This study aimed at investigating the pathophysiological effects of crude seed powders of Irvingia gabonensis and Irvingia wombolu combined in different proportions on alloxan-induced diabetic rats. Both species of Irvingia were analysed for phytochemical content. A total of 72 male albino rats were used for the experiment. A latin square design of six treatment groups replicated thrice, with each replicate having 4 albino rats each was used for the experiment. Diabetes was induced in 5 groups of the experimental animals by the intraperitoneal administration of alloxan in a dose of 120mg/kg body weight, while one group served as the positive control. Crude seeds of Irvingia gabonensis and Irvingia wombolu combined in 3 different proportions (80% I.gabonensis: 20% I.wombolu (IgIw1), 20% I. gabonensis: 80% I. wombolu(IgIw2) and 50% I. gabonensis : 50% I.wombolu (IgIw3)) were administered to 3 groups of the diabetic animals while the remaining 2 diabetic groups served as the diabetic and standard drug control. Body weight, blood glucose levels and serum levels of low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and malondialdehyde (MDA) were monitored at 7 days interval for 21 days. Pancreas, kidney and liver were harvested from a rat in each group at the end of the experiment for histological studies. Phytochemical analysis of both species of Irvingia reveals that I. wombolu has more flavonoids, alkaloids, glycosides, terpenoids and steroids than I. gabonensis. There was no significant difference (P < 0.05) in the body weight of the diabetic animals compared to the positive control. Administration of combinations crude seed powders of Irvingia gabonensis and Irvingia wombolu and the standard drug glibenclamide significantly (P < 0.05) lowered blood glucose levels of the diabetic rats. Serum TC, TG and LDL-C decreased significantly (P < 0.05) while HDL-C increased significantly (P < 0.05) in the treated animals compared to the diabetic control. There was also a significant decrease (P < 0.05) in serum levels of ALT, AST and ALP of the treated rats compared to the diabetic control. However, there was no significant difference (P < 0.05) between the serum levels of MDA of the treated animals and the diabetic control. The histopathological damage on the pancreas, kidney and liver of the diabetic rats was not entirely revised within the period of experiment. This study therefore suggests that Irvingia gabonensis and Irvingia wombolu combined has blood glucose lowering effect and may also protect against dislipidemia. Hence, Irvingia gabonensis and Irvingia wombolu may be used as ingredients in health and functional food to ameliorate diabetes.
TABLE OF CONTENTS
Title page – – – – – – – – – – -i
Approval page – – – – – – – – – – -ii
Dedication – – – – – – – – – – -iii
Acknowledgement – – – – – – – – – -iv
Table of Contents – – – – – – – – – vi
List of Tables – – – – – – – – – – ix
List of Figures – – – – – – – – – x
List of Plates – – – – – – – – – – xi
Abstract – – – – – – – – – – xii
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction – – – – – – – – – -1
1.1.2 Justification of Study – – – – – – – – -6
1.1.3 Objectives of Study – – – – – – – – -7
1.2 Literature Review – – – – – – – – -8
1.2.1 Medicinal Plants – – – – – – – – – -8
1.2.2 Food Plants and their biochemical components – – – – -10
1.2.3 Biological Activities of Phytochemicals – – – – – -11
1.2.4 Anti-diabetic phytochemicals – – – – – – -13
1.4.5 Aetiology and Pathophysiology of Diabetes Mellitus – – – -15
1.2. 5.1 Aetiological Classification of Diabetes Mellitus – – – – – -17
1.2.6 The Genus Irvingia – – – – – – – – – 19
1.2. 6.1 Ecology and Biology of Irvingia species – – – – – -22
1.2. 6.2 Morphological Traits and their Variation – – – – – -23
1.4.7 Bioactive Constituents and Health Effects of Irvingia Species – – -23
1.4.8 Mechanism of Alloxan Action – – – – – – – -28
CHAPTER TWO: MATERIALS AND METHODS
2.1 Procurement of Irvingia Seeds – – – – – – -32
2.2 Procurement of Experimental Drugs – – – – – – -32
2.3 Procurement and Management of Experimental Animals – – – -32
2.4 Preparation of Irvingia Seeds – – – – – – – -33
2.5 Phytochemical Analysis – – – – – – – -33
2.6 Toxicity Test – – – – – – – – – -35
2.7 Diabetes Induction in Rats – – – – – – – -35
2.8 Experimental Design – – – – – – – – -36
2.9 Blood Sample Collection – – – – – – – -38
2.10 Fasting Blood Glucose Analysis – – – – – – -38
2.11 Serum Lipid Profile – – – – – – – – -38
2.12 Liver Enzyme Assay – – – – – – – -40
2.13 Assessment of Malondialdehyde (MDA) concentration of plasma – – – 41
2.14 Histopathological Studies of the Pancreas, Kidney and Liver – – -41
Statistical Analysis – – – – – – – – – -41
CHAPTER THREE: RESULTS
3.1 Qualitative and Quantitative Phytochemical Composition of Crude Seed Powder of Irvingia gabonensis – – – – – – – – – -42
3.2 Qualitative and Quantitative Phytochemical Composition of Crude Seed Powder of Irvingia wombolu – – – – – – – – – -42
3.3 Acute Toxicity Test of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu – – – – – – – – – -45
3.4 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on the Body Weights (BWs) of Alloxan-Induced Diabetic Rats – – -45 – –
- 5 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Fasting Blood Glucose levels (FBGL) of Alloxan-Induced Diabetic Rats -46 – –
- 6 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Total Cholesterol (TC) levels of Alloxan-Induced Diabetic Rats – -46
- 7 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on High Density Lipoprotein Cholesterol (HDL-C) levels of Alloxan-Induced Diabetic Rats – – – – – – – – – -47
- 8 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Total Triglyceride (TG) levels of Alloxan-Induced Diabetic Rats – -51
- 9 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Low Density Lipoprotein-Cholesterol (LDL-C) levels of Alloxan-Induced Diabetic Rats – – – – – – – – – – -51
- 10 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Alanine Aminotransferase (ALT) levels of Alloxan-Induced Diabetic Rats -52
- 11 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Aspartate Aminotransferase (AST) levels of Alloxan-Induced Diabetic Rats -54
- 12 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Alkaline Phosphatase (ALP) levels of Alloxan-Induced Diabetic Rats – -54
- 13 Effects of Combinations of Crude Seed Powders of Irvingia gabonensis and Irvingia wombolu on Malondialdehyde (MDA) levels of Alloxan-Induced Diabetic Rats – -56
3.14 Histopathological Features of Pancreases, Kidneys and Liver in Alloxan-Induced Diabetic Rats – – – – – – – – – -58
CHAPTER FOUR: DISCUSSION
4.1 Discussion – – – – – – – – – 82
INTRODUCTION AND LITERATURE REVIEW
Glucose is an indispensable fuel for the brain and other tissues (Whitney et al., 2007). However, chronic amounts of circulating glucose cause toxic effects on the structure and function of organs, including pancreatic islets (Matsinkou et al., 2012). Therefore, to regulate glucose in the body, the body produces insulin to drive glucose into cells for use or storage; and glucagon, epinephrine and other hormones to bring glucose out of storage again for use during extreme starvation. Chief among these hormones produced for glucose regulation is insulin; which is produced by the beta cells of the pancreatic islets of Langerhans. Insufficient production or resistance of body cells to insulin results in a disease known as diabetes mellitus; characterized mainly by hyperglycemia (chronic amounts of circulating glucose) and most times hyperlipidemia (chronic amounts of ‘bad’ fats in circulation) (Kavishankar et al., 2011; Matsinkou et al,. 2012; Saravanamuttu and Sudarsanam, 2012).
