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.
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
Theobroma cacao belongs to the family of Malvaceae (alternatively Sterculiaceae), and is characterized by three main cultivar groups: Criollo, Forastero and Trinitario, which are widespread in the (sub)humid tropics. All cultivated species originated from America. Cocoa is one the world’s most valuable crops, cultivated worldwide on 8.2 million hectares, playing an important role in the social and economic life of more than 5 million households, and affecting 25 million people in poor rural areas. Ivory Coast, Ghana, Nigeria, Indonesia and Brazil are the most important cocoa producers (Pohlan and Perez, 2011). The cocoa plant is classified thus;
Kingdom Plantae
Subkingdom Viridaeplantae
Infrakingdom Streptophyta
Division Tracheophyta
Subdivision Spermatophytina
Infradivision Angiospermae
Class Magnoliopsida
Superorder Rosanae
Order Malvales
Family Malvaceae
Genus Theobroma L.
Species cacao
Integrated Taxonomic Information System (ITIS) (version 2011)
Plate 1: Seeds from the cacao pod
Plate 2: A cacao tree with the pods on it
1.1.2 Significance of Theobroma cacao in cardiovascular health
Cocoa (Theobroma cacao) is one of the major cash crops in Nigeria (Awe et al., 2012). Cocoa has polyphenolic flavonoid constituents and is consumed as an unsweetened drink of raw, dried cocoa powder, traditionally by native Indians (Grivetti and Howard-Yana, 2009). Sugar was added, after it was brought to Europe, and other processing steps became common in order to reduce bitterness and to provide European taste and flavor. These modifications resulted in reduction in flavanol content which is the likely polyphenolic with potential antioxidant effects (Hristova et al., 2012). The medicinal value of cocoa beans was noted by American researchers when they noticed that residents of the island Kuna in South America drank large amounts of home-prepared cocoa, rich in flavanoids, and remained hypertension free. The Kuna on the mainland however, consumed commercial cocoa devoid of flavonoid and developed hypertension and cardiovascular diseases (Fisher and Hollenberg, 2005). On monitoring the renal hemodynamics, the Kuna island people demonstrated high NO levels, consistent with a favorable antioxidant effect of cocoa flavanoids on the endothelium (Fisher et al., 2005). The main flavonoids present in cacao beans are flavanols, especially catechins and epicatechins (Steinberg et al., 2003). The antioxidant activity of cocoa and chocolate has been shown to be correlated with their catechin and procyanidin contents (Wan et al., 2001). These are strong anti-oxidants and beneficially modulate the cardiovascular system (Ding et al., 2006; Corti et al., 2009; and Desch et al., 2010). The prophylactic and therapeutic cardiovascular claims associated with cocoa intake are growing (Corti et al., 2009; Ella et al., 2012). In a recent Meta analysis, high levels of chocolate consumption were associated with a reduction of cardiovascular disease by 37%, diabetes by 31% and stroke by 29% (Adriana et al., 2011). Cocoa appears to be beneficial against the risk of insulin resistance, hypertension, stroke, coronary artery disease (CAD), metabolic syndrome, cognitive function and dementia (Henderson et al., 2007; Taubert et al., 2007; Armitage et al., 2009; Reid et al., 2010; Desch et al., 2010; Tokede et al., 2011; Hooper et al., 2012; Larsson et al., 2012; Skelhon et al., 2012). Cocoa consumption exerts several beneficial effects on cardiovascular health (Corti et al., 2009). Many researchers have shown that polyphenols and/or polyphenol-rich foods have an important role in health preservation due their antioxidant properties (Han et al., 2007; Cooper et al., 2008; Awe et al., 2013). Epidemiologically, its consumption is associated with a reduction in cardiovascular diseases and all cause mortality and inversely correlated with blood pressure (BP), (Buijsse et al., 2006). This has been recently confirmed in a large population-based study (Buijsse et al., 2010). Endothelial dysfunction is a pathophysiological condition, associated with premature atherothrombotic disease (Oemar et al., 1998). Diminished NO bioavailability and increased oxidative stress are among the most important features of endothelial dysfunction (Munzel et al., 2010). Congestive heart failure (CHF) is a prevalent condition, representing the final stage of most cardiovascular diseases and is associated with high morbidity and mortality (Swedberg et al., 2005). Patients with CHF typically show endothelial dysfunction, increased oxidative stress, and baroreceptor dysfunction (Katz et al., 2005). Moreover, patients with impaired Flow Mediated Dilatation (FMD) are at increased risk for cardiovascular events and death (Katz et al., 2005); complications of atherosclerosis that involve increased platelet activation. Indeed, many complications in Heart Failure (HF) are thrombus-related and increased platelet activation has been observed in CHF (Gibbs et al., 2001). Cocoa acutely improve NO-dependent vasodilatation in healthy humans (Fisher et al., 2003) and in patients with cardiovascular risk factors, including diabetes, both in the forearm circulation (Heiss et al., 2003; Grassi et al., 2005a; Balzer et al., 2008) and in coronary arteries (Flammer et al., 2007). Congestive heart failure is a very late stage of most forms of cardiovascular disease. At that stage, many cardiovascular alterations are irreversible. For instance, lowering of low-density lipoproteins by 3-hydroxyl-3-methyl-glutaryl (HMG) coenzyme reductase inhibitors no longer reduce the event rates even in patients with ischemic cardiomyopathy (Kjekshus et al., 2007; Tavazzi et al., 2008). Thus, alternative treatment options have to be explored in these high-risk patients.
