International Journal of Radiation Biology, December 2014; 90(12): 1184–1190 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.926040

Synergestic effect of aqueous purslane (Portulaca oleracea L.) extract and fish oil on radiation-induced damage in rats Afrag. S. H. Abd El-Azime, Elham M. Hussein & Omaima M. Ashry

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Radiation Biology Department, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo, Egypt

O2-, hydroxyl radical (HO-), hydrogen peroxide (H2O2) that overwhelm the levels of antioxidants resulting in oxidative stress (Spitz et  al. 2004) that are known to cause abnormal chemical reactions in living substances, such as suppressing the immune system inducing birth defects, liver and kidney dysfunction, hypercholesterolemic effects, cancer and other serious diseases (Liochev and Fridovich 1999). Free radicals will oxidize anything in their way by punching holes in cell membranes, destroying key enzymes and fracturing DNA (Cook et al. 2003). One free radical may cause a new formation of more free radicals by breaking a stable bond, leaving the two atoms of the formerly stable molecule to share one unpaired electron. These free radicals lead to lipid peroxidation and the stimulation of apoptosis (Tuppo and Forman 2001, Afanasev 2005, Ashry et al. 2012). Ionizing radiation is accompanied by oxidative-stress, proteins and lipids oxidative damage, elevated proteincarbonyl level, injured membranes of blood cells and impaired immune functions (Song et al. 2006). The over production of reactive oxygen species (ROS) in cells and tissues increases oxidative stress. The uncontrolled ROS production could induce damages to lipids, protein oxidation, DNA and RNA (Ornoy 2007). Purslane provides a rich plant source of nutritional benefits (Sudhakar et  al. 2010). The plant contains many biologically active compounds, including free oxalic acids, several alkaloids (oleraceins A, B, C, D, and E), omega-3 fatty acids involved in strengthening the immune system (Simopoulos 2004), glutathione (Simopoulos et  al. 1992), coumarins, and several flavonoids (kaempferol, apigenin, myricetin, quercetin and luteolin) (Mohammad et al. 2004, Xin et  al. 2008). Meanwhile, its a-tocophrol, ascorbic acid, b-carotene content ranges from 22–30 mg/g fresh mass in leaves and reported to be a health food for patients with cardiovascular diseases (Liu et al. 2000). The seeds also contain s a high percentage of a-linolenic acid (ALA) (Ezekwe et al. 1999) which plays an important role in human growth, deve­ lopment and disease prevention (Galli et al. 1994). Purslane seeds contain a variety of vitamins including A, C and B as

Abstract Purpose: To evaluate the impact of oral administration of purslane (Portulaca oleracea) extract or fish oil and their co-treatments in the modulation of radiation-induced damage. Material and methods: Purslane (P) (400 mg/kg body weight) or fish oil (Fo) (60 mg/kg body weight) was administrated to male albino rats via gastric intubation for 15 days after whole body exposure to a single dose of 6 Gy gamma rays. The animals were sacrificed after the elapse of 15 days. Results: The results revealed that irradiation induced a significant elevation of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), and atherogenic index: TC/high density lipoprotein cholesterol (HDL-c) in addition to aspartate and alanine transaminase (AST, ALT), alkaline phophatase (ALP), bilirubin, as well as urea, creatinine and uric acid. Moreover, liver, kidney and heart malondialdehyde (MDA) was significantly elevated, whereas nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT) and HDL-c were depressed. Purslane and/or fish oil treatment significantly attenuated lipids alteration, liver and kidney functions as well as oxidative stress in irradiated rats. The results pointed out that dietary fish oil supplementation, at adequate doses, may provide a cushion for a prolonged therapeutic option against radiation-induced damage without harmful side-effects. Conclusion: It could be concluded that purslane extract and fish oil may have therapeutic potential to improve hepatic and renal functions as well as oxidative stress in irradiated rats. Moreover, their co-administration showed a better improved liver function. Keywords: Purslane, fish oil, oxidative stress, g-radiation

Introduction Exposure to radiation has been shown to induce the formation of senescence markers possibly contributing to the permanent reduction in tissue functionality (Le et al. 2010). Radiation damage is largely caused by the overproduction of reactive oxygen species (ROS) including Superoxide anion

