hepatoprotective effect of hesperidin on...

International Journal of Environmental Science and Ecotechnology1(1) January-June 2011; pp. 55-66

*Corresponding Author: E-mail: [email protected]

HEPATOPROTECTIVE EFFECT OF HESPERIDIN ON NORMAL AND STREPTOZOTOCIN-INDUCED DIABETIC RATS: BIOCHEMICAL AND HISTOLOGICAL EVIDENCES

M. Ilankeswaran1*, G. Senthil Kumar1, M. Rajadurai2, and R. Stephan3

1Department of Biotechnology, V.M.K.V. Engineering College, Vinayaka Mission’s University, Salem-636308,Tamil Nadu, India.2Post Graduate and Research Department of Biochemistry, Muthayammal College of Arts and Science,Rasipuram-637408, Tamil Nadu, India.3Department of Botany, Government Arts College, Ariyalur - 621713, Tamil Nadu, India.

Abstract: We investigated the hepatoprotective effect of hesperidin on biochemical and histologicalparameters in normal and streptozotocin (STZ)-induced diabetic rats. Diabetes was induced byintraperitonial injection of STZ (45 mg/kg) to male Wistar rats. Rats induced with STZ, showed a significantincrease in the levels of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (HP)in plasma and liver with subsequent decrease in the activities enzymatic antioxidants such as superoxidedismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in liverand the levels of non-enzymatic antioxidants such as reduced glutathione (GSH), vitamin C and E inplasma and liver. R ats treated with hesperidin (25 and 50 mg/kg) for a period of 35 days to STZ-inducedrats showed a significant reduction in the levels of lipid peroxidative products and improved both enzymaticand non-enzymatic antioxidants. In addition to the biochemical parameters, our histopathological findingsof the liver also support the hepatoprotective role of hesperidin in STZ-induced diabetic rats. The effectat a dose of 50 mg/kg of hesperidin was more pronounced than 25 mg/kg. Thus the results of our studyindicates that hesperidin possess hepatoprotective effect in STZ-induced oxidative stress.

Keywords: Antioxidants, Diabetes, Hesperidin, Lipid peroxidation, Streptozotocin

1. INTRODUCTION

The prevalence o diabetes for all age groups worldwide was estimated to be 2.8% in 2000 and4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in2000 to 366 million in 2030 (Trivedi et al., 2004). Diabetes is a complex chronic disordercharacterized by hyperglycemia, which via several mechanisms leads to an increase inproduction of reactive oxygen species (ROS) and oxidative stress. The increased generation offree radicals in diabetes, impaired generation of naturally occurring antioxidants also resultsin increased oxidative injury, by failure of protective mechanisms. Antioxidant defense systemappears to be compromised in diabetic patients. Recent findings suggest that the same pathwaysused in the activation of glucose dependent insulin secretion can dramatically enhance ROSproduction and manifestations of oxidative stress and possibly apoptosis (Kholeand et al.,2009). Thus free radicals play a significant role in the pathogenesis of chronic diabetic

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complications (Segal, 2004). Considerable attention has been placed on understanding thepathophysiology of diabetes because of its importance in human health.

Streptozotocin (STZ), is a broad spectrum antibiotic, widely used to induce experimentaldiabetes. High dose of STZ, induces β-cell necrosis and diabetes, because it causes selectivedamage to insulin secreting β-cells (Bassirat and Khalil, 2000). Free radical production inanimal cells is well known, with the increasing acceptance of free radical as common placeand important biochemical intermediates, they have been implicated in large number of humandisease, including diabetes. Therefore, the STZ induced diabetic model has been widelyemployed in experimental animals. It has been reported that STZ-diabetic animals may exhibitmost of the diabetic complications mediated through oxidative stress, and the involvement offree radicals in pancreatic cell destruction (Lukic et al., 1998). The STZ-diabetic rat serves asan excellent model to study the molecular, cellular and morphological changes in tissues duringdiabetes (Latha and Pari, 2004).

