ABSTRACT
The study examined the proximate and phytochemical (saponins, tannins, phytate and flavonoid) composition of roasted cream coloured African yam bean (AYB) flour and the lipid profile of hypercholesterolemic rats fed diets supplemented with African yam bean. Different diets were formulated using the AYB flour. Group 1 consisted of the normal control rats (untreated rats), group 2 consisted of the hypercholesterolemic group of rats that were untreated. Groups 3-6 consisted of rats fed rat chow with 5%, 10%, 15% and 20% AYB flour supplementation, respectively. Proximate analysis was done using AOAC methods. Phytochemical and biochemical analyses were done in duplicates using standard methods. The proximate analysis showed that AYB flour contain 4.94% moisture, 27.66% protein,
4.29% fat, 3.66% ash, 9.57% crude fibre and 49.88% carbohydrate. The phytochemical analysis showed 0.77mg/100g saponins, 42.07mg/100g tannins, 9.08mg/100g phytate and
1.01mg/100g flavonoid. Total cholesterol level for the different supplementation groups showed significant decrease (p<0.05) with 20% supplementation showing the highest decrease (38.70%). Triglyceride levels decreased significantly (p<0.05) across all the groups. The 5% AYB supplementation showed the highest decrease (35.63%) and 20% supplementation the lowest decrease (28.01%). Low density lipoprotein (LDL) levels of the rats significantly (p<0.05) decreased across the groups. The control group also showed
50.34%  decrease  in  LDL  but  10%  AYB  supplementation  showed  the  highest  decrease (61.29%).  Serum  high  density  lipoprotein  (HDL)  concentration  increased  significantly (p<0.05)  after  supplementation  with  AYB.  The  20%  AYB  supplementation  showed  the highest percentage  increase  (22.85%)  relative  to the  other groups. There were significant decreases  in serum  total  cholesterol,  LDL  cholesterol,  triglyceride  and  increase  in HDL cholesterol for the groups with AYB supplementation.
CHAPTER ONE INTRODUCTION
1.1 Background to the Study
Hypercholesterolemia is the presence of high levels of cholesterol in the blood, and is a form of “hyperlipidemia” (elevated levels of lipids in the blood) and “hyperlipoproteinemia” (elevated levels of lipoproteins in the blood) (Durrington, 2003).This is a major risk factor for cardiovascular disease especially coronary heart disease (Mudabmbi & Rajagopal, 2007). Cholesterol is the main sterol found in body tissues. Since cholesterol is insoluble in water, it is transported in the blood plasma bound to protein particles (lipoproteins). Lipoproteins are classified by their density; very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) (Biggerstaff & Wooten, 2004). All the lipoproteins carry cholesterol, but elevated levels of the lipoproteins other than HDL (termed non-HDL cholesterol), particularly LDL-cholesterol is associated with an increased risk of atherosclerosis and coronary heart disease (Carmena, Duriez & Fruchart, 2004). Dietary intervention trials have shown that the reduction of serum total and LDL cholesterol concentration is beneficial for the reduction of coronary atherosclerosis in people with or without coronary heart disease (Gould, 1992; Ornish, 1998). Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels (arteries and veins) (Maton, 1993). It refers to any disease that affects the cardiovascular system, principally cardiac disease, vascular diseases of the brain and kidney, and peripheral arterial disease (Kelly & Fuster, 2010). According to the World Health Organization, chronic diseases are responsible for 63% of all deaths in the world, with cardiovascular disease as the leading cause of death (WHO, 2011). An estimated 17.3 million people died from CVD’s in
2008. Over 80% of CVD deaths take place in low- and middle-income countries. By 2030, almost 23.6 million people will die from CVDs (WHO, 2011). There are many  causes of cardiovascular  disease but the most common are atherosclerosis  and/or hypertension.  The different  types  of  cardiovascular   disease  include  coronary  heart  disease   (CHD), cardiomyopathy,   hypertensive  heart  disease,  heart  failure,  cor  pulmonare,   cardiac dysrhythmias,  inflammatory  heart  disease  (endocarditis,  inflammatory  cardiomegaly,  and myocarditis),  valvular  heart disease, stroke or  cerebrovascular  accident, peripheral  arterial disease. CVD typically involves the coronary arteries and thus is frequently termed coronary heart disease (CHD) or  coronary artery disease (CAD). The risk factors of cardiovascular disease  are  in  addition  to  high  fat  diet;  age,  gender,  high  blood  pressure,  high  blood cholesterol levels, tobacco, smoking, excessive alcohol consumption, family history, obesity, lack of physical activity, psychosocial factors and diabetes mellitus (Kelly & Fuster, 2010). Evidence shows that the Mediterranean diet improves cardiovascular  outcomes (Walker  & Reamy,  2009).  The  Mediterranean  diet  is  made  up of high  olive  oil consumption,  high consumption of legumes, unrefined cereals, fruits, and vegetables, moderate consumption of dairy products, and moderate to high consumption of fish, low consumption of meat and meat products  and  moderate  wine  consumption.  A 10-year  study published  in the  Journal  of American Medical Association (JAMA) found that  adherence to a Mediterranean diet and healthful  lifestyle  was  associated  with  more  than  a  50%  lowering  of  early  death  rates (Knoops et al., 2004).
