Ameliorative Effect of Pumpkin Oil (Cucurbita Pepo L.) Against Alcohol-Induced Hepatotoxicity and Oxidative Stress in Albino Rats

beni-suef university journal of basic and applied sciences 3 (2014) 178e185

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Full Length Article Ameliorative effect of pumpkin oil (Cucurbita pepo L.) against alcohol-induced hepatotoxicity and oxidative stress in albino rats

Howida Sayed Abou Seif

Medical Physiology Department, National Research Center, Cairo, Egypt article info abstract

Article history: Objective: The aim of the present study was to evaluate the protective role of pumpkin oil on Received 19 April 2014 experimental alcohol e induced hepatotoxicity. Received in revised form Materials and methods: Rats are divided into three groups of 10 animals each. Group one (G1) 31 July 2014 was the control group is orally given distilled water for 4 weeks. Group two (G2) is given Accepted 13 August 2014 absolute ethyl alcohol (10%) in drinking water for 4 weeks. Group three (G3) alcohole Available online 1 November 2014 administered rats were pretreated with pumpkin oil (50 mg/kg body weight) three times per week for three weeks and alcohol (10%) three times per week (at the ﬁrst two weeks of Keywords: the experiment). Pumpkin oil-hepatotoxicity Results: Alcohol caused a marked rise in serum alanine aminotransferase (ALT), aspartate Rattus norvegicus aminotransferase (AST), alkaline phosphatase (ALP) and gamma glutamyl transferase Oxidative stress (gGT) activities. Concerning oxidative stress and antioxidant defense system, the depleted hepatic glutathione content, glutathione-S-transferase and catalase activities of alcohol- administered rats were potentially increased above normal levels as a result of pretreatment with pumpkin oil. However, while elevated lipid peroxidation was noticed in alcohol treated rats, pretreatment with pumpkin oil produced a detectable decrease in lipid peroxidation level. Conclusion: The natural plant components found in pumpkin could improve the liver against alcohol-induced liver toxicity and oxidative stress. However, further clinical studies are required to assess the safety and beneﬁts of pumpkin oil in human beings. Copyright 2014, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved.

and causes disease and toxicity. Accumulating evidence sug- 1. Introduction gest that intermediates of oxygen reduction may be associated with the development of alcoholic disease (Calabrese Alcohol is the most psychoactive substance used after et al., 2002). Ethanol or its metabolites can prompt a sharp caffeine. Chronic alcoholism is a major public health problem increase of free radicals in the human body (e.g. hepatic cells)

E-mail address: [email protected]. Peer review under the responsibility of Beni-Suef University. http://dx.doi.org/10.1016/j.bjbas.2014.08.001 2314-8535/Copyright 2014, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved. beni-suef university journal of basic and applied sciences 3 (2014) 178e185 179

