Protective Effect of Plectranthus amboinicus Leaf Extract Containing Luteolin Flavanoid in Isoproterenol Hydrochloride Induced Myocardial Infarction in Rats - An Assessment on Biochemical and Cellular Changes

 

S. Raghavi1, V.G. Karthigeyan1 and S. Prabhu2*

1Second Year M. Tech, Sri Venkateswara College of Engineering, Pennalur, Irungattukotai, Sriperumbudur- 602117

2Associate Professor, Sri Venkateswara College of Engineering, Pennalur, Irungattukotai, Sriperumbudur- 602117

*Corresponding Author Email: sprabhu@svce.ac.in

 

ABSTRACT:

Almost all ages and in all countries, diseases of cardiovascular system are the major cause for death and disability. Among the cardiovascular diseases, myocardial infarction appears to be the number one killer disease in affulent societies till today. Oxygen free radical is implicated as a mediator of tissue injury in cardiovascular pathology. Recently, attention has been focused on many medicinal plants and plant derived phytochemicals such as the flavonoids, alkaloids, polyphenols and xanthones as potential therapeutic agents in the prevention and management of cardiovascular diseases due to their antioxidant nature. The present study aims to develop experimental MI (Myocardial Infarction) in laboratory animals (rats) to understand the morphology, biochemistry, electrophysiology and mechanical properties of the damaged myocardium and to test the efficacy of plant derived therapeutic agent in treating MI in rats.The main objective of the study is to evaluate and explore the cardio protective effect of luteolin flavanoid in isoproterenol induced experimental myocardial infarcted rats. The flavanoid will be isolated and purified by analytical methods. The optimum dosage of the drug will also be studied. In myocardial infarcted rats, the oxidative damage induced by isoproterenol will be studied. In order to strengthen the antioxidant and the free radical scavenging activity of this flavonoid (luteolin), the antioxidant effect of the drug will be analyzed in myocardial tissue by assaying the biochemical parameters in cytosol, mitochondria, lysosomes and serum of the animals. Then the therapeutic efficacy of luteolin for the possible use as a carditonic drug for cardiovascular diseases will be evaluated. If the result is found good then the prospects of this drug for human beings could be assessed.

 

KEYWORDS: Luteolin flavanoid, Myocardial Infarction, Plectranthus amboinicus

 


INTRODUCTION:

Among the many health predictions for the new millennium, the most alarming is cardiovascular disease (CVD) - heart disease and stroke, topping the list for death and disability (Padmavathi 2002; Manson et al, 1993). It kills victims faster than most malignant neoplasmís (Siltanen, 1990). Among the cardiovascular diseases, coronary heart disease (CHD) appears to be the most prominent one.

 

Demographic shift in population age-increase profile combined with lifestyle related increase in cardiovascular risk factors are accelerating CHD epidemic in India (Reddy and Yusuf, 1998) and the social economic indices of epidemiological transition explain the increasing CHD prevalence (Gupta and Singhal, 1997). In urban population, it is increased from 3.5% in 1960ís to 9.5% in 1990ís. In rural areas, it is increased from 2% in 1970ís to 4% (Gupta and Gupta, 1996). Population based surveys pointed out that there are great regional variations (Padmavathi, 2002) while there are undoubted regional differences between the developed countries and other economies, the predictions for India by 2015 show a steady increase since 1985. The projected rate is higher than that for other causes such as cancer (Padmavathi, 2002). The Framingham study also showed that myocardial infarction (MI), sudden ischemic cardiac deaths or acute coronary insufficiency were the first manifestations of 70% of men and 50% of women (Maseri, 1995).

 

Oxygen free radical is implicated as a mediator of tissue injury in cardiovascular pathology (Kakreja and Hess, 1992). In this current generation, the different environmental factor dominates the life style of human population. Due to variety of physiological reason, free radicals are continuously produced in the body mostly by biochemical redox reactions involving oxygen which occur as part of normal cell metabolism, by phagocytes, as part of an uncontrolled inflammatory reaction, occasionally in response to exposure to ionizing radiation, UV light, environmental pollution, cigarette smoke, hyperoxia, excess exercise and ischaemia. Examples of free radicals are ground state O2, singlet O2, super oxide anion radical, hydroxyl, carbon centered radical, alkoxyl, peroxyl, thiyl, and nitroxyl free radicals. Among these super oxide anion and hydroxyl radical are the common radicals in living systems and have been implicated as cause or contributing factor in a variety of types of cell injury and ischemia (Slater, 1984; Hess et al, 1982).

 

Majid Ali and Omar Ali propose that ischemic heart disease (IHD) is caused by "AA oxidopathy"-a state of accelerated oxidative molecular injury to blood corpuscles and plasma components (Ali and Ali, 1997). There is substantial evidence that ischemic tissue generates oxygen-derived free radicals (oxygen radicals), i.e., oxygen molecules containing an odd number of electrons, making them chemically reactive, and often leading to chain reactions (Hammond and Hess, 1985; Ambrosio et al, 1987). World Health Organization (WHO) has defined primary prevention of coronary heart disease as prevention of its first events, beginning early in childhood and continuing throughout childhood, youth and adult life.

