- Email: [email protected]
Combined effects of quercetin and α-tocopherol on lipids and glycoprotein components in isoproterenol induced myocardial infarcted Wistar rats
Chemico-Biological Interactions 181 (2009) 322–327
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Combined effects of quercetin and ␣-tocopherol on lipids and glycoprotein components in isoproterenol induced myocardial infarcted Wistar rats V.R. Punithavathi, P. Stanely Mainzen Prince ∗ Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar-608002, TamilNadu, India
a r t i c l e
i n f o
Article history: Received 22 April 2009 Received in revised form 24 June 2009 Accepted 2 July 2009 Available online 10 July 2009 Keywords: Quercetin ␣-Tocopherol Isoproterenol Lipids Glycoprotein components Myocardial infarction
a b s t r a c t This study was aimed to evaluate the combined effects of quercetin and ␣-tocopherol on lipid metabolism and glycoprotein components in isoproterenol induced myocardial infarction in Wistar rats. Myocardial infarction in rats was induced by isoproterenol (100 mg/kg) at an interval of 24 h for 2 days. Quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) were given to rats as pretreatment for 14 days orally using an intragastric tube. Quercetin and ␣-tocopherol significantly reduced the levels of cholesterol, triglycerides and free fatty acids in the serum and heart and serum phospholipids and significantly increased the levels of heart phospholipids in isoproterenol induced rats. They also significantly decreased the activity of plasma and liver 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and increased the activity of plasma and liver lecithin cholesterol acyl transferase in isoproterenol treated rats. In addition to this, they also significantly reduced the levels of hexose, hexosamine, fucose and sialic acid in the serum and heart of isoproterenol treated rats. Quercetin and ␣-tocopherol also showed significant decrease in plasma lipid peroxidation products (thiobarbituric acid reactive substances and lipid hydroperoxides). Pretreatment with quercetin alone and ␣-tocopherol alone showed significant effect in all the biochemical parameters in myocardial infarcted rats. But, combined pretreatment with quercetin and ␣-tocopherol normalized all the above mentioned biochemical parameters in isoproterenol treated myocardial infarction in rats. Thus, the experiment clearly showed that quercetin and ␣-tocopherol prevented the accumulation of lipids and glycoprotein components in myocardial infarcted rats by their anti-lipid peroxidative effect. This study also showed that combined pretreatment was better than single pretreatment. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Myocardial infarction (MI) is the condition of necrosis of the myocardium that occurs as a result of imbalance between coronary blood supply and myocardial demand. A catecholamine, isoproterenol (ISO) is used to study the protective effect of various drugs on cardiac function. MI induced by ISO, a -adrenergic agonist has been reported to show many metabolic and morphologic aberrations in the heart tissue of the experimental animals similar to those observed in human MI [1]. It induces myocardial necrosis by a multiple step mechanism [2]. It has been reported that accumulation of lipids in the myocardium might be due to the enhanced activity of adenylate cyclase in ISO treated rats [3]. A growing body of evidence is emerging which suggests that reactive oxygen derived free radicals play a crucial role in the pathogenesis of ISO induced MI [4]. Chagoya de sanchez et al. [5] have reported that ISO causes an increase in the levels of circulatory and myocardial lipids. It also
∗ Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141. E-mail address: ps [email protected] (P.S.M. Prince). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.07.002
increases the glycoprotein components in the circulation and heart. Furthermore, ISO promotes lipolysis in the myocardium [6]. Many plant species and their constituents are used in Indian Ayurvedic Medicine for the treatment of MI. Recently, there has been renewed interest in medicinal plants and food products derived from medicinal plants that have been found to have certain preventive actions in the treatment of cardiovascular diseases (CVD). Flavonoids are ubiquitous compounds, occurring in various plants such as tea, herbs, citrus fruits and red wine and many of them have been shown to be strong free radical scavengers and antioxidants [7]. Several epidemiological studies have supported the hypothesis that the antioxidant actions of flavonoids may reduce the risk of developing CVD [8]. Quercetin (3,3 ,4 ,5,7 pentahydroxy flavone) belongs to the family of flavonoids, a large class of naturally occurring, low molecular weight plant metabolites that display a wide range of pharmacological properties [9]. It is found in many foods, including vegetables, tea, fruits and wine [10]. The antioxidant properties of quercetin might be due to its ability to chelate transition metal ions, such as Fe2+ and Cu2+ , and scavenge free radicals [11]. It is accumulated and maintained in the body more easily than other flavonoids,
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
even though it is hydrophilic. It exhibits a broad range of pharmacological activities such as anti-inflammatory [12], anti-tumour [13], anti-ulcer [14], anti-mutagenesis [15], immuno modulatory [16] and vasodilation [17]. It also prevents oxidation of low density lipoproteins in vitro [18]. Vitamin E (␣-tocopherol) is a lipid soluble antioxidant that protects poly unsaturated fatty acids and other components of the cell and organelle membranes from oxidation of reactive free radicals [19]. It is found only in foods of plant origin. Wheat germ is the richest source of the vitamin. Vegetable oils and whole grains are additional rich sources of this nutrient. Green leafy vegetables, nuts, peanut butter, salad dressings and vegetable oils are also good sources of vitamin E. Epidemiological studies have shown that intake of vitamin E is associated with decreased incidence of CVD [20]. Certain natural dietary products and supplements can reduce lipids and glycoprotein components in experimental myocardial infarcted rats [21,22]. According to Medical Practitioners, a combination of drugs exhibit augmented protective efficacy than a single drug. Circulating lipids and glycoprotein components play an important role in the pathogenesis of MI. To achieve the greatest possible reduction in coronary heart diseases (CHD) risk, treatment strategies should be aimed at reducing the elevated levels of circulatory lipids and glycoprotein components. In this research paper, we studied whether combined pretreatment with quercetin and ␣-tocopherol exerts better effect than quercetin alone or ␣-tocopherol alone on the levels of lipids, enzymes associated with lipid metabolism, glycoprotein components and lipid peroxidation products in ISO treated rats. 2. Materials and methods 2.1. Experimental animals All the experiments were carried out with male albino Wistar rats weighing 180–200 g, obtained from The Central Animal House, Rajah Muthiah Institute of Health Sciences, Annamalai University, Tamilnadu, India. They were housed in polypropylene cages (47 cm × 34 cm × 20 cm) lined with husk, renewed every 24 h under a 12:12 h light/dark cycle at around 22 ◦ C. The rats had free access to tap water and food. The rats were fed on a standard pellet diet (Pranav Agro Industries Ltd., Maharashtra, India). The pellet diet consisted of 22.02% crude protein, 4.25% crude oil, 3.02% crude fibre, 7.5% ash, 1.38% sand silica, 0.8% calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins, and 56.17% nitrogen free extract (carbohydrates). The experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Animal Ethical Committee of Annamalai University (Approval No.: 546: 20.3.2008). 2.2. Drug and chemicals Quercetin, ISO and digitonin were purchased from Sigma Chemical Co., St. Louis, MO, USA. ␣-Tocopherol, hydroxylamine hydrochloride, dextran sulphate, sodium meta arsenate, thiobarbituric acid, cysteine hydrochloride, sodium bisulphite, sodium sulphite, dimethyl sulfoxide (DMSO) and potassium tetraborate were purchased from Himedia, Mumbai, India. Ammonium molybdate, amino naphthol sulfonic acid, orcinol and diphenyl carbazide were purchased from S.D. Fine Chemicals, Mumbai, India. All other chemicals used were of analytical grade. 2.3. Induction of experimental myocardial infarction ISO (100 mg/kg) was dissolved in saline and subcutaneously injected to rats at an interval of 24 h for 2 days.