Diabetes mellitus is a lifestyle disorder that is rapidly becoming a major threat to populations all over the globe. Over the past 30 years, the status of diabetes has changed from being considered as a mild lifestyle disorder of the elderly to one of the major causes of morbidity and mortality, affecting people of all ages (Saravanamuttu and Sudarsanam, 2012). Diabetes mellitus is one of the most common health problems worldwide (Shukla et al. 2012), and the prevalence of this disease is rapidly increasing, leading to microvascular (retinopathy, neuropathy and nephropathy) and macrovascular (heart attack, stroke and peripheral vascular disease) complications (Umar et al., 2010). The number of individuals with diabetes is increasing due to population growth, aging, urbanization and increasing prevalence of obesity and physical inactivity (Firdous, 2014).
Essentially, insulin deficiency results in faulty glucose utilization, causes hyperglycemia and mobilization of fatty acids from adipose tissue. In diabetes blood glucose is not utilized by the tissues and this condition leads to hyperglycemia. Hyperglycemia in diabetes is associated with long term damage, dysfunction and failure of various organs (Lyra et al., 2006). The fatty acids from adipose tissue are mobilized for energy purpose, as a result of inability of the cells to make use of insulin. Hence, excess fatty acids are accumulated in the liver, which are converted to triglyceride (Shih et al., 1997). Accumulation of triglyceride leads to increase in the formation of low density lipoproteins (LDL) and a reduction in high density lipoproteins (HDL).
Multiple biochemical pathways and mechanisms of action of glucose toxicity have been suggested. Reactive oxygen species (ROS) attack unsaturated fatty acids of the membrane phospholipids to initiate lipid peroxidation, causing severe damages to the membrane structure with sequential fluidity. Diabetes mellitus is usually accompanied by impaired antioxidant capacity (Matsinkou et al., 2012). There is a considerable evidence that hyperglycemia results in the generation of ROS (Punitha et al., 2006), ultimately leading to increased oxidative stress in a variety of tissues (Evans et al., 2002), variations in its fluidity and ability to function correctly (Shukla et al., 2012). In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes overwhelmed (redox imbalance), leading to the activation of stress-sensitive intracellular signaling pathways (Evans et al., 2002). All of these pathways produce ROS in excess and which over time causes chronic oxidative stress, which in turn causes defective insulin gene expression and insulin secretion as well as increased apoptosis (programmed cell death) of the beta cells (Robertson, 2004).
Free radicals are formed disproportionately in diabetes by glucose oxidation, non enzymatic glycation of proteins and subsequent oxidative degradation of glycation proteins (Kangralkar et al., 2010). These free radicals produced as a result of normal biochemical reactions in the body, are implicated in contributing to cancer, atherosclerosis, aging, immunosuppression, inflammation, ischemic heart disease, hair loss and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease (Olutayo et al., 2013).
Although the body possesses innate defence mechanisms to counter free radicals in the form of enzymes such as superoxides dismutate (SOD), catalase and glutathione peroxidase, this increased free radical generation along with declined antioxidant defense system may damage enzymes, cellular organelles, which leads to lipid peroxidation (Kangralkar et al., 2010). All these activities can cause or exacerbate diabetes mellitus. This may happen as a result of decreased insulin production (Type I) or insufficient insulin utilization (Type II) (Marshal and Bangert, 2004; Matsinkou et al,. 2012). Insulin deficiency contributes to increased serum levels of transaminase enzymes due to easy availability of amino acids which leads to enhanced occurrence of gluconeogenesis and ketogenesis processes during diabetes inducing hyperglycemia and hyperlipidemia (Punitha et al,. 2006, Dzuefiet et al., 2009; Shukla et al., 2012).
The hyperlipidemic condition certainly contributes to a major risk factor for atherosclerosis and cardio vascular diseases (Punitha et al., 2006). The saturated fatty acids present in fat could increase the production of triglycerides and cholesterols by the liver and could decrease the catabolism of LDLs by the repression of their receptors (Shukla et al., 2012). Actually, insulin deficiency inactivates the lipoprotein lipase, which promotes the conversion of free fatty acid into phospholipids and cholesterol in the liver, and they finally get discharged into the blood resulting into elevated serum phospholipids.
It is believed that any medicinal plant that can work as a potential antioxidant together with having anti-diabetic property could prevent or reduce diabetic complication more effectively than the conventionally used anti-diabetic drug (Bhattaram et al., 2002). This is because most synthetic drugs have been associated with so many side effects. Plant secondary metabolites such as flavonoids and terpenoids etc. have been shown to play important role in the defence against free radicals (Devasagayan and Sainis, 2002; Govindarajan et al., 2005). Hence, several plants have been found useful in ameliorating diabetic complication.
Currentlly available therapy for diabetes includes insulin and various oral hypoglycemic agents such as sulfonylureas, metformin, glucosidasae inhibitors, troglitazone, etc. But these are reported to produce serious adverse side effects such as liver problems, lactic acidosis and diarrhea (Ngondi et al., 2006). In addition they are not suitable for use during pregnancy (Kangralkar et al. 2010). More so, although many anti-diabetic drugs are already available which have been commercially used by diabetic people, none of these have the dual properties of reducing blood glucose level and scavenging free radicals. Moreover the side effects and cost of the drugs are not affordable by all. Again, taking too much insulin can cause hyperglycemia (Whitney et al. 2007), by causing blood glucose level to fall so rapidly that it triggers the liver to release counter regulatory hormones (insulin antagonists) which may raise the blood glucose higher than the available insulin can handle.
Many traditional treatments for diabetes are used throughout the world (Ngondi et al,. 2006; Oduola et al., 2007). Some medicinal plants have been reported to be useful in diabetes treatment and have been used empirically as antihyperglycemic and antihyperlipidemic remedies (Bhattaram et al,. 2002). Most of these plants contain glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc. that have been shown to have antidiabetic effects (Loew and Kaszkin, 2002; Haque et al., 2012; Firdous et al., 2014). The attributed antihyperglycemic effect of the plants is due to their ability to restore the function of the pancreatic tissues by causing an increase in insulin output or by inhibiting the intestinal absorption of glucose or to increase the facilitation of metabolites in insulin dependent process (Ngondi et al., 2006).
