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
a r t i c l e i n f o
Article history:Received 19 April 2014 Received in revised form 31 July 2014
Accepted 13 August 2014
Available online 1 November 2014
Keywords:
Pumpkin oil-hepatotoxicity Rattus norvegicus
Oxidative stress
a b s t r a c t
Objective: The aim of the present study was to evaluate the protective role of pumpkin oil on experimental alcohole induced hepatotoxicity.
Materials and methods: Rats are divided into three groups of 10 animals each. Group one (G1) was the control group is orally given distilled water for 4 weeks. Group two (G2) is given absolute ethyl alcohol (10%) in drinking water for 4 weeks. Group three (G3) alcohole 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 the experiment).
Results: Alcohol caused a marked rise in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma glutamyl transferase (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 pretreat-ment with pumpkin oil. However, while elevated lipid peroxidation was noticed in alcohol treated rats, pretreatment with pumpkin oil produced a detectable decrease in lipid per-oxidation 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.
1.
Introduction
Alcohol is the most psychoactive substance used after caffeine. Chronic alcoholism is a major public health problem
and causes disease and toxicity. Accumulating evidence sug-gest that intermediates of oxygen reduction may be associ-ated with the development of alcoholic disease (Calabrese et al., 2002). Ethanol or its metabolites can prompt a sharp increase of free radicals in the human body (e.g. hepatic cells)
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by acting as a prooxidant or by reducing antioxidant levels and contributing to the progression of a variety of chronic diseases (Clemens and Jerrells, 2004). Reactive oxygen species (ROS) are highly reactive and can damage lipids, proteins and DNA (Arteel, 2003). The ROS, the main culprit, the other one being reactive nitrile species (RNS) are capable of damaging several cellular components such as proteins, lipids and DNA (Koneru et al., 2011).
Liver is the primary organ for the metabolism of ingested alcohol (Shanmugam et al., 2010).The liver is the largest, important organ and the site for essential biochemical re-actions in the human body. It has the function to detoxify toxic substances and synthesize useful biomolecules. Therefore, damage to the liver leads to grave consequences. Alcohol induces oxidative stress which is known to cause liver injury that is many biochemical metabolic reactions occur as a result of it. Some of these include redox state changes, production of reactive acetaldehyde, damage to the mitochondria of cells, cell membrane damages, hypoxia, ef-fects on immune system, altered cytokine production, and induction of CYP2E1 and mobilization of iron (Baskaran et al., 2010). Alcoholic liver disease is a worldwide health problem which has three manifestations in form of fatty liver/stea-tosis, alcoholic hepatitis and liver cirrhosis. At least 80% of chronic alcoholic consumers may develop steatosis, 10e35% alcoholic hepatitis and approximately 10% liver cirrhosis. Intake of alcohol causes accumulation of reactive oxygen species (ROS) like superoxide, hydroxyl radical and hydrogen peroxide in the hepatic cells that oxidize the glutathione which leads to lipid peroxidation of cellular membranes, oxidation of protein and DNA resulting in hepatic damage (Muhammad et al., 2009). Initially, in the liver alcohol is metabolized into the highly toxic acetaldehyde by the enzyme alcohol dehydrogenase. Acetaldehyde is then oxidized to acetate by acetaldehyde oxidase or xanthine ox-idase giving rise to ROS via cytochrome P450 2E1. Prolonged consumption of alcohol increases nitric oxide (NO) level which leads to formation of toxic oxidant peroxynitrite. Low capacity of antioxidants in this situation leads to damage of the cells of the hepatic cells and the cell organelles with the release of reactive aldehydes and ROS (Saalu et al., 2012). Treatment options available for common liver diseases such as cirrhosis, fatty liver and chronic hepatitis are inadequate in modern medicine. Conventional drugs used in the treat-ment of liver diseases such as corticosteroids, antiviral, immunosuppressant may lead to serious adverse effects; they may even cause hepatic damage on prolonged use. Therefore, alternative drugs in the form of herbal medicines which are now used for the treatment of liver diseases are sought instead of currently used drugs of doubtful efficacy and safety (Vetriselvan et al., 2010).
Natural antioxidants are found in many compounds classified as secondary plant metabolites, e.g. in poly-phenols (phenolic acids, flavonoids) and terpenoids (carot-enoids), and the consumption of foods which contain these compounds in large quantities seems to play an important role in prophylaxis against many diseases. Epidemiological studies have revealed that the incidence of some cardio-vascular and cancer diseases is less frequent when fruit and vegetables are consumed regularly (Horubała, 1999),
which should be attributed to the large quantities of the phenolics present. Phenolic compounds belong to a numerous group of antioxidants and act via different modes, e.g. by ‘scavenging’ free radicals. Phenolic com-pounds can also enhance the activity of other antioxidants, for example that of fat-soluble vitamins (Dru_zy n ska et al., 2008).