1.2 Cardiovascular diseases
Ischemic heart disease is a major cause of death and disability in the world (World Health Organization, 2008). Common therapies, such as primary coronary angioplasty and thrombolysis, are applied to restore blood supply to the heart, limit infarct size and reduce mortality. However, the restoration of blood supply would generate reactive oxygen species in damaged sites of the myocardium, intensifying the damage to the cardiac tissues (Chan et al., 2011). Myocardial infarction (MI) can be recognized by clinical features, including electrocardiographic (ECG) findings, elevated values of biochemical markers (biomarkers) of myocardial necrosis, and by imaging, or may be defined by pathology. It is a major cause of death and disability worldwide. MI may be the first manifestation of coronary artery disease (CAD) or it may occur, repeatedly, in patients with established disease. From the epidemiological point of view, the incidence of MI in a population can be used as a proxy for the prevalence of CAD in a population. Myocardial infarction is one of the leading health problems in the world and it is an outcome measure in clinical trials, observational studies and quality assurance programmes. These studies and programmes require a precise and consistent definition of MI. In studies of disease prevalence, the World Health Organization (WHO) defined MI from symptoms, ECG abnormalities and cardiac enzymes. In 2000, the First Global MI Task Force presented a new definition of MI, which implied that any necrosis in the setting of myocardial ischemia should be labelled as MI (The Joint European Society of Cardiology/American College of Cardiology Committee, 2000). These principles were further refined by the Second Global MI Task Force, leading to the Universal Definition of Myocardial Infarction Consensus Document in 2007, which emphasized the different conditions which might lead to an MI (Thygesen et al., 2007). This document, endorsed by the European Society of Cardiology (ESC), the American College of Cardiology Foundation (ACCF), the American Heart Association (AHA), and the World Heart Federation (WHF), has been well accepted by the medical community and adopted by the WHO (Mendis et al., 2011).
1.3 Pathological characteristics of myocardial infarction
MI 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). It is defined in pathology as myocardial cell death due to prolonged ischemia. After the onset of myocardial ischemia, histological cell death is not immediate, but takes a finite period of time to develop—as little as 20 min, or less in some animal models (Jennings and Ganote, 1974). It takes several hours before myocardial necrosis can be identified by macroscopic or microscopic post-mortem examination. Complete necrosis of myocardial cells at risk requires at least 2–4 h, or longer, depending on the presence of collateral circulation to the ischemic zone, persistent or intermittent coronary arterial occlusion, the sensitivity of the myocytes to ischemia, preconditioning, and individual demand for oxygen and nutrients (Thygesen et al., 2007). The entire process leading to a healed infarction usually takes at least 5–6 weeks. Reperfusion may alter the macroscopic and microscopic appearance.