Irvingia gabonensis and Irvingia wombolu are highly valuable and extensively utilised tropical African trees. They are local fruit trees with wide distribution across West and Central Africa. Irvingia kernels are used in soup making as they form an important part of the West and Central African diet. The yellow fruit pulp of Irvingia gabonensis is edible having yellow pulp. However, the fruit pulp of Irvingia. wombolu, is bitter and has turpentine taste, so it is not edible. Fat extracted from the kernels can be used for food applications, such as in margarine or cooking oil, and is also suitable for soap, cosmetics and pharmaceuticals. Flour can be produced from the kernels. The seeds contain oil used in different culinary purposes. The wood is hardy and has resistance to termites. The bark is green in colour and is used as medicine for arthritis, rheumatism, dropsy, swellings, oedema, gout, eye treatment, fabrifuges, stomach trouble and venereal diseases (Singh, 2007). The only part of the Irvingia plant that doesn’t seem to have other medicinal application is its pulp.
Fractions of Irvingia gabonensis seeds were reported to have hypoglycemic effect (Ngondi et al., 2006; Dzuefiet et al., 2009; Omonkhua and Onoagbe, 2011).
Fruits of both Irvingia species possessed all five phytochemicals (alkaloids, flavonoids, saponins, tannins and glucosides). However, whilst both species had the same amounts of flavonoids and glycosides, I. wombolu possessed relatively higher amounts of alkaloids, saponins and tannins than I. gabonensis. Irvingia wombolu may be the preferred choice if domestication would be based on phytochemicals. In like manner, I. gabonensis may be the preferred choice for domestication if taste, weight and size of fruits are the parameters of interest (Etebu, 2013).
Several researches have been carried out to check for the hypolipidemic and hypoglycemic effects of both species of Irvingia (Ngondi et al., 2005, 2006, 2009). The results of these researches showed that Irvingia species have antidiabetic effects. However, a comparison of the nutritive qualities of the kernels of both species conducted by Ndoye et al. (1997) indicated that I. wombolu is more energy-rich due to its higher percentage fat although both species are a good source of oil. The fat from I. wombolu kernel has lower iodine and saponification values. Therefore, there is a possibility that the differences in percentage of phytochemical composition of both species could lead to differences in their degree of biochemical activities in the body. Hence, the aim of this present research is to evaluate the pathophysiological effects of various fractions obtained from combinations of the seeds of Irvingia gabonensis and Irvingia wombolu harvested in Nigeria, on alloxan-induced diabetic rats.
1.1.2 Justification of study
This work investigated the hypoglycemic and hypolipidemic effect of the two species of Irvingia found in Nigeria. Two major factors that lead to diabetes includes; genetic factors and lifestyle. Oftentimes therefore, diabetes is usually managed to maintain blood glucose level at a healthy range and prevent further diabetic complications. The three basic management procedures for diabetes include diet, exercise and medication. Hence, there has been increase in the search for both medicinal and food plants that can be used to manage diabetes with little or no side effects. Food plants with antidiabetic effects are very useful in diabetes management, since they can easily be added in daily meal, eschewing the phobia of using only drugs and the side effects of injecting excessive insulin after a meal.
Irvingia gabonensis and Irvingia wombolu are highly valuable and extensively used tropical African trees. Irvingia seeds are used in soup making as they form an important part of West and Central African diets. It has been reported from several studies that various parts of Irvingia species harvested from other West African countries like Cameroun have hypoglycemic and hypolipidemic effects. However, it has also been discovered that both species of Irvingia vary in both phytochemical and proximate content. Hence, there is need to evaluate the pathophysiological effects of various combinations of crude seed powders of Irvingia gabonensis and Irvingia wombolu harvested in Nigeria, and compare this with glibenclamide, a standard diabetic drug, so as to determine the best and as well help dieticians in planning adequate diet management for diabetics.
1.1.3 Objectives of study
The objectives of the study were to determine the effects of combinations of crude seed powders of I. gabonensis and I. wombolu combined on:
- fasting blood glucose level in alloxan induced diabetic rats,
- body weight in alloxan induced diabetic rats,
- serum lipid profile (total cholesterol, triglycerides, low density lipoprotein (LDL), and high density lipoprotein (HDL)) in alloxan induced diabetic rats,
- liver enzyme activity in alloxan induced diabetic rats,
- malondialdehyde (MDA) concentration of plasma, liver and kidney and
- histopathology of the pancreas, liver and kidney in alloxan induced diabetic rats.
1.4 Literature Review
1.2.1 Medicinal plants
Medicinal plants are those plants (parts, extract etc) that are used in treating and preventing specific ailments and diseases that affect human beings (Nwachukwu et al. 2010). Hence the important role of medicinal plants in health care delivery (services) cannot be over emphasized. Demand for medicinal plants is increasing in both developing and developed countries. Hence, research on medicinal plants is one of the leading areas of research globally (Soetan et al. 2009; Omonkhua and Onoagbe, 2012; Olutayo et al. 2013).
The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. Most plant parts (extract) identified e.g. ( bark root, seeds, fruit, leaf) serve as major source of active ingredient and products of secondary metabolites like alkaloid, terpenoids etc used in curing diseases, production of drugs as well as in maintaining good health by both the traditional and orthodox medical practitioners, although it has been opined that plant leaves are about 51% more favorable for storing active ingredients, as compared to other parts of the medicinal plants (Mannan et al. 2014). Medicinal plants are either “wild plant species” those growing spontaneously in self maintaining populations in natural or semi-natural ecosystems and could exist independently of direct human actions or the contrasting “domesticated plants species” those that have arisen through human actions such as selection or breeding and depend on management for their existence.
Medicinal plants contain physiologically active prhytochemicals that over the years have been exploited in traditional medicine for the treatment of various ailments (Adebajo et al., 1983). The drugs contained in medicinal plants are known as active principles. Cowman (1999) and Banso and Olutimayin (2001) reported that plants contain a wide variety of active principles. In the case of most drugs, herbs, ethnomedicines, essential oils and cosmetics are derived from the secondary products of plant metabolism such as the alkaloids, terpenoids and flavaonoids (Alaribe, 2008). These substances have evolved as responses of plants to stress, predation and competition constituting to what is regarded as the vast chemical library of biological systems. It is usually “extracts” not the plants themselves or their parts such as fruits, seeds leaves etc; that are used for medicinal effects. However, medicinal plants possess what is referred to as pathological niche and they assume pathogenomic structure. This means that medicinal herbs can be used for different ailments with respect to its on human physiology.
Some examples of indigenous medicinal plants in Nigeria and their active parts, according to recent researches include; Azadirachta indica (neem/dogoyaro) leaves, roots and bark, Aspilia africana (haemorrhage plant) leaves, Gongronema latifolia (Utazi) leaves, stem, fruit and root, Costus afar (Ginger lily/Bush cane) leaves, Psidium guajava (Guava) leaves, stem, fruit and root, Carica papaya (Paw-paw) seeds (Eyo et al., 2013a)root, bark, and leaves, Vernonia amagdalina (Bitter leaf) leaves (Eyo et al., 2013b), Ocimum gratissimum (Scent leaf) leaves, (Nwachukwu et al., 2010 Eyo et al., 2014), Entandrophragma angolense Welw (Meliaceae) stem-bark, Khaya senegalensis Desr. (Meliaceae), Anogeissus leiocarpus (Combretaceae), Pavetta crassipes, K.Schum (Rubiaceae) flowers and Abrus precatorius Linn.(Leguminosaceae) (Olutayo et al., 2013), Allium cepa L. (onion): (Liliaceae), Allium sativum L. (garlic): (Liliaceae) (Kavishankar et al., 2011; Eyo et al., 2011; Ozougwu et al., 2014; Ozougwu and Eyo, 2014). These plants have been shown to contain active phytochemicals which are useful in curing many human diseases.