Pumpkin seeds (Cucurbita pepo L.) are a rich source of unsaturated fatty acids, antioxidants and fibers, known to have anti-atherogenic and hepatoprotective activities (Makni et al., 2008). Pumpkin is one such plant that has been frequently used as functional food or medicine (Caili et al., 2006). Some of its common uses in most countries are for diabetes where it is used internally, as well as externally for management of worms and parasites. Treatment of sponta-neously hypertensive rats with felodipine or captopril mono-therapy or combined with pumpkin seed oil produced improvement in the measured free radical scavengers in the heart and kidney (Al-Zuhair et al., 2000).
Being rich in unsaturated fatty acids especially linoleic and oleic acid and tocopherols and with very high oxidative stability, pumpkin seed oil is suggested to be a healthy addition towards human diet and have potential suitability for food and industrial applications (Stevenson et al., 2007). In addition to the carotenoids and gamma aminobutyric acids (GABA) found in the fruits (Liu, 2001), there are other biologically active ingredients, which are found in pump-kins (Gossell-Williams et al., 2008) such as, sterols, proteins and peptides, polysaccharides, para-aminobenzoic acid and fixed oils. Essential fatty acids are necessary for human health but the body cannot make them; so they must be taken through food (Eynard et al., 1992). Pumpkin seed oil's main nutrients are: essential fatty acid-omega 6, omega 9, phytosterols, and antioxidants such as carotenoids, vitamin A and vitamin E (Murkovic et al., 1996). Linoleic acid, a polyunsaturated fatty acid present in pumpkin seed oil, is known to increase membrane fluidity and allows for osmosis, intracellular and extra cellular gaseous exchange (Lovejoy, 2002). Pumpkin seed oil includes fatty acids: pal-mitic (C 16:0), stearic (C 18:0), oleic (C 18:1) and linoleic (C 18:2) (Kulaitiene et al., 2007). Antioxidants are the sub-stances that when present in low concentration signifi-cantly delay or reduce the oxidation of the substrate (Halliwell, 2000).
Compared pumpkin oil with another oil, Jia et al. (2011)
evaluated the protective effects of almond oil against acute hepatic injury induced by carbon tetrachloride in rats. These result demonstrated that almond oil has potent hep-atoprotective effects, and could be developed as a functional food for the therapy and prevention of liver damage. It is well known that almonds contain a wide variety of phenolic acids and flavonoids and the consumption of almonds has been associated with a reduced risk of chronic diseases. (Milbury et al., 2006). The almond contains as much as 50% oil (Zhang et al., 2009). As one of the most popular vegetable oils, almond oil is rich in mono and poly-unsaturated fatty acids, with oleic and linoleic acids as the major constituents, and a number of minor components such as tocopherols and phenolic compounds. Monounsaturated (MUFA) and poly-unsaturated fatty acids (PUFA), as well as minor lipid
components, play an important role in human nutrition health (Turan et al., 2007). Diets rich in these compounds can decrease blood pressure and total blood cholesterol levels, prevent oxidative stress and maintain body weight in humans. Some studies have shown the antioxidant activity and free radical scavenging capacity of almond oil in vitro. The antioxidant activities of minor components of almond oil have also been reported. (Espı´n et al., 2000; Li et al., 2007; Ramadan and Moersel, 2006).
Based on these previous studies, the present work aimed to assess the protective and antioxidant effects of pumpkin oil against alcohol induced hepatotoxicity and oxidative stress in albino rats.
2.
Materials and methods
2.1. Experimental animalsAdult male albino rats (Rattus norvegicus) weighing 120e150 g were used in the present study. The animals were obtained from the animal house in the Ophthalmology Research Center, Giza, Egypt. They were kept under obser-vation for two weeks before the onset of the experiment to exclude any intercurrent infection. The animals were kept at room temperature and exposed to natural daily lightedark cycles. Rats were fed ad libitum and clean water was continuously available. All animal procedures are in accor-dance with the recommendations for the proper care and use of laboratory animals stated by the Canadian Council on Animal Care (CCAC, 1993).
2.2. Chemicals
Pumpkin oil was purchased from El Captin pharmaceutical company. All other chemicals used for the investigation were of analytical grade.