1.4 Polyphenols
Phenolic compounds are abundant micronutrients in our diet with an average consumption of about 1 g/day (Scalbert et al., 2005). The phenolic content of fruits, vegetables and other plant foods varies considerably, not only between different types but also between cultivars of the same type and can even depend on growing condition and the time of harvest. Fruits and vegetables are particularly rich sources of polyphenols, and many polyphenols are well known for their antioxidant activity (Ghosh, 2005). Polyphenols are group of chemical substances, characterized by the presence of more than one phenolic group whereas the phenolic acids are phenols with only one ring. Polyphenols belong to one of the major classes of plant secondary metabolites and includes flavonoids, lignans, stilbenes, coumarins and tannins (Harborne, 1993). Polyphenols are large and heterogeneous group of biologically active secondary metabolites in plants, where they act as cell wall support materials, colorful attractants for birds and insects, and defense mechanisms under different environmental stress conditions (wounding, infection, excessive light, or UV irradiation) (Hakkinen, 2000). Based on the number of phenolic rings and on the structural elements that link these rings, they are divided into four groups: phenolic acids, lignans (recognized as phytoestrogens; flaxseed and flaxseed oil are the main source), flavonoids (the most abundant polyphenols in human diets), and stilbenes (resveratrol is under investigation for its anticarcinogenic properties). Flavonoid group is subdivided into: anthocyanins, flavonols, flavanols (catechins in tea, red wine, and chocolate), flavanones (citrus fruit are the main source), flavones, and isoflavones (main source is soya) (Tomas-Barberan, 2012). Several thousand polyphenols have been identified in edible plants and they are divided into different groups according to their structure and complexity (Shahidi and Naczk, 1995). Flavonoids are the largest group of phenolic compounds and have a basic skeleton composed of three rings. They are classified into six families according to their substitution pattern, and include anthocyanins, flavones, isoflavones, flavonols, flavanones and flavanols. Even though the term, ‘polyphenol’ encompasses more than 8000 different structures, only a limited number have been studied pharmacologically. For example, red wine contains the natural phytoalexin resveratrol and the flavonoids quercetin, delphinidin and (+)-catechin, which have been well studied and shown to possess the pharmacological properties explaining the beneficial effects of moderate red wine consumption against the onset of cardiovascular disease (Naissides et al., 2006; Kiviniemi et al., 2007; Kirimlioglu et al., 2008). Three main groups of polyphenols in unfermented cocoa bean are flavan-3-ols or catechins, anthocyanins, and proanthocyanidins (Misnawi, et al., 2004a). Main polyphenol compound in fresh cocoa bean is (−)-epicatechin, followed by (+)-catechin, and dimers and trimers of these compounds (Jalil and Ismail, 2008). The brown and purple color of cocoa bean, are due to the altered and complex products of catechin and tannins (Jalil and Ismail, 2008). The polyphenol content of cocoa beans is determined by the genetic makeup of the species (Saltini et al., 2013). In addition, crop season and country of origin have impact on polyphenols in cocoa beans. Cocoa bean processing also affects polyphenol content. During fermentation, polyphenols diffuse with cell liquid from storage cells and are subjected to oxidation (both nonenzymatic and polyphenol-oxidase-catalyzed), polymerisation, and reactions with proteins (Wollgast and Anklam, 2000; Misnawi et al., 2004b). Anthocyanins are hydrolysed to anthocyanidins and sugar component, leucocyanidins are dimerised (Misnawi et al., 2004a), and (−)-epicatechin and soluble polyphenol contents are reduced to 10–20% (Wollgast and Anklam, 2000). Hurst et al. (2011) reported that unripe and ripe cacao pods contain solely (−)-epicatechin and (+)-catechin, however, during fermentation, levels of both of these compounds were reduced, but (−)-catechin was formed due to heat-induced epimerization. During drying, additional loss of polyphenol occurs, mainly due to nonenzymatic browning reactions. Roasting results in significant loss of polyphenols due to thermolabile flavanols (Rusconi and Conti, 2010) and oxidation of epicatechin and catechin to quinones which complex with amino acids and proteins, and polymerize with other polyphenols (Li et al., 2012). Antioxidant properties of polyphenols highly depend on the arrangement of functional groups around the nuclear structure. Free radical scavenging capacity is primarily attributed to hydroxyl groups, and aglycones are more potent antioxidant than their responding glycosides (Heim et al., 2002). Polyphenols can act as proton donor-scavenging radicals (Rice-Evans et al., 1997), inhibitors of enzymes that increase oxidative stress, chelate metals, bind carbohydrates, and proteins (Heim et al., 2002). These properties enable them to act as anticarcinogenic, antiinflammatory, antihepatotoxic, antibacterial, antiviral, and antiallergenic compounds (Rice-Evans et al., 1997; Arts and Hollman, 2005; Vita, 2005; Zaveri, 2006). During the processing of cocoa, (−)-epicatechin and (+)-catechin are lost and this could partly be attributed to heat-induced epimerization to (−)-catechin (Hurst et al., 2011). Alkalization, high temperatures and presence of oxygen used during the process of chocolate making have been shown to destroy polyphenols and significantly reduce antioxidant activity of cocoa powder (Wollgast and Anklam, 2000). All these processes (fermentation, roasting, drying) are needed to develop characteristic cocoa aroma. Polyphenols give astringent and bitter aroma to cocoa and contribute to reduced perception of “cocoa flavor” by sensory panel (Misnawi et al., 2004b). However, there have been recent advancements in cocoa processing aimed at preserving the polyphenol content whilst maintaining satisfactory aroma (Schinella et al., 2010). This is in recognition of the health benefits inherent in cocoa polyphenols, thus making cocoa a potential functional food.
