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Shairibha and Rajadurai eISSN 2278-1145
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International Journal of Integrative sciences, Innovation and Technology (A Peer Review E-3 Journal of Science Innovation Technology)
Section A – Basic Sciences; Section B –Applied and Technological Sciences; Section C – Allied Sciences
Available online at www.ijiit.net
Research Article
ANTI-DIABETIC EFFECT OF p-COUMARIC ACID ON LIPID PEROXIDATION,
ANTIOXIDANT STATUS AND HISTOPATHOLOGICAL EXAMINATIONS IN
STREPTOZOTOCIN-INDUCED DIABETIC RATS
S. M. REXLIN SHAIRIBHA1, M. RAJADURAI*
1Research Scholar, Department of Biochemistry, Muthayammal College of Arts & Science, Rasipuram-637 408, Namakkal.
*Principal, Sri Venkateshwara College of Arts & Science, Dharmapuri- 636 809, Tamil Nadu, India.
*Corresponding Author email: [email protected]
ABSTRACT Diabetes Mellitus is a chronic widely spread metabolic disorder. Chronic hyperglycemia in diabetes is associated with
oxidative stress mediated tissue damage. The present study was designed to investigate the effect of p-coumaric acid
on lipid peroxidative products (thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxide), antioxidant
status and histopathological examinations in streptozotocin (STZ)-induced diabetic rats. STZ induction (45 mg/kg/i.p)
caused a hyperglycemic state that lead to various physiological and biochemical alterations. Albino Wistar rats were
divided in to 4 groups, p-coumaric acid (100 mg/kg/day) was dissolved in 10% propylene glycol and administered to
rats for 45 days. Diabetic rats had elevated levels of TBARS and hydroperoxide and decreased antioxidant activities
(both enzymatic and non-enzymatic) when compared with normal control rats. Treatment with p-coumaric acid shows
the decreased level of lipid peroxides and increased levels of antioxidants in STZ-induced diabetic rats. These results
demonstrated that p-coumaric acid has strong antidiabetic effects thus; it may have beneficial properties in the
prevention of diabetes. KEY WORDS: Hyperglycemia, Lipid peroxidation, Antioxidant, Streptozotocin, p-Coumaric acid.
INTRODUCTION Diabetes mellitus (DM) is a widespread disease that is
associated with high morbidity and health care costs
(Mednieks, et al., 2009). DM is a generalized
common chronic metabolic disorder characterized by
hyperglycemia resulting from defects in insulin
secretion and/or action (Mitrovic, et al., 2006; Unger
and Parkin, 2010) and certain abnormalities in
carbohydrate, fat, electrolyte, and protein metabolism
(Kumar and Clark, 2002). Prolonged periods of
hyperglycemia lead to glucose degradation within
living tissues, causing an accumulation of glucose
within organ cells, which result in various micro and
macrovascular complications of DM such as coronary
heart disease, retinopathy, nephropathies, and
neuropathies (Chaiyavat, et al., 2010). World Health
Organization (WHO) estimates that more than 220
million people worldwide have diabetes and this
number is likely to double by 2030 (Aragao, et al.,
2010). According to the Diabetes atlas 2009, India has
the largest number of people with diabetes in the
world, with an estimated 50.8 million people diabetic
and this may rise to 87 million by the year 2030 (IDA,
2009).
Streptozotocin (STZ), an antibiotic produced by
Streptomyces achromogenes, is frequently used to
induce DM in experimental animals through its toxic
effects on pancreatic β-cells (Kim, et al., 2003). It is
taken into the pancreatic β-cells by glucose transporter
2 (GLUT-2) (Vessal, et al., 2003). The cytotoxic
action of STZ is associated with the generation of
ROS causing oxidative damage (Szkudelski, 2001).
Substances with antioxidant properties and free
radical scavenging properties may help in the
regeneration of β-cells and protect the pancreatic islets
against the cytotoxic effects of STZ (Coskun, et al.,
2005). Oxidative stress caused by the production of free
radical is a recognized participant in the development
and progression of diabetes and its complications
(Ceriello, 2000). These free radicals also destroy
pancreatic β-cells that produce and secrete insulin
(Laybutt, et al., 2002). Most of the diabetic
complications are due to generation of reactive
oxygen species (ROS), which lead to endothelium
dysfunction (Potenza, et al., 2009). There are many
protective enzymes against ROS, such as superoxide
dismutase (SOD), glutathione peroxidase (GPx) and
catalase (Soto, et al., 2003).
