Nelumbo Nucifera Leaf Extract Ameliorates Lipidperoxides and Antioxidants in Isoproterenol- Induced Myocardial Infarction: Biochemical and Histological Examination

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Rajakumaran Subashiniet al. / Journal of Pharmacy Research 2012,5(7),3878-3882 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Nelumbo nucifera leaf extract ameliorates lipidperoxides and antioxidants in isoproterenol- induced myocardial infarction: biochemical and histological examination

Rajakumaran Subashini*1 Ponusamy Ponmurugan2 Murugan Rajadurai3 1Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar-608002, Tamil Nadu, India. 2Department of Biotechnology, K.S.Rangasamy college of Technology,Tiruchengodu-637215, Tamil Nadu, India. 3Department of Biochemistry, Muthayammal College of Arts & Science, Rasipuram - 637408, Tamil Nadu, India. Received on:07-04-2012; Revised on: 12-05-2012; Accepted on:16-06-2012

ABSTRACT Screening cardioprotective activity of a drug against ISO-induced cardiac damage is meaningful and the study will rationalize the traditional use of the Nelumbo nucifera leaf extract (NNE) extract in the treatment of cardiovascular disease. This study explains the cardioprotective role of NNE on lipid peroxides, antioxidants and histopathological findings in isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Subcutaneous injection of ISO (85 mg/kg) to rats showed a significant increase in the levels of thiobarbituric acid reactive substances and lipid hydroperoxides in plasma and the heart with subsequent decrease in the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in the heart and the levels of reduced glutathione, vitamin C and vitamin E in plasma and heart and ceruloplasmin in plasma. Oral pretreatment with NNE (400 mg/kg) to ISO- induced rats daily for a period of 21 days showed a significant decrease in the levels of lipid peroxidative products and improved the antioxidant. In addition to the biochemical parameters the histopathological findings also supports our study. Thus, administration of NNE posses cardioprotective effect in ISO-induced rats.

Key words: Nelumbo nucifera; isoproterenol; myocardial infarction; lipid peroxides; antioxidants.

INTRODUCTION The Indian subcontinent (including India, Pakistan, Bangladesh, Sri Lanka, changes [6]. Ideally, animal models of human pathological conditions are and Nepal) has 20% of world’s population and considered as one of the mimic the cellular and physiological processes responsible for the regions with the highest burden of cardiovascular disease (CVD) in the pathological conditions in man. Rona et al. (1959) [7] reported that the world [1]. The world health organization (WHO) predicts that deaths due administration of ISO to rats produces "infarct-like" myocardial necrosis in to circulatory system diseases are projected to double between 1985 and the absence of significant coronary artery lesions. 2025, particularly in developing countries like India [2]. The lack of blood supply is caused by closure of the artery (coronary artery) that supplies It is well known that, the administration of ISO produces a positive inotropic that particular part of the heart muscle with blood, which occurs in the and chronotropic effects on the heart. In skeletal muscle arterioles, this closed or narrowed arteries by atherosclerosis. produces vasodilatation. The isopropyl amine group in ISO makes it selective for ß-receptors. The free catechol hydroxy groups keep it susceptible to Oxidative stress resulting from increased production of free radicals enzymatic metabolism [8]. It’s inotropic and chronotropic effects elevate associated with decreased levels of antioxidants in the myocardium plays a the systolic BP, while its vasodilatory activity tends to lower diastolic BP. major role in CVD such as ischemic heart disease (IHD), atherosclerosis, The mechanism of increased vascular reactivity in, which may play a major congestive heart failure, cardiomyopathy and arrhythmias [3]. Myocardial in the development of hypertension, has not been defined. The adverse infarction (MI) is invariably followed by several biochemical alterations, effects of isoproterenol are also related to the drug's cardiovascular effects. such as lipid peroxidation, free radical damage, and prolonged hyperglycemia, The metabolite of ISO, adrenochrome causes deliterios effect of cardiac hyperlipidemia, leading to qualitative and quantitative alterations of cells. ISO undergo biotransformation and its metabolite adrenochrome is myocardium. Various factors keep contributing to the epidemic scope of responsible for its toxic effect. CVD, which involves the physical, emotional, environmental, and chemical stresses of modern life [4]. Biologically active plant chemicals other than traditional nutrients that have a beneficial effect on human health have been termed “phytochemicals” [9]. Isoproterenol (ISO), a synthetic catecholamine and b-adrenergic agonist, Phytochemicals are naturally occurring, non-nutritive chemicals, which used as a model to study several cardiac dysfunctions [5]. The deleterious appear to work alone and in combination, and perhaps in conjunction, with effects of ISO on heart is well known, and also associated not only with vitamins and other nutrients in food to prevent, halt, or lessen disease. For functional alterations, but also with numerous morphological and biochemical example, onions and corn are rich in phytochemicals [10]. Therefore, foods in our diet that can aid in prevention of these diseases are of major interest to both the scientific community and the general public. *Corresponding author. R. Subashini Nelumbo nucifera has been reported to treat obesity3, hepatotoxicity, Assistant Professor, arrhythmia4 and hyperlipidemia. Traditionally, leaves are used to treat Department of Biochemistry and Biotechnology, diarrhea fever and inflammatory skin conditions. Young leaves are useful in Annamalai University, many varieties of raktapitta, or bleeding disorders. The presences of various Annamalainagar-608002, alkaloids have been reported from the entire plant including nuciferine, neferine, lotusine, and isoliensinine. The ether extract of the petals and Tamil Nadu,India. stamens yielded quercitin; the aqueous extract of the leaves yielded

Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3878-3882 Rajakumaran Subashiniet al. / Journal of Pharmacy Research 2012,5(7),3878-3882 flavonoids, quercitin, isoquercitrin and leukodelphinidin. This study was The heart tissue was dissected out immediately and washed in ice-cold designed to evaluate the efficacy of Nelumbo nucifera leaves extract (NNE) saline. 100 mg of tissue was weighed accurately and homogenized in 5 ml of in the treatment of MI by studying the levels of lipoperoxidative products 0.1 M Tris-HCl buffer (pH 7.4) in ice-cold condition. The homogenate was and assaying the activities of enzymatic and the levels of non-enzymatic centrifuged and the clear supernatant solution was taken for analysis. antioxidants in ISO-induced Wistar rats. Histopathology MATERIALS AND METHODS Heart tissue obtained from all experimental groups were washed immediately with saline and then fixed in 10% buffered neutral formalin solution. After Experimental animals fixation, the heart tissue was processed by embedding in paraffin. Then, the Adult male albino rats of Wistar strain weighing 150-200 g were purchased heart tissue was sectioned and stained with hematoxylin and eosin (H&E) from Venkateswara Enterprises, Bangalore, Karnataka, India. The experiment and examined under high power microscope (100 x) and photomicrographs was carried out according to the guidelines of the Committee for the Purpose were taken. of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Animal Ethical Committee of Vinayaka Biochemical Estimations Missions University (IAEC NO : P.Cog-1/06). They were housed in Plasma thiobarbituric acid reactive substances (TBARS) were estimated by polypropylene cages (47x34x20 cm) lined with husk, renewed every 24 h the method of Yagi (1987) [12]. TBARS in the heart was estimated by the under a 12:12 h light/dark cycle at around 22°C and had free access to tap method of Fraga et al. (1988) [13]. Estimation of plasma and cardiac tissue water and food. The rats were fed on a standard pellet diet (Pranav Agro lipid hydroperoxides (HP) was done by the method of Jiang et al. (1992) Industries Ltd., Maharashtra, India). The pellet diet consisted of 22.02% [14]. Superoxide dismutase (SOD) activity in the myocardium was assayed crude protein, 4.25% crude oil, 3.02% crude fibre, 7.5% ash, 1.38% sand by the method of Kakkar et al. (1984) [15]. The activity of catalase in silica, 0.8% calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins and myocardium was assayed by the method of Sinha (1972) [16]. Estimation 56.17% nitrogen free extract (carbohydrates). The diet provided of GSH in plasma and the heart tissue was done by the method of Ellman metabolisable energy of 3, 600 kcal. (1959) [17]. GPx activity was assayed by the method of Rotruck et al. (1973) [18]. The activity of GST was assayed by the method of Habig and Chemicals Jakoby (1981) [19]. (±) Isoproterenol hydrochloride, reduced nicotinamide adenine dinucleotide (NADH), were purchased from Sigma Chemical Company, St. Louis, MO, Vitamin C in plasma and the heart tissue was estimated by the method of USA. Thiobarbituric acid (TBA), 1, 1’ ,3 , 3’ tetramethoxy propane, butylated Omaye et al. (1979) [20]. The levels of Vitamin E in plasma and the hydroxy toluene (BHT), xylenol orange, dithionitro bis benzoic acid (DTNB), concentration in cardiac tissue were estimated by the method of Baker et al. ascorbic acid, 2, 2’ dipyridyl, p-phenylene diamine and sodium azide were (1980) [21]. Ceruloplasmin levels in plasma were estimated by the method