1.2.2 Food plants and their biochemical components
Plant foods have remained the ultimate source of nutrients for larger population of the world. They are simply described as irreplaceable food resources for humans, which exclude animal sources. These foods contain many chemical compounds needed for metabolic functions in varying proportions. Some of these chemical compounds are non-nutrients that are beneficial to man while some others provoke some adverse reactions (e.g oxalic acid found in Amaranthus species). However, the level of adverse effect produced by these non-nutrients depends on the levels of intake, interrelationships of nutrients and food habits. Plant foods are classified as cereals, roots and tubers, legumes, vegetables and fruits (Aremu and Ibrahim, 2014). According to the major nutrients they provide, plant foods are classified into three main categories namely; macronutrients (carbohydrate, protein, fat and water), micronutrients (minerals and vitamins) and non-nutrient components (dietary fibre, phytochemicals, anti-nutrients, food toxicants and additives) (Lutz and pryztulski, 2008).
The bioactive compounds or secondary metabolites are the non-nutrient components in plant foods. They have some nutritional effects and health benefits. They are those substances contained in foods which supply no nutrients. They could contain some compounds that are beneficial to health or toxic to humans and/or act as antagonists to nutrients in foods. These include tannins and other phenolic compounds (phenols, flavonoids, isoflavonoids), saponins, glucosinolates, alkaloids (Drewnowski and Gomez-Carneros, 2000), phytate and dietary fibre (Gibson, 2007). These chemical compounds are found in different classes and parts of plant foods in varying amounts. They are more concentrated in plant storage organs (leaves and seeds) than in other parts of the plants (Chan et al. 2012). These constituents have their individual health-promoting qualities that compel people to combine the different food sources to achieve healthy eating and maintain good health. Several authors have studied therapeutic potentials and metabolic effects of foods rich in dietary fibre and phytochemical constituents (Jenkins et al. 2003; McCarty, 2004; Soetan, 2008). These include lower risk of colon cancer (Ricciardiello et al., 2011), promotion of early satiety and normal laxation (Hossain et al., 2012), moderation of post-prandial blood glucose responses and improved insulin sensitivity (Ngondi et al., 2006; Hossain et al., 2012), reduction in total and low density lipoprotein (LDL)-cholesterol (Ngondi et al. 2006; Baldeon et al. 2012) and regulation of appetite and enhancement of sodium and fluid balance (Camargo et al., 2004). They are also used to treat constipation and prevent development of diverticulosis and diverticulitis (Wintola et al., 2010). Diets adequate in dietary fibre are usually rich in micronutrients and phytochemicals, and frequently less calorically dense and lower in fat and added sugars. However, environmental factors, cultural food habits and insufficient nutritional information about health benefits of traditional plant foods still pose a problem to healthy food choices. Drewnowski and Gomez-Carneros (2000) reported that most of the bioactive compounds are bitter, acrid or astringent and aversive to the consumer and may be wholly incompatible with consumer acceptance. These factors have caused increasing epidemic of diet-related diseases across the regions. They suggested the need to take sensory properties and food preferences into account when advocating for increased consumption and diversification of rich sources of these secondary metabolites in plant foods. The challenge of achieving adequate supply of energy and nutrient intake as well as the health-promoting compounds from plant-based foods/diets without compromising the health of an individual forms the basis for current dietary recommendations aimed at promoting consumption of plant foods to reduce diet-related non-communicable diseases.
1.2.3 Biological activities of phytochemicals
The most commonly encountered secondary metabolites of plants (phytochemicals) are saponins, tannins, flavonoids, alkaloids, anthraquinones, cardiac glycosides, cyanogenic glycosides, phlobatannins, resins, balsam and volatile oils (Soetan et al., 2008; Olutayo et al., 2013). The presence of these secondary metabolites in plants probably explains the various uses of plants for traditional medicine, because most of them play important role in the defence against free radicals. The pharmacological and other beneficial effects of antinutritional factors in plants have been reviewed by Soetan (2008). Plants rich in chemical constituents like phenols, coumarins, monoterpenes, glycosides, alkaloids and xanthenes have been found to be protective to the liver (Bhavna and Sharma, 2012; Ozougwu and Eyo, 2014; Ozougwu et al.,2014). Hence the hepatoprotective potency of the plant could be attributed to its antioxidant property.
Saponins are glycosides of both triterpenes and steroids having hypotensive and cardiac depressant properties (Olaleye, 2007). Saponins bind to cholesterol to form insoluble complexes. Dietary saponins in the gut of monogastrics combine with endogenous cholesterol excreted via the bile. This prevents cholesterol reabsorption and results in a reduction of serum cholesterol. Saponins have been found to be potentially useful for the treatment of hypercholesterolaemia which suggests that saponins might be acting by interfering with intestinal absorption of cholesterol.
Tannins are complex phenolic polymers which can bind to proteins and carbohydrates resulting in reduction in digestibility of these macromolecules and thus inhibition of microbial growth (Nwogu et al., 2008). Tannins from the bark, roots and other parts of many plants especially Euphorbiaceae are used to treat cells that have gone neoplastic. Tannins are also reported to have astringent properties on mucous membranes (Egunyomi et al., 2009).
Flavonoids are a group of phytochemicals found in varying amounts in foods and medicinal plants which have been shown to exert potent anti-oxidant activity against the superoxide radical. Its consumption has been documented not to be associated with mortality due to coronary heart disease. This may be as a result of its antioxidant activity and subsequent inhibitions of low density lipoproteins (LDL) oxidation known to have been attributed to the dietary and supplemental intake of flavonoids and other micronutrients. Epidemiologic studies indicate an inverse relationship between intake of dietary flavonoids and coronary artherosclerotic disease.
Alkaloids are basic natural products occurring primarily in many plants. They are generally found in the form of salts with organic acids and they are haemolytically active and are also toxic to microorganisms. Alkaloids, comprising a large group of nitrogenous compounds are widely used as therapeutic agents in the management of cancer. Alkaloids also interfere with cell division. An alkaloid isolated from Hibiscus sabdariffa demonstrated its ability to prevent mutagenesis.
Cardiac glycosides are cardioactive compounds belonging to triterpenoids class of compounds. Their inherent activity resides in the aglycone portions of their sugar attachment. Their clinical effects in cases of congestive heart failure are to increase the force of myocardiac contraction. They exert their hypotensive effect by inhibiting Na+ -K+ ATPase. They also act directly on the smooth muscle of the vascular system. They exert a number of effects on neural tissue and thus indirectly influence the mechanical and electrical activities of the heart and modify vascular resistance and capacitance (Olaleye, 2007).