 

Drug therapy is an alternative option for prevention of myocardial infarction (WHO Study Group, 1985). The various indigenious systems such as siddha, ayurveda, unani and sometimes allopathy use several plant species for degenerative diseases (Rabe and staden, 1997). The prophylactic and therapeutic effect of many plant foods and plant derived compounds in reducing cardiovascular disease has been reviewed (Walker, 1996). Like dietary antioxidants some non-nutrient based antioxidants from plants such as sulphur containing compounds in garlic, phyto-estrogens in soy, green tea, anthocyanins in red berries, lycopene in tomatoes, red wine and white wine from grape seeds are increasingly being recognised as potential health promoters in reducing the risk of cardiovascular disease and atherosclerosis.

 

Plectranthus amboinicus:

Plectranthus amboinicus belongs to the family Lamiaceae is a large succulent herb, fleshy and highly aromatic, much branched, possessing short soft erect hairs, with distinctive smelling leaves (Kaliappan et.al., 2008). In Tamil it is called Karpuravalli .The leaves have also had many traditional medicinal uses, especially for the treatment of coughs, sore throats and nasal congestion, but also for a range of other problems such as infections, rheumatism and flatulence. The plant is cultivated in home-gardens throughout India for use in traditional medicine, being used to treat malarial fever, hepatopathy, renal and vesical calculi, cough, chronic asthma, hiccough, bronchitis, helminthiasis, colic, convulsions, and epilepsy. It contains Luteolin as a major phytochemical constituent.

 

Fig.1: Plectranthus amboinicus plant

 

Luteolin:

Luteolin is a widely distributed flavonoid, one of a group of naturally occurring polyphenolic compounds found in many fruits and vegetables (Li et al., 2009 & Lin et al., 1997). It has been reported in the literature that luteolin has a wide range of biological and pharmacological properties, including antineoplastic (Chang et al., 2005, Li et al., 2001 & Yee et al., 2003), antihepatotoxic, antiallergic, antiosteoporotic (DiCarlo et al., 1999) antidiabetic (Zarzalo et al., 1996), anti-inflammatory (Wu et al., 2005), antiplatelet and vasodilatory activity (Lin et al., 1997) as well as antioxidant effects (Chen et al., 1990).

 

 

Molecular Formula: C15H10O6

Fig. 2 Structure of luteolin

 

Patients who have survived previous heart attacks and those would have subjected to the subsequent acute myocardial infarction is readily understood by little experimental research in myocardial infarction studies in animals. So the present study will deal with the efficacy of a Plectranthus amboinicus leaf extract containing Luteolin flavonoid in preventing myocardial infarction in rats as an experimental study using isoproterenol as a free radical inducing cardiac injury to assess on Biochemical and Cellular Changes.

 

METHODOLOGY:

Animals:

The Department animal house is approved by CPCSEA- New Delhi (CPCEA Reg. Number- 1398/a/10/CPCSEA). All experiments will be carried out according to the guidelines of Institutional Animal Ethics Committee (IAEC- SVCE). Animals will be obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai. They will be fed with standard diet and water ad libitum and housed under standard environmental conditions.

 

Table 1: Animals Group and specification:

Group

Specification

Group 1 :

Normal control rats

Group 2:

 

Rats will be induced with standardized dose of Isoproternol hydrochloride

Group 3:

 

Rats will be treated with standardized dose of the flavanoid

Group 4:

 

Rats will be treated with both the drug luteolin and inducing agent Isoproternol hydrochloride

 

Isoproternol Hydrochloride:

Isoproterenol hydrochloride will be received from Sigma Aldrich company. Isoproterenol induced myocardial infarction is well a standardized model to study the cardio protective effect of many drugs and cardiac function. Eventhough, an accurate method of standardization of dosage of isoproterenol administration will be carried out by analysis of serum and cardiac tissue marker enzymes and heart weight to body weight ratio analysis. Similarly average survival of rats will be noted under different doses of isoproterenol hydrochloride.

 

Isolation of luteolin from Plectranthus amboinicus:

The leaves of P. amboinicus (Lour) Spreng were collected from the fields of Kanchipuram, Tamil Nadu. Then the shade-dried leaves were powered to get a coarse powder. The powdered material was successively extracted with petroleum ether, chloroform, ethanol and water by cold maceration in increasing order of their polarities.In addition, the fresh powder was defatted with petroleum ether and extracted with 95% ethanol (72 h) and water (24 h) separately. The extracts were filtered with muslin cloth and the solvent was distilled off. Final traces of solvent were removed under vacuum. Luteolin flavanoid will be isolated from Plectranthus amboinicus leaf using standard analytical instruments such as Mass Spectrometers and the purity of the compound will be checked by C- 18 column using HPLC. After confirmation of the existence of active compounds, the toxicity and optimum dosage of the drug will be studied to reveal the plantís cardioprotective activity.

