Karthiga K et al. / Journal of Pharmacy Research 2010, 3(12),

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1 Research Article ISSN: Available online through Protective Role of Sesbania grandiflora on Oxidative Stress Status during Alcohol and PUFA Induced Hepatotoxicity Karthiga K, Kumaravel M, Keerthana R, Rukkumani R*,Raviteja. V. Department of Biochemistry and Molecular biology, Pondicherry University, Kalapet, Puducherry-65, India. Received on: 5-6-; Revised on: 8-8-; Accepted on:3-9- ABSTRACT Alcohol consumption has become a global burden both in terms of morbidity and mortality. Alcohol induces a state of oxidative stress when taken at higher concentration. This leads to membrane damage by lipid peroxidation, alteration of enzyme function and formation of acetaldehyde protein adducts. Many alcoholics along with alcohol consume fried foods that are normally made up of polyunsaturated fatty acid (PUFA). The toxic effects of alcohol are further enhanced by the consumption of PUFA. In our study, we analyzed the effect of Sesbania grandiflora on alcohol and PUFA induced oxidative stress in male albino Wistar rats. The levels of lipid peroxidative markers thiobarbutric acid reactive substances (TBARS) and lipid hydroperoxides (HP), the levels of enzymatic (catalase, superoxide dismutase, glutathione peroxidase) and non enzymatic (reduced glutathione, Vitamin E, vitamin C) antioxidants were analyzed in liver to evaluate the effects of S. grandiflora. The levels of TBARS and HP were significantly increased in alcohol+pufa group, which were found to be reduced on treatment with S. grandiflora. The levels of both enzymatic and non enzymatic antioxidants were decreased in alcohol+pufa group, which were found to be restored on treatment with S. grandiflora. From this we conclude that S. grandiflora protects the liver against alcohol and PUFA induced oxidative stress. Key words: Alcohol, Heated PUFA, Liver, Oxidative stress, Liver marker enzyme, S.grandiflora. INTRODUCTION Liver is the essential organ of our system due to its extraordinary metabolic machinery. Ethanol a potent inducer of oxidative stress causes liver injury. A number of pathways are known to contribute to ethanol induced oxidative stress such as induction of cytochrome P5 (CYP E), production of ROS by mitochondrial electron transport chain, ethanol mobilization of iron, production of reactive product acetaldehyde, altered cytokine production and ethanol induced hypoxia. Alcoholics after heavy consumption of alcohol have the habit of consuming fried food items that are normally made up of PUFA. PUFA aggravates adverse effect of ethanol. Apart from its beneficial effects, studies have shown that the excess consumption of PUFA is detrimental to health 3. Repeated heating of oil alters the nutritional properties of the oil, resulting in the production of toxic metabolites, which cause membrane damage. The combined intake of PUFA and alcohol induces the generation of free radicals that can initiate lipid peroxidation. Sesbania grandiflora commonly known as agathi (tamil) belongs to the family, leguminosae. It is known to possess active biomolecules to treat various ailments. In folk medicine it is used as a medicine for bruises, dysentery, fever, headache, small pox and sore throat. Studies have revealed the use of this as an antioxylytic, anticonvulsive, antiulcer, antiurolithiatic, antioxidant, antimicrobial, antipyretic and analgesic 5. Hence in our study we tested the effect of crude extract of Sesbania grandiflora against alcohol and PUFA induced hepatotoxcity. MATERIALS AND METHODS Animals Male albino rats, Wistar strain of body weight ranging from -6g, bred in the Central Animal House, Pondicherry University, Puducherry, India were used in this study. The animals were fed on the standard pellet diet (Hindustan Lever Limited, Mumbai, India). Water was given ad libitum. The standard pellet diet comprised % protein, 5% lipids, % crude fiber, 8% ash, % calcium,.6% phosphorus, 3.% glucose, % vitamins and 55% nitrogen free extract (carbohydrates). *Corresponding author. Dr. R. Rukkumani Assistant Professor, Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry- 65. India. The animals were housed in plastic cages under controlled condition of h light / h dark cycles, 5% humidity and at 3 ± C. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council for Medical Research, Hyderabad, India and approved by the Animal Ethical Committee, Pondicherry University. Materials Used Absolute ethanol [AR] was obtained from Hayman Private Limited, England. Sunflower oil marketed by Gold Winner was purchased from local market, Puducherry, India. Sunflower oil was subjected to heating at 8 C for 3 minutes, twice. The oil was analyzed by gas chromatography and found to contain altered fatty acid composition. All chemicals and solvents used were of the highest purity and analytical grade. Preparation of Plant Extract The plants were collected from the fields near Puducherry. Leaves were separated and dried at room temperature, powdered, sieved and stored prior to further use. The powder ( g) was homogenized with water (8 ml) and the homogenate was kept in a shaker at ºC for h and then filtered using Whatman No. paper. The filtrate was evaporated to dryness under reduced pressure. The yield of the aqueous extract was 56 g of leaf powder. The extract was resuspended in a known volume of distilled water and used for different experiments. Experimental Design The animals were divided into groups of 6 rats each. Group rats served as the control. Group rats were given % ethanol (7.9g/Kg body weight) 6 orally, using an intragastric tube and 5% heated sunflower oil mixed with the diet. Group 3 rats were given % ethanol orally, using an intragastric tube, 5% heated sunflower oil mixed with the diet and aqueous extract (leaf) of S. grandiflora ( mg/kg body weight) using an intragastric tube. Group rats were given aqueous extract (leaf) of S. grandiflora ( mg/kg body weight) orally, using an intragastric tube. At the end of the experimental period (5 days), the rats were sacrificed after an overnight fast by cervical dislocation. Liver tissues were removed, cleared off blood and immediately transferred to ice-cold containers containing.9% NaCl for various estimations. A known amount of tissue was weighed and homogenized in appropriate buffer (%) for the estimation of various biochemical parameters. Preparation of Plasma Blood was collected in heparinised tubes and plasma was separated by centrifugation at g for 5 minutes for the estimation of GGT and ALP.

2 Biochemical Investigations The activity of plasma Gamma-glutamyl transferase (GGT) was assayed by the method of Fiala et al., 7. The activity of alkaline phosphatase (ALP) was assayed by the method of King and Armstrong using a reagent kit 8. The concentrations of Thiobarbituric acid reactive substances (TBARS) was estimated by the method of Niehaus and Samuelsson 9 and Lipid hydroperoxides (LH) by the method of Jiang et al.,. Ascorbic acid was estimated by the method of Roe and Kuether and vitamin E by the method of Baker and Frank. Reduced glutathione (GSH) content in the tissue was determined by the method of Ellman 3. The activity of glutathione peroxidase (GPx) was assayed by the method of Rotruck et al.,, superoxide dismutase (SOD) by the method of Kakkar et al., 5, and catalase (CAT) by the method of Sinha 6. For histopathological study, two animals from each group were perfused with formalin (%) and the tissues were separated and stored in % formalin. They were latter sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin section, and stained with hemotoxylin and Eosin (H & E). Statistical Analysis Statistical analysis was done by analysis of variance (ANOVA) followed by Tukey s test. The values were considered statistically significant when p<.5. RESULTS Figure and show the changes in the activities of GGT and ALP. The activities of these liver marker enzymes were increased significantly in plasma of alcohol + DPUFA group when compared to normal. Co-administration of S.grandiflora significantly reduced their activity. S.grandiflora control group showed no significant change in the activities when compared to normal. Figure 3 and show the changes in the levels of TBARS and lipid hydroperoxides in liver. The levels of TBARS and HP were increased significantly in Figure : Changes in the activities of GGT in plasma 3.5 IU/L.5 3 ALC+ PUFA+ differ significantly at P <.5 Figure : Changes in the activities of ALP in plasma 3 5 IU/L Alc+ PUFA+ differ significantly at P <.5 alcohol + PUFA group when compared to normal. Treatment with S.grandiflora significantly decreased their levels. S.grandiflora control group did not show any significant change in the levels when compared to normal. The changes in the levels of vitamin C, vitamin E and GSH in liver are given in Figure 5, 6 and 7 respectively. There was a significant decrease in the levels Figure 3: Levels of