Legumes have benefits in terms of reduced risk of CHD. Legumes or dry beans have been shown to improve  serum lipid profiles  in patients with CHD (Anderson &  Major, 2002; Bazzano,  Thompson,  Tees,  Nguyen  & Winham,  2009)  and  a growing  body of evidence supports the positive effects dietary legume consumption confers on  health, particularly in relation  to risk of CHD.  Epidemiological  studies  support the  cardio protective  effects  of legumes as part of a healthy diet. In particular, one study examined the relationship between beans consumption and occurrence of CVD and reported that 1 serving per day of beans was associated  with  a  38%  lower  risk  of  myocardial  infarction  (Kabagambe,  Baylin,  Ruiz- Narvarez, Siles & Campos, 2005). A second study by Bazzano, He & Ogden (2001) reported that individuals consuming legumes at least four times per week had a  22% lower risk of heart  disease  than  individuals  consuming  legumes  less than  once  per week.  Majority of legume studies have examined the relationship  between soybeans and heart disease. Some studies using navy beans and chickpeas have been conducted. In an early report, Anderson and Major  (2002) demonstrated  that  consumption  of navy beans  in tomato  sauce (baked beans)  for  21  days  decreased  serum  total  cholesterol  (TC)  and  low-density  lipoprotein cholesterol  (LDL-C)  concentrations  in  hypercholesterolemic  men  (Anderson,  Gustafson, Spencer, Tetyen & Bryant, 1990). A research by Finley, Burrel, and Reeves (2007) reported that pinto beans consumption is also favourable. Cowpea commonly consumed in Nigeria has been  linked  to  present  significant  reductions  in  plasma  total  cholesterol  and  non-HDL cholesterol (Frota, Mendonca, Saldiva, Cruz, & Areas, 2008).  As Liu (2006) reported, diets with low glycemic index are associated with a reduced risk for the development of diabetes mellitus, obesity and cardiovascular disease.
All legumes contain phytochemicals which play metabolic roles in humans who frequently consume these foods. Phytochemicals, also referred to as phytonutrients, are found in fruits, vegetables, whole grains, legumes, beans, herbs, spices, nuts, and seeds and are classified according to their chemical structures and functional properties. Phytochemicals are non- nutritive plant chemicals that have protective or disease preventive properties. There are many phytochemicals and each works differently. Presence of phytochemical components such as phytohemagglutinins, tannins, phytic acid, saponins, protease inhibitors, oligosaccharides and phytoestrogens in food legumes has both health benefits and adverse effects. These are some possible action; antioxidant, hormonal action, stimulation of enzymes, interference with DNA replication, anti-bacterial effect and physical actions. These
biologically active compounds in food legumes also have immense potential in biomedical application. On the other hand, phytochemicals have adverse effects as they limit the digestibility of proteins and carbohydrates or reduce the bioavailability of certain nutrients, interfere with normal growth, reproduction and flatulence production. Moreover, phytoestrogens have been linked with infertility problems. The synergistic or antagonistic effects of mixtures of these phytochemicals from food legumes, their interaction with other components of the diet and the mechanism of their action have remained a challenge with regard to understanding the role of phytochemicals in health and diseases. These have been associated with numerous health benefits, including reduced cardiovascular and renal disease risks, health care treatments including anti-aging, enhancement of brain function, and lower glycemic index for persons with diabetes, increased satiation and cancer prevention. Dietary intake of phytochemicals may provide health benefits, protecting against numerous diseases or disorders, such as coronary heart disease, diabetes, high blood pressure and inflammation (Bouchenak & Lamri-Senhadj, 2013).