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The pharmacological agents currently used for the
treatment of type 2 diabetes include sulfonylurea,
biguanide, thiazolidinedione and α-glycosidase
inhibitors. These agents, however, have restricted
usage due to several undesirable side effects and fail
to significantly alter the course of diabetic
complications (Rang and Dale, 1991). The high
prevalence of diabetes as well as its long term
complications has led to an ongoing search for
hypoglycemic agents from natural sources (Nicasio, et
al., 2005). Herbal remedies have been used since
ancient times for the treatment of diabetes mellitus.
About 90% of the world population in rural areas of
developing countries relies solely on traditional
medicines for their primary health care (Hassan, et al.,
2010). p-Coumaric acid (4-hydroxyphenyl-2-propenoic acid)
is a phenolic acid widely distributed in plants and
form a part of human diet (Fig 1) (Scalbert and
Williamson, 2000). The main dietary phenolic acids
sources are fruits and beverages such as tea, coffee,
wine, chocolate, and beer (King and Young, 1999).
The mechanisms of antioxidant effect of phenolics
include binding of metal ions, scavenging of reactive
oxygen species (ROS); reactive nitrogen species
(RNS); or their precursors, up-regulation of
endogenous antioxidant enzymes, or the repair of
oxidative damage to biomolecules (Ursini, et al.,
1999). The antioxidant activity of natural phenolic
acids depends on the number and relative position of
the hydroxyl groups on the ring, which give reducing
properties and hydrogen donating abilities (Rice-
Evans, et al., 1996). Recent interest in food phenolics
has increased owing to their roles as antioxidants and
scavengers of free radicals and their implication in the
prevention of many pathologic diseases such as
cardiovascular disease (Ursini, et al., 1999) and
certain types of cancer (Hudson, et al., 2000). The aim
of the present study is to investigate the anti-diabetic
effect of p-coumaric acid on lipid peroxidation and
antioxidant status in STZ-induced diabetic rats.
MATERIALS AND METHODS Animals and diet Male albino Wistar rats (170-200 g) were obtained
from Venkateswara Enterprises, Bangalore and used
in this study. They were housed in polypropylene
cages (47×34×20 cm) lined with husk, renewed every
24 hours under a 12:12 hours light/dark cycle at
around 22˚C and had free access to tap water and
food. The rats were fed on a standard pellet diet
(Pranav Agro Industries Ltd., Maharashtra, India).
The pellet diet consisted of 22.02% crude protein,
4.25% crude oil, 3.02% crude fibre, 7.5% ash, 1.38%
sand silica, 0.8% calcium, 0.6% phosphorus, 2.46%
glucose, 1.8% vitamins and 56.17% nitrogen free
extract (carbohydrates). The diet provided
metabolisable energy of 3,600 kcal. The experiment
was carried out in according with the guidelines of the
Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA),
New Delhi, India and approved by the Institutional
Animal Ethics Committee (IAEC) of Muthaymmal
College of Arts & Science, Rasipuram
(MCAS/Ph.D.,/02/2012-2013).
Chemicals p-Coumaric acid, reduced nicotinamide adenine
dinucleotide (NADH), and phenazine methosulphate
(PMS) were purchased from Sigma Chemical
Company, St. Louis, MO, USA. Streptozotocin
(STZ), thiobarbituric acid (TBA), 1, 1’ ,3 , 3’
tetramethoxy propane, butylated hydroxy toluene
(BHT), xylenol orange, dithionitro bis benzoic acid
(DTNB), ascorbic acid, 2, 2’ dipyridyl, p-phenylene
diamine and sodium azide were obtained from
Himedia laboratory, Mumbai, India. All other
chemicals were obtained under analytical grade.
Induction of experimental diabetes Streptozotocin (STZ) was used to induce diabetes
mellitus in normoglycemic male albino Wistar rats. A
freshly prepared solution of STZ (45 mg/kg body
weight) in 0.1 M citrate buffer, pH 4.5 was injected
intraperitoneally in a volume of 1 ml/kg body weight
in overnight fasted rats (Pari and Venkateswaran,
2002).
Experimental design In the experiment, a total of 24 rats (12 diabetic
surviving rats, 12 control rats) were used in the study.
The rats were divided into 4 groups of 6 rats in each
group. p-Coumaric acid was dissolved in 10%
propylene glycol and administrated to rats orally using
an intragastric tube daily for a period of 45 days. Group 1 = Normal control rats. Group 2 = Normal + p-coumaric acid (100 mg/kg)
Group 3 = Diabetic control rats
Group 4 = Diabetic + p-coumaric acid (100 mg/kg)
At the end of the treatment period, all rats were
anaesthetized with pentobarbital sodium (35 mg/kg)
and sacrificed by cervical decapitation. Blood
collected using potassium oxalate and sodium fluoride
as anticoagulant and plasma was separated. The
tissues (pancreas, liver and kidney) were dissected
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out, washed in ice-cold saline, and patted dry. The
tissues were weighed and homogenized and 10%
tissue homogenate was used for estimation of various
biochemical parameters.