1.2.4 Anti-diabetic phytochemicals
Plants can provide biologically active molecules and structural compounds for the development of modified derivatives with enhanced activity and reduced toxicity (Mannan et al., 2014). Plants have chemical compounds which demonstrate alternative and safe effects on diabetes mellitus. Most of plants contain glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc., that are frequently implicated as having antidiabetic effect (Malviya et al., 2010). Galega officinalis is a plant from which hypoglycemic drugs was obtained traditionally (oseph and Jini, 2011). Insulin, biguanides, sulfonylurease and thiazolinediones are mordern pharmacotherapeutics, but still except glycemic control with insulin, there is need to look for new drugs for more efficacious agents with less side effects is needed.
Several studies have shown that tannins and saponins found in most plants are the components responsible for their hypoglycemic effect (Ngondi et al., 2005, 2006; Omonkhua and Onoagbe, 2010; Eyo et al., 2011). The administration of different plant extracts in diabetic rats may act by a direct stimulation of insulin secretion in remaining β- cells. This effect could be attributed to compounds like glycosides, alkaloids, flavonoids, anthocyanin, tannins (Matsinkou et al., 2012). Their action may involve insulin-like extrapancreatic mechanisms such as the stimulation of glucose utilisation and the reduction of hepatic gluconeogenesis (Hossain et al., 2012).
Polyphenols have also been suggested to decrease the oxidative stress in human especially through inhibition of the LDL-cholesterol oxidation (Fuhrman and Aviram, 2001). Flavonoids found in the pulp extracts may inhibit the oxidative stress by: i) scavenging free radicals by acting as reducing agent, hydrogen atom donating molecules or singlet oxygen quenchers; ii) chelating metal ions; iii) sparing other antioxidants (e.g. carotene, vitamin C and E); and iv) preserving HDL associated serum paraoxonase activity (Fuhrman and Aviram, 2001). Antioxidant properties of polyphenols are related to their chemical structure and depend on the number and arrangement of their phenolic hydroxyl groups. The amount of phenolics varies considerably in the different pulp extracts.
Some plant constituents appear to be disease specific. Plants with considerable hypoglycaemic property have beenreported. Drewnowski and Gomez-Carneros (2000) and Noor et al (2013) reported phenols and polyphenols, flavonoids, isoflavones, terpenes and glucosinolates in vegetables and fruits. Several studies have published similar effects with dietary fibre (non-starch polysaccharides (NSPs)) (Jekins et al. 2003). A new classification of dietary fibre (water-soluble and insoluble dietary fibre) was based on their solubility characteristics (Gray, 2003). The soluble dietary fibre is highly viscous and has added viscosity as functional property in the evaluation food/diets. These NSPs lower blood glucose level by impeding glucose absorption from the gastrointestinal tract and reduce post-prandial hyperglycaemia (Hossain et al., 2012). The water-insoluble NSP are mainly obtained from structural carbohydrates (cellulose and lignin of the cell walls) of starchy roots/tubers and cereals. The water-soluble NSP are obtained from storage carbohydrates (gum and hemicellulose) of legumes and as pectin from fruits and vegetables (Busch, 2015).
Ngondi et al. (2006) observed that high fiber diet has been shown to work better in controlling diabetes. On the basis roles of phytochemicals and antioxidant constituents of plant foods, it is believed that they hold good promise for diabetes.
1.2.5 Aetiology and pathophysiology of diabetes
Diabetes mellitus is a chronic life-long disease, which has been known to mankind for over 2000 years. It requires careful monitoring and control. Diabetes is a chronic metabolic disorder, characterized by high blood glucose (hyperglycemia), associated with impaired carbohydrate, fat and protein metabolism, resulting from either insufficient or no release of insulin by pancreas in the body (American Diabetes Association, 2012).
Evans et al. (2002) has reported that pathogenesis of diabetes is better appreciated and should be discussed in line with hyperglycaemia and elevated free fatty acid in the blood in relation to oxidative stress and production of free radicals; where oxidative stress refers is the imbalance between production and removal of reactive oxygen species (ROS) and free radicals.
Diabetes is usually accompanied by impaired antioxidant capacity (Matsinkou et al., 2012). Several reports have shown that there are alterations in the endogenous antioxidant enzymes in diabetic condition (Preet et al., 2005), especially the anti-oxidative defense system, like superoxide dismutase and catalase, which are lowered in diabetic subjects. There is a considerable evidence that hyperglycemia results in the generation of reactive oxygen species (Punitha et al., 2006), ultimately leading to increased oxidative stress in a variety of tissues (Evans et al., 2002). ROS attack unsaturated fatty acids of the membrane phospholipids to initiate lipid peroxidation, causing severe damages to the membrane structure with sequential variations in its fluidity and ability to function correctly (Shukla et al., 2012). In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes overwhelmed (redox imbalance), leading to the activation of stress-sensitive intracellular signaling pathways (Evans et al., 2002). Multiple biochemical pathways and mechanisms of action of glucose toxicity have been suggested. All of these pathways produce ROS in excess and which over time causes chronic oxidative stress, which in turn causes defective insulin gene expression and insulin secretion as well as increased apoptosis (programmed cell death) (Robertson, 2004).
Again, free radicals are formed disproportionately in diabetes by glucose oxidation, non enzymatic glycation of proteins and subsequent oxidative degradation of glycation proteins (Kangralkar et al., 2010; Matsinkou et al., 2012). These free radicals, produced as a result of normal biochemical reactions in the body are implicated in contributing to cancer, atherosclerosis, aging, immunosuppression, inflammation, ischemic heart disease, diabetes, hair loss and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease (Olutayo et al., 2013). Although the body possesses innate defence mechanisms to counter free radicals in the form of enzymes such as superoxides dismutate, catalase and glutathione peroxidase, increased free radical generation along with declined antioxidant defense system may damage enzymes, cellular organelles, cause lipid peroxidation and increase diabetic abnormalities (Kangralkar et al., 2010). All these activities can cause or exacerbate diabetes. This happens as a result of decreased insulin production (Type I) or insufficient insulin utilization (Type II) (Marshal and Bangert, 2004; Matsinkou et al., 2012). Insulin deficiency contributes to increased serum levels of transaminase enzymes due to easy availability of amino acids which leads to enhanced occurrence of gluconeogenesis and ketogenesis processes during diabetes inducing hyperglycemia and hyperlipidemia (Punitha et al., 2006; Shukla et al., 2012).
The hyperlipidemic condition certainly contributes to a major risk factor for atherosclerosis and cardio vascular diseases (Punitha et al., 2006). The saturated fatty acids present in the fat could increase the production of triglycerides and cholesterols by the liver and could decrease the catabolism of LDCs by the repression of their receptors (Shukla et al., 2012). Also, insulin deficiency inactivates the lipoprotein lipase, promoting the conversion of free fatty acid into phospholipids and cholesterol in the liver and finally it gets discharged into the blood resulting into elevated serum phospholipids (Hossain et al., 2012).
188.8.131.52 Aetiological classification of diabetes
Diabetes mellitus includes type I diabetes (immune-mediated and idiopathic), type II diabetes, gestational diabetes and other specific types (American Diabetes Association, 2012). However, types I and II diabetes mellitus appeared to have gained much more popularity among researchers and have generally been considered as the two major categories.