by acting as a prooxidant or by reducing antioxidant levels and which should be attributed to the large quantities of the contributing to the progression of a variety of chronic diseases phenolics present. Phenolic compounds belong to a (Clemens and Jerrells, 2004). Reactive oxygen species (ROS) are numerous group of antioxidants and act via different highly reactive and can damage lipids, proteins and DNA modes, e.g. by ‘scavenging’ free radicals. Phenolic com- (Arteel, 2003). The ROS, the main culprit, the other one being pounds can also enhance the activity of other antioxidants, reactive nitrile species (RNS) are capable of damaging several for example that of fat-soluble vitamins (Dru_zynskaetal., cellular components such as proteins, lipids and DNA (Koneru 2008). et al., 2011). Pumpkin seeds (Cucurbita pepo L.) are a rich source of Liver is the primary organ for the metabolism of ingested unsaturated fatty acids, antioxidants and fibers, known to alcohol (Shanmugam et al., 2010).The liver is the largest, have anti-atherogenic and hepatoprotective activities (Makni important organ and the site for essential biochemical re- et al., 2008). Pumpkin is one such plant that has been actions in the human body. It has the function to detoxify frequently used as functional food or medicine (Caili et al., toxic substances and synthesize useful biomolecules. 2006). Some of its common uses in most countries are for Therefore, damage to the liver leads to grave consequences. diabetes where it is used internally, as well as externally for Alcohol induces oxidative stress which is known to cause management of worms and parasites. Treatment of sponta- liver injury that is many biochemical metabolic reactions neously hypertensive rats with felodipine or captopril mono- occur as a result of it. Some of these include redox state therapy or combined with pumpkin seed oil produced changes, production of reactive acetaldehyde, damage to the improvement in the measured free radical scavengers in the mitochondria of cells, cell membrane damages, hypoxia, ef- heart and kidney (Al-Zuhair et al., 2000). fects on immune system, altered cytokine production, and Being rich in unsaturated fatty acids especially linoleic induction of CYP2E1 and mobilization of iron (Baskaran et al., and oleic acid and tocopherols and with very high oxidative 2010). Alcoholic liver disease is a worldwide health problem stability, pumpkin seed oil is suggested to be a healthy which has three manifestations in form of fatty liver/stea- addition towards human diet and have potential suitability tosis, alcoholic hepatitis and liver cirrhosis. At least 80% of for food and industrial applications (Stevenson et al., 2007). chronic alcoholic consumers may develop steatosis, 10e35% In addition to the carotenoids and gamma aminobutyric alcoholic hepatitis and approximately 10% liver cirrhosis. acids (GABA) found in the fruits (Liu, 2001), there are other Intake of alcohol causes accumulation of reactive oxygen biologically active ingredients, which are found in pump- species (ROS) like superoxide, hydroxyl radical and hydrogen kins (Gossell-Williams et al., 2008) such as, sterols, proteins peroxide in the hepatic cells that oxidize the glutathione and peptides, polysaccharides, para-aminobenzoic acid and which leads to lipid peroxidation of cellular membranes, fixed oils. Essential fatty acids are necessary for human oxidation of protein and DNA resulting in hepatic damage health but the body cannot make them; so they must be (Muhammad et al., 2009). Initially, in the liver alcohol is taken through food (Eynard et al., 1992). Pumpkin seed oil's metabolized into the highly toxic acetaldehyde by the main nutrients are: essential fatty acid-omega 6, omega 9, enzyme alcohol dehydrogenase. Acetaldehyde is then phytosterols, and antioxidants such as carotenoids, vitamin oxidized to acetate by acetaldehyde oxidase or xanthine ox- AandvitaminE(Murkovic et al., 1996). Linoleic acid, a idase giving rise to ROS via cytochrome P450 2E1. Prolonged polyunsaturated fatty acid present in pumpkin seed oil, is consumption of alcohol increases nitric oxide (NO) level known to increase membrane fluidity and allows for which leads to formation of toxic oxidant peroxynitrite. Low osmosis, intracellular and extra cellular gaseous exchange capacity of antioxidants in this situation leads to damage of (Lovejoy, 2002). Pumpkin seed oil includes fatty acids: pal- the cells of the hepatic cells and the cell organelles with the mitic (C 16:0), stearic (C 18:0), oleic (C 18:1) and linoleic (C release of reactive aldehydes and ROS (Saalu et al., 2012). 18:2) (Kulaitiene et al., 2007). Antioxidants are the sub- Treatment options available for common liver diseases such stances that when present in low concentration signifi- as cirrhosis, fatty liver and chronic hepatitis are inadequate cantly delay or reduce the oxidation of the substrate in modern medicine. Conventional drugs used in the treat- (Halliwell, 2000). ment of liver diseases such as corticosteroids, antiviral, Compared pumpkin oil with another oil, Jia et al. (2011) immunosuppressant may lead to serious adverse effects; evaluated the protective effects of almond oil against acute they may even cause hepatic damage on prolonged use. hepatic injury induced by carbon tetrachloride in rats. These Therefore, alternative drugs in the form of herbal medicines result demonstrated that almond oil has potent hep- which are now used for the treatment of liver diseases are atoprotective effects, and could be developed as a functional sought instead of currently used drugs of doubtful efficacy food for the therapy and prevention of liver damage. It is well and safety (Vetriselvan et al., 2010). known that almonds contain a wide variety of phenolic acids Natural antioxidants are found in many compounds and flavonoids and the consumption of almonds has been classified as secondary plant metabolites, e.g. in poly- associated with a reduced risk of chronic diseases. (Milbury phenols (phenolic acids, flavonoids) and terpenoids (carot- et al., 2006). The almond contains as much as 50% oil (Zhang enoids), and the consumption of foods which contain these et al., 2009). As one of the most popular vegetable oils, compounds in large quantities seems to play an important almond oil is rich in mono and poly-unsaturated fatty acids, role in prophylaxis against many diseases. Epidemiological with oleic and linoleic acids as the major constituents, and a studies have revealed that the incidence of some cardio- number of minor components such as tocopherols and vascular and cancer diseases is less frequent when fruit phenolic compounds. Monounsaturated (MUFA) and poly- and vegetables are consumed regularly (Horubała, 1999), unsaturated fatty acids (PUFA), as well as minor lipid 180 beni-suef university journal of basic and applied sciences 3 (2014) 178e185