Biochemical estimations
The level of plasma TBARS was estimated by the
method Yagi (1987), and the tissue TBARS was
estimated by the method of Fraga, et al. (1988). The
levels of lipid hydroperoxides (HP) were estimated by
the method of Jiang, et al. (1992). The activity of
SOD, catalase and GPx was assayed according to the
procedures of Kakkar, et al. (1984), Sinha (1972) and
Rotruck, et al. (1973), respectively. The level of
ceruloplasmin was estimated by the method of Ravin
(1961). The level of GSH was estimated by the
method of Ellman (1959). The level of vitamin C and
E was estimated by the methods of Omaye, et al.
(1979) and Baker, et al. (1980), respectively.
Histopathology
Tissues (liver and kidney) obtained from all
experimental groups were washed immediately with
saline and then fixed in 10% buffered neutral formalin
solution. After fixation, the tissues were processed by
embedding in paraffin. Then, the tissues were
sectioned and stained with hematoxylin and eosin
(H&E) and examined under high power microscope
(400x) and photomicrographs were taken.
Statistical analysis
Statistical analysis was performed by one way
analysis of variance (ANOVA) followed by Duncan’s
Multiple Range Test (DMRT) using Statistical
Package for the Social Sciences (SPSS) software
package version 16.00. P values <0.05 were
considered significant.
RESULTS
Effect of p-coumaric acid on TBARS and
hydroperoxides in plasma and tissues
The levels of TBARS and hydroperoxides in plasma
and tissue of diabetic and control rats are presented in
Tables 1 & 2. STZ-induced diabetic rats had elevated
levels of TBARS and hydroperoxides in plasma and
tissue. Treatment with p-coumaric acid (100 mg/kg
for 45 days) in STZ-induced diabetic rats showed
reversal of these parameters to near normal levels.
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Effect of p-coumaric acid on antioxidants in
plasma and tissues
The activities of enzymatic antioxidants such as
superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPx) in the pancreas, liver
and kidney of normal and STZ-induced diabetic
animals were shown in Tables 3, 4 & 5. STZ-induced
diabetic rats showed significant reduction in the
activity of SOD, CAT and GPx. Oral administration
of p-coumaric acid exerted a significant effect on the
antioxidants in STZ-induced diabetic rats.
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The activity of non-enzymatic antioxidants such as
reduced glutathione (GSH), ceruloplamin, vitamin C
and vitamin E in the plasma, pancreas, liver and
kidney of normal and STZ-induced diabetic rats were
shown in Tables 6, 7, 8 & 9. The levels of
ceruloplasmin, GSH, vitamin C and E were found to
be significantly reduced in plasma, pancreas, liver and
kidney of diabetic rats. Oral administration of p-
coumaric acid significantly increased the levels of
ceruloplasmin, GSH, vitamin C and E in STZ-induced
diabetic rats.
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Effect of p-coumaric acid on histopathology of
diabetic rat liver and kidney
The effects of p-coumaric acid on histopathology of
diabetic rat liver are shown in Fig 2. Normal control
rats showed normal hepatic structure. Normal rats
treated with p-coumaric acid (100 mg/kg) showed
portal triad and normal architecture of the hepatic
tissue. STZ-induced diabetic control rats showed
dilation of hepatic sinusoids and inflammatory cells.
Rats treated with p-coumaric acid (100 mg/kg)
showed near normal appearance with few
inflammatory cells.
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The effects of p-coumaric acid on histopathology of
diabetic rat kidney are shown in Fig 3. Normal control
rats showed normal architecture of mesangial cells
and glomeruli. Normal rats treated with p-coumaric
acid (100 mg/kg) showed normal glomeruli and
tubules. STZ-induced diabetic control rats showed
severe epithelial atrophy and mesangial cell
proliferation and cloudy swelling of the tubules. Rats
treated with p-coumaric acid (100 mg/kg) showed
showing mild glomerular changes with normal tubules
.
DISCUSSION
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Streptozotocin (STZ) is a nitrosourea analogue,
preferentially uptaken by pancreatic β-cells via
GLUT-2 glucose transporter and causes DNA
alkylation followed by the activation of poly ADP
ribosylation leading to depletion of cytosolic
concentration of NAD+ and ATP. Enhanced ATP
dephosphorylation after STZ treatment provides
substrate for xanthine oxidase resulting in the
formation of superoxide radicals. Further, nitric oxide
moiety is liberated from STZ leading to the
destruction of β-cells by necrosis (Szkudelski, 2001).
Hence, STZ-induced experimental diabetic animal
model is chosen for the present study.
