Protective Effect of Neferine Against Isoproterenol ...

Protective Effect of Neferine Against Isoproterenol-Induced Cardiac Toxicity

Gurusamy Lalitha, Paramasivan Poornima, Arjunan Archanah & Viswanadha Vijaya Padma Cardiovascular Toxicology ISSN 1530-7905 Volume 13 Number 2 Cardiovasc Toxicol (2013) 13:168-179 DOI 10.1007/s12012-012-9196-5

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Author's personal copy Cardiovasc Toxicol (2013) 13:168–179 DOI 10.1007/s12012-012-9196-5

Protective Effect of Neferine Against Isoproterenol-Induced Cardiac Toxicity Gurusamy Lalitha • Paramasivan Poornima • Arjunan Archanah • Viswanadha Vijaya Padma

Published online: 30 December 2012 Ó Springer Science+Business Media New York 2012

Abstract The present study was designed to investigate the cardioprotective effect of neferine against isoproterenol-induced myocardial infarction. Neferine was given orally for 30 days, and isoproterenol was injected subcutaneously for 2 days. Histopathological examination of heart tissue of isoproterenol-treated rats showed myocardial necrosis. Biochemical analysis of isoproterenol-treated rats showed significant increase in the serum marker enzymes—creatine kinase, lactate dehydrogenase, and aspartate transaminase and increased serum glycoprotein components with a concomitant decrease in the heart tissue homogenate when compared to control. Increased lipid peroxidation and decreased antioxidants reduced glutathione, superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase and altered lipid profile in serum and tissue was also recorded in the isoproterenoltreated rats, whereas the rats which received neferine pretreatment followed by isoproterenol injection showed minimal histological changes, absence of inflammation, and a significant decrease in the serum marker enzymes and serum glycoprotein components with a concomitant increase in the heart tissue homogenate when compared to isoproterenol group. Neferine pre-treatment restored the altered biochemical parameters and lipid profile to near normal. The results of the present study showed that neferine exerts strong antioxidant property against isoproterenol-induced oxidative stress and can be used as a potent

G. Lalitha P. Poornima A. Archanah V. V. Padma (&) Animal Tissue Culture Laboratory, Department of Biotechnology, School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamilnadu, India e-mail: [email protected]

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cardioprotective agent against isoproterenol-induced myocardial infarction. Keywords Neferine Isoproterenol Marker enzymes Oxidative stress Lipid peroxidation Myocardial infarction Abbreviations CVD Cardiovascular diseases MI Myocardial infarction MN Myocardial necrosis CK Creatine kinase LDH Lactate dehydrogenase AST Aspartate transaminase HDL High-density lipoprotein LDL Low-density lipoprotein VLDL Very low-density lipoprotein SOD Superoxide dismutase CAT Catalase GSH Reduced glutathione GST Glutathione-S-transferase GPx Glutathione peroxidase

Introduction Cardiovascular diseases (CVDs) have a high prevalence in developing and developed countries, and myocardial infarction accounts for majority of deaths and disabilities [1]. Myocardial infarction (MI) is caused due to an interruption in blood supply to any part of heart, resulting in death of cardiac tissue (myocardial necrosis; MN). Acute MI is characterized by varying degree of chest pain, sweating, weakness, vomiting, arrhythmia, loss of consciousness, and even sudden death [2].

