THE EFFECT OF ETHANOL EXTRACT OF THEOBROMA CACAO POLYPHENOL ON ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION IN WISTAR RATS

ABSTRACT
This study investigated the effect of cocoa polyphenol on isoproterenol-induced myocardial infarction in Wistar rats. Polyphenol was extracted from cocoa using a hydroalcohol solvent. Forty albino rats divided into six groups of four rats each were used for the study. Groups 3, 4, 5 and 6 were pretreated with the extract (300, 500 and 700 mg/kg) and 100 mg/kg of atenolol (standard drug) respectively before administration with isoproterenol. Group 1 served as normal control while group 2 was administered with isoproterenol without any treatment. The activities of marker enzymes such as aspartate amino transferase (AST), alanine amino transferase (ALT) and lactate dehydrogenase (LDH) in both serum and heart tissue homogenate were assayed and serum concentrations of total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and malondialdehyde (MDA) were used to determine success of isoproterenol induction and response to treatment. Enzyme assays carried out on the serum and heart tissue homogenates showed increased activities in the serum and low activities in the heart homogenate in the untreated group when compared to the normal control. Histological studies carried out on the heart tissues revealed marked degeneration of the myocardium in the untreated group and in groups that received lower concentrations (300 and 500 mg/kg) of the cocoa extract. This corresponded with increased activities of the marker enzymes in the serum and low activities in the heart homogenate in these same groups. However, almost a total cardioprotection was observed in the group that received 700 mg/kg extract as revealed by the activities of marker enzymes in serum and heart tissue homogenate, concentrations of lipid profile parameters, level of lipid peroxide product and histological observations that showed heart tissue morphology very similar to that of the normal group. The results showed that consumption of cocoa as a functional food could reduce the risk of cardiovascular diseases and improve treatment outcomes.

TABLE OF CONTENTS
Title Page……………………………………………………………………………………………………i
Certification……………………………………………………………………………………iii
Dedication…………………………………………………………………………………iv
Acknowledgement………………………………………………………………………………v
Abstract………………………………………………………………………………….vi
Table of Contents……………………………………………………………………vii
List of Figures……………………………………………………………………………x
List of Tables……………………………………………………………………xi
CHAPTER ONE: INTRODUCTION
1.1 Theobroma cacao…………………………………………………………..1
1.1.1 Scientific classification of Theobroma cacao………………….2
1.1.2 Significance of Theobroma cacaoin cardiovascular health……………………………5
1.2 Cardiovascular diseases………………………………………………….6
1.3 Pathological characteristics of myocardial infarction…………….7
1.4 Polyphenols……………………………………………………………………..8
1.5 Bioavailability of cocoa polyphenols…………………………………10
1.6 Influence of cocoa polyphenls on health………………………………………11
1.6.1 The vascular endothelium…………………………………………………..13
1.6.2 Nitric oxide (NO)……………………………………………………………………13
1.7 Biomarker detection of myocardial injury with necrosis………………15
1.8 The model of myocardial induction using isoprpterenol……………………………………………15
1.9 Beta blockers and CVD health…………………………………………….16
1.10 Atenolol……………………………………………………………………………………18
1.11.1 Aim of Research…………………………………………………………………….19
1.11.2 Specific research objectives…………………………………………..19
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials…………………………………………………………………………….20
2.1.1 Plant material…………………………………………………………………..20
2.1.2 Animals………………………………………………………………………20
2.1.3 Equipment………………………………………………………………..20
2.1.4 Chemicals and reagents………………………………………………………21
2.2 Methods………………………………………………………………………21
2.2.1 Preparation of plant materials……………………………………………..21
2.2.2 Extraction of plant material and preparation of cocoa polyphenol extract……………………21
2.2.3 Determination of polyphenol content………………………………..22
2.2.4 Preparation of normal saline……………………………………….22
2.2.5 Preparation of drug solutions…………………………………………22
2.2.6 Induction of myocardial infarction………………………………………22
2.2.7 Acute toxicity test………………………………………………………..22
2.2.8 Sample collection………………………………………………23
2.2.9 Experimental design…………………………………………………..23
2.2.10 Determination of serum total cholesterol concentration………………….24
2.2.11 Determination of serum triacylglcerol concentration…………………..25
2.2.12 Determination of serum low density lipoprotein-cholesterol concentration………………….27
2.2.13 Determination of serum high density lipoprotein-cholesterol concentration………………..28
2.2.14 Assayof serum Alanine aminotransferases (ALT) activity………………………………………..29
2.2.15 Assayof serum Lactate dehydrogenase (LDH) activity…………………………………………….30
2.2.16 Assayof serum Aspartate aminotransferase (AST) activity……………………………………….31
2.2.17 Determination of lipid peroxide (malondialdehyde) concentration……………………………..32
2.2.18 Histological studies……………………………………………………32
2.3 Statistical analysis…………………………………………………………..33
CHAPTER THREE: RESULTS
3.1 Polyphenol yield at different extraction stages………………………34
3.2 Acute toxicity (LD50) test of T. cacao seeds…………………….35
3.3 Effect of polyphenol extract of T. cacaopretreatment on marker enzymes in the serum of isoproterenol-induced myocardial infarct rat…………………….36
3.4 Effect of polyphenol extract of T. cacaopretreatment on marker enzymes in the heart tissue homogenate of isoproterenol-induced myocardial infarct rat…………………………….37
3.5 Effect of polyphenol extract of T. cacaopretreatment on serum lipid profile of isoproterenol- induced myocardial infarct rat……………………………….38
3.6 Effect of polyphenol extract of T. cacaopretreatment on malondialdehyde concentration in isoproterenol-induced myocardial infarct rat…………………………..39
3.7 Effect of polyphenol extract of T. cacaopost-treatment on marker enzymesin the serum of isoproterenol-induced myocardial infarct rat ………………………………………………………40
3.8 Effect of polyphenol extract of T. cacaopost-treatment on marker enzymes in the heart tissue homogenate of isoproterenol-induced myocardial infarct rat…………………………….41
3.9 Effect of polyphenol extract of T. cacaopost-treatment on lipid profile of isoproterenol- induced myocardial infarct rat………………………………….42
3.10 Effect of polyphenol extract of T. cacaopost-treatment on malondialdehyde conentration in isoproterenol-induced myocardial infarct rat………………………………………………………..43
3.11.1 Phase I histological results (treatment before induction)………………………44
3.11.2 Phase II histological results (induction before treatment)………….45
CHAPTER FOUR: DISCUSSION
4.1 Discussion…………………………………………………………………..46
4.2 Conclusion ………………………………………………………………………………..50
4.3 Suggestions for Further Studies……………………………..50
References………………………………………………………………………..51
Appendices………………..…………….…………………………………………………………67