Oxidative stress in diabetes coexisted with a reduction
in the antioxidant capacity, which could increase the
deleterious effects of free radicals. The accumulation
of free radical observed in diabetic rats is attributed to
chronic hyperglycemia that alters antioxidant defense
system (Hong, et al., 2004). Free radicals may also be
formed through the auto-oxidation of unsaturated
lipids in plasma and membrane lipids. They may react
with polyunsaturated fatty acids in cell membrane
leading to lipid peroxidation (Lery, et al., 1999).
Lipid peroxidation is one of the characteristic features
of diabetes mellitus. The decline of antioxidant
defense mechanisms associated with an excess of free
radicals lead to damage of cellular organelles and
enzymes, increased lipid peroxidation, and
development of insulin resistance (Maritim, et al.,
2003). Lipid peroxidation results in damage to the cell
membrane, caused by ROS (Das, et al., 2000).
Measurement of plasma thiobarbituric acid reactive
substances (TBARS) was used as an index of lipid
peroxidation and it helps to assess the extent of tissue
damage (Gutteridge, 1995). Lipid peroxides and
hydroperoxides are the oxidative stress markers that
are elevated as a result of the toxic effect of reactive
oxygen species (ROS) produced during chronic
hyperglycemia (Evans, et al., 2002). Several studies
have reported an increase in TBARS and
hydroperoxides in plasma and tissues in experimental
diabetes mellitus (Pari and Venkateswaran, 2002).
Oral administration of p-coumaric acid (100 mg/kg
for a period of 45 days) significantly decreases the
levels of TBARS and hydroperoxides in plasma and
tissues. This shows that p-coumaric acid has the
ability to protect organism from lipid peroxidation and
reduces the risk of tissue damage.
Cells and tissues contain an array of antioxidant
machinery, which averts the buildup of ROS and
maintain the redox balance. Chronic oxidative stress is
associated with decrease in the antioxidant
competence, which can further increase the
deleterious effects of free radicals (Atli, et al., 2004).
Antioxidant enzymes such as SOD, CAT and GPx,
form the first line of defense against ROS in the
organism, play an important role in scavenging the
toxic intermediate of incomplete oxidation.
Superoxide dismutase is an antioxidative defence
enzyme, protects from oxygen free radicals by
catalyzing the removal of superoxide radical, which
damage the membrane and biological structures. SOD
destroys the superoxide radical through dismutation
and generates hydrogen peroxide, which is
consecutively reduced by the activities of catalase or
glutathione peroxidase. Several studies have reported
a reduced activity of SOD in experimental diabetes
(Meghana, et al., 2007; Bagri, et al., 2009). Thus the
reduced activity of SOD could be the result of
superoxide anion over accumulation in the cell (Raha
and Robinson, 2000), and its inactivation by hydrogen
peroxide (Ravi, et al., 2004) or by glycation of the
enzyme (Meghana, et al., 2007).
Catalase, another enzymatic antioxidant
predominantly present in peroxisomes, ameliorated
the deleterious effect of hydrogen peroxide, which
was produced by SOD, into water and non-reactive
oxygen species. It restrained the generation of
hydroxyl radical and protected the cells from
oxidative damage. In diabetic conditions, the
uncontrolled production of hydrogen peroxide due to
the auto-oxidation of glucose, protein glycation and
lipid oxidation led to a marked decline in the catalase
activity (Rajasekaran, et al., 2005; Farombi and Ige,
2007). Recently, the decreased activity of catalase in
the protection of pancreatic β-cells from oxidative
stress during diabetic conditions has been reported
(Rajasekaran, et al., 2005).
Another antioxidant enzyme, glutathione peroxidase
(GPx) was a selenium-containing tetrameric
glycoprotein plays a primary role in minimizing
oxidative damage. GPx is involved in the
detoxification of hydrogen peroxide into water and
molecular oxygen. During diabetic conditions, the
activity of glutathione peroxidase is decreased as a
result of radical-induced inactivation and glycation of
the enzyme (Zhang and Tan 2000).
Reduced activities of these antioxidant enzyme in
pancreas, liver and kidney was observed in diabetic
rats and this activity may resulting a number of
deleterious effects due to accumulation of superoxide
anion (O) and hydrogen peroxide (H2O2), which in
turn generate hydroxyl radicals (OH), resulting in
initiation and propagation of LPO. Treatment with p-
coumaric acid showed a significant increase in SOD,
CAT and GPx activities of the diabetic rats. This
means that p-coumaric acid can reduce the potential
glycation of enzymes or they may reduce reactive
oxygen free radicals and improve the activities of
antioxidant enzymes.
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Reduced glutathione, a ubiquitous tripeptide thiol, is
an important intracellular metabolite. It acts as an
antioxidant and provides secondary line of defence
against intracellular free radicals and peroxides
generated by oxidative stress (Mohammed, 2008).
