G Model JMAU-115; No. of Pages 9 ARTICLE IN PRESS Journal of Microscopy and Ultrastructure xxx (2016) xxx–xxx CORE Metadata, citation and similar papers at core.ac.uk Contents lists available at ScienceDirect Provided by Elsevier - Publisher Connector Journal of Microscopy and Ultrastructure jo urnal homepage: www.elsevier.com/locate/jmau Experimental Study Bio-tea prevents membrane destabilization during Isoproterenol-induced myocardial injury ∗ Reema Orison Lobo , Chandrakala K. Shenoy Department of Biosciences, Mangalore University, Mangalagangothri, Mangalore, Karnataka, 574199, India a r t i c l e i n f o a b s t r a c t Article history: The present study was undertaken to determine the membrane-stabilizing effect of Bio- Received 25 March 2016 tea in the prevention of myocardial injury caused by isoproterenol in rats. The efficiency of Received in revised form 27 August 2016 Bio-tea pretreatment was compared against black tea pretreatment and the positive con- Accepted 17 September 2016 trol (rats with isoproterenol-induced myocardial infarction) and negative control (normal Available online xxx control rats). For this purpose, biochemical analysis of the in vivo antioxidants (super- oxide dismutase, catalase, and reduced glutathione), glycoprotein components (hexose, Keywords: hexosamine, sialic acid, and fucose), lipids (total, ester and free cholesterol, triglycerides, free fatty acids + + free fatty acids, and phospholipids), and transmembrane protein activities (Na /K ATPase, hexose 2+ 2+ Kombucha Ca ATPase, and Mg ATPase) was carried out along with the histological and ultrastruc- Na+/K+ ATPase tural study of the myocardial tissue. Induction of myocardial infarction using isoproterenol phospholipids resulted in a significant decrease in tissue antioxidants and an increase in the levels of superoxide dismutase total, ester and free cholesterol, triglycerides, free fatty acids, and glycoprotein compo- transmembrane proteins nents in plasma and heart. The phospholipid content showed an increase in plasma and a + + simultaneous decrease in the heart tissue, while the Na /K ATPase activity decreased and 2+ 2+ Ca ATPase and Mg ATPase activities increased, resulting in destabilization of the mem- branes. Pretreatment with Bio-tea was able to bring these components to near normal, indicating its reactive-oxygen-species-scavenging, lipid-lowering, membrane-stabilizing and glycoprotein-modulating effects and lending credibility to the regular use of Bio-tea. © 2016 Saudi Society of Microscopes. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction interruption of the blood supply to an area of the heart [2]. The period of ischemia is followed by reperfusion, where Cardiovascular disease is a term used for a heteroge- blood supply returns to the tissue, resulting in inflamma- neous group of disorders that affects the heart and blood tion and oxidative stress. Isoproterenol (ISO) is a synthetic vessels. Myocardial infarction (MI) is one of the main causes catecholamine and ␤-adrenergic agonist, which causes of cardiovascular death, which is estimated to reach 23.3 severe stress in the myocardium due to the generation of million by 2030 [1]. MI results from prolonged myocardial free radicals, and stimulates lipid peroxidation, which is a ischemia, which causes necrosis of the myocytes due to causative factor of irreversible damage to the myocardial membrane [3]. The study of the cardioprotective effects of natural prod- ucts has shown an upsurge in recent years as they have ∗ Corresponding author. Department of Biosciences, Mangalore fewer side effects than synthetic drugs. Kombucha, known University, Mangalagangothri, Mangalore, Karnataka, India, 574199. as Bio-tea in Karnataka, India, is one such natural prod- Tel.:+91 988 621 4118. E-mail address: [email protected] (R.O. Lobo). uct, produced by the fermentation of sugared black tea http://dx.doi.org/10.1016/j.jmau.2016.09.001 2213-879X/© 2016 Saudi Society of Microscopes. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Lobo RO, Shenoy CK. Bio-tea prevents membrane destabilization during Isoproterenol- induced myocardial injury. J Microsc Ultrastruct (2016), http://dx.doi.org/10.1016/j.jmau.2016.09.001 G Model JMAU-115; No. of Pages 9 ARTICLE IN PRESS 2 R.O. Lobo, C.K. Shenoy / Journal of Microscopy and Ultrastructure xxx (2016) xxx–xxx using a Kombucha pellicle, which is a combination of acetic placed on it and incubation was carried out at room tem- acid bacteria and yeast. It is a traditional drink that is said perature under aerobic conditions for 7 days. After 7 days, to regulate gastric, intestinal, and glandular activities, and the Bio-tea obtained was filtered and sterilized before relieve joint rheumatism, gout, and hemorrhoids. It has a refrigeration. positive influence on the cholesterol level, arteriosclerosis, diabetes, and aging problems [4]. Efficacy