Type I diabetes insulin dependent diabetes mellitus)
Type I diabetes is also known as insulin dependent diabetes mellitus (IDDM), juvenile onset diabetes. In this type of diabetes, the pancreas becomes completely unable to or insufficiently produces insulin, due to autoimmune destruction of the pancreatic beta cells (Whitney et al., 2007). This results in variation in blood glucose levels, which makes the patient prone to ketoacidosis; characterized by hyperglycemia and ketones, and hypoglycemia (Lutz and Przytulski, 2008).
In type 1 diabetes, glucose absorbed from the digestive tract remains in the blood, even though the cells are starved of it. Insulin is therefore injected regularly to assist the cells in taking up the needed glucose; hence, the descriptive term insulin dependent (Whitney et al., 2007).
Since no glucose enters the cells (in the case of untreated type I diabetes), excessive hunger and overeating (polphagia), the cells breakdown protein and fat in order to generate the needed energy fuels. Consequently, weight loss occurs and ketones are produced for energy resulting in ketonemia (ketones in the blood, which causes acidiosis) and ketones in the urine (ketonuria). Ketonuria is a sign that diabetes has gone out of hand.
Researchers have also found that people with diabetes type I have certain genes associated with immune response. However, not everyone with this gene expresses clinical diabetes.
Type I diabetes occurs in about five to ten percent of all the cases of diabetes and is frequently develop in childhood, although some cases arise in adulthood (Lutz and Przytulski, 2008).
Type II diabetes (non-insulin dependent diabetes)
Type II diabetes has also been called non-insulin dependent diabetes mellitus (NIDDM) and adult onset diabetes. In this case, the pancreas produces insulin but the cells are less sensitive to it. Though the pancreas may respond by making more insulin, glucose uptake is still inadequate to meet the cells need. Over time, the pancreas produces less insulin; hence, the blood glucose rises.
Due to inadequate energy fuels for the cells, the cells hunger for energy which results in overeating and consequently more increase in blood glucose. The blood then converts excess glucose into fat; hence, overweight occurs. This is why obesity is commonly associated with type 2 diabetes. Overweight and obesity leads to adverse metabolic effects like high blood pressure and hyperlipidemia (WHO, 2003). Obesity or increased body fat (especially central or abdominal obesity) leads to increased insulin resistance, due to the secretion of a group of hormones that may possibly impair glucose tolerance; and also increase in the serum resistin level which in turn correlates to insulin resistance. It is also believed that fat tissues reduce insulin receptors thereby causing insulin insensitivity in the cells (Lutz and Przytulski, 2008).
Type II diabetes occurs in 90 to 95 percent of all the cases and it develops in people of over 40 years of age.
1.2.6 Genus Irvingia
Species: Irvingia gabonensis, Irvingia wombolu (Kengni et al., 2011)
The genus irvingia is commonly known as Bush mango, African mango, Dika nut, or wild mango. Two species of this genus are widely distributed in West and Central African namely, I. gabonensis and I. wombolu. They are found in their natural range in humid forest zones in Angola, Cameroun, Nigeria, Ghana, Senegal, etc. In Nigeria, two varieties of this species were identified in 1974, I. gabonensis var. gabonensis (with sweet edible fruit) and I. gabonensis var. excelsa (with bitter fruit). In a revision of the taxonomy of the Irvingiaceae family, Harris renamed the bitter variety I. wombolu vermoesen and the sweet variety I. gabonensisAubry-Lecomte ex O’Rorke. Other species of the same genera are I. excelsa, I. robur, I. smithii and I. grandifolia. The kernels of these species also have various local names: in Nigeria, they are ‘ogbono’ in Ibo and ‘apon’ in Yoruba, ‘goron, or ‘biri’ in Hausa. Igbo people of Nigeria distinguish between kernels from I. gabonensis and I. wombolu, referring to the former as ‘ugiri’ and the latter ‘ogbono’.
Figure 1: (a) fruits of Irvingia gabonensis (b) fruits of Irvingia wombolu (c) Seeds of Irvingia gabonensis (d) Seeds of Irvingia gwombolu (e) seed powder of Irvingia gabonensis (f) seed powder of Irvingia gabonensis
The fruits of I. wombolu and I. gabonensis are similar in appearance to that of cultivated mango (Mangifera indica) (Tchoundjeu et al. 2005) and their color varies from green to yellow when mature. I. gabonensis flowers in February-March and fruits of the rainy season (July- September) while I. wombolu flowers in October and fruits in the dry season (January-March).
The kernels of both species are used as a condiment in soups, increasing their viscosity and drawability (sliminess), but I. wombolu is preferred due to its better sliming qualities (Awono et al., 2009). They form an important diet providing carbohydrates, oils and proteins to enhance health and nutrition (Fajimi et al., 2007). Kengni (2003) reported the composition of seeds from Cameroon to be 68.5% fat, 6.1% total carbohydrate, 2.7% ash, 6.2% crude protein, 6.9% soluble fibre, 17.3% insoluble fibre, 0.1% phenolic compounds. The fruit of Irvingia gabonensis has a sweet mesocarp and it is eaten fresh while that of Irvingia wombolu is sour and is not consumed locally (Fajimi et al., 2007; Awono et al. 2009). A comparison of the nutritive qualities of the kernels of both species indicates that I. wombolu is more energy-rich due to its higher percentage fat although both species are a good source of oil. The fat from I. wombolu kernel has lower iodine and saponification values. However, the percentage of crude protein is low for the two species (near 7%).
184.108.40.206 Ecology and biology of Irvingia Species
Both bush mango species occur at altitudes between 200 – 500 m, with mean annual temperature of 25 – 32ºC. Sweet bush mango grows best in a dense moist forest on well- drained acidic soils, with mean annual rainfall of 1500 – 3000 mm. Bitter bush mango can tolerate a wider range of soils, growing in swamps and seasonally flooded forest as well as dry land forest, where annual rainfall is1500 – 2500 mm (Kengni et al., 2011). Sweet bush mango can reach 40 m in height under good conditions while bitter bush mango can reach 25 m in height.
220.127.116.11 Morphological traits and their variation
The two species are very similar in their morphological traits, except that sweet bush mango is taller and has a more elongated crown than bitter bush mango. The crown is dense and compact. The bark is grey and smooth or very slightly scaly. Leaves are green, simple and elliptic (Kengni et al., 2011). They are placed alternately along the twigs. Petiole length is 5 – 10 cm. Flowers are yellowish to greenish-white, and grow in slender, clustered racemes or small panicles above the leaves. Individual flower stalks are slender, about 6 mm long and petals bend right back. Fruit of I. gbonensis is yellow when ripe, broadly ellipsoid and varies in size from 5 to 20 cm long, and 4 to 11 cm wide. Fruit consists of yellow, fibrous pulp surrounding a large seed. Variation has been noted in flowering and fruiting phenology, crown shape, flower colour, fruit production, precocity, fruit characteristics (shape, quality, colour, size, pulp colour, sweetness, fibrousnesses) and seed hardness. Many of these traits are important to farmers. Depending on the degree to which the variation in these traits is inherited, there may be opportunities for substantial improvement in a breeding and domestication programme.
