EVALUATION OF ANTIHYPERGLYCAEMIC EFFECT OF ETHANOL LEAF EXTRACT OF ANNONA SENEGALENSIS PERSOON (ANNONACEAE) IN ALLOXAN-INDUCED DIABETIC RATS

ABSTRACT

Annona senegalensis is an evergreen shrub that is used for  several ethnomedicinal purposes in Nigeria, including the management of diabetes mellitus. The present study was carried out to investigate the antihyperglycaemic activity of the leaf extract of A. senegalensis in alloxan-induced diabetic rats. The toxicological potential of the ethanolic leaf extract was investigated by determining the acute toxicity and the effect of the plant extract on body weights. A total of eighteen adult mice were  used  to  determine  LD50.  A  total  of  forty  two  adult  Wistar  rats  were  used  for  other investigations. The animals were divided into seven groups, each group contained six rats. Group 1 was the normal control, group 2 was the positive control, group 3 was the standard drug control and groups 4, 5, 6, and 7 served as the extract-treated groups. The blood glucose levels were assayed. Their  serum  samples  were  analyzed  after  two  weeks  of  treatment,  to  ascertain  the  alkaline phosphatase (ALP), aspartate transaminase (ALT), aspartate transaminase (AST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. malondialadehyde (MDA), reduced glutathione (GSH), total protein, total bilirubin, creatinine and urea levels were also tested and also, some blood electrolyte concentrations (sodium ion, potassium ion, chloride ion and bicarbonate ion) were investigated. Lipid profile (total cholesterol, TAG, HDL, LDL, and VLDL levels), as well as some haematological indices (platelets, PCV, Hb, RBC, and WBC) were also investigated. The phytochemical screening of the ethanol extract showed the presence of bioactive compounds; alkaloids, terpenoids, flavonoids and saponins, which were found to be abundantly present, while glycosides, phenol, and carotenoids were slightly present in the leaf extract. The LD50 of the extract indicated a very high safety profile with no mortality. There was significant (P <0.05) increase in the weights of the animals after treatment except for the positive control (induced but not treated) which exhibited no significant (P >0.05) difference from day 0 up to day 14.The blood glucose levels of the extract-treated groups after treatment showed significant (p<005) decreases when compared with the positive control group (group 2). Generally, there were significant (P <0.05) decreases in the activities of AST, ALT, an ALP, as well as the total bilirubin levels in the treated groups when compared to group 2 (induced but not treated). However, total protein levels in the treated groups showed significant (p<0.05) increases. There were no significant (p>0.05) changes in creatinine and urea concentrations in the treated groups relative to the positive control group and normal control group. Na+ concentration in group 2 was significantly (p<0.05) lower when compared to treated groups. The k+ levels in treated groups exhibited significant (p<0.05) increases when compared to group 2.There was no significant difference (p>0.05) in the concentrations of CL- and bicarbonate ions in treated groups when compared to the group 2.The SOD, GPx, CAT activities and GSH levels in treated groups were significantly (p<0.05) higher when compared with group 2.The MDA levels in the treated groups showed significant (p<0.05) decreases when compared to the positive control group. The concentrations of serum total cholesterol and LDL in treated groups were observed to be significantly (p<0.05) lower when compared to group 2.TAG and VLDL levels in group 2 were not significantly different when compared to treated groups. However, the concentrations of HDL-cholesterol in treated groups were significantly (p<0.05) higher when compared with the group 2. Platelets, PCV, Hb, RBC, and WBC in the treated groups showed no significant   (p>0.05) changes when compared with normal control. The results in this study showed that the leaf extract of A. senegalensis significantly reduced the hyperglycaemia in alloxan-induced diabetic rats and also restored some altered haematological indices as well as some biochemical parameters in alloxan-induced diabetic rats. The investigation provides evidence for the use of A. senegalensis leaf extract in folkloric treatment of diabetes and its associated complications.