323
2.4. Experimental design A pilot study was done with quercetin (5 and 10 mg/kg) pretreatment alone, ␣-tocopherol (5 and 10 mg/kg) pretreatment alone and combined pretreatment with quercetin (5 and 10 mg/kg) and ␣-tocopherol (5 and 10 mg/kg) showed significant (P < 0.05) effect on cardiac marker enzyme, serum creatine kinase in ISO treated rats. But after 14 days of study, the effect of quercetin (10 mg/kg) alone, ␣-tocopherol (10 mg/kg) alone and combined pretreatment of quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) showed the highest significant effect than the lower dose (5 mg/kg). Hence, we have taken only 10 mg/kg for our study. In our experiment, a total of forty-eight rats were used. The rats were divided into eight groups of six rats each. Group I: normal control rats; Group II: rats were orally treated with quercetin (10 mg/kg) alone for 14 days using an intragastric tube; Group III: rats were orally treated with ␣-tocopherol (10 mg/kg) alone for 14 days using an intragastric tube; Group IV: rats were orally treated with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) for 14 days using an intragastric tube; Group V: rats were subcutaneously injected with ISO alone (100 mg/kg) at an interval of 24 h for 2 days (15th and 16th day); Group VI: rats were pretreated with quercetin (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days; Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days; Group VIII: rats were pretreated with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days. Normal control and ISO control rats were received DMSO (0.5%) alone for 14 days of the experimental period. Quercetin and ␣-tocopherol were dissolved in DMSO and administered to rats orally using an intragastric tube daily for a period of 14 days. At the end of the experimental period, after 12 h of second ISO injection, all the rats were sacrificed by cervical decapitation after injecting anesthetic. Blood was collected in two different tubes, i.e., one with anticoagulant for the separation of plasma and another without anticoagulant for the serum. Plasma and serum were separated by centrifugation. Heart and liver tissues were excised immediately and rinsed in ice-chilled normal saline. Known weights of the tissues were homogenized in 5.0 ml of 0.1 M Tris–HCl buffer (pH 7.4) solution. The homogenate was centrifuged and the supernatant was used for the estimation of various biochemical parameters. 2.5. Biochemical estimations Activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured in the serum using standard commercial kits. The level of plasma thiobarbituric acid reactive substances (TBARs) was estimated by the method of Yagi [23] and lipid hydroperoxides (LOOH) in the plasma was estimated by the method of Jiang et al. [24]. Lipids were extracted from the serum and heart by the method of Folch et al. [25]. The levels of total cholesterol, triglycerides (TGs), free fatty acids (FFAs) and phospholipids (PLs) were estimated by the methods of Zlatkis et al. [26], Fossati and Prencipe [27], Falholt et al. [28] and Zilversmit and Davis [29], respectively. The ratio between 3-hydroxy-3-methylglutarylcoenzyme-A (HMG-CoA) and mevalonate in the tissue was taken as an index of the activity of HMG-CoA reductase as described by the method of Rao and Ramakrishnan [30]. The activity of lecithin cholesterol acyl transferase (LCAT) was also assayed by the method of Hitz et al. [31]. Glycoprotein components such as hexose, hexosamine, fucose and sialic acid were estimated by the methods of Dubois and Gilles [32], Wagner [33], Dische and Shettles [34] and Warren [35], respectively. Protein content in the tissue homogenate was estimated by the method of Lowry et al. [36].
324
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
Fig. 1. Levels of serum cardiac marker enzymes. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
2.6. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using Software Package for the Social Science (SPSS) software package version 12.00. Results were expressed as mean ± S.D. for six rats in each group. P values <0.05 were considered significant. 3. Results Fig. 1 shows the activities of serum CK-MB and LDH in normal and experimental rats. ISO treated rats showed significant increase in the activities of CK-MB and LDH in the serum compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) daily for a period of 14 days normalized the activities of these enzymes in the serum compared to ISO alone treated rats (P < 0.05). ISO treated rats showed significant increase in the levels of plasma TBARs and LOOH when compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of TBARs and LOOH in ISO treated rats when compared to ISO alone treated rats (Figs. 2 and 3) (P < 0.05). Rats treated with ISO showed significant increase in the levels of total cholesterol, TGs, FFAs and PLs in the serum compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of
Fig. 2. Level of plasma TBARs. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 3. Level of plasma LOOH. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
total cholesterol, TGs, FFAs and PLs in the serum compared to ISO alone treated rats (Fig. 4) (P < 0.05). Rats treated with ISO showed significant (P < 0.05) increase in the levels of total cholesterol, TGs and FFAs in the heart tissue homogenate and significant decrease in the level of PLs in the heart tissue homogenate compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of total cholesterol, TGs and FFAs and PLs in the heart tissue homogenate compared with ISO alone treated rats (Figs. 5 and 6) (P < 0.05). ISO treated rats showed significant (P < 0.05) increase in the activity of HMG-CoA reductase in the plasma and liver tissue homogenate compared to normal control rats. Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the activity of this enzyme in the plasma and liver compared to ISO control rats (P < 0.05). Lower ratio of HMGCoA/mevalonate indicates higher enzyme activity and vice versa (Fig. 7). ISO treated rats showed significant decrease in the activity of LCAT in the plasma and liver tissue homogenate compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the activity of LCAT compared to ISO alone treated rats (Fig. 8) (P < 0.05). Figs. 9 and 10 show the levels of glycoprotein components such as hexose, hexosamine, fucose and sialic acid in the serum and heart of normal and experimental rats. ISO treated rats showed significant increase in the levels of these glycoprotein components compared
Fig. 4. Levels of serum lipids. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg)+ ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
325
Fig. 5. Levels of lipids in the heart. Group I: Normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 8. Activity of LCAT in the plasma and liver. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 6. Level of free fatty acids in the heart. Group I: Normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 9. Levels of serum glycoprotein components. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
with normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of these glycoprotein components in the serum and heart tissue homogenate compared to ISO alone treated rats (P < 0.05). For all the biochemical parameters studied, pretreatment with quercetin (10 mg/kg) alone and ␣-tocopherol (10 mg/kg) alone showed significant effect in all the biochemical parameters in ISO treated rats. Combined oral pretreatment with quercetin
Fig. 7. Activity of HMG-CoA reductase in the plasma and liver. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercet in (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT). Lower ratio indicates higher enzyme activity and vice versa.
(10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized all the biochemical parameters in ISO treated rats. Combined pretreatment with quercetin and ␣-tocopherol showed the highest effect than pretreatment with quercetin alone or ␣-tocopherol alone. Treatment with quercetin (10 mg/kg) alone, ␣-tocopherol (10 mg/kg) alone and combined treatment with quercetin (10 mg/kg) and ␣tocopherol (10 mg/kg) to normal control rats did not show any significant effect in all the biochemical parameters studied.