1.2.7 Bioactive constituents and health effects of Irvingia species
The bioactive compounds or secondary metabolites are the non-nutrient components in plant foods. They have some nutritional effects and health benefits. They are those substances contained in foods which supply no nutrients. They could contain some compounds that are beneficial to health or toxic to humans and/or act as antagonists to nutrients in foods (Soetan, 2008). They include tannins and other phenolic compounds (phenols, flavonoids, isoflavonoids), saponins, glucosinolates, alkaloids (Drewnowski and Gomez-Carneros, 2000), phytate and dietary fibre (Gibson, 2007).
Plant contains useful extractable substances in their storage organs (leaves and seeds/roots) in quantities sufficient to be economically useful as raw materials for various scientific technological and commercial applications.
Evidences from several researches have shown that irvingia spp contain many phytochemicals.
Singh (2007) summaried the physicochemical properties of I. gabonensis at 39 ºC – 40 ºC as follows; saponification value 212 – 220, smoking point 213 – 220, free fatty acid value 0.25 – 0.30 iodine value 1.99mg/gm, acid value 1.36, total lipid content. In another study, Nangue et al. (2011) observed that I. gabonensis contains two main saturated fatty acids namely; lauric acid (40.7%) and mystric acid (49.05%). These two fatty acids was claimed to be associated with capric acid (1.54%), palmitic acid (5.06%) and stearic acid (2.38%), making saturated fatty acids in I. gabonensis up to 98.86% of the whole fatty acid content. Oleic acid was found to be the only unsaturated acid in the sample. Therefore, I. gabonensis was found to increase the serum triglycerides in the treated rats except for HDL. It was claimed that lauric acid was found to modify the activities in hepatic lipid metabolism, leading to triglycerides accumulation. It was also said that the fat in I. gabonenesis may have also slowed down protein synthesis, either by interrupting the activities of liver enzymes or by the detoriation initiated by the fatty acids which may be in excess in the liver. Surprisingly, in the male rats, they observed that this effect may have been countered by the secretion of regulatory hormones. However, several studies (Ngondi et al., 2006; Omokhua and Onoagbe, 2011; Hossain et al., 2012), have suggested that intake of I. gabonensis reduced serum lipids but increased HDL. It could be that the reduction of ‘bad’ serum lipids in these researches resulted from the body defense system of the model animals, which may have been ellicted by the I. gabonenesis. Moreover, the diets of the experimental rats used by Nangue et al. (2011) contain high concentrations of I. gabonensis fat. On the other hand, Nangue et al. (2011) suggested that the mystric acid found in I. gabonensis could be beneficial for of cellular metabolism, since the roles of mystric acid includes; participation in a ‘switch’ mechanism, permitting the protein to cycle in a regulated manner between membranes and cytosol and influencing protein conformation leading to protein stability or ligand binding).
A study carried out by Matsinkou et al. (2012), revealed that the pulp extract of Irvingia wombolu fruits is rich in polyphenols. This is in agreement with the result of Oviasogie et al. (2009). Polyphenols are usually known to act as antioxidants. They are thought to rid the body of harmful molecules known as free radicals, which can damage a cell’s DNA and may trigger some forms of cancer and other diseases (American Cancer Society, 2013). Matsinkou et al. (2012) suggested that the polypenols decrease the oxidative stress in humans, especially through the inhibition of LDL cholesterol oxidation. They observed that the amount of phenolics varies considerably in the fruit pulp extract. Polyphenol content in the aqueous extract was found to be 1.94 times higher than that of hydroethanolic extract. This showed the influence of the type of solvent used on the polyphenol content.
Joyal (2012) indicated that I. gabonensis extracts has beneficial effects on a variety of metabolic targets involved in carbohydrate metabolism thus: inhibitory effect on glycerol-3-phosphate dehydrogenase, a key enzyme involved in conversion of glucose to stored fat, inhibitory effect on alpha-amylase, a key enzyme involved in the digestion of dietary complex carbohydrates into maltose and dextrin,
beneficial impact on PPAR-gamma, a key enzyme involved in both adipogenesis (new fat cells metabolism) as well as insulin sensitivity, up-regulation of adipopectin, a key protein hormone involved in enhancing insulin sensitivity and endothelial function, and enhancing leptin sensitivity and therefore decreasing leptin sensitivity.
Irvingia gabonensis seeds have been suggested to have delayed stomach emptying effect, thus leading to a more gradual absorption of dietary sugar (Dzeufiet et al., 2009; hossain et al., 2012). This effect led to reduced the elevation of blood sugar levels that is typical after a meal. Ngondi et al. (2006) attributed this hypoglycemic effect to the high dietary fibre content of the seed. They also observed that Irvingia gabonensis seeds protein have anti-amylase activity. Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starch from being digested by pancreatic amylase.
According to Bard (2010), consumption of Irvingia has been linked to increased` effective fat loss via multiple pathways:
- Reducing glucose levels and insulin induced lipogenesis (A.K.A reducing insulin sensitivity)
- Reducing the absorption of sugar (inhibiting amylase activity)
iii. Inhibiting the conversion of glycerol to triglycerides (reducing fat cell triglycerides and glucose-3-phosphate dehydrogenase enzyme)
- Reducing leptin resistance (reducing CRP binding to leptin)
- Lowering serum leptin levels (leptin unable to be used by cells)
- Increasing anti-inflamatory, anti-atherogenic and anti-diabetic effects (increasing adipopetin levels).
In a similar research, Ngondi et al. (2009) found that IGOB131, a relatively rich plant-derived protein, extracted from I. gabonensis, safely and significantly reduced body weight in overweight and /or obese subjects and had favourable impact on a variety of other metabolic parameters. IGOB131 administration was associated with increases in plasma adipopectin levels and decreases leptin and CRP level. Plasma leptin levels are closely related and correlated with the levels of adipose tissue. Hence, decrease in plasma leptin levels associated with the decrease of adipose tissue was a consequence of weight loss.
Irvingia gabonensis extract was also found to significantly reduce body weight of rabbits (Ngondi et al., 2005; Oben et al., 2008; Omonkhua and Onoagbe, 2011). It was suggested that the significant reduction in subcutaneous fat of the animals implies that weight reduction was as a result of loss of fat deposits and not muscle wasting (Oben et al., 2008). The weight lowering effect may as a result of the hypoglycemic effect of I. gabonensis, since obesity is a predisposing factor to diabetes and loss of weight has been shown to improve insulin sensitivity. Omonkhua and Onoagbe (2011) attributed this effect to presence of phytochemicals such as tannins, saponins and high fibre content. Dzeufiet et al. (2009) also observed that defatted seeds of I. gabonensis consumed by diabetic rats produced a more positive effect on body weight. This they said could probably be due to its fat free composition. Fatty diets as have been observed are usually less consumed due to its high energy content (Dzeufiet et al., 2009). Nangue et al., (2011) opined that the oil extracted from I. gabonensis seed is made of 90 percent saturated fatty acids. Saturated fatty acids are known to increase cholesterol concentration and expose us to the risk of cardiovascular diseases.