LIST OF FIGURES
Figure 1: Seeds from the cocoa pod…………………………………………………………………………….4
Figure 2: Cocoa tree with pods on it…………………………………………4
Figure 3: Phase I histological examination of heart tissue sections in control and treated animals (Hematoxylin and Eosin)……………………44
Figure 4: Phase II histological examination of heart tissues sections in control and experimental animals (Hematoxylin and Eosin)……………………………..45

LIST OF TABLES
Table 1: Test procedure for the determination of serum triacylglycerol…………………………………26
Table 2: ALT activity reference table………………………………………..30
Table 3: AST activity reference table……………………………………..32
Table 4: Polyphenol yield at different extraction stages…………………….34
Table 5:Phase I and II of the acute toxicity (LD50) test of T. cacao seeds……………………………..35
Table 6: Activities of marker enzymes in theserum ofcontrol and pretreated rats………………….36
Table 7: Activities of marker enzymes from heart tissue homogenates in control and pretreated rats…………………………………………………..37
Table 8: Levels of CHOL, LDL, HDL and TAG in the serum of control and pretreated rats…………………………………………………………..38
Table 9: Levels of MDA in both serum and heart tissue homogenate in control and pretreated rats……………………………………………………..39
Table 10:Activities of marker enzymes inthe serum of control and post-treated rats……………….40
Table 11: Marker enzyme activities from heart tissue homogenates in control and post-treated rats……………………………………………………………..41
Table 12:Levels of CHOL, LDL, HDL and TAG in the serum of control and post-treated rats…………………………………………………………..42
Table 13: Levels of MDA in both serum and heart tissue homogenate in control and post-treated rats………………………………………………………………..43

