Protective Effect of Punica granatum Peel and Vitis vinifera Seeds on DEN-Induced Oxidative Stress and Hepatocellular Damage in Rats

Protective Effect of Punica granatumPeel and Vitis vinifera Seeds on DEN-Induced OxidativeStress and Hepatocellular Damage in Rats

Ashok K. Kumar & Vijayalakshmi K

Received: 6 May 2014 /Accepted: 23 September 2014# Springer Science+Business Media New York 2014

Abstract This study was designed to find out the efficacy of ethanol extracts of Punicagranatum peel and Vitis vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stressand hepatocellular damage in Wistar rats. Rats were divided into four groups. The first groupserved as normal control, and the second group received DEN at a dose of 200 mg/kg bodyweight by single intraperitoneal administration. The third one received DEN as in DEN-treatedgroup and co-treated with 400 mg/kg P. granatum peel extract. The final group also receivedDEN and co-treated with 400 mg/kg V. vinifera seed extract. DEN administration to ratsresulted in significantly elevated levels of serum SGPT, SGOT, ALP, and GGT which isindicative of hepatocellular damage. DEN-induced oxidative stress was confirmed by elevatedlevels of lipid peroxides and decreased activities of superoxide dismutase, catalase, andglutathione peroxidase in the serum and liver tissues. The status of non-enzymatic antioxidantslike vitamin C, vitamin E, and reduced glutathione were also found to be decreased in serumand tissues of DEN-administered rats. Co-treatment with the P. granatum peel and V. viniferaseed extracts orally for 12 weeks significantly reversed the DEN-induced alterations in theserum and liver tissues.

Keywords Punica granatum .Vitis vinifera . Antioxidant . Diethylnitrosamine . Hepatocellulardamage

Introduction

Hepatocellular carcinoma (HCC) is one of the most common and frequent malignancythroughout the world and is the fifth most common cancer and the third leading cause ofcancer mortality in the world [1] with an estimated 560,000 newly diagnosed cases per year [2,3]. HCC is the malignant transformation of hepatic cells and is a common complication of

Appl Biochem BiotechnolDOI 10.1007/s12010-014-1276-5

A. K. Kumar (*)Department of Biotechnology, Arulmigu Meenakshi Amman College of Engineering, Vadamavandal, 604410 Thiruvanamalai District, Tamil Nadu, Indiae-mail: [email protected]

V. KDepartment of Biochemistry, Bharathi Women’s College, 600 108 Chennai, Tamil Nadu, India

chronic hepatitis mainly caused by hepatitis B virus (HBV) and hepatitis C virus (HCV)infection in China [4]. The factor that induces the formation of HCC includes exposure toenvironmental carcinogens, iron overload, fatty liver diseases, and alcohol abuse [5].

Diethylnitrosamine is an N-nitroso alkyl compound, categorized as a potent hepatotoxinand hepatocarcinogen in experimental animals, intiating reproducible tumors on administration[6–8]. As oxidative stress plays a central role in diethylnitrosamine-induced hepatotoxicity, theuse of antioxidants would offer better protection to counteract liver damage [9]. Since theincrease in the use of synthetic chemicals in cancer therapy has led to many side effects andundesirable hazards, there is a worldwide trend to use natural resource which is therapeuticallyeffective, culturally acceptable, and economically within the reach of the poor people [10]. Thereduced cancer risk and lack of toxicity associated with high intake of natural products suggestthat specific concentrations of phytochemicals from these plant sources may produce cancerchemopreventive effects without causing significant levels of toxicity.

Diets rich in fruits and vegetables have been associated with reduced risk of developingchronic diseases such as cardiovascular disease, cancer, and diabetes [11]. The Punicagranatum and Vitis vinifera are common fruits consumed throughout the world. The peeland seeds of the mentioned fruits respectively are the waste materials of food processingindustries. Both the fruit wastes are rich source of polyphenols, potent antioxidants that includephenols, flavonoids, and tannins. Flavonoids and phenolic compounds widely distributed inplants which have been reported to exert multiple biological effect, including antioxidant, freeradical scavenging abilities, and anti-inflammatory and anticarcinogenic effects [12].