Author's personal copy Cardiovasc Toxicol (2013) 13:168–179

Isoproterenol (L-b-(3,4-dihydroxyphenyl)-a-isopropyl amino ethanol hydrochloride), a synthetic catecholamine and a sympathomimetic b-adrenergic receptor agonist, causes severe stress to the myocardium resulting in an infarct-like necrosis of heart muscle [3]. MI induced by isoproterenol shows many metabolic and morphologic changes in the heart tissue of the experimental animals similar to those observed in human MI [4]. Experimental and clinical studies on heart failure have shown that there is increased generation of reactive oxygen species such as superoxide anion (O2-) and hydroxyl radicals (OH-) which are involved in the formation of lipid peroxides, cell membrane damage, and destruction of antioxidative defense system [5–7]. Isoproterenol is also well known to generate free radicals and to stimulate lipid peroxidation which may be a causative factor for irreversible damage to the myocardial membrane [8]. The rat model of isoproterenol-induced MN serves as a well-standardized model to evaluate several cardiac dysfunctions [9] and to study the efficacy of various natural and synthetic cardioprotective agents [10]. Recently, attention has been focused on phytochemicals and polyphenols such as the flavonoids, alkaloids, and xanthones derived from different plant species as potential therapeutic agents in the prevention and management of cardiovascular diseases due to their antioxidant nature [11]. Lotus (Nelumbo nucifera Gaertn) is a perennial aquatic crop with stout creeping yellowish white rhizomes. Rhizomes, leaves, seeds, pods, stolons, and petals are included in the diet of many Asian countries, and all parts of N. nucifera are used for various medicinal purposes in oriental medicine. In particular, the embryos have been used widely in traditional Chinese medicine for nervous disorders, insomnia, high fevers with restlessness, relieving cough pulmonary fibrosishmic, and CVDs such as hypertension and arrhythmia [12]. The major phytoconstituents present in the seeds of N. nucifera are alkaloids like dauricine, lotusine, nuciferine, pronuciferine, liensinine, isoliensinine, roemerine, nelumbine, and neferine [13, 14]. Of these, neferine is a major bisbenzylisoquinoline alkaloid derived from the embryos of N. nucifera Gaertn., along with liensinine and isoliensinine [15]. This abundant alkaloid in the green embryos possesses a variety of biological activities, including anti-arrhythmia, anti-hypertensive [16], relaxant [17, 18], anti-diabetic [19], cholinesterase inhibitory [20, 21], sedative [21], anticancer [22], and anti-multidrug resistance [23]. Neferine has shown to have anti-arrhythmic action and also significantly inhibits platelet aggregation in rabbits [24, 25] and also inhibits Cu2?-mediated LDL oxidation [26]. Therefore, the present study has been designed to assess whether oral pre-treatment with neferine could exert any protective action against isoproterenol-induced myocardial infarction.

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Materials and Methods Chemicals Isoproterenol was obtained from M/s. Sigma Private Limited, Bangalore. Neferine was extracted from embryo of the lotus seeds as previously described by Wu et al. [27]. All other chemicals used were of analytical grade. Animals Wistar strain male albino rats, purchased from Kerala agricultural university, Mannuthy, weighing 180 ± 10 g each, were selected for the present study. After a week of acclimatization, animals were grouped six per cage and maintained at a temperature of 28 ± 2 °C, with a normal 12-h light/dark cycle. The animals were fed with commercially available pelleted rat chow (Sai Durga private limited, Bangalore) and water ad libitum. The experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and regulations, and the study was approved by the Institutional Animals Ethics Committee, India. Administration of Dosage Neferine (10 mg/kg body weight/day) dissolved in 0.1 ml of 50 % ethanol was made up to 0.3 ml with water so that the final concentration of ethanol is about 16 % which was administered to rats by oral intubation method; this treatment was continued for 30 days. MI was induced in experimental rats by injecting isoproterenol (85 mg/kg body weight/day) dissolved in physiological saline subcutaneously for 2 days [28]. Experimental Design After a week of acclimatization, the experimental animals were divided into five groups, comprising six rats each. Rats in group I (normal control) received standard diet for a period of 30 days. To group II animals (vehicle control), 0.3 ml of 16 % ethanol was given orally on a daily basis for 30 days. In group III, animals received 0.3 ml of neferine (10 mg/kg body weight/day) dissolved in 16 % ethanol for 30 days. Group IV animals were injected with isoproterenol (85 mg/kg body weight/day) dissolved in physiological saline subcutaneously (on 31st and 32nd) for 2 days at 24-h interval for the induction of myocardial infarction. In group V, the animals were pretreated with neferine (10 mg/kg body weight/day) for 30 days and then injected with isoproterenol at the dose mentioned above. At the end of the experimental period, that is, 12 h after last injection of isoproterenol, the experimental animals were anesthetized by exposing to diethyl ether and then killed by

