Project Description

CHAPTER ONE

INTRODUCTION

Pancreatic ß-cell and insulin receptor cells have emerged as a target of oxidative stress mediated tissues damage, which can lead to diabetes mellitus. Under diabetic conditions reactive oxygen species (ROS) generated from non-enzymatic glycosylation reaction, electron transport chain in mitochondria and hexosamine pathways increase in various tissues and involved in the development of diabetic complications (Kim et al.,2006). Research has equally shown that the presence of antioxidant in adequate quantity could help in mopping up these free radicals that can lead to oxidative stress, thus supplementing of endogenous antioxidant either from fruits or vegetables is encouraged.

Recently the use of herbal products for medicinal benefits has played an important role in nearly every culture on earth and for many years, the search for anti-diabetic products will continue to focus on plants and other natural resources (Osinubi et al., 2006). The cost of administrating modern antidiabetic drugs is beyond the reach of most people in the low income group and those living in the rural areas, hence the use of plants for the treatment of common diseases such as diabetes are very common. In line with the WHO (1999) expert committee on diabetes which recommends that traditional methods of management of diabetes should be further investigated. Also considering the economic resource constraints and cheapness of these herbal products, this present study was designed to determine the effects of increasing dosages of fresh onion and tomato juice on alloxan-induced diabetic Rattus novergicus and its possible mechanisms of action, for possible use in the control of hyperglycaemia in alloxan- induced diabetes mellitus

1.1       Diabetes

1.1.1    Diabetes mellitus (DM): History, Path physiology and classification

Despite periods of feeding and fasting, in normal individuals plasma glucose remains in a narrow range between 4 and 7 mM reflecting the balance between: (i) the release of glucose into the circulation by either absorption from the intestine or the breakdown of stored glycogen in the liver and (ii) the uptake and metabolism of blood glucose by peripheral tissues (Saltiel and Kahn 2001). These processes are controlled by a set of metabolic hormones. For decades diabetes had been viewed from a bi-hormonal perspective of glucose regulation involving insulin (discovered in the 1920s; released by pancreatic β-cells) and glucagon (discovered in the 1950s; released by the pancreatic α-cells) (Vilsboll et al., 2001). In the mid-1970s several gut hormones, the incretins, were identified. One of these, glucagon-like peptide-1 (GLP-1), was recognized as another important contributor to the maintenance of glucose homeostasis. Subsequently the discovery in 1987, of a second pancreatic β-cell hormone, amylin, whose role complemented that of insulin, led to the view of glucose homeostasis involving multiple hormones. Amylin, like insulin is found to be deficient in people with diabetes. Diabetes mellitus is commonly referred to as diabetes, is a group of metabolic disease in which there are high blood sugar levels over a long period of time. Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. Diabetes mellitus describes as metabolic disorder of multiple aetiology characterized by chronic hyperglycemia with distribution of carbohydrate, fats and protein metabolism resulting from defects in insulin secretion, insulin action or both (WHO, 1999). In 2006 according to the World Health Organization, 171 million people worldwide suffer from diabetes. The incidence is increasing rapidly and it is estimated that by the year 2030 the number will double (ADA, 2005).

Diabetes mellitus is dated back to 1500 BC (Leonid 2009). The term diabetes was first used in 230 BC by Greek Appollonius of Memphis. The first described cases are believed to be of type 1 diabetes. Type 1 and 2 diabetes were identified as separate conditions for first time by the Indian physicians Sushruta and Charaka in 400-500 AD with type 1 associated with youth and type 2 with being overweight. Effective treatment was not developed until the early part of the 20th century when the Canadians Fredrick Banting and Charles best developed insulin in 1921 and 1922 this was followed by the development of long acting insulin NPH (neutral protamine hagedorn) in the 1940s. In a healthy person the liver convert glycogen into glucose which is then released into the blood stream. Beta cells found in the islets of langerhans in the pancrease releases insulin in response to rising levels of blood glucose after eating .insulin is the principal hormone that regulates uptake of glucose from the blood into the most cells (primarily muscle and fats cells, but not central nervous system cells) for use as fuel, for conversion to other needed molecules or for storage as glycogen in the liver and muscles cells. Therefore deficiency of insulin or the insensitivity of it receptors plays a central role in all forms of diabetes mellitus.

