Hepatoprotective role and antioxidant capacity of pomegranate

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Hepatoprotective role and antioxidant capacity of pomegranate (Punica granatum) .... antioxidants have been isolated from the fruit juice and have been found to ...
Food and Chemical Toxicology 47 (2009) 145–149

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Hepatoprotective role and antioxidant capacity of pomegranate (Punica granatum) flowers infusion against trichloroacetic acid-exposed in rats Ismail Celik *, Atilla Temur, Ismail Isik Department of Biology, Science and Letters Faculty, Faculty of Arts and Sciences, Yuzuncu Yil University, 65080 Van, Turkey

a r t i c l e

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Article history: Received 5 August 2008 Accepted 24 October 2008

Keywords: Punica granatum Serum marker enzymes Antioxidant defense system Malondialdehyde Rat

a b s t r a c t This study was designed to investigate the protective and antioxidant properties of Punica granatum (PG) beverage against trichloroacetic acid (TCA)-exposure in rats. The hepatopreventive and antioxidant potential of the plant’s infusion was evaluated by measuring level of serum enzymes, antioxidant defense systems (ADS) and lipid peroxidation content in various organs of rats. Three experimental groups: A (untreated = control), B (only TCA-treated) and C (TCA+PG treated). According to the results, while the levels of AST and ALT increased significantly in B groups’ they decreased significantly in the C groups’. LDH and CK did not change significantly in B groups’ whereas decreased significantly in the C groups’. Liver, brain, kidney and heart tissues MDA content significantly increased in B groups’, whereas no significant changes were observed in the C groups’. On the other hand, SOD decreased significantly in liver of the B group but did not change significantly in the C groups’. GST activity increased significantly in liver, brain and spleen of C group while significant decrease was observed for kidney as compared to those of control. Hence, the study reveals that constituents present in PG impart protection against carcinogenic chemical induced oxidative injury that may result in development of cancer during the period of a 52-day protective exposure. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The reactive oxygen species (ROS) are known to play a major role in either the initiation or progression of carcinogenesis by inducing oxidative stress (Sun, 1990; Gulcin 2006a,b). Peroxides and superoxide anion (O 2 ) produce cytotoxicity/genotoxicity in cellular system (Perchellet and Perchellet, 1989; Gulcin 2006a, b). Reactive oxygen and nitrogen species are formed in the human body and endogenous antioxidant defenses are not always sufficient to counteract them completely. A large number of studies support the hypothesis that oxidative damage to DNA, lipids and proteins may contribute to the development of cardiovascular disease, cancer and neurodegenerative diseases (Halliwell, 1996; Gulcin et al., 2006; Ak and Gulcin, 2008; Gulcin et al., 2008). Diet-derived antioxidants may therefore be particularly important in protecting against chronic diseases (Vendemiale et al., 1999; Halliwell, 1996). Hopefully the genotoxic effects of the toxicants can be minimized by modulation of the physiological detoxification processes. Many naturally occurring compounds with antioxidative action are now known to protect cellular components from oxidative damage and prevent diseases. A number of such compounds can activate the phase II detoxification enzymes, which can remove the toxic elements from our system. Exposure to such * Corresponding author. Tel.: +90 432 2251083x2278; fax: +90 432 2251114. E-mail addresses: [email protected], [email protected] (I. Celik). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.10.020

phytochemicals is therefore beneficial to human health. (Saha and Das, 2003). In addition, many natural compounds are now known to have a modulator role on physiological functions and biotransformation reactions involved in the detoxification process, thereby affording protection from cytotoxic, genotoxic, and metabolic actions of environmental toxicants (Saha and Das, 2003). The most commonly pursued treatments are antibacterial, antiviral, antitumor, anti-inflammatory, antihypertensive, antioxidant properties. Today’s world is increasingly seeking ways to replace the synthetic drugs with the therapeutic power of natural products. The traditional folk medicine had already found the secret of healing in the nature. Medicinal plants have been used for therapeutic purposes since the beginning of civilization. Following a recent period in western medicine when plant medicines were shunned, there has been a resurgence of interest in plant compounds with beneficial pharmacological properties (Schulman and Heather, 2003). Punica granatum Linn. (Punicaceae), commonly known as pomegranate, is a shrub or a small tree, native to the Mediterranean region. PG flower is consumed worldwide as Turkey and is in popularity as a beverage. The plant possesses a number of biological activities such as such as antitumour (Afaq et al., 2005), antibacterial (Prashanth et al., 2001), antidiarrhoeal (Das et al., 1999), antifungal (Dutta et al., 1998), antiulcer (Gharzouli et al., 1999) have been reported with various extracts/constituents of different parts of this plant. Also, PG is now gaining importance because of its potent antioxidant activity. Some effective