CHAPTER ONE

INTRODUCTION

Diabetes mellitus (DM) is a common disorder characterised by the presence of chronic hyperglycaemia that results from defects in insulin secretion, insulin action or both (Maryam and Rahimi,  2015).  DM  is  a  prevalent  disease  affecting  the  citizens  of  both  developed  and developing countries (Mahsud et al., 2010). It is predicted that about 366 million people are likely to be diabetic by the year 2030 (WHO, 2011). It is a major health concern because it is frequently associated with debilitating health complications such as blindness, kidney function failure, cardiovascular diseases, amputations, and subsequently death. The classic symptoms of untreated diabetes are weight loss, polyuria. Other symptoms include blurry vision, headache, fatigue, slow healing of cuts and itchy skin (WHO, 2008). The therapeutic cure for diabetes mellitus  has  remained  elusive  despite  the  discovery  of  an  array  of  medications  that  can ameliorate the outcome of the disease. Therefore, it has become imperative to investigate alternative sources of medicament, especially those that are cheap and easily sourced. In traditional practices, medicinal plants are used to control diabetes mellitus in many African countries.  In  Nigeria,  herbal  therapies  occupy  a  very  special  position  in  health  delivery, especially  among  the rural  populace.  Easy  accessibility  and  low  cost of treatment  enhance patronage (Iwueke and Nwodo, 2008). Recent findings revealed that over 80% of the African population relies on medicinal plant species for their primary healthcare (Ngbolua et al., 2016). The hypoglycemic effects of some medicinal plants used as antidiabetic remedies have been reported (Odoemena et al., 2010; Maryam and Rahimi, 2015). Annona senegalensis, commonly known as “ubulu ocha” in Igbo (Nigeria) is one of such plants used traditionally in the management of diabetes mellitus. All the plant parts of A. senegalensis are useful in several diseases in Nigeria. The leaves are used in treating yellow fever, tuberculosis, and small pox (Mustapha et al., 2013).The root is used in conditions such as difficulty in swallowing, gastritis, snake bites, male sexual impotence, erectile dysfunction, tuberculosis, and as antidote for deadly toxins; the root bark is effective in infectious diseases (Adzu et al., 2005). A. senegalensis posseses antiparasitic activity on a resistant strain of Plasmodium falciparum (Fall et al., 2003). A. senegalensis has been reported to possess anti-inflammatory and antipyretic properties (Madièye et al., 2017).

1.1 Botanical description of Annona senegalensis Persoon plant.

Annona senegalensis Persoon is a tropical plant species also known as ‘wild custard apple’. It is a shrub (2–6 m) or small tree (11 m) under some suitable ecological conditions. The bark is smooth to roughish, silver grey or grey-brown. The leaves of this medicinal plant are alternate, simple, oblong, or elliptic, green to bluish green, with brownish hairs on lower surface. The flowers are up to 3 cm in diameter on stalks, 2 cm long, solitary or in groups of 2–4, arising above the leaf axils.

The fruits are formed from many fused carpels, fleshy, lumpy; egg shaped, unripe fruit green, turning yellow to orange on ripening. Wild fruit trees of this species are found in semi-arid to sub-humid regions of Africa.It is native to tropical east and north-east, west and west- central, and southern Africa, as well as southern subtropical Africa, and islands in the western Indian Ocean. The species occur along river banks, fallow land, swamp, forests and at the coast. (Orwa et al., 2009).

1.1.1Taxonomy of Annona senegalensis Persoon plant. Kingdom:                              Plantae

Subkingdom:                        Tracheobionta Superdivision:                       Spermatophyta Division:                                Magnoliophyta Class:                                     Magnoliopsida Subclass:                               Magnoliidae Order:                                    Magnoliales Family:                                  Annonaceae Genus:                                   Annona

Species:                                  A. senegalensis (Mustapha, 2013).

1.1.2 Common Names

English: wild custard apple, wild sour sap

Igbo: Ubulu ocha

French: Annone, pomme cannelle du senegal

Swahili: Mchekwa, mkonokona, e.t.c (Mustapha, 2013)

1.1.3 Medicinal Uses Annona senegalensis Persoon

Annona senegalensis Persoon. (Annonaceae) is a multipurpose plant with many traditional and medicinal uses. Traditionally, the plant is used as stimulant, pain reliever, food etc. As for the traditional medicine practices, all the plant parts of A. senegalensis are useful in several diseases.The leaves have been used in treating yellow fever, tuberculosis, and small pox (Mustapha et al., 2013). The stem bark has been used in snake-bite and hernia treatment. The root is used in conditions such as difficulty in swallowing, gastritis, snake-bites, male sexual impotence, erectile dysfunction, tuberculosis, and as antidote for deadly toxins; the root bark is effective in infectious diseases and others (Noumi and Safiatou, 2015). Many of the plant parts are used as antidotes for venomous bites and in the treatment of malaria (Traore et al., 2013). A. senegalensis  has  been  employed  in  the  management  of  diabetes  by  herbal  practitioners  ( Ahombo et al., 2012).These medicinal  properties of the plant have been attributed to important phytochemical constituents such as  triterpenes, anthocyanes, glucids, coumarins, flavonoids and alkaloids etc. (Samuel et al., 2016).