components, play an important role in human nutrition Pumpkin oil dose was adjusted to 50 mg/kg and were given health (Turan et al., 2007). Diets rich in these compounds can daily for 4 weeks (Al-Zuhair et al., 2000). decrease blood pressure and total blood cholesterol levels, prevent oxidative stress and maintain body weight in humans. Some studies have shown the antioxidant activity 2.5. Blood and organ sampling and free radical scavenging capacity of almond oil in vitro. The antioxidant activities of minor components of almond oil Rats were anesthetized with diethyl ether and after decapi- have also been reported. (Espıń et al., 2000; Li et al., 2007; tation and dissection, blood samples were collected and liver Ramadan and Moersel, 2006). was excised for physiological and biochemical assays as well Based on these previous studies, the present work aimed to as for histopathological studies. Blood sample was collected assess the protective and antioxidant effects of pumpkin oil from jugular vein of each animal (5 ml) in a centrifuge tube against alcohol induced hepatotoxicity and oxidative stress in and left to clot at room temperature for 45 min. Sera were albino rats. separated by centrifugation at 3000 r.p.m. at 30 C for 15 min and kept frozen at 30 C for various physiological and biochemical analyses. 2. Materials and methods Liver from each animal was excised after dissection. One part was fixed in buffered formalin for 24 h, trimmed and then 2.1. Experimental animals transferred into 70% alcohol for histopathological examination. 0.5 g was homogenized in 5 ml 0.9% NaCl (10% w/v) using Adult male albino rats (Rattus norvegicus) weighing teflon homogenizer (Glas-Col, Terre Haute, USA). 120e150 g were used in the present study. The animals were obtained from the animal house in the Ophthalmology 2.6. Biochemical analyses Research Center, Giza, Egypt. They were kept under obser- vation for two weeks before the onset of the experiment to Serum total bilirubin concentration was determined by a exclude any intercurrent infection. The animals were kept at colorimetric procedure using kits obtained from Bio- room temperature and exposed to natural daily lightedark diagnostic (Egypt) according to Walter and Gerad (1970). cycles. Rats were fed ad libitum and clean water was Serum ALT and AST activities were determined by using kits continuously available. All animal procedures are in accor- obtained from Biodiagnostic (Egypt) according to the dance with the recommendations for the proper care and use methods of Reitman and Frankel (1957). Serum ALP and GGT of laboratory animals stated by the Canadian Council on were determined by using kits obtained from Biodiagnostics Animal Care (CCAC, 1993). according to the method of Belfield and Goldberg (1971) and Szasz (1969), respectively. Serum total protein was deter- 2.2. Chemicals mined by using kits obtained from Biodiagnostics according to the method of Young (2001). Serum LDH was estimated Pumpkin oil was purchased from El Captin pharmaceutical according to the method described by Bu¨ hl and Jackson company. All other chemicals used for the investigation were (1978), using reagent kit purchased from Stanbio Labora- of analytical grade. tories (Texas, USA). Liver glutathione (GSH) was determined according to the method of Beulter et al. (1963). Liver lipid 2.3. Doses and treatment peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS) according to the method of Alcoholism was experimentally induced in animals by giving Preuss et al. (1998). Liver glutathione-s-transferase activity absolute ethyl alcohol (10%) in drinking water for 15 days was assayed using the method of Matkovics et al. (1998) and before the experiment (Hekmatpanah et al., 1994). Pumpkin Mannervik and Gutenberg (1981). Liver catalase activity oil dose was adjusted to 50 mg/kg and were given daily for 4 were assayed following the method of Kar and Mishra weeks (Al-Zuhair et al., 2000). (1976).