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cervical decapitation. Blood was collected and the serum was separated and used for biochemical assays. The heart tissue was dissected out, washed in ice-cold saline, patted dry, weighed, and stored at -80 °C for further analysis. Histopathology Heart was dissected out soon after killed, cleaned thoroughly in 0.9 % saline, and a small portion of heart tissue was fixed in 10 % neutral buffered formalin and processed by standard procedure for paraffin embedding, and serial sections of about 5 lm size were cut and were stained with hematoxylin and eosin (H&E) dyes.

Cardiovasc Toxicol (2013) 13:168–179

homogenizer. The homogenate was used for the assay of lipid peroxidation, endogenous anti-peroxidative enzymes [superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione-S-transferase (GST), and glutathione peroxidase (GPx)]. Lipid peroxidation was estimated by the method of [40]. SOD was determined by the method of [41]. Catalase was estimated by the method given by [42]. GSH and GSSG were determined according to the methods of [43, 44]. GPx and GST were estimated by the methods described by [45, 46], respectively. The marker enzymes CK, LDH, and AST were also assayed in the heart tissue homogenate. Extraction and Estimation of Lipids in Heart Tissue

Biochemical Parameters in Serum Assay of Marker Enzymes The collected serum was used for the estimation of serum marker enzymes creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate transaminase (AST). Serum CK activity was determined by the method of [29]; LDH activity was assayed according to the method of [30], and AST activity was assayed according to the method of [31]. Lipid Profile in Serum Lipid profile including total cholesterol, phospholipids, triglycerides, and lipoproteins (HDL, LDL, VLDL) was estimated in the serum. Total cholesterol, phospholipids, and triglycerides in the serum were estimated by the methods of [32–34], respectively. Serum lipoproteins were estimated by the method of [35].

Lipids were extracted from the heart tissue by the method of [47], and the lipid extract was estimated for total cholesterol, phospholipids, and triglycerides. Extraction and Estimation of Glycoproteins in Tissue A known weight of the tissue was homogenized in 7 ml of methanol. The contents were filtered and homogenized with 14 ml of chloroform. This was filtered, the residue was subsequently homogenized in chloroform–methanol (2:1 v/v), and each time the extract was filtered. The residue (defatted tissues) was obtained and the filtrate discarded. A weighed amount of defatted tissue was suspended in 3 ml of 2 N HCl and heated at 90 °C for 4 h. The sample was cooled and neutralized with 3 ml of 2 N NaOH. Aliquots from this were used for estimation of hexose, hexosamine, fucose, and sialic acid.

Extraction and Estimation of Glycoproteins in Serum

Statistical Analysis

The glycoprotein components including hexose, hexosamine, fucose, and sialic acid were also estimated in the serum. To 0.1 ml of serum, 5 ml of methanol was added, mixed well, and centrifuged for 10 min at 3,0009g. The supernatant was decanted, and the precipitate was again washed with 5 ml of 95 % ethanol, recentrifuged, and the supernatant was decanted to obtain the precipitate of glycoproteins. Hexose, hexosamine, fucose, and sialic acid were estimated by the methods of [36–39], respectively.

Data analyses were performed using SPSS 17. All data were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance followed by Tukey’s multiple comparison test. Differences between treatments were considered statistically significant at P \ 0.001.

Biochemical Parameters in Heart Tissue

Effects of Neferine Pre-treatment on Heart Weight to Body Weight Ratio in Isoproterenol-Induced Myocardial Infarction in Rats

Lipid Peroxidation, Reduced Glutathione, and Antioxidant Enzymes The excised heart tissue was homogenized in chilled Tris– HCl buffer (0.1 M) pH 7.4 in Potter-Elvehjem Teflon

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Results

The heart weight, body weight, and heart weight to body weight ratio are depicted in the Table 1. There was no significant difference in the body weight of the animals between


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Table 1 Effect of neferine on heart weight, body weight, and heart weight to body weight ratio in isoproterenol-treated rats Parameters