1.5 Bioavailability of cocoa polyphenols
Generally, bioavailability of polyphenols is affected by chemical structure of polyphenols, food matrix, factors related to food processing, and interactions with other constituents in diet, as well as with some host related factors (genetic aspects of individuals, gender and age, disorders and physiological condition, and microbiota metabolism and enzyme activity in the colon (D’Archivio et al., 2010; Tomas-Barberan, 2012). The most important food sources of polyphenols are vegetables and fruits, green and black tea, red wine, coffee, chocolate (cocoa), olives, and some herbs and spices, as well as nuts and algae (Quinones et al., 2013). Some polyphenols are specific to particular food and some are found in all plant products, so that, generally, food is considered to contain complex mixtures of polyphenols (D’Archivio et al., 2010). Isoflavones and phenolic acids have highest absorption, followed by catechins, flavanones, and quercetin glucosides, whereas proanthocyanidins, anthocyanidins, and galloylated tea catechins are poorly absorbed (Han et al., 2007). Once absorbed, polyphenols are conjugated to glucuronide, sulphate, and methyl groups in the gut mucosa and inner tissues, where epicatechin and epigallocatechin are mostly present as the glucuronide and sulfate conjugates. Absorption of epicatechin and catechin in the intestine averages between 22% and 55%, while dimers and trimers are poorly absorbed (less than 0.5%). Procyanidins cross intestinal barrier and are transported to liver, where they undergo methylation, glucuronidation, and sulfation which result in antioxidant capacity (Han et al., 2007). Polyphenols that reach colon are fermented by microflora to phenolic acids of low molecular weight (Han et al., 2007). Epicatechin from chocolate is rapidly absorbed by humans, with plasma levels detected after 30min of oral digestion, peaking after 2-3 h and returning to baseline after 6–8 h. Generally, it can be stated that the smaller the polyphenol, the higher the concentration in blood and the higher the chance it will reach its target organ in the body (Cooper et al., 2008). Chirality might also influence bioavailability of polyphenols,—(+)-form of catechin is almost 10 times more absorbed than (−)-form (Cooper et al., 2008). Presence of sugars and oils generally increases bioavailability of polyphenols, while proteins, on the other hand, decrease it (Tomas-Barberan, 2012). Research by Neilson et al. (2010) showed that milk proteins and sucrose modulate metabolism, plasma pharmacokinetics, and bioavailability of catechins from chocolate confections. They found that milk proteins reduce bioavailability of epicatechin in chocolate confectionary. Serafini et al. (2003) reported inhibition of in vivo antioxidant activity of chocolate by addition of milk either during manufacturing process or during ingestion. However, this effect was not observed in chocolate beverages (Neilson et al., 2010). Study of interactions of cocoa polyphenols with milk proteins by proteomic techniques demonstrated that protein-polyphenol complex formation involves covalent binding of free SH-group of the free cysteine residue of protein. This was supported by the fact that alkylated form of peptide did not react with flavanols, while lactosylation did not prevent polyphenol binding. Since only small portion of protein interacts with polyphenol, bioavailability of polyphenols is not significantly influenced (Gallo et al., 2013). This was supported by researches of Keogh et al. (2007) and Roura et al. (2007) who reported that milk does not affect bioavailability of cocoa powder flavonoids in healthy adults. Sucrose increased bioavailability of polyphenols, but formulation also influenced the extent of sucrose impact. Schramm et al. (2003) observed enhanced uptake of aglycone flavanols when they were consumed immediately after carbohydrate-rich meal. Peters et al. (2010) concluded that sucrose addition to green tea resulted in delay of catechin absorption, partly due to viscosity increase, but it also improved catechin uptake by the intestine.
