in-vivo and in-vitro antioxidant potential of fruits of...

Journal of Pharmacy Research Vol.5 Issue 4.April 2012

Mehta D.K et al. / Journal of Pharmacy Research 2012,5(4),2031-2034

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Research ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author.Dinesh Kumar MehtaSri Sai College of Pharmacy,Badhani,Pathankot -145001,Punjab,India

INTRODUCTION

In-Vivo and In-Vitro Antioxidant Potential of Fruits of Zanthoxylum armatum D.C1*Mehta D.K, 2Bhandari A, 3Satti N.K, 3Singh S, 1Das R and 3Suri K.A

1Sri Sai College of Pharmacy, Badhani, Pathankot(pb)- 145001, India.,2JNU, Jodhpur (Raj), India.3IIIM, Jammu (J&K), India.

Received on:06-01-2012; Revised on: 14-02-2012; Accepted on:23-03-2012

ABSTRACTIn the present study in vitro and in vivo antioxidant activity of methanolic extract (ME) of Zanthoxylum armatum ( ZA) fruit was investigated.The studywas carried out using male wistar rats (180-230g). In the present study, antioxidant potential of the methanol extract of ZA fruit was evaluated by usingDPPH method for in-vitro and in- vivo antioxidant activity was investigated using wistar albino rats.The crude methanolic extract prepared from the ZAfruits showed significant (P< 0.05) in- vivo antioxidant activity. After seven days oral feeding of the methanol extract (100 and 200 mg/kg b.wt) resultedin the significant (P< 0.05) reduction of hepatic thiobarbutiric acid reacting substances (TBARS) levels which were elevated by CCl4 challenge in rats.Antioxidant enzymes viz. glutathione peroxidase, catalase, super oxide dismutase levels in liver were increased significantly (P< 0.05) and increased in theformation of malondialdehyde (MDA) in untreated positive control animals. The ME showed significant activities in DPPH radical scavenging assaycompared to the reference antioxidant ascorbic acid in a dose dependent manner. ME of ZA fruit at a dose level of 100mg/kg and 200mg/kg producesignificant (P<0.05) antioxidant activity by decreasing the activity of lipid peroxidation, while they significantly increased the levels of glutathioneperoxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) in a dose dependent manner. The effects of ME of ZA were comparable to that ofstandard drug, Silymarin . From this study, it can be concluded that the ME of ZA fruit possesses significant (p<0.05) antioxidant activity.

Key words: Zanthoxylum armatum ,methanolic extract, Antioxidant activity, TBARS, Antioxidant enzymes

Antioxidants are supposed to protect cell membranes against free radicaloxidative damages [1, 2]. In plants, the term antioxidant often refers to a widerange of phenolic compounds that vary from simple phenolic acids to highlypolymerized compounds such as tannins. Phenolic compounds, or polyphe-nols, are categorized into 15 main classes with over 8000 identified com-pounds. The largest category is the flavonoid group, comprising 13 classeswith over 5000 compounds [3, 4, 5]. In plants, polyphenols are important forstructural support, as ant herbivorous substances, for attracting pollina-tors, for protection from ultraviolet radiation and for wound repair (4). Thehuman body also synthesizes endogenous antioxidants such as superoxidedismutases, glutathione peroxidases, alpha-tocopherol and melatonin tocounteract cellular damage by active oxygen and free radicals [6, 7]. In thegiven literature consumption of a variety of plant foods may provide addi-tional health benefits [7, 8]. Strict legislation on the use of synthetic foodadditives and consumer preferences has also shifted the attention of manu-facturers from synthetic to natural antioxidants [9].

Zanthoxylum Armatum (TUMBURU) Tumburu consists of dried fruitof Zanthoxylum armatum DC. Syn. Z. alatum Roxb. Zanthoxylum armatumDC. belongs to family Rutaceae. Commonly known as ‘timur’ or ‘NepaliDhania’. An armed or erect shrub or small tree found in Punjab along withvalleys of the Himalayas at an altitude of 1000 to 2100m at Poonch district(J&K) in Khasia hills at 600 to 1800 m, and in the Ghats in peninsular India.Kumaon 5000-7000 ft, eastward to Bhutan [10]. To the fruit is attached aportion of stalk [11]. Fruits of Z. armatum are a well-known ayurvedic medi-cine. The fruit and seeds are employed as aromatic and tonic, in fever,

