Journal of Pharmacology and Pharmacotherapeutics

RESEARCH PAPER
Year
: 2010  |  Volume : 1  |  Issue : 1  |  Page : 32--37

Cardioprotective potential of Punica granatum extract in isoproterenol-induced myocardial infarction in Wistar rats


Mahalaxmi Mohan1, Pankaj Patankar1, Prakash Ghadi2, Sanjay Kasture3,  
1 Department of Pharmacology, M. G. V's Pharmacy College, Panchavati, Nashik, Maharashtra, India
2 Clinipath, Computerised Laboratory, 1-Shriram Niketan, Opp. Kulkarni Garden, Sharanpur Road, Nashik, Maharashtra, India
3 Pinnacle Biomedical Research Institute, Bhopal-462 003, India

Correspondence Address:
Mahalaxmi Mohan
Department of Pharmacology, M. G. VSQs Pharmacy College, Panchavati, Nashik, Maharashtra 422 003
India

Abstract

Objective: To determine the protective role of Punica granatum L. (Punicaceae) seed juice extract and its butanolic fraction on heart rate, electrocardiographic patterns, vascular reactivity to catecholamines, cardiac marker enzymes, antioxidant enzymes together with morphologic and histopathological changes in isoproterenol-induced myocardial infarction in male Wistar rats. Materials and Methods: The effects of Punica granatum seed juice extract (100 mg/kg, p.o. and 300 mg/kg, p.o.) and butanolic fraction of Punica granatum seed juice extract (100 mg/kg., p.o.) on cardiac parameters were studied. Isoproterenol hydrochloride was used to induce myocardial infarction in Wistar rats. At the end of the experiment, heart rate, ECG, pressure rate index and cardiac marker enzyme levels were assessed. Results: Rats treated with isoproterenol (85 mg/kg, administered subcutaneously twice at an interval of 24 h) showed a significant increase in heart rate, ST elevation in ECG, pressure rate index and a significant increase in the levels of cardiac marker enzymes- lactate dehydrogenase, and creatine kinase in serum. Isoproterenol significantly reduced superoxide dismutase and catalase activity and increased vascular reactivity to various catecholamines. Pretreatment with PJ (100 mg/kg, p.o. and 300 mg/kg, p.o.) and B-PJ (100 mg/kg., p.o.) for a period of 21 days significantly inhibited the effects of ISO on heart rate, PRI, ECG patterns, levels of LDH, CK, SOD, CAT, and vascular reactivity changes. Treatment with PJ (100 mg/kg and 300 mg/kg) and B-PJ (100 mg/kg., p.o.) alone did not alter any of the parameters as compared to vehicle-treated Wistar rats. Punica granatum-treated animals showed a lesser degree of cellular infiltration in histopathological studies. Conclusion: Punica granatum ameliorates cardiotoxic effects of isoproterenol and may be of value in the treatment of MI.



How to cite this article:
Mohan M, Patankar P, Ghadi P, Kasture S. Cardioprotective potential of Punica granatum extract in isoproterenol-induced myocardial infarction in Wistar rats.J Pharmacol Pharmacother 2010;1:32-37


How to cite this URL:
Mohan M, Patankar P, Ghadi P, Kasture S. Cardioprotective potential of Punica granatum extract in isoproterenol-induced myocardial infarction in Wistar rats. J Pharmacol Pharmacother [serial online] 2010 [cited 2020 Oct 25 ];1:32-37
Available from: http://www.jpharmacol.com/text.asp?2010/1/1/32/64533


Full Text

 Introduction



An increased production of free radicals or generation of toxic reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide and hydroxyl radical gives rise to oxidative stress, which plays a major role in cardiovascular diseases such as ischemic heart disease, atherosclerosis, congestive heart failure, cardiomyopathy and arrhythmias. [1] Isoproterenol {4-[1-hydroxy-2-(1-methylethylamino) ethyl] benzene-1, 2-diol} (ISO), a synthetic catecholamine and β-adrenergic agonist has been found to cause severe stress in the myocardium resulting in infarct like necrosis of the heart muscles. Catecholamines rapidly undergo auto-oxidation and it has been suggested that the oxidative products of catecholamines are responsible for changes in the myocardium. [2] High concentrations of catecholamines have been reported to cause necrotic lesions in the heart resulting in myocardial infarction in experimental animals. [3]