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antioxidants have been isolated from the fruit juice and have been found to be bioavailable affect (Cerda et al., 2003). PG fruit, fruit juice, peel extracts, seed oil and seed extracts have been found to possess a potent antioxidant activity (Schubert et al., 1999; Gil et al., 2000; Noda et al., 2002; Singh et al., 2002; Kaur et al., 2006). Also, PG juice has also been shown to modulate the expression of oxidation-sensitive genes in cultured endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice (de Nigris et al., 2005). PG fruit juice flavonoids have also been found to prevent low-density lipoprotein (LDL) oxidation and hence are antiarthrogenic (Wang et al., 2004). PG wine inhibits nuclear factor k B (NFkB), a transcription factor activated by ROS and hence implicated in physiopathology of numerous diseases (Schubert et al., 2002). Further, Faria et al (2007) found that antioxidant enzymatic activities such as GSH-PX, GST, GR, SOD and catalase were found to be decreased by PG juice treatment. However, no studies have so far been reported on antioxidant activity of PG flowers as beverage. Thus, in the present study, we have extensively studied the antioxidant activity of beverage extract of pomegranate flowers using in vivo models. For this aim, the treatment of PG infusion was done orally as drinking water because the effect of plants represents a well characterized in nutrition and widely used as a beverage by human in our country and world wide. The enzymes were chosen due to their importance as index of liver and other tissues cell injury. The protective effects of crude and pure products of these two plants on some phase II detoxification enzymes, lipid peroxidation and serum marker enzymes were evaluated during exposure to the chemical carcinogen TCA in rats.

2. Materials and methods 2.1. Chemicals Thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA), reduced glutathione (GSH), metphosphoric acid, 5,50 dithiobis-(2-nitrobenzoic acid) (DTNB), trihydroxymethyl aminomethane (Tris), 1-chloro-2, 4-dinitrobenzene (CDNB), oxidized glutathione (GSSG), b-Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), potassium dihydrogen phosphate (KH2PO4) and sodium chloride (NaCl) of technical grade used in this study were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Kits for antioxidant enzymes analysis were supplied by Randox Laboratories Ltd. 2.2. Animals Rats (Sprague–Dawley albino) 4 months of age with an average weighing 150– 200 g were provided by the animal house of the Medical School of Yuzuncu Yil University, and were housed in 3 groups, each group containing 6 rats. The animals were housed at 20 ± 2 °C in a daily light/dark cycle. All animals were fed a group wheat-soybean-meal-based diet and water ad libitum in stainless cages, and received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and published by the National Institutes of Health (WMA, 2000). The ethic regulations were followed in accordance with national and institutional guidelines for the protection of animal welfare during experiments. This study was approved by The Ethic Committee of the Yüzüncü Yıl University. 2.3. Preparation of plant feeds Authenticated by Dr Murat Unal dry plant of PG (Nar çiçeg˘i) was purchased from a local herb store. A voucher specimen has been kept in the VANF herbarium (Yuzuncu Yil University, Faculty of Arts and Sciences, Department of Biology herbarium) for future reference. 2.4. Dose selection for treatment Plant infusions were prepared by soaking 2.5 g dry plant flower leaves in 1 L boiled water for 5 min to simulate actual brewing conditions for tea consumed by human adults. Dose of TCA was selected on the basis of a 0.2% (0.012 M) concentration at which caused carcinogen in vivo, as drinking water was administered orally to rats ad libitum for 52 days continuously (ACGIH, 1991).

