EFFECT OF POMEGRANATE JUICE AND PEEL ON ANTIOXIDANT ENZYMES AND LIPID PROFILE IN CARBON TETRACHLORIDE-INDUCED HYPERLIPIDEMIC RATS

Doaa M. Genena 1 and Neveen F. Agamy 2 . 1. Fellow of Nutrition, Medical Research Institute University of Alexandria 2. Assistant Professor of food analysis, High Institute of Public HealthUniversity of Alexandria ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Lipid peroxidation is a crucial step in the pathogenesis of free radicalrelated disease states, including glomerulonephritis, rheumatoid arthritis, gastrointestinal and cardiovascular diseases. Antioxidants play a significant role in protecting living organism from the toxic effect of various chemicals by preventing free radical formation. Pomegranate is a good source of polyphenols and other antioxidants. The purpose of the present study is to evaluate the effect of pomegranates juice and peel administration on antioxidant enzymes activities and lipid profile in carbon tetrachloride-induced hyperlipidemic rats. Hyperlipidemic rats were treated with pomegranates juice and peel for 8 weeks. Results showed that all hyperlipidemic rats administrated juice and peel had significant decrease in TBARS concentration and increase in GSH, GPx, GST, CAT and SOD in plasma compared to CCl4 group. Lipid profile parameters (except HDL-C) decreased significantly with juice and peel administration compared to CCl4 group. In conclusion, consumption of pomegranate juice and peel showed significant improvement of lipid parameters and antioxidant enzyme activities in hyperlipidemic rates suggesting therapeutic potential of using pomegranate as a medicinal plant for managing hyperlipidemia and modifying the risk of CVD.

Introduction: -
Human are continuously exposed to different kindsof chemicals such as food additives, industrialchemicals, pesticides and other undesirablecontaminants in the air, food and soil (Stavric, 1994).These substancesmostly induce a freeradical-mediated lipid peroxidation leading todisruption of biomembranes and cellular dysfunction. Lipid peroxidation is thought to play an important role in the pathogenesis of free radical-related disease states, including glomerulonephritis, rheumatoid arthritis, gastrointestinal and cardiovascular diseases (Cho et al., 2003). Oxidative stress has been linked to an altered lipid profile and endothelial damage which is a crucial event in the most common pathological processes underlying cardiovascular diseases (CVD) (Halperin et al., 2006). Additionally, the wellknown side effects of the current synthetic lipid-lowering drugs have increased the need to move toward alternative treatment for the prevention of hyperlipidemia including medicinal plants and natural antioxidants (Pahan, 2006).
Antioxidants play a significant role in protecting living organism from the toxic effect of various chemicals by preventing free radical formation (Sheweita et  1710 Group (2):Ten rats were given the standard diet and treated with CCL 4 alone (1 ml/Kg BW) twice a week for 8 weeks. Group(3):Ten rats were given the standard diet and treated with combination of CCL 4 (1ml/kg BW) and pomegranate juice(5 ml/Kg BW) for 8 weeks. Group (4): Ten rats were treated with combination of CCL 4 (1 ml/Kg BW) andpomegranate peel alone added to standard diet by 5% for 8 weeks. Group (5): Ten rats were treated with combination of CCL 4 (1 ml/KgBW), pomegranate juice (5 m1/Kg BW) andpomegranate peel by 5% for 8 weeks.