Fig. 10. Levels of glycoprotein components in the heart. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
326
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
4. Discussion Serum CK-MB and LDH are diagnostic markers of MI. These marker enzymes are released from the heart into the blood during myocardial damage due to ISO induced necrosis in the myocardium. When myocardial cells containing CK-MB and LDH are damaged or destroyed, the cell membrane becomes permeable or may rupture, which results in the leakage of these enzymes. This accounts for the increased activities of serum CK-MB and LDH in ISO treated rats [37]. Prior combined treatment with quercetin and ␣-tocopherol normalized the activities of these enzymes in ISO treated rats. ISO administration is associated with increased levels of lipid peroxidation as evidenced by increased levels of plasma TBARs and LOOH in this study. This leads to oxidative damage of cell components (e.g. proteins, lipids and nucleic acids). A possible explanation for the enhancement of lipid peroxidation products (TBARs and LOOH) concentration in ISO treated rats may be due to decreased levels of antioxidant system. Combined pretreatment with quercetin and ␣-tocopherol inhibited lipid peroxidation in ISO treated rats. This effect revealed the anti-lipid peroxidative effect of quercetin and ␣-tocopherol. ISO induced MI is associated with increased levels of lipids in the serum. In this study, we observed increased levels of total cholesterol, TGs, FFAs and PLs in the serum and myocardium of myocardial infarcted rats. Hypercholesterolemia is a risk factor for the development of MI. Increased levels of blood cholesterol and their accumulations in the heart are well associated with myocardial damage [38]. Strong evidence suggests that hypercholesterolemia induces oxidative stress by causing a reduction in the enzymatic antioxidant defense potential of tissues and generation of oxygen free radicals like superoxide anions. As a result of these metabolic events peroxidation reactions are accelerated leading to cellular injury [39]. Anandan et al. [40] have reported that the increase in the myocardial cholesterol content in ISO treated rats is due to increased uptake of low density lipoprotein (LDL)-cholesterol from the blood by myocardial membranes. Accumulation of TGs is one of the risk factors of CVD. The mechanism of observed increase in TGs after MI may be due to elevated flux of fatty acids and impaired removal of very low density lipoprotein (VLDL) from the serum. Combined pretreatment with quercetin and ␣-tocopherol decreased the levels of TGs in myocardial infarcted rats. Under severe ischemic conditions, oxidation of carbohydrates and FFAs will cease. Thus, circulating FFAs may be increased in myocardial infarcted rats. There is a report showing that increased levels of blood FFAs may depress cardiac function, promote arrhythmias and further increase the extent of myocardial damage [41]. Crass et al. [42] also reported that after addition of catecholamine to heart homogenate resulted in the release of FFAs. The heart derives a significant portion of its fatty acid substrates as FFAs derived by lipolysis from adipose tissue. Although lipid availability is important for the heart, excess levels of fatty acids in myocytes can be deleterious [43]. Combined pretreatment with quercetin and ␣-tocopherol lowered the levels of FFAs in myocardial infarcted rats. Membrane PLs are essential factors in maintaining cell function and degradation of PLs by phospholipase A2 and other phospholipases have been implicated as a vital factor in the genesis of ischemic cellular injury [44]. In this context, Franson et al. [45] have reported that ISO increases the activity of phospholipase A2 . Thus, the observed decreased content of PLs in ISO induced myocardium is due to accelerated membrane degradation of PLs by phospholipases. Combined pretreatment with quercetin and ␣-tocopherol showed a stabilizing effect on myocardial phospholipids. This effect is due to the ability of quercetin and ␣-tocopherol to prevent peroxidation of membrane phospholipids. HMG-CoA reductase plays a major role in the regulation of cholesterol metabolism and a rate limiting enzyme in the path-
way of cholesterol biosynthesis. We observed significant increase in the activity of HMG-CoA reductase in the plasma and liver of ISO induced rats. Enhanced activity of HMG-CoA reductase might be due to increased lipid peroxidation in ISO treated rats. In this context, we observed increased levels of lipid peroxidation products (TBARs and LOOH) in ISO treated rats. An increase in HMG-CoA reductase activity leads to excessive production and accumulation of cholesterol and resulting in the formation of foam cell, a prerequisite step in the development of atherosclerosis [46]. Quercetin and ␣-tocopherol pretreatment normalized the activity of HMGCoA reductase in ISO treated rats. Thus, the observed decrease in HMG-CoA reductase in quercetin and ␣-tocopherol pretreated ISO induced rats might be due to the inhibition of lipid peroxidation. LCAT is an enzyme responsible for the conversion of cholesterol to cholesterol esters on the surface of high density lipoproteins (HDLs). The esterification of cholesterol by LCAT leads to the remodeling of the lipoprotein, HDL and results in the formation of large HDL particles that are known to offer protection against CVD. Decreased activity of LCAT inhibits the esterification of cholesterol in ISO treated rats. This leads to high concentration of lipids and lipoproteins in circulation, which are at high risk of atherosclerosis and MI. Increased oxidative stress resulted in the deficiency of LCAT in ISO treated rats. Quercetin and ␣-tocopherol pretreatment increased the activity of LCAT in ISO induced rats. Quercetin and ␣-tocopherol increased the activity of LCAT which increases the concentration of good cholesterol (HDL) in ISO induced rats. Thus, the observed increase in LCAT might be due to the blocking of lipid peroxidation in quercetin and ␣-tocopherol pretreated ISO induced rats. The function of glycoproteins in stabilizing the tissue may be involved in maintaining the structural stability of collagen fibrils. Glycoproteins are important components of intracellular matrix, cell membrane and membranes of sub-cellular organelles [47]. Judd and Wexler [48] suggested that glycoproteins are involved in the myocardial necrosis and repair. In this study, we have observed a significant increase in the levels of hexose, hexosamine, fucose and sialic acid in the serum and heart of ISO induced rats. Mathew et al. [49] reported increased levels of glycoprotein components in the serum and heart in ISO induced MI rats. The elevation in the levels of serum glycoprotein components might be due to secretion from cell membrane glycoconjugates into the circulation [50]. The observed increase in the levels of glycoprotein components in ISO induced rats may also be due to increased deposition of macromolecular components, which is a physiological adjustment to the pathological process. Quercetin and ␣-tocopherol pretreatment decreased the levels of glycoprotein components in the serum and heart in ISO induced rats. Thus, the observed combined effects of quercetin and ␣-tocopherol on lipids and glycoprotein components might be due to the inhibition of lipid peroxidation.
5. Conclusion It is clear that combined pretreatment with quercetin and ␣tocopherol normalized the levels of lipids in ISO induced MI rats. This could be due to the ability of quercetin and ␣-tocopherol by inhibiting the activity of the HMG-CoA reductase and increasing the activity of LCAT. They also normalized the levels of glycoprotein components in ISO induced MI rats. The antioxidant property of quercetin and ␣-tocopherol indirectly helps to lower the levels of lipids and glycoprotein components by reducing or inhibiting lipid peroxidation. Thus, quercetin and ␣-tocopherol protects the myocardium against the accumulation of lipids and glycoprotein components in ISO induced MI rats. Pretreatment with quercetin alone and ␣-tocopherol alone showed significant effect on lipids, glycoprotein components and lipid peroxidation products in ISO
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
treated rats. Our study also showed that combined pretreatment (quercetin and ␣-tocopherol) was better than individual pretreatment (quercetin or ␣-tocopherol alone). Further studies are needed to find out the exact mechanism of action of quercetin and ␣tocopherol. Conflicts of interest We declare that there are no conflicts of interest. Acknowledgement We thank Annamalai University for the financial assistance in the form of “University Research Fellowship” to Ms.V.R. Punithavathi. References [1] C. Nirmala, R. Puvanakrishnan, Protective role of curcumin against isoproterenol induced myocardial infarction in rats, Mol. Cell. Biochem. 159 (1996) 85–93. [2] L.V. Ravichandran, R. Puvanakrishanan, K.T. Joseph, Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats, Biochem. Int. 22 (1990) 387–396. [3] S. Dhawan, N.K. Kapoor, S. Nityanand, Effect of isoprenaline on lipid profile and cardiac enzymes in rats, Ind. J. Exp. Biol. 16 (1978) 376–378. [4] C. Nirmala, R. Puvanakrishnan, Effect of curcumin on certain lysosomal hydrolases in isoproterenol-induced myocardial infarction in rats, Biochem. Pharmacol. 51 (1996) 47–51. [5] V. Chagoya de Sanchez, R. Hernandez-Munoz, F. Lopez-Barrera, L. Yanez, S. Vidrio, J. Suarez, M.D. Cota-Garza, A. Aranda-Fraustro, D. Cruz, Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced by isoproterenol in rats: a long-term and integrative study, Can. J. Physiol. Pharmacol. 75 (1997) 1300–1311. [6] S. Sushama Kumari, A. Varghese, D. Muraleedharan, V.P. Menon, Protective action of aspirin in experimental myocardial infarction induced by isoproterenol in rats and its effect on lipid peroxidation, Ind. J. Exp. Biol. 28 (1990) 480–485. [7] Y. Chen, X. Li, B. Zhao, R. Zheng, W. Xin, Superoxide and hydroxyl radical sca, venging activities of rutin and other natural products studied by ESR, Biophys. Acta 5 (1989) 253–260. [8] H.D. Sesso, J.M. Gaziano, S. Liu, J.E. Buring, Flavonoid intake and the risk of cardiovascular disease in women, Am. J. Clin. Nutr. 77 (2003) 1400–1408. [9] V. Cody, E. Middleton Jr., J.B. Harbone, A. Beretz (Eds.), Plant Flavonoids in Biology and Medicine. II. Biochemical, Cellular and Medicinal Properties, AR. Liss. Inc., NewYork, 1988, pp. 1–461. [10] M.G. Hertog, H.B. Bueno-de-Mesquita, A.M. Fehily, P.M. Sweetnam, P.C. Elwood, D. Kromhout, Fruit and vegetable consumption and cancer mortality in the Caerphilly study, Cancer Epidemiol. Biomarkers Prev. 5 (1996) 673–677. [11] P. Sestili, A. Guidarelli, M. Dacha, O. Cantoni, Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide: free radical scavenging versus iron chelating mechanism, Free Radic. Biol. Med. 25 (1998) 196–200. [12] K. Taguchi, Y. Hagiwara, K. Kajiyama, Y. Suzuki, Phamacological studies of Houttuyniae herba: the anti-inflammatory effect of quercitrin, Yakuga Zasshi 113 (1993) 327–333. [13] G.J. Soleas, L. Grass, P.D. Josphy, D.M. Goldberg, E.P. Diamandis, A comparison of the anti-carcinogenic properties of four red wine polyphenols, Clin. Biochem. 35 (2002) 119–124. [14] M.J. Martin, V. Motilva, A.D.L. Lastra, Quercetin and naringenin; effects on ulcer formation and gastric secretion in rats, Phytother. Res. 7 (1993) 150–153. [15] M.J. Wargovich, V.W.S. Eng, H.L. Newmark, Inhibition by plant phenols of benzo[a] pyrene-induced nuclear aberrations in mammalian intestinal cells: a rapid in vivo assessment method, Food Chem. Toxicol. 23 (1985) 47–49. [16] M. Russo, R. Palumbo, I. Tedesco, G. Mazzarella, P. Russo, G. Iacomino, G.L. Russo, Quercetin and anti-CD95 (Fas/APO1) enhance apoptosis in HPB-cell line, FEBS Lett. 462 (1999) 322–328. [17] J. Durate, F.P. Vizcaino, P. Utrilla, J. Jimenez, J. Tamarago, A. Zarzuelo, Vasodilatory effects of flavonoids in rat aortic smooth muscles; structure–activity relationships, J. Pharmacol. 24 (1993) 857–862. [18] C.V. De Whalley, S.M. Rankin, J.R. Hoult, W. Jessup, D.S. Leake, Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages, Biochem. Pharmacol. 39 (1990) 1743–1750.
