Pharmacodynamic interaction of allium sativum with captopril and metformin in rats with diabetes and myocardial dysfunction



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PHARMACODYNAMIC INTERACTION OF ALLIUM SATIVUM WITH CAPTOPRIL AND METFORMIN IN RATS WITH DIABETES AND MYOCARDIAL DYSFUNCTION”



  1. BRIEF RESUME OF THE INTENDED WORK


Need of study:

Cardiovascular disease is one of the leading causes of death in the western world and Diabetes mellitus, which alters the vascular responsiveness to several vasoconstrictors and vasodilators, is a major factor underlying its development1. Metformin belongs to bigunide insulin-sensitizing class of anti-diabetic drugs, widely used for the treatment of type 2 diabetes. In the UKPDS (United Kingdom Prospective Diabetes Study), overweight and obese patients randomized to initial monotherapy with metformin experienced significant reduction in myocardial infarction and diabetes-related deaths2,3. However, till now there is no report exploring the role of metformin in preventing the myocardial damage in type two diabetic animals.

Angiotensin-converting enzyme (ACE) inhibitors are currently used as standard medical therapy in the management of congestive heart failure, regardless of etiology. In addition to their afterload-decreasing effects secondary to decreased synthesis of the potent vasoconstrictor angiotensin II from angiotensin I, these drugs appear to be cardioprotective at a cellular level, as demonstrated by their ability to decrease myocardial injury both in vitro and in vivo4,5. It is known that captopril (CAP) can ameliorate the deleterious effects of elevated renin and angiotensin II levels in patients with acute myocardial infarction. The results of several important clinical trials have shown that ACE inhibitors significantly reduce cardiovascular morbidity and mortality by attenuation of the left ventricular enlargement and heart failure, and also by reductions in the occurrence of acute coronary artery disease-related events6. Although, earlier studies in our lab demonstrated remarkable protective ability of captopril during hyperlipidemia7 and ischemic damage to myocardium8, but there is no experimental demonstration of cardioprotective potential of captopril in cardiovascular complications of diabetic injury.

During the recent past, a dramatic rise in the use of herbs and herbal remedies has been witnessed in many parts of the world9. While such products had been used with apparent safety in traditional societies for many centuries, when they are being combined with pharmacological agents, posses the possibility of potential interaction between the two groups of substances. Reports indicate that about 15–20% of individuals on prescription medications also use herbal supplements and less than 40% of patients disclose to their physicians the usage of herbal remedies, even if they experience severe side effects—because of the fear of censure or rebuke10. The problem is further compounded by the fact that many physicians are themselves not always familiar with the potential for herb–drug interactions11. Hence it is imperative to promote credible research on the safety and efficacy of combined herb-drug treatment for variety of ailments including cardiovascular diseases. Garlic enjoys the great benefit of enhanced efficacy at times of stress. It is used in folklore for the treatment of variety of conditions including cardiovascular diseases and metabolic disorders. There is established evidence that supports the inverse relationship between consumption of garlic and progression of cardiovascular complications12,13. Recently we reported enhanced cardioprotective potential of combined therapy of garlic with captopril during isoproterenol induced myocardial damage in rat heart14. However, there is no report that explains the benefits of this combined regimen in presence of metformin in animals with diabetes. Therefore present study is designed to determine the role of adding garlic to antidiabetic therapy of metformin in alloxan induced diabetic rats and cardioprotective treatment of captopril in ischemia-reperfusion injury as cardiac complication in diabetic rats.


Review of Literature:

Metformin (1,1-dimethylbiguanide) is an orally administered drug used to lower blood glucose concentrations in patients with type 2 diabetes mellitus15. Metformin has various beneficial metabolic effects15, including antihyperglycemic actions by suppressing hepatic glucose output and increasing insulin-mediated glucose disposal, without weight gain. It also improves the lipid profile by reducing hyper-triglyceridemia, lowering plasma fatty acids and LDL-cholesterol, and raising HDL-cholesterol in some patients. Moreover, metformin improves insulin sensitivity by decreasing endogenous and exogenous insulin requirements and reducing basal plasma insulin concentrations. The exact mechanisms of action of metformin, is poorly understood16, but it includes suppression of endogenous glucose output by liver and increased sensitivity in skeletal muscle17. Metformin lowers blood pressure in certain human patients18 but not in others19. Metformin lowers blood pressure in fructose fed rats20, OLETF rats21, SHR22 and insulin-resistant rats23. Recent data suggests that AMP-activated protein kinase (AMPK) is the intracellular target of metformin action. AMPK acts as an intracellular energy sensor that maintains energy balance within cells. Among currently available oral hypoglycemic agents, metformin is unique in decreasing macro-vascular diseases24. It is intriguing to see if metformin has anti-atherosclerotic effect beyond glucose lowering.