2.4. Experimental design 2.7. Histopathological studies

Animals were divided into three groups comprising ten ani- Fixed liver tissue samples were embedded after dehydration mals each: in parafﬁn wax, sectioned at thickness of 5 mm and stained with hematoxylin and Eosin (HandE) for general histopatho- 1 Group 1 (normal control) is orally given the equivalent logical examination using the light microscope. volume of the vehicle 1 (distilled water) daily for 4 weeks. 2.8. Statistical analysis 2 Group 2 (positive”þve”control) is given absolute ethyl alcohol (10%) in drinking water for 4 weeks (Hekmatpanah The data were analyzed using the one-way analysis of variance et al., 1994). (ANOVA) (PC-STAT, 1985) followed by LSD analysis to compare 3. Group 3 (treated with pumpkin oil and alcohol) is given various groups with each other. Results were expressed as absolute ethyl alcohol (10%) in drinking water for 15 days mean ± standard error (SE). F-probability, obtained from one- before the experiment (Hekmatpanah et al., 1994). way ANOVA, expresses the effect between groups. beni-suef university journal of basic and applied sciences 3 (2014) 178e185 181

Table 1 e Protective effect of pumpkin oil on serum liver function activities in alcohol treated rats. Treatments Parameters ALT (U/ml) % change AST (U/ml) % change ALP (IU/L) % change g e GT (U/L) % change

G1 39.00 ± 0.815d e 185.00 ± 0.001b e 429.5 ± 6.75b e 9.28 ± 0.746a e Normal G2 56.50 ± 0.289a 44.87 202.00 ± 0.575a 9.19 781.5 ± 6.95a 81.96 12.15 ± 5.79a 30.93 (Alcohol) G3 47.00 ± 0.575c 16.81 201.50 ± 0.289a 0.741 386.0 ± 4.62b 50.61 2.23 ± 0.281a 81.65 Pumpkin oil þ Alcohol F-Probability P < 0.0001 e P < 0.0001 e P < 0.0001 e P < 0.0771 e LDS at 5% level 2.88 e 9.84 e 16.06 ee e LDS at 1% level 4.04 e 13.79 e 22.51 ee e

Data are expressed as mean ± standard error. Number of animals in each group is ten. Mean, which have the same superscript symbol(s), are not signiﬁcantly different. Percentage changes (%) were calculated by comparing normal group with alcohol treated group and pre-treated alcohol group with alcohol treated group.

These results are in agreement with Padmanabhan and 3. Results and discussion Jangle (2014) who illustrated that alcohol feeding causes eleva- tion in serum activities of AST, ALT, ALP, gGT and LDH enzymes One-way ANOVA revealed that the effect on the variables of which are markers of liver damage. In the study reported by < oxidative stress and antioxidant system was of p 0.001 be- Vidhya et al. (2009) ethanol treated group resulted in significant tween groups. increase in AST and ALT activities, which is an indication of Changes in different serum variables related to liver hepatocellular damage in rats, whereas treatment with quer- function are presented in Tables 1 and 2. Concerning serum cetin reduced ethanol einduced toxicity as indicated by the enzymes related to liver function, the alcohol-administered lowering of marker enzymes. In alcohol intoxication as a result < rats exhibited significant increase (p 0.01; LSD) in ALT, AST of structural changes, an increase in the cell membrane and ALP activities. The pre-treatment with pumpkin oil suc- permeability to ions results. The increase in membrane cessfully ameliorated the elevated activities of ALT and ALP, permeability causes leakage of ALT and AST into blood circu- g although recorded non-significant changes on AST and GT lation as shown by abnormally high levels of serum hepatic activities. Serum total bilirubin concentration was elevated in markers. The observations by Vermaulen et al. (1992) which alcohol-administered rats but recording a significant decrease indicated that ALT, AST, gGT and ALP are normally located in in pumpkin oil pretreated rats. Serum total protein level was the cytoplasm and released into circulation after hepatic increased as a result of administration of alcohol alone, while cellular damage. Nkosi et al. (2005) and Oboh (2005) observed it was significantly decreased in animals pretreated with that the administration of pumpkin seeds proteins after CCl4 pumpkin oil. Serum LDH activity was significantly increased intoxication resulted in significantly reduced activity levels of in alcohol administered rats, although have a non-significant lactate dehydrogenase, alanine transaminase, aspartate trans- increase in pumpkin oil pretreatment. aminase and alkaline phosphatase.