Flavonoids are a group of naturally occurring polyphenolic compounds ubiquitously foundin fruits and vegetables, which are the component of human diet and intake, may reach 800mg/day (Ana et al., 2009). Flavonoids are widely recognized as naturally occurring antioxidantsthat inhibit lipid peroxidation in biological membranes (Amic et al., 2003). Hesperidin is aflavanone glycoside, abundantly found in lemon and oranges. The peel and membranous partof these fruits have the highest hesperidin concentration. Sweet oranges (Citrus sinensis) andtangelos are the richest dietary sources of hesperidin. Hesperidin is also know as hesperidin7-rhamnoglucoside. Hesperidin exhibits various biological and pharmacological propertiessuch as anti-inflammatory, anticancer, lipid-lowering, antioxidant, vasoprotective andantihypertensive and protect against ischemia-reperfusion tissue damage (Jung et al., 2004).

In our laboratory, we previously reported the effect of hesperidin blood glucose, plasmainsulin and carbohydrate metabolic enzymes in STZ-induced diabetic rats (Ilankeswaran etal., 2009). In the present study, we evaluated the protective effect of hesperidin on lipidperoxides, enzymatic, non-enzymatic antioxidants and histopathological findings in normaland STZ-induced diabetes in male albino Wistar rats.

2. MATERIALS AND METHODS

2.1 Experimental Animals

All the experiments were carried out with male albino Wistar rats weighing 140-160 g, wereobtained from Venkateshwara enterprises, Bangalore, India. They were housed in polypropylenecages (47 cm × 34 cm × 20 cm) lined with husk, renewed every 24 h under a 12:12 h light darkcycle at around 22ºC and had free access to tap water and food. The rats were fed on a standardpellet diet (Pranav Agro Industries Ltd., Maharashtra, India). The pellet diet consisted of 22.02%crude protein, 4.25% crude oil, 3.25% 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 3000 kcal. The experiment wascarried out according to the guidelines of the Committee for the Purpose of Control and

HEPATOPROTECTIVE EFFECT OF HESPERIDIN ON NORMAL AND STREPTOZOTOCIN- ... / 57

Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by theAnimal Ethical Committee of Vinayaka Missions University (Approval No: IAECNo: Biotech/02/2008).

2.2 Drugs And Chemicals

Streptozotocin, butylated hydroxyl toluene, nitroblue tetrazolium, phenazine methosulphateand glutathione were purchased from Sigma Chemical Company, St. Louis, MO, USA.Hesperidin was purchased from Himedia laboratories Pvt. Ltd., Mumbai. All other biochemicaland chemicals used in the study were of analytical grade.

2.3 Induction of Experimental Diabetes

A freshly prepared solution of streptozotocin (STZ) (45 mg /kg) in 0.1 M citrate buffer, pH 4.5was injected intraperitonially in a volume of 1 ml/kg in overnight fasted rats. 48 h afterSTZ-administration rats with moderate diabetes having glycosuria and hyperglycemia(with glucose levels of 200-300 mg/dL) were taken for the experiment.

2.4 Experimental Procedure

In the experiment, a total of 36 rats (18 diabetic surviving rats, 18 control rats). The rats weredivided into 6 groups of 6 rats each. Hesperidin was dissolved in carboxy methyl cellulose(CMC) and administrated to rats orally using an intragastric tube daily for a period of 35 days(Ilankeswaran et al., 2009).

Group 1: Normal control rats

Group 2: Control rats administrated orally with hesperidin 25 mg/kg

Group 3: Control rats administrated orally with hesperidin 50 mg/kg

Group 4: Diabetic control rats

Group 5: Diabetic rats treated orally with hesperidin 25 mg/kg

Group 6: Diabetic rats treated orally with hesperidin 25 mg/kg

Fig. 1: Control Rat Liver Shows Normal Architecture


Fig. 2: Normal Rat Treated with Hesperidin 25 mg/kg Liver Shows Few Hepatocyteswith Vesicular Appearance

Fig. 3: Normal Rat Treated with Hesperidin 50 mg/kg Liver Shows SinusoidalDilation Only No Paranchymal Damage