Correspondence: Prof. Omaima Mohamed Ashry, National Center of Radiation Research and Technology, Radiation Biology, 3 Al ahlam St, Nasr City Cairo, Egypt. E-mail: [email protected] (Received 13 October 2013; revised 9 May 2014; accepted 15 May 2014)

1184

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Effect of purslane extract and fish oil on radiation damage  1185 well as minerals iron, calcium and protein (Dunmire and Tierney 1997). Its co-administration with fish oil attenuate gentamicin nephrotoxicity (Hozayen et  al. 2011). Several biological properties have been attributed to purslane, such as: antispasmodic, diuretic, vermifuge (Xiang et  al. 2005), anti-scorbutic, antibacterial, wound-healing (Lim and Quah 2007), analgesic, and anti-inflammatory activities. It is used as an anti-viral agent, and for the treatment of viral hepatitis and in diabetes management (Hu et al. 2003). Fish oil is rich in omega-3 fatty acids, mainly ecosapentaenoic (EPA) and decosahexaenoic (DHA) and when taken by man they compete with the precursors of arachidonic acid. Their actions include reduction in plasma triglycerides (TG), cholesterol (TC) and low density lipoprotein cholesterol (LDL), and increase in high density lipoprotein cholesterol (HDL) (Guo et al. 2005), anti-inflammatory action, a fibrinolytic and anti-platelet effect (Meydani et al. 1991). In view of these considerations, the current study aimed to evaluate the effects of purslane rich in ALA and fish oil rich in EPA and DHA on liver and kidney functions, their hypocholesterolemic as well as antioxidant effects.

Material and methods Experimental animals Adult male albino rats weighing 130–160 g were obtained from the animal house belonging to the National Center for Nuclear Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt. Animals were kept under room temperature, and were provided with tap water and rodent diet (from the local market) ad libitum. All animal treatments were conducted according to the Ethics Committee of the National Research Centre and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (HN publication No. 85-23, 1996).

Radiation facility Irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt, using Gamma Cell-40 (137Cesium) biological irradiator manufactured by Canada Ltd, Ottawa, Ontario, Canada. In order to induce drastic changes animals were whole body irradiated at acute single dose of 6 Gy gamma rays delivered at a dose rate of 1 Gy/1.5 min calculated according to the dosimeter department in NCRRT.

Plant extract Purslane was purchased from local market. The aqueous extract of the purslane (stem and leaves) were boiled in the traditional way. Briefly, herbs were minced and seeped in boiling water in the proportion of 1:10 (w/v) for 3 h. This was repeated two additional times for 3 h of boiling. After boiling, the resulting crude extract was filtered and the filtered extract was evaporated to dryness under reduced pressure at 40°C and a yield of 24–28% (w/w) was obtained. The dried powder was kept at 4°C for future use (Zhang et al. 2007). It was suspended in distilled waters and administrated via gastric intubation at the dose level of 400 mg/kg body weight/day for

15 days. This dose was effective for Diabetes mellitus (Gong et  al. 2009) and for gentamicin induced toxicity (Hozayen et al. 2011).

Fish oil Fish oil from menhaden fish (Product No. F 8020) is a crude source of omega-3 fatty acids eicosapentaeoic and docosahexaenoic acids and was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and administrated via gastric intubation at dose level of 60 mg/kg body weight, a dose that was found to alter eicosanoid synthesis and serum lipid profiles (Sinha et al. 1990) for 15 successive days.

Animal groups Forty-two adult male albino rats were randomly categorized into seven equal groups (n  6). 1. Control rats received saline via gastric intubation; 2. Rats administrated aqueous extract of purslane (P) by gastric intubation at a dose level of 400 mg/kg body weight/day for 15 days; 3. Rats orally administrated fish oil (FO) by gastric intubation at a dose level of 60 mg/kg body weight for 15 days; 4. Rats subjected to a single dose of 6 Gy whole body gamma irradiation and received saline by gastric intubation for 15 days; 5. Rats orally administrated aqueous extract of purslane (P) at a dose of 400 mg/kg body weight for 15 days by gastric intubation after 6 Gy gamma irradiation; 6. Rats orally administrated fish oil (Fo) at a dose of 60 mg/kg body weight for 15 days by gastric intubation after 6 Gy gamma irradiation; and 7. Rats were subjected to 6 Gy gamma rays and orally treated with purslane and fish oil (R P  FO). The weight, food and water consumption of the different groups were monitored and showed no unexpected changes in general behavior in mice during 15 days of treatment.