Reduced state of cell is maintained by high level of
GSH/GSSG ratio. When the level of GSSG, the
oxidized form of GSH increased in the presence of
persistently elevated ROS then the redox state of cell
get affected and it may result in the development of
diabetic complications. Measurement of intracellular
GSH/GSSG ratio may provide valuable information
about the redox status of the cell (Veerapan, et al.,
2004). Diabetic rats showed the decreased levels of
GSH and this may be due to the decrease of GSH
synthesis or an increase of its degradation induced by
STZ oxidative stress. Treatment with p-coumaric acid
showed a significant increase in the levels of GSH.
This may be due to the increase in GSH biosynthesis
or reduce the oxidative stress leading to less
degradation of GSH.
Ceruloplasmin is also a sialoglycoprotein. Removal of
caruloplasmin from blood is triggered by the loss of
sialic acid units (Osaki, et al., 1966). Increase in sialic
acid level may resialate the desialated, ceruloplasmin
which may also be responsible for the decreased
removal and thereby increasing the level of
ceruloplasmin in hyperlipidemic patients with or
without diabetes (Kaviarasan, et al., 2005). The level
of ceruloplasmin was significantly decreased in
diabetic rats, which may facilitate the scavenging
action on peroxyl radicals. Administration of p-
coumaric acid showed significant reversal of
ceruloplasmin level in plasma of diabetic rats.
Vitamin E is the most ancient antioxidant in the lipid
phase (Ingold, et al., 1987). Apart from enzymic
antioxidants, non-enzymic antioxidants play a vital
role in protecting cells from oxidative changes.
Vitamin E neutralizes the free radicals, preventing the
chain reaction that contributes to oxidative damage
(Sun, et al., 1999). It has the potential to quench lipid
peroxidation and protects cellular structure from the
attack of free radicals (Bertoni-Freddari, et al., 1995).
The decreased level of vitamin E observed in diabetic
rat (Murugan and Pari, 2006).
Vitamin C (ascorbic acid) is the most widely cited
form of water-soluble antioxidant. It prevents
oxidative damage to the cell membrane induced by
aqueous radicals. Vitamin C can act as a co-oxidant
by regenerating α-tocopherol from the α-tocopheroxyl
radical produced during scavenging of free radicals. It
is also possible that the correction of hyperglycemia
has a sparing effect on vitamin C, and this increases
the potential to recycle vitamin E (Chan, 1993; Stah
and Sies, 1997). We also observed significant
decrease in the levels of vitamin C in diabetic rats
could be due to increased utilization of vitamin C as
an antioxidant defense against reactive oxygen species
(Ceriello, et al., 1998). Oral administration of p-
coumaric acid improved the level of vitamin C and E
level in plasma and tissues of STZ-induced diabetic
rats. This may be due to the antioxidant properties of
the plant phenolics.
Histopathology of liver in control rats showed normal
hepatic structure. Diabetic control liver showed
dilation of hepatic sinusoids and inflammatory cells.
Diabetic rats treated with p-coumaric acid showed
mild sinusoidal dilation and few inflammatory cells.
Normal rats treated with p-coumaric acid showed
portal triad and normal architecture of the hepatic
tissue.
Histopathology of kidney in control rats revealed
normal architecture of the mesangial cells and
glomeruli. Diabetic control rats showed severe
epithelial atrophy and mesangial cell proliferation and
cloudy swelling of the tubules. Diabetic rats treated
with p-coumaric acid showed mild glomerular
changes with normal tubules. Normal rats treated with
p-coumaric acid showed normal glomeruli and
tubules. This could be due to free radical scavenging,
antioxidant and membrane stabilizing properties of p-
coumaric acid. The liver and kidney exhibits
numerous morphological and functional alterations
during diabetes.
CONCLUSION
The results of the present investigation indicate that p-
coumaric acid ameliorates hyperglycemia mediated
oxidative stress in STZ-induced diabetic rats, which
was evidenced by improved glycemic as well as
antioxidant status. Thus, we can conclude that p-
coumaric acid is a promising adjuvant agent in
diabetes mellitus therapy.
ACKNOWLEDGEMENT
The authors gratefully acknowledge Dr. N.
Pazhanivel, Associate Professor, Department of
Veterinary Pathology, Madras Veterinary College,
Chennai for his help to carryout histopathological
studies.
REFERENCES 1. Mednieks, M.I., Szczepanski, A., Clark. B. and
Hand, A.R. 2009. Protein expression in salivary
glands of rats with streptozotocin diabetes. Int. J.
Exp. Pathol., 90(4): 412-422.