2.3. Doses and treatment
Alcoholism was experimentally induced in animals by giving absolute ethyl alcohol (10%) in drinking water for 15 days before the experiment (Hekmatpanah et al., 1994). Pumpkin oil dose was adjusted to 50 mg/kg and were given daily for 4 weeks (Al-Zuhair et al., 2000).
2.4. Experimental design
Animals were divided into three groups comprising ten ani-mals each:
1 Group 1 (normal control) is orally given the equivalent volume of the vehicle 1 (distilled water) daily for 4 weeks.
2 Group 2 (positive”þve”control) is given absolute ethyl alcohol (10%) in drinking water for 4 weeks (Hekmatpanah et al., 1994).
3. Group 3 (treated with pumpkin oil and alcohol) is given absolute ethyl alcohol (10%) in drinking water for 15 days before the experiment (Hekmatpanah et al., 1994).
Pumpkin oil dose was adjusted to 50 mg/kg and were given daily for 4 weeks (Al-Zuhair et al., 2000).
2.5. Blood and organ sampling
Rats were anesthetized with diethyl ether and after decapi-tation and dissection, blood samples were collected and liver was excised for physiological and biochemical assays as well as for histopathological studies. Blood sample was collected from jugular vein of each animal (5 ml) in a centrifuge tube and left to clot at room temperature for 45 min. Sera were separated by centrifugation at 3000 r.p.m. at 30C for 15 min and kept frozen at 30 C for various physiological and biochemical analyses.
Liver from each animal was excised after dissection. One part was fixed in buffered formalin for 24 h, trimmed and then transferred into 70% alcohol for histopathological examina-tion. 0.5 g was homogenized in 5 ml 0.9% NaCl (10% w/v) using teflon homogenizer (Glas-Col, Terre Haute, USA).
2.6. Biochemical analyses
Serum total bilirubin concentration was determined by a colorimetric procedure using kits obtained from Bio-diagnostic (Egypt) according to Walter and Gerad (1970). Serum ALT and AST activities were determined by using kits obtained from Biodiagnostic (Egypt) according to the methods ofReitman and Frankel (1957). Serum ALP and GGT were determined by using kits obtained from Biodiagnostics according to the method ofBelfield and Goldberg (1971)and
Szasz (1969), respectively. Serum total protein was deter-mined by using kits obtained from Biodiagnostics according to the method ofYoung (2001). Serum LDH was estimated according to the method described by Bu¨hl and Jackson (1978), using reagent kit purchased from Stanbio Labora-tories (Texas, USA). Liver glutathione (GSH) was determined according to the method ofBeulter et al. (1963). Liver lipid peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS) according to the method of
Preuss et al. (1998). Liver glutathione-s-transferase activity was assayed using the method ofMatkovics et al. (1998)and
Mannervik and Gutenberg (1981). Liver catalase activity were assayed following the method of Kar and Mishra (1976).
2.7. Histopathological studies
Fixed liver tissue samples were embedded after dehydration in paraffin wax, sectioned at thickness of 5 mm and stained with hematoxylin and Eosin (HandE) for general histopatho-logical examination using the light microscope.
2.8. Statistical analysis
The data were analyzed using the one-way analysis of variance (ANOVA) (PC-STAT, 1985) followed by LSD analysis to compare various groups with each other. Results were expressed as mean± standard error (SE). F-probability, obtained from one-way ANOVA, expresses the effect between groups.
3.
Results and discussion
One-way ANOVA revealed that the effect on the variables of oxidative stress and antioxidant system was of p< 0.001 be-tween groups.
Changes in different serum variables related to liver function are presented inTables 1 and 2. Concerning serum enzymes related to liver function, the alcohol-administered rats exhibited significant increase (p< 0.01; LSD) in ALT, AST and ALP activities. The pre-treatment with pumpkin oil suc-cessfully ameliorated the elevated activities of ALT and ALP, although recorded non-significant changes on AST and g GT activities. Serum total bilirubin concentration was elevated in alcohol-administered rats but recording a significant decrease in pumpkin oil pretreated rats. Serum total protein level was increased as a result of administration of alcohol alone, while it was significantly decreased in animals pretreated with pumpkin oil. Serum LDH activity was significantly increased in alcohol administered rats, although have a non-significant increase in pumpkin oil pretreatment.