Fig. 4: Diabetic Control Rat Liver Shows Microvesicular Fat Filled Hepatocytes and Hepatic Necrosis

Fig. 5: Diabetic Rat Treated with Hesperidin 25 mg/kg Liver Shows FewHepatocytes with Vesicular Appearance


At the end of treatment period all the rats were anaesthetized with pentobarbital sodium(35 mg/kg) and sacrificed by cervical decapitation, the blood was collected by using potassiumoxalate and sodium fluoride as anticoagulant for estimation of fasting blood glucose. The liverand kidney were dissected out, washed in ice-cold physiological saline, patted dry and weighed.Plasma and serum were separated by centrifugation. The liver and kidney were weighed and10% tissue homogenate was prepared with appropriate buffer was used for bio-chemical estimations.

2.5 Biochemical Assays

Plasma thiobarbituric acid reactive substances (TBARS) were estimated by the method ofYagi (1987). TBARS were quantitated by their reactivity with thiobarbituric acid (TBA) inacidic conditions to generate a pink coloured chromophore, which was read at 530 nm. TBARSin the heart was estimated by the method of Fraga et al. (1988). In this method, malondialdehydeand other TBARS were measured by their reactivity with TBA in acidic conditions to generatea pink coloured chromophore, which was read at 535 nm. Estimation of plasma and cardiactissue lipid hydroperoxides (HP) was done by the method of Jiang et al. (1992). In this method,oxidation of ferrous ion (Fe2+) under acidic conditions in the presence of xylenol orange led tothe formation of a chromophore, which was read at 560 nm.

Superoxide dismutase (SOD) activity in the myocardium was assayed by the method ofKakkar et al. (1984). Superoxide radicals react with nitroblue tetrazolium in the presence ofreduced nicotinamide adenine dinucleotide and produce formazon blue. SOD removes thesuperoxide radicals and inhibits the formation of formazon blue. The intensity of the colour isinversely proportional to the activity of the enzyme and read at 560 nm. The activity of catalasein myocardium was assayed by the method of Sinha (1972). In this method, dichromate inacetic acid is converted to perchromic acid and then to chromic acetate when heated in thepresence of hydrogen peroxide. The chromic acetate formed was measured at 620 nm.

Estimation of GSH in plasma and the heart tissue was done by the method of Ellman(1959). This method is based on the development of yellow colour, when dithionitro benzoicacid is added to compounds containing sulfhydryl groups. The colour developedwas read at

Fig. 6: Diabetic Rat Treated with Hesperidin 50 mg/kg Liver Shows MildDilation and Near Normal Architecture


412 nm. The activity of GPx was assayed by the method of Rotruck et al. (1973). A knownamount of enzyme preparation was allowed to react with hydrogen peroxide and GSH for aspecified time period. The GSH content remaining after the reaction was measured by Ellman’sreaction. The activity of GST was assayed in the cardiac tissue following the increase in theabsorbance at 340 nm using 1-chloro-2,4- dinitro benzene as substrate by the method of Habigand Jakoby (1981).

Vitamin C in plasma and the heart tissue was estimated by the method of Omaye et al.(1979). The ascorbic acid is converted into dehydroascorbic acid in the presence of thiourea, amild reducing agent and then coupled with 2,4-dinitrophenyl hydrazine (DNPH). The coupledDNPH is converted into a red coloured complex when treated with sulphuric acid, which wasread at 530 nm. The levels of Vitamin E in plasma and the concentration in cardiac tissue wereestimated by the method of Baker et al. (1980). This method involves the reduction of ferricion to ferrous ion by -tocopherol and the formation of red coloured complex with 2,2-dipyridyl.The absorbance of the chromophore was measured at 520 nm.

Protein in the enzyme extract was determined by the method of Lowry et al. (1951). TheCO–NH group (peptide bond) present in the protein molecule reacts with copper sulphate inalkaline medium to give a blue colour, which was read at 620 nm.

