection of the functional -cells from further
deterioration so that they remain active and produce insulin.
Diabetic rats induced by STZ show an increased sensitivity to
oxygen free radicals and hydrogen peroxide, the breakdown
products of the liver, which impose oxidative stress in diabetes
and would damage inner endothelial tissue; this would eventually be directly responsible for high blood glucose (Reddi and
Bollineni, 2001). The experimentally induced diabetes significantly (p < 0.05) increased the fasting blood glucose level by
148% of the control level. However, the treatment of STZinduced diabetic rats with the genistein and ISP reduced their
blood glucose levels by 19.6% and 24.9%, respectively, in
comparison to the diabetic group. The genistein and ISP supplementations improved glucose tolerance in diabetic rats. It
seems that the hypoglycemic effects of genistein and ISP are
due to the increased level of serum insulin and the enhancement
of peripheral metabolism of glucose (Skim et al., 1999).
Supplement with ISP was found to increase the plasma insulin
level more effectively than genistein. It seems that ISP contains
one or more constituents that could increase the bioavailability
of genistein. Further study will be necessary to elucidate the
mechanism of blood glucose regulation by genistein and ISP.
HbAIC was found to increase in diabetic rats up to 176%.
And this increase is directly proportional to the fasting blood
glucose levels (Saravanan and Pugalendi, 2005). Since the
diabetic animals had prior higher blood glucose levels, elevated
levels of HbAIC were observed. Genistein and ISP fed diabetic
rats showed the decrease in HbAIC level, which seems to be the
reduction of blood glucose level.
Insulin increases hepatic glycolysis by increasing the activity
and amount of several key enzymes including glucokinase, phosphofructokinase and pyruvate kinase. In the liver, glucokinase is
an important regulator of glucose storage and disposal (Saravanan
and Pugalendi, 2005). In this study, glucokinase activity was
decreased in the liver of diabetic fats which may be due to a
deficiency of insulin. Genistein and ISP fed diabetic rat showed an
elevated activity of glucokinase, which may be associated with
reduced blood glucose. Therefore, the ISP appears to be more
potent than the genistein.
Insulin decreases gluconeogenesis by decreasing the activities
of key enzymes such as glucose-6-phosphatase, fructose 1,6bisphosphatase, phosphoenolpyruvate carboxykinase and pyru-

vate carboxykinase (Murray et al., 2000). Glucose-6-phosphatase

plays an important role in glucose homeostasis in liver and kidney
(Berg et al., 2002). In this study, the increased activity of glucose6-phosphatase in liver of diabetic rats seems to be insulin deficiency. In genistein and ISP fed diabetic rats, the activity of that
enzyme was significantly reduced, which may be responsible for
the improved glycemic control. The ISP seems to be more potent
than the genistein, and contains other active constituents that
could potentiate the effect.
Hyperglycemia also generates reactive oxygen species which
in turn cause lipid peroxidation and membrane damage in this
study (Hunt et al., 1988). In the current study, the concentration
of hepatic TBARS was significantly increased after treatment of
STZ in the rats. The increased concentration of TBARS suggests
that an increase in oxygen free radicals could be due to either
their increased production or decreased destruction (Kakkar
et al., 1995). The level of hepatic TBARS in genistein and ISP
supplemented rats showed a significant reduction, which indicates a decreased rate of lipid peroxidation. Several studies have
shown increased lipid peroxidation in clinical and experimental
diabetes (Sundaram et al., 1996; Kakkar et al., 1998). The
present results show increased lipid peroxidation in tissues of the
diabetic group. The increase of oxygen free radicals in diabetes
could be due to an increase of blood glucose level, since an
autoxidation generates free radicals (Ivorra et al., 1989). STZ has
been shown to produce oxygen free radicals. Lipid peroxidemediated tissue damages have been observed in the development
of type I and type II mellitus. Previous studies have reported that
lipid peroxidation in liver, kidney, and brain of diabetic rats was
increased (Latha and Pari, 2003; Venkateswaran and Pari, 2002).
Concerning to the changes in lipid peroxidation, the diabetic
tissue showed decreased activity of the key antioxidants SOD,
CAT, glutathione, GPx, and glutathione S-transferase, which play
an important role in scavenging the toxic intermediate of incomplete oxidation. The decrease in the activity of these antioxidants can lead to an excess availability of the superoxide anion
(O2) and hydrogen peroxide in biological systems, which in turn
generate hydroxyl radicals resulting in initiation and propagation
of lipid peroxidation. Administration of genistein and ISP
increased the activity of enzymes and may help to control free
radicals (Kumuhekar and Katyane, 1992).
The induction of SOD activity by genistein and ISP may be
attributed to inhibition of the generation of active oxygen species from autoxidation of glucose generation from the action of
STZ. The increased activity of SOD accelerates dismutation of
superoxide radicals to H2O2, which is removed by CAT. This
indicates that the genistein and ISP supplements have altered the
SOD, CAT, and GSH-Px activities and reduced oxidative stress
in the diabetic rats, resulting in a lower TBARS concentration.
The diabetic hyperglycemia induces the elevation of plasma
levels of urea and creatinine which are considered as significant markers of renal dysfunction (Almdal and Vilstrup,
1988). In this study plasma urea in the diabetic groups increased by 88.8% of the control level. While after the supplement of genistein and ISP to the diabetic rats, the level of urea
was significantly ( p < 0.05) decreased in plasma by 30.0% and
32.5%, respectively, compared with the diabetic group. This

J.-S. Lee / Life Sciences 79 (2006) 15781584

result indicates that genistein and ISP are capable of ameliorating the impaired diabetic kidney function in addition to its
hypoglycemic control.
Increased activities of AST and ALT are used as indices of
liver damage. When the liver is damaged, these enzymes leak out
of liver cells in large quantities, so their concentrations in the
blood are increased (Tawta et al., 2000). Possible explanation for
the differential effects of genistein and ISP on the activities of
AST and ALT in plasma is that genistein and ISP may inhibit the
liver damage induced by STZ.
Genistein and ISP would appear to contribute to alleviating
the adverse effect of diabetes mellitus by enhancing the lipid
metabolism as well as the hepatic antioxidant defense system.
Genistein and ISP supplements may be beneficial for correcting
the hyperglycemia and preventing diabetic complications. The
ISP appears to be more potent than the genistein. Hence further
biochemical and pharmacological studies are being carried out
to elucidate their mechanism of action.
Acknowledgements
The author is grateful for the financial support provided by
the foundation of Kosin University.
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