Group I control

Group II ethanol

Group III neferine

Group IV isoproterenol

Heart weight (g)

0.57 ± 0.03a

0.54 ± 0.02a

0.58 ± 0.04a

0.95 ± 0.03b

Body weight (g)

a

a

a

a

Heart weight to body weight ratio

249.67 ± 4.51

a

0.226 ± 0.02

245.01 ± 5.01

a

0.220 ± 0.01

247.33 ± 2.52

a

0.228 ± 0.01

242.33 ± 3.79

Group V NEF ? ISO 0.71 ± 0.02c 246.98 ± 5.29a

b

0.383 ± 0.005

0.283 ± 0.006c

Values are mean ± SD of six animals a,b,c

Means bearing different superscripts on the same row differ significantly at P \ 0.001. One-way ANOVA followed by Tukey’s multiple comparison test

Fig. 1 Histopathology of heart tissue: a control group showing normal myocardial fibers, b neferine group showing muscle bundles with near-normal architecture, c isoproterenol group showing

myocardial necrosis, d Nef ? Iso group showing small focus of inflammation with absence of necrotic damage. Magnification9100

the five groups. Heart weights of the isoproterenol-treated rats were significantly increased, though there was no significant change in the body weight compared to that of control animals. In neferine pre-treated rats followed by isoproterenol injection, the heart weight and the heart weight to body weight ratio decreased significantly when compared to isoproterenol-alone-treated rats. There was no significant difference in the heart weight and heart weight to body weight ratio in the ethanol- or neferine-alone-treated rats when compared to control.

Histopathological Studies Histological examination of the myocardial tissue of control rats showed normal myocardial fibers with clear integrity of myocardial membrane (Fig. 1a). The heart tissue of rats treated with neferine alone showed nearnormal architecture (Fig. 1b). In isoproterenol-treated rats, extensive myofibrillary degeneration with inflammation and necrosis was found (Fig. 1c). The rats pre-treated with neferine followed by isoproterenol injection showed

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Fig. 2 a Effect of neferine on serum creatine kinase activity, b tissue creatine kinase activity, c serum lactate dehydrogenase activity, d tissue lactate dehydrogenase activity, e serum aspartate transaminase activity, f tissue aspartate transaminase activity against

myocardial infarction. Each bar represents mean ± SEM of six animals. One-way ANOVA followed by Tukey’s multiple comparison test. a,b,cMeans not sharing a superscript letter differ significantly at P \ 0.001

significantly decreased inflammation and absence of MN compared to rats treated with isoproterenol alone (Fig. 1d).

treated rats while the activities of these enzymes were decreased significantly in the heart tissue of these animals when compared to control animals. In the rats pre-treated with neferine followed by isoproterenol administration, the activities of the marker enzymes CK, LDH, and AST in the serum were decreased significantly, whereas in the heart tissue, the activities of these enzymes were increased significantly compared to rats treated with isoproterenol alone. The activities of these enzymes did not show any significant difference in the ethanol- or neferine-alone-treated rats when compared to control (Fig. 2).

Effect of Neferine Pre-treatment on the Activities of CK, LDH, and AST in Serum and Heart Tissue of Isoproterenol-Injected Rats The activities of the serum marker enzymes CK, LDH, and AST were significantly increased in the isoproterenol-

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Table 2 Effect of neferine pre-treatment on lipid peroxidation, endogenous antioxidant levels, and the activities of antioxidant enzymes in isoproterenol-induced myocardial infarction in rats Parameters