dyspepsia and expelling round worms (14). Fruit, branches and thorn areused to cure toothache and other diseases of teeth. It is considered good forAsthma (12). Bark powder mixed with honey gives relief against gum bleed-ing (13). Previous investigations on Z. armatum fruits have revealed thepresence of the major constituents are linalool methyl cinnamate, ß-phelendrene(15,16) Undecane 2-one and Flavonoids. Alkaloids, terpenoids, (17)

volatile oil which is about 10-12% known as wartara oil. The plant has beenreported to possess antioxidant[18], antinociceptive[18], anti-inflamma-tory[18,19], natural pisciside [20], Antimicrobial [21], antihelmintic [21],Hepatoprotective [22],Antiproliferative[24], and antifungal activities[25].

But, since no systematic study of the antioxidant activity of the Z. armatumfruits have been reported so far. Keeping this in view, the present study hasbeen undertaken to investigate antioxidant power of methanol extract offruits of Z.A DC, which was performed in vitro by the DPPH and in vivoantioxidant activity in CCl4 induced hepatotoxicity model in rats.

MATERIALS AND METHODS:Fruits of Zanthoxylum armatum were collected from, Poonch district, J&K,India and plant material was identified by Dr. S.N Sharma of Department ofBotany, IIIM, Jammu. A voucher specimen (20536) was deposited in theDepartment for future records. The fruit was dried under shade and make topowder.

Preparation of Methanolic extract:The shade dried coarse powder of the fruits of Zanthoxylum armatum (1kg) was extracted with methanol by cold maceration process at room tem-perature for three days. Then the extract was filtered and concentrated witha rotary evaporator and kept in a refrigerator.

Phytochemical Test:The freshly prepared ME of Z.armatum was subjected to standard phy-tochemical screening tests for various constituents.

Animals:Male wistar rats (180-230g) are used. The animals were housed under stan-



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dard environmental conditions. The animals were grouped and housed inpolyacrylic cages (38 x23 x10 cm) with not more than six animals per cageand maintained under standard laboratory conditions (temperature 25 ±20C) with dark and light cycle (12/12 h). The animals were fed with stan-dard pellet diet supplied by Hindustan Lever Ltd. Kolkata, India and freshwater ad libitum. All the animals were acclimatized to laboratory conditionfor a week before commencement of experiment.All experiment were car-ried out after getting the approval from the committee for the purpose ofcontrol and supervision of experimental animals (CPCSEA) having theregistration number is 1349/AC/10/CPCSEA.

Drugs: DPPH (1, 1-diphenyl, 2-picrylhydrazyl) was obtained from SigmaChemical Co. USA, Ascorbic acid was obtained from SD Fine Chem.Ltd.,Silymarin was purchased from Micro labs,Tamilnadu. India, Thiobarbituricacid was purchased from Central Drug House, New Delhi, India and therest of the chemicals utilized were of analytical grade and were obtainedfrom Sisco research laboratories,Mumbai, India.

Antioxidant Evaluation:In-vitro Antioxidant activity using DPPH method

DPPH radical scavenging activity: In order to measure antioxidant activ-ity DPPH free radical scavenging assay method was used and comparedwith (IC50) of Ascorbic acid as standard. This assay measures the freeradical scavenging capacity of the extract under investigation

Hydrogen-Donating Activity Hydrogen donating activity was quantifiedin presence of stable DPPH radical as per the process described by Blois etal [26], on the basis of Blois method. Briefly, the methanol extract of Zan-thoxylum armatum was diluted to final concentrations of 50,100,150,200,250and 300 µg/ml in methanol. One ml of DPPH solution (0.2 mM/ml inmethanol) was added to 2.5 ml of sample solutions at different concentra-tions. Reaction mixture was shaken and after 30 min at room temperature,the absorbance values were measured at 518 nm and converted into per-centage of antioxidant activity (% AA). Ascorbic acid was used as standard.The degree of discoloration indicates the scavenging efficacy of the extract,was calculated by the following equation:% AA = 100 – {[(Abssample – Absblank) x 100] / AbsDPPH}

In- Vivo Antioxidant activity:Animals and Experimental design:Antioxidant activity was evaluated on male wistar albino rats (150-200 g).Animals were randomly allocated into five groups of six animals each.Group I was regarded as Normal control receiving 0.5% CMC (1ml/kg). Allother groups received CCl4 (3 ml/kg, i.p) mixed with an equal volume ofolive oil (50%, by volume). Animals from group III were treated withsuspension of the methanol extract (100 mg/kg/day, p.o. during seven days),whereas animals from group IV were treated with suspension of the metha-nol extract (200 mg/kg/day, p.o. during seven days). Animals from group Vreceived silymarin (25 mg/kg) as the reference drug. The treatment schedulewas once daily for 7 days. All groups except the normal control, weretreated with CCl4 (3ml/kg,p.o.) once daily for 7 days. After treatment, allanimals were sacrificed by cervical dislocation. Further on, the liver wasquickly removed and fixed in a 10% buffered neutral formalin solution andstored under refrigeration.