obtained from S.D. Fine Chemicals, Mumbai, India. Methanol was of Ravin (1961) [22]. Protein in the enzyme extract was determined by the purchased from Anilax chemicals, USA. All other chemicals used for the method of Lowry et al. (1951) [23]. experiment is of analytical grade. All the chemicals used in this experiment were of analytical grade. Statistical Analysis Statistical analysis was done by one-way analysis of variance (ANOVA) Drugs followed by Duncan’s multiple range test (DMRT). Using SPSS software Leaves of Nelumbo nucifera were purchased from local market, Chennai, package, version 9.05. P values <0.05 were considered as significant. Tamilnadu, India, and were authenticated by National Institute of Herbal Science Plant Anatomy Research Centre, West Tambaram, Chennai, RESULTS Tamilnadu, India. Authentication No: PARC/2010/596. Effect of NNE on lipid peroxides Extract Preparation Table 1 shows the levels of thiobarbituric acid reactive substances (TBARS) Dried leaves of Nelumbo nucifera were coarsely powdered and 1kg of this and hydroperoxides (HP) in plasma and the heart of normal and ISO- powdered plant material was extracted with the help of the soxhlet apparatus induced rats. Rats induced with ISO showed a significant increase in the using methanol as a solvent. The solvent from the methanolic extract was levels of TBARS and HP in plasma and the heart when compared to normal removed under vacuum distillation; dried material (brown colored, yield control rats. Oral pretreatment with NNE to ISO-induced rats significantly 11.25% w/w with respect to dry starting material) was kept in a desiccators. decreased the levels of TBARS and HP in plasma and the heart when This methanolic extract was dissolved in distilled water for further compared with ISO-alone induced rats. experiments. Table 1. Effect of Nelumbo nucifera leaf extract (NNE) on the levels of Induction of Experimental Myocardial Infarction thiobarbituric acid reactive substances (TBARS) and hydroperoxides Isoproternol (85 mg/kg) was dissolved in normal saline and injected (HP) in plasma and the heart in normal and isoproterenol (ISO)- subcutaneously to rats at an interval of 24 hours for 2 days (Subashini and induced myocardial infarction (MI) in rats. Rajadurai, 2011) [11]. Groups Plasma Plasma HP Heart TBARS Heart HP TBARS (values x 10-5 (mM/100 g (mM/100g Experimental Design (nM/ml) mM/dL) wet tissue) wet tissue) A total number of 24 rats were used in the experiment, 6 rats of each group. Normal control rats 4.02 ± 0.26a 10.05 ± 0.61a 0.63 ± 0.03a 18.13 ± 1.12a Group 1 Normal control rats Normal rats + NNE 3.95 ± 0.18a 10.11 ± 0.76a 0.61 ± 0.05a 17.55 ± 1.02a Group 2 Normal rats + NNE (400 mg/kg) (400 mg/kg) Group 3 ISO control rats ISO control rats 6.85 ± 0.31b 19.17 ± 1.35b 1.72 ± 0.11b 36.54 ± 2.51b Group 4 NNE (400 mg/kg) + ISO NNE (400 mg/kg) + ISO 4.77 ± 0.30c 13.22 ± 0.59c 0.86 ± 0.06c 22.70 ± 1.93c Nelumbo Nucifera leaf extract (NNE) was dissolved in distilled water and Each value is mean ± S.D. for 6 rats in each group. Values not sharing a administered to rats orally for a period of 21 days. At the end of the common superscript (a, b and c) differ significantly with each other (P<0.05, DMRT). experimental period, after 12 h of second ISO-injection, all the rats were anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and sacrificed by Effect of NNE on antioxidants defense system cervical decapitation. Blood was collected to separate serum and plasma. The activities of superoxide dismutase (SOD) and catalase in the heart of

Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3878-3882 Rajakumaran Subashiniet al. / Journal of Pharmacy Research 2012,5(7),3878-3882 normal and ISO-induced rats are shown in Table 2. Rats induced with ISO, when compared with normal control rats. Oral pretreatment with NNE to exhibited a significant decrease in the activities of these antioxidant enzymes ISO-induced rats significantly increased the levels of vitamin C, vitamin E in the heart on comparison with normal control rats. Pretreatment with in plasma and the heart and ceruloplasmin in plasma when compared with NNE to ISO-induced rats significantly increased the activities of these ISO-alone induced rats. enzymes when compared with ISO-alone induced rats. Table 2. Effect of Nelumbo nucifera leaf extract (NNE) on the activities Effect of NNE on histopathology The histopathological studies showed that ISO treatment caused of superoxide dismutase (SOD) and catalase in the heart of normal haemorrhagic necrosis to the heart tissue in rats. Marked tissue injury with and isoproterenol (ISO)-induced myocardial infarction (MI) in rats. subendocardial loss of muscles and accumulation of acute inflammatory Groups SOD (Units/mg protein) Catalase (Units/mg protein) cells surrounded by mild edema was seen in ISO group. Pretreatment with NNE preserve the functional cytoarchitecture of the entire heart tissue. Normal control rats 14.21 ± 0.79a 9.11 ± 0.44a Normal rats + NNE (400 mg/kg) 14.52 ± 1.02a 9.27 ± 0.59a NNE to normal rats didn’t show any pathological alterations. ISO control rats 8.08 ± 0.46b 4.24 ± 0.23b NNE (400 mg/kg) + ISO 12.05 ± 0.91c 7.05 ± 0.60c In all the parameters studied, oral administration of NNE (400 mg/kg) to Units of enzyme activity expressed as follows: SOD - one unit is defined as the normal rats for a period of 21 days showed minor effects but none were enzyme concentration required to inhibit the OD at 560 nm of chromogen statistically significant, NNE at a dose of 400 mg/kg showed significant production by 50% in one minute; Catalase - mmoles of H2O2 consumed/ effect in ISO-induced rats. min/mg protein. Each value is mean ± S.D. for 6 rats in each group. Values not sharing a common superscript (a, b and c) differ significantly with each other (P<0.05, DMRT).