327
[19] A.L. Tappel, VitaminE and free radical peroxidation of lipid, Ann. N. Y. Acad. Sci. 203 (1972) 12–28. [20] K.F. Gey, P. Puska, P. Jordan, U.K. Moser, Inverse correlation between plasma vitamin E and mortality from ischaemic heart disease in cross-cultural epidemiology, Am. J. Clin. Nutr. 53 (Suppl.) (1991) S326–S334. [21] M. Rajadurai, P. Stanely Mainzen Prince, Preventive effect of naringin on lipids, lipoproteins and lipid metabolic enzymes in isoproterenol-induced myocardial infarction in Wistar rats, J. Biochem. Mol. Toxicol. 20 (2006) 191–197. [22] M. Rajadurai, P. Stanely Mainzen Prince, Preventive effect of naringin on isoproterenol-induced cardiotoxicity in Wistar rats: an in vivo and in vitro study, Toxicology 232 (2007) 216–225. [23] K. Yagi, Lipid peroxides and human diseases, Chem. Phys. Lipids 45 (1987) 337–351. [24] Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein, Anal. Biochem. 202 (1992) 384–389. [25] J. Folch, M. Lees, S.G.H. Sloane, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [26] A. Zlatkis, B. Zak, A.J. Boyle, A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med. 41 (1953) 486–492. [27] P. Fossati, L. Prencipe, Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide, Clin. Chem. 28 (1982) 2077–2080. [28] K. Falholt, B. Lund, W. Falholt, An easy colorimetric method for routine determination of free fatty acids in plasma, Clin. Chim. Acta 46 (1973) 105–111. [29] D.B. Zilversmit, A.K. Davis, Microdetermination of plasma phospholipids by trichloro acetic acid precipitation, J. Lab. Clin. Med. 35 (1950) 155–160. [30] A.V. Rao, S. Ramakrishnan, Indirect assessment of hydroxymethylglutaryl-CoA reductase activity in liver tissue, Clin. Chem. 21 (1975) 1523–15228. [31] J. Hitz, J. Steinmetz, G. Siest, Plasma lecithin cholesterol acyl transferasereference values and effects of xenobiotics, Clin. Chim. Acta 133 (1983) 85–96. [32] M. Dubois, K.A. Gilles, Methods in Enzymology, Academic Press, New York, 1956, pp. 83–85. [33] W.D. Wagner, A more sensitive assay discriminating galactosamine and glucosamine in mixtures, Anal. Biochem. 94 (1979) 394–396. [34] Z. Dische, L.B. Shettles, Specific colour reactions of methylpentoses and spectrophotometric micromethod for their determination, J. Biol. Chem. 175 (1948) 595–603. [35] L. Warren, The thiobarbituric acid assay of sialic acids, J. Biol. Chem. 234 (1959) 1971–1975. [36] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with folinphenol reagent, J. Biol. Chem. 193 (1951) 265–275. [37] S. Mathew, P.V. Menon, P.A. Kurup, Effect of administration of vitamin A, ascorbic acid and nicotinamide adenine dinucleotide + flavin adenine dinucleotide on severity of myocardial infarction induced by isoproterenol in rats, Ind. J. Exp. Biol. 23 (1985) 500–504. [38] A.M. Salter, D.A. White, Effects of dietary fat on cholesterol metabolism; regulation of plasma LDL concentration, Nutr. Res. Rev. 9 (1996) 241–257. [39] C. Gokkusu, T. Mostafazadeh, Changes of oxidative stress in various tissues by long-term administration of vitamin E in hypercholesterolemic rats, Clin. Chim. Acta 328 (2003) 155–161. [40] R. Anandan, S. Mathew, T.V. Sankar, P.G. Viswanathan Nair, Protective effect of n3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats, Prostaglandins Leukot. Essent. Fatty Acids 76 (2007) 153–158. [41] G. Jacksen, Metabolic agents for stable angina, Heart Metabol. 1 (1998) 10–11. [42] M.F. Crass, J.C. Shipp, G.M. Pieper, Effects of catacholamines on myocardial endogenous substrates and contractility, Am. J. Physiol. 228 (1975) 618–627. [43] H. Vik-Mo, O.D. Mjos, Influence of free fatty acids on myocardial oxygen consumption and ischemic injury, Am. J. Cardiol. 48 (1981) 361–365. [44] K.R. Chien, S.C. Sherman, Mittnacht F Jr.S., J.L. Farber, Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase, Arch. Biochem. Biophys. 205 (1980) 614–622. [45] R. Franson, M. Wiate, W. Weglicki, Phospholipase A activity of lysosomes of rat myocardial tissue, J. Biochem. 11 (1972) 472–476. [46] H. Esterbauer, J. Gebicki, H. Puhl, G. Jurgens, The role of lipid peroxidation and antioxidants in oxidative modification of LDL, Free Radic. Biol. Med. 13 (1992) 341–390. [47] B. Zachariah, D. Basu, Surface carbohydrates in cell-biology, Ind. J. Biochem. Biophys. 30 (1993) 422–425. [48] J.T. Judd, B.C. Wexler, Myocardial connective tissue metabolism in response to injury. II. Investigation of the mucopolysaccharides involved in isoproterenolinduced necrosis and repair in rat hearts, Circ. Res. 26 (1970) 101–109. [49] S. Mathew, P.V. Menon, P.A. Kurup, Changes in glycoproteins in isoproterenolinduced myocardial infarction in rats, Ind. J. Biochem. Biophys. 19 (1982) 41– 43. [50] M.A. Crook, K. Earle, A. Morocutti, J. Yip, G. Viberti, J.C. Pickup, Serum sialic acid, a risk factor for cardiovascular disease, is increased in IDDM patients with microalbuminuria and clinical proteinuria, Diabetes Care 17 (1994) 305–310.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Combined effects of quercetin and ␣-tocopherol on lipids and glycoprotein components in isoproterenol induced myocardial infarcted Wistar rats V.R. Punithavathi, P. Stanely Mainzen Prince ∗ Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar-608002, TamilNadu, India
a r t i c l e
i n f o
Article history: Received 22 April 2009 Received in revised form 24 June 2009 Accepted 2 July 2009 Available online 10 July 2009 Keywords: Quercetin ␣-Tocopherol Isoproterenol Lipids Glycoprotein components Myocardial infarction
a b s t r a c t This study was aimed to evaluate the combined effects of quercetin and ␣-tocopherol on lipid metabolism and glycoprotein components in isoproterenol induced myocardial infarction in Wistar rats. Myocardial infarction in rats was induced by isoproterenol (100 mg/kg) at an interval of 24 h for 2 days. Quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) were given to rats as pretreatment for 14 days orally using an intragastric tube. Quercetin and ␣-tocopherol significantly reduced the levels of cholesterol, triglycerides and free fatty acids in the serum and heart and serum phospholipids and significantly increased the levels of heart phospholipids in isoproterenol induced rats. They also significantly decreased the activity of plasma and liver 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and increased the activity of plasma and liver lecithin cholesterol acyl transferase in isoproterenol treated rats. In addition to this, they also significantly reduced the levels of hexose, hexosamine, fucose and sialic acid in the serum and heart of isoproterenol treated rats. Quercetin and ␣-tocopherol also showed significant decrease in plasma lipid peroxidation products (thiobarbituric acid reactive substances and lipid hydroperoxides). Pretreatment with quercetin alone and ␣-tocopherol alone showed significant effect in all the biochemical parameters in myocardial infarcted rats. But, combined pretreatment with quercetin and ␣-tocopherol normalized all the above mentioned biochemical parameters in isoproterenol treated myocardial infarction in rats. Thus, the experiment clearly showed that quercetin and ␣-tocopherol prevented the accumulation of lipids and glycoprotein components in myocardial infarcted rats by their anti-lipid peroxidative effect. This study also showed that combined pretreatment was better than single pretreatment. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Myocardial infarction (MI) is the condition of necrosis of the myocardium that occurs as a result of imbalance between coronary blood supply and myocardial demand. A catecholamine, isoproterenol (ISO) is used to study the protective effect of various drugs on cardiac function. MI induced by ISO, a -adrenergic agonist has been reported to show many metabolic and morphologic aberrations in the heart tissue of the experimental animals similar to those observed in human MI [1]. It induces myocardial necrosis by a multiple step mechanism [2]. It has been reported that accumulation of lipids in the myocardium might be due to the enhanced activity of adenylate cyclase in ISO treated rats [3]. A growing body of evidence is emerging which suggests that reactive oxygen derived free radicals play a crucial role in the pathogenesis of ISO induced MI [4]. Chagoya de sanchez et al. [5] have reported that ISO causes an increase in the levels of circulatory and myocardial lipids. It also
∗ Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141. E-mail address: ps [email protected] (P.S.M. Prince). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.07.002
increases the glycoprotein components in the circulation and heart. Furthermore, ISO promotes lipolysis in the myocardium [6]. Many plant species and their constituents are used in Indian Ayurvedic Medicine for the treatment of MI. Recently, there has been renewed interest in medicinal plants and food products derived from medicinal plants that have been found to have certain preventive actions in the treatment of cardiovascular diseases (CVD). Flavonoids are ubiquitous compounds, occurring in various plants such as tea, herbs, citrus fruits and red wine and many of them have been shown to be strong free radical scavengers and antioxidants [7]. Several epidemiological studies have supported the hypothesis that the antioxidant actions of flavonoids may reduce the risk of developing CVD [8]. Quercetin (3,3 ,4 ,5,7 pentahydroxy flavone) belongs to the family of flavonoids, a large class of naturally occurring, low molecular weight plant metabolites that display a wide range of pharmacological properties [9]. It is found in many foods, including vegetables, tea, fruits and wine [10]. The antioxidant properties of quercetin might be due to its ability to chelate transition metal ions, such as Fe2+ and Cu2+ , and scavenge free radicals [11]. It is accumulated and maintained in the body more easily than other flavonoids,
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
even though it is hydrophilic. It exhibits a broad range of pharmacological activities such as anti-inflammatory [12], anti-tumour [13], anti-ulcer [14], anti-mutagenesis [15], immuno modulatory [16] and vasodilation [17]. It also prevents oxidation of low density lipoproteins in vitro [18]. Vitamin E (␣-tocopherol) is a lipid soluble antioxidant that protects poly unsaturated fatty acids and other components of the cell and organelle membranes from oxidation of reactive free radicals [19]. It is found only in foods of plant origin. Wheat germ is the richest source of the vitamin. Vegetable oils and whole grains are additional rich sources of this nutrient. Green leafy vegetables, nuts, peanut butter, salad dressings and vegetable oils are also good sources of vitamin E. Epidemiological studies have shown that intake of vitamin E is associated with decreased incidence of CVD [20]. Certain natural dietary products and supplements can reduce lipids and glycoprotein components in experimental myocardial infarcted rats [21,22]. According to Medical Practitioners, a combination of drugs exhibit augmented protective efficacy than a single drug. Circulating lipids and glycoprotein components play an important role in the pathogenesis of MI. To achieve the greatest possible reduction in coronary heart diseases (CHD) risk, treatment strategies should be aimed at reducing the elevated levels of circulatory lipids and glycoprotein components. In this research paper, we studied whether combined pretreatment with quercetin and ␣-tocopherol exerts better effect than quercetin alone or ␣-tocopherol alone on the levels of lipids, enzymes associated with lipid metabolism, glycoprotein components and lipid peroxidation products in ISO treated rats. 2. Materials and methods 2.1. Experimental animals All the experiments were carried out with male albino Wistar rats weighing 180–200 g, obtained from The Central Animal House, Rajah Muthiah Institute of Health Sciences, Annamalai University, Tamilnadu, India. They were housed in polypropylene cages (47 cm × 34 cm × 20 cm) lined with husk, renewed every 24 h under a 12:12 h light/dark cycle at around 22 ◦ C. The rats had free access to tap water and food. The rats were fed on a standard pellet diet (Pranav Agro Industries Ltd., Maharashtra, India). The pellet diet consisted of 22.02% crude protein, 4.25% crude oil, 3.02% crude fibre, 7.5% ash, 1.38% sand silica, 0.8% calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins, and 56.17% nitrogen free extract (carbohydrates). The experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Animal Ethical Committee of Annamalai University (Approval No.: 546: 20.3.2008). 2.2. Drug and chemicals Quercetin, ISO and digitonin were purchased from Sigma Chemical Co., St. Louis, MO, USA. ␣-Tocopherol, hydroxylamine hydrochloride, dextran sulphate, sodium meta arsenate, thiobarbituric acid, cysteine hydrochloride, sodium bisulphite, sodium sulphite, dimethyl sulfoxide (DMSO) and potassium tetraborate were purchased from Himedia, Mumbai, India. Ammonium molybdate, amino naphthol sulfonic acid, orcinol and diphenyl carbazide were purchased from S.D. Fine Chemicals, Mumbai, India. All other chemicals used were of analytical grade. 2.3. Induction of experimental myocardial infarction ISO (100 mg/kg) was dissolved in saline and subcutaneously injected to rats at an interval of 24 h for 2 days.