Punica granatum (Punicaceae) fruit (commonly called Pomegranate) is considered as a heart healthy fruit juice. [4] Pomegranate is rich in antioxidant of polyphenolic class which includes tannins and anthocynins [5] and flavonoids. [6] The soluble polyphenol content in pomegranate juice is between 0.2 and1.0 %, depending on variety and include mainly tannins, ellagic tannins, anthocyanins, catechins, gallic and ellagic acids. [7] Interaction of flavonoids with various biological systems is well documented. [6] As per the epidemiological studies, cardiovascular mortality and flavonoid intake appear to be inversely proportional. [8] Despite the fact that Punica granatum has antioxidant properties, its cardioprotective activity against ISO induced myocardial infarction (MI) has not been studied. In view of this, the present study was designed to investigate if oral administration of P.granatum seed juice extract (PJ) and its butanolic fraction (B-PJ) for 21 days has any protective action against ISO-induced myocardial injury. Histopathological and biochemical changes induced by ISO have been monitored and their modulation with PJ and B-PJ were evaluated.

 Materials and Methods



Experimental animals- Male adult albino Wistar rats, weighing 150-200 g were obtained from Serum Institute Pune, India. They were housed in polypropylene cages lined with husk, renewed every 48 h under 12:12 h light dark cycle and maintained at 25 o ± 2 o C. They were fed with commercial rat pellet (AMRUT Laboratory animal feed, Pranav Agro Industries, Sangli) and given water ad libitum. The experiments were 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 Institutional Animal Ethical Committee.

Drugs and chemicals- Isoproterenol hydrochloride (ISO), adrenaline (Adr), noradrenaline (NA), phenylepherine (PE), serotonin (5-HT), 1,1-diphenyl, 2- picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, Mumbai. All other chemicals used in the study were of analytical grade. All drug solutions were freshly prepared in saline before each experiment. The extracts were dissolved in distilled water and administered orally.

Preparation of extract- Pomegranates (1 kg) were purchased from the local market of Nashik. The seeds were separated and ground to obtain juice. The juice was then filtered and dried under reduced pressure to get pomegranate juice extract (PJ). The yield was found to be 3.2% w/w. The seeds were separated from the fruits and were refluxed with 2 M HCl for 1 h at 100 0 C. This gave a red color extract from pomegranate which was cooled and filtered. The acidic extract was extracted with ethyl acetate to remove the flavones. This ethyl acetate fraction was heated at 80 0 C for 3 min to remove the last traces of ethyl acetate and cooled. It was then extracted exhaustively with n- butanol. The n-butanol extract (B-PJ) (yield 1.6% w/w) was concentrated by distillation. It gave a concentrated red colored pigment which was subjected to thin layer chromatography using TLC silica gel 60 F 254 plates (Merck) and BAW[ n-butanol, acetic acid and water (4:1:5)], forestal [Concentrated HCl, acetic acid and water (3:30:10)] and formic acid [Concentrated HCl, formic acid and water (2:5:3)] as mobile phases. Iodine vapors were used for visualizing the spots on the plates and Rf values were calculated. [9] The phytochemical testing and the total flavonoid and phenolic contents of the extracts were also determined. [10],[11],[12]

Experimental design- ISO (85 mg/kg) was dissolved in normal saline and injected subcutaneously to rats at an interval of 24 h for 2 days to induce experimental MI. Animals were divided into eight groups of five each. Group I received saline, group II received ISO (85 mg/kg s.c., on 20 th and 21 st day), group III received PJ extract (100 mg/kg, p.o., for 3 weeks), group IV received PJ extract (300 mg/kg, p.o., for 3 weeks), group V received B- PJ extract (100 mg/kg, p.o., for 3 weeks), group VI received PJ extract (100 mg/kg, p.o., for 3 weeks) and ISO (85 mg/kg s.c., on 20 th and 21 st day), group VII received PJ extract (300 mg/kg, p.o., for 3 weeks) and ISO (85 mg/kg s.c., on 20 th and 21 st day), and group VIII received B-PJ extract (100 mg/kg, p.o., for 3 weeks) and ISO (85 mg/kg s.c., on 20 th and 21 st day). After 12 h following the last dose of ISO, heart rate, ECG, PRI and changes to vascular reactivity to various catecholamines were recorded using Powerlab 4SP (AD Instrument, Australia). The serum of animals of all groups was assessed for their cardiac marker enzyme levels. The heart tissue of animals of all groups was excised and subjected to superoxide dismutase (SOD) and catalase (CAT) measurements, histopathological and staining studies.