Figure 1: Annona senegalensis Persoon plant

1.1.4 Other traditional usesAnnona senegalensis Persoon.

The ashes from the wood is added to chewing or snuff tobacco and also is a solvent in soap production in some areas in Africa and the Leaves are sometimes used in filling mattresses and pillows, and in Sudan a perfume is made from the boiled leaves. In South Africa, the roots are said to cure madness, and in Mozambique, they are fed to small children to induce them to forget the breast and thus hasten weaning. It has also been claimed that the leaves picked on a thursday morning and thrown over the right shoulder bring good luck (Samuel et al., 2016).

1.1.5 Previous scientific investigations.

Review of documented literature showed that the plant has anticancer (Sowemimo et al., 2007), antimalarial and cytotoxic (Ajaiyeoba et al., 2006), anticonvulsant (Ezugwu and Odoh, 2003), analgesic and anti-inflammatory (Adzu et al., 2003) properties. The spermatogenic (Oladele et al., 2014), antioxidant (Ajboye et al., 2010), anthelmintic (Alawa et al., 2003) and Antitrypanosomal (Ogbadoyi et al., 2007) activities of A. senegalensis plant have also been investigated.

1.2 Phytochemicals

Phytochemicals  are  secondary  metabolites  produced  by  plants;  they  give  plants  its  colour, flavour, smell and are part of a plant’s natural defence system (Okwu, 2005) . Phytochemicals are chemical compounds formed during the plant normal metabolic processes.They are made up of several classes of compounds which include: alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Okwu, 2005). These compounds have been linked to human health by contributing to protection against deteriorative diseases (Dandjesso et al., 2012). Phytochemicals are present in varieties of plants  and are utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables (Okwu, 2005). Different mechanisms have been suggested for the action of phytochemicals. They may act as antioxidants, or modulate gene expression and signal transduction pathways (Dandjesso et al., 2012).The medicinal values of these plants lie in their component phytochemicals, which produce the definite physiological actions on human body.

1.3 Glucose homeostasis

Glucose is the main fuel for most tissues. Glucose metabolism is critical to normal physiological functioning. Glucose acts both as a source of energy and as a source of starting material for nearly all types of biosynthetic reactions. Oxidative metabolism of glucose allows production of adenosine triphosphate (ATP), which is an energy source for many chemical reactions in the body (Wolfe, 1998). Plasma glucose concentration is a reflection of the balance between glucose uptake in the gut, endogenous production, utilization and storage in the body (Woerle et al.,

2003). Endogenous production of glucose is through gluconeogenesis and glycogenolysis, while tissue utilization is mainly mediated via glycolysis and to a lesser extent, the pentose phosphate pathway. Following ingestion of a meal, a third of glucose is taken up by the brain tissue where it undergoes complete aerobic break down to water and carbon dioxide, 2 to 3 hours later; another third is used by the muscle where it is converted to glycogen orcatabolized to lactate or water and carbon dioxide; and the rest is taken up by the liver where it is mainly converted to glycogen and stored (Morifuji et al., 2005). The liver, therefore, plays an essential role in the maintenance of blood glucose. It maintains blood glucose levels by maintaining a balance between glucose uptake and its conversion to glycogen and the release of glucose into circulation via glycogenolysis and gluconeogenesis (Saadat et al., 2004). The role of other tissues such as the

kidney in  endogenous  glucose production  is  minimal.  Nevertheless,  the contribution  of the kidney is significant during prolonged fasting.