Table 3 illustrates the effect of NNE on the activities of myocardial GPx and GST and the levels of GSH in plasma and the heart in normal and ISO- induced rats. Rats induced with ISO showed a significant decrease in the activities of these antioxidant enzymes and the levels of GSH on comparison with normal control rats. Oral pretreatment with NNE to ISO-induced rats significantly increased the activities of these antioxidant enzymes and the levels of GSH when compared with ISO-alone induced rats.

Table 3. Effect of Nelumbo nucifera leaf extract (NNE) on the activities Fig. 1. Light micrograph of rat heart Fig. 2 - NNE alone treated rats shows of myocardial glutathione peroxidase (GPx), glutathione-S- shows normal cardiomyocytes. normal heart tissue architecture with no transferase (GST) and the levels of reduced glutathione (GSH) in pathological changes. plasma and the heart in normal and isoproterenol (ISO)-induced myocardial infarction (MI) in rats.

Groups GPx (mg of GSH GST (nmoles of Plasma GSH Heart GSH consumed/ CDNB conjugated) (mg/dL) (mM/g wet min/mg protein) /min/mg protein tissue)

Normal control rats 5.12 ± 0.31a 615.5 ± 25.1a 18.35 ± 1.12a 6.53 ± 0.43a Normal rats + NNE 5.33 ± 0.37a 623.4 ± 33.2a 18.89 ± 1.31a 6.77 ± 0.61b (400 mg/kg) ISO control rats 2.83 ± 0.22b 428.7 ± 24.5b 10.18 ± 0.70b 3.73 ± 0.20b NNE (400 mg/kg) +ISO 4.66 ± 0.31c 566.3 ± 31.3c 16.69 ± 1.48C 5.89 ± 0.48c GSH – Reduced glutathione; CDNB-1-chloro 2, 4- dinitrobenzene.Each value is mean ± S.D. for 6 rats in each group. Values not sharing a common superscript (a, b and c) differ significantly with each other (P < 0.05, DMRT). Fig.3. Rat treated with ISO showing Fig.4. NNE treatment shows inhibition subendocardial loss of muscles with Table 4 shows the effect of NNE on the levels of plasma and the heart of necrosis and reduced inflammation inflammatory cells surrounded by in ISO induced rats. vitamin C, vitamin E and plasma ceruloplasmin in normal and ISO-induced edema and muscle necrosis. rats. Rats induced with ISO exhibited a significant decrease in the levels of vitamin C, vitamin E in plasma and the heart and ceruloplasmin in plasma DISCUSSION Table 4. Effect of Nelumbo nucifera leaf extract (NNE) on the levels of Reactive oxygen species (ROS) may attack various biomolecules like proteins, nucleic acids and carbohydrates, but their main target is vitamin C, vitamin E in plasma and the heart and ceruloplasmin in polyunsaturated fatty acids (PUFA), which is the precursor of lipid peroxide plasma in normal and isoproterenol (ISO)-induced myocardial formation. Lipid peroxidation and its product play very important role in infarction (MI) in rats. liver, kidney, heart and brain toxicity [24]. It involves the formation and Groups Plasma Plasma Heart Heart Plasma propagation of lipid radicals, the uptake of oxygen and rearrangement of Vitamin C Vitamin E Vitamin C Vitamin E Ceruloplasmin double bonds in unsaturated lipids results in destruction of membrane (mg/dL) (mg/dL) (µmoles/mg (µmoles/mg (mg/dL) protein) protein) lipids. Biological membranes are often rich in unsaturated fatty acids and bathed in oxygen-rich metal containing fluid. Therefore, it is not surprising Normal control rats 1.83 ± 0.09a 1.66 ± 0.10a 0.86 ± 0.06a 0.67 ± 0.04a 28.72 ± 1.68a that membrane lipids are susceptible to peroxidative attack. Normal rats + 1.95 ± 0.11a 1.71 ± 0.09a 0.89 ± 0.06a 0.70 ± 0.05a 30.05 ± 2.10a NNE (400 mg/kg) ISO control rats 0.79 ± 0.06b 0.63 ± 0.03b 0.43 ± 0.02b 0.36 ± 0.03b 17.63 ± 1.33b Free radicals, which are produced continuously in cells either during NNE (400 mg/kg) 1.55 ± 0.10c 1.46 ± 0.11c 0.69 ± 0.05c 0.59 ± 0.04c 25.18 ± 1.91c phagocytosis or accidentally as by-product metabolites. The balance of + ISO oxidant-antioxidant system must exist in the cell, while the disturbance of Each value is mean ± S.D. for 6 rats in each group. Values not sharing a antioxidant-proxidant balance causes oxidative stress [25]. Lipid common superscript (a, b and c) differ significantly with each other (P < 0.05, peroxidation in vivo has been identified as important and basic deteriorative DMRT). Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3878-3882 Rajakumaran Subashiniet al. / Journal of Pharmacy Research 2012,5(7),3878-3882 reactions in cellular mechanisms of myocardial ischemia [26]. ISO- Reduced glutathione (GSH) protects the myocardium against free radical administration in rats leads to increased levels of lipid peroxidation and damage, which leads to reduction in cellular GSH levels and impair the recov- extensive necrosis of cell membranes by elevating the levels of thiobarbituric ery after a short period of ischemia. Decreased GSH levels may be associated acid reactive substances (TBARS) and hydroperoxides (HP). ISO-injection with an enhanced protective mechanism of oxidative stress in MI. GPx produces free radicals, which involved in the membrane damage, leading to catalyzes the reduction of hydrogen peroxides and hydroperoxides to non- elevated levels of TBARS and HP [27]. toxic products. GST catalyzes the conjugation of both hydroquinones and epoxides of polycyclic aromatic hydrocarbons with GSH for their excre- Pretreatment with NNE to ISO-induced rat significantly decreased the levels tion. It also shows low activity towards organic hydroperoxides for their of TBARS and HP in plasma and heart. This could be due to detoxification from cells/tissues [36]. The decreased level of GSH, GPx and antilipoperoxidative and free radical scavenging properties of NNE. Various GST were observed in heat of ISO-induced rats. Decreased GSH levels in medicinal plants are reported to possess free radical scavenging properties ISO-induced rats might be due to its increased utilization in protecting in ISO-induced MI in rats. sulphur containing proteins from free radicals. Decreased levels and avail- ability of GSH could reduce the activities of GPx and GST in ISO-induced Antioxidant defense mechanisms involve both enzymatic and non-enzymatic MI [37]. strategies. Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase (SOD), catalase, In our study; we have observed decreased levels of vitamin C and vitamin E glutathione peroxidase (GPx), and glutathione reductase (GRD). in plasma and heart and ceruloplasmin in plasma of ISO-induced rats. Vita- Antioxidants have the capacity to inhibit oxidation of subsequent molecules, min C involved in the scavenging of singlet oxygen, superoxide and hy- which is a chemical reaction that transfers an electron from a substance to droxyl radicals. It reduces the risk of CVD by reducing blood pressure, an oxidizing agent [28]. Antioxidants terminate these chain reactions by blood cholesterol and formation of oxidized LDL cholesterol [38]. Vitamin removing free radical intermediates and inhibit other oxidation reactions. E involved in the inhibition of lipid peroxidation and regenerates the re- Low levels of antioxidants, or inhibition of the antioxidant enzymes, leads duced vitamin C and GSH. By protecting myocardial membranes and inhib- to oxidative stress and may damage or kill cells [29,30]. iting the oxidation of lipoproteins, vitamin E inhibits membrane peoxidative damage and atherogenesis [38]. Ceruloplasmin is an extra cellular antioxi- ROS are generated from the leakage of electrons into oxygen from various dant which has ferroxidase and copper binding capacity, which is involved systems in our body and the endogenous antioxidant enzymatic defense is in scavenging of superoxide radicals and inhibits ferritin dependent lipid a very important source to neutralize the oxygen free radical mediated peroxidation by catalyzing the oxidative reincorporation of released iron injury. Although the physiologic significance of these antioxidant markers into ferritin [39]. This could be due to increased utilization of the non- in vivo has yet to be fully understood, these markers are widely used as the enzymatic antioxidants in ISO-mediated free radicals and lipid peroxidation. first step in evaluating the role of in vivo antioxidants in disease-associated conditions [31]. Pretreatment with NNE showed increased activities of antioxidant enzymes such as SOD, catalase, GPx, and GST and increased the levels of nonenzy- Biological system has an effective mechanism to prevent and neutralize the mic antioxidants like GSH, vitamin C and E and ceruloplasmin in ISO- free radical induced damage. This is accomplished by a set of endogenous induced rats. NNE pretreatment controls the cardiac oxidative stress via antioxidant enzymes such as superoxide dismutase (SOD), catalase, glu- reducing the formation and ROS and enhance antioxidant defense by in- tathione peroxidase (GPx), and glutathione-S-transferase (GST). The bal- creasing GSH retention and restoring the activity of antioxidant enzymes. ance between ROS and antioxidant defense is lost, the oxidative stress Thus the NNE scavenges superoxide radicals and hydrogen peroxide pro- results, which through a series of events deregulates the cellular functions duced by ISO and reduces myocardial damage. These pharmacological prop- leading to various diseased conditions [32]. Organs cannot continue its erties of NNE may contribute in the antioxidant machinery. function without adequate blood flow, and if a vital organ such as heart or brain is severely compromised, death is inevitable. Following ischemia, Bioassay-guided fractionation and repeated chromatography of Nelumbo ROS are produced during reperfusion phase [33]. The extent of ROS- nucifera has led to the isolation and identification of various phyto con- induced oxidative damage can be exacerbated by a decreased efficiency of stituents including, quercetin 3-O-alpha-arabinopyranosyl-(1,2)-beta- antioxidant defense mechanisms [34]. There is evidence that antioxidants galactopyranoside, rutin, (+)-catechin, hyperoside, isoquercitrin, quercetin can protect against free radical production, which is responsible for and astragalin. It also contain flavonoids like hyperin, isoquercetin and reperfusion-induced damage and lipid peroxidation, and may thereby in- astragalin. Its widely accepted that falvonoids posses free radical scaveng- hibit thrombosis, myocardial damage and arrhythmias during MI. ing and antioxidant properties by its hydroxyl and carbonyl groups. NNE having these constituents may be responsible for the protective effect. In this study, we have observed decreased activities of SOD and catalase in ISO-induced rats. Free radical-scavenging enzymes such as SOD and cata- CONCLUSION lase are the first line of cellular defense against oxidative injury, decompos- Based on the present investigation’s biochemical and histopathological findings, NNE administration offers significant protection to the myocardium ing superoxide radicals and H2O2 before interacting to form the more reac- . in ISO-induced MI in rats. The protective action of NNE could be due to tive hydroxyl radical. Superoxide radicals (O2 -) are eliminated by SOD in cytosol (Cu/Zn-SOD) and in mitochondria (Mn-SOD) and the resultant prevention or inhibition of lipid peroxidation and antioxidant effect (pres- H O is removed by catalase [35]. ence of flavonoids, alkaloids and other phenolic groups). Hence, NNE pos- 2 2 sess cardioprotective effect in ISO-induced MI in rats. 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Journal of Pharmacy Research Vol.5 Issue 7.July 2012 3878-3882