• Group I: Normal control Carboxy Methyl Cellulose (CMC) 0.5%, 1ml/kg.• Group II: Positive control treated with CCl4 3ml/kg p.o. ip• Group III: Treated with alcoholic extract 100 mg/kg p.o + CCl4 3ml/kg p.o• Group IV: Treated with alcoholic extract 200 mg/kg p.o + CCl4 3ml/kg p.o.• Group V: Treated with standard, silymarin 25 mg/kg p.o + CCl4 3ml/kg p.o.

Liver Homogenates Preparation and Biochemical Estimation:Frozen liver samples were homogenized (with a Potter-Elvehjem homog-enizer) in a Tris-HCl buffer or in a phosphate buffer solution (PBS) to givea 20% homogenate. To evaluate levels of lipid peroxidation (TBARS), thehomogenate was centrifuged at 1700 rpm/min for 10 min at 4 oC. To assesscatalase activity (CAT), the homogenate was centrifuged at 3500 rpm/minfor 15 min at 4 oC and then diluted up to 5%. Further on, the supernatantwas again centrifuged either at 10,000 rpm/min for 1 min and diluted to 2%for measurement of glutathione peroxidase activity (GPX) or at 30,000rpm/min for 10 min before extraction of tissue superoxide dismutase (SOD) activity with 20% ethanol.

Assay of catalase (CAT)Antioxidant enzyme catalase was assayed by the method of Sinha et al. [27]

This activity was expressed as micromoles of hydrogen peroxide utilized/min/mg of protein and recorded in Table: 2 Fig: 5

Table: 1 Free radical scavenging capacity of methanolic extract of Z. armatumConcentration DPPH Scavenging %(µg/ml) Methanol Ascorbic Acid

Extract

50 22.37±1.25 95.17±1.15100 38.25±3.42 -150 65.42±0.85 -200 88.19±3.62 -250 108.29±2.51 -300 122.53±1.36 -IC50 117.4 -

Values are means ± SEM of six determinations

Table: 2 InVivo Antioxidant activity of Zanthoxylum armatum fruit.

Treatment Catalase Superoxide Glutathione TBARS(CAT) dismutase Peroxide

(SOD) (GPX)

Normal Control CMC 272.8±5.6 87.83±5.1 0.87±0.03 1.36±0.0210ml/kg CCl4 151.8±3.0a 28.5±3.4a 0.68±0.02a 1.80±0.03a

( Positive control) 1ml/kgExtract (100 mg/kg oral ) 184.5±3.9b 58.17±3.3b 0.72±0.03 1.64±0.01Extract (200mg/kg oral) 256.2±2.4b 84.17±3.1b 0.89±0.04b 1.34±0.03b

Silymarin Standard 262.7±3.1b 79.17±3.8b 0.87±0.02b 1.37±0.03b

(25mg/kg)

Values are expressed as mean ± SEM, n = 6 in each group. aP<0.05(Statistically significantcompared with normalcontrol),bP<0.05 (Statistically significant compared with CCL4 treated group. One way analysis ofvariance (ANOVA)

Fig.1. In Vitro antioxidant activity of Zanthoxylum armatum fruit

CMC= (Normal control), conc1=Extract (100 mg/kg oral), conc2=Extract (100 mg/kg oral),Std=Silymarin . Values are expressed as mean ± SEM, n = 6 in each group. aP<0.05(Statisticallysignificant compared with norma lcontrol), bP<0.05 (Statistically significant compared withCCL4 treated group. One way analysis of variance (ANOVA)



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Assay of superoxide dismutase (SOD)The antioxidant status was measured by method of Kakkar et al. [28]

Activity was expressed as units/min/mg of protein and recorded in table: 2,Fig: 3

Assay of thiobarbutiric acid substances (TBARS):The malondialdehyde (MDA) content, a measure of lipid peroxidation,was assayed in the form of TBARS by the method of Okhawa et al. [30]

The level of lipid peroxidation was expressed as nono mole ofmalondialdehyde (MDA) per mg of protein and recorded in Table: 2, Fig: 2

Catalase (CAT)(mg/liver protein)-1

Fig.5. Catalase (CAT) levels in the plasma of rats after administration ofmethannolic extract of Zanthoxylum armatum.