1.1.2 Types of Diabetes Mellitus

1.1.2.1 Types 1 diabetes mellitus

This type of diabetes result, due to the failure to produce enough insulin, this form was previously referred to as insulin dependent diabetes mellitus (IDDM).The majority of type 1 diabetes is of the immune mediated nature where beta cell loss its T-cell mediated autoimmune attack (Rother, 2007). Type 1 diabetes can affect children or adults but was termed juvenile diabetes because it represents a majority of the diabetes cases in children.

1.1.2.2 Type 2 diabetics’ mellitus

This type begins with insulin resistance a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also develop. This form was previously referred to as non insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes. The primary cause is excessive body weight and not enough exercise. Globally as of 2010 it was estimated that there are 285 million diabetics, with type 2 making up about 90% of the case (Bailey, 1999).

1.1.2.3 Gestational diabetes

It is the third main form and occurs when pregnant women without a previous history of diabetes this occurs in about 2-5% of all the pregnancies and may improve or disappear after delivery. It is treatable but requires careful medical supervision throughout the pregnancy.

1.1.3 Complication of diabetes mellitus

The complication of diabetes mellitus are far less common and less severe in people who have well controlled blood sugar levels (Nathan et al., 2005). Acute complications of diabetes mellitus including the following: The classic symptoms of untreated diabetes are weight loss, polyuria, polydipsia and polyphagia. Symptoms may develop rapidly (weeks or months) in type 1 DM while they usually develop much in slowly and subtle or absent in type 2 DM, if diabetes is left untreated diabetes can lead to complications. Acute complications can include diabetic ketoacidosis, nonketotic, hyperosmolar coma or death. Serious long term complications include heart disease, stroke, chronic kidney failure, foot ulcer and damage to the eyes (Fong et al., 2003).

Diabetic ketoacidosis (DKA): One of the most prominent features of insulin deficiency is rapid mobilization of fatty acids from adipose tissue. In IDDM, excessive lipolysis during insulin deficiency is the combined result of insulin lack and insulin resistance (Singh et al., 1987). One of the consequences of excessive mobilization of fatty acids in IDDM is the production of ketone bodies (acetoacetate, 3-hydroxybutyrate and acetone) in liver. Fatty acids taken up by the liver, after conversion to their CoA esters, are either esterified to glycerolipid or oxidized to acetyl-CoA in mitochondria. A high proportion of the acetyl-CoA formed is converted to acetoacetate and 3-hydroxybutyrate. The rate of transfer of fatty acyl units to the mitochondria is regulated by the activity of carnitine palmitoyltransferase I, which faces the intermitochondrial membrane space and catalyses the first step specific to mitochondrial fatty acid oxidation. Carnitine palmitoyltransferase I is regulated by malonyl-CoA, an intermediate in fatty acid synthesis, and it is also regulated by a phosphorylation mechanism. Malonyl-CoA decreases the affinity of the enzyme for its fatty acyl-CoA substrate while phosphorylation increases the affinity for substrate (Harano et al., 1985). In insulin deficiency, the rate of fatty acid synthesis in liver declines and consequently the concentration of malonyl-CoA also decreases, and the affinity of carnitine palmitoyltransferase for malonyl- CoA also decreases (Gamble and Cook, 1985), thus relieving the inhibition of carnitine palmitoyltransferase by malonyl-CoA. Changes in the kinetic properties of detergent-solubilized carnitine palmitoyltransferase following exposure of liver cells to glucagon (activation) or insulin (inactivation) have also been observed (Harano et al., 1985), indicating that additional mechanisms contribute to activation (or deinhibition) of carnitine palmitoyltransferase in insulin deficiency, thereby favouring increased transfer of long-chain fatty acids into mitochondria. The utilization of acetoacetate and 3-hydroxybutyrate as oxidative fuels (or lipogenic substrates) by a variety of tissues is well established (Robinson and Williamson, 1980) and increases with blood ketone body concentration in the fed-to-fasted transition. In muscle, at ketone body concentrations attained in prolonged starvation or diabetic ketosis, the rate of uptake reaches saturation. Consequently, with increasing ketone body production and thereby plasma concentration, there is a progressive decrease in total fractional clearance (Fery and Balasse, 1985). Although the biochemical routes of metabolism of acetoacetate and 3-hydroxybutyrate are well established, the conversion of acetoacetate to acetone and its subsequent excretion or metabolism has only recently received attention. In man, plasma (acetone) correlates with, and is generally higher than, acetoacetate in fasting and diabetic ketosis (Owen et al., 1982) and the production rate is estimated to be about half the rate of ketogenesis.