1.6 Influence of cocoa polyphenols on health
Unlike vitamins, polyphenols are not essential components of human diet. Nevertheless, they are consumed on daily basis due to their ubiquitous presence in fruits and vegetables. Many researchers have shown that polyphenols and/or polyphenol-rich foods have an important role in health preservation due to antioxidant properties (Han et al., 2007; Cooper et al., 2008; Awe et al., 2013). The antioxidant activity of cocoa and chocolate was shown to be correlated with their catechin and procyanidin contents (Wan et al., 2001). Polyphenols and especially flavonoids are well acknowledged for their antioxidant and protective effects in circumstances of oxidative stress (Terao et al., 2008; Boots et al., 2008). Qiu et al. (2012) reported that in acute situations of oxidative stress quercetin and epigallocatechin gallate were the most potent antioxidants amongst other flavonoids which they tested. They have also been recognized to interact with cell death-survival signaling pathways which depending on the dose may promote or inhibit apoptosis, exhibiting chemopreventive or cytoprotective effects, respectively (Mandel et al., 2004; Ramos 2007). The short- and long-term ingestion of cocoa and dark chocolate, particularly rich in flavanols (a subclass of flavonoids), has been shown to induce a consistent and striking peripheral vasodilation in healthy people (Fisher et al., 2006; Bayard et al., 2007). Polyphenols, in particular flavanols in cocoa products, have been shown to increase the formation of endothelial nitric oxide, which promotes vasodilation and consequently may lower blood pressure (Karim et al., 2000; Fisher et al., 2003; Fisher and Hollenberg, 2006). The flavanols present in cocoa and chocolate include the monomers, (–)-epicatechin and (+)-catechin as well as the oligomers of these monomeric units, procyanidins. In a recent long-term (12 wk) intervention study in women (18–65 years), the intake of a high flavanol (329 mg) cocoa drink was associated with a significant increase in blood flow in cutaneous and subcutaneous tissue, as well as an improved resistance against UV-induced erythema (Neukam et al., 2007). This increase in cutaneous blood flow associated with cocoa intake is consistent with other human trials which report enhanced flow mediated dilation (FMD) of conduit arteries and augmented microcirculation after the ingestion of flavanol-rich cocoa (Heiss et al., 2003; Schroeter et al., 2006). Decreasing blood pressure and cardiovascular mortality has also been demonstrated with cocoa consumption in a substudy of the Zutphen population (Buijsse et al., 2006). Another study in smokers also demonstrated improved flow-mediated vasodilation after a cocoa rich diet (Heiss et al., 2005). An association of decreased cerebral perfusion with dementia has been recently highlighted (Francis et al., 2006) and the prospect of increasing cerebral perfusion with cocoa flavanols is extremely promising. A recent pilot study evaluated the relationship between cerebral blood flow (CBF) and a single acute dose (450 mg) of flavanol-rich cocoa and found this treatment to increase local CBF to grey matter by up to 60 % by 2–3 h postconsumption (Francis et al., 2006). Researchers from the University Hospital of Cologne pooled data from five studies regarding the effects of cocoa on blood pressure involving 173 participants and found that the consumption of cocoa had significant positive effects on reducing blood pressure (Taubert et al., 2007). Similar to cocoa containing drinks, another flavanol-rich food, dark chocolate (100 g containing 500 mg polyphenols) has been shown to be able to lower blood pressure and improve insulin sensitivity in healthy people (Grassi et al., 2005b). In a randomized, sham procedure-controlled, crossover study, 100 g of dark chocolate (75 % cocoa) eaten on two separate days (Vlachopoulos et al., 2005) resulted in an increase in the diameter of the resting brachial artery, increased arterial blood flow and a significantly increased heart rate. Though, this effect might be due to the presence of significant amounts of caffeine which can affect blood flow, heart rate and cognition independently of the polyphenol content.
1.6.1 The Vascular Endothelium
The endothelium, once considered a simple monolayer of cells covering the entire inner surface of all the blood vessels, has recently been established as a strategically-located multifunctional organ. It lies between circulating blood and the vascular smooth muscle and plays many pivotal roles in the regulation of vascular tone and endothelial integrity, as well as in the maintenance of blood fluidity and homeostasis. To perform such a wide range of functions, the endothelium synthesizes or releases several vasoactive substances, including the vasodilators NO, prostacyclin and endothelium-derived hyperpolarising factors (EDHFs) and the vasoconstrictors angiotensin II and endothelin-1. Under physiological conditions, the endothelium acts as an inhibitory regulator of vascular contraction, leukocyte adhesion, vascular smooth muscle cell growth and platelet aggregation (Cooke, 2000). However, the characteristics of the endothelium change in response to local or systemic changes such as trauma, hyperglycemia or dyslipidaemia and dysfunction of endothelium is considered present when normal organ function can no longer be preserved either in the basal state or in response to any given physical, humoral or chemical stimuli.