CHAPTER ONE

INTRODUCTION
1.1 Theobroma cacao
Cocoa (Theobroma cacao) is a major economic tree crop in Nigeria (Alamu, 2013). T. cacao grows in the subtropical areas of the world. Although it grows widely from the southeastern Mexico to the Amazon basin, two thirds of the world’s production comes from four West African countries, the Ivory Coast, Ghana, Nigeria and Cameroon. Ivory Coast is the world’s largest exporter of cocoa (Agarwal, 2013). The cocoa tree produces ‘cauliflory’ flowers in clusters directly on the trunk and older branches. After pollination, ‘cacao pod’ fruits are produced. Each pod contains about 20 to 60 seeds, called “cocoa beans”, embedded in a white pulp. These cocoa beans were considered divine by the Mayans, as they were presumably discovered by the god Quetzalcoatl. They believed that cocoa beans make one strong and invincible. Spaniards noted that cocoa consumption allowed the Aztecs greater stamina and they could walk long distances without fatigue. The Olmec, Mayan and some Mexican tribes also recognized the medicinal value of these beans (Agarwal, 2013). Cocoa products have been enjoyed by humans for centuries. Chocolate is made from cocoa (Theobroma cacao). Consumed world over for its pleasant taste and its pleasurable and stimulating effects, epidemiological and scientific studies have repeatedly demonstrated significant health benefits with its intake. Kuna Indians living on Panama’s San Blas Islands drink more than four cups of cocoa per week and rarely develop age related high blood pressure or heart disease. Bioactive compounds from plant sources such as phenolics have gained substantial interest in recent years owing to their unique functions and nutritional values including antioxidant, antimicrobial, antimutagenic and antitumor activities (Nsor-Atindana et al., 2012). Cocoa is rich in flavonoids which protect against cardiovascular diseases through their antioxidant, antiplatelet, and anti-inflammatory effects. Flavonoids may also lower blood pressure, increase high density lipoprotein cholesterol, positively modify insulin sensitivity and improve endothelial function (Agarwal, 2013). Cocoa and cocoa derivatives are recognized as major dietary source of antioxidants because of their high phenolic (procyanidins and flavanols mainly) content (Tomas-Barberan et al., 2007). Several groups of polyphenols are found in fruits, whereas the most important are the flavanols which can be further subdivided into the monomers epicatechin and catechin (Arts et al., 2000) and their dimers, oligomers and polymers, the so-called procyanidins (Lazarus et al., 1999; Adamson et al., 1999) responsible for the bitterness of cacao, through the formation of the complexes with salivary proteins (Manach et al., 2004). Numerous dietary intervention studies in humans and animals indicate that flavanol-rich foods and beverages exert cardioprotective effects with respect to vascular function and platelet reactivity (Carl et al., 2005). The consumption of flavanol-rich cocoa has been reported to improve endothelial function (Wang-Polagruto et al., 2006) and reduce the incidence of atherosclerotic diseases (McCullough et al., 2006).
Cardiovascular disease (CVD) is one of the main causes of death worldwide and most common in industrial societies (Chiva-Blanch et al., 2013). It is developed by a multifactorial process. Most CVDs are due to atherosclerosis, a degenerative process of the arteries that is induced by oxidative stress and chronic inflammatory status. The risk factors of this disease are smoking, diabetes mellitus, arterial hypertension, abnormalities in serum levels of total cholesterol and its fractions, overweight/obesity, family history of early CVD and physical inactivity, amongst others (Chiva-Blanch et al., 2013). Myocardial infarction is the acute condition of necrosis of the myocardium that occurs as a result of imbalance between coronary blood supply and myocardial demand (Boudina et al., 2002). Ischemic tissues generate oxygen derived free radicals which have been implicated in cardiac diseases and metabolic disorder (Prabhu et al., 2006). The model of isoproterenol-induced myocardial ischemia is considered as one of the most widely used experimental model to study the beneficial effects of many drugs on cardiac function (Grimm et al., 1998). The pathophysiological changes following isoproterenol administration are comparable to those taking place in human myocardial ischemia/infarction (Wexler, 1978). Increases in the formation of reactive oxygen species during ischemia/reperfusion and the adverse effects of oxyradicals on myocardium have been well established by both direct and indirect measurements (Wexler, 1978). Many epidemiological studies associate an increased consumption of foods and beverages rich in flavonoids, with a reduced risk of cardiovascular death (Kris-Etherton and Keen, 2002).
1.1.1 Scientific classification of Theobroma cacao