Nonketotic hyperosmolar coma. This results when blood levels is above 300 mg/dl (16 mmol/l). In this case, water is osmotically drawn out of cells into the blood and the kidneys eventually begin to dump glucose into the urine. This results in loss of water and an increase in blood osmolarity.

Hypoglycemia: this is also known as abnormally low blood glucose is an acute complication of several diabetes treatments. The patient may become agitated, sweaty and weak and have many symptoms of sympathetic activation of the autonomic nervous system resulting in feelings akin to dread and immobilized panic. Consciousness can be altered or even lost in extreme cases leading to coma, seizures or even brain damage and death.

Chronic complications of diabetes mellitus include the following:

Diabetic neuropathy, abnormal and decreased sensation, usually in a “glove and stocking” distribution, starting with the feet but potentially in other nerve, later often finger and hands, when combined with damaged blood vessels can lead to diabetic foot.

Diabetic retinopathy, resulting from the growth of friable and poor quality new blood vessels in the retina as well as macular oedema which can lead to severe vision loss or blindness. Diabetic retinopathy is the most frequent cause of new cases of blindness among adult aged 20-70 years (Fong et al., 2003). Diabetic nephropathy, damage to the kidney which can lead to chronic renal failure and eventually requiring dialysis (WHO, 1999).

1.1.4 Management of Diabetes Mellitus

Diabetes mellitus is a chronic disease which cannot be cured except in very specific situations. Management concentrates on keeping blood sugar levels as close to normal as possible without causing hypoglycemia. This can usually be accomplished with diet, exercise and use of appropriate medications. Patient education, understanding and participation is vital since the complications of diabetes are far less common and less severe in people who have well managed blood sugar levels (Nathan et al., 2005). Attention is also paid to other health problems that may accelerate the deleterious effect of diabetes. These include smoking, elevated cholesterol levels, obesity, high blood pressure and lack of regular exercise (NHS, 2009). Currently the available therapies for diabetes includes insulin and various oral anti-debetic agents such as sulfonylurea, thiazolidinedione. These drugs are used as monotherapy or in combination to achieve better glycaemic control. Each of the above oral antidiabetic agents is associated with a number of serious adverse effects (Kasari and Gupta, 2006). Hence the treatment of diabetes has shifted to the use of natural plant sources that has minimal side effects. Plants play a major role in the introduction of new therapeutic agents and have received much attention as sources of biologically active substance including antioxidants, hypoglycaemic and hypolipidaemic agents (Eross et al., 1984).

1.2 Effect of Diet on Glucose Level

Diabetes mellitus is characterized by recurrent or persistent hyperglycaemia and is diagnosed by demonstrating any of the following (WHO, 1999):

• Blood sugar analyzed at any time of the day without any prior preparation is called random blood sugar and at or above 11.2 mmol/l (200 mg/dl) indicates hyperglycaemia.
• Blood sugar estimated in the morning before taking breakfast (12 hours fasting) is fasting blood sugar its ranges from3.9 to7.2 mmol/l (70-125 mg/dl) indicates hyperglycaemia.
• Blood sugar estimated 2 hours after a good meal is post-prandial blood sugar should be less than10 mmol/dl (180 mg/dl) indicates hyperglycaemia.

Diagnosis of diabetes mellitus should not be based on a single random test alone, it should be repeated. Type 1diabetes patients can be confirmed by blood test that identifies antibodies directed against pancreas. However, there is no test for type 2 diabetes beyond measurement of blood sugar (American Diabetes Association, 2011).

