Membrane stabilization effect and histological changes in the heart in experimental myocardial rats with Zanthoxulym armatum fruit

a Department of Biochemistry, PSG College of Arts & Science, Coimbatore641014, Tamilnadu, India. b Department and Graduate Institute of Applied Chemistry, Chaoyang University of Technology, No.16, Jifeng East Road, Wufeng District, Taichung City – 41349, Taiwan (R.O.C). c Raman Research Laboratory, PG & Research Department of Physics, Government Arts College, Tiruvannamalai-606603, Tamilnadu, India d Department of Chemistry, Sri Vijay Vidyalaya College of Arts and Science (Women’s college), Dharmapuri, Periyar University, Tamilnadu, India. e Department of Chemistry, Sri Ramakrishna College of Engineering, Perambalur, Tamil Nadu 621113, India


Introduction
Myocardial damage is thought to be an essential trigger for the pathological process of coronary illness.

Determination of membrane related enzyme like ATPases
show the changes in the membrane under obsessive conditions. ATPases of cardiovascular cells play a vital role in the maintenance of normal ion levels with on the heart and are involved in the contraction and relaxation of cardiac muscle [1]. Na + /K + ATPase catalyze the dynamic uptake of K + and expulsion of Na + to the detriment of ATP [2]. Maintenance of these ionic gradients is essential for all cells; a low intracellular Na + : K + ratio is imperative for enzyme activation in metabolic pathways, maintenance of cell volume, intracellular pH regulation and both secretory and absorptive function [3].
Ca 2+ is a basic component in ordinary vascular tissue physiology. It regulates blood pressure, ion fluxes and the binding of a receptor to the membrane [4].
To examine the possible protective effect of drugs on the myocardial damage from MI, a generally used model is the induction of infarction by the administration of ISO in rats. ISO, a β-adrenergic agonist is a notable inducer of myocardial necrosis and interstitial fibrosis [9].
Mohanty et al, [10] have demonstrated that ISO prompts myocardial necrosis characterized ny expanded enddiastolic volume, end-diastolic pressure and left ventricular wall thickness. Oxidative stress has been embroiled in the pathogenesis of myocardial ischemia [11]. It discourages the sarcolemmal Ca 2+ transport and results in the development of intracellular Ca 2+ overload and ventricular dysfunction [12]. Ca 2+ overcharge inside the cell is identified with the enactment of the adenylate cyclase and exhaustion of adenosine triphosphate (ATP) levels [13].
Our previous studies have shown that Z. armatum fruit is a potent blocker of lipid peroxide formation and scavenger of superoxide anions and hydroxyl radicals.
Apart from antioxidant activities, the Z. armatum fruit has cardiac glycosides, phenols, flavonoids and alkaloids which may substantially aid to its cardioprotective action. Based on these onservations, the present investication was aimed to evaluate the membrane-stabilizing action of Z. armatum fruit by analyzing the levels of transmembrane protein activity and electrolytes in myocardial tissue. This study could be helpful in the mechanism based therapeutic effect of Z. armatum fruit on heart injury by ischemia.

Collection of plant material and preparation of extract
The plant Z. armatum fruit was collected from Then it was filtered, dired in a rotary vacuum evaporator and used for analysis.

Induction of MI
ISO was used to induce MI in rats. Animals were injected subcutaneously with freshly prepared ISO in sterile normal saline at a dose of 8.5mg/100g body weight.

Experimental design
Animals were divided into six groups of six rats in each group and the grouping of animal is shown in table-1.
At the end of the experimental period i.e, 12hr after the second dose of ISO injection, all the rats were sacrificed by cervical dislocation under mild chloroform anesthesis. The heart tissue was excised immediately and thoroughly washed with ice-cold physiological saline and it was used for various biochemical estimations. subcutaneously twice at an interval of 24hr on 28 th and 29 th day. GVI-Rats pretreated with standard drug + ISO Standard drug verapamil (1mg/kg body weight for 30 days) + ISO subcutaneously twice at an interval of 24hr on 28 th and 29 th day.

Assay of sodium potassium dependent adenosine triphoaphatase (Na + /K + ATPase)
Na + /K + ATPase transport Na + /K + against concentration gradient at the cost of ATP molecule liberating inorganic phosphate (Pi). Na + /K + ATPase activity was estimated by the method of Bonting [14]. The The amount of Pi liberated was estimated by the method of Fiske and Subbarow [15]. The enzyme activity is expressed as μmoles of Pi liberated/min/mg protein.

Assay of magnesium dependent adenosine triphosphatase (Mg 2+ ATPase)
The activity of Mg 2+ ATPase was assayed by the method of Ohnishi et al [16]. The assay was initiated by the addition of 0.1ml of tissue homogenate to an incubation medium containing 0.1ml of 2mM ATP, 0.1ml of double distilled water respectively in total incubation volume of 0.5ml. the contents were incubated at 37 o C for 15 min. the reaction was arrested by adding 0.5ml of cold 10% TCA. The amount of Pi liberated was estimated by the method of Fiske and Subbarow [15]. The enzyme activity is expressed as μmoles of Pi liberated/min/mg of protein.