Superoxide dismutase (SOD) (mg/liver protein)-1

Fig.3. Superoxide dismutase (SOD) levels in the plasma of rats afteradministration of methannolic extract of Zanthoxylum armatum.

Assay of glutathione peroxidase (GPX)Glutathione peroxidase was assayed by the method of Rotruck et al [29] withsome modification. The activity was expressed as units/ mg of protein andrecorded in Table: 2 Fig: 4


Glutsthione peroxide (GPAX)(mg/liver protein)-1

CMC= (Normal control), conc1=Extract (100 mg/kg oral), conc2=Extract (100 mg/kg oral),Std=Silymarin . Values are expressed as mean ± SEM, n = 6 in each group. aP<0.05(Statisticallysignificant compared with norma lcontrol), bP<0.05 (Statistically significant compared withCCL4 treated group. One way analysis of variance (ANOVA)Fig.4. Glutathione peroxidase (GPx) levels in the plasma of rats afteradministration of methannolic extract of Zanthoxylum armatum.

Thiobarbutiric acid substances (TBARS)(mg/liver protein)-1

Fig.2. Malondialdehyde (MDA) in the plasma of rats after administration ofmethannolic extract of Zanthoxylum armatum.


Statistical analysisThe experimental results were expressed as the Mean ± SEM for six ani-mals in each group. The biochemical parameters were analysed statisticallyusing one-way analysis of variance ANOVA, followed by Dunnett’s mul-tiple comparison test (DMCT). P value of < 0.05 was considered as statis-tically significant.

RESULT AND DISCUSSION:The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity ofZ. armatum is given in Fig.1 This activity was increased by increasing theconcentration of the sample extract. DPPH antioxidant assay is based onthe ability of 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radi-cal, to decolorize in the presence of antioxidants. The DPPH radical con-tains an odd electron, which is responsible for the absorbance at 518 nm andalso for a visible deep purple color. When DPPH accepts an electron do-nated by an antioxidant compound, the DPPH is decolorized, which can bequantitatively measured from the changes in absorbance. The IC50 value ofthe extract was 117.40µg/ml, as opposed to that of ascorbic acid (IC50 95.17µg/mL), which is a well known antioxidant.

In-Vivo Anti-oxidant activity:Liver injury induced by CCl4 is the best characterized system of chemical-induced hepatotoxicity and is a commonly used model for screeninghepatoprotective drugs [31].

Many studies have investigated the role of antioxidant drugs and plant-derived compounds in the prevention of oxidative stress. In the presentstudy, the methanolic extract of Z armatum fruits was evaluated for in vivoantioxidant activity using CCl4-intoxicated rats. CCl4 is a widely used toxi-cant for experimental induction of liver toxicity in laboratory animals [32]. Itinduces lipid peroxidation in experimental animals and causes severe he-patic injury [33]. CCl4 is converted to the reactive trichloromethyl radical(CCl3), which readily interacts with molecular oxygen to form thetrichloromethyl peroxy radical (CCl3O2). These free radicals bind covalentlyto macromolecules and induce peroxidative degradation of the membranelipids of the endoplasmic reticulum rich in polyunsaturated fatty acids.This leads to the formation of lipid peroxides. Both trichloromethyl and itsperoxy radicals are capable of binding to proteins or lipids, initiating lipidperoxidation and liver damage, thus playing a significant role in the patho-



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Source of support: Nil, Conflict of interest: None Declared

genesis of diseases [34, 35]. Carbon tetrachloride produces significant reduc-tion in catalase, SOD and peroxidise with a significant increase in MDAformation. CCl4 causes changes in liver marker enzymes by damaging theliver parenchymatous cells. It was found that administration of CCl4 3ml/kgp.o.once daily for 7 consecutive days, produced a marked fall in antioxidantenzymes and increased lipid peroxidation in vehicle-treated positive con-trol animals. These effects indicate the extent of damage caused to hepato-cellular parenchyma. CCl4 intoxication significantly lowered hepatic pro-tein levels. These hepatic protein levels significantly (P<0.05, P<0.001)increased in the drug- treated groups when compared with the hepatotoxiccontrol group.