Group I control

Group II ethanol

Group III neferine

Group IV isoproterenol

Group V NEF ? ISO

LPO

3.67 ± 0.13a

3.83 ± 0.10a

3.62 ± 0.09a

5.74 ± 0.12b

4.10 ± 0.15c

SOD

5.0 ± 0.22

a

a

4.95 ± 0.15

a

b

4.67 ± 0.14c

6.26 ± 0.17

a

6.25 ± 0.09

a

b

5.84 ± 0.08c

3.83 ± 0.11

a

3.84 ± 0.08

a

b

3.44 ± 0.20c

723.37 ± 1.92

a

725.00 ± 1.49

a

b

715.90 ± 1.77c

CAT GPx GST

4.81 ± 0.15

a

6.09 ± 0.15

a

3.70 ± 0.12

a

722.23 ± 1.61

2.54 ± 0.13 3.71 ± 0.15 2.24 ± 0.12 514.70 ± 1.96

Values are mean ± SD of six animals a,b,c

Means bearing different superscript letter on the same row differ significantly at P \ 0.001. One-way ANOVA followed by Tukey’s multiple comparison test. Units are expressed as follows: LPO-nm of TBA reactants/mg of protein; SOD-U/mg of protein; CAT-nm of H2O2 decomposed/min/mg of protein; GPx-lg of GSH utilized/min/mg of protein; GST-nm of CDNB conjugated/min/mg of protein

Fig. 3 Effect of neferine on GSH and GSSG levels and GSH/GSSG ratio of isoproterenol-treated rats. Each bar represents mean ± SEM of six animals. One-way ANOVA followed by Tukey’s multiple

comparison test. a,b,c,p,q,rMeans not sharing a superscript letter differ significantly at P \ 0.001

Effects of Neferine Pre-treatment on Lipid Peroxidation, Endogenous Antioxidant Levels, and the Activities of Antioxidant Enzymes in Isoproterenol-Induced Myocardial Infarction in Rats

there was no significant difference in the LPO and GSH levels and the activities of the antioxidant enzymes when compared to control. When comparing the GSH/GSSG ratio of the isoproterenol-alone-treated group with control, there was a significant decrease, whereas the neferine pre-treatment could increase the GSH/GSSG ratio significantly than the isoproterenol-treated group (Fig. 3).

The levels of LPO and the activities of the antioxidant enzymes GST, GPx, SOD, and CAT were represented in the Table 2. In the rats treated with isoproterenol, there was a significant increase in the TBA reactive substances, the end product of lipid peroxidation when compared to control, whereas in the rats pre-treated with neferine followed by isoproterenol injection, there was a significant decrease in the LPO level when compared to isoproterenol-alone-treated rats. GSH level and the activities of the antioxidant enzymes GPx, GST, SOD, and CAT were decreased significantly in the isoproterenol-treated rats when compared to control. However, in the neferine pre-treated followed by isoproterenol-administered rats, there was a significant increase in the GSH content and the activity of antioxidant enzymes GPx, GST, SOD, and CAT when compared to isoproterenoltreated rats. In the ethanol- or neferine-alone-treated rats,

Effect of Neferine on Total Cholesterol, Phospholipid, and Triglyceride Levels in Serum and Heart Tissues of Isoproterenol-Induced Myocardial Infarction Rats Total cholesterol, phospholipids, and triglycerides levels in serum and tissue were represented in Table 3. The levels of total cholesterol and triglycerides in serum and tissue were found to be significantly increased in isoproterenol-treated rats when compared to control. The serum phospholipids were found to be significantly increased with concomitant decrease in the tissue phospholipid content in isoproterenoltreated rats when compared to control. The rats which received neferine pre-treatment followed by isoproterenol

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Table 3 Effect of neferine pre-treatment on serum and tissue total cholesterol, triglycerides, and phospholipids in isoproterenol-induced myocardial infarction in rats Parameters

Group I (control)

Group II (ethanol)

Group III (neferine)

Group IV (isoproterenol)

Group V (NEF ? ISO)