323
2.4. Experimental design A pilot study was done with quercetin (5 and 10 mg/kg) pretreatment alone, ␣-tocopherol (5 and 10 mg/kg) pretreatment alone and combined pretreatment with quercetin (5 and 10 mg/kg) and ␣-tocopherol (5 and 10 mg/kg) showed significant (P < 0.05) effect on cardiac marker enzyme, serum creatine kinase in ISO treated rats. But after 14 days of study, the effect of quercetin (10 mg/kg) alone, ␣-tocopherol (10 mg/kg) alone and combined pretreatment of quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) showed the highest significant effect than the lower dose (5 mg/kg). Hence, we have taken only 10 mg/kg for our study. In our experiment, a total of forty-eight rats were used. The rats were divided into eight groups of six rats each. Group I: normal control rats; Group II: rats were orally treated with quercetin (10 mg/kg) alone for 14 days using an intragastric tube; Group III: rats were orally treated with ␣-tocopherol (10 mg/kg) alone for 14 days using an intragastric tube; Group IV: rats were orally treated with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) for 14 days using an intragastric tube; Group V: rats were subcutaneously injected with ISO alone (100 mg/kg) at an interval of 24 h for 2 days (15th and 16th day); Group VI: rats were pretreated with quercetin (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days; Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days; Group VIII: rats were pretreated with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) for 14 days and then subcutaneously injected with ISO (100 mg/kg) for 2 days. Normal control and ISO control rats were received DMSO (0.5%) alone for 14 days of the experimental period. Quercetin and ␣-tocopherol were dissolved in DMSO and administered to rats orally using an intragastric tube daily for a period of 14 days. At the end of the experimental period, after 12 h of second ISO injection, all the rats were sacrificed by cervical decapitation after injecting anesthetic. Blood was collected in two different tubes, i.e., one with anticoagulant for the separation of plasma and another without anticoagulant for the serum. Plasma and serum were separated by centrifugation. Heart and liver tissues were excised immediately and rinsed in ice-chilled normal saline. Known weights of the tissues were homogenized in 5.0 ml of 0.1 M Tris–HCl buffer (pH 7.4) solution. The homogenate was centrifuged and the supernatant was used for the estimation of various biochemical parameters. 2.5. Biochemical estimations Activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured in the serum using standard commercial kits. The level of plasma thiobarbituric acid reactive substances (TBARs) was estimated by the method of Yagi [23] and lipid hydroperoxides (LOOH) in the plasma was estimated by the method of Jiang et al. [24]. Lipids were extracted from the serum and heart by the method of Folch et al. [25]. The levels of total cholesterol, triglycerides (TGs), free fatty acids (FFAs) and phospholipids (PLs) were estimated by the methods of Zlatkis et al. [26], Fossati and Prencipe [27], Falholt et al. [28] and Zilversmit and Davis [29], respectively. The ratio between 3-hydroxy-3-methylglutarylcoenzyme-A (HMG-CoA) and mevalonate in the tissue was taken as an index of the activity of HMG-CoA reductase as described by the method of Rao and Ramakrishnan [30]. The activity of lecithin cholesterol acyl transferase (LCAT) was also assayed by the method of Hitz et al. [31]. Glycoprotein components such as hexose, hexosamine, fucose and sialic acid were estimated by the methods of Dubois and Gilles [32], Wagner [33], Dische and Shettles [34] and Warren [35], respectively. Protein content in the tissue homogenate was estimated by the method of Lowry et al. [36].
324
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
Fig. 1. Levels of serum cardiac marker enzymes. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
2.6. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using Software Package for the Social Science (SPSS) software package version 12.00. Results were expressed as mean ± S.D. for six rats in each group. P values <0.05 were considered significant. 3. Results Fig. 1 shows the activities of serum CK-MB and LDH in normal and experimental rats. ISO treated rats showed significant increase in the activities of CK-MB and LDH in the serum compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) daily for a period of 14 days normalized the activities of these enzymes in the serum compared to ISO alone treated rats (P < 0.05). ISO treated rats showed significant increase in the levels of plasma TBARs and LOOH when compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of TBARs and LOOH in ISO treated rats when compared to ISO alone treated rats (Figs. 2 and 3) (P < 0.05). Rats treated with ISO showed significant increase in the levels of total cholesterol, TGs, FFAs and PLs in the serum compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of
Fig. 2. Level of plasma TBARs. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 3. Level of plasma LOOH. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
total cholesterol, TGs, FFAs and PLs in the serum compared to ISO alone treated rats (Fig. 4) (P < 0.05). Rats treated with ISO showed significant (P < 0.05) increase in the levels of total cholesterol, TGs and FFAs in the heart tissue homogenate and significant decrease in the level of PLs in the heart tissue homogenate compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of total cholesterol, TGs and FFAs and PLs in the heart tissue homogenate compared with ISO alone treated rats (Figs. 5 and 6) (P < 0.05). ISO treated rats showed significant (P < 0.05) increase in the activity of HMG-CoA reductase in the plasma and liver tissue homogenate compared to normal control rats. Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the activity of this enzyme in the plasma and liver compared to ISO control rats (P < 0.05). Lower ratio of HMGCoA/mevalonate indicates higher enzyme activity and vice versa (Fig. 7). ISO treated rats showed significant decrease in the activity of LCAT in the plasma and liver tissue homogenate compared to normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the activity of LCAT compared to ISO alone treated rats (Fig. 8) (P < 0.05). Figs. 9 and 10 show the levels of glycoprotein components such as hexose, hexosamine, fucose and sialic acid in the serum and heart of normal and experimental rats. ISO treated rats showed significant increase in the levels of these glycoprotein components compared
Fig. 4. Levels of serum lipids. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg)+ ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
325
Fig. 5. Levels of lipids in the heart. Group I: Normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 8. Activity of LCAT in the plasma and liver. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 6. Level of free fatty acids in the heart. Group I: Normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
Fig. 9. Levels of serum glycoprotein components. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
with normal control rats (P < 0.05). Combined pretreatment with quercetin (10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized the levels of these glycoprotein components in the serum and heart tissue homogenate compared to ISO alone treated rats (P < 0.05). For all the biochemical parameters studied, pretreatment with quercetin (10 mg/kg) alone and ␣-tocopherol (10 mg/kg) alone showed significant effect in all the biochemical parameters in ISO treated rats. Combined oral pretreatment with quercetin
Fig. 7. Activity of HMG-CoA reductase in the plasma and liver. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercet in (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT). Lower ratio indicates higher enzyme activity and vice versa.
(10 mg/kg) and ␣-tocopherol (10 mg/kg) normalized all the biochemical parameters in ISO treated rats. Combined pretreatment with quercetin and ␣-tocopherol showed the highest effect than pretreatment with quercetin alone or ␣-tocopherol alone. Treatment with quercetin (10 mg/kg) alone, ␣-tocopherol (10 mg/kg) alone and combined treatment with quercetin (10 mg/kg) and ␣tocopherol (10 mg/kg) to normal control rats did not show any significant effect in all the biochemical parameters studied.
Fig. 10. Levels of glycoprotein components in the heart. Group I: normal control rats, Group II: rats were treated with quercetin (10 mg/kg), Group III: rats were treated with ␣-tocopherol (10 mg/kg), Group IV: rats were treated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg), Group V: ISO treated rats (100 mg/kg), Group VI: rats were pretreated with quercetin (10 mg/kg) + ISO (100 mg/kg), Group VII: rats were pretreated with ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg), Group VIII: rats were pretreated with quercetin (10 mg/kg) + ␣-tocopherol (10 mg/kg) + ISO (100 mg/kg). Each column is mean ± S.D. for six rats in each group; values not sharing a common letter (a, b, c, d) differ significantly with each other (P < 0.05; DMRT).