The antioxidant enzymes SOD, catalase and glutathione peroxidise consti-tute a mutually supportive team of defense mechanisms against ROS. Per-oxidase is an enzyme that catalyzes the reduction of hydroperoxides, in-cluding hydrogen peroxides, and protects the cell from peroxidative damage[36, 37]. Peroxidase levels significantly increased (P<0.05) in the methanolicextract- and silymarin-treated groups. Catalase is a hemoprotein containingfour heme groups. It catalyzes the decomposition of hydrogen peroxide towater and oxygen [38]. Under oxidative stress conditions, the catalase en-zyme activity will be reduced. Levels of catalase enzyme increased signifi-cantly in the groups treated with silymarin and methanolic extract, 100 mg/kg and 200mg/kg (P<0.05) Figure-5

CCl4 challenge caused a marked elevation in MDA or TBARS levels in liver.The increased MDA content might have resulted from an increase of reac-tive oxygen species (ROS) as a result of stress due to CCl4 intoxication.Increased MDA content is an important indicator of lipid peroxidation [39].Seven days oral feeding of 100 and 200 mg/kg b.wt methanolic extractresulted in a significant decrease (P<0.05) in TBARS levels in liver overCCl4 control (Figure-2).

From the above results and discussion it can be concluded that the methanolextract of Z.armatum possesses the potent antioxidant substances whichmay be responsible for its anti-inflammatory activity.

The methanol extract of Z.armatum exhibited significant in vitro and in vivoantioxidant activity in terms of reducing liver TBARS levels, increasingSOD, catalase and glutathione peroxidase levels. Phytochemical character-istics of this extract may lead to isolation and characterization of activeprinciple.

ACKNOWLEDGEMENT:Authors are grateful to Dr. S.N.SHARMA, Botanist, IIIM (CSIR), Jammu,for identification of fruits of Zanthoxylum armatum.

REFERENCES1) SRJ Maxwell. Prospects for the use of antioxidant therapies Drugs.

(1995) 49: 345.2) B Halliwell. The Antioxidant Paradox. Lancel. (2000) 355: 1179-

1180.3) Fine AM, CPA, Candidate ND. Oligomeric proanthocyanidin com-

plexes: history, structure, and phytopharmaceutical applications. Altern.Med. Rev. (2000) 5:144-151.

4) Harborne JB, Turner BL. Plant Chemosystematics, Academic Press,Orlando, FL. (1984). 362-373.

5) Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM, BinkoskiAE, Hilpert KF,Griel AE, Etherton TD. Bioactive compounds in foods,their role in the prevention of cardiovascular disease and cancer. Am. J.Med. (2002) 113:71-88.

6) Manchester LC, Tan DX, Reiter RJ, Park W, Monis K, Qi W. Highlevels of melatonin in the seeds of edible plants possible function ingerm tissue protection. Life Sci. (2000) 67: 3023-3029.

7) Oktay M, Gülçin I, Küfrevioglu OI. Determination of in vitro antioxi-dant activity of fennel (Foeniculum vulgare) seed extracts.Lebensmittel-Wissenschaft Technologie. (2003) 36:263-271.

8) Parr AJ, Bolwell GP. (2000) Review: Phenols in the plant and in man,the potential for possible nutritional enhancement of the diet by modi-

fying the phenols content or profile. J. Sci. Food Agric. 80:985-1012.9) A Dapkevicius, R Venskutonis, TA Van Beek, JPH Linssen . Antioxidant

activity of extracts obtained by different isolation procedures fromsome aromatic herbs grown in Lithuania. J Sci Food Agric. (1998)77:140-146.

10) Nadkarni, A.K. Indian Materia Medica. (1954) Vol-I, (3rd Ed.,) PopularBook Depot. Bombay

11) Singh, R.S. Vanausadhi Nirdeshika. (2002)2nd Ed. U.P. Hindi Sansthan.Lucknow.

12) Swami Sada Shiv Tirtha. The Ayurvedic Encyclopedia. (1998) Sri SatguruPublication.Delhi.

13) Lehankar, M. A. and Rehman, M.K. (1960) Deerghayu Interna-tional.6:5.

14) Ramchandran, R. and Ali M. Hamdard Medicus. (1996) XLII (4) : 51-54.