Total cholesterol Serum

83.5 ± 0.3a

83.92 ± 0.5a

82.09 ± 0.4a

116.5 ± 0.7b

87.4 ± 0.4ac

Tissue

a

a

a

b

12.2 ± 0.2ac

10.3 ± 0.1

11.4 ± 0.1

10.4 ± 0.3

20 ± 0.2

Triglycerides Serum

36.2 ± 0.3a

35.1 ± 0.1a

34.1 ± 0.2a

Tissue

a

a

a

15.3 ± 0.3

16.4 ± 0.3

17.4 ± 0.3

64.1 ± 0.08b 33.9 ± 1.2

b

37.3 ± 0.5ac 19.2 ± 0.2ac

Phospholipids Serum Tissue

98.4 ± 0.01a 31.5 ± 0.2

a

103.8 ± 0.01a

101.1 ± 0.01a

170.7 ± 0.01b

a

a

b

30.2 ± 0.03

34.3 ± 0.01

15.2 ± 0.18

89.0 ± 0.27ac 32.9 ± 0.2ac

NEF neferine, ISO isoproterenol Values are expressed as mean ± SD of mean of 6 animals. One-way ANOVA followed by Tukey’s multiple comparison test. a b c Means bearing different superscript within each row differ significantly at P \ 0.001. Serum values are expressed as milligrams per deciliter, and tissue values are expressed as milligrams per gram of heart tissue.

HDL when compared to isoproterenol group. There was no significant difference in either ethanol- or neferine-alonetreated rats (Fig. 4). Effect of Neferine Pre-treatment on the Levels of Glycoprotein Components in Serum and Heart of Isoproterenol-Induced Myocardial Infarction in Rats

Fig. 4 Effect of neferine on serum lipoprotein levels in isoproterenol-induced cardiac toxicity in rats. Each bar represents mean ± SEM of six animals. One-way ANOVA followed by Tukey’s multiple comparison test. a,b,cMeans not sharing a superscript letter differ significantly at P \ 0.001

administration showed significant decrease in the total cholesterol and triglyceride levels in both serum and tissue while serum phospholipids levels were significantly decreased with a significant increase in the tissue phospholipid content. No significant changes were observed in the ethanol- or neferine-alone-treated groups. Effect of Neferine Pre-treatment on Serum Lipoproteins of Isoproterenol-Induced Myocardial Infarction in Rats The serum lipoproteins LDL and VLDL were found to be significantly increased and HDL was found to be significantly decreased in the isoproterenol-treated rats when compared to control, whereas the rats which received neferine pre-treatment showed significantly decreased levels of LDL, VLDL, and significantly increased level of

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The glycoprotein components hexose, hexosamine, fucose, and sialic acid were found to be significantly increased in the serum of isoproterenol-injected rats with a concomitant decrease in the heart tissue when compared to control, whereas neferine pre-treatment significantly decreased the levels of glycoprotein components with a significant increase in the heart tissue when compared to isoproterenol-injected rats. No significant difference was observed either in the ethanol- or neferine-alone-treated rats when compared to control (Fig. 5).

Discussion Isoproterenol, a synthetic b-adrenergic agonist, by its positive inotropic and chronotropic actions increases the myocardial oxygen demand that leads to ischemic necrosis of myocardium in rats similar to that seen in human MI [48]. Histological examination of the heart tissue of isoproterenol-treated rats showed MN with separated myocardial fibers and inflammation which become globular or polygonal with dark eosinophilic cytoplasm. The rats which received neferine pre-treatment followed by isoproterenol administration showed minimal histological changes.


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Fig. 5 Effect of neferine pre-treatment on the levels of glycoprotein components in serum and heart tissue of isoproterenol-induced cardiac toxicity in rats. Each bar represents mean ± SEM of six

animals. One-way ANOVA followed by Tukey’s multiple comparison test. a,b,cMeans not sharing a superscript letter differ significantly at P \ 0.001

