Cardioprotective Interaction of Allium Sativum with Enalapril in Isoproterenol Induced

“CARDIOPROTECTIVE INTERACTION OF ALLIUM SATIVUM WITH ENALAPRIL IN ISOPROTERENOL INDUCED MYOCARDIAL DAMAGE IN RATS”

a)BRIEF RESUME OF THE INTENDED WORK:

Need of study:

The use of complementary and alternative medicines is burgeoning globally, especially in developed countries including US1. It is interesting to note that herbs are often administered in combination with therapeutic drugs, raising the potential of herb-drug interactions2. Certain herbal supplements can cause potentially dangerous side effects when taken with prescription drugs and the number of cases reported for the emerging herb-drug interactions are already on the rise3. Hence it is widely accepted that in-depth and appropriate studies on drug-herb interactions should be carried out to confirm the efficacy of combined drug-herb treatments.

Myocardial infarction is the commonest single cause of death in many parts of the world. Its therapy includes administration of thrombolytic agents, anticoagulants, -blockers etc., or surgical angioplasty or coronary bypass surgery4. It has been shown that cardiac renin angiotensin system (RAS) rather than the circulatory system may play an important role in myocardial infarction. Recent experimental and limited clinical trials strongly indicate a role for ACE inhibitors in limiting myocardial ischemia-reperfusion induced injury 5-8. Coronary vasodilation due to interference with kininase II or complex changes in systemic haemodynamics that reduce oxygen demand or inhibition of cardiac angiotensin II formation may account for cardioprotection5. In addition, altered prostaglandin production or oxygen free radical scavenging properties of ACE inhibitors have been postulated to reduce myocardial infarction5. One of the most commonly used ACE inhibitor is enalapril. Enalapril is used in the treatment of hypertensionand some types of chronic heart failure. Enalapril was the first member of the group of ACE inhibitors known as the dicarboxylate-containing ACE inhibitors. Enalapril is a prodrug that is converted by deesterification to converting enzyme inhibitor, enalaprilat, with effects similar to those of captopril. Enalaprilat itself is available only for intravenous use, primarily for hypertensive emergencies. The consensus study of enalapril therapy in heart failure demonstrated that an ACE inhibitor could prolong survival in patients with advanced heart failure9. Further, reduction of reperfusion arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme inhibitors was also demonstrated10.

In traditional medicine, garlic (Allium sativum) and its preparations have been widely recognized as agents for the prevention and treatment of cardiovascular and other metabolic diseases, such as atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes11. Previous studies have demonstrated that garlic has a significant anti-arrhythmic effect in both ventricular and supraventricular arrhythmias12. These beneficial effects have been proposed to be due to its alteration on cardiac electrophysiology including effective refractory period (ERP) prolongation, Ca2+ influx suppression, as well as its free radical scavenging activity12.

Garlic enjoys the great benefit of potentiating concurrently administered conventional cardioprotectives in ischemia reperfusion induced myocardial damage13. However, there is no reported interaction of garlic with enalapril. Hence the present study is designed to evaluate the pharmacodynamic interaction of garlic with enalapril in isoproterenol induced acute and chronic myocardial damage in rats.

Review of literature

It is also demonstrated by epidemiologic studies that there is an inverse correlation between garlic consumption and progression of cardiovascular disease14. Garlic juice mimics beta-blocking property by inhibiting norepinephrine-induced contractions of rabbit and guinea pig aortic rings15. Earlier reports on the drug interaction studies of garlic with calcium channel blocker indicate that it produces concentration dependent synergistic effect by its calcium blocking property16. Recently we reported improved cardiac recovery from isoproterenol-induced ischemia when garlic was combined with captopril.

Biochemical, pharmacological and molecular biological data provide evidence for the presence of a cardiac RAS17. The potential usefulness of ACE inhibitors in myocardial ischemia may be explained by intracardiac suppression of angiotensin II generation and bradykinin degradation17. Renal RAS is salt sensitive. Renal renin secretion is directly proportional to sodium excretion through kidneys. In healthy Na+replete animals and humans, a single dose of ACE inhibitor has little or no effect on supine or erect systemic blood pressure whereas the same lowers blood pressure substantially in normal subjects when they have been depleted of Na+18.

In the early 1980s, angiotensin converting enzyme inhibitors were introduced into the clinical arena. Since then, this class of drug has emerged as one of the most important cardiovascular therapeutic agents in modem medicine. At about the same cellular and molecular biologic techniques were introduced to cardiovascular research. Molecular research has enabled physiologists and biochemists to re-examine, in greater depth, many of the basic concepts of cardiovascular regulation. One of the outcomes of molecular research is an improved understanding of the biology of the renin angiotensin system19. It is now appreciated that angiotensin is not only synthesized in the circulation but also locally in many tissues. The concept of an autocrine-paracrine mechanism of renin angiotensin action has evolved from the traditional endocrine concept20. Inhibitors of renin angiotensin system, such as the angiotensin converting enzyme (ACE) inhibitor may exert much of their pharmacological effects via the blockade of local angiotensin production21. This mechanism may explain the general efficacy of this class of drug in the treatment of several major cardiovascular disorders, irrespective of the plasma renin level of the patients. Clinical and experimental data to date suggest that ACE inhibition might reduce coronary heart disease risk in hypertensive subjects, improve survival in patients with heart failure, and may influence coronary ischaemia and reperfusion injury.