1.3 Insulin Hormone Action in Man

Insulin is a key anabolic hormone that is secreted from pancreatic β-cells in response to increased blood glucose and amino acids following ingestion of a meal. Insulin, through its action on the insulin receptor decreases blood sugar levels by: (i) increasing glucose uptake in muscle and fat through triggering the translocation of the intracellular glucose transporter GLUT4 to the plasma membrane (ii) stimulating the storage of glycogen and fat in muscle, liver and adipose tissues through stimulation of the synthesis of glycogen, fat and protein and (iii) reducing glucose production and release by the liver through inhibition of glycogen breakdown. Insulin signaling also inhibits the breakdown of fat and protein. The extent to which primary or secondary defects in insulin receptor activity can explain cellular insensitivity to insulin is still hotly debated. The insulin receptor is well characterized (Kahn and Gushman, 1985) and its gene has been cloned (Ullrich et al., 1985). It comprises two 135 kDa α a subunits which are extracellular and contain insulin-binding sites, and are linked by disulphide bonds to two 95kDa ß subunits. The ß subunit has a hydrophobic transmembrane region, and an intracellular domain which has several tyrosine residues, a tyrosine kinase and an ATP-binding site. Insulin binding to α subunits activates the ß subunit tyrosine protein kinase and brings about phosphorylation of tyrosine residues on the ß subunit. Activation of the ß subunit kinase may be involved in transmission of the insulin signal, perhaps by initiating a phosphorylation/dephosphorylation cascade (Denton et al., 1981). Kinase activity of the insulin receptor can be decreased by cyclic AMP-dependent protein kinase phosphorylation of serine or threonine sites on the ß subunit, and this could conceivably underlie catecholamine-induced insulin resistance (Roth and Beaudoin, 1987). In states of extreme insulin resistance (Grigorescu et al., 1987; Grunberger et al., 1984) and NIDDM (Freidenberg et al., 1987; Caro et al., 1986) the process of signal transmission from the receptor α subunit insulin-binding site to activate the kinase appears to be defective at one or more sites. Amino acid substitution in the ATP-binding region or the tyrosine kinase region (Chou et al., 1986) abolishes insulin action. Whether or not the kinase is involved in mediating all actions of insulin remains uncertain. Some antireceptor antibodies simulate insulin action without changing kinase activity (Simpson and Hedo, 1984). Not all actions of insulin are modulated in parallel by treatment of insulin resistant states, suggesting that divergent pathways of intracellular insulin signal transmission may be separately affected (Boden et al., 1983).

1.4 Oxidative stress and antioxidant

Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbance in the normal redox state of tissue (such as the attraction of free radical to another molecule) can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA (Mate et al., 1999). Oxidative stress involves excess formation and insufficient removal of highly reactive molecules such as reactive oxygen species and reactive nitrogen species by antioxidant, such as glutathione etc (Ahamed, 2005; Rahimi et al., 2005). Free radicals are unstable, highly reactive molecules that loss an electron as a result of their interaction with other molecules within the cells, this can cause oxidative damage to proteins, membranes and genes. There are several sources by which reactive oxygen species are generated most reactive oxygen species come from the endogenous source as by product of normal and essential metabolic reaction, such as energy generation from mitochondria or the detoxification reaction involving the liver cyctochrome P-450 enzyme system. Exogenous source include exposure to cigarette smoke, environmental pollutants such as emission from automobile and industries, consumption of alcohol in excess, exposure to ionizing radiation and bacterial, fungal or viral infections. The ROS includes free radicals such as superoxide anion, hydroxyl radical, peroxyl radical, hydroperoxyl radical and hydrochlorous acid (D Souza et al., 2009). Of these reactive molecules, NO and ONOO^– are the most widely studied species and play important role in the diabetic cardiovascular complications (Evans, 2002). Reactive oxygen species can be beneficial as they are used by the immune system as a way to attack and kill pathogens since they are produced at a low level by normal aerobic metabolism. Under physiological conditions, they are involved to some extent as signaling molecules and defense mechanism as seen in phagocytosis, neutrophil function and shear sets induced vasorelaxation, excess generation in oxidative stress has pathological consequence including damage of protein, lipids and DNA (Jennings, 1987). The effects of oxidative damage depend upon the size of these changes, with a cell been able to overcome small perturbation and regain its original state. Short term oxidative stress may be important in prevention of aging by induction of a process named mitohormesis (Valko et al., 2007). However, more severe stress can cause cell death and even moderate oxidation can trigger apoptosis while more intense stress may cause necrosis. Antioxidant means “against oxidation”, they are molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that can produce free radicals leading to chain reactions that may damage cells. Antioxidant are substance that either directly or indirectly protect cell against the adverse effect of xenobiotics, drugs, carcinogens and toxic radical reaction. Free radicals reside primarily in the mitochondria of cells. when free radical are released from the mitochondria in numbers sufficient to overwhelm the protective biochemical system of the body, they become a threat to some cellular structures such as lipids, proteins, carbohydrates and nucleic acids in cell membranes. Compromised cellular structure alters cellular function and may lead to the initiation of disease process. In severe oxidative stress, cell death may occur. Antioxidant reacts with the free radical before they are able to react with other molecules thus providing protection from oxidation reactions. Antioxidants are found in many forms the principal vitamins with antioxidant properties are vitamin E, C and beta carotene (Dunnet, 2003). Antioxidant involves a variety of component both endogenous and exogenous in origin that can function interactively and synergistically to neutralize free radical.