These results are in agreement with Padmanabhan and Jangle (2014)who illustrated that alcohol feeding causes eleva-tion in serum activities of AST, ALT, ALP, gGT and LDH enzymes which are markers of liver damage. In the study reported by Vidhya et al. (2009) ethanol treated group resulted in significant increase in AST and ALT activities, which is an indication of hepatocellular damage in rats, whereas treatment with quer-cetin reduced ethanol einduced toxicity as indicated by the lowering of marker enzymes. In alcohol intoxication as a result of structural changes, an increase in the cell membrane permeability to ions results. The increase in membrane permeability causes leakage of ALT and AST into blood circu-lation as shown by abnormally high levels of serum hepatic markers. The observations byVermaulen et al. (1992)which indicated that ALT, AST, gGT and ALP are normally located in the cytoplasm and released into circulation after hepatic cellular damage.Nkosi et al. (2005)andOboh (2005)observed that the administration of pumpkin seeds proteins after CCl4 intoxication resulted in significantly reduced activity levels of lactate dehydrogenase, alanine transaminase, aspartate trans-aminase and alkaline phosphatase.
Table 1e 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 Normal 39.00± 0.815d e 185.00± 0.001b e 429.5± 6.75b e 9.28± 0.746a e G2 (Alcohol) 56.50± 0.289a 44.87 202.00± 0.575a 9.19 781.5± 6.95a 81.96 12.15± 5.79a 30.93 G3
Pumpkin oilþ Alcohol
47.00± 0.575c 16.81 201.50± 0.289a 0.741 386.0± 4.62b 50.61 2.23± 0.281a 81.65 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 e e e LDS at 1% level 4.04 e 13.79 e 22.51 e e 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.
Table 2e 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 Normal 2.50± 0.289ab e 6.80± 0.349a e 261.48± 18.46c e G2 (Alcohol) 3.55± 0.685a 42.0 7.20± 0.423a 5.88 343.00± 21.08ab 31.18 G3
Alcoholþ Pumpkin oil
1.03± 0.132c 70.99 6.58± 0.61a 8.61 367.13± 8.16a 7.03
F-Probability P< 0.004 e P< 0.529 e P< 0.0011 e
LDS at 5% level 1.22 e e e 43.81 e
LDS at 1% level 1.71 e e e 61.42 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.
Table 3show the effect of the tested pumpkin oil on the liver oxidative stress markers and antioxidant defense system of normal and alcohol-administered rats. The pre-treatment with pumpkin oil produced a potential increase (p < 0.01; LSD) of the glutathione level, glutathione-S-transferase and catalase activities when compared to alcohol treated group which exhibited significant decrease in GSH level, glutathione-S-transferase and catalase activities as compared to normal control group. Liver lipid peroxidation recorded a significant increase as a result of alcohol administration while significantly decreased in pumpkin oil treated rats corre-sponding to control group.
The current results are in agreement with Brown et al. (2004), Mallikarjuna et al. (2008); Shanmugam et al. (2011)
who found that GSH acts as an antioxidant and a powerful nucleophile, critical for cellular protection, such as detoxifi-cation of reactive oxygen species (ROS), conjugation and excretion of toxic molecules and control of inflammatory cytokine cascade (Brown et al., 2004). It has been mentioned that the antioxidant activity of plants might be due to their phenolic compounds (Hatapakki et al., 2005). The levels of GSH were significantly decreased in alcohol-treated rats, as well as earlier published reports, which showed that the GSH concentration decreases during alcohol ingestion (Mallikarjuna et al., 2008; Shanmugam et al., 2011). It is easily susceptible to lipid peroxidation. Pumpkin seed oil has shown to possess strong antioxidant properties in experimental an-imals (Dreikorn et al., 2002).Ahmed et al. (2009)reported that pumpkin seed oil administration showed a significant eleva-tion in the striatum and serum glutathione level at all-time intervals in adult male albino rats. Pumpkin seeds of Cucur-bita pepo L. a herbaceous plant of CucurCucur-bitaceae family, contain 40.4e55.6% of linoleic acid: LA; 18:2 n-6, x-6 fatty acid (Al-Khalifa, 1996; Ganzera et al., 1999; Jamieson, 1943). They may have beneficial effects in health and may prevent chronic diseases (Al-Khalifa, 1996; Kumar et al., 2006).
Catalase acts as a preventive antioxidant and plays an important role in the protection against the deleterious effects of LPO (lipid hydroperoxide). Reports have shown that there is
a significant decrease in the activities of catalase in alcoholic subjects (Husain and Somani, 1997). A decreased activity of CAT was due to exhaustion of the enzyme as a result of oxidative stress induced by the alcohol. Presumably, a decrease in CAT activity could be attributed to cross-linking and inactivation of the enzyme protein in the lipid peroxides.