2.6 Histopathological Examination

The liver tissue obtained from all experimental groups were washed immediately with salineand then fixed in 10% buffered neutral formalin solution. After fixation, the liver tissue wasprocessed embedding in paraffin. Then, the tissue was sectioned and stained with hematoxylinand eosin (H & E) and examined under high power microscope (320X) and photomicrographswere taken.

2.7 Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed byDuncan’s multiple range test (DMRT) using SPSS software package 9.05. Results wereexpressed as mean ± S.D. from six rats in each group. P values < 0.05 were consideredas significant.

3. RESULTS

Table 1 shows the levels of TBARS and HP in plasma and liver of normal and STZ-induceddiabetic rats. Rats induced with STZ, showed a significant (P < 0.05) increase in the levels ofTBARS and HP in plasma and liver. Rats treated with hesperidin (25 and 50 mg/kg) toSTZ-induced diabetic rats daily for a period of 35 days significantly (P < 0.05) decreased thelevels of TBARS and HP in plasma and liver when compared with diabetic rats. The activitiesof SOD and catalase are shown in Table 2. Rats induced with STZ, showed a significant(P < 0.05) decrease in the activities of these enzymes in liver. Hesperidin treated diabetic ratssignificantly (P < 0.05) increased the activities of these enzymes.


Table 3 illustrates the effect of hesperidin on the levels of plasma and liver GSH and theactivities of liver GPx and GST in liver in normal and STZ-induced diabetic rats. Rats inducedwith STZ, showed a significant (P < 0.05) decrease in the levels of GSH in plasma and liverand the activities of GPx and GST in liver. Rats treated with hesperidin significantly (P < 0.05)increased the levels and activities of these enzymatic and non-enzymatic antioxidants.

Table 2Effect of Hesperidin on the Activities of Superoxide Dismutase (SOD) and Catalase in Liver and

Kidney in Normal and Streptozotocin (STZ)-induced Diabetic Rats

Groups LiverSOD Catalase

(Unitsa / mg protein) (Unitsb / mg protein)

Normal control 10.2 ± 0.9a 95.6 ± 5.7a

Normal + hesperidin (25 mg/kg) 10.4 ± 0.5a 96.1 ± 6.4a

Normal + hesperidin (50 mg/kg) 10.9 ± 0.5a 96.8 ± 8.0a

Diabetic control (45 mg/kg) 4.25 ± 0.3b 45.6 ± 4.5b

Diabetic + hesperidin (25 mg/kg) 7.2 ± 0.7c 72.2 ± 6.7c

Diabetic + hesperidin (50 mg/kg) 9.5 ± 0.6d 88.2 ± 8.5d

Each value is mean ± S.D. for six rats in each group. Values not sharing a common superscript (a-d)differ significantly with each other (P < 0.05, DMRT).

Table 1Effect of Hesperidin on the Levels of Thiobarbituric Acid Reactive Substances (TBARS) and

Hydroperoxides (HP) in Plasma and Liver in Normal and Streptozotocin (STZ)-induced Diabetic Rats

Groups TBARS (nmol/ml) Hydroperoxides

Plasma Liver Plasma Liver(mM/dL) (mM/100g tissue)

Normal control 2.3 ± 0.08a 0.95 ± 0.07a 8.33 ± 0.71a 72.4 ± 4.8a

Normal + hesperidin(25 mg/kg) 2.5 ± 0.09a 0.92 ± 0.07a 8.19 ± 0.56a 72.5 ± 3.2a


Diabetic control(45 mg/kg) 3.5 ± 0.10b 2.12 ± 0.20b 17.56 ± 1.07b 112.4 ± 8.5b

Diabetic + hesperidin(25 mg/kg) 2.7 ± 0.08c 1.85 ± 0.17c 13.28 ± 0.82c 95.3 ± 6.6c

Diabetic + hesperidin(50 mg/kg) 2.2 ± 0.07d 1.25 ± 0.10d 10.12 ± 0.74d 80.4 ± 6.7d

Each value is mean ± S.D. for six rats in each group. Values not sharing a common superscript (a-d)differ significantly with each other (P < 0.05, DMRT).