326
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
4. Discussion Serum CK-MB and LDH are diagnostic markers of MI. These marker enzymes are released from the heart into the blood during myocardial damage due to ISO induced necrosis in the myocardium. When myocardial cells containing CK-MB and LDH are damaged or destroyed, the cell membrane becomes permeable or may rupture, which results in the leakage of these enzymes. This accounts for the increased activities of serum CK-MB and LDH in ISO treated rats [37]. Prior combined treatment with quercetin and ␣-tocopherol normalized the activities of these enzymes in ISO treated rats. ISO administration is associated with increased levels of lipid peroxidation as evidenced by increased levels of plasma TBARs and LOOH in this study. This leads to oxidative damage of cell components (e.g. proteins, lipids and nucleic acids). A possible explanation for the enhancement of lipid peroxidation products (TBARs and LOOH) concentration in ISO treated rats may be due to decreased levels of antioxidant system. Combined pretreatment with quercetin and ␣-tocopherol inhibited lipid peroxidation in ISO treated rats. This effect revealed the anti-lipid peroxidative effect of quercetin and ␣-tocopherol. ISO induced MI is associated with increased levels of lipids in the serum. In this study, we observed increased levels of total cholesterol, TGs, FFAs and PLs in the serum and myocardium of myocardial infarcted rats. Hypercholesterolemia is a risk factor for the development of MI. Increased levels of blood cholesterol and their accumulations in the heart are well associated with myocardial damage [38]. Strong evidence suggests that hypercholesterolemia induces oxidative stress by causing a reduction in the enzymatic antioxidant defense potential of tissues and generation of oxygen free radicals like superoxide anions. As a result of these metabolic events peroxidation reactions are accelerated leading to cellular injury [39]. Anandan et al. [40] have reported that the increase in the myocardial cholesterol content in ISO treated rats is due to increased uptake of low density lipoprotein (LDL)-cholesterol from the blood by myocardial membranes. Accumulation of TGs is one of the risk factors of CVD. The mechanism of observed increase in TGs after MI may be due to elevated flux of fatty acids and impaired removal of very low density lipoprotein (VLDL) from the serum. Combined pretreatment with quercetin and ␣-tocopherol decreased the levels of TGs in myocardial infarcted rats. Under severe ischemic conditions, oxidation of carbohydrates and FFAs will cease. Thus, circulating FFAs may be increased in myocardial infarcted rats. There is a report showing that increased levels of blood FFAs may depress cardiac function, promote arrhythmias and further increase the extent of myocardial damage [41]. Crass et al. [42] also reported that after addition of catecholamine to heart homogenate resulted in the release of FFAs. The heart derives a significant portion of its fatty acid substrates as FFAs derived by lipolysis from adipose tissue. Although lipid availability is important for the heart, excess levels of fatty acids in myocytes can be deleterious [43]. Combined pretreatment with quercetin and ␣-tocopherol lowered the levels of FFAs in myocardial infarcted rats. Membrane PLs are essential factors in maintaining cell function and degradation of PLs by phospholipase A2 and other phospholipases have been implicated as a vital factor in the genesis of ischemic cellular injury [44]. In this context, Franson et al. [45] have reported that ISO increases the activity of phospholipase A2 . Thus, the observed decreased content of PLs in ISO induced myocardium is due to accelerated membrane degradation of PLs by phospholipases. Combined pretreatment with quercetin and ␣-tocopherol showed a stabilizing effect on myocardial phospholipids. This effect is due to the ability of quercetin and ␣-tocopherol to prevent peroxidation of membrane phospholipids. HMG-CoA reductase plays a major role in the regulation of cholesterol metabolism and a rate limiting enzyme in the path-
way of cholesterol biosynthesis. We observed significant increase in the activity of HMG-CoA reductase in the plasma and liver of ISO induced rats. Enhanced activity of HMG-CoA reductase might be due to increased lipid peroxidation in ISO treated rats. In this context, we observed increased levels of lipid peroxidation products (TBARs and LOOH) in ISO treated rats. An increase in HMG-CoA reductase activity leads to excessive production and accumulation of cholesterol and resulting in the formation of foam cell, a prerequisite step in the development of atherosclerosis [46]. Quercetin and ␣-tocopherol pretreatment normalized the activity of HMGCoA reductase in ISO treated rats. Thus, the observed decrease in HMG-CoA reductase in quercetin and ␣-tocopherol pretreated ISO induced rats might be due to the inhibition of lipid peroxidation. LCAT is an enzyme responsible for the conversion of cholesterol to cholesterol esters on the surface of high density lipoproteins (HDLs). The esterification of cholesterol by LCAT leads to the remodeling of the lipoprotein, HDL and results in the formation of large HDL particles that are known to offer protection against CVD. Decreased activity of LCAT inhibits the esterification of cholesterol in ISO treated rats. This leads to high concentration of lipids and lipoproteins in circulation, which are at high risk of atherosclerosis and MI. Increased oxidative stress resulted in the deficiency of LCAT in ISO treated rats. Quercetin and ␣-tocopherol pretreatment increased the activity of LCAT in ISO induced rats. Quercetin and ␣-tocopherol increased the activity of LCAT which increases the concentration of good cholesterol (HDL) in ISO induced rats. Thus, the observed increase in LCAT might be due to the blocking of lipid peroxidation in quercetin and ␣-tocopherol pretreated ISO induced rats. The function of glycoproteins in stabilizing the tissue may be involved in maintaining the structural stability of collagen fibrils. Glycoproteins are important components of intracellular matrix, cell membrane and membranes of sub-cellular organelles [47]. Judd and Wexler [48] suggested that glycoproteins are involved in the myocardial necrosis and repair. In this study, we have observed a significant increase in the levels of hexose, hexosamine, fucose and sialic acid in the serum and heart of ISO induced rats. Mathew et al. [49] reported increased levels of glycoprotein components in the serum and heart in ISO induced MI rats. The elevation in the levels of serum glycoprotein components might be due to secretion from cell membrane glycoconjugates into the circulation [50]. The observed increase in the levels of glycoprotein components in ISO induced rats may also be due to increased deposition of macromolecular components, which is a physiological adjustment to the pathological process. Quercetin and ␣-tocopherol pretreatment decreased the levels of glycoprotein components in the serum and heart in ISO induced rats. Thus, the observed combined effects of quercetin and ␣-tocopherol on lipids and glycoprotein components might be due to the inhibition of lipid peroxidation.
5. Conclusion It is clear that combined pretreatment with quercetin and ␣tocopherol normalized the levels of lipids in ISO induced MI rats. This could be due to the ability of quercetin and ␣-tocopherol by inhibiting the activity of the HMG-CoA reductase and increasing the activity of LCAT. They also normalized the levels of glycoprotein components in ISO induced MI rats. The antioxidant property of quercetin and ␣-tocopherol indirectly helps to lower the levels of lipids and glycoprotein components by reducing or inhibiting lipid peroxidation. Thus, quercetin and ␣-tocopherol protects the myocardium against the accumulation of lipids and glycoprotein components in ISO induced MI rats. Pretreatment with quercetin alone and ␣-tocopherol alone showed significant effect on lipids, glycoprotein components and lipid peroxidation products in ISO
V.R. Punithavathi, P.S.M. Prince / Chemico-Biological Interactions 181 (2009) 322–327
treated rats. Our study also showed that combined pretreatment (quercetin and ␣-tocopherol) was better than individual pretreatment (quercetin or ␣-tocopherol alone). Further studies are needed to find out the exact mechanism of action of quercetin and ␣tocopherol. Conflicts of interest We declare that there are no conflicts of interest. Acknowledgement We thank Annamalai University for the financial assistance in the form of “University Research Fellowship” to Ms.V.R. Punithavathi. References [1] C. Nirmala, R. Puvanakrishnan, Protective role of curcumin against isoproterenol induced myocardial infarction in rats, Mol. Cell. Biochem. 159 (1996) 85–93. [2] L.V. Ravichandran, R. Puvanakrishanan, K.T. Joseph, Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats, Biochem. Int. 22 (1990) 387–396. [3] S. Dhawan, N.K. Kapoor, S. Nityanand, Effect of isoprenaline on lipid profile and cardiac enzymes in rats, Ind. J. Exp. Biol. 16 (1978) 376–378. [4] C. Nirmala, R. Puvanakrishnan, Effect of curcumin on certain lysosomal hydrolases in isoproterenol-induced myocardial infarction in rats, Biochem. Pharmacol. 51 (1996) 47–51. [5] V. Chagoya de Sanchez, R. Hernandez-Munoz, F. Lopez-Barrera, L. Yanez, S. Vidrio, J. Suarez, M.D. Cota-Garza, A. Aranda-Fraustro, D. Cruz, Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced by isoproterenol in rats: a long-term and integrative study, Can. J. Physiol. Pharmacol. 75 (1997) 1300–1311. [6] S. Sushama Kumari, A. Varghese, D. Muraleedharan, V.P. Menon, Protective action of aspirin in experimental myocardial infarction induced by isoproterenol in rats and its effect on lipid peroxidation, Ind. J. Exp. Biol. 28 (1990) 480–485. [7] Y. Chen, X. Li, B. Zhao, R. Zheng, W. Xin, Superoxide and hydroxyl radical sca, venging activities of rutin and other natural products studied by ESR, Biophys. Acta 5 (1989) 253–260. [8] H.D. Sesso, J.M. Gaziano, S. Liu, J.E. Buring, Flavonoid intake and the risk of cardiovascular disease in women, Am. J. Clin. Nutr. 77 (2003) 1400–1408. [9] V. Cody, E. Middleton Jr., J.B. Harbone, A. Beretz (Eds.), Plant Flavonoids in Biology and Medicine. II. Biochemical, Cellular and Medicinal Properties, AR. Liss. Inc., NewYork, 1988, pp. 1–461. [10] M.G. Hertog, H.B. Bueno-de-Mesquita, A.M. Fehily, P.M. Sweetnam, P.C. Elwood, D. Kromhout, Fruit and vegetable consumption and cancer mortality in the Caerphilly study, Cancer Epidemiol. Biomarkers Prev. 5 (1996) 673–677. [11] P. Sestili, A. Guidarelli, M. Dacha, O. Cantoni, Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide: free radical scavenging versus iron chelating mechanism, Free Radic. Biol. Med. 25 (1998) 196–200. [12] K. Taguchi, Y. Hagiwara, K. Kajiyama, Y. Suzuki, Phamacological studies of Houttuyniae herba: the anti-inflammatory effect of quercitrin, Yakuga Zasshi 113 (1993) 327–333. [13] G.J. Soleas, L. Grass, P.D. Josphy, D.M. Goldberg, E.P. Diamandis, A comparison of the anti-carcinogenic properties of four red wine polyphenols, Clin. Biochem. 35 (2002) 119–124. [14] M.J. Martin, V. Motilva, A.D.L. Lastra, Quercetin and naringenin; effects on ulcer formation and gastric secretion in rats, Phytother. Res. 7 (1993) 150–153. [15] M.J. Wargovich, V.W.S. Eng, H.L. Newmark, Inhibition by plant phenols of benzo[a] pyrene-induced nuclear aberrations in mammalian intestinal cells: a rapid in vivo assessment method, Food Chem. Toxicol. 23 (1985) 47–49. [16] M. Russo, R. Palumbo, I. Tedesco, G. Mazzarella, P. Russo, G. Iacomino, G.L. Russo, Quercetin and anti-CD95 (Fas/APO1) enhance apoptosis in HPB-cell line, FEBS Lett. 462 (1999) 322–328. [17] J. Durate, F.P. Vizcaino, P. Utrilla, J. Jimenez, J. Tamarago, A. Zarzuelo, Vasodilatory effects of flavonoids in rat aortic smooth muscles; structure–activity relationships, J. Pharmacol. 24 (1993) 857–862. [18] C.V. De Whalley, S.M. Rankin, J.R. Hoult, W. Jessup, D.S. Leake, Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages, Biochem. Pharmacol. 39 (1990) 1743–1750.