Samples collection and biochemical analysis After 15 days animals were anesthetized with ether obtained from SDFL (SD Fine Chemicals Ltd, Industrial State 248, Mumbai, India). Blood was collected by heart puncture in non- heparinized tubes, centrifuged at 10,000 g for 10 min to separate the serum and stored frozen until assayed. Liver, kidney and heart were removed, weight and homogenized immediately to give 50% weight/volume (w/v) tissue homogenate in ice-cold medium containing 50 mM Tris-HCl and 300 mM sucrose (Tsakiris et al. 2004). The homogenate was centrifuged at 10,000 g for 15 min at 4°C. The supernatant (10%) was used for the various biochemical determinations. Serum TC was measured according to Allain et al. (1974), TG according to Fossati and Prencipe (1982) and HDL-c according to Demacker et  al. (1980) using Stabino Laboratory Kit (Boerne, USA). LDL-c level was calculated by use of the Friedwald equation (Friedwald 1972) LDL-c  (TC-HDLc -TG/5). Atherogenic index (TC/HDL- c) was calculated. For assay of liver function, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum were determined according to the method of Reitman and

1186  A. S. H. Abd El-Azime et al. Table I. Effect of purslane extract and/or fish oil on lipid profile in serum of rats 15 days post 6 Gy g-irradiation. Athero.index Parameter/groups TC (mg/dl) TG (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) TC/HDL-c Control Purslane (P) Fish oil (Fo) Irradiated (R) RP R  Fo R  P  Fo

88.7  3.4 88.2  3.3 89.1  3.3 135.3  5.9C 97.6  4.2R 98.0  4.9R 94.3  3.8R

65.3  2.2 65.1  2.1 65.6  2.3 89.3  3.3C 70.4  3.1R 71.3  3.3R 69.7  2.5R

43.6  2.2 43.9  2.2 44.3  2.2 30.5  2.5C 35.2  2.1R 36.9  2.1R 38.1  2.3R

32.0  1.2 30.7  1.6 31.1  1.6 78.6  3.8C 44.3  2.5R 44.8  2.5R 38.4  1.8RZ

2.03  0.1 2.02  0.1 2.01  0.1 3.71  0.1C 2.49  0.1R 2.52  0.1R 2.25  0.1R

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­ alues are expressed as mean of 6 rats  standard error. Csignificantly different compared to control; Rsignificantly different V compared to irradiated group; Zsignificantly different compared to irradiated animals treated with P or Fo.

Frankel (1957) using Plasmatec Laboratory Products Ltd (Bridport, UK). Serum alkaline phosphatase (ALP) activity was determined according to the method of Kind and King (1954) using Biodiagnostic kit (Giza, Egypt) and total and direct bilirubin were determined according to the method described by Jendrassik and Grof (1938). For kidney function, serum urea, creatinine and uric acid were assayed using Stabino Kit according to the methods of Patton and Crouch (1977), Henry (1975) and Fossati et al. (1980), respectively. All chemicals and reagents used were pure chemicals from Sigma-Aldrich Chemical Company (St Louis, MO, USA). Liver, kidney and heart tissues malondialdehyde (MDA), an end product of lipid peroxidation, was determined according to Yoshioka et al. (1979), and nitric oxide (NO) according to Cortas and Wakid (1990). Superoxide dismutase (SOD) activity was assayed by the method of Minami and Yoshikawa (1979) and catalase (CAT) activity was determined according to the method described by Aebi (1984) using T60 UV/VIS spectrophotometer (PG Instruments, London, UK).

Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Knapp and Miller (1992) as post hoc ANOVA test. Differences were considered significant at p values   0.05.