1.5       Dietary (Non–enzymatic) Antioxidant

1.5.1    Vitamin A

Vitamin A is a group of saturated nutritional organic compounds that includes retinol, retinal, retinoic acid and several provitamin A carotenoids (most notably beta carotene).Vitamin A (retinol) is required by humans for the normal functioning of the visual system (Tee, 1993). Retinol is transported to ocular tissue and to the retina of the eye by intracellular binding and transport protein in which it plays an important part in the formation of rhodopsin, an important visual pigment, particularly for dim-light vision. All-trans retinol is converted to retinaldehyde, isomerised to the 11-cis form and bound to opsin to form rhodopsin. When there is insufficient amount of retinol available, rhodopsin synthesis is affected and night blindness may result. The condition can, however, also be due to a lack of other nutrients which are critical to the regeneration of rhodopsin such as protein and zinc. The second main function of vitamin A is in the maintenance of growth and epithelial cellular integrity and immune function in the body. Thus, in vitamin A deficiency, the numbers of goblet cells are reduced in epithelial tissues, resulting in a reduction in mucous secretions with their antimicrobial components. Cells lining protective tissue surfaces flatten and accumulate keratin because they fail to regenerate and differentiate. All these changes result in diminished resistance to invasion by potentially pathogenic organisms. The immune system is also adversely affected by direct interference with production of some types of protective secretion and cells. As these changes in internal epithelial tissues occur, the external reflections of such changes are seen in the classical eye changes in xerophthalmia and xerosis. vitamin A is found almost exclusively in animal products. Liver from various animals are extremely rich source of vitamin A (Tee et al., 1997).

1.5.2    Vitamin C (Ascorbic acid)

Vitamin C (ascorbic acid) is a monosaccharide oxidation reduction (redox) catalyst found in both animals and plants (Weiss, 2005). Vitamin C is a water soluble antioxidant and is found in the water compartments of the body where it interacts with free radicals. ascorbate function as a reductant for many free radical thereby minimizing the damage caused by oxidative stress. As an antioxidant ascorbate can directly scavenge oxygen free radicals with and without enzyme catalysts and can indirectly scavenge them by recycling tocopherol to the reduced form. ascorbate react with superoxide, hydrogen peroxide or the tocopheroxyl radical to form monodehydroascorbic acid or dehydroascorbic acid. The reduced forms are recycled back to ascorbic acid by monodehydroascorbate reductase and dehydroascorbate reductase using reducing equivalents from NADPH or glutathione respectively (Halliwell, 1992). Vitamin C can recycle tocopherol radical (Ogugua and Ikejiaku, 2005).

Tocopherol→ Semidehyro ascorbate + Tocopherol – OH

The rate at which damage is removed is dependent on the level of repair enzymes. The body’s ability to produce antioxidants is controlled by the genetic makeup and influenced by exposure to environmental factors such as diet and smoking. Changes in our lifestyle, which include more environmental pollution like paints fumes and less quality diets increases the exposure to free radicals more than ever before. It has been shown that short term supplementation of vitamin C lasting two to four hours weeks can significantly reduce the level of free radicals in the body (Weiss, 2005). Dietary vitamin C is absorbed primarily by active transport in the small intestine, with absorption decreasing as intake increases. Approximately 70 to 90% vitamin C is absorbed when dietary intake is between 30 to 180 mg/day. The kidneys excrete excess dietary vitamin C in urine, but excrete virtually no vitamin is very low. After absorption in the small intestine vitamin C is transported in the blood to cells in its reduced form, ascorbic acid or ascorbate (Valko et al., 2004).

1.5.3 Vitamin E (d-Alpha tocopherol)

Vitamin E is a fat-soluble antioxidant which means it is stored in the body fat and works within the lipid portion of the cell membranes to provide an alternative binding site for free radicals, preventing the oxidation of polyunsaturated fatty acid (Padh, 1990). Vitamin E is a family of eight compounds synthesized by plants in nature: four tocopherols (alpha, beta, gamma, delta) each has different levels of bioactivity in the body over quite a wide range, but generally speaking, alpha tocopherol has greater bioactivity than gamma-tocopherol, which has greater bioactivity than delta tocopherol. Digestion and absorption of vitamin E is greatly improved consumption is accompanied with dietary lipids or fats.Vitamin E may be stored in liver, adipose tissues and skeletal muscle. When needed vitamin E places itself in cell membrane. A diet that is excessively low in fat may negatively affect vitamin E absorption as well as fat soluble nutrients. Properly extracted and protected vegetable oils are major source of vitamin E (Valko et al., 2007).