2. Mitrovic, M., Pantelinac, P., Radosavljevic, J.,
Bajkin, I. and Todorovic-Dilas L. 2006. The role
of insulin analogues in the current treatment of
diabetes mellitus. Med. Pregl., 59(11-12): 539-
544.
3. Unger, J. and Parkin, C.G. 2010. Type 2 diabetes:
an expanded view of pathophysiology and
therapy. Postgrad. Med., 122(3): 145-157.
4. Kumar, P. and Clark, M. 2002. Diabetes mellitus
and other disorders of metabolism. In: Clark
Shairibha and Rajadurai eISSN 2278-1145
10 Int. J. Int sci. Inn. Tech. Sec. B, Oct. 2014, Vol.3, Iss 5, pg 01-11
PKM, editor. Clinical medicine. WB Saunders,
1069-1121.
5. Chaiyavat, C., Winthana, K., Narissara, L.,
Peerasak, L., Maitree, S. and Somdet, S. 2010.
Effects of phenolic compounds of fermented thai
indigenous plants on oxidative stress in
streptozotocin-induced diabetic rats. Evidence
Based Comp. Alt. Med., 2011: 1-10.
6. Aragao, D.M.O., Guarize, L., Lanini, J., Da
Costa, J.C., Garcia, R.M.G. and Scio E. 2010.
Hypoglycemic effects of Cecropia pachystachya in
normal and alloxan-induced diabetic rats. J.
Ethnopharmacol., 128: 629-633.
7. IDA. Diabetes Atlas. 4th ed. 2009.
8. Kim, M.J., Ryu, G.R., Chung, J.S., Sim, S.S., Min
Dos Rhie, D.J., Yoon, S.H., Hahn, S.J., Kim, M.S.
and Jo, Y.M. 2003. Protective effect of epicatechin
against the toxic effects of STZ on rat pancreatic
islets: in vivo and in vitro. Pancreas, 26: 292-299.
9. Vessal. M., Hemmati, M. and Vasei, M. 2003.
Antidiabetic effects of quercetin in streptozocin-
induced diabetic rats. Comp. Biochem. Physiol.
Toxicol. Pharmacol., 135: 357-364.
10. Szkudelski T. 2001. The mechanism of alloxan and
streptozotocin action of β-cells of the rat
pancreas. Physiol. Res., 50: 537-546.
11. Coskun, O., Kanter, M., Korkmaz, A. and Oter, S.
2005. Quercetin, a flavonoid antioxidant, prevents
and protects streptozotocin-induced oxidative
stress and β-cell damage in rat pancreas.
Pharmacol. Res., 51: 117-123.
12. Ceriello A. 2000. Oxidative stress and glycemic
regulation. Met., 49: 27-29.
13. Laybutt, D.R., Kaneto, H., Hasenkamp, W., Grey,
S., Jonas, J.C., Sgroi, D.C., Groff, A., Ferran, C.,
Bonner-Weir, S., Sharma, A., and Weir, G.C.
2002. Increased expression of antioxidant and
antiapoptotic genes in islets that may contribute to
beta-cell survival during chronic hyperglycemia.
Diab., 51: 413-423.
14. Potenza, M.A., Gagliardi, S., Nacci, C., Carratu,
M.R. and Montagnani, M. 2009. Endothelial
dysfunction in diabetes: from mechanisms to
therapeutic targets. Curr. Med. Chem., 16: 94-
112.
15. Soto, C., Recoba, R., Barron, H., Alvarez, C. and
Favari, L. 2003. Silymarin increases antioxidant
enzymes in alloxan-induced diabetes in rat
pancreas. Comp. Biochem. Physiol., 136: 205-
212.
16. Rang, H.P. and Dale, M.M. 1991. The Endocrine
System Pharmacology, second ed. Longman
Group Ltd, United Kingdom, 504-508.
17. Nicasio, P., Aguilar-Santamaria, L., Aranda, E.,
Ortiz, S. and Gonzalez, M. 2005. Hypoglycemic
effect and chlorogenic acid content in two
Cecropia species. Phytother. Res., 19: 661-664.
18. Hassan, Z., Yam, M.F., Ahmad, M. and Yusof,
A.P. 2010. Antidiabetic properties and mechanism
of action of Gynura procumbens water extract in
streptozotocin-induced diabetic rats. Molecules,
15: 9008–9023.
19. Scalbert, A. and Williamson, G. 2000. Dietary
intake and bioavilability of polyphenols. J. Nutr.,
130(8): 2073-2085.
20. King, A. and Young, G. 1999. Characteristics and
occurrence of phenolic phytochemicals. J. Am.
Diet Assoc., 99(2): 213-218.
21. Ursini, F., Tubaro, F., Rong, J. and Sevanian, A.
1999. Optimization of nutrition: polyphenols and
vascular protection. Nutr. Rev., 57(8): 241-249.