Results Table I summarized the data of serum lipid levels of rats in all groups. There was no significant difference in lipid profile between control and purslane or fish oil-treated groups. 6 Gy gamma irradiation induced a significant (p  0.05) elevation of serum TC, TG and LDL-C and atherogenic indices, while HDL-C value was significantly (p  0.05) depressed compared to control group. Irradiated rats that received purslane

extract (400 mg/kg body weight/day) or fish oil (60 mg/kg body weight/day) showed significantly reduced TC, TG and LDL-C and atherogenic indices besides elevation of HDL-C compared to the irradiated group. Dual treatment showed a (p  0.05) significant improvement in lipid profile post gamma rays compared to the irradiated group. However, regarding LDL-C the improvement was more pronounced compared to purslane or fish oil treatment alone. As shown in Table II, there was no significant differences in AST, ALT, ALP, (total and direct) bilirubin between control and purslane or fish oil-treated group. g-irradiation induced significant increase of the enzymes compared to the control rats. Treatment with purslane extract or with fish oil as well as their co-administration post irradiation exerts a significant decrease (p  0.05) of serum AST, ALT, ALP and (total and direct) bilirubin as compared to irradiated group. However, the dual treatment with purslane and fish oil together exerted a more pronounced significant decrease in the previous parameters as compared to each treatment alone. Results in Table III showed no significant differences in serum levels of urea, creatinine and uric acid of purslane or fish oil-treated group compared to the control. Irradiation of rats induced a significant increase (p  0.05) in serum levels of urea, creatinine and uric acid as compared to the control. Treatment with purslane extract, or fish oil and their dual treatment post-irradiation exerts a significant decrease (p  0.05) of serum urea, creatinine and uric acid compared to the irradiated group. Rats receiving purslane and/or fish oil showed no significant change in nitric oxide and MDA levels in liver, kidney and heart tissues (p  0.05) as compared to the control group. Nitric oxide levels of liver, kidney and heart tissues of rats 15 days post 6 Gy g-irradiation were significantly decreased (p  0.05), whereas MDA levels were significantly increased as compared to the control group. Administration

Table II. Effect of purslane extract and/or fish oil on liver function of rats 15 days post 6 Gy g-irradiation in different groups. Parameter /groups ALT (U/L) AST (U/L) ALP (U/L) T. Bilr. (mg/dl) D. Bilr. (mg/dl) Control Purslane (P) Fish oil (Fo) Irradiated (R) RP R  Fo R  P  Fo

21.9  1.9 22.2  1.7 21.5  1.2 49.4  3.7C 34.5  2.1R 35.9  2.6R 28.1  3.0RZ

48.3  2.2 48.4  2.0 48.8  2.3 95.2  5.2C 71.4  3.9R 77.8  3.7R 55.9  3.0RZ

139.4  5.2 139.9  4.9 140.0  4.9 219.1  6.8C 167.4  4.6R 173.3  5.9R 144.6  5.2RZ

1.62  0.1 1.53  0.1 1.50  0.03 4.33  1C 2.7  0.6R 2.9  0.7R 2.1  0.6RZ

0.61  0.1 0.58  0.1 0.56  0.1 1.91  0.1C 1.43  0.1R 1.56  0.1R 1.05  0.1RZ

­ alues are expressed as mean of 6 rats  standard error. Csignificantly different compared to control; Rsignificantly different V compared to irradiated group; Zsignificantly different compared to irradiated animals treated with P or Fo.

Effect of purslane extract and fish oil on radiation damage  1187 Table III. Effect of purslane extract and/or fish oil on kidney function of rats 15 days post 6 Gy g-irradiation in different groups. Parameter/ Urea Creatinine Uric acid groups (mg/dl) (mg/dl) (mg/dl) Control Purslane (P) Fish oil (Fo) Irradiated (R) R  P R  Fo R  P  Fo

33.8  1.9 33.9  1.8 33.7  1.2 53.8  2.9C 40.9  2.5R 41.3  5.8R 38.1  6.3R

9.6  0.4 9.7  0.4 9.9  0.4 16.7  0.4C 12.8  0.4R 13.3  0.4R 11.8  0.4R

6.3  0.1 6.5  0.1 6.8  0.4 14.9  1.5C 9.9  0.7R 10.6  1.0R 8.0  0.5R

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­ alues are expressed as mean of 6 rats  standard error. Csignificantly different V compared to control; Rsignificantly different compared to irradiated group; Zsignificantly different compared to irradiated animals treated with P or Fo.

of purslane, fish oil or the dual treatment post-irradiation showed significant increase (p  0.05) of nitric oxide and significant decrease of MDA of liver, kidney and heart tissues compared to the irradiated group. Combined treatment of purslane and fish oil showed higher decrease of liver MDA as compared to each treatment alone (Table IV). Data represented in Table V showed no significance differences in liver, kidney and heart SOD and CAT activities between control and purslane or fish oil-treated group. The activities of SOD and CAT were significantly (p  0.05) decreased in liver, kidney and heart of rats 15 days post 6 Gy g-irradiation compared to the control group. SOD and CAT activities were significantly restored in the liver, kidney and heart of the rat group treated with purslane, fish oil or their co-administration for 15 days post 6 Gy g-irradiation. However, the supplementation of purslane with fish oil induced a more pronounced increase of liver SOD activity compared to each treatment alone.