1.6 Enzymatic antioxidant

Enzymatic antioxidants are substances that when present at very low concentration inhibits the oxidation of a molecule. It has the capacity to nullify the ill effects of oxidation caused by free radicals in the living organisms. The unpaired electrons of these free radicals are highly reactive and neutralize the harmful reactions of human metabolism. Protection of the body against free radicals is provided by some enzymes which come under a distinctive group, concerned solely with the detoxification of these radicals. Superoxide dismutase (SOD) and catalase (CAT) are the key enzymatic antioxidants of this defense system by which the free radicals that are produced during metabolic reactions are removed (Halliwell and Gutteridge, 1995).

1.6.1 Superoxide dismutase (SOD)

Superoxide dismutase (SOD) is a prime antioxidant enzyme found in two forms. One, complex with zinc and copper, localized in the cytosol, and the other bound with manganese found in the mitochondrial matrix (Soliman, 2008). Both forms of this metallo enzyme catalyze the inactivation of destructive superoxide anion by converting them to hydrogen peroxide which is then transformed to water and oxygen by the enzyme catalase (Sharma et al., 2011). superoxide anion can cause mutation in DNA or attack enzymes that make amino acids and other essential molecules.

.O₂ + O₂           SOD          O₂+ H₂O₂

To combat this potential danger, SOD is widely distributed to protect cells against the toxic effects of superoxide anion. Superoxide dismutase is present in all aerobic organisms and most sub cellular compartments that generate activated oxygen. It is therefore assumed that SOD has a central role in the defense against oxidative stress.

1.6.2 Catalase (CAT)

Catalase (CAT) is an enzymatic antioxidant widely distributed in all animal tissues and the highest activity is found in the red blood cells and in the liver (Switala and Loewen, 2002). CAT decompose H₂O₂ and protects the tissue from highly reactive OH therefore, the reduction in the activity of these enzyme may result in an accumulation of O₂ radicals and H₂O₂ (Deepak et al., 2007).

2 H₂O₂   Catalase    2 H₂O + O₂

Hence, catalase has been considered an important regulator of oxidative stress (Dsouza et al., 2008). Catalase, localized in peroxisomes, has two enzymatic activities depending on the concentration of H₂O₂. If the concentration of H₂O₂ is high catalase acts catalytically, that is it removes H₂O₂ by forming H₂O and O₂. However at low concentration of H₂O₂ and in the presence of suitable hydrogen donor e.g., ethanol, methanol, phenol and others catalase acts peroxidiclly, removing H₂O₂ but oxidizing its substrate (Scibior and Czeczot, 2006).

1.7 Lipid Peroxidation

Lipid peroxidation is an autocatalytic free radical mediated destructive process whereby poly-unsaturated fatty acids in cell membranes undergo degradation to from lipid hydroperoxides (Raha and Robinson, 2000). It is the process in which free radicals steal electrons from the lipids cell membrane, resulting in cell damage. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lies methylene (-CH₂) groups that possess especially reactive hydrogen. This process proceeds by a free radical chain reaction mechanism. Lipid peroxidation triggers the loss of membrane integrity, causing increase cell permeability, enzyme inactivation and structural damage to DNA and cell death (Halliwell, 1992).

Cellular membrane is vulnerable to oxidation by ROS due to the presence of high concentration of unsaturated fatty acids in their lipid components. ROS reactions with membrane lipids cause lipid peroxidation, resulting in formation of lipid hydroperoxide (LOOH) which can further decomposed to an aldehyde such as malondialdhyde (MDA), 4-hydroxy nonenal (4-TINE) or fro cyclic endoperoxide, and hydrocarbon. By product of lipid peroxidation such as conjugated dienes and malondialdehyde are increased in the circulation of diabetes mellitus patients. MDA is generated as relatively stable end product from the oxidative degradation has been causatively implicated in the aging process, atherosclerosis, Alzhemers disease and cancer (Niki et al., 2005). Serum MDA has been used as a biomarker of lipid peroxidation and has served as an indicator of free radical damage. Additionally MDA can interact with several functional group on proteins and lipoproteins altering their chemical behavior and possibly contributing to carcenogensis and mutagenesis (Ogugua and Ikejiaku 2005). Due to its highly reactive nature, malondialdehyde also function as an electrophile that can cause toxic stress within the cell and is therefore a potent marker for measuring the overall level of oxidative stress within an organism (Soliman, 2008).