The present study aims to carry out a systematic investigation on the protective effect ofP. granatum peel and V. vinifera seeds on diethylnitrosamine (DEN)-induced oxidative stressand hepatocellular damage by analyzing serum glutamate pyruvate transaminase (SGPT),serum glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) levels along with the non-enzymatic and enzymatic antioxidants.

Materials and Methods

Plant Material

The P. granatum and V. vinifera belongs to families Lythraceae and Vitaceae, respectively.These fruits were collected from the Koyambedu Market, Chennai, India from the samecultivar. The plants were identified and authenticated by Dr. P. Jayaraman, Director of NationalInstitute of Herbal Science, Plant Anatomy Research Centre, Chennai. These voucher speci-mens are maintained in Plant Anatomy Research Centre, Chennai (PARC/2009/360 andPARC/2009/361).

Drugs and Chemicals

Diethylnitrosoamine, phenobarbital, hydrogen peroxide, ascorbic acid, α-tocopherol acetate,and glutathione were obtained from Sigma Chemicals. All other chemicals used for this studywere of analytical grade.

Preparation of Extract

P. granatum peel and V. vinifera seeds were removed from the fruits and washed thoroughlyusing sterilized water. The selected plant materials were shade-dried and pulverized to fine

Appl Biochem Biotechnol

powder individually in a mechanical grinder. One hundred grams of coarse powder wasweighed and extracted with ethanol by keeping it for 24 h in the orbital shaker at roomtemperature [13]. These extracts were collected after filtration using Whatman No. 1 filterpaper. The solvent was concentrated under reduced pressure in a rotary evaporator until all thesolvent is completely evaporated from the extract. The alcoholic extracts obtained were usedfor further studies.

Selection of Animal

Male Wistar rats (150–200 g) were obtained from Kings Institute, Chennai, India, and usedfor the studies. The animals were housed in cages and maintained under standard condi-tions of humidity, temperature (25.2 °C), and light (12-h light/12-h dark) at BRULAC,Saveetha University, Chennai, India. They were fed with standard pelleted diet (M/sPranav Agro Industries Ltd., India) and had free access to water. Experimental animalswere handled according to the university and institutional legislation, regulated by thecommittee for the purpose of Control and Supervision of Experiments on Animals(CPCSEA), Ministry of Social Justice and Empowerment, Government of India (IAECNo. Biochem BWC.004/2009).

Experimental Design

Hepatic cancer was induced in rats using DEN (200 mg/kg body weight by i.p.), and after2 weeks, the carcinogenic effect was promoted by 0.05 % phenobarbital, which was supple-mented to the experimental animals through drinking water for 20 successive weeks.

The experimental animals were divided in to four groups of seven each.

Group I Normal vehicle controlGroup II Hepatic cancer induced in rats using DEN (200 mg/kg body weight by single i.p.

injection). Phenobarbital, which was supplemented to the experimental animalsthrough drinking water for 20 successive weeks

Group III Cotreatment with P.granatum peel extract (EPGP) (400 mg/Kg body weight) byoral gavage for 12 weeks.

Group IV Cotreatment with V. vinifera seed extract (EVVS) (400 mg/kg body weight) byoral gavage for 12 weeks

Collection of Samples

Collection of Blood and Preparation of Liver Homogenate

All the experimental animals were sacrificed by cervical decapitation after the experimentalperiod. Blood was collected, and serum was separated by centrifugation before the sacrifice ofanimal and used for further studies. Liver homogenate was prepared according to the methoddescribed by El-Demerdash et al. [14]. The abdominal cavity of rats was dissected immedi-ately after sacrifice, and the liver was rapidly removed. This liver were excised, washed withice-cold 0.9 % sodium chloride (w/v) to remove the blood, cut in to small pieces by finescissors, and then homogenized (10 % w/v) separately in ice-cold 1.15 % potassium chloride,0.01 M sodium phosphate buffer, pH 7.4 with a Potter–Elvehjem glass homogenizer. The


homogenate was centrifuged at 10,000×g 20 min at 4 °C. Supernatant of the liver homogenatewas collected into sterilized tubes and stored at −20 °C for further studies.

