Shairibha and Rajadurai eISSN 2278-1145 International ... · Shairibha and Rajadurai eISSN 2278-1145 1 Int. J. Int sci. Inn. Tech. Sec. B, Oct. 2014, Vol.3, Iss 5, pg 01-11 International

Shairibha and Rajadurai eISSN 2278-1145

1 Int. J. Int sci. Inn. Tech. Sec. B, Oct. 2014, Vol.3, Iss 5, pg 01-11

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



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.



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.



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.



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.



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



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.



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.

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Shairibha and Rajadurai eISSN 2278-1145 International ... · Shairibha and Rajadurai eISSN 2278-1145 1 Int. J. Int sci. Inn. Tech. Sec. B, Oct. 2014, Vol.3, Iss 5, pg 01-11 International