1.8 Glutathione

Glutathione is an important antioxidant in plant, animals, fungi and some bacteria and archaea. GSH is capable of preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides and heavy metals (Traber and Atkinson, 2007). It is tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and the amine group of cysteine, and the carboxyl group of cysteine is attached by normal peptide linkage to a glycine. Glutathione (GSH) can be used to detoxify reactive oxygen species such as H₂O₂ a process in which GSH is oxidized to the dimer glutathione disulfide (GSSG) in a reaction catalyzed by glutathione peroxidase (GPx). Glutathione plays an important role in protecting hemoglobin, red blood cell enzymes and biological cell membrane against oxidative damage by increasing the level of GSH in the process of aerobic glycolysis ( Traber and Alkinson, 2007 ).Deficiency of the enzyme may result in mild to moderately severe hemolytic anemia upon exposure to certain drugs or chemical ( Otitoju and Onwurah, 2005).

1.9 Tomato (Lycopersico esculentum)

Tomato is edible red fruit of commonly known as a tomato plant which belongs to the nightshade family. Numerious varieties of tomato are widely grown in temperate climates across the world greenhouse allowing its production throughout the year and in cooler areas. Dietary intake of lycopene predominantly comes from the consumption of tomatoes. In addition the red color found in ripe tomatoes is due to lycopene content thus unripe tomatoes are relatively low in lycopene (Di Mascio et al., 1989). Furthermore consumption of carotenoid rich food like tomatoes has been associated with several health benefits including their ability to prevent oxidative damage that result to diabetes mellitus (Olmedilla et al., 1997). Tomatoes can be used in the management of diabetes. Controlling blood sugar level is important part of diabetes management for diabetics; because of the low carbohydrate content in tomatoes it can play a big role in controlling blood sugar level. Interestingly over the year’s dietary consumption of vegetables and fruits (tomatoes) rich in carotenoid has been recommended for diabetic patients to be a protective factor against hyperglycemia (Suzuki et al., 1989).

1.9.1 Benefits of Tomatoes

In the distant past tomatoes were thought to be toxic in nature and a contributing factor in the development of conditions such as cancer, brain fever, and appendicitis. Research, however, has shown evidence to the contrary. Tomatoes have been found to be rich in vitamin A, containing 15% of the daily requirement, vitamin C, these vitamins also called antioxidants, are known to fight off the effects of free radicals, known to cause cell damage in the body. Tomatoes are an excellent food for aiding in vision improvement due to its high concentration of vitamin A. Tomatoes also contain a high amount of chromium which has been proven to be helpful in controlling your body’s blood sugar level. Thus diabetics will certainly benefit from consuming tomatoes, the presence of potassium and vitamin B help to lower high cholesterol levels and blood pressure this will aid the prevention of heart attacks and strokes. (Blot et al., 1993). Tomatoes are packed full of the valuable mineral known as chromium. It works effectively to help diabetics keep their blood sugar levels under better control. One of the main reasons for diabetes is the deficiency of vitamin C in our body. Supplements of vitamin C are beneficial to cure the diabetes as they help in processing of insulin and glucose. It can be cured by regular intake of vitamin c.