Biochemical Studies

Determination of Serum Markers of Liver Damage

SGOT and SGPT activities were determined in rats using the method described by Mohun andCook [15]. Serum alkaline phosphatase activity was assayed by the method of King andArmstrong [16]. GGT level in serum was estimated using kit supplied from Agappe Diag-nostics, Kerala, India, and the assay was carried out following standard procedure provided inthe kit manual.

Lipid Perioxides and Antioxidant Enzyme Determination

Lipid peroxides content was measured using a thiobarbituric acid assay as reported anddescribed earlier by Ohkawa et al. [17] and expressed as level of malondialdehyde (MDA).The activity of superoxide dismutase was determined by the method of Marklund andMarklund [18]. The catalase activity was assayed by the method of Sinha [19]. The activityof GPx was assayed by the method of Rotruck et al. [20]. The concentration of reducedglutathione was measured by the method of Moron et al. [21]. The concentration of ascorbicacid and Vitamin E were estimated by the methods of Omaye et al. [22] and Desai [23].

Statistical Analysis

Data were analyzed with SPSS version 20 (IBM, USA). The test values were analyzed usingone-way analysis of variance (ANOVA), followed by least significant difference (LSD) test.Statistical significance was defined as p<0.05. All results were expressed as mean±standarddeviation. Graphs for this study were plotted using GraphPad Prism version 6.02.

Result and Discussion

Lipid peroxidation (LPO) brings peroxidative loss of unsaturated lipids, thus bringing aboutcellular lipid degradation and membrane disordering. Reactive oxygen species (ROS) resultsin lipid peroxidation and subsequently increase in thiobarbituric acid reactive substances(TBARS) levels. Elevated lipid peroxidation causes degradation of cellular macromoleculesleading to tissue damage [24]. The increased level of LPO in group II cancer-bearing animalsmay be due to free radicals produced by DEN administration [25]. Administration of DEN hasbeen reported to generate LPO products in general [26], and phenobarbital enhanced theformation of the activated oxygen species in the preneoplastic nodules [27] in rat liver. Theadministration of DEN and phenobarbital has shown to increase the levels of LPO during ofhepatocarcinogenesis. MDA, which is a major end-product and an index of LPO, cross-linkswith protein and nucleotides on the same and opposite strands. Figures 1 and 2 show the effectof extracts on LPO level in liver and serum of animals. Nevertheless, administration of twoselected extracts, significantly decreased the level of LPO in group III (p<0.001 in liver andp<0.01 in serum) and group IV (p<0.001 in liver and p<0.05 in serum) animals whencompared to group II animals. The level of LPO was increased in HCC-induced animals of


group II when compared to control group. LPO can be prevented at the initiation stage by freeradical scavengers and antioxidants defense system of cells [28].

From the qualitative and quantitative phytochemical analysis, it was clear that both the fruitextracts selected for studies contain relatively high amount of flavonoids which may inhibit theproduction of LPO. The flavonoid content of P. granatum peel and V. vinifera seeds werefound to be 144.27±5.34 and 59.59±3.48 mg/g, respectively [29, 30].

Gamma-glutamyl transferase plays a key role in the production and degradation of gluta-thione and may also be involved in drug and xenobiotic detoxification [31]. Gamma-glutamyltransferase is located in the membranes of the cells of many tissues, and active site is presenton the outer surface of the membrane. Chemical carcinogens that enter the liver may initiatesome systematic effects thereby synthesize certain tumor markers in the system which maylead to the induction of gamma-glutamyl transferase synthesis [32]. An elevation in gamma-glutamyl transferase was observed in cancerous condition [33]. This elevation of gamma-

Fig. 1 Effect of the EPGP andEVVS on the lipid peroxidationin the liver of control and experi-mental animals. Values areexpressed as mean±SD; n=7 ani-mals. Comparisons are made be-tween group I (a, control rats) andgroup II (b, DEN-treated rats).Statistical significance: *p<0.001and **p<0.01; NS non-significant

Fig. 2 Effect of the EPGP andEVVS on the lipid peroxidationin serum of control and experi-mental animals. Values areexpressed as mean±SD; n=7 ani-mals. Comparisons are made be-tween group I (a, control rats) andgroup II (b, DEN-treated rats).Statistical significance: *p<0.001,**p<0.01, and ***p<0.05


glutamyl transferase activities shows the stages of carcinogenic process, since its levels arecorrelated with growth rate, histological differentiation, and survival time of the host [34].