Phytic acid (phytate), a type of phytochemical classified as anti-nutrient is found in many types of plant foods, such as grains, legumes (including peanuts and soybeans), nuts, and seeds. In legumes and seeds, phytic acid resides almost entirely in the endosperm (Schlemmer, Frolich, Prieto & Grases, 2009). Phytic acid has shown some capacity to reduce cholesterol and triglycerides, and positively impact the glycemic response of certain foods (Lee, Park and Chun, 2007). In some cases, phytic acid seems to have an ability to slow down a potential blood sugar spike following the ingestion of certain high-carbohydrate foods. Again, this may explain why high-fiber foods have been associated with improved blood sugar control. The potential benefits of phytic acid occur in instances with high dietary phytic acid intake. However, a high intake has also been associated with reduced mineral absorption. So, in order for us to get the best of both worlds, it’s important to discover some ways in
which we can minimize the negative effects while maximizing the beneficial effects. One way we can do this (specifically in regards to iron) is by incorporating more vitamin C (ascorbic acid) into our diet. These two work well together, with vitamin C placing iron in a chemical state that is more readily absorbed by the body (Hummers and Offeman,
1958).Preparation methods such as soaking, germinating, or fermenting can be very effective in reducing the amount phytic acid present in foods.
Tannin is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids. Tannins have traditionally been considered anti-nutritional but it is now known that their beneficial or anti-nutritional properties depend upon their chemical structure and dosage. Recent studies have demonstrated that products containing chestnut tannins included at low dosages (0.15–
0.2%) in the diet of chickens may be beneficial (Schiavone, Guo and Tassone, 2008). Most legumes contain tannins. Red-coloured beans contain the most tannins, and white-coloured beans have the least (Reed, 1995). There is a growing consensus for the hypothesis that the specific intake of food and drink containing relatively high concentrations of polyphenols may play a meaningful role in reducing the risk of cardiovascular disease (Ashutosh, 2003).
Saponins are a class of chemical compounds found in abundance in various plant species. Saponins are attracting considerable interest as a result of their diverse properties, both deleterious and beneficial. Clinical studies have suggested that these health-promoting components, saponins, affect the immune system in ways that help to protect the human body against cancers, and also lower cholesterol levels. Saponins decrease blood lipids, lower cancer risks, and lower blood glucose response. The detergent qualities of saponins allow them to bind to bile and prevent its reabsorption. Once bound to saponins, cholesterol leaves the body in waste. A lower cholesterol level means less risk of heart attack or stroke.
Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow, their color in nature) are a class of plant secondary metabolites. Flavonoids were referred to as Vitamin P (Benthsath, Rusznyak and Szent-Gyorgyi, 1937) probably because of the effect they had on the permeability of vascular capillaries. Flavonoids have been shown to have a wide range of biological and pharmacological activities in in vitro studies. Examples include anti-allergic, anti-inflammatory, antioxidant, anti-microbial (antibacterial, antifungal, and antiviral), anti- cancer, and anti-diarrheal activities (Yamamoto and Gaynor, 2001; Cushnie and Lamb, 2011; Friedman, 2007; Manner et al., 2013). Inflammation has been implicated as a possible origin of numerous local and systemic diseases, such as cancer, cardiovascular disorders, diabetes mellitus, and celiac disease (Manach, Mazur and Scalbert, 2005; Babu, Liu and Gilbert, 2013; Ravishankar, Rajora, Greco and Osborn, 2013).