Xu (2000)who found that pumpkin polysaccharide could increase the SOD and GSH-Px activity and reduce the MDA content in tumor mice serum (Xu, 2000).Pandanaboina et al. (2012)who observed a significant increase in lipid peroxida-tion during alcohol consumpperoxida-tion, as reported by earlier studies (Das and Vasudevan, 2006). The alcohol intoxication increases lipid peroxide production (LPO) in various tissues, and is indicative of tissue oxidative stress. Nearly 60e80% ingested alcohol is metabolized in the liver, and this makes it more vulnerable than other organs to alcohol-induced oxidative stress (Lieber, 2003). This concept is supported by the larger extent of an increase in LPO production in the liver, as compared to other organs. Plasma and liver contain en-zymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), which showed contribute to the antioxidant defense mechanism (Lee et al., 2002).Mary et al. (2002) and Visavadiya and Narasimhacharya (2007)
observed a decrease in the activities of the antioxidant en-zymes SOD, CAT and GPx in hypercholesterolemic rats. Such decreases may be associated to the production of a-, b-un-saturated aldehydes during lipid peroxidation. These com-pounds have the ability to increase oxidative stress by promoting the cellular consumption of glutathione and by inactivating selenium-dependent glutathione peroxidase (Kinter and Roberts, 1996). GSH serves as a substrate for the enzyme GPx, and it had been suggested that, through its ac-tivity, GSH protects plasma against oxidative damage. More-over PUFAs (linoleic acid“LA” and alpha-linolenic acid “ALA”) have displayed protection against lipid peroxidation increasing the levels of several cellular antioxidants such as ascorbic acid, a-tocopherol and GSH (Nagao et al., 2005).
Fig. 1showed the histology of liver of normal control. There was no histopathological alteration observed and the normal
Table 3e 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 U/g % change MDA nmol/g % change G1 Normal 73.70± 1.09a e 6.04± 0.146b e 1.90± 0.001a e 36.31± 0.255b e G2 (Alcoholic) 54.53± 1.12d 39.58 3.63± 0.154c 9.77 0.371± 0.001b 80.47 50.97± 0.921a 28.76 G3
Alcoholþ Pumpkin oil
68.93± 1.55b 54.49 6.98± 0.049a 92.29 1.71± 0.162a 360.92 23.48± 0.363c 35.33
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
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.
histological structure of the central vein and surrounding hepatocytes.
The portal area in ethyl alcohol administered rats (G2) showed severe dilatation and congestion in the portal vein 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 pumpkin oil pretreated rats.
The current study was in agreement withPadmanabhan and Jangle (2014).who found that the higher levels of alcohol intake cause cirrhosis and liver damage by enhancing lipid peroxidation in the liver. Acetaldehyde the toxic metabolite of alcohol depresses liver GSH by conjugating with sulphydryl groups of GSH (Maruthaappan and Shree, 2009). In both acute
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 in accumulation of hepatic lipids, lipid peroxides leading to the autooxidation of hepatic cells acting as pro-oxidants and decreasing antioxidant levels thereby resulting in a note-worthy hepatotoxicity. (Kumar et al., 2013).
4.
Conclusion
The present results demonstrated that pumpkin oil may play an important role in the protection against alcohol induced hepatotoxicity and oxidative stress. Pretreatment with pumpkin oil may protect the liver from the hepatotoxic effect and oxidative stress caused by alcohol. Further clinical studies are required to assess the benefits and safety of pumpkin oil Fig. 2e 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.
Fig. 3e Fatty change was observed in diffuse manner (d) all over the hepatocytes in ethyl alcohol treated group (G2). £400.
Fig. 4e The hepatocytes showed fatty change in diffuse manner in pumpkin pretreated group (G3).£400. Fig. 1e A transverse section of a liver of normal control
(distilled water) rat showing a central vein (CV), hepatic cords (h) and sinusoids in between.£400.
before use in human beings and approval by Food and Drug administration (FDA).
r e f e r e n c e s
Ahmed HH, Abdel-Rahman M, Ali RS, Abdel Moniem AE. Protective effect of Ginkgo biloba extract and pumpkin seed oil against neurotoxicity of rotenone in adult male rats. J Appl Sci Res 2009;5(6):622e35.
Al-Khalifa AS. Physicochemical characteristics, fatty acid composition, and lipoxygenase activity of crude pumpkin and melon seed oils. J Agric Food Chem 1996;44:964e6.