The effect of hesperidin on the levels of plasma and liver vitamin C and E in normal andSTZ-induced diabetic rats is shown in Table 4. Rats administered with STZ, exhibited asignificant (P < 0.05) decrease in the levels of vitamin C and E in plasma and liver. Oraladministration of hesperidin to STZ-induced rats diabetic significantly (P < 0.05) increasedthe levels of vitamin C and E in plasma and the liver when compared with STZ- induceddiabetic rats.

Table 3Effect of Hesperidin on the Levels of Reduced Glutathione (GSH) and the Activities of Glutathione

Peroxidase (GPx) and Glutathione-S-transferase (GST) in Liver inNormal and Streptozotocin (STZ)-induced Diabetic Rats

Groups Reduced glutathione (GSH) Liver

Plasma Liver GPx GST(mg/dL) (mg/100g tissue) (Unitsa /min/ (Unitb/min/

protein) mg protein)







Each value is mean ± S.D. for six rats in each group. Values not sharing a common superscript (a-d) differsignificantly with each other (P < 0.05, DMRT).

Table 4Effect of Hesperidin on the Levels of Vitamin C and E in Plasma and Liver in Normal and

Streptozotocin (STZ)-induced Diabetic Rats

Groups Vitamin C ( mol/mg) Vitamin E ( mol/mg)Plasma Liver Plasma Liver







Each value is mean ± S.D. for six rats in each group. Values not sharing a common superscript (a-d) differ significantlywith each other (P < 0.05, DMRT).


Histopathological changes were observed in the liver of normal STZ-induced rats. Theliver samples of STZ induced animals showed sinusoidal-dilation, fatty changes of microvesicular type and infiltration of inflammatory cells along the portal triad. On the other handSTZ with hesperidin (25 and 50 mg/kg) treated rat liver exhibited a few hepatocytes withvesicular appearance, mild dilation and limited fatty changes and near normal architecture.Administration of hesperidin to normal rats did not have any histopathological changes in theliver. This indicates that hesperidin does not possess any adverse effects under normal conditions.

For all the parameters studied, oral administration of hesperidin (25 and 50 mg/kg) tonormal rats for a period of 35 days showed a minor effect, but it was not statistically significant(P < 0.05). Hesperidin at a dose of 50 mg/kg showed a better effect than 25 mg/kg inISO-induced MI in rats.

4. DISCUSSION

Streptozotocin (STZ), N-[methylnitrocarbamoyl]-D-glucosamine is a potent methylating agentfor DNA and acts as nitric oxide donor in pancreatic β-cells and thus β-cells are more sensitiveto damage by nitric oxide and free radical scavenging enzymes. STZ specifically induces DNAstrand breakage in β-cells causing diabetes (Calabresi and Chabner, 1987). Oxygen derivedfree radicals generated in excess in response to various stimuli can be cytotoxic to severaltissues. Most of the tissue damage considered to be mediated by these free radicals by attackingmembranes through peroxidation of poly unsaturated fatty acids (PUFA). The increase in oxygenfree radicals in diabetes could be primarily due to increase in blood glucose levels, which uponautoxidation generate free radicals in diabetes (Ivorra et al., 1989).

In this study, we have observed elevated levels of lipid peroxides (TBARS and HP) inplasma and liver in STZ-induced diabetic rats is due to an increase in the generation of freeradicals by STZ. Auto-oxidation of glucose and the increased susceptibility of the tissues ofthe diabetic animals may be due to the activation of the lipid peroxidation system and observedremarkable increase in the concentration of TBARS and HP (Prince and Menon, 2001).Treatment with hesperidin to diabetic rats significantly decreased the levels of TBARS and HPin plasma and liver. It is well known that, administration of flavonoids decreases the levels oflipid peroxides in oxidative stress conditions. In this study, the decreased levels of lipidperoxidative products could be due to free radical scavenging property of hesperidin.