2.5. Experimental groups In the treatment groups, while the exposed rats with TCA were given TCA solution orally as drinking water the treatment groups with TCA+plants infusions received TCA+PG infusion by orally with selected doses of aqueous. Control group was fed boiled and cooled water only. Treatments were initiated synchronistically. 2.6. Preparation of tissues supernatant and erythrocytes homogenate At the end of the treatments, the rats were anesthetized by inhalation of diethyl ether, and after blood and tissues samples were obtained, they were sacrificed. The blood samples were obtained from a cardiac puncture using syringe for the determination of serum enzyme levels and biochemical analysis. For serum enzyme levels, blood samples were put immediately into ice-chilled siliconzed disposable glass tubes. The serum samples were obtained by centrifuging blood samples at 4000g for 15 min. at 4 °C, and enzyme levels were measured in these serum samples. For biochemical analysis, blood samples were put immediately into silicon disposable glass tubes with EDTA as an anticoagulant. Blood samples were centrifuged at 4000g for 15 min at 4 °C and erythrocyte pellets were obtained. Then the pellets were washed tree times with physiological saline (0.9% NaCl). The GSH concentration in erythrocytes and tissues were measured just after the animals were sacrificed because of tremendous loss of GSH. The level of GSH, content of MDA and the activity of SOD, GR, GSH-PX and GST in erythrocytes were measured in the pellets. The tissues were dissected and put in Petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples were taken and kept at 78 °C during the analysis. The tissues were homogenized for 5 min in 50 mM ice-cold KH2PO4 solution (1:5 w/v) using a glass-porcelain homogenizer (20 KHz frequency ultrasonic, Jencons Scientific Co.) for 5 min. and then centrifuged at 7000g for 15 min. All processes were carried out at 4 °C. Supernatants and homogenates were used to determine antioxidant defense systems and MDA. 2.7. Biochemical analysis The erythrocyte and tissues MDA concentration was determined using the method described by Jain et al. (1989) based on TBA reactivity. Briefly, 0.2 ml erythrocyte pellets or supernatant obtained from tissues, 0.8 ml phosphate buffer (pH 7.4), 0.025 ml BHT and 0.5 ml 30% TCA were added to the tubes and mixed. After 2 h incubation at 20 °C, the mixture was centrifuged (400g) for 15 min. After this, 1 ml supernatant was taken and added to each tube, and then 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% TBA were added. These tubes with Teflon-lined screw caps were incubated at 90 °C in a water bath for 15 min and cooled to room temperature. The optical density was measured at 532 and 600 nm in a spectrophotometer for erythrocyte MDA, and the optical density was measured at 532 for tissues MDA concentration (Novaspec II Pharmacia-Biotech, Biochrom Ltd., UK). The erythrocyte and tissues GSH concentration was measured using the method described by Beutler et al. (1963). Briefly, 0.2 ml fresh erythrocyte pellet or supernatant was added to 1.8 ml distilled water. Three ml of the precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml distilled water) was mixed with haemolysate. The mixture was allowed to stand for approximately 5 min and then filtered (Whatman N 42). Two ml of filtrate was taken and added into another tube, and then 8 ml of the phosphate solution (0.3 M disodium phosphate) and 1 ml DTNB were added. A blank was prepared with 8 ml of phosphate solution, 2 ml diluted precipitating solution (three parts to two parts distilled water), and 1 ml DTNB reagent. A standard solution of the glutathione was prepared (40 mg/100 ml). The optical density was measured at 412 nm in the spectrophotometer. GST (EC 2.5.1.18) was assayed at 25 °C spectrophotometrically by following the conjugation of glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm as described by Mannervik and Guthenberg (1981). GR (EC 1.6.4.2) activity was assayed at 37 °C and 340 nm by following the oxidation of NADPH by GSSG (Beutler, 1984). To determine the activity of GSH-PX (EC 1.11.1.9), t-butyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GR and NADPH and the activity of GSH-PX was assayed at 37 °C and 340 nm by calculating the difference in absorbance values during the oxidation of NADPH (Beutler, 1984). SOD (EC 1.15.1.1) activity was measured at 505 nm and 37 °C and calculated using inhibition percentage of formazon formation (McCord and Fridovich, 1969). 2.8. Measurement of enzyme levels Serum enzyme activities [AST (EC 2.6.1.1), ALT (E.C 2.6.1.2), LDH (EC 1.1.1.27), CK (EC 2.7.3.2) and ALP (EC 3.1.3.1)] were measured by an auto analyzer (BM/HITACHI-911), using the kits (Randox Laboratories Ltd). 2.9. Analysis of data All data were expressed as mean ± standard deviation (SD). The statistical analyses were made using the Minitab 13 for windows packet program. Means and Standard deviations were calculated according to the standard methods for all