Heart rate, ECG and PRI - Needle electrodes were placed and changes in Lead II were recorded 12 h after the second dose of isoproterenol, on an electrocardiograph (Powerlab, AD Instrument, Australia). Heart rate and electrocardiograph (ECG) recordings were made in anesthetized animals for 1 min for every 5 min. The type of alterations (ST-segment elevation or depression) in normal and experimental animals was considered. [13] Pressure rate index [14] (PRI), a parameter used as an index of myocardial oxygen demand, was calculated as the product of mean arterial blood pressure (MABP) Χ HR/1000.

Vascular reactivity to catecholamines- After completion of the treatment schedule, rats from each group was anesthetized with urethane (1200 mg/kg). Femoral vein was cannulated with fine polyethylene catheter for administration of the drug. Tracheostomy was performed and blood pressure (BP) was recorded from left common carotid artery using pressure transducer by direct method on Chart data system. Heparinised saline (100 IU/ ml) was filled in the transducer and in the fine polyethylene catheter cannulated to the carotid artery to prevent clotting. After 30 min of stabilization, mean change in BP to NA (1 μg/kg), Adr (1 μg/kg), Ang II (25 ng/kg) and 5-HT (1 μg/kg) were recorded. [15]

Assay of marker enzymes- At the end of vascular reactivity study, blood was collected from retro-orbital plexus from the inner canthus of the eye using capillary tubes. Serum was separated using R-24 research centrifuge (Remi Instruments Ltd, Mumbai, INDIA) at 4000 rpm for 5 min. The activities of lactate dehydrogenase-LDH [16] and creatine kinase-CK [17] were assayed in serum using commercial kits purchased from Aspen Labs, (Baddi) India and measured spectrophotometrically (Shimadzu).

Antioxidant enzymes- The heart was dissected out, immediately washed in ice-cold saline and weighed. Ten percent homogenate was prepared in 0.1 M Tris-buffer, pH 7.4. The homogenate was centrifuged at 10,000 Χ g for 20 min. The supernatants were used for measuring activity of enzymes -superoxide dismutase (SOD) [18] and catalase (CAT). [19]

Determination of myocardial necrosis by direct staining- Myocardium of rat was frozen immediately after removal. When the tissue was firm, the heart was sliced into 3 - 5 mm thick slice from the apex toward the atrioventricular groove and incubated in 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffer saline with pH 7.4 at 37 o C for 20 min. The sections were examined under light and photographs were taken. [20]

Histopathological examination- The hearts were excised and immediately fixed in 10% buffered formalin. The ventricular mass was sectioned from the apex to the base of the heart, which was embedded in paraffin after being dehydrated in alcohol and subsequently cleared with xylene. Five-micrometer thick serial histological sections were obtained from the paraffin blocks and stained with hematoxylin and eosin. The sections were examined under light microscope and photomicrographs were taken. [21]

Statistical analysis- Data are presented as mean ± SEM. The data were analyzed by one-way ANOVA followed by Dunnett's test.

 Results



Phytochemical investigations of PJ and B-PJ- The total flavonoid content of PJ-extract and its n-B-PJ extract was found to be 455.7 ± 6.33 μg and 222.7 ± 2.88 μg rutin equivalent/mg of extract, respectively. The total phenolic content of PJ- extract and its n-B-PJ extract was found to be 94.15 ± 9.92 μg and 56.56 ± 3.58 μg gallic acid equivalent/mg of extract, respectively. The phytoconstituents present in the PJ extract were flavonoids and tannins whereas B-PJ extract showed only flavonoid positive. The Rf values for B-PJ using BAW, forestal and formic acid were 0.86, 0.71 and 0.40, respectively, which indicates the presence of anthocynidins.