Superoxide dismutase and catalase are the two major scavenging enzymes that remove thetoxic free radicals in vivo. We have observed decreased activities of SOD and catalase werefound to be lower in STZ-induced diabetic rats. SOD scavenges the superoxide radical byconnecting it to H

2O

2 and molecular oxygen. Catalase is a hemeprotein, which catalyzes the

reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxy radicals(Chance, 1952). The decreased activities of SOD and catalase in the liver have been observedduring diabetes and this may results in a number of deleterious effects, due to the accumulationof superoxide radicals and hydrogen peroxides (Rajasekaran et al., 2005). Hesperidin treatmentto STZ-induced diabetic rats significantly increased the activities of these enzymes in liver.This could be due to antioxidant property of hesperidin, which scavenges the free radical andthereby, increased the levels of antioxidants.


In our study, we observed decreased concentration of GSH in plasma and liver and decreasedactivities of glutathione dependent enzymes such as GPx and GST in the liver of STZ-induceddiabetic rats. GSH is an abundant and ubiquitous antioxidant, a tripeptide and essential biofactorsynthesized in all living cells. It is an effective intracellular reductant (Rahman and MacNee,1999). GSH involved in the protection of the biological membranes from free radical mediateddamage caused by drugs and ionizing radiation. It acts as an important substrate for the enzymesGPx, GST, which is involved in the scavenging action free radicals (Jagetia et al., 2004). GPxis a major enzymatic mechanism for the disposal of peroxides, and a prolonged depression ofthis enzyme may lead to the accumulation of intracellular peroxide. GST involved in the removalof stable peroxides from the system, which results in the reduction of peroxide-induced damage(Jagetia et al., 2004). Decreased GSH levels in diabetic rat liver might be due to increasedutilization in protecting sulphur containing proteins from lipid peroxides. The reducedconcentration or unavailability of GSH leads to decreased activities of GPx and GST. Ratstreated with hesperidin significantly increased the levels of GSH in plasma and the liver andthe activities of GPx and GST in STZ-induced diabetic rats.

In this study, we have observed decreased levels of vitamin C and E in plasma and liver inSTZ-induced diabetic rats. Vitamin C is a water-soluble vitamin that directly involved in thescavenge action of singlet oxygen, superoxide and hydroxyl radicals (Benidich et al., 1986).Vitamin E interrupts the chain reaction of lipid peroxidation by reacting with lipid peroxylradicals, thus protecting the cell structures against damage. It inhibits lipid peroxidation andregenerates reduced vitamin C and GSH. The decreased levels of ascorbic acid and α-tocopherolcould be due to increased utilization of antioxidant vitamins as an antioxidant defense againstincreased ROS or to a decrease in the GSH level, since GSH is required for the recycling ofascorbic acid (Rajadurai and Prince, 2006). Hesperidin treated rats significantly increased thelevels of vitamin C, vitamin E in plasma and liver of STZ-induced diabetic rats, which couldbe due to antioxidant property of hesperidin.

The liver samples of STZ induced rats showed sinusoidal-dilation, fatty changes of microvesicular type and infiltration of inflammatory cells along the portal triad. The rats treatedwith hesperidin exhibited a few hepatocytes with vesicular appearance, mild dilation and limitedfatty changes, which is near normal architecture of rat liver. This could be due to membranestabilizing capacity of hesperidin, which helps to protect the biological membrane fromSTZ-induced oxidative damage. Administration of hesperidin to normal rats did not cause anyhistopathological alterations in the liver, which indicates that hesperidin does not possess anyadverse effects under normal conditions.

5. CONCLUSION

In conclusion, the results obtained from our study indicate that hesperidin offers protection tothe liver by decreasing the levels of lipid peroxides and maintaining the levels and activities ofnon-enzymatic and enzymatic antioxidants in STZ-induced diabetic rats. This could be due toits free radical scavenging, antioxidant as well as membrane-stabilizing property of hesperidin.Our histopathological studies on liver also support these biochemical findings in STZ-induced


rats. Thus, this study may have a significant impact on the clinical treatment of diabetes. Onthe basis of our previous and present findings, we report that hesperidin could be an effectiveagent against STZ-induced diabetes in rats.

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