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serum enzymes, antioxidant defense systems and lipid peroxidation contents of blood and various tissues samples from control and treated rats. The results of experiment showed that the treatment of rats with TCA and TCA+the plant infusion caused changes in the levels of serum enzymes (Table 1), MDA content and ADS constituent (Table 2) in comparison to control rats. According to the results, the increased levels of AST and ALT enzymes due to hepatic damage induced by TCA were found to be decreased in plants-treated groups. Also, LDH and CK activities did not change in exposure to TCA group whereas decreased the TCA+PG infusion

3. Results Following the exposure of experimental groups, the body weights of TCA-exposed rats were found to reduce slightly. The effects of TCA and the plant infusion administrations on liver damages index and antioxidative role were evaluated as marker

Table 1 Determination hepatoprotective capacity of Punica granatum infusion against trichloroacetic acid-exposed in rats (Mean ± SD). Parameters

Control X ± SD

Body weight (g)

Beginning 196 ± 27

AST(U/L) ALT (U/L) LDH (U/L) CK (U/L) ALP (U/L)

TCA X ± SD Finally 207 ± 19

Beginning 174 ± 16

TCA+PG X ± SD Finally 162 ± 14

220.8 ± 54.9a 63.6 ± 8.4a 2061.2 ± 412.9 860.8 ± 381.6 500.8 ± 38.5

155.8 ± 22.9 48.8 ± 4.7 1991.4 ± 247.3 918.8 ± 278.7 558.8 ± 90.9

Beginning 188 ± 20

Finally 197 ± 13

148.2 ± 17.5b 49.6 ± 2.4b 952.6 ± 335.5a,b 406.2 ± 77.1a,b 542.8 ± 37.8

Each value represents the mean ± SD. a Significantly different from control. b Significantly different from TCA exposed rats at p < 0.05 (One way ANOVA).

Table 2 Determination antioxidant capacity of Punica granatum infusion against trichloroacetic acid-exposed in rats. Tissue

Parameters

Control X ± SD

TCA X ± SD

TCA+PG X ± SD

Erythrocyte

GSH mg/ml MDA nmol/ml GST U/ml GPx U/ml GR U/ml SOD U/ml GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g GSH mg/g MDA nmol/g GST U/g GPx U/g GR U/g SOD U/g

3.62 ± 2.4 1.43 ± 0.19 5.58 ± 0.71 93.55 ± 9.17 0.27 ± 0.07 1333.61 ± 8.11 32.92 ± 7.83 35.23 ± 13.84 70.27 ± 7.54 80.12 ± 2.54 0.24 ± 0.13 1238.22 ± 74.46 24.17 ± 3.01 12.30 ± 5.14 35.18 ± 5.91 54.21 ± 3.93 0.15 ± 0.02 1133.49 ± 35.1 34.09 ± 4.89 68.90 ± 14.23 20.17 ± 6.67 91.24 ± 5.40 0.28 ± 0.07 1271.98 ± 21.01 38.11 ± 14.4 11.78 ± 4.01 46.46 ± 7.58 93.71 ± 3.32 0.17 ± 0.04 1315.05 ± 4.8 59.4 ± 28.5 13.92 ± 5.39 16.28 ± 2.40 147.55 ± 6.9 0.42 ± 0.17 2623.03 ± 7.2 58.44 ± 19.2 2.33 ± 1.62 21.10 ± 4.09 95.66 ± 3.51 0.30 ± 0.04 1306.39 ± 6.6