Discussion The interaction of ionizing radiation with the biological system resulted in generation of ROS (Gracy et  al. 1999). ROS significantly affects the cellular membrane and induces peroxidation of lipids, thereby producing damaging effects to the cells (Agrawal et al. 2001). ROS also affects the haemopoietic system and considerably decreases its cellular components and affects the antioxidant defense mechanisms. Whole body exposure of male rats to 6 Gy gamma radiation induced significant increase in TC, TG, LDL-c and atherogenic index accompanied by a significant decrease in HDL-c due to irradiation. The hyper cholesterolemia conditions was attributed to the stimulation of cholesterol synthesis in the

liver after gamma irradiation (Ladan et al. 2008). Free radicals destruct cell membranes and enhanced cholesterol release and increase lipid peroxidation (Karbownik and Reiter 2000). Rats treated with purslane and/or fish oil exhibited significant decrease in TC, TG, LDL-c and atherogenic index accompanied by significant increase in HDL-c compared to the irradiated group. The increase in HDL-c may slow the atherosclerotic process and atherogenic indices that was believed to be important risk factors of atherosclerosis (Nofer et al. 2002). Amelioration of lipid profile may be attributed to the purslane herb, which is effective as an antioxidant agent (Nancai et al. 2006). The anti-oxidative properties of purslane herb aqueous extracts (PHAS), being examined in different models, contain pharmacologically active substances including 28% flavonoids that are nearly exclusively flavonolO-glycosides, 8% terpenoids (known as ginkgolides A, B, C and bilobalide), 6–12% organic acids, and  0.5% proanthocyanidins defined as flavonoid-based polymers (Honggui et al. 2004, Xiyun et al. 2006). Hozayen et  al. (2011) attributed the hypocholesterolemic effect of aqueous purslane to its high content of a-linolenic acid. Barakat and Mahmoud (2011) described anti-atherogenic, anti-hypolipidemic and immuonomodulatory effects of purslane to be mediated by unsaturated fatty acids (including a-linolenic acid) whereas Sudhakar et  al. (2010) considered purslane to be a rich vegetable source of omega-3 fatty acids. However, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the predominant omega-3 polyunsaturated fatty acids (PUFA) in fish oil. It could be suggested that Omega-3 exert their fat-lowering effect through extensive regulation of lipid metabolism via: (1) Inhibiting lipogenesis (Tai and Ding 2010) through decreasing the expression of lipogenic genes (Nakatani et  al. 2003); (2) enhancing lipolysis through increasing the expression of hormone sensitive lipase and decreasing the expression of perilipin (Wang et  al. 2009); and (3) enhancing fatty acid oxidation which is mediated by increasing the oxidation-related enzyme activities (Guo et al. 2005). The current study revealed that 6 Gy whole body gamma irradiated-rats induced significant elevation of serum AST, ALT, ALP total and direct bilirubin. These results were attributed to irradiation increased serum enzymatic activities that may be due to the damage of liver paranchymal cells (a hypoxia state) and extrahepatic tissues, followed by a release of the intracellular enzymes into the circulation

Table IV. Effect of purslane extract and/or fish oil on nitric oxide (NO) and malondialdehyde (MDA) levels in liver, kidney and heart tissues of rats 15 days post 6 Gy g-irradiation in different groups. NO (mmol/g tissue) Parameter/groups Control Purslane (P) Fish oil (Fo) Irradiated (R) R P R Fo R P  Fo

MDA (nmol/g tissue)