1.3.1 Glucose transport into cells

The lipid nature of cell membranes offers a barrier against free glucose entry into cells. There are

2 major types of glucose transporters that overcome this membrane barrier. Sodium-glucose co- transporters are mainly located in the kidney and intestines; these actively transport glucose into cells against concentration gradient by transporting sodium. GLUT transporters are a family of glucose transporters that transport glucose into cells passively, down a concentration gradient. They includeGLUT-l transporters which are located in the brain, erythrocytes and endothelial cell; GLUT-2, located in the kidney, small intestines, liver and pancreatic tissues; GLUT-3, expressed in the neurons and placenta; GLUT-4, expressed in the skeletal, cardiac and adipose tissues. Transport of glucose via GLUT-4 into muscle and adipose tissue is the rate-controlling step in insulin-mediated glucose disposal and this disposal is compromised in conditions of insulin-resistance (Herman  and  Kahn,  2006).  GLUT-5  transporters are  located in  the small intestine, sperm, brain, kidney and muscle (Shepherd and Kahn, 1999).

1.3.2 Roles of kidney in glucose metabolism

Kidney participates in glucose homeostasis maintaining by taking part in gluconeogenesis, glucose filtration, reabsorption and utilization. Kidneys have an important influence on maintaining body metabolic glucose homeostasis both in physiologic and pathologic conditions (Marsenic, 2009). Kidneys not only take part in glycolysis and gluconeogenesis, but also perform essential functions of glucose filtration in glomeruli, and reabsorption in tubules. In these processes two types of transporters are involved. GLUT’s (glucose transporters), which are glucose specific protein carriers, and SGLT s (sodium-dependent glucose cotransporters), which require sodium ions for work (Hummel et al., 2011 and Gerich, 2010). Every of these processes may be disturbed in diabetes (Flordellis et al., 2005).

1.4 The role of the pancreas.

The pancreas is a large gland that nestles under the stomach, plays an important part in glucose regulation and is unusual having both an exocrine and endocrine function. As an exocrine gland

it produces several digestive enzymes that are secreted into the duodenum via the pancreatic duct (Pandol et al., 2011). Over 90 percent of the pancreas is devoted to its exocrine and diagestive function. As an endocrine gland, the pancreas secretes a variety of hormones that are concerned with the regulation of blood glucose, including insulin, glucagon, and somatostatin (Guthrie, 2002).

1.4.1 Synthesis and Release of Insulin

Insulin is produced by the beta cells of the pancreas; it is the principal hormone regulating glucose metabolism. Its presence enhances glucose uptake and metabolism by various tissue cells including skeletal muscle, white adipose tissue and the liver (Barros et al., 2006). During non-pathological states, glucose is the stimulus for insulin secretion in pancreatic beta cells. Glucose entry into the pancreatic cells is through GLUT-2 transporters (Herman and Kahn,

2006). Oxidation of glucose and the subsequent increase in ATP/ADP ratio in the cells triggers closure of ATP-sensitive potassium channels (Doyle and Egan, 2003). Inhibition of potassium efflux results in cell depolarization leading to influx of voltage dependent calcium ions that stimulate extrusion of insulin. Physiological  insulin secretion  consists of a basal  and bolus component. During fasting, basal insulin secretion maintains glucose levels between 70 and 120 mg/dl, and bolus secretion keeps glycaemic levels below 180 mg/dl after a meal (Fonseca, 2006).

1.4.2 Insulin signaling pathway

The Insulin function involves various signaling cascades, which are initiated by insulin binding to its receptor (IR) of target cells, stimulating autophosphorylation of the receptor, eliciting the activation of receptor tyrosine kinases and subsequently stimulating the tyrosine phosphorylation of   insulin   receptor   substrates   (IRSs).   This   activates   tyrosine   kinase   which   further phosphporylates insulin receptor substrates (IRS) leading to a series of pathways that result into insulin  signal  transduction  (Sale  and  Sale,  2008).  Depending  on  the  target  tissue  cell,  the outcome of the signal transduction might be movement of GLUT 4; for instance, from intracellular pools towards the cell surface of skeletal muscle and white adipose tissues, or reducing expression of gluconeogenesis genes in the liver (LeRoith and Gavrilova, 2006) or indeed, increasing levels and activities of glycogen synthase and glycogen phosphorylase in the liver (Barros et al., 2006).