 

Biochemical changes:

Histopathological and electron microscopic studies will be carried out to find out the degenerative changes of the heart on isoproterenol induction and to assess the prophylactic efficacy of luteolin pretreatment in maintaining the normal architecture of the heart myocardium. Electrophoresis of serum LDH and plasma proteins will be carried out in order to find out the necrotic action of isoproterenol and cardio tonic action luteolin. Oxidant and antioxidant status (both enzymic and non-enzymic) will be assessed to observe the free radical damage of isoproterenol and to establish the antioxidant nature of the luteolin. Studies on lipids and lipoprotein profile will be carried out to find out the metabolic alterations in isoproterenol induced myocardium and the protective efficacy of luteolin. General parameters such as blood glucose, heart tissue glycogen, DNA and RNA studies will be carried out to access the myocardial damage induced by isoproterenol and to access the protective action of luteolin.

 

 

Table 2: Biochemical studies on markers:

S.No

Name of the Marker Enzyme

Method Followed

1

Aspartate Transaminase

Mohur and Cooke (1975)

2

Alanine Transaminase

Mohur and Cooke (1975

3

Creatinine Kinase

Norman and Marlene (1967)

4

Glutathione reductase

Carlberg (1975)

5

Superoxidase mutase

Misra and Fridovichi (1972)

6

Glutathione S transferase

Habig and Jackoby (1981)

7

Catalase

Sinha(1972)

 

 

Cellular Changes:

At cellular level the alteration in the activity of mitochondrial enzyme ETC components, antioxidant enzymes activities and lipid levels were assessed to find out the efficacy of the drug in protecting the mitochondrial structure from isoproterenol induced mitochondrial damage. Analysis of membrane bound enzyme studies such as ATPases, proteases and phospholipases were carried out to observe the mitchondrial membrane protection by luteolin pretreatment from isoproterenol induced damage. The mitochondrial electrolyte levels will also be estimated to find out the electrophysiological nature of the myocardium. The activity of lysosomal enzymes will be assessed in serum and heart lysosome subcellular organelle to find out the protection of luteolin from isoproterenol induced damage.

 

Statistical calculation:

The significance of difference among the groups of experimental rats will be assessed using one way analysis of variance (ANOVA) followed by Least Significant multiple (LSD) comparison test (Fisher, 1958). F value at the level of 5% will be considered statistically significant and will be calculated as

††††††††††††††††††††††

††††††††††† ††††††††††

From the table for the degree of freedom 3, 20 the F value 3.10 will be calculated.

 

DISCUSSION AND CONCLUSION:

Almost at all ages and in all countries, diseases of cardiovascular system are the major cause for death and disability. Among the cardiovascular diseases, myocardial infarction appears to be the number one killer disease in affluent societies till today. Experimental models of MI have been developed in laboratory animals to understand the morphology biochemistry, electro-physiology and mechanical properties of the damaged myocardium and to test the efficacy of various therapeutic agents. Isoproterenol, a synthetic catecholamine and beta-adrenergic agonist, has been found to cause a severe stress to the myocardium resulting in infarct like necrosis of the heart muscle. Isoproterenol induced myocardial infarction is a well-standardized model to study the cardiac function and to find out the beneficial effects of many drugs in curing the disease.

 

Epidemiological studies support the hypothesis that the medicinal plants and plant derived antioxidants may play a beneficial role in the prevention of cardiovascular disorders. Recently, attention has been focused on many medicinal plants and plant derived phytochemicals such as the flavonoids, alkaloids, poly phenols and xanthones as potential therapeutic agents in the prevention and management of cardiovascular diseases due to their antioxidant nature. With technological advancement of science, the isolation, identification and elucidation of the chemical principles from medicinal plants have become much simpler and have contributed significantly to the development of new drugs for almost all type of diseases including cardiovascular diseases. Based on the literature and the scientific interest in the possible use of natural plant products as a prophylactic / therapeutic measure, an attempt will be made in the present investigation to evaluate the cardio protective role Plectranthus amboinicus leaf extract containing luteolin a flavanoid on experimental myocardial infarction in rats.

 

Due to its antioxidant, cardio-tonic and other medicinal properties, luteolin flavanoid, is chosen as a prophylactic agent against isoproterenol induced experimental myocardial infarction in rats. The present study may definitely throw more light in identifying the biochemical mechanism behind the prophylactic basis of cardio protection offered by the flavanoid luteolin against isoproterenol induced experimental myocardial infarction.

 

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Received on 25.08.2013††††††† †††††††††††††††††††† Accepted on 01.09.2013††††††††

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Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 195-198