Figure 3 shows the activities of marker enzymes in serum of test and control animals. Theactivities of marker enzymes were elevated (p<0.001) in serum of animals in group II whencompared to control (group I). The activities of liver marker enzymes (SGPT, SGOT, ALP, andGGT—p<0.001) were significantly decreased in group III and group IV animals whencompared to group II animals. Increased transaminases activity in HCC is reported by [35].Several studies reported elevation in the activities of SGOT and SGPT after DEN administra-tion [36]. Administration of aqueous extract of Andrographis paniculata to mice sufferingfrom liver damage induced by benzene hexacholorocyclohaxane significantly lowered theraised enzymes [37]. V. vinifera root extract showed hepatoprotective effect on carbon tetrachloride (CCl4)-treated rats. The elevated level of marker enzymes SGPT, SGOT, and ALP inCCl4-treated rats were significantly reversed when treated with V. vinifera root extract [38].Lagerstroemia speciosa extract showed hepatoprotective activity against CCl4-inducedhepatotocixity and significantly lowered the levels of SGPT, SGOT, and ALP enzymeselevated in CCl4 toxicity [39]. This study indicates that the selected two extracts prevent liverdamage by maintaining the integrity of the plasma membrane, thereby suppressing the leakageof enzymes through membranes, exhibiting hepatoprotective activity. This might be the reasonfor the restoration in the activities of the marker enzymes on administration of EPGP andEVVS.

Studies has shown that hepatic metabolism of DEN generates reactive oxygen species(ROS) resulting in oxidative stress and cellular damage [40]. Free radicals are regularlyproduced in vivo as a result of carcinogen treatment causing oxidative stress that leads todamage of nucleic acids, proteins, and lipids resulting in chromosomal instability, mutations,loss of organelle function, and membrane damage which play an important role in thedevelopment of cancer [41]. In the present investigation, DEN induces hepatocellular damageand this is clearly evidenced by the marked decrease in activity of serum superoxide dismutase(SOD), catalase, and glutathione peroxidase (GPx). Table 1 presents the activities of enzymaticantioxidants in serum of control and experimental animals. The enzymatic antioxidants such asSOD, catalase, and GPx were significantly (p<0.001) reduced in group II when compared withgroup I. The extract-treated groups III and IV were shown to possess markedly increasedactivity of antioxidant enzymes when compared to group II.

The V. vinifera seeds show hepatoprotective action against acetaminophen-induced toxicityand the decreased level of enzymatic antioxidants SOD, catalase, and GPx which wereincreased on treating with V. vinifera seeds [42]. The SOD and catalase activities increasedon treatment with L. speciosa roots against CCl4-induced hepatotoxicity [40]. The endogenous

Fig. 3 Effect of EPGP and EVVSon the activities of marker en-zymes in serum of control and ex-perimental animals. Values areexpressed as mean±SD; n=7 ani-mals. Comparisons are made be-tween group I (a, control rats) andgroup II (b, DEN-treated rats).Statistical significance: *p<0.001


antioxidant system may counteract the ROS and reduce the oxidative stress with the enzymaticantioxidants like SOD, catalase, and GPx. The SOD dismutase superoxide radicals (O2

−) werecatalyzed into hydrogen peroxide (H2O2) and O2. Catalase further detoxifies H2O2 to H2O.Thus SOD, catalase, and GPx act mutually and constitute the enzymatic antioxidant defensemechanism against reactive oxygen species [43]. The decrease in the activities of theseenzymes in the present study could be attributed to the excessive utilization of these enzymesin deactivating the free radicals generated during the metabolism of DEN. Figures 4 and 5show changes in activities of enzymatic antioxidants in the liver tissues of experimentalgroups. The enzymatic antioxidants such as SOD, catalase, and GPx were significantly(p<0.001) reduced in group II animal when compared with group I animals. P. granatum-and V. vinifera-treated groups showed increased activities (p<0.001) of enzymatic antioxidantscompared with group II animals.