Al-Zuhair H, Abdel-Fattah AA, El-Sayed MI. Pumpkin-seed oil modulates the effect of felodipine and captopril in spontaneously hypertensive rats. Pharmacol Res 2000;41(5):555e63.
Arteel GE. Oxidants and antioxidants in alcoholic liver disease. Gastroenterol 2003;124:778e90.
Baskaran M, Periyasamy L, Rajagopalan R. Effect of Phyllanthus niruri on alcohol and polyunsaturated fatty acid induced oxidative stress in Liver. Int J Pharm Pharm Sci
2010;2(4):58e62.
Belfield A, Goldberg DM. Normal ranges and diagnostic value of serum 5'nucleotidase and alkaline phosphatase activities in infancy. Arch Dis Child 1971 Dec;46(250):842e6.
Beutler E, Duron O, Kelly BM. Improved method for determination of blood glutathione. J Lab Clin Med 1963;61:882e8.
Brown LA, Harris FL, Ping XD, Gauthier TW. Chronic ethanol ingestion and the risk of acute lung injury: a role for glutathione availability? Alcohol 2004;33:191e7.
Bu¨hl SN, Jackson KY. Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C. Clin Chem 1978 May;24(5):828e31.
Caili F, Huan S, Quanhong L. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods Hum Nutr 2006;61:73e80.
Calabrese V, Scapagnini G, Latteri S, Colombrita C, Ravagna A, Catalano C, et al. Long term ethanol administration enhances age-dependent modulation of redox state in different brain regions in the rat: Protection by acetyl carnitine. Int J Tissue React 2002;24(3):97e104.
Canadian Council on Animal Care (CCAC). Guide to the care and use of experimental animals, vol. 2. Ottawa. Ontario: Canada; 1993. p. 1e298. CCAC.
Clemens DL, Jerrells TR. Ethanol consumption potentiates viral pancreatitis and may inhibit pancreas regeneration: preliminary findings. Alcohol 2004;33:183e9.
Das SK, Vasudevan DM. Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver. Indian J Biochem Biophysics 2006;43(5):306e11.
Dreikorn K, Berges R, Pientka L, Jonas U. Phytotherapy of benign prostatic hyperplasia. Current evidence-based evaluation. Urologe 2002;41(5):447e51.
Dru_zyn ska B, Strzecha I, Wołosiak R, Worobiej E. Zawartosc wybranych zwia˛zkow biologicznie aktywnych w ekstraktach z suszonych moreli oraz ich własciwosci przeciwutleniaja ce [The contents of selected biologically active compounds in the extracts of the dried apricots and their antioxidant properties]. Z_ywnosc Nauka Technol Jakosc 2008;6(61):77e87.
Espı´n JC, Soler-Rivas C, Wichers HJ. Characterization of the total free radical scavenger capacity of vegetable oils and oil fractions using 2, 2- diphenyl-1-picrylhydrazyl radical. J Agric Food Chem 2000:648e56.
Eynard AR, Cejas V, Silva R, Quiroga P. Mu~noz S. Histopathology of essential fatty acid-deficient mice. Nutrition 1992;8:37e40.
Ganzera M, Croom Jr , Khan IA. Determination of the fatty acid content of pumpkin seed, pygeum, and saw palmetto. J Med Food 1999;2:21e7.
Gossell-Williams M, Lyttle K, Clarke T, Gardner M, Simon O. Supplementation with pumpkin seed oil improves plasma lipid profile and cardiovascular outcomes of female non-ovariectomized and non-ovariectomized Sprague-Dawley rats. Phytotherapy Res 2008;22(7):873e7.
Halliwell B. The antioxidant paradox. Lancet 2000;355(9210):1179e80.
Hatapakki BC, Suresh HM, Bhoomannavar V, Shivkumar SI. Effect of Cascia auriculata Linn flowers against alloxan induced diabetes in rats. A J Nat Remedies 2005;5:132e6.
Hekmatpanah J, Haghighat N, Adams CR. Alcohol consumption by nursing rats and its the cerebellum of the offspring. Alcohol 1994;29(5):535e47.
Horubała A. Pojemnosc przeciwtleniaja˛ca i jej zmiany w procesach przetwarzania owocow [Antioxidants capacity and its changes in the processing of fruit]. Przem Ferment i Owocowo-Warzywny 1999;3:30e2.
Husain K, Somani SM. Interaction of exercise and ethanol on hepatic and plasma antioxidant system in rat.