22. Rice-Evans, C.A., Miller, N.J. and Paganga, G.
1996. Structure antioxidant activity relationships
of flavonoids and phenolic acids. Free Rad. Biol.
Med., 20: 933-956.
23. Hudson, E.A., Dinh, P.A., Kokubun, T.,
Simmonds, M.S. and Gescher, A. 2000.
Characterization of potentially chemopreventive
phenols in extracts of brown rice that inhibit the
growth of human breast and colon cancer cells.
Cancer Epidemiol. Biomarkers Prev., 9(11):
1163-1170.
24. Pari, L. and Venkateswaran, S. 2002. Antioxidant
effect of Phaseolus vulgaris in streptozotocin-
induced diabetic rats. Asia Pacific J. Clin. Nutr.,
11: 206-209.
25. Yagi, K. 1987. Lipid peroxidase and human
disease. Chem. Phys. Lipids, 45: 37-51.
26. Fraga, C.G., Leibovitz, B.E. and Toppel, A.L.
1988. Lipid peroxidation measured as TBARS in
tissue characterization and comparison with
homogenates and microsomes. Free Radic. Biol.
Med., 4: 155-161.
27. Jiang, Z.J., Hunt, J.V. and Wolf, S.D. 1992.
Ferrous ion oxidation in the presence of xylenol
orange for detection of lipid hydroperoxide in low
density lipoprotein. Anal Biochem., 202: 384-389.
28. Kakkar, P., Dos, B. and Vishwanathan, P.N. 1984.
A modified spectrophotometric assay of
superoxide dismutase. Ind. J. Biochem. Biphys.,
21: 130-132.
29. Sinha, K.A. 1972. Colorimetric assay of catalase.
Anal Biochem., 47: 389-394.
30. Rotruck, J.T., Pope, A.L., Ganther, H.E. and
Swanson, A.B. 1973. Selenium: Biochemical roles
as a component of glutathione peroxidase. Sci.,
779: 588-590.
31. Ravin, H.A. 1961. An improved colorimetric
enzymatic assay of ceruloplasmin. J. Lab. Med.,
58: 161-168.
32. Ellman, G.L. 1959. Tissue sulfhydryl groups.
Arch. Biochem. Biophys., 82: 70-77.
33. Omaye, S.T., Turbull, T.P. and Sauberlich, H.C.
1979. Selected methods for determination of
ascorbic acid in cell tissues and fluids. Meth.
Enzymol., 6: 3-11.
34. Baker, H., Frank, O., Angelis, B. and Feingold, S.
1980. Plasma α-tocopherol in man at various
times after ingesting free of acetylated tocopherol.
Nutr. Rep., 21: 531-536.
35. Szkudelski, T. 2001. The mechanism of alloxan
and streptozotocin action in β-cells of the rat
pancreas. Physiol. Res., 50(6), 537-546.
36. Hong, J., Bose, M., Ju, J., Ryuhm, J., Chenm, X.,
and Sang, S. 2004. Modulation of arachidonic
acid metabolism by curcumin and related β-
diketone derivatives: effects on cytosolic
phospholipase A2, cyclooxygenases and 5-
lipoxygenase. Carcinogenesis, 25: 1671-1679.
37. Lery, V., Zaltzber, H., Ben-Amotz, A., Kanter, Y.
and Aviram, M. 1999. β-Carotene affects
Shairibha and Rajadurai eISSN 2278-1145
11 Int. J. Int sci. Inn. Tech. Sec. B, Oct. 2014, Vol.3, Iss 5, pg 01-11
antioxidant status in non-insulin dependent
diabetes mellitus. Pathophysiol., 6: 157-162.
38. Maritim, A.C., Sanders, R.A. and Watkins, J.B.
2003. Diabetes, oxidative stress, and antioxidants:
a review. J. Biochem. Mol. Toxicol., 17: 24-38.
39. Das, S., Vasisht, S., Snehalata, K., Das, N. and
Srivastava, L.M. 2000. Correlation between total
antioxidant status and peroxidation in
hypercholesterolemia. Cur. Sci., 78: 486-487.
40. Gutteridge, J.M.C. 1995. Lipid peroxidation and
antioxidants as biomarkers of tissue damage.
Clin. Chem., 14: 1819-1828.
41. Evans, J.L., Goldfine, I.D., Maddux, B.A. and
Grodsky, G.M. 2002. Oxidative stress and stress
activated signaling pathways: a unifying
hypothesis of type 2 diabetes. Endocr. Rev., 23:
599-622.
42. Atli, T., Keven, K., Avci, A., Kutlay, S., Turkcapar,
N., Varli, M., Aras, S., Ertug, E. and Canbolat, O.