1.6.2 Nitric Oxide (NO)
NO is generated along with L-citrulline from the cationic amino acid L-arginine by a class of enzymes known as nitric oxide synthases (NOS) in the presence of molecular oxygen and NADPH (Hibbs et al., 1987). NOS contain both flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and require several co-factors including tetrahydrobiopterin (H4B) and reduced glutathione for activity (Hevel and Marletta, 1992). Three isoforms of NOSs have so far been identified, all of which are the products of separate genes which share approximately 60% homology at amino acid level (Nathan and Xie, 1994). NOSs are divided into two classes with regard to the nature of their expression and requirement of Ca2+ for their enzymatic activity. Both endothelial type (eNOS or NOS3) and neuronal type (nNOS or NOS1) NOS are constitutively expressed and Calcium ion- dependent, while the inducible type (iNOS or NOS2) is expressed in response to several stimuli including cytokines, and does not require Ca2+ for its activity. Although NOS3 is constitutively expressed, many pathophysiological stimuli regulate its expression. Indeed, chronic fluid shear stress (Woodman et al., 1999), exercise (Sessa et al., 1994) and sex hormones (Hishikawa et al., 1995) elicit an increase in NOS3 gene expression while tumor necrosis factor (Yoshizumi et al., 1993) and hypoxia (McQuillan et al., 1994) downregulate its expression on mRNA and/or protein levels. The current data on the molecular regulation of NOS3 in diabetic animals (Lund et al., 2000; Zanetti et al., 2000) and in endothelial cells grown under hyperglycemic conditions suggest a defect in its gene regulation (Chakravarthy et al., 1998). NOS3 is expressed in abundance in cardiac myocytes and coronary microvascular endothelial cells and is therefore considered as the main source of NO within the vascular endothelium (Bayraktutan et al., 1998). Endothelium-derived NO is known to be the most potent endogenous vasodilator in the body. It is synthesized and released by the endothelium in response to a wide range of chemical, physical and humoral stimuli including thrombin, hormones, local autacoids, alterations in oxygen tension and shear stress (Furchgott and Zawadzki, 1980). After synthesis, NO is released into the subendothelial space and vascular lumen where it directly causes the underlying vascular smooth muscle to relax by binding to the heme moiety of soluble guanylate cyclase, thereby increasing the production of intracellular cyclic 3’-5’-guanosine monophosphate (cGMP) (Rapoport and Murad, 1983). Endothelial secretion of NO counterbalances the direct vasoconstrictive effects of norepinephrine, serotonin, angiotensin II and endothelin on the vascular smooth muscle (Rubanyi, 1993). NO has also been shown to reduce oxygen consumption (Shen et al., 1994) and plays a critical role in the pathogenesis of atherosclerosis due to its inhibitory effects on platelet aggregation (Radomski et al., 1987), leukocyte adhesion (Kubes et al., 1991), DNA synthesis (Nakaki et al., 1990) and vascular smooth muscle cell proliferation (Garg and Hassid, 1989). In addition to its roles mentioned above, NO plays a significant role in the regulation of blood pressure. Indeed, NOS3 gene knockout mice develop severe hypertension, and blood vessels isolated from these mice do not relax when exposed to endothelium-derived vasodilators such as acetylcholine (Huang et al., 1995). It has also been shown that the inhibition of NO synthesis leads to significant peripheral vasoconstriction and elevation of blood pressure (Rees et al., 1989; Tresham et al., 1991).
1.7 Biomarker detection of myocardial injury with necrosis
There are a lot of biomarkers that could tell the state of the myocardium but Cardiac troponin I and T, components of the contractile apparatus of myocardial cells expressed almost exclusively in the heart, give the most specific and sensitive assay results (Thygesen et al., 2007). Myocardial injury is detected when blood levels of sensitive and specific biomarkers such as cardiac troponin or the MB fraction of creatine kinase (CKMB) (Thygesen et al., 2007); AST, ALT, LDH, lipid profile tests and lipid peroxidation products are increased. Although elevations of these biomarkers in the blood reflect injury leading to necrosis of myocardial cells, they do not indicate the underlying mechanism (Jaffe et al., 2006). Various possibilities have been suggested for release of structural proteins from the myocardium, including normal turnover of myocardial cells, apoptosis, and cellular release of troponin degradation products, increased cellular wall permeability, formation and release of membranous blebs, and myocyte necrosis (White, 2011). Regardless of the pathobiology, myocardial necrosis due to myocardial ischemia is designated as Myocardial Infarction. Also, histological evidence of myocardial injury with necrosis may be detectable in clinical conditions associated with predominantly non-ischemic myocardial injury. Detection of a rise and/or fall of the measurements is essential to the diagnosis of acute MI (Jaffe et al., 2006).