Measured parameters and techniques:
At the end of the experiment, rats were fasted overnight and anesthetized with chloroform. The blood samples were collected in tubes with heparin (anti-coagulant). The heparinated blood samples were placed immediately in ice. Plasma samples were obtained by centrifugation at 860xg for 20min, and were stored at -20°C until analyses.Liver was immediately removed at the end of the experiment; weighed and washed using chilled saline solution. Tissues were minced and homogenized (10% w/v) in ice-cold sodium potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl in a Potter-Elvehjem typehomogenizer. The homogenate was centrifuged at 10,000 g for 20 min at 4°C. The resultant supernatant of the organs was used for the measurement of different enzyme activities. Statistical analysis:-Data were analyzed using SPSS software package version 20 (SPSS, Chicago, IL, USA). P value (< 0.05) was considered significant. The data wereassessed for normality using Kolmogorov-Smirnov test. The data were normally distributed and presented as mean ± standard deviation. One way ANOVA test was used to compare the mean for different measures among the different experimental groups, followed by post-hoc pairwise comparison with adjustment of P value using Bonferroni correction. Table 1 shows the effects of pomegranate juice and peel on antioxidant enzymes activities in CCl4-induced hyperlipidemic rats. CCl4 significantly (p<0.05) increased TBARS and decreased GSH levels as well as GPx, GST, CAT and SOD activities in plasma of the rat group treated only with CCl4 as compared to control group. In other words, CCl4 induced oxidative stress in plasma as manifested by the alterations observed in antioxidant defense systems both enzymatic and non-enzymatic. Lipid peroxidation is one of the principal causes of CCl4 toxic effect and is mediated by the free radicals' derivatives of CCl4. Our results are in consistence with the results of Shih et al. (2005) who confirmed that the hepatic activity of superoxide dismutase was markedly decreased by CCl4 treatment. Also,Comporti (1985) indicated a substantial increase in ROS, MDA levels and depletion of GSH levels in CCl4 intoxicated rats.

Results and Discussion:-
On the other hand, our study demonstrated that treatment of hyperlipidemic rats with combination of CCL4, pomegranate juice and peel caused significant (p<0.05) decrease in TBARS concentration and increase in GSH, GPx, GST, CAT and SOD activities. In other words, pomegranate juice and peel reduced the toxic effects of CCl4 (Table1). The significant elevation in GSH, GST, SOD, GPx and catalase of plasma indicates the protection offered 1711 by juice and peel against CCl4 induced toxicity. It is known that some flavonoids are able to reduce xenobioticinduced toxicity in animals and counteract the damaging effects of oxidative stress, cooperating with natural systems like glutathione and other endogenous protective enzymes (Kadarian et al., 2002). These results are in agreement with Mohieldin et al. (2011) whorevealedthattreating rats with carbon tetrachloride andpomegranate peel ethanolic extract had significantly decrease the GST, catalase and SOD activity, and significantly increase the GST, catalase and SOD activity when compared with CCl4-treated group during the hepatoprotective and curative periods.Our findings are also confirmed by the findings ofAviram et al., (2004)who reported that total antioxidant status in serum of patients with carotid artery stenosis was substantially increased, by 130% after 12 months of juice consumption. The effect of juice may be related to its potent tannins and anthocyanins which scavenge wide spectrum of free radicals. (Gil et al., 2000), (Aviram et al., 2000).
Pomegranate peel administration showed insignificant higher levels of plasma GSH, GPx, GST, CAT, and SOD when compared to juice, which is may be due to the slightly higher content of polyphenols in peel than in juice. Aviram et al., (2000) found that the inner and outer peels contain 20-30 fold more polyphenols than do the aqueous fractions of the seeds and Juice (566 and 739 μmol polyphenols/mg compared with 22 and 25 μmol polyphenols/mg, respectively).
The decrease of TBARS in plasma of the hyperlipidemic rat groups treated with juice and peel (Table 1) Table 2 shows the effect of pomegranate juice and peel on lipid profile of CCl4-induced hyperlipidemic rats. Treatment of rats with CCl4 caused a significant increase (p<0.05) in plasma TG, TC, LDL-C and VLDL-C, while HDL-C was significantly decreased (p<0.05) compared to control group. These findings are in agreement with the results of Uličná1 et al. (2003) who reported that the long-term intraperitoneally administration of CCl4 to experimental animals induced, as expected, pathological changes in the liver. CCl4 treatment of rats also caused a severe increase of hepatic triacylglycerol, total cholesterol and Malondialdehyde. The highly reactive free radical (trichloromethyl, CCl3°-) in the presence of oxygen leads to auto-oxidation of fatty acids and causes functional and morphological changes in the cell membrane. Scavenging of free radicals is one of the major antioxidation mechanisms to inhibit the chain reaction of lipid peroxidation. ROS are highly reactive and can react with many intracellular molecules, mainly unsaturated fatty acids (phospholipids, glycolipids, glycerides and sterols) and transmembrane proteins with oxidizable amino acids. The oxidation of these molecules causes the increase in the cellular membrane permeability. ROS can attack the unsaturated bonds of the membrane lipids in an autocatalytic process, with the genesis of peroxides, alcohol and lipidic aldehydes as by-products of the reaction. Thus, the increase of free radicals in cells can induce the lipid peroxidation by oxidative breakdown of polyunsaturated fatty acids in membranes of cells (Henke et al., 2005).