1.57 ± 0.28 1.54 ± 0.35 4.06 ± 0.92a 81.55 ± 11.26a 0.31 ± 0.03 1325.87 ± 3.2 41.31 ± 4.48 69.99 ± 17.99a 73.32 ± 8.10 85.52 ± 8.46 0.26 ± 0.06 1135.13 ± 41.68a 22.64 ± 6.89 20.36 ± 5.57a 60.81 ± 12.84a 58.09 ± 3.7 0.12 ± 0.01 1128.18 ± 33.8 27.89 ± 7.40 80.51 ± 7.94 24.69 ± 5.03 96.31 ± 3.27 0.26 ± 0.10 1294.60 ± 14.7 38.39 ± 16.2 19.45 ± 3.95a 32.27 ± 9.58a 90.68 ± 4.1 0.22 ± 0.06 1296.33 ± 11.3 57.96 ± 15.62 17.89 ± 6.12 22.52 ± 3.42a 147.32 ± 1.9 0.51 ± 0.07 2635.22 ± 3.7 43.99 ± 8.74 5.56 ± 2.49a 21.65 ± 2.93 95.93 ± 5.00 0.28 ± 0.01 1292.65 ± 24.1

1.42 ± 0.26 1.32 ± 0.34 4.51 ± 0.52a 92.48 ± 4.33 0.35 ± 0.10 1314.7 ± 11.0a 39.80 ± 4.53 52.69 ± 16.64 66.43 ± 14.08 90.59 ± 4.73a 0.26 ± 0.11 1247.3 ± 125.3 25.77 ± 6.68 15.02 ± 3.4b 35.48 ± 15.04b 48.91 ± 1.15a 0.10 ± 0.04 1068.74 ± 124.9 34.24 ± 9.48 43.13 ± 11.53b 26.19 ± 9.79 96.83 ± 4.26 0.22 ± 0.05 1241.33 ± 32.8 35.84 ± 9.7 8.16 ± 1.17b 39.20 ± 5.39 88.51 ± 10.3 0.25 ± 0.06 1289.89 ± 7.5a 61.36 ± 26.35 10.03 ± 3.48b 18.23 ± 5.68 182.36 ± 32.7a 0.46 ± 0.09 2644.83 ± 17.5a 47.52 ± 6.30 3.49 ± 1.86 40.99 ± 5.84 95.10 ± 4.41 0.25 ± 0.04 1298.67 ± 28.2

Liver

Brain

Lungs

Kidney

Spleen

Heart

Each value represents the mean ± SD. a Significantly different from control. b Significantly different from TCA exposed rats at p < 0.05 (One way ANOVA).

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supplement groups’ rats. With regard to MDA content and ADS constituent, the results of experiment showed that the treatment of rats with TCA and TCA+the plant infusion caused changes in MDA concentration and antioxidative defense systems such as GSH, GSH-PX, GST, GR, and SOD in erythrocyte, liver, brain, lungs, spleen, heart and kidney in comparison to control rats. According to the results, the increased MDA content due to oxidative stress induced by TCA were found to be decreased in plants-treated groups. Namely, MDA content increased significantly in the liver, brain, kidney and heart tissues of exposure to TCA group whereas decreased the C groups’ rats. Further, the MDA content significantly decreased in the, brain, lungs, and kidney and spleen tissues of C groups’ as compared to those of TCA-exposed rats. With regard to antioxidant defense systems, GST activity increased significantly in liver, brain and spleen of C group while a significant decrease was observed for kidney. While SOD decreased significantly in liver of the B group did not change significantly in the C groups’ as compared to those of control and TCA-exposed rats. On the other hand, GSH level, GSH-PX and GR activity did not a significant change in the all groups’ tissues as compared to those of control rats.