1.8 The model of MI induction using isoproterenol
Chronic isoproterenol (ISO; isoprenaline) administration produces a rapid, highly reproducible rodent model of cardiac hypertrophy (Heather et al., 2009). Isoproterenol is a dual β1-β2-adrenergic receptor agonist that has acute positive chronotropic and inotropic effects on the heart (Kitagawa et al., 2004). When administered chronically or at high doses, isoproterenol has deleterious effects on the heart, inducing hypertrophy (Beznak, 1962), necrosis, fibrosis, apoptosis, oxidative damage and inflammatory cell infiltration (Li et al., 2006). Histological analysis has shown that isoproterenol induces an infarct-like lesion at the apex of the myocardium (Beznak and Hacker, 1964; Grimm et al., 1998). While in some studies the effects of isoproterenol are reversible after cessation of the drug (Kitagawa et al., 2004). In other studies, rats go on to develop mild heart failure months after cessation of the drug, as shown by increased end-diastolic pressure, preserved hypertrophy and scar remodeling (Beznak and Hacker, 1964; Grimm et al., 1998). Isoproterenol can be administered subcutaneously inducing consistent cardiac hypertrophy over a relatively short period of time with less variability in the damage produced, compared with invasive (surgical) infarct model (Heather et al., 2009). Heather et al. (2009) observed that isoproterenol infusion impaired in vivo cardiac function and induced hypertrophy, dilation and fibrosis, mainly within the apical regions of the heart. There was a downregulation of fatty acid metabolism, with reductions in fatty acid oxidation, myocardial triglyceride concentrations, mitochondrial enzyme activities and fatty acid transporter levels. Isoproterenol-induced cardiomyopathy is simple and rapid to reproduce, requiring minimal or no surgery and leaving the pericardium intact. Isoproterenol administration consistently produced cardiac dysfunction and left ventricular dilation similar to those found in the moderate severity infarcted rat heart (Heather et al., 2009). Furthermore, isoproterenol infused hypertrophied hearts had decreased fatty acid and glucose metabolism, similar to the infarcted rat heart and other models of non-ischemic heart failure (Heather et al., 2006; Murray et al., 2006).
1.9 Beta Blockers and CVD Health
β-adrenergic antagonists (β-blockers) comprise a group of drugs that are mostly used to treat cardiovascular disorders such as hypertension, cardiac arrhythmia, or ischemic heart disease. Each of these drugs possesses at least one chiral center, and an inherent high degree of enantio-selectivity in binding to the β-adrenergic receptor (Mehvar and Brocks, 2001). β-blockers are antiarrhythmic agents and standard therapy to control the ventricular rate in chronic atrial fibrillation (Opie, 2013). β-blockers bind selectively to the β-adrenoceptors producing a competitive and reversible antagonism of the effects of β-adrenergic stimuli on various organs (Lopez-Sendon et al., 2004). β-1 receptors are primarily found in myocardial tissue, and receptor stimulation results in an increase in the rate of contraction (Lyden et al., 2014). β-1-adrenergic stimulation facilitates calcium influx into cardiac myocytes by increasing the levels of cyclic adenosine monophosphate (cAMP), which in turn upregulates the opening of L-type calcium channels. Formation of cAMP results in phosphorylation of these channels, with resultant opening and calcium entry into myocardial cells (Shepherd, 2006). β-blockers were first developed by Sir James Black at the imperial chemical industries in the United Kingdom in 1962 (Aijaz and Upendra, 2009). They are among the proven medication in cardiovascular medicine, reducing both the morbidity as well as the mortality. Most beta-blockers are well-absorbed after oral intake. Currently, beta-blockers are employed in a number of cardiovascular conditions. The strongest evidence for their use is in systolic heart failure, post- myocardial infarction (myocardial protection) and in prevention and treatment of ventricular arrhythmias in post MI patients. In acute myocardial infarction, current recommendation (based on COMMIT/CC S-2 trial (Clopidogrel and Metoprolol in Myocardial Infarction Trial/Second Chinese Cardiac Study)) is to avoid early use of beta blockers in patients with hemodynamic instability or who are at risk of cardiogenic shock. Once stable, beta blockade is strongly recommended in patients of myocardial infarction. Beta-blockers are not currently favored as the first line anti-hypertensive therapy, particularly in the elderly, unless there are specific indications (Aijaz and Upendra, 2009). A number of mechanisms have been suggested for beta blockers in heart failure. It is shown that beta blockers increase myocardial efficiency since the left ventricle can improve stroke work index without increasing oxygen uptake. A more efficient aerobic metabolism is achieved after long term beta-blockade indicated by a switch from myocardial release to uptake of lactate (Andersson et al., 1991). Normalization of the phosphocreatine/ATP ratio (PCr/ATP) after one month treatment with metoprolol in rats with post-myocardial infarction heart failure, which parallels improved ejection fraction, suggests that this is an important mechanism for the improvement of restoration of energy balance (Omerovic et al., 2000). Marked reduction in sudden cardiac death in beta-blocker could be explained by a combination of central and peripheral effects. Reduction in ventricular volume and wall stress reduce the stretch which can provoke arrhythmias whereas improved subendocardial flow will reduce the ischemia, another important trigger for arrhythmias. Moreover, the long-term central nervous effect of beta-blockers has been shown to reduce sympathetic outflow to the heart and increase vagal tone and this reduces the risk of ventricular fibrillation, the most important cause of sudden death (MERIT-HF Study Group, 1999). Beta-blockers antagonize the effects of sympathetic nerve stimulation or circulating catecholamines at beta-adrenoceptors which are widely distributed throughout the body systems.