Effect of PJ extract on heart rate, pressure rate index and ST elevation in ECG- The heart rate, PRI and ECG in vehicle-treated animals was recorded as 282.5 ± 8.54 beats/ min, 25.65 ± 1.97 mm Hg/min and 0.029 ± 0.001 mV, respectively. ISO-treated rats showed a significant (P P P P P [22] The toxic effects of catecholamines could be accounted due to the oxidation of hydroxyl groups in catecholamines leading to the conversion into quinones and the subsequent formation of adrenochromes which cause cell necrosis and contractile failure in the rat's heart. Highly toxic oxygen-derived free radicals are produced during this phase which is detrimental to extra- and intracellular enzymes and proteins. [23] ECG-abnormalities is an important tool for the accurate diagnosis of MI. [24] It is reported that ST elevation correlates well with the leak of CK from the myocardium and the degree of damage observed histologically. [25] Pressure rate index was calculated as an index of oxygen demand in all groups. Our results are in congruence with the previous study as reported by Kela et al and Satish. et al.[26],[27] Vascular reactivity to NA, Adr, AngII and 5-HT was measured by invasive blood pressure technique in all groups of rats, as described earlier by Balaraman et al. Endogenous enzymes such as catalase and superoxide dismustase are the first line cellular defense free radical scavenging enzymes against oxidative injury. [28] Increased activity of SOD and CAT indicates increased removal of superoxide radicals thereby reducing myocardial damage caused by free radicals.

It has been reported that, TTC forms a red formazan precipitate with LDH of the viable myocardial tissue in the presence of mitochondrial dehydrogenase enzyme system, whereas areas of necrosis lack mitochondrial dehydrogenase activity and do not stain. Consequently, areas not stained with TTC correspond to areas of total necrosis. [29]

On histopathological examination, ISO-treated group, demonstrated thrombus formation, contraction band necrosis and inflammation. Pretreament with PJ extract and B-PJ reversed these changes. It is concluded that PJ extract and its butanolic fraction has a potential to inhibit the cardiotoxic effects induced by ISO and possesses a significant therapeutic value in the prophylactic treatment of MI.

 Acknowledgments



The authors acknowledge the technical assistance provided by Mr. Pradeep for histopathology study and Dr. Meena Kulkarni, HOD, Dept of histopathology, KBH Dental College, Nashik.