TBARS in liver mm/g tissue mm/g tissue mm/g tissue Alc+ PUFA Alc+ PUFA+ differ significantly at P <.5 Figure : Levels of Hydroperoxides in liver 8 6 Alc + PUFA 8 Alc+PUFA+ 6 3 differ significantly at P <.5 of vitamin C, vitamin E and GSH in alcohol + PUFA groups when compared to normal. The levels were significantly increased in S.grandiflora treated groups. S.grandiflora control group showed no significant change in their levels when compared to normal. The activities SOD (Figure 8), CAT (Figure 9), GPx (Figure ) showed a significant decrease in liver of alcohol + PUFA group. Treatment with S.grandiflora significantly increased their activities. Administration of Figure 5: Levels of Vitamin C in liver differ significantly at P <.5

3 Figure 6: Levels of Vitamin E in liver Figure 9: Activities of Catalase in liver mm/g tissue mm/g tissue differ significantly at P <.5 Figure 7: Levels of Reduced Glutathione in liver Alc+ PUFA + differ significantly at P <.5 S.grandiflora alone did not alter the activities of these enzymic antioxidants. In histopathological observations (Figure ), the liver of alcohol+ heated PUFA treated rats showed fibrosis (Fig B), and Sesbania grandiflora treated rats showed only sinusoidal dilatation (Fig C). The histology of liver was not Figure 8: Activities of Superoxide Dismutase in liver units/mg protein units/mg protein Alc + PUFA Alc + PUFA + Values sharing a common superscript do not differ significantly at P <.5 Units - µm of H O liberated/ minute. Figure : Activities of Glutathione Peroxidase in liver Alc + PUFA Alc + PUFA + Values sharing a common superscript do not differ significantly at P <.5 Units - mmoles of glutathione liberated / minute. affected in both normal (Fig A) and Sesbania grandiflora control (Fig D) rats. DISCUSSION Alcoholic liver disease is progressive and major cause of morbidity and mortality. Ethanol stimulates free radical formation that causes lipid peroxidation. Figure : Histopathology of liver 8 6 units/mg protein Alc + PUFA Alc + PUFA + Fig.A. Liver (X),Liver showing normal histology with central vein( ) Values sharing a common superscript do not differ significantly at P <.5 Units - enzyme reaction which gives 5% inhibition of NBT reduction / minute Fig.B.Alcohol + PUFA Liver (X) Fibrotic changes ( )

The resultant comparatively long lived carbon radical species may undergo reaction with molecular oxygen to form hydroperoxy fatty acids which can decompose to form aldehydic products such as

4 The resultant comparatively long lived carbon radical species may undergo reaction with molecular oxygen to form hydroperoxy fatty acids which can decompose to form aldehydic products such as malondialdehye (MDA), hydroxyalkenals, -hydroxy nonenal (HNE) and -hydroyhexanal (HHE) and other aldehydes. These products can react with TBA and hence the levels of TBARS and the secondary products of LPO, hydroperoxides are increased during ethanol and PUFA ingestion. Chronic alcohol consumption can increase oxidative stress through several mechanisms. In normal metabolism there is a balance between the generation of free radicals and antioxidant defense mechanism. This balance is disturbed by the chronic consumption of alcohol. Studies have shown that ethanol consumption results in increased formation of lipid peroxides and free radicals leading to depletion of antioxidants present in the biosystems 3. Fig.C.Alcohol +PUFA+ Liver (X) Sinusoidal dilatation ( ) Fig.D. Liver (X) Liver showing normal histology with central vein ( ) Consumption of PUFA along with alcohol, primes the toxic effects of alcohol. Hence, we investigated the effect of Sesbania grandiflora (crude extract) on alcohol and PUFA induced toxicity. The excess amount of alcohol consumed is metabolized in liver, causing severe liver injury. Acetaldehyde, the major metabolite of alcohol metabolism binds to hepatocyte membrane affecting the membrane structure. Acetaldehyde form adducts with membrane proteins and these adducts are also involved in alcohol related liver injury 7. Ethanol alters membrane fluidity by modulating fatty acid composition affecting the membrane function. Due to structural changes and functional alterations, the hepatocytes become leaky to liver markers. Heating of PUFA brings structural, physical and chemical changes leading to compositional diversities due to degradation reaction such as autooxidation, thermal polymerization, cyclisation and hydrolysis. Highly oxidized oils produce poly aromatic hydrocarbons