Garlic (Allium Sativum) is used traditionally as a complementary therapy in the treatment of several diseases such as diabetes, several forms of cancer and neurodegenerative conditions such as ischemic stroke25,26. In addition, garlic has been reported to possess a range of cardiovascular effects such as lowering of plasma cholesterol27; inhibition of platelet aggregation as well as lowering of arterial blood pressure28.

Captopril is an orally effective angiotensin I converting enzyme inhibitor and is used in the treatment of hypertension and congestive heart failure. Captopril has a relatively short elimination half-life in plasma with estimates in man ranging from 1.6 to 1.9 h29. Food may decrease oral absorption of captopril by up to 25–40%30.



Objective of study:

The objective of the present research is to carryout pharmacodynamic interaction of Allium sativum with captopril and metformin in rats with diabetes and myocardial dysfunction.


SPECIFIC OBJECTIVES

  • To standardize the alloxan dose for induction of diabetes.

  • To collect and authenticate the cloves of garlic.

  • To prepare raw garlic homogenate (GH) and determine their phytochemical constituents.

  • To arrive at the therapeutic doses for captopril and metformin after acute toxicity studies following WHO guidelines.

  • To standardize modified Langendorff preparation for studying ischemia-reperfusion injury.

  • To explore the role of captopril and metformin in presence/absence of garlic during ischemia reperfusion injury induced myocardial derangement in diabetic and non-diabetic rats.




  1. 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 ad libitum. These studies will be carried out in intact animal as well as on isolated tissues using perfused heart that will be supported by biochemical data and histopathological studies.


Method of Collection of Data:

Chemicals and reagents will be procured from standard companies. Following models will be used for interactive studies:



  • Induction of diabetes by alloxan treatment.

  • Ischemia-reperfusion induced (IRI) myocardial damage in isolated rat heart

Suitable biochemical and histological investigation will be carried out in each animal model. The data collected will be based on animal experimentation as per the parameters studied under each animal model.
EXPERIMENTAL MODELS

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 animal13. GH will be administered within 30 min of preparation. Among the two doses of garlic, best hypoglycemic dose will be selected for the study.


Oral Glucose tolerance test (OGTT)

The oral glucose tolerance test will be performed on overnight fasted Sprague dawley rats. This test will be performed in diabetic group of rats which is specified in treatment protocol.


Alloxan induced diabetes in adult rats

Serum glucose level will be checked after 72 hrs of alloxan injection. Animals with serum glucose levels >/300 mg/100 ml will be considered diabetic. Animals with established hyperglycemia will be chosen for the treatment.


Experimental protocol

The cardioprotective role of combined therapy of metformin and captopril with garlic will be determined in diabetic and non diabetic rats. The Sprague dawley rats of either sex will be divided into two sets (Normal and Diabetic) comprising the following groups consisting of six animals each :




  • Group-I- animals kept as control without pretreatment

  • Group-II- Garlic (30 days oral treatment)

  • Group-III- Metformin (10 days, p.o.).

  • Group-IV- Captopril (10 days, p.o.).

  • Group-V- Garlic (30 days oral treatment) + Metformin(10 days, p.o.).

  • Group-VI- Garlic (30 days oral treatment) + Captopril(10 days, p.o.).

  • Group-VII-Metformin (10 days, p.o.) + Captopril (10 days, p.o.).

  • Group-VIII-Metformin (10 days, p.o.) + Captopril (10 days, p.o.) + Garlic (30 days oral treatment)


Ischemia-reperfusion induced (IRI) myocardial dysfunction21

At the end of treatment as specified above, heart will be excised from deeply anesthetized rat (35 mg/kg sodium pentobarbitone, i.p) and perfused with Kreb-Henseleit solution gassed with carbogen at 37o C at a constant flow rate of 5 ml/min by one-way circulation using modified Langendorff’s preparation. Measurement of contractile force will be done with displacement transducer and recorded on a grass electromechanical recorder. After a short period of equilibrium (15 minutes), records will be taken for a control period of 15 minutes, followed by 15 minutes global ischemia and reperfusion. Recovery in terms of inotropic and chronotropic effect will be studied and the extent of cardioprotection due to prophylactic therapies will be evaluated by measuring the developed tension. Perfusate will be collected both during pre and post ischemic periods and will be subjected for biochemical estimations of LDH and CPK. 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. Biochemical findings will be supported by histopathological and electron microscopic studies.


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 animal model.
Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed.




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  2. Krentz AJ, Bailey CJ. Oral antidiabetic agents: current role in type 2 diabetes mellitus. Drugs 2005;65:385–411.

  3. UK Prospective Diabetes Study. Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). Lancet 1998;352:854–865.

  4. Westlin W, Mullane K. Does captopril attenuate reperfusion-induced myocardial dysfunction by scavenging free radicals?. Circulation 1988;77:30–39.

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  11. Fugh-Berman A. Herb-drug interaction. Lancet 2000;355:134-138.

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  13. Asdaq SM, Inamdar MN. The potential for interaction of hydrochlorothiazide with garlic in rats. Chem-Biol Interac 2009;181:472–479.

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