Assay of calcium dependent triphosphatase (Ca 2+ ATPase)
The incubation mixture contained 0.1ml of 75mM Tris-Hcl buffer pH 7.6, 0.1ml of 5mM calcium chloride, 0.1ml of 2mM ATP, 0.1ml of double distild H2O and 0.1ml of tissue homogenate [17]. The contents were incubated at 37 o C for 15min. the reaction was the arrested by the addition of 0.5ml of cold 10%TCA. The amount of phosphorous liberated was estimated by the method of Fiske and Subbarow [15]. The enzyme activity is expressed as μmoles of Pi liberated/min/mg/protein.

Estimation of electrolytes in heart tissue
The concentration of Na + and K + ions in heart homogenate was estimated using commercial kits purchased from Lab-Care Diagnostics Private Limited, India. The level of Ca 2+ ion in the heart was measured by the O-cresolphthalein complexone method using a reagent kit purchased from Span Diagnostics Limited, India. The result was expressed as nmol/mg protein.

Histopathological analysis
The heart tissue were fixed in 10% formalin, underwent a dehydration process with isopropanol for 1hr then embedded in molten paraffin wax using the tissue-embedding centre, which later cooled down to formed blocks of paraffin. Each block was trimmed then sectioned about 5μm by using a microtome. For the heart tissue, the specimens were stained with Haematoxylin and Eosin (H&E) dye to observe the elastic fibers which mounted with DPX for microscopic examinations.

Statistical analysis
The results were articulated as mean ± standard deviation. Statistical analysis was carried between the experimental groups using one way analysis of variance (ANOVA) employing statistical package for social science (SPSS Version 16.0). Post hoc testing was performed for inter-group comparisons using Fisher's least significant difference (LSD) tests. The level of significance was set as (p<0.05).

Results and Discussion
The activity of Na + /K + ATPase significantly decreased and the activities of Ca 2+ ATPase and Mg 2+ ATPase significantly increased in the heart tissue of the ISO induced rats (Gropu-III), compared to the normal control rats (Group-I). Oral pretreatment with Z. armatum fruit (200 and 400mg/kg) for a period of 30 days significantly regulated the activities of Na + /K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase in the heart of MI induced rats.
There was no significant difference between standard drug treated rats (Group-VI) and Z. armatum fruit treated rats at a dose of 400mg/kg (Group-V). When comparing the control rats and plant alone treated rats (Group-II) did not show any significant effect and is given in table-2.
ATPases are integral membrane proteins which require thiol groups and phospholipids (PL) to keep up their structure and function [18]. Na + /K + ATPase, a widely considered individual from the P-type ATPase family, is associated with the regulation of cell volume, development of membrane potential and transport of supplements in tissues [19]. Na + /K + ATPase is a lipid dependent enzyme contains (-SH) group. Mg 2+ ATPase plays the main role in Ca 2+ influx and cell bursting [20].
Oxidants are known to initiate lipid peroxidation and accordingly demolish PL which is required for the typical action of membrane bound protein. 7.05 ± 0.04 a,b 6.92 ± 0.12 a,b All the values are mean + SD of six samples in each group. a,b,c-significant at 5% level (p<0.05). Group comparison: a-GI vs GII, GIII, GIV, GV, GVI: b-GIII vs GIV,GV,GVI: c-GVI vs GIV, GV.   In the cell, ATPases are personally connected with the plasma membrane and participate in the energydependent transport of Na + , K + and Ca 2+ translocation [24].
An increase in Na + and Ca 2+ along with the decrease in K + was seen in ISO injected rats. ISO administration triggers lipolysis and generation of ROS which destabilize the myocardial membrane [25]. Modification in ATPase activity results in the alteration of electrolytes concentration. The changes in the levels of Na + and K + in ISO induced rats might be attributable to loss of cellular integrity, inhibition of the Na + /K + ATPase function because of energy depletion and changes in the proportion of intracellular -to-extracellular volume that indicate the severity of degenerative heart infection [26,27].
Pretreatment with hydroethanolic extract of Z. armatum fruit significantly (p<0.05) prevented the ISO induced alteration in Na + , K + and Ca 2+ ion levels in the heart tissue. The transport of Na + and K + amongst intra and extracellular pools and the maintenance of the transmembrane gradients are essential to cell function and integrity. Z. armatum fruit sustained the membrane bound ATPases which are essential to control the electrolyte levels in ISO induced MI rats.  preventing cardiac dysfunction in rats, conceivably due to membrane stabilizing the property by maintain the levels of electrolytes and ATPases and cytoprotective activity by maintain the normal architecture of heart tissue.