Pathophysiology 1997;4:69e74.
Jamieson GS. Vegetable fats and oils. 2nd ed. New York: Reinhold Publishing Corp.; 1943.
Jia ZY, Zhang QA, Zhang ZQ, Wang Y, Yuan IF, Wang HY, et al. Hepatoprotective effects of almond oil against carbon tetrachloride induced liver injury in rats. Food Chem 2011;125:673e8.
Kar M, Mishra D. Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence. American Society of Plant Biologists; 1976.
Kinter M, Roberts RJ. Glutathione consumption and glutathione peroxidase inactivation in fibroblast cell lines by 4-hydroxil-2-nonenal. Free Radic Biol Med 1996;21:457e62.
Koneru A, Sathyanarayana S, Mukkanti K, Khan KA. In vitro antioxidant activity of Itrifal Kishneezi: a Unani formulation. Am J Drug Discov Dev 2011;1:121e8.
Kulaitiene J, Jariene E, Danilcenko H, Kita A, Venskutoniene E. Oil pumpkins seeds and their quality. Pol J Food Nutr Sci 2007;57(4B):349e52.
Kumar SA, Sudhahar V, Varalakshmi P. Protective role of eicosapentaenoate lipoate (EPAeLA) derivative in combating oxidative hepatocellular injury in hypercholesterolemic atherogenesis. Atherosclerosis 2006;189:115e22.
Kumar EK, Harsha KN, Shabana S, Rao NN, Babu GN. Evaluation of inevitro antioxidant activity and in vivo hepatoprotective activity of Moringa oleifera seeds extract against ethanol induced liver damage in wistar rats. Iosr J Pharm 2013;3(1):10e5.
Lee MK, Bok SH, Jeong TS, Moon SS, Lee SE, Yong BP, et al. Supplementation of naringenin and its synthetic derivative alters antioxidant enzyme activities of erythrocyte and liver in high cholesterol-fed rats. Bioorg Med Chem 2002;10:2239e44.
Li L, Rong T, Yang Y, Kramer JKG, Hernandez M. Fatty acid profiles, tocopherol contents and antioxidant activities of hearnut (Juglans ailanthifolia Var cordiformis) and Persian walnut (Juglans regia L.). J Agric Food Chem 2007;55:1164e9.
Lieber CS. Relationships between nutrition, alcohol use, and liver disease. Alcohol Res Health 2003;27:220e31.
Liu HW. Determination of 4-aminobutyric acid in pumpkin powder by high performance liquid chromatography. Chin J Chromatogr 2001;19(6):532e3.
Lovejoy JC. The influence of dietary fats in insulin resistance. Curr Diab Rep 2002;2(5):430e40.
Makni M, Fetoui H, Gargouri NK, ElM Garoui, Jaber H, Makni J, et al. Hypolipidemic and hepatoprotective effects of flax and pumpkin seed mixture rich inu-3 and u-6 fatty acids in hypercholesterolemic rats. Food Chem Toxicol
2008;46:3714e20.
Mallikarjuna K, Sahitya Chetan P, Sathyavelu Reddy K, Rajendra W. Ethanol toxicity: rehabilitation of hepatic antioxidant defense system with dietary ginger. Fitoterapia 2008;79(3):174e8.
Mannervik B, Gutenberg C. Glutathione transferase (Human placenta). Meth Enzymol 1981;77:231e5.
Maruthappan V, Shree S. Hepatoprotective effect of Azadirachta indica (Neem) leaves against alcohol induced liver injury in albino rats. J Pharm Res 2009;2(4):655e9.
Mary NK, Shylesh BS, Padikkala J. Antioxidant and hypolipidemic activity of a herbal formulation-liposem. Indian J Exp Biol 2002;40:901e4.
Matkovics B, Kotorman M, Sz Varga I, Hai DQ, Varga C. Oxidative stress in experimental diabetes induced by streptozotocin. Acta Physiol Hung 1998;85(1):29e38.
Milbury PE, Chen CY, Dolnikowski GG, Blumberg JB. Determination of flavonoids and phenolics and their distribution in almonds. J Agric Food Chem 2006;54:5027e33.
Muhammad HR, Mahmood T, Salim T, Afzal N, Nasir A, Iqbal J, et al. Effect of silymarin and serum levels of ALT and GGT in ethanol induced hepatotoxicity in albino rats. J Ayub Med Coll Abbottabad 2009;21(4):73e5.