Data listed in
Meanwhile, our study reported that treatment of hyperlipidemic rats with pomegranate juice caused a significant decrease (p<0.05) in plasma lipids except HDL-C, which was significantly increased (p<0.05) compared to control group.These results are in agreement with Esmaillzadeh et al., (2006) who found that pomegranate juice significantly reduced TC, LDL-C, the ratio of LDL-C/HDL-C, and the ratio of TC to HDL-C. Another study on mice fed pomegranate juice, plasma lipid peroxidation was markedly lower, and this effect was dependent on concentration (Aviram et al., 2000). Furthermore, Aviram et al., (2004) showed that pomegranate juice consumption for 3 years on patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and LDL-C oxidation. Khoo et al., (1990) reported that LDL-C aggregation was inhibited in vitro by pomegranate juice; this inhibition may be related to hydrophobic interactions between constituents of pomegranate juice and the lipoprotein.Pomegranate juice was indeed shown to possess impressive antioxidative properties due to its polyphenolics, tannins and anthocyanins (Gil et al., 2000).Additionally, phenolic compounds derived from pomegranate, vitamins C and E, melatonin and lycopene have been used as antioxidant agents to prevent various lipid-peroxidation-induced damages in different organs (Sönmez et al., 2005) The decrease in plasma lipid profile of hyperlipidemic rats treated with peel which is showed in the present study is in agreement with Hossin(2009)who evaluated the pomegranate peel powder as a dietary fiber source for the treatment of hypercholesterolemia and atherosclerosis and found that dietary supplementation with peel powder at a concentration of 5, 10 and 15 g/100 g for a period of four weeks significantly reduced serum TC, TG, LDL-C, VLDL-C and lipid peroxidation levels in Hypercholesterolemic rats. Results of the present study confirm thesefindings(Table2).He alsoreported that all Hypercholesterolemic groups administered with different level of pomegranate peel extract (1, 2 and 3%) had significant decrease in serum TC, LDL-C-, VLDL-C, lipid peroxidation and atherogenic index and triglycerides comparing with control positive. Additionally, Ibrahim (2010)noticed that feeding the rats on standard diet containing 400 or 800 mg/Kg BW pomegranate peel extract for 4 weeks obviously decreased (P<0.05) levels of TC, TG and TL.Pomegranate peel polyphenolic extract is effective in lowering serum and hepatic lipids (Cheng et al., 2005).
In the current study pomegranate peel was shown to be more influential in lowering level of lipid profile than juice, which is consistent with the findings of Aviram et al., (2000) who compared between the juice and the peel in terms of total polyphenol content. The researchers analyzed the antioxidant properties of pomegranate constituents other than the juice; they prepared aqueous solutions of the inner and outer peels and the crushed seeds and found that the aqueous extracts of the inner and outer peels were more powerful antioxidants than the juice, suggesting that the inner and outer peels may contain more potent antioxidant polyphenols. The aqueous extract obtained from the crushed seeds was found to be a weak antioxidant against LDL-Coxidation.
Based on the previous results, the authors concluded that the consumption of pomegranate juice and peel caused significant improvement of lipid parameters and antioxidant enzyme activities in hyperlipidemic rats suggesting therapeutic potential of using pomegranate as a medicinal plant for managing hyperlipidemia and modifying the risk of CVD.