Upon administration of isoproterenol, the heart weight increased significantly with relatively unchanged body weight resulting in the increase in heart weight to body weight ratio when compared to control. Increase in heart weight might be attributed to increased water content and edematous intramuscular space [49]. These results are consistent with the previous report of [50]. Pre-treatment with neferine significantly reduced the heart weight to body weight ratio when compared to isoproterenol-treated group. This may be due to the decrease in the water content in the myocardium. This is in line with the previous study that melatonin pre-treatment decreased the heart weight to body weight ratio significantly in the isoproterenol-treated rats when compared to control [51]. Isoproterenol, the cardiotoxic agent, damages the myocardial cells resulting in the loss of membrane integrity, and as a result, the cytosolic enzymes creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and transaminases (AST and ALT) are released into the blood stream and serve as diagnostic markers of the myocardial membrane damage [52, 53]. The diagnostic marker enzymes, CPK, LDH, and AST, increased significantly in the isoproterenol-treated rats when compared to control rats, whereas a significant decrease in the activity of these marker enzymes was observed in the heart tissue homogenate. This may be due to the leakage of the marker enzymes into the blood stream due to lipid peroxidemediated membrane damage. This is in accordance with the previous study of [54]. The rats pre-treated with neferine showed a significant reduction in the serum marker enzymes CPK, LDH, and AST with a concomitant increase in the heart tissue homogenate when compared to the isoproterenol-treated rats. This may be due to the antioxidant property of neferine which protects the myocardium from

the oxidative damage and helps in maintaining the myocardial cell membrane integrity, thereby preventing the leakage of these enzymes from the myocardium into the blood stream. Previous studies have shown that neferine has a wide range of biological activities, including antihypertensive, anti-arrhythmic, anti-agglutinating, antithrombotic, antioxidant, negative inotropic effect, and vascular smooth muscle relaxation [55, 56]. Thus, the antioxidant property of neferine might have played a significant role in maintaining the membrane integrity. Increased generation of cytotoxic free radicals is one among the several mechanisms proposed to explain the isoproterenol-induced MN [57]. Large number of studies has demonstrated that free radicals initiate lipid peroxidation resulting in alteration of membrane integrity, fluidity, and permeability [58]. In the present study, significantly increased levels of TBA reactive substances have been recorded in the heart tissue homogenate of the animals treated with isoproterenol when compared to control. This may be due to the excessive formation of free radicals and the activation of lipid peroxidation system, thus resulting in the irreversible myocardial membrane damage. The observed increase in the level of lipid peroxides in the isoproterenol-treated rats was in accordance with the previous reports of [59, 60]. The rats pre-treated with neferine showed a significant decrease in the TBA reactive substances when compared to isoproterenol-treated group. Previous studies have reported that hydroalcoholic extract of N. nucifera seeds possesses strong antioxidant and free radical scavenging activity due to the presence of phenolics, alkaloids, and saponins [61]. So the reduced LPO level in the heart tissue of neferine pre-treated rats may be due to the antioxidant and free radical scavenging activity of the alkaloid neferine.

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The equilibrium between antioxidants and free radicals is an important process for the effective removal of oxidative stress in intracellular organelles. However, in pathological conditions like myocardial infarction, the generation of reactive oxygen species can dramatically disturb this balance with an increased demand for the antioxidant defense system [57]. SOD is a class of enzymes, which catalyzes the dismutation of two superoxide radicals to form hydrogen peroxide and molecular oxygen. Thus, generated H2O2 is inactivated by either catalase or by the GSH redox system consisting of GPx and glutathione reductase which require reduced glutathione as the cofactor [62]. The activities of antioxidant enzymes (SOD, catalase GPx, and GST) were decreased significantly along with a decline of GSH levels in the heart tissue of isoproterenolinjected animals when compared to control animals which concurs with the previous study of [63]. The decrease in the activities of these enzymes is due to the increased generation of reactive oxygen radicals, such as superoxide and hydrogen peroxide, which in turn leads to the inhibition of these enzymes [51, 52]. Decreased GSH levels in isoproterenol-induced rats might be due to its increased utilization in protecting –SH group containing proteins from free radicals [64]. Lowered activities of GPx and GST in the heart of isoproterenol-treated rats may be due to decreased availability of GSH [65]. The rats pre-treated with neferine showed a marked increase in the activities of antioxidant enzymes and nonenzymic antioxidant GSH when compared to the isoproterenol-treated group. The reversal of GSH/GSSG ratio in the neferine pre-treated group indicates that the neferine could decrease the alterations in the redox system of isoproterenol-treated rat myocardium. This may be due to the antioxidant activity of neferine owing to its hydroxyl group in the structure. The results of the present study are in accordance with a previous report where neferine served as a free radical scavenger, enhancing SOD activity and inhibiting lipid peroxidation [66]. Lipids play an important role in MN. An excess of lipids is considered to accelerate the development of atherosclerosis which is a risk factor in MI [67]. Increased levels of cholesterol and triglycerides are well associated with cardiovascular disturbances, and isoproterenol promotes lipolysis in myocardium [68]. The present study showed a significant increase in the serum total cholesterol, triglycerides, and phospholipid levels in isoproterenol-treated rats when compared to control. Increased triglycerides after MI may be due to elevated flux of fatty acids and impaired removal of very low-density lipoproteins from the serum [69]. The increase in serum phospholipids may be due to an increased peroxidation of membrane phospholipids released through phospholipase A2 [70].