Liver

Kidney

Heart

Liver

Kidney

Heart

52.7  4.0 52.6  4.1 52.9  4.2 23.3  2.7C 44.3  3.3R 45.8  3.4R 48.2  4.1R

31.8  2.4 32.1  2.6 32.4  2.7 15.9  1.6C 27.1  2.9R 27.6  3.1R 28.9  3.2R

29.8  2.5 29.3  2.7 29.6  2.7 13.7  1.3C 22.9  2.0R 22.0  2.1R 25.8  2.4R

180.8  4.9 181.2  5.3 181.6  5.4 241.0  6.6C 201.2  8.5R 207.2  8.5R 187.1  7.4RZ

136.5  7.7 136.9  7.7 137.1  7.8 196.9  9.1C 149.4  8.7R 151.4  8.2R 142.8  8.7R

89.3  4.7 89.4  4.7 89.8  4.9 136.1  7.6C 99.1  5.0 101.2  5.4R 95.1  4.8R

­ alues are expressed as mean of 6 rats  standard error. Csignificantly different compared to control; Rsignificantly different V compared to irradiated group; Zsignificantly different compared to irradiated animals treated with P or Fo.

1188  A. S. H. Abd El-Azime et al. Table V. Effect of purslane and/or fish oil on superoxide dismutase (SOD) and catalase (CAT) activities in liver, kidney and heart tissues of rats 15 days post 6 Gy g-irradiation in different groups. SOD (U/g) CAT (U/g) Parameter /groups Control Purslane (P) Fish oil (Fo) Irradiated (R) R  P R  Fo R  P  Fo

Liver

Kidney

229.3  6.7 229.8  6.8 230.2  6.9 156.5  4.4C 202.0  5.9R 206.5  5.7R 220.6  5.7RZ

139.3  4.2 139.9  3.9 139.0  3.6 106.2  3.9C 127.9  3.3R 127.3  3.1R 132.6  4.0R

Heart 71.4  2.8 71.8  2.8 71.0  2.8 52.4  2.1C 62.9  2.6R 63.3  2.7R 65.7  3.0R

Liver

Kidney

Heart

135.4  4.8 135.9  4.8 135.1  4.9 97.6  3.6C 126.8  4.4R 125.9  4.4R 129.5  4.7R

151.1  5.8 151.8  5.9 151.4  5.9 109.1  3.7C 141.2  4.7R 140.9  4.6R 145.3  4.7R

59.3  2.8 59.7  2.9 59.2  2.7 36.3  1.5C 48.5  2.7R 48.0  2.5 51.0  2.93R

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­ alues are expressed as mean of 6 rats  standard error. Csignificantly different compared to control; Rsignificantly different V compared to irradiated group; Zsignificantly different compared to irradiated animals treated with P or Fo.

(El-Khafif et  al. 2003). Bilirubin is a chemical breakdown product of hemoglobin that is conjugated with glucuronic acid in hepatocytes to increase its water solubility. Elevated total and direct bilirubin post-irradiation indicate liver degeneration induced by hepatocytes (Taysi et al. 2002). The results showed that purslane extract exerted a hepatoprotective effect. Restoration of serum enzymes was proven by Annush et  al. (2011) and the decrease in liver function indicates its protective role against liver damage (Dkhil et al. 2011). Purslane contain omega-3, omega-6 and phenolic compounds (Ivo et al. 2009) which act against oxidative stress (Cherukui et al. 2013). The serum activities of ALT; AST and ALP as well as the level of bilirubin in animals treated with fish oil after irradiation showed the protective effect of fish oil on irradiation-induced hepatotoxicity in rats (El-Daley 1996). Radiation-induced oxidative stress in the kidney was associated with a significant increase in the level of serum urea, creatinine and uric acid. It could be referred to increased ammonia formed by deamination of amino acids in the liver converted to urea (Ganong 1999). Reduction-oxidation imbalances lead to kidney injury and proteinuria (Nistala et al. 2008). The impaired detoxification function of the liver by irradiation could also contribute in the increase of urea in the blood (Robbins et  al. 2001). Significant elevation in serum uric acid of irradiated rats might be related to the breakdown of nucleotides into uric acid (Ganong 1999). The increase in the degree of injury in kidney caused by irradiation may lead to a significant increase in the serum urea and creatinine concentration. Zhao et  al. (2006) reported that radiation-induced normal tissue injury in late-responding organs, such as the kidney, involves complex and dynamic responses between multiple cell types that not only lead to targeted cell death but also to acute and chronic alterations in cell function. Treatment with purslane and/or fish oil showed their ability to restore the normal functional status of the kidney. Decreased levels of urea, uric acid and creatinine in the purslane and/or fish oil-treated animals, may be due to the antioxidative potential of purslane (Shirwaikar et  al. 2003). Gholamreza et al. (2010) proved that the aqueous extract of purslane possesses marked nephroprotective activity and could have a promising role in the treatment of acute renal injury induced by nephrotoxins. Besides, treatment of rats with fish oil decreased serum urea, creatinine, serum and uric acid levels due to its ability to treat nephrotoxicity (Karahan