2004. Oxidative stress and antioxidant status in
elderly diabetes mellitus and glucose intolerance
patients. Arch. Gerontol. Geriatr., 39: 269-275.
43. Meghana, K., Sanjeev, G. and Ramesh, B. 2007.
Curcumin prevents streptozotocin-induced islet
damage by scavenging free radicals: a
prophylactic and protective role. European J.
Pharmacol., 577: 183-191.
44. Bagri, P., Ali, M., Aeri, V., Bhowmik, M. and
Sultana, S. 2009. Antidiabetic effect of Punica
granatum flowers: effect on hyperlipidemia,
pancreatic cells lipid peroxidation and
antioxidant enzymes in experimental diabetes.
Food Chem. Toxicol., 47: 50-54.
45. Raha, S. and Robinson, B.H. 2000. Mitochondria,
oxygen free radicals, disease and ageing. Trends
Biochem. Sci., 25: 502-508.
46. Ravi, K., Ramachandran, B. and Subramanian, S.
2004. Protective effect of Eugenia jambolaa seed
kernel on tissue antioxidants in streptozotocin-
induced diabetic rats. Bio. Pharma. Bulletin, 27:
1212-1217.
47. Rajasekaran, S., Sivagnanam, K. and
Subramanian, S. 2005. Antioxidant effect of Aloe
vera gel extract in streptozotocin-induced diabetic
rats. Pharmacol. Rep., 57: 90-96.
48. Farombi, E.O. and Ige, O.O. 2007. Hypolipidemic
and antioxidant effects of ethanolic extract from
dried calyx of Hibiscus sabdariffa in alloxan-
induced diabetic rats. Fund. Clin. Pharmacol., 21:
601-609.
49. Zhang, X.F. and Tan, B.K. 2000.
Antihyperglycemic and antioxidant properties of
Andrographis paniculata in normal and diabetic
rats. Clin. Exp. Pharmacol. Physiol., 27: 358-363.
50. Mohammed, A.H. 2008. Antidiabetic and
antioxidant activity of Jasonia montana extract in
streptozotocin-induced diabetic rats. Saudi
Pharma. J., 16: 3-4.
51. Veerapan, R.M., Senthil, S., Ramakrishna Rao,
M., Ravikumar, R. and Pugalendi, K.V. 2004.
Redox status and lipid peroxidation in alcoholic
hypertensive patients with diabetes. Clinica.
Chimica. Acta., 340: 207-212.
52. Osaki, S., Johnson, D.A. and Frienden, E. 1966.
The possible significance of the ferroxidase
activity of ceruloplasmin in normal human serum.
J. Biol. Chem., 241: 2746-2751.
53. Kaviarasan, K., Arjunan, M.M. and Pugalendi,
K.V. 2005. Lipid profile, oxidant-antioxidant
status and glycoprotein components in
hyperlipidemic patients with/without diabetes.
Clinica. Chimica. Acta., 362: 49-56.
54. Ingold, K.U., Webb, A.C., Witter, D., Burton,
G.W., Metcalf, T.A. and Muller, D.P. 1987.
Vitamin E remains the major lipid soluble, chain
breaking antioxidant in human plasma even in
individuals suffering from severe vitamin E
deficiency. Arch. Biochem. Biophys., 259: 224-
225.
55. Sun, F., Iwaguchi, K., Shudo, R.Y., Nagaki, Y.,
Tanaka, K. and Ikeda, K. 1999. Change in tissue
concentrations of lipid hydroperoxides, vitamin C
and vitamin E in rats with streptozotocin-induced
diabetes. Clin. Sci., 96: 185-190.
56. Bertoni-Freddari, C., Fattoretti, P., Caselli, U.,
Paoloni, R. and Meier-Ruge, W. 1995. Vitamin E
deficiency as a model of precocious brain aging:
assessment by X-ray microanalysis and
morphometry. Scanning Microscopy, 9(1): 289-
301.
57. Murugan, P. and Pari, L. 2006. Effect of tetra
hydro curcumin on plasma antioxidants in
streptozotocin-nicotinamide experimental
diabetes. J. Basic clin. Physiol. Pharmacol.,
17(4): 231-244.
58. Chan, A.C. 1993. Partners in defense, vitamin E
and vitamin C. Can. J. Physiol. Pharmacol., 71:
725-731.
59. Stah, I.W. and Sies, H. 1997. Antioxidant defense:
Vitamin E and C and carotenoids. Diab., 46: 14-
18.
60. Ceriello, A., Bortolotti, N. and Crescenti, A. 1998.
Antioxidant defenses are reduced during the oral
glucose tolerance test in normal and non-insulin
dependent diabetic subjects. Eur. J. Clin. Invest.,
28: 329-333.