The increased superoxide radical levels in tumor cells as compared with normal cells mayexplain the decrease in the enzymatic activity in malignant tissues than in normal tissues. In thepresent study, the decreased level of SOD as observed in cancer-bearing animals may be due tothe utilization of the enzyme to scavenge H2O2 radicals. GSH is converted to oxidizedglutathione (GSSG) by the enzyme GPx for scavenging accumulated H2O2. The reductionof GSSG to GSH is catalyzed by glutathione reductase (GR) using NADPH as reducingpotential. Catalase is a hemoprotein that requires NADPH for its regeneration to active form[44]. Catalase is mainly peroxisomal, whereas the GR/GPx cycle is active in the cytoplasm.The antioxidant phytochemicals such as flavonoids, alkaloids, tannins, sterols, phlobotannins,and glycosides present in the selected extracts may act as free radical scavengers.

Table 1 Effect of EPGP and EVVS on the activities of enzymatic antioxidants in serum of control andexperimental animals

S. No Groups SOD Catalase GPx

1 G1 4.91±0.04 34.48±0.70 4.13±0.05

2 G2 3.06±0.07 a* 19.26±1.04 a* 3.12±0.10 a*

3 G3 4.33±0.05 a*, b* 28.63±1.26 a*, b* 3.94±0.06 a*, b*

4 G4 4.16±0.07 a*, b* 26.21±1.16 a*, b* 3.66±0.09 a*, b*

Values are expressed as mean±SD; n=7 animals. Comparisons are made between group I (a, control rats) andgroup II (b, DEN-treated rats). Units: SOD—U/mg of protein, GPx—μ mol of GSH/min/mg of protein, andCatalase—μmol of H2O2 consumed/min/mg of protein

*p<0.001

Fig. 4 Effect of EPGP and EVVSon the activities of SOD and GPxin the liver tissue of control andexperimental animals. Values areexpressed as mean±SD; n=7 ani-mals. Comparisons are made be-tween group I (a, control rats) andgroup II (b, DEN-treated rats).Statistical significance: *p<0.001and **p<0.01. Units: SOD—U/mg of protein, GPx—μmol ofGSH/min/mg of protein


Glutathione is one of the most copious compounds in the body which act as a cellularreductant, a metabolic controller, and sensitive marker of health, and its biological functionsdepends mainly due to the thiol group of its cysteinyl residue [45]. GSSG is formed by fusingtwo reduced glutathione molecules by their –SH groups. Glutathione peroxidase converts thereduced form of glutathione (GSH) to the oxidized form (GSSG) of glutathione. Reducedglutathione (GSH) has the potential to scavenge the free radicals, and enzymes like GST andGPx also scavenge the hydroxyl radicals and thereby prevent the initial damage to themacromolecules [46]. When there is a decrease in the levels of glutathione, the levels of theascorbic acid and alpha-tocopherol are also decreased and thereby, the antioxidant status alsoget diminished, and this leads to the enhanced lipid peroxidation which may further lead to cellsuicide by the process called apoptosis [47]. Glutathione and these vitamins are tightly linkedto each other in a way that it helps to replenish ascorbic acid which in turn regenerates alpha-tocopherol. In the present investigation, Glutathione level was decreased in the DEN-inducedcancer-bearing animals. The groups treated with the selected extracts showed significantincrease in the concentration of glutathione when compared with HCC-induced groups. Thelevel of intracellular GSH was significantly increased on treatment with L. speciosa rootswhich was depleted in the CCl4-induced hepatotoxicity in rats [39]. V. vinifera seeds showhepatoprotective action against acetaminophen-induced toxicity. The level of GSH

Fig. 5 Effect of EPGP and EVVSon the catalase activity in the livertissue of control and experimentalanimals. Values are expressed asmean±SD; n=7 animals. Compar-isons are made between group I (a,control rats) and group II (b, DEN-treated rats). Statistical signifi-cance: *p<0.001

Table 2 Effect of EPGP and EVVS on the levels of non-enzymatic antioxidants in serum of control andexperimental animals