et al. 2005). Priyamvada et al. (2008) attributed the decreases in kidney function to the presence of omega-3, a constituent of fish oil, in gentamicen nephrotoxicity. In addition, an important factor in the nephroprotective activity of any drug is the ability of its constituents to inhibit the aromatase activity of cytochrome P-450 (Speck et al. 1990) which could be attributed to fish oil. Whole body gamma-irradiation of 6 Gy showed a significant decrease of nitric oxide accompanied by elevation of MDA in different tissues (liver, kidney and heart) compared to irradiated group, which coincides with Gorbunov et al. (2000). Exposure of animals to ionizing radiation may increase the generation of ROS, which induce the intracellular Ca2  over load (Dhalla et  al. 2000). The elevation of cellular Ca2  activates the nitric oxide synthase (NOS) enzyme resulting in local NO production which transiently inhibit mitochondrial ATP production and in turn decrease myocardial contractility (Kanai et  al. 2001). In the present study, the marked increase in MDA levels in liver, kidney and heart tissues of rats could be due to free radicals production. MDA, which is a secondary product of lipid peroxidation, is known to cause cross linkage of membrane component containing amino groups, which makes the membrane fragile (Manda and Bhatia 2003). In the present study, NO level in the liver, kidney and heart were significantly low in the irradiated group. NO plays an important role in diverse range of physiological processes. It reacts with the super oxide anion to generate peroxynitrite, which is a selective oxidant, and nitrating agent that interacts with numerous biological molecules, causing their damage (Choi et  al. 2006). In the present study, oral administration of purslane and/or fish oil significantly increased NO liver, kidney and heart tissues compared to irradiated group. That may be attributed to their high content of omega-3 that plays an important role in the protection of cells against ROS by increasing the activity of antioxidant enzymes manufactured by the body (Bhattacharya et al. 2007). The results showed that oral administration of purslane and/or fish oil significantly decrease MDA liver, kidney and heart tissues. The decrease may be due to the role of purslane in slowing down oxidative stress related to lipid peroxidation and in addition, to the presence of beneficial health effects against various pathologies (Simopoulos 1991) including, cancers, inflammatory and immune renal disorders (Thakkar et al. 2000).

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Effect of purslane extract and fish oil on radiation damage  1189 Data obtained in the present study showed that whole body gamma-irradiation of 6 Gy showed a significant decrease in CAT and SOD in different tissues (liver, kidney and heart) as compared to the control group. Free radical scavenging enzymes such as SOD and CAT are the first line of defense against oxidative injury. The inhibition of antioxidant system may cause the accumulation of H2O2 or products of its decomposition (Sabitha and Shyamaladevi 1999). Decrease in SOD activity might be due to loss of mitochondria, leading to a decrease in total SOD activity or due to a feedback inhibition or oxidative inactivation of enzyme protein caused by ROS generation (Yilmaz et al. 2004). The results showed an increase of SOD and CAT activities in liver, kidney and heart tissues of rats treated with purslane. This might be because purslane is also reported as an excellent source of the antioxidant vitamins: a-tocopherol, ascorbic acid and b- carotene, as well as glutathione and a rich source of many amino acids like isoleucine, leucine, lysine, methionine, cystine, phenylalanine, tyrosine, threonine and valine (Hao et al. 2009). The elevation of SOD and CAT by fish oil is attributed to its content of a-tocopherol, as an antioxidant which protects membrane lipids against peroxidation. Gokkusu and Mostafazadeh (2003) reported that long-term administration of vitamin E activated the antioxidant enzymes, scavenged toxic free radicals, and in turn protected cellular structures against hypercholesterolemia induced peroxidation reactions.

Conclusion Based on the results obtained in the current study, it appears that purslane extract and fish oil may have therapeutic potential to improve hepatic and renal functions as well as oxidative stress in irradiated rats. Moreover, their co-administration showed a better improved liver function. Therefore further studies are needed to clarify the mechanism(s) responsible for the beneficial effects of purslane and fish oil observed in this study that may rank them among the important foods in the future.­­

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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