Preliminary studies indicate that flavonoids may affect anti-inflammatory mechanisms via their ability to inhibit reactive oxygen or nitrogen compounds (Izzi et al., 2012). Flavonoids have also been proposed to inhibit the pro-inflammatory activity of enzymes involved in free radical production, such as cyclooxygenase, lipoxygenase or inducible nitric oxide synthase, and to modify intracellular signaling pathways in immune cells (Izzi et al., 2012). Procyanidins, a class of flavonoids, have been shown in preliminary research to have anti- inflammatory mechanisms including modulation of the arachidonic acid pathway, inhibition of gene transcription, protein expression and activity of inflammatory enzymes, as well as secretion of anti-inflammatory mediators (Martinez-Micalo et al., 2012). Among the most intensively studied of general human disorders possibly affected by dietary flavonoids, preliminary cardiovascular disease research has revealed the following mechanisms under investigation in patients or normal subjects (van Dam, Naidoo and Landberg, 2013; Tangney and Rasmussen, 2013):
inhibit coagulation, thrombus formation or platelet aggregation
reduce risk of atherosclerosis
reduce arterial blood pressure and risk of hypertension
reduce oxidative stress and related signaling pathways in blood vessel cells
modify vascular inflammatory mechanisms
improve endothelial and capillary function
modify blood lipid levels
regulate carbohydrate and glucose metabolism
modify mechanisms of aging
Other components of the legume such as the protein, carbohydrate, fat, and dietary fibre have also been shown to improve CVD risk factors. The low content of saturated fatty acids and high content of fiber make pulses a good choice for a healthy diet. Dry beans are essentially fat-free and act to displace fat from the diet. Many of them have moderate amounts of oil that is predominantly unsaturated. Their complex carbohydrates together with dietary fiber result in low glycemic indexes of legumes and legume products, and play an important role in the prevention of diabetes. Dry beans contain a mixture of soluble fiber, which significantly lowers cholesterol and blood sugar concentrations, and insoluble fiber, which aids in gastrointestinal function. Dietary fiber has major protective effects against atherosclerotic cardiovascular disease (Anderson, Deakins, Floore, Smith & Whitis, 1990). Epidemiologic data suggest that intake of complex carbohydrates and dietary fiber is inversely related to coronary artery disease (Anderson et al., 1990). Dietary fiber intake also slows development of atherosclerosis in animal models. Whereas soluble fiber clearly decreases serum cholesterol and LDL-cholesterol concentrations (Anderson et al., 1990), the inverse relation between dietary fiber intake and coronary artery disease appears to be independent of serum cholesterol concentration. Findings from epidemiologic studies show a significant
relationship between increased protein intake and lower risk of hypertension and coronary heart disease. In experimental animal models feeding a variety of different plant proteins is associated with lower serum cholesterol concentrations than with feeding a variety of different animal proteins (Carroll, 1982). Legume proteins appear to have specific hypocholesterolemic effects. Less is known about the effects of other commonly consumed varieties, especially African yam beans (AYB) on heart diseases (Anderson & Major, 2002; Anderson, Smith, & Washnock, 1999).
African Yam bean (AYB) (Sphenostylis stenocarpa) is an herbaceous leguminous plant occurring throughout tropical Africa (Porter, 1992) and grows during the wet season (Allen & Allen, 1981). It is often cited among the lesser-known and underexploited species (Ene- Obong & Okoye, 1992; Bennett-Lartey, Ayensu, Monma, & Ito, 1993). It is cultivated both for the seeds and tubers because of its valuable and prominent source as plant protein (Nwodo & Nwinyi, 2012). Okeke, Ene-Obong, Uzoegbuna, Ozioko and Kuhnlein (2008) reported that AYB has medicinal value. According to Ngwu, Ndiokwelu, Ibaro and Nwachi (2012), AYB showed a significant reduction in the fasting blood sugar, blood pressure, appetite, urination, foot numbness and itching of diabetics in a rural community. Uguru and Madukaife (2001) who did a nutritional evaluation of forty-four genotypes of AYB reported that the crop is well balanced in essential amino acids and has higher amino acid content than pigeon pea, cowpea and bambara groundnut. The nutritional values of its whole and dehulled seeds have been reported (Masdac & Jerums, 2004). Nutritionally, AYB compares favourably with the cowpea bean which has been found to reduce blood cholesterol. Cowpea bean contains 53.3% carbohydrates, 25.9% proteins, and 2% fat (Frota et al., 2008) while AYB has
62.6% carbohydrate, 19.5% protein and 2.5% fat (Okigbo, 1973). The aim of this study is to investigate the proximate and phytochemical constituents of AYB and its effect on hypercholesterolemia.