Table 2 e Protective effect of pumpkin oil on serum total bilirubin, total protein levels and lactate dehydrogenase activity in alcohol treated rats. Treatments Parameters Total bilirubin (mg/dl) % change Total protein (g/dl) % change LDH (U/L) % change

G1 2.50 ± 0.289ab e 6.80 ± 0.349a e 261.48 ± 18.46c e Normal G2 3.55 ± 0.685a 42.0 7.20 ± 0.423a 5.88 343.00 ± 21.08ab 31.18 (Alcohol) G3 1.03 ± 0.132c 70.99 6.58 ± 0.61a 8.61 367.13 ± 8.16a 7.03 Alcohol þ Pumpkin oil F-Probability P < 0.004 e P < 0.529 e P < 0.0011 e LDS at 5% level 1.22 ee e43.81 e LDS at 1% level 1.71 ee e61.42 e

Table 3 show the effect of the tested pumpkin oil on the a significant decrease in the activities of catalase in alcoholic liver oxidative stress markers and antioxidant defense system subjects (Husain and Somani, 1997). A decreased activity of of normal and alcohol-administered rats. The pre-treatment CAT was due to exhaustion of the enzyme as a result of with pumpkin oil produced a potential increase (p < 0.01; oxidative stress induced by the alcohol. Presumably, a LSD) of the glutathione level, glutathione-S-transferase and decrease in CAT activity could be attributed to cross-linking catalase activities when compared to alcohol treated group and inactivation of the enzyme protein in the lipid peroxides. which exhibited significant decrease in GSH level, Xu (2000) who found that pumpkin polysaccharide could glutathione-S-transferase and catalase activities as compared increase the SOD and GSH-Px activity and reduce the MDA to normal control group. Liver lipid peroxidation recorded a content in tumor mice serum (Xu, 2000). Pandanaboina et al. significant increase as a result of alcohol administration while (2012) who observed a significant increase in lipid peroxida- significantly decreased in pumpkin oil treated rats corre- tion during alcohol consumption, as reported by earlier sponding to control group. studies (Das and Vasudevan, 2006). The alcohol intoxication The current results are in agreement with Brown et al. increases lipid peroxide production (LPO) in various tissues, (2004), Mallikarjuna et al. (2008); Shanmugam et al. (2011) and is indicative of tissue oxidative stress. Nearly 60e80% who found that GSH acts as an antioxidant and a powerful ingested alcohol is metabolized in the liver, and this makes it nucleophile, critical for cellular protection, such as detoxifi- more vulnerable than other organs to alcohol-induced cation of reactive oxygen species (ROS), conjugation and oxidative stress (Lieber, 2003). This concept is supported by excretion of toxic molecules and control of inflammatory the larger extent of an increase in LPO production in the liver, cytokine cascade (Brown et al., 2004). It has been mentioned as compared to other organs. Plasma and liver contain en- that the antioxidant activity of plants might be due to their zymes such as catalase (CAT), superoxide dismutase (SOD) phenolic compounds (Hatapakki et al., 2005). The levels of and glutathione peroxidase (GPx), which showed contribute to GSH were significantly decreased in alcohol-treated rats, as the antioxidant defense mechanism (Lee et al., 2002). Mary well as earlier published reports, which showed that the GSH et al. (2002) and Visavadiya and Narasimhacharya (2007) concentration decreases during alcohol ingestion observed a decrease in the activities of the antioxidant en- (Mallikarjuna et al., 2008; Shanmugam et al., 2011). It is easily zymes SOD, CAT and GPx in hypercholesterolemic rats. Such susceptible to lipid peroxidation. Pumpkin seed oil has shown decreases may be associated to the production of a-, b-un- to possess strong antioxidant properties in experimental an- saturated aldehydes during lipid peroxidation. These com- imals (Dreikorn et al., 2002). Ahmed et al. (2009) reported that pounds have the ability to increase oxidative stress by pumpkin seed oil administration showed a significant eleva- promoting the cellular consumption of glutathione and by tion in the striatum and serum glutathione level at all-time inactivating selenium-dependent glutathione peroxidase intervals in adult male albino rats. Pumpkin seeds of Cucur- (Kinter and Roberts, 1996). GSH serves as a substrate for the bita pepo L. a herbaceous plant of Cucurbitaceae family, enzyme GPx, and it had been suggested that, through its ac- contain 40.4e55.6% of linoleic acid: LA; 18:2 n-6, x-6 fatty acid tivity, GSH protects plasma against oxidative damage. More- (Al-Khalifa, 1996; Ganzera et al., 1999; Jamieson, 1943). They over PUFAs (linoleic acid “LA” and alpha-linolenic acid “ALA”) may have beneficial effects in health and may prevent chronic have displayed protection against lipid peroxidation diseases (Al-Khalifa, 1996; Kumar et al., 2006). increasing the levels of several cellular antioxidants such as Catalase acts as a preventive antioxidant and plays an ascorbic acid, a-tocopherol and GSH (Nagao et al., 2005). important role in the protection against the deleterious effects Fig. 1 showed the histology of liver of normal control. There of LPO (lipid hydroperoxide). Reports have shown that there is was no histopathological alteration observed and the normal