4. Discussion Overexposures to oxidative stress caused by environmental pollutants are thought to increase the risk from cancer. Hence efforts are needed to provide effective protection from the damaging agents. Experimental studies have implicated the influence of a number of plant chemicals in this regard. Based on the concept that agents that can activate the biochemical pathways for detoxification of hazardous compounds are potential chemopreventive agents for cancer, we have investigated a number of plants in search of new sources for candidate compounds that will be able to protect from irreversible DNA damage and carcinogenesis. The first aim of this study was to investigate whether PG could prevent hepatotoxic of TCA, decrease content of the MDA and increase the antioxidant defense system in rats. As shown in Table 1, TCA caused a significant elevation in the levels of AST and ALT whereas the plant’s infusion supplement caused a significant decrease the serum marker enzymes in comparison to control rats. The reasons for such effect of TCA and the plant’s infusion are not understood at present certainly. However, it is known that several of soluble enzymes of blood serum such as these enzymes have been considered as indicators of the hepatic dysfunction and damage. Also, the increase in the activities of AST and ALT in plasma of rats treated with TCA is mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (Navarro et al., 1993). Further, ALT and AST levels are also of value indicating the existence of liver diseases, as this enzyme is present in large quantities in the liver. ALT increases in serum when cellular degeneration or destruction occurs in this organ (Hassoun and Stohs, 1995). On the other hand, phosphatases and dehydrogenases are important and critical enzymes in biological processes too. They are responsible for detoxification, metabolism and biosynthesis of energetic macromolecules for different essential functions. Any interference in these enzymes leads to biochemical impairment and lesions of the tissue and cellular function (Khan et al., 2001). Yamaguchi et al. (1981) and Yousef et al. (2007) reported that the changes in the activities of these enzymes in SnCl2-treated rats were regarded as the biochemical manifestation of the toxic action of inorganic tin. Also, Rahman et al. (2000) suggested that the increase in the activities of AlP and AcP in plasma might be due to the increased permeability of plasma membrane or cellular necrosis, and this showed the stress condition of the treated animals. Also, the increase in plasma LDH activity may be due to the hepatocellular necrosis leading to leakage of

the enzyme to the blood stream (Wang and Zhai, 1988). Thus, when TCA may lead to the release of these enzymes into plasma as result of autolytic breakdown or cellular necrosis, the plant’s infusion supplement impart protection against carcinogenic chemical induced oxidative injury that may result in development of liver damage. As shown in Table 2, the present study demonstrated that the rats treated with plant’s beverage could have antioxidative role in rats. This was obvious from our observation that, by the consequence of plants infusion with additional treatment in vivo, the concentration of MDA in the tissues differed from that of TCA-exposed group. According to the obtained the results, while MDA concentrations appreciable increased in the liver, brain, kidney and heart of rats treated with TCA, the increased MDA contents were lesser in TCA+ plant’s infusion supplemented groups. The reasons for such effect of TCA and plant’s beverage are not understood at the present. But, the increased MDA content might have resulted from an increase of reactive oxygen species (ROS) as a result of stress condition in the rats with TCA intoxication. It is known that the elevation of lipid peroxidation after some xenobiotic is consumed, and followed often by the superoxide overproduction, which after dismutation produce singlet oxygen and hydrogen peroxide, and it can be easily converted later into the reactive OH. Both single oxygen and OH radical have a high potential to initiate free radicals chain reactions of lipid peroxidation. Also, it is known that .OH can initiate lipid peroxidation in tissues (Halliwell, 1996) and MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation (Freeman and Crapo, 1981). The results in the present study also showed that antioxidant enzyme activity such as SOD decreased significantly in liver of the TCA-exposed group whereas almost remained in the plant’s infusion additional groups in comparison with control and TCA-treated rats. Meanwhile, the ancillary enzyme GR, GSH-PX activity and GSH levels whether almost remained or did not change at appreciable level in the experimental groups’. The drug metabolizing enzyme GST increased significantly in liver, brain and spleen of the TCA-exposed group whereas whether almost remained or decreased in the plant’s infusion additional groups in comparison with control and TCA-treated rats. The reasons for such effect of plant’s beverage are not understood at the present. But, oxidative stress can affect the activities of protective enzymatic antioxidants in organisms exposed to TCA. The decreased activity of GST may lead to decreased protection against oxidants (Amstad et al., 1991). Doyotte et al. (1997) pointed out that a decreased GST response might accompany a first exposure to pollutants, which can be followed by an induction of antioxidative systems. Thus, the existence of an inducible antioxidant system may reflect an adaptation of organisms. Also, the reasons for such effect of plant’s infusion may due to the flavonoids present in the plants have strong antioxidant and metal-chelating properties and may therefore protect cells and tissues against free oxygen radicals. So far, no study examining the preventive role of PG infusion in vivo have been made on rat serum marker enzymes levels, antioxidant defense systems and MDA content as a drinking water of infusion. Therefore, we had no chance to compare our results with the previous ones. In addition, because of high variability in analyzing serum enzymes-chemicals interaction in vitro and in vivo, and inconsistent factors like treatment time and manner, the setting of studies and species tissue differences etc., it is difficult to compare the present data to different studies regarding the chemo preventive properties. However, Cerda et al. (2003) investigated that the isolated from the fruit juice have antioxidative properties. On the other hand, antioxidant activities of PG fruit, fruit juice, peel extracts, seed oil and seed extracts have been found to possess a potent antioxidant activity (Schubert et al., 1999; Gil et al., 2000; Noda et al., 2002; Singh et al., 2002; Kaur et al.,