1.10 Onion (Allium cepa)

Allium cepa belongs to the family Liliaceae and is probably native of south west Asia and is widely cultivated throughout the world (Ikram, 1971). It has aglobose bulb that is an underground part of the stem and is so often treated as a single household vegetable. A.cepa has been used medicinally for hundreds of years (Ikram, 1971). Its most popular modern uses is to lower blood pressure (Ikram, 1971), antiseptic, hypoglycaemic and hypocholesterlemic properties (Mathew and Augusti, 1975). The active ingredient in A.cepa is allyl propyl disulfide (APDS), though other active sulphurous compounds are present (Kumari et al., 1995). Onions contain 89% of water, 4% sugar, 1% protein, 2% fiber and 0.1% fat. Onion contain low amount of essential nutrient and are low in fats, and have an energy value of 166 kj (40kcal) per 100g (3.5oz). They contribute their flavor to savory dish without rising caloric content appreciably. Freshly cut onions often cause a stinging sensation in the eyes of people nearby and often uncontrollable tears. This is caused by the release of a volatile gas syn-propanethial –s-oxide, which stimulate nerves in the eye creating a stinging sensation (Jain, 1976). This gas is produced by a chain of reactions which serve as a defense mechanism. Chopping an onion cause damage to cell which release enzymes called alliinases. Onion contain high amount of organosulphur which is one of their health benefit they make between 1to 5% of the dry weight of the bulb (Cho et al., 2002). The most important sulphur containing substance is the amino acid cystein and its derivatives, especially the S-substituted cystein sulphoxides and the gamma peptides. Onion extract posses some lipid-lowering effect and in higher concentration also have hypoglycaemic effect.

 

 

 

Fig. 2: Onion (Allium cepa)

1.11 Glibenclamide

Glibenclamide also known as glyburide is an antidiabetic drug in a class of medication known as sulfonylureas. It was developed in 1966 in a co-operative study (Kunte et al., 2007). It is used in the treatment of type 2 diabetes as of 2003 in the United States. Side effects and contradictions this drug is a major cause of drug induced hypoglycemia the risk increased against other sulfonylureas (Yamada and Inagaku 2005). glibenclamide may not be recommended in those with G6PD deficiency as it may cause acute haemolysis (Kunte et al., 2007).  This drug works by binding to and inhibiting the ATP sensitive potassium channels (^kATP) inhibitory regulatory subunits sulfonylurea receptor I (SURI) in pancreatic beta cells. This inhibition causes cell membrane depolarization, opening voltage dependent calcium channels (Nistico et al., 2007). This result in an increase in the intracellular calcium in the beta cell and subsequent stimulation of insulin release. After a cerebral ischemic insult, the blood brain barrier is broken and glibenclamide can reach the central nervous system (Simard et al., 2012). Glibenclamide has been shown to bind more efficiently to the ischemic hemisphere. Moreover under ischemic condition SURI the regulatory subunit of the ^kATP and the NC_ca ATP channels is expressed in neurons astrocytes, oligodendrocytes, and endothelial cells and by reactive microglia. glibenclamide improves outcome in animal stroke models by preventing brain swelling and enhancing neuroprotection (Xavier and Costa, 2009).

1.12 Alloxan

Hyperglycaemia, hyperlipidaemia and depressed antioxidants are the main common clinical features of the autoimmune disease, diabetes mellitus. Diabetes could be induced in experimental animals by chemical which selectively destroy pancreatic beta cells. The most common diabetogenic agents are alloxan or Streptozotocin (Baynes, 1991). Alloxan (2, 4, 5, 6- tetraoxypyrimidine, 5,6-dioxyuracil) was first described by Brugnatell in 1818 but Wohler and Liebig first called it alloxan and described its synthesis (Lenzen and Panten 1988). The cytotoxic action of alloxan is mediated by reactive oxygen species (Bromme et al., 2006) alloxan and the product of it reduction, dialuric acid establishes a redox cycle with the formation of superoxide radicals. Alloxan is an oxygenated pyrimidine derivative. It is present as alloxan hydrate in aquous solution (Frode and Medeiros, 2008). Alloxan is a toxic glucose analogue which selectively destroys insulin-producing cells in the pancreas when administered to rodents and many other animal species (Szkudelski, 2001) .This cause insulin dependent mellitus (called “alloxan diabetes”) in these animals, with characteristic similar to type I diabetes in humans. Human islets are considerably more resistant to alloxan than those of other animals (Mythill et al., 2004).

 

 

 

1.13 Aim and Objectives of the Study

1.13.1 Aim of the Study

The aim of this study work was to determine the synergistic effects of tomato and onion in alloxan-induced diabetic rats.

1.13.2  Specific Objectives of the Study

• To determine the median lethal dose (LD₅₀) of tomato and onion extract.
• To determine the effect of combining tomato and onion on blood glucose concentration in alloxan-induced diabetic rat.
• To determine the effect of tomato and onion on Vitamins A, C and E    concentrations in alloxan-induced diabetic rats.
• To determine the effect of tomato and onion extract on lipid peroxidation and some enzyme antioxidant in alloxan-induced diabetic rat.

DOWNLOAD (CHAPTER 1-5)