Table 3 e Protective effect of pumpkin oil on liver glutathione level, glutathione-s-transferase and catalase activities and lipid peroxidation level in alcohol treated rats. Treatments Parameters GSH mg/gm % change GST U/gm % change Catalase % change MDA % change U/g nmol/g

G1 73.70 ± 1.09a e 6.04 ± 0.146b e 1.90 ± 0.001a e 36.31 ± 0.255b e Normal G2 54.53 ± 1.12d 39.58 3.63 ± 0.154c 9.77 0.371 ± 0.001b 80.47 50.97 ± 0.921a 28.76 (Alcoholic) G3 68.93 ± 1.55b 54.49 6.98 ± 0.049a 92.29 1.71 ± 0.162a 360.92 23.48 ± 0.363c 35.33 Alcohol þ Pumpkin oil F-Probability P < 0.0001 e P < 0.0001 e P < 0.0001 e P < 0.0001 e LDS at 5% level 4.65 e 0.479 e 0.256 e 1.72 e LDS at 1% level 6.51 e 0.672 e 0.359 e 2.42 e

Fig. 1 e A transverse section of a liver of normal control Fig. 3 e Fatty change was observed in diffuse manner (d) all (distilled water) rat showing a central vein (CV), hepatic over the hepatocytes in ethyl alcohol treated group (G2). cords (h) and sinusoids in between. £400. £400.

Fig. 4 e The hepatocytes showed fatty change in diffuse manner in pumpkin pretreated group (G3). £400. Fig. 2 e The portal area showed sever dilatation and congestion in the portal vein (PV) associated with periductal fibrosis (f) surrounding the bile ducts (b) in ethyl alcohol treated group (G2). £400. and chronic administration of alcohol causes formation of cytokines in large amounts, particularly TNFea by hepatic Kupffer cells, which plays a major role in causing hepatic damage. Moreover, chronic administration of alcohol results histological structure of the central vein and surrounding in accumulation of hepatic lipids, lipid peroxides leading to hepatocytes. the autooxidation of hepatic cells acting as pro-oxidants and The portal area in ethyl alcohol administered rats (G2) decreasing antioxidant levels thereby resulting in a note- showed severe dilatation and congestion in the portal vein worthy hepatotoxicity. (Kumar et al., 2013). associated with periductal fibrosis surrounding the bile ducts (Fig. 2). Alcohol administered rats also recorded fatty change in diffuse manner all over the hepatocytes (Fig. 3). Fig. 4 showed fatty change in diffuse manner in the hepatocytes in 4. Conclusion pumpkin oil pretreated rats. The current study was in agreement with Padmanabhan The present results demonstrated that pumpkin oil may play and Jangle (2014).who found that the higher levels of alcohol an important role in the protection against alcohol induced intake cause cirrhosis and liver damage by enhancing lipid hepatotoxicity and oxidative stress. Pretreatment with peroxidation in the liver. Acetaldehyde the toxic metabolite of pumpkin oil may protect the liver from the hepatotoxic effect alcohol depresses liver GSH by conjugating with sulphydryl and oxidative stress caused by alcohol. Further clinical studies groups of GSH (Maruthaappan and Shree, 2009). In both acute are required to assess the benefits and safety of pumpkin oil 184 beni-suef university journal of basic and applied sciences 3 (2014) 178e185

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