S. No Groups GSH Vitamin C Vitamin E

1 G1 5.29±0.07 1.70±0.07 1.99±0.05

2 G2 3.23±0.26 a* 1.12±0.07 a* 1.14±0.09 a*

3 G3 4.56±0.16 a*, b* 1.52±0.05 a*, b* 1.77±0.06 a*, b*

4 G4 4.29±0.16 a*, b* 1.50±0.08 a*, b* 1.60±0.08 a*, b*

Values are expressed as mean±SD; n=7 animals. Comparisons are made between group I (a, control rats) andgroup II (b, DEN-treated rats). Units: GSH—μg/mg proteins, vitamin C and vitamin E—mg/dL

*p<0.001


significantly increased on treatment with V. vinifera seeds in acetaminophen-induced toxicity[38]. DEN was proved to be a potent liver carcinogen which depletes the level of glutathione.There are numerous reports that fruits, vegetables, several herbs, and plants have the diversi-fied pharmacological functions which can inhibit this process [48–50]. In this present study,treatments with both the selected extracts have significantly augmented the glutathione level.Vitamin E is otherwise known as alpha-tocopherol. It is a well-recognized, important biolog-ical free radical scavenger in the cell membrane [51]. Alpha-tocopherol stabilizes the biolog-ical membranes by preventing the apoptotic process and act on all steps of membraneoxidative damage [52, 53]. During the antioxidant reaction, alpha-tocopherol is converted toan alpha-tocopheroxyl radical by the donation of labile hydrogen to a lipid or lipid peroxylradical. Alpha-tocopherols can either initiate or inhibit apoptosis [54]. Alpha-tocopherolcontent in group II was significantly decreased, and this might be due to the excessiveutilization of this antioxidant for quenching enormous free radicals produced in these condi-tions. Alpha-tocopherol has the ability to penetrate to an exact site into the membrane, whichmay be the important feature of defense against highly reactive radicals [55]. The in vitroantioxidant assay showed both the selected extracts possess antioxidant potential againstHepG2 cell lines, and GC-MS analysis shows presence of alpha-tocopherol-beta-D-mannoside,

Fig. 6 Effect of EPGP and EVVSon the levels of vitamin C andvitamin E in the liver of controland experimental animals. Valuesare expressed as mean±SD; n=7animals. Comparisons are madebetween group I (a, control rats)and group II (b, DEN-treated rats).Statistical significance: *p<0.001

Fig. 7 Effect of EPGP and EVVSon the level of GSH in the liver ofcontrol and experimental animals.Values are expressed as mean±SD;n=7 animals. Comparisons aremade between group I (a, controlrats) and group II (b, DEN-treatedrats). Statistical significance:*p<0.001; NS non-significant


a vitamin E derivative in both the selected extracts which might plays a major role in itsantioxidant potential and increased level of vitamin E in extract-treated experimental animals[56, 57].

Vitamin C, otherwise known as ascorbic acid, is a strong reductant which protects themembrane of the cell and also prevents the lipoprotein particles from oxidative damage andreplenishes the antioxidant from vitamin E. Ascorbic acid and alpha-tocopherol can also spareglutathione and prevent its oxidation [58]. Ascorbic acid removes free radicals from cytosol byreacting directly with them [59]. In the present study, the significantly decreased level(p<0.001) of ascorbic acid found in group II (DEN-induced) animals may be due to theutilization of antioxidant to scavenge the free radicals. The availability of ascorbic acid is adetermined factor in controlling and potentiating many aspects of host resistance againstcancer. But these non-enzymatic antioxidants levels were found to be significantly increased(p<0.001) in both serum (Table 2) and liver homogenate (Figs. 6 and 7) of the animals treatedwith EPGP and EVVS (group III and group IV) when compared to group II animals. Thetreatment has enhanced various cellular antioxidant contents in tissues, which in turn reducesfree radical-mediated cellular damage. The selected extracts possess the antioxidant property,and it could have been attributed to the maintenance of the tocopherol, ascorbic acid, andendogenous glutathione antioxidant balance against DEN-induced hepatic carcinoma.

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