In the Kidney: Blockade of β1 receptors inhibits the release of renin from juxta-glomerular cells and thereby reduces the activity of the renin-angiotensin-aldosterone system.
In the Heart: Blockade of β1 receptors in the sino-atrial node reduces heart rate (negative chronotropic effect) and blockade of β1 receptors in the myocardium decrease cardiac contractility (negative inotropic effect).
In the Central and peripheral nervous system: Blockade of beta-receptors in the brainstem and of prejunctional beta-receptors in the periphery inhibits the release of neurotransmitters and decreases sympathetic nervous system activity.
All beta-blockers occupy the β receptor and counter the effects of catecholamines on the cardiovascular tissues. β1 receptors are located on the cardiac sarcolemma and belong to the G-protein coupled adenyl cyclase system. When catecholamines stimulate the receptor, Gs protein couples the activated receptor to adenyl cyclase and generates cAMP. cAMP, the second messenger, activates protein kinase A (PKA) which phosphorylates the membrane calcium channel and increases calcium entry into the cytosol. PKA also increases calcium release from the sarcoplasmic reticulum. The calcium loading accounts for the positive inotropic effect (inotropy refers to force of myocardial contraction or cardiac contractility which is a cardiac cell’s ability to transform an electrical signal originating at sino-atrial node into mechanical action). PKA also phosphorylates Troponin I (decreases affinity of myosin head to actin) and phospholamban (increased calcium reuptake by sarcoplasmic reticulum). This accounts for the lusitropic effect (lusitropy refers to the active phase of relaxation of the cardiac muscles). Increased If (funny current or pacemaker current) in the sinus node leads to positive chronotropic effect (chronotropy refers to automaticity i.e. the ability to initiate its own heart beat by firing of sinoatrial node of the heart or heart rate and rhythmicity of cardiac contraction). Accelerated conduction across atrio-ventricular node (AVN) and conduction tissue causes the positive dromotropic effect (dromotropy refers to electrophysiological properties of the heart such as conduction velocity of atrio-ventricular node or rate of electrical impulses) (Opie, 2009).
1.10 Atenolol
Although more than 100 beta-blockers have been developed, only about 30 are available for clinical use (Frishman, 2003). Water-soluble beta-blockers (Atenolol, Nadolol) tend to have longer half-lives and are eliminated via the kidney. Lipid-soluble beta-blockers (metoprolol, propranolol) are metabolized mainly in the liver and have shorter half-lives (Koch-Weser and Frishman, 1981). Three types of beta-receptors (β1, β2, β3) are variably distributed in tissues (Gauthier et al., 1996). β1 receptors are mainly located in the heart while β2 receptors are found in vascular and bronchial smooth muscle. β3 receptors are located in the adipocytes and heart (Frishman, 2003). Cardio-selective beta-blockers (metoprolol, atenolol) exhibit greater affinity for β1 versus β2 receptors at usual drug levels (Helfand et al., 2009). This selectivity is lost at higher drug doses (Andersson, 1991). Atenolol acts by antagonizing the stimulation of β-1-adrenergic receptors by catecholamines such as epinephrine, norepinephrine and isoproterenol (a synthetic catecholamine).
1.11.1 Aim of Research
This study was aimed at determining the effect of ethanol extract of Theobroma cacao seeds polyphenol on cardiovascular health.
1.11.2 Specific Research Objectives
The specific research objectives were:
- Determination of total polyphenol content
- Induction of myocardial ischemia/infarct in rats using Isoproterenol.
- Determination of the effect of treatment with polyphenol-rich cacao extracts on lipid profile
of control and treated rats.
- Quantification of lipid peroxidation product (TBARS) in control and treated rats
- Assay of marker enzymes using serum and the heart tissue homogenate.
6. Histological studies on heart tissue