that have carcinogenic effect 8.Thus in our study ingestion of PUFA and alcohol might have disturbed the structure, fluidity and functions of hepatocytes, releasing the membrane bound and subcellular enzymes into the circulation. In every cell the amount of oxygen consumed is reduced in a specific way and yields highly reactive intermediates. These highly reactive molecules are capable of independent existence with one or more unpaired electrons. Free radicals attack healthy cells, disrupting the cell membranes and subjecting the cells to infection, genetic damage and mutations 9. ROS including superoxide anion, hydrogen peroxide, hydroxyl radical, alkoxy radical, peroxyl radical, peroxy nitrate, hypocholorous acid, nitiric oxide implicate ethanol induced oxidative stress. Various pathways play a role in ethanol induced oxidative stress, including changes in cellular oxidized nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide redox ratio, production of acetaldehyde protein adducts, induction of cytochrome P5 E (CYPE), formation of -hydroxy ethyl free radicals, ethanol mediated mitochondrial damage, endotoxin derived activation of kupffer cells and subsequent production of tumor necrosis factor? and decrease in the cellular antioxidant defense. High intake of PUFA also causes lipid peroxidation, specifically polyunsaturated fatty acid (PUFA) oxidation makes the membrane more susceptible to damage. The membranes are made up of lipid conjugates. The carbon adjacent to the double bond is highly susceptible to attack by ROS, which extracts a proton leaving behind a free radical form of acyl chain. The methylene protons of membrane fatty acids are more susceptible to attacks by radicals. PUFA can also cause deterioration of the antioxidant status due to their liability to become oxidized. A high intake of PUFA has been demonstrated to increase the formation of lipid radicals and to deplete endogenous antioxidants. In our study, the levels of enzymatic and non - enzymatic antioxidants were decreased in alcohol and PUFA group. The inactivation and depletion is due to the excess amount of reactive oxygen species produced. Sesbania garandiflora is considered a wonder plant with a variety of potential. It is known to contain phenolic and anthocyanins 5. These compounds have hydrogen donating ability, forming aryloxyl radicals. It has been proposed that hydroxyl and hydroperoxy radicals initiate H + abstraction from a free phenolic substrate to form phenoxy radical that can rearrange to quinonemethide radical intermediate, which is excreted via bile. These molecules are considered to scavange free radicals and inhibit lipid peroxidation. In our study, the administration of crude extract of Sesbania grandiflora has reduced the lipid peroxidation and restored the antioxidant status. This may be because of its effective antioxidant property. our histopathological observations were also in correlation with our biochemical parameters. Studies have shown that the leaf juice of Sesbania grandiflora scavanges nitric oxide and - diphenyl - picryl hydrazyl free radicals 5. The crude extract of Sesbania grandiflora effectively protected the liver and prevented the release of liver markers and decreased the extent of lipid peroxidation and reactivated the antioxidant machinery. Hence this wonder plant Sesbania grandiflora can be considered as a hepatoprotector against alcohol and PUFA induced hepatotoxicity. REFERENCES. Alessandro C. Alcohol induced fatty liver and inflammation: Where do kupffer cells act?, J. Hepatol. 3,, Aytacoglu BN, Suku N, Kose N. Alcohol induced lung damage and oxidative stress. 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5 TNF? and modifies acute hepatic inflammation in rats. J. Gastroenterol. 39,, 7.. Yasuo V, Furutera A. Malondialdehyde generated from peroxidized linolenic acid protein modification in heat stressed plants. Plant Physiol. Biochem. 6, 8, Spiteller G. Review on the chemistry of oxidative stress. J. Lipid Mediators. 7, 993, Patil B, Kodiliwadamath MV. Correlation between lipid peroxidation and non enzymatic antioxidants in pregnancy induced hypertension. Ind. J. Clinical Biochem. 3, 8, Kathleen H, Donough MC. Antioxidant nutrients and alcohol. Toxicology, 89, 3, Doddla S, Pasupulati H. Evaluation of Sesbania grandiflora for antiurolithiatic and antioxidant properties. Nat. Medicines. 6, 8, Source of support: Nil, Conflict of interest: None Declared

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