Murkovic M, Hillebrand A, Winkler J, Pfannhauser W. Variability of vitamin E content in pumpkin seeds (Cucurbita pepo L.). Eur Food Res Technol 1996;202:275e8.
Nagao T, Komine Y, Soga S, Meguro S, Hase T, Tanaka Y, et al. Ingestion of a tea rich in catechins leads to a reduction in body fat and malondialdehyde modified LDL in men. Am J Clin Nutr 2005;81(1):122e9.
Nkosi CZ, Opoku AR, Terblanche SE. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats. Phytother Res 2005;19:341e5.
Oboh G. Hepatoprotective property of ethanolic and aqueous extracts of fluted pumpkin (Telfairia occidentalis) leaves against garlic-induced oxidative stress. J Med Food 2005;8(4):560e3.
Padmanabhan P, Jangle SN. Hepatoprotective activity of herbal preparation (HP-4) against alcohol Induced hepatotoxicity in mice. Int J Appl Sci Biotechnol 2014;2(1):50e8.
Pandanaboina SC, Kondeti SR, Rajbanshi SL, Kunala PN, Pandanaboina S, Pandanaboina MM, et al. Alterations in antioxidant enzyme activities and oxidative damage in alcoholic rat tissues: protective role of Thespesia populnea. Food Chem 2012;132:150e9.
PC-STAT. One-way analysis of variance. Version 1A (C) copyright. The University of Georgia. Programs coded by Roa, M.; Blane K. and Zonneberg, M. University of Georgia, USA. 1985.
Preuss HG, Jarrel ST, Scheckenbach S, Anderson RA. Coparative effects of chromium, vanadium and Gymnema sylvestre on sugar induced blood pressure elevations in SHR. J Coll Nutr 1998;17(2):116e23.
Ramadan MF, Moersel JT. Screening of the antiradical action of vegetable oils. J Food Compos Analysis 2006;19:838e42.
Reitman A, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Amer J Clin Path 1957:28e56.
Saalu LC, Ogunlade B, Ajayi GO, Oyewopo AO, Akunna GG, Ogummodede OS. The hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-toxicity in wistar rat. Am J Biotechnol Mol Sci.
2012;2(1):6e14.
Shanmugam KR, Ramakrishna CH, Mallikarjuna K, Reddy SK. Protective effect of ginger against alcohole induced renal damage and antioxidant enzymes in male albino rats. Indian J Exp Biol 2010;48:143e9.
Shanmugam KR, Mallikarjuna K, Nishanth K, Chien H, Kuo W, Hua C, Sathyavelu K. Ginger feeding protects against renal oxidative damage caused by alcohol drinking in rats. J Ren Nutr 2011;21(3):263e70.
Stevenson DG, Eller FJ, Wang L, Jane JL, Wang T, Inglett GE. Oil and tocopherol content and composition of pumpkin seed oil in 12 cultivars. J Agric Food Chem 2007;55(10):4005e13.
Szasz G. A kinetic photometric method for serum gamma glutamyl transpeptidase. Clin Chem 1969;15(2):124e36.
Turan S, Topcu A, Karabulut I, Vural H, Hayaloglu AA. Fatty acid, triacylglycerol, phytosterol, and tocopherol variations in kernel oil of Malatya Apricots from Turkey. J Agric Food Chem 2007;55:10787e94.
Vermaulen NPE, Bessems JGM, Van de straat R. Molecular aspects of paracetamole induced hepatotoxicity and its mechanism ebased prevention. Drug Met Dev 1992;24:367e407.
Vetriselvan S, Rajamanikkam V, Parimala Devi, Subasini Arun G. Comparative evaluation of hepatoprotective activity of andrographis paniculata and silymarin in ethanol induced hepatotoxicity in albino wistar rats. Der Pharm Lett 2010;2(6):52e9.
Visavadiya NP, Narasimhacharya AVRL. Hypocholesteremic and antioxidant effects of Withania somnifera (Dunal) in hypercholesterolemic rats. Phytomedicine 2007;14:136e42.
Walter M, Gerarde H. Ultramicromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchem J 1970;15:231e6.
Xu GH. A study of the possible antitumour effect and immunom petence of pumpkin polysaccharide. J Wuhan Prof Med Coll 2000;28(4):1e4.
Young DS. Effect of disease on clinical lab. Test. 4th ed. AACC; 2001.
Zhang QA, Zhang ZQ, Yue XF, Fan XH, Li T, Chen SF. Response surface optimization of ultrasound-assisted oil extraction from autoclaved almond powder. Food Chem 2009;116:513e8.