15) Sah, N.C Chemical composition of the pericarp oil of Zanthoxylumarmatum DC. J. Ess.Oil. Res. (1991) 3(6): 467-468.

16) Ramidi R, Ali M, Velasco-Negueruelea A. and Prez-Alonso M (1998)J.Ess.Oil Res.10: 127-130.

17) Rick Burett and Bob morikawa .(2006) Zanthoxylum propagationstudy in Northern Thailand.

18) Tao Guo et al. Antinociceptive and anti-inflammatory activities ofethyl acetate fraction from Zanthoxylum armatum in mice.Fitoterapia. (2011) 82:347–351.

19) S. C. Sati, M. D. Sati, Rakesh Raturi , P.Badoni. Anti-Inflammatory andAntioxidant Activities of Zanthoxylum Armatum Stem Bark. Globaljournal of researches in engineering. (2011) vol-11(5):21-23.

20) S.N. Ramanujam and B.K. Ratha. Effect of alcohol extract of a naturalpiscicide - Fruits of Zanthoxylum armatum DC. on Mg2+- and Na+, K+-ATPase activity in various tissues of a freshwater air-breathing fish.Heteropneustes fossilis, Agriculture. (2008) 283: 77-82.

21) Mehta M.B, Kharya M.D, Shrivastva R, Verma K.C . Antimicrobial andantihelmintic activities of the essential oil of Zanthoxylum alatumRoxb. India Perfum. (1981) 25:19-21.

22) Lalit singh ranawat et al. Hepatoprotective activity of ethanolic ex-tract of bark of zanthoxylum armatum DC in CC14 induced hepaticdamage in rats. Journal of ethnopharmacology. (2010)127: 777-780.

23) Glossary of Indian Medicinal Plants by R.N Chopra (1956) vol -1,87:423

24) S. Kumar and K. Miller . Inhibition of keratinocyte growth by differentNepalese Zanthoxylum species. Phytother. Res. (1999)13: 214.

25) A. Dikshit and A. Husain . Antifungal action of some essential oilsagainst animal pathogens.Fitoterapia. (1984)55:171

26) Blois M S. Antioxidant determinations by the use of a stable freeradical. Nature. (1958) 181: 1199-1200.

27) Sinha A. K. (1972) colorimetric assay of catalase, anal biochem. 47,389.28) Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric

assay of superoxide dismutase. Ind J Biochem Biophys . (1984)21:131-132

29) Rotruck J.T, Pope A.L, Ganther H.E, Swanson A.B, Hafeman D.G, andHoekstra W.G .Science. (1973) 179:588-590.

30) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissuesby thiobarbituric acid reaction.Analytical Biochemistry. (1979) 95:351-358.

31) Brent JA, Rumack BH. Role of free radicals in toxic hepatic injury. II.Are free radicals the cause of toxin induced liver injury? Journal ofToxicology Clinical Toxicology. (1993)31: 173-196.

32) Tsukamoto H, Matsuoka M, French SW. Experimental models of he-patic fibrosis a review. Semin Liver Dis. (1990) 10(1): 56–65.

33) Terblanche J, Hickman R. Animal models of fulminant hepatic failure.Digestive Diseases and Sci. (1991) 36(6): 770–774.

34) Recknagel, R.O., E.A. Glende, J.R. Dolak J.A. Waller. Mechanisms ofcarbon tetrachloride toxicity. Pharmacol Ther. (1989)43(1): 139-154.

35) Kanter M, Meral I, Dede S, Cemek M, Ozbek H, Uygan I, Gunduz H.Effects of Nigella sativa L. and Urtica dioica L. on lipid peroxidation,antioxidant enzyme systems and some liver enzymes in CCl4-treatedrats. J Vet Medi. (2003) 50(5): 264-268.

36) Halliwell, B., Gutteridge, J. M. C. (1989) Free radicals in biology andmedicine, 2nd Edn, Clarendon press: Oxford. 86.

37) Sandhir R, Gill KD. Hepatoprotective effects of Liv-52 on ethanolinduced liver damage in rats. Indian J Exp Biol. (1999)37 (8): 762-766.

38) Chelikani P, Fita I, Loewen PC. Diversity of structures and propertiesamong catalases. Cell Mol Life Sci. (2004)61 (2): 192–208.

39) Freeman BA, Crapo JD. Hyperoxia increases oxygen radical produc-tion in rat lung and lung mitochondria. Journal of Biological Chemis-try. (1981) 256: 10986-10992.

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