Objective of study

The objective of the present research is to carry out cardioprotective interaction of Allium sativum with enalapril in isoproterenol induced myocardial damage in rats.

SPECIFIC OBJECTIVES:

To collect and authenticate the cloves of garlic.
To prepare and carry out phytochemical evaluation of raw garlic homogenate.
To standardized acute and chronic models of isoproterenol for induction of myocardial damage.
To select the dose of enalapril.
To study the biochemical and antioxidant profile in serum and heart tissue homogenate upon chronic administration of garlic homogenate with or without enalapril.
To explore the role of enalapril in presence/absence of garlic during isoproterenol induced acute myocardial derangement in rats.
To study the benefits of adding garlic to cardioprotective therapy of enalapril in isoproterenol mediated chronic myocardial damage in rats.

b)MATERIALS AND METHODS:

Source of Data:

Data will be obtained from laboratory based studies by using Sprague dawley rats of either sex weighing between 150-200 gms maintained at room temperature having free access to food (std pellet diet), tap water adlibitum. These studies will be carried out in intact animal that will be supported by biochemical data and histopathological studies.

Method of Collection of Data:

Chemicals and reagents will be procured from standard companies. Isoproterenol induced myocardial damage will be used as model to evaluate cardioprotective efficacy. Suitable biochemical and histological investigation will be carried out in animal model. The data collected will be based on animal experimentation as per the parameters studied under each animal model.

Preparation of garlic homogenate

Garlic (Allium sativum, family: Lilliaceae) bulbs will be purchased from the local vegetable market. The cloves will be peeled, sliced and ground into a paste and suspended in distilled water. Two different concentrations of the garlic homogenate (GH) were prepared, 0.1 and 0.2 gm/ml, corresponding to 250 mg and 500 mg/kg body weight of animal22. GH will be administered within 30 min of preparation.

Experimental protocol

The cardioprotective role of combined therapy of enalapril with garlic will be determined in isoproterenol induced ischemia-reperfusion rats. The Sprague dawley rats of either sex will be divided into following groups consisting of six animals each:

Group-I- animals kept as control without pretreatment
Group II- ISO control
Group-III- Garlic (30 days oral treatment GH 250 mg/kg)
Group-IV- Garlic (30 days oral treatment GH 500 mg/kg)
Group-V- Enalapril (10 mg/kg, orally for ten days)23
Group-VI- Garlic (250 mg/kg 30 days oral treatment) + Enalapril (10 mg/kg, orally for ten days)
Group-VII- Garlic (500 mg/kg 30 days oral treatment)+ Enalapril (10 mg/kg, orally for ten days)

Isoproterenol induced chronic myocardial damage in rats

During 30 days of GH, ten days of enalapril and incorporation of enalapril in the last ten days of 30 days of GH treatment in their respective groups, animals in all groups except group I will receive isoproterenol 3 mg/kg/day subcutaneously24. During treatment, haemodynamic parameters such as body weight, physiological changes and electrocardiographic observations will be noted. At the end of treatment, blood samples will be collected and serum will be separated. Heart tissue homogenate will be prepared in 0.25 M sucrose (10%) and both serum and heart tissue homogenate will be studied for CK-MB, LDH, SOD, Catalase, TBARS etc. Microscopic slides will be prepared to study histopathological and electron microscopic changes.

Isoproterenol (ISO) induced acute myocardial necrosis in rats25

At the end of treatment of animals as mentioned in experimental protocol, blood samples will be collected and serum will be separated. Influence of chronic therapy of garlic, in presence and absence of enalapril will be determined by estimating LDH, CK-MB and TBARS in serum. Subsequently, ISO (150 mg/kg, s.c) will be administered for two consecutive days. Symptoms and mortality in each group will be recorded and compared with those of the rats given ISO alone. After anesthetizing the rat with a combination of ketamine hydrochloride (75mg/kg, i.p) and xylazine (8.0mg/kg, i.p), leads will be attached to the dermal layer of both the front paws and hind legs and recording will be made on polygraph with the help of electrodes ECG system (subcutaneous lead II method). Blood samples will be collected and separated serum will be evaluated again for estimating LDH, CK-MB and TBARS. Forty-eight hours after the first ISO administration, the rats will be sacrificed and autopsied. Three excised hearts will be homogenized to prepare heart tissue homogenate (HTH) using sucrose (0.25 M). The endogenous biological markers such as LDH, CK-MB and antioxidants (Superoxide dismutase and catalase) will be determined in heart tissue homogenate. Microscopic slides of remaining three hearts will be prepared for evaluating histopathological and electron microscopic changes.

Statistical analysis

The statistical significance will be assessed using one-way analysis of variance (ANOVA) followed by Dunnet comparison test. The values will be expressed as mean ± SEM and p < 0.05 will be considered significant.

Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

YES

Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animals.

Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed.

c)List of References

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