of Bio-tea against 2.5. Experimental protocol hypoglycemic and hypolipidemic effects in diabetes con- dition, and its hepatoprotective properties have been well The present study was carried out using four groups studied [5,6]. of six rats each in the following treatment groups: Group Although there is a paucity of data regarding the car- 1: normal control rats; Group 2: ISO-induced control rats; dioprotective effect of Bio-tea, a previous study in our Group 3: tea pretreated + ISO-induced rats; and Group laboratory has shown that its consumption protects the 4: Bio-tea pretreated + ISO-induced rats. Tea and Bio-tea heart against myocardial injury, probably by preventing were administered daily to the respective groups orally at membrane destabilization [7]. Thus, the present study was a concentration of 1.71 mL/kg [8] for a period of 30 days. designed to investigate the membrane-stabilizing action MI was induced by subcutaneous administration of ISO of Bio-tea by analyzing the levels of antioxidants, lipids, (85 mg/kg) on Days 29 and 30 at a 24-hour interval [9]. At lipid peroxidation products, glycoprotein components, the end of the study period, the animals were anesthetized transmembrane protein activity, and the histopathological using ketamine (22–24 mg/kg intramuscularly) and blood analysis of the myocardial tissue. collected by heart puncture for biochemical estimations. The animals were sacrificed by cervical dislocation and the 2. Materials and Methods heart excised. 2.1. Experimental animals and diet 2.6. Estimation of lipid peroxidation products and antioxidants in heart All the experiments were carried out as per the guidelines of the Committee for the Purpose of Control The heart thiobarbituric acid reactive substances and Supervision of Experiments on Animals (CPCSEA), (TBARS) were estimated as per the protocol of Fraga et al New Delhi, India after approval by the Institutional Ani- [10] against 1,1’,3,3’-tetra-methoxy propane as standard. mal Ethics Committee (IAEC) of Mangalore University The values were expressed as mmol/100 g wet tissue (MU/AZ/99/2013-14/IAEC, dated 2 April 2013). The study weight. Superoxide dismutase (SOD) in the heart sam- was carried out using 24 male albino Wistar rats (Rattus ples was assayed according to Kakkar et al [11] by using norvegicus) aged 4 months, which were maintained in the phenazine methosulfate and nitroblue tetrazolium along Animal House of the Department of Biosciences, Manga- with reduced nicotinamide adenine dinucleotide. The lore University, Karnataka, India. The rats were housed enzyme activity was expressed as units/mg protein, where, in polypropylene cages, lined with husk, renewed every one unit of enzyme activity was defined as the enzyme con- 24 hours under a 12: 12 hour light: dark cycle at around centration required to inhibit the optical density at 560 nm ◦ 22 C, food and water was supplied ad libitum, and they of chromogen production by 50% in 1 minute. The catalase were fed a standard pellet diet (Pranav Agro Industries enzyme activity was estimated by the method of Sinha [12], Limited, Maharashtra, India). with dichromate–acetic acid reagent and hydrogen per- oxide as standard. The activity of catalase was expressed 2.2. Drugs and chemicals as ␮mol hydrogen peroxide consumed/min/mg protein. The estimation of reduced glutathione (GSH) was carried Isoproterenol hydrochloride, 1,1’,3,3’-tetra methoxy out by the method of Ellman [13] using 5,5’-dithiobis-(2- propane, cholesterol, palmitic acid, galactose, mannose, nitrobenzoic acid) in 1% sodium citrate. The results were galactosamine hydrochloride, N-acetyl neuraminic acid, expressed as mmol/g wet tissue weight of heart. adenosine triphosphate (ATP), and cysteine hydrochloride were purchased from Sigma Chemical Company (St. Louis, 2.7. Extraction of lipids and glycoprotein components MO, USA), while all the other chemicals used were of ana- lytical grade. Lipids were extracted from plasma and tissues by the method of Folch et al [14]. The tissues were rinsed thor- 2.3. Preparation of ISO oughly in ice-cold physiological saline and dried using filter paper. The samples were homogenized in cold ISO was prepared at a dose of 85 mg/kg body weight in chloroform–methanol (2:1 v/v) and the contents were cold saline. extracted after 24 hours. The extraction protocol was repeated until all the lipids were extracted. The filtrates 2.4. Preparation of Bio-tea were combined and washed with 0.7% potassium chloride in a separating funnel. The lower lipid layer was made up Tea was prepared using 10% (w/v) commercial sucrose to a known volume with chloroform and used for various and 7.5 g/L (w/v) tea leaves (Brooke Bond Red Label). The estimations. The defatted samples were suspended in 3 mL ◦ ◦ tea decoction was cooled to 30 C and filtered into clean 2N hydrochloric acid and digested by heating at 90 C for glass bottles.