1.4.3 Insulin regulatory and counter-regulatory hormones

Cells are located in the islets of Langerhans in the pancreas. Other cell types present in islets include alpha and pancreatic polypeptide (PP) cells. Glucagon, the catecholamines epinephrine and norepinephrine, glucocorticoids, and growth hormone all act to raise plasma glucose levels. Their actions generally oppose those of insulin, and as a group, they are called counter regulatory hormones (Hussain et al., 2000). Glucagon increases glycaemia by mediating hepatic glycogenolysis (Robertson and Harmon, 2006). Studies have shown that diabetics have basal or increased  plasma  glucagon  concentrations.  Furthermore,  suppression  of  this  hormone  is associated with reduction of plasma glucose concentration (Cherrington et al., 1976). Cells also secrete  somatostatin  which  plays  an  inhibitory  role  in  the  secretion  of  several  hormones, including insulin (Robertson and Harmon, 2006).

Counter-regulatory effects of epinephrine are due to its direct effects on target tissues and its effects on other participating hormones. For example, Epinephrine is released by the adrenal medulla in response to hypoglycemia (as well as other stress stimuli), and Norepinephrine is released from sympathetic neurons. Both catecholamines have significant roles in maintaining glucose levels in exercise (especially in supporting the massive increase in glucose use by the muscle), and in stress-related conditions. Catecholamines stimulate glucagon release and inhibit insulin release, causing a decrease in insulin (Guy et al., 2005). Due to its effects on muscle, epinephrine also decreases insulin mediated glucose uptake as a result of glucose-6-phosphate- dependent  inhibition  of  hexokinase  (Raz  et  al.,  1991).  In  addition  adrenaline  compromises insulin-induced glycogen synthesis attributed to its inhibition of glycogen synthase (Raz et al., 1991).

Glycogenolysis and inhibition of glycogen synthesis, by both epinephrine and glucagon, are mediated via increased generation of cAMP which activates protein kinase A. Protein kinase A inactivates glycogen synthase and activates phosphorylase (Hodis, et al., 2007). Glucocorticoids (primarily cortisol in humans) are released from the adrenal cortex in response to stress; one such stress is a decrease in plasma glucose. Glucocorticoids stimulate gluconeogenesis and glycogen synthesis in the liver, and reduce muscle and adipose tissue glucose uptake. They also acutely inhibit insulin release, and over longer term, insulin action; prolonged cortisol hypersecretion can result in diabetes (Liu et al., 2005). In addition, glucocorticoids enhance hepatic gluconeogenesis

and impair effects of insulin to reduce glucose production in type II diabetes (Liu et al., 2005). In states  of  intensive  glycaemic  control  resulting  in  hypoglycaemia,  hormones  antagonistic  to insulin   action   are   released   to   restore   normoglycaemia   (Ross   et   al.,   2005).   Hence, counteregulatory  hormones  may  offer  benefits  in  specific  conditions.  Adiponectin  is  an adipocyte derived peptide which improves insulin sensitivity via several mechanisms. It modulates fatty acid oxidation, inhibits fatty acid synthesis and uptake in the liver and enhances glucose and fatty acid oxidation in the muscle (Mlinar et al., 2007). These effects are mediated through stimulation of perixosome proliferator gamma receptors (PP AR-y) nuclear receptors and activation of AMP kinase (Mlinar et al., 2007). Like adiponectin, leptin is an adipocyte derived-hormone that circulates in plasma in free and bound form. Acting through its receptors located in the hypothalamus, hippocampus, cortex, cerebellum, thalamus and choroids plexus, leptin decreases levels of circulating neuropeptide resulting in reduced appetite (Mantzoros,

1999).

Leptin also modulates glucose homeostasis (Levy and Stevens, 2001). In insulin deficientrats, administration of leptin restores normal glucose levels, enhancesglucose metabolism in the post absorptive stages and improves hepatic insulin sensitivity during glucose clamp (Chinookoswong et  al.,  1999).  Leptin,  therefore,  acts  agonistically  to  effects  of  insulin.  Moreover,  leptin modulates release of thyroid hormones which playa homeostatic role in glucose metabolism (Wang et al., 2000). Thyroid hormones secreted by the thyroid glands also exert glucose homeostatic functions in synergy as well as antagonistic to insulin action. Thyroid hormones may increase glucose production via hepatic gluconeogenesis, to meet increased energy requirements by the body (Lenzen and Bailey, 1984). Thyroid hormones, however, enhance transcription of insulin sensitive glucose transporter-4 (GLUT-4) (Chidakel, et al.,2005). This allows increased uptake of glucose by tissue cells thereby offsetting initial increase in blood glucose (Lenzen and Bailey, 1984).