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2006). Despite treatment time and manner and the different setting of studies the results of the above-mentioned studies are in agreement with our results. As a conclusion, the observations presented here led us to conclude that while administration of subacute TCA promotes MDA concentration fluctuates in the antioxidative systems and elevates tissue damage serum marker enzymes the plant beverage supplement impart protection against carcinogenic chemical induced liver injury and oxidative stress. The observations, along with changes, also might suggest that such a test will also be of value in chemopreventive studies, and also be of interest to understand molecular basis of refractoriness of plant’s protective role. Nature is, in fact, the richest botanic pharmacy created for living beings and scientific efforts will certainly be more oriented toward the ‘discovery of nature’ in future. Also, we wish to study this further with destructive chemical dose of the infusion furthermore before coming to any conclusion. Nevertheless the results do suggest that regular intake of naturally occurring combination of anticarcinogenic agents possessing varied biological activities should also be considered potentially useful for prevention of chronic degenerative liver diseases. Conflict of interest statement This study was approved by the ethic committee of faculty of medicine of Yuzuncu Yil University. Funding source This study was supported by the University Grant Commission of Yuzuncu Yil University. References ACGIH, 1991. Documentation of the threshold limits values and biological exposure indices, sixth ed. Cincinnati, OH. In: American Conference of Governmental Industrial Hygienists. Afaq, F., Saleem, M., Krueger, C.G., Reed, J.D., Mukhtar, H., 2005. Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NFkappaB pathways and inhibits skin tumorigenesis in CD-1 mice. Int. J. Cancer 113, 423–433. Ak, T., Gulcin, L., 2008. Antioxidant and radical scavenging properties of curcumin. Chem. Biol. Interact. 174, 27–37. Amstad, P., Peskin, A., Shah, A.G., Mirault, M.E., Moret, R., Zbinden, I., Cerutti, P., 1991. The balance between Cu, Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress. Biochemist 30, 9305–9313. Beutler, E., 1984. Red cell metabolism. A Manual of Biochemical Methods. Third ed.. Grune and Startton, New York. pp. 105–106. Beutler, E., Dubon, O.B., Kelly, M., 1963. Improved method for the determination of blood glutathione. J. Lab Clin. Med. 61, 882–888. Cerda, B., Llorach, R., Ceron, J.J., Espin, J.C., Tomas-Barberan, F.A., 2003. Evaluation of the bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from pomegranate juice. Eur. J. Nutr. 42, 18–28. Das, A.K., Mandal, S.C., Banerjee, S.K., Sinha, S., Das, J., Saha, B.P., Pal, M., 1999. Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. J. Ethnopharmacol. 68, 205–208. De Nigris, F., Williams-Ignarro, S., Lerman, L.O., Crimi, E., Botti, C., Mansueto, G., D’Armiento, F.P., De Rosa, G., Sica, V., Ignarro, L.J., Napoli, C., 2005. Beneficial effects of pomegranate juice on oxidationsensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress. Proc. Natl. Acad. Sci. USA 102, 4896–4901. Doyotte, A., Cossu, C., Jacquin, M.C., Babut, M., Vasseur, P., 1997. Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus. Aquat. Toxico. 39, 93–110. Dutta, B.K., Rahman, I., Das, T.K., 1998. Antifungal activity of Indian plant extracts. Mycoses 41, 535–536. Freeman, B.A., Crapo, J.D., 1981. Hyperoxia increases oxygen radical production in rat lung and lung mitochondria. J. Biol. Chem. 256, 10986–10992. Gharzouli, K., Khennouf, S., Amira, S., Gharzouli, A., 1999. Effects of aqueous extracts from Quercus ilex L. root bark, Punica granatum L. fruit peel and Artemisia

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