327
[19] A.L. Tappel, VitaminE and free radical peroxidation of lipid, Ann. N. Y. Acad. Sci. 203 (1972) 12–28. [20] K.F. Gey, P. Puska, P. Jordan, U.K. Moser, Inverse correlation between plasma vitamin E and mortality from ischaemic heart disease in cross-cultural epidemiology, Am. J. Clin. Nutr. 53 (Suppl.) (1991) S326–S334. [21] M. Rajadurai, P. Stanely Mainzen Prince, Preventive effect of naringin on lipids, lipoproteins and lipid metabolic enzymes in isoproterenol-induced myocardial infarction in Wistar rats, J. Biochem. Mol. Toxicol. 20 (2006) 191–197. [22] M. Rajadurai, P. Stanely Mainzen Prince, Preventive effect of naringin on isoproterenol-induced cardiotoxicity in Wistar rats: an in vivo and in vitro study, Toxicology 232 (2007) 216–225. [23] K. Yagi, Lipid peroxides and human diseases, Chem. Phys. Lipids 45 (1987) 337–351. [24] Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein, Anal. Biochem. 202 (1992) 384–389. [25] J. Folch, M. Lees, S.G.H. Sloane, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [26] A. Zlatkis, B. Zak, A.J. Boyle, A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med. 41 (1953) 486–492. [27] P. Fossati, L. Prencipe, Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide, Clin. Chem. 28 (1982) 2077–2080. [28] K. Falholt, B. Lund, W. Falholt, An easy colorimetric method for routine determination of free fatty acids in plasma, Clin. Chim. Acta 46 (1973) 105–111. [29] D.B. Zilversmit, A.K. Davis, Microdetermination of plasma phospholipids by trichloro acetic acid precipitation, J. Lab. Clin. Med. 35 (1950) 155–160. [30] A.V. Rao, S. Ramakrishnan, Indirect assessment of hydroxymethylglutaryl-CoA reductase activity in liver tissue, Clin. Chem. 21 (1975) 1523–15228. [31] J. Hitz, J. Steinmetz, G. Siest, Plasma lecithin cholesterol acyl transferasereference values and effects of xenobiotics, Clin. Chim. Acta 133 (1983) 85–96. [32] M. Dubois, K.A. Gilles, Methods in Enzymology, Academic Press, New York, 1956, pp. 83–85. [33] W.D. Wagner, A more sensitive assay discriminating galactosamine and glucosamine in mixtures, Anal. Biochem. 94 (1979) 394–396. [34] Z. Dische, L.B. Shettles, Specific colour reactions of methylpentoses and spectrophotometric micromethod for their determination, J. Biol. Chem. 175 (1948) 595–603. [35] L. Warren, The thiobarbituric acid assay of sialic acids, J. Biol. Chem. 234 (1959) 1971–1975. [36] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with folinphenol reagent, J. Biol. Chem. 193 (1951) 265–275. [37] S. Mathew, P.V. Menon, P.A. Kurup, Effect of administration of vitamin A, ascorbic acid and nicotinamide adenine dinucleotide + flavin adenine dinucleotide on severity of myocardial infarction induced by isoproterenol in rats, Ind. J. Exp. Biol. 23 (1985) 500–504. [38] A.M. Salter, D.A. White, Effects of dietary fat on cholesterol metabolism; regulation of plasma LDL concentration, Nutr. Res. Rev. 9 (1996) 241–257. [39] C. Gokkusu, T. Mostafazadeh, Changes of oxidative stress in various tissues by long-term administration of vitamin E in hypercholesterolemic rats, Clin. Chim. Acta 328 (2003) 155–161. [40] R. Anandan, S. Mathew, T.V. Sankar, P.G. Viswanathan Nair, Protective effect of n3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats, Prostaglandins Leukot. Essent. Fatty Acids 76 (2007) 153–158. [41] G. Jacksen, Metabolic agents for stable angina, Heart Metabol. 1 (1998) 10–11. [42] M.F. Crass, J.C. Shipp, G.M. Pieper, Effects of catacholamines on myocardial endogenous substrates and contractility, Am. J. Physiol. 228 (1975) 618–627. [43] H. Vik-Mo, O.D. Mjos, Influence of free fatty acids on myocardial oxygen consumption and ischemic injury, Am. J. Cardiol. 48 (1981) 361–365. [44] K.R. Chien, S.C. Sherman, Mittnacht F Jr.S., J.L. Farber, Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase, Arch. Biochem. Biophys. 205 (1980) 614–622. [45] R. Franson, M. Wiate, W. Weglicki, Phospholipase A activity of lysosomes of rat myocardial tissue, J. Biochem. 11 (1972) 472–476. [46] H. Esterbauer, J. Gebicki, H. Puhl, G. Jurgens, The role of lipid peroxidation and antioxidants in oxidative modification of LDL, Free Radic. Biol. Med. 13 (1992) 341–390. [47] B. Zachariah, D. Basu, Surface carbohydrates in cell-biology, Ind. J. Biochem. Biophys. 30 (1993) 422–425. [48] J.T. Judd, B.C. Wexler, Myocardial connective tissue metabolism in response to injury. II. Investigation of the mucopolysaccharides involved in isoproterenolinduced necrosis and repair in rat hearts, Circ. Res. 26 (1970) 101–109. [49] S. Mathew, P.V. Menon, P.A. Kurup, Changes in glycoproteins in isoproterenolinduced myocardial infarction in rats, Ind. J. Biochem. Biophys. 19 (1982) 41– 43. [50] M.A. Crook, K. Earle, A. Morocutti, J. Yip, G. Viberti, J.C. Pickup, Serum sialic acid, a risk factor for cardiovascular disease, is increased in IDDM patients with microalbuminuria and clinical proteinuria, Diabetes Care 17 (1994) 305–310.