1.2 Statement of the Problem
This study is hinged on the problem of hypercholesterolemia and its adverse effects. Little effort has been made on the management of hypercholesterolemia through diet diversification programs especially using indigenous crops. A diet-based systemic approach to abolish these health disparities is desirable because it will assure the long-term solutions against the existing menace (Butt, Shahzadi, Sharif & Nasir, 2007). Recent studies suggest plant-based diets may be more effective for weight loss, blood sugar control, and reduction of cardiovascular risk factors, particularly blood cholesterol. Legumes have benefits in terms of reduced risk of coronary heart disease due to their many components such as phytochemicals. There are many phytochemicals and each work differently. The synergistic or antagonistic effects of mixtures of these phytochemicals from food legumes, their interaction with other components of the diet and the mechanism of their action have remained a challenge with regard to understanding the role of phytochemicals in health and diseases. Dietary intake of phytochemicals may provide health benefits, protecting against numerous diseases or disorders, such as coronary heart disease, diabetes, high blood pressure and inflammation (Bouchenak & Lamri-Senhadj, 2013).
High levels of lipids in the form of cholesterol  (LDL)  cause  heart disease,  stroke,  poor circulation and kidney disease. High levels of lipids in the blood are due to the consumption of foods high in fat, or due to an inherited  disorder.  It could also be  caused by medical conditions  such  as  diabetes,  obesity,  hyperthyroidism,  alcoholism,  kidney  disease,  liver disease and stress. The best way to reduce lipid levels in the blood is to eat less fat, exercise regularly and loose weight if necessary (American Academy of Family Physicians, 1998). AYB is highly nutritious with low fat, high protein, mineral and dietary fiber (Ene-Obong & Carnovale, 1992). The potential role of AYB consumption on the prevention and treatment of chronic diet-related non communicable diseases such as obesity, diabetes, hypertension and cardiovascular diseases has been suggested (Oshodi, Ipinmoroti & Adeyeye, 1997; Onyechi & Nwachi, 2008; Alozie, Udofia, Lawal & Ani, 2009).
This study seeks to determine the proximate and phytochemical composition of AYB and its effect on hypercholesterolemic rats.
1.3 Objective of the study
1.3.1 General Objective: The broad objective of the study is to determine the proximate and phytochemical composition of African Yam Bean and its effect on the lipid profile of hypercholesterolemic rats.
1.3.2 Specific Objectives: The specific objectives of the study were to:
1. assess the proximate composition of the flour;
2. assess some phytochemical constituents of the flour (saponins, phytate, tannins and flavonoid);
3. determine the serum total cholesterol, triglyceride, low density lipoprotein, and high density lipoprotein of adult Wister rats and
4. determine the effect of roasted AYB flour on the lipid profile of the adult Wister rats.
1.4 Significance of the study
The result of this study will generate scientific evidence that will be used by dietitians and other health care professionals in dietary counseling and for prevention and management of non communicable diseases (NCDs). This will be a means of diversification and upgrading AYB. It will provide basic data and information for sustainable commercial exploitation and increased use of the legume. The findings would increase the food use of AYB which will consequently increase the production of AYB by farmers and strengthen the food security of the people. It will create more areas of research based on AYB. It will also revitalize the place of AYB in the food system.
This material content is developed to serve as a GUIDE for students to conduct academic research
PROXIMATE AND PHYTOCHEMICAL COMPOSITION OF AFRICAN YAM BEAN (SPHENOSTYLIS STENOCARPA) AND ITS EFFECT ON THE LIPID PROFILE OF HYPERCHOLESTEROLEMIC RATS>
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