Bhavna and Sharma (2012) investigated the in–vitro hepatoprotective effect of methanol extract of I. gabonensis on CC14-induced liver cell damage as well as the possible antioxidant mechanism involved in this protective effect. Phytochemical analysis of the extract showed the presence of seven compounds identified as: 3-friedelanone, betulinic acid, oleanolic acid, 3, 3’, 4’-tri-O-trimethylellagic acid, methyl gallate, hardwickiic acid and 3-β-acetoxyursolic acid. It was found that compounds such as oleanolic acid, 3-β-acetoxyursolic acid, methyl gallate and betulinic acid showed significant hepatoprotective activity as indicated by their ability to prevent liver cell death and LDH leakage during CCl4 intoxication compounds oleanolic acid, methyl gallate and 3-β-acetoxyursolic acid showed significant antioxidant effects involving radical scavenging action, inhibition of microsomal lipid peroxidation, β-CLAMS and FRAP assays.
Furthermore Hossain et al. (2012), reported that the oral consumption of food incorporated with I. gabonensis crude powder resulted in reduced serum glucose level. In addition to its high dietary fibre, I. gabonensis posses insulinomimetic or insulin sensitizing effect. Based on this study I. gabonensis was suggested not to have changed liver glycogen contents significantly. Hence, it seems that lowering of blood glucose by I. gabonensis seeds may not have been accomplished through glycogenesis.
Phytochemicals from other plants have also been found to have hypoglycemic effect. Cooked Lupinus mutabilis (a legume) and its purified extract were found to have hypoglycemic effects on subjects with type 2 diabetes (Baldeon et al. 2012). Shukla et al. (2012) also found that lepidine and semilepidine found in Lepidium sativum linn (garden grass) had anti-diabetic effect against alloxan-induced diabetic rats, probably through the reduction of oxidative damage and modulating antioxidant enzymes. They claimed that the possible mechanism L. sativum seed total alkaloid brought about its antihyperglycemic action may be by potentiation of pancreatic secretion of insulin from the remaining islet beta cells.
1.2.8 Mechanism in alloxan action
Alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyura- cil) is an oxygenated pyrimidine derivative which is present as alloxan hydrate in aqueous solution (Rohilla and Ali, 2012). Alloxan is a hydrophilic and unstable substance. Its half-life at neutral pH and 37 °C is about 1.5 minutes and is longer at lower temperatures.
Alloxan was first described by Brugnatelli in 1818. Wöhler and Liebig used the name “alloxan” and described its synthesis by uric acid oxidation. The diabetogenic properties of this drug were reported many years later by Dunn et al. (1943), who studied the effect of its administration in rabbits and reported a specific necrosis of pancreatic islets. Since then, alloxan diabetes has been commonly utilized as an animal model of insulin-dependent diabetes mellitus (IDDM). The name Alloxan emerged from the merging of two words, i.e., allantoin and oxaluric acid. Allantoin is a product of uric acid excreted by the foetus in the allantois and oxaluric acid has been derived from oxalic acid and urea that is found in urine. Alloxan was originally prepared by the oxidation of uric acid by nitric acid. It has been regarded as a strong oxidizing agent that forms a hemiacetal with its reduced reaction product; dialuric acid, in which a carbonyl group is reduced to a hydroxyl group, that is called alloxantin (Rohilla and Ali, 2012).
Alloxan exerts its diabetogenic action when it is administered parenterally: intravenously, intraperitoneally or subcutaneously. The dose in alloxan required for inducing diabetes depends on the animal species, route of administration and nutritional status. Human islets are considerably more resistant to alloxan than those of the rat and mouse. The most frequently used intravenous dose of this drug to induce diabetes in rats is 65 mg/kg body weight. When alloxan is given intraperitonealy or subcutaneously its effective dose must be 2 – 3 times higher. The intraperitoneal dose below 150 mg/kg b.w. may be insufficient for inducing diabetes in the rat. Fasted animals are more susceptible to alloxan (Szkudelski et al. 2001), whereas increased blood glucose provides partial protection (Szkudelski et al., 2001).
Alloxan evokes a sudden rise in insulin secretion in the presence or absence of glucose which appeared just after alloxan treatment (Lachin and Reza, 2012). This particular alloxan-induced insulin release occurs for short duration followed by the complete suppression of the islet response to glucose even when high concentrations of glucose were used. Further, the alloxan action in the pancreas is preceded by its rapid uptake by pancreatic beta cells that have been proposed to be one of the important features determining alloxan diabetogenicity. Moreover, in pancreatic beta cells, the reduction process occurs in the presence of different reducing agents like reduced glutathione (GSH), cysteine, ascorbate and protein-bound sulfhydryl (-SH) groups. Alloxan reacts with two -SH groups in the sugar binding site of glucokinase resulting in the formation of the disulfide bond and inactivation of the enzyme. As a result in alloxan reduction, dialuric acid is formed which is then re-oxidized back to alloxan establishing a redox cycle for the generation of reactive oxygen species and superoxide radicals (Das et al., 2012). The superoxide radicals liberate ferric ions from ferritin and reduce them to ferrous and ferric ions. In addition, superoxide radicals undergo dismutation to yield hydrogen peroxide (H2O2) in the presence of superoxide dismutase. As a result, highly reactive hydroxyl radicals are formed according to the Fenton reaction in the presence of ferrous and H2O2. Another mechanism that has been reported is the effect of ROS on the DNA of pancreatic islets. The fragmentation of DNA takes place in the beta cells exposed to alloxan that causes DNA damage, which stimulates poly ADP-ribosylation, a process participating in DNA repair (Rohilla and Ali, 2012). Antioxidants like superoxide dismutase, catalase and the non- enzymatic scavengers of hydroxyl radicals have been found to protect against alloxan toxicity (Ebelt et al., 2000). In addition, the disturbance in intracellular calcium homeostasis has also been reported to constitute an important step in the diabetogenic action in alloxan. It has been noted that alloxan elevates cytosolic free Ca2+ concentration in the beta cells of pancreatic islets. Calcium influx is resulted from the ability in alloxan to depolarize pancreatic beta cells that further opens voltage dependent calcium channels and enhances calcium entry into pancreatic cells. The increased concentration of Ca2+ ion further contributes to supraphysiological insulin release that along with ROS has been noted to ultimately cause damage of beta cells of pancreatic islets (Szkudelski, 2001; Etuk, 2010).
Induction of diabetes in the laboratory animals by alloxan injection is the result of selective uptake in alloxan via GLUT2 into pancreatic beta cell (Elsner et al. 2000). This is because alloxan is an unstable chemical which also have a similar shape with glucose.
The chemical induction of diabetes appears to be the most popularly used procedure in inducing diabetes mellitus in experimental animals. The foremost drug-induced diabetic model is the alloxan diabetes that is capable of inducing type I diabetes mellitus in experimental animals. The surgical and genetic methods of diabetes induction are associated with a high percentage of animal morbidity and mortality. Hence, Rohilla and Ali (2012) are of the opinion that alloxan induced diabetes model appears to be the most reliable and easily reproducible method of inducing diabetes mellitus in experimental animals.
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