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The present study showed significantly increased total cholesterol and triglyceride levels followed by a decreased phospholipid level in the heart tissues of isoproterenoltreated rats when compared to control animals. An increased synthesis of triglycerides in the heart tissue could be due to accumulation of acyl CoA and an augmented production of glycerol by increased glycolytic flux [71]. The significant increase observed in the lipid profiles except phospholipids in the rats treated with isoproterenol alone could be due to enhanced lipid biosynthesis by cardiac cAMP. Increase in membrane cholesterol content reduces membrane fluidity, affects permeability to ions, activities of membrane-bound enzymes, and increased degradation of phospholipids [72]. Pre-treatment with neferine restored the altered levels of cholesterol, triglycerides, and phospholipids in the serum and tissue to nearnormal levels. Lipoproteins are independent risk factor for cardiovascular disease. Lipid peroxidation plays an important role in lipoprotein modification, which makes them susceptible to atherosclerosis, and this is one of the mechanisms of isoproterenol-mediated cardiotoxicity [73]. Increased LDL, VLDL, and decreased HDL level was observed in the isoproterenol-treated rats when compared to control. Neferine pre-treatment prevented the altered lipid profile. These results are in line with the study of [74]. Previous studies [75] suggested that glycoproteins are involved in the MN and repair. There was a significant increase in the serum glycoprotein components hexose, hexosamine, fucose, and sialic acid in the isoproterenolinjected rats. This may be due to leakage of protein-bound components from the damaged myocardium into the systemic circulation. The reduction in the hexose, hexosamine, fucose, and sialic acid content observed in heart tissue of isoproterenol-induced MI might be due to inhibition of glycoprotein synthesis. Oxidation of protein is a common phenomenon mediated by highly reactive oxygen species generated during MI condition, and the oxidized proteins, in turn, act as potential mediators of the pathogenesis. This protein oxidation might also be a possible reason for the decline noted in the protein and glycoprotein levels in the heart tissue of isoproterenol-administered rats. Oral pre-treatment with neferine prevented the isoproterenol-induced alterations in the levels of protein content and glycoprotein components in serum and heart tissue when compared to isoproterenol-administered rats. Experimental studies demonstrated that seed of N. nucifera has hepatoprotective and antifertility activities as well as free radical scavenging activity. Neferine isolated from the seeds of lotus exerts anti-arrhythmic effect on the cardiovascular system in vivo and in vitro models. With its antioxidant property, neferine could effectively trap the lipid peroxyl radical to inhibit the free radical-initiated lipid peroxidation as


well as curb the cellular antioxidant pool depletion. The results of the present study confirm that neferine isolated from the embryos of lotus seed possesses cardioprotective effect against isoproterenol-induced MI in rats. Neferine pre-treatment protects the myocardium from the membrane damage and histological changes by maintaining the myocardial antioxidant status and significant restoration of the altered biochemical parameters. These findings suggest that lotus seeds predominantly containing the alkaloid neferine could be considered as a functional food to prevent myocardial infarction. Acknowledgments The authors express their sincere gratitude to Mr. Kumaresh, Sanjeevini Herbals, Salem, India, for providing lotus seeds for the isolation of neferine. Conflict of interest of interest.

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The authors declare that they have no conflict 17.

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