References

1Das DK, Maulik N. Protection against free radical injury in the heart and cardiac performance. In: Sen CK, Packer L, Hannineren O, editors. Exercise and Oxygen Toxicity. Amsterdam: Elsevier Science; 1995. p. 359-88.
2Yates JC, Dhalla NS. Induction of necrosis and failure in the isolated perfused rat heart with oxidized Isoproterenol. J Mol Cell Cardiol 1975;7:807-16.
3Knufman NM, van der Laarse A, Vliegen HW, Brinkman CJ. Quantification of myocardial necrosis and cardiac hypertrophy in Isoproterenol-treated rats. Res Commun Chem Pathol Pharmacol 1987;57:15-32.
4Basu A, Penugonda K. Pomegranate juice: A heart-healthy fruit juice. Nutr Rev 2009;67:49-56.
5de Nigris F, Balestrieri ML, Williams-Ignarro S, D'Armiento FP, Fiorito C, Ignarro LJ, et al. The influence of Pomegranate fruit extract in comparison to regular pomegranate juice and seed oil on nitric oxide and arterial function in obese Zucker rats. Nitric Oxide 2007;17:50-4.
6Sudheesh S, Presannakumar G, Vijayakumar S, Vijayalakshmi NR. Hypolipidemic effect of flavonoids from Solanum melongena. Plant Foods Hum Nutr 1997;51:321-30.
7Ben Nasr C, Ayed N, Metche M. Quantitative determination of the polyphenolic content of pomegranate peel. Z Lebensm Unters Forsch 1996;203:374-8.
8Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavanoids and risk of coronary heart disease. The Zupthen elderly study. Lancet 1993;342:1007-11.
9Harborne JB. A guide of modern technique of plant analysis. 3 rd ed. 2007. p. 75-6.
10Woisky RG, Salatino A. Analysis of propolis: some parameters and procedures for chemical quality control. J Agric Res 1998;37:99.
11Slinkard K, Singleton VL. Total phenol analyses: Automation and comparison with manual methods. Am J Enol Viticult 1977;28:49-55.
12Kokate CK. Practical Pharmacognosy. In: Kokate C.K, Editor. Pharmacognosy, 3rd ed. Vallabh Prakashan; New Delhi, 1994. p. 107
13Maroko PR, Libby P, Sobel BE, Bloor CM, Sybers HD, Shell WE, et al. Effect of glucose-insulin-potassium infusion on myocardial infarction following experimental coronary artery occlusion. Circulation 1972;45:1160-75.
14Nossuli TO, Hayward R, Scalia R, Lefer AM. Peoxynitrite reduces myocardial infarct size and preserves coronary endothelium after ischaemia and reperfusion in cats. Circulation 1997;96:2317-24.
15Balaraman R, Hingorani N, Rathod SP. Studies on the antihypertensive effect of abana in rats. Indian J Pharmacol 1993;25:209-14.
16Henry JB. Clinical Diagnosis and management by Laboratory Methods. Philadelphia, PA: Saunders WB and Company; 1979. p. 365.
17Wagner GS, Roe CR, Limbird LE, Rosati RA, Wallace AG. The importance of identification of the myocardial-specific isoenzyme of creatine phosphokinase (MB Form) in the diagnosis of acute myocardial infarction. Circulation 1973;47:263-9.
18Saggu H, Cooksey J, Dexter D, Wells FR, Lees A, Jenner P, et al. A selective increase in particulate Superoxide dismutase activity in Parkinsonin subtansia nigra. J Neurochem 1989;53:692-7.
19Beers RF Jr, Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952;15:133-40.
20Khalil PN, Siebeck M, Huss R, Pollhammer M, Khalil MN, Neuhof C, et al. Histochemical assessment of myocardial infarction using 2,3,5,-triphenyltetrazolium chloride in blood-perfused porcine hearts. J Pharmacol Toxicol Methods 2006;54:307-12.
21Zhou R, Xu Q, Zheng P, Yan L, Zheng J, Dai G. Cardioprotective effect of fluvastatin on isoproterenol-induced myocardial infarction in rat. Eur J Pharmacol 2008;586:244-50.
22Wheatley AM, Thandroyen FT, Opie LH. Catecholamine induced myocardial cell damage: catecholamines or adrenochrome. J Mol Cell Cardiol 1985;17:359-79.
23Thompson JA, Hess ML. The oxygen free radical system: a fundamental mechanism in the production of myocardial necrosis. Prog Cardiovasc Dis 1986;28:449-62.
24Miller DD. Acute Myocardial infarction. In: Miller DD, Editor. Acute Myocardial Infarction, New York; CLS Inc Press; 1991. p.45-76.
25Maroko PR, Libby P, Sobel BE, Bloor CM, Sybers HD, Shell WE, et al. Effect of glucose-insulin-potassium infusion on myocardial infarction following experimental coronary artery occlusion. Circulation 1972;45:1160-75.
26Kela AK, Reddy LP, Thombre DP. ECG findings in normal rats and after administration of isoproterenol. Indian J Physiol Pharmacol 1980;24:84-90.
27Sathish V, Vimal V, Ebenezar KK, Devaki T. Synergistic effect of nicorandil and amlodipine on mitochondril functions during isoproterenol-induced myocardial infraction in rats. J Pharm Pharmacol 2002;54:133-7.
28Sharma M, Kishore K, Gupta SK, Joshi S, Arya DS. Cardioprotective potential of ocimum sanctum in isoproterenol induced myocardial infarction in rats. Mol Cell Biochem 2001;225:75-83.
29Lie JT, Pairolero PC, Holley KE, Titus JL. Marcroscopic enzyme mapping verification of large, homogenous, experimental myocardial infarcts of predictable size and location in dogs. J Thorac Cardiovasc Surg 1975;69:599-605.