1.4.4 Insulin resistance

Insulin resistance largely caused by central obesity is a key component in the pathogenesis of type II diabetes mellitus. It is defined  as the inability of insulin to  curtail hepatic glucose

production and promote glucose disposal in the peripheral tissues (Guillermo and Luigi, 2013). Insulin resistance constitutes defective insulin receptor function, insulin receptor signal transduction, glucose transport and phosphorylation, glucose uptake and glycogen synthesis in adipose, skeletal and hepatic tissues (Panunti et al., 2004).

Adipose tissue secretes large quantities of tumour necrosis factor (TNF)-a. According to Mlinar et al. (2007), TNF–a is the main factor that stimulates increased levels of circulating free fatty acids into circulation. Increased intraportal free fatty acids impair insulin clearance. The mechanisms by which this is mediated are still unclear (Kahn and Flier, 2000). It is, however, suggested that accumulation of intramyocellular lipids in striated muscles suppresses glucose.

1.5.0 Diabetes mellitus

Diabetes mellitus is a group of metabolic disorders with different underlying aetiologies, each characterised by chronic hyperglycaemia owing to over production and/or underutilization of glucose in the cellular system. It is the condition in which the body does not properly process food for use as energy. Most of the food is turned into glucose, or sugar, for our bodies to use for energy. The pancreas, an organ that lies near the stomach, makes a hormone called insulin to help glucose get into the cells of the bodies. Diabetes, occurs when our bodyeither doesn’t make enough insulin or can’t use its own insulin as well as it should. This causes sugars to build up in the blood (ADA, 2012).

1.5.1 Signs and symptoms of diabetes mellitus.

The common signs and symptoms of diabetes include: Frequent urination, Excessive thirst, Unexplained weight loss, Extreme hunger, Sudden vision changes, Tingling or numbness in hands or feet, Feeling very tired much of the time, Very dry skin, Sores that are slow to heal andMore infections than usual (Rother, 2007).

1.5.2 Types of Diabetes mellitus

The major types of diabetes mellitus are type 1 and type 2, the former arising from inadequate production of insulin due to pancreatic p-cell dysfunction, and the latter arising from reduced sensitivity of insulin in target tissues and / or inadequate insulin secretion (Zhao, 2011).

1.5.3 Type 1 Diabetes mellitus

Type 1 diabetes mellitus, previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes,  may account for 5 percent to 10 percent of all diagnosed cases of diabetes. Insulin is necessary for the body to be able to use sugar. Sugar is the basic fuel for the cells in the body, and insulin takes the sugar from the blood into the cells.” Type I diabetes without any production of insulin is according to this understanding a severe problem for the body and will lead definitely to death (Kaufmanet al., 2012). Risk factors are less well defined for Type 1 diabetes than for Type 2 diabetes, but autoimmune, genetic, and environmental factors are involved in the development of this type of diabetes. There are two major type I diabetes, namely, Immune-mediated and Idiopathicdiabetes. (Soumya and Srilatha, 2011).

1.5.4 Immune-mediated diabetes mellitus

Immune-mediated type 1 diabetes mellitus, previously known as insulin dependent diabetes mellitus (IDDM), constitutes 5 to 10% of all diabetic cases (American Diabetes association,

2004). The body’s immune system can mistakenly destroy the insulin-producing beta cells of the pancreas. The causes of autoimmune diabetes are poorly understood, but genetics and family history play a role, and viruses or other environmental factors are believed to figure in (Kukreja and Maclaren, 1999).

Risk factors for pathogenesis of type 1 diabetes include genotype, diet and viral infections e.g. rubella, coxsackie, mumps (Wei, et al.,2006). Coxsackie viral infections are a type of environmental factors that do not initiate, but enhance development of type I diabetes in genetically prone victims (Serreze, et al., 2000). It is proposed that coxsackieviral infections probably provide a molecular mimic during replication that triggers a cross-reactive CD4+ T-cell response against the candidate cell autoantigen (Serrezeet al., 2000). Ultimately cell death occurs by apoptosis (Kurrer, et al., 1997).

Figure 2: auto-immune diabetes disorder mechanism.. (William et al., 2011). In addition, low birth weight is considered a risk factor for type I diabetes. Low birth weight is indicative of exposure of foetus to maternal viral infection and the interaction of immune cross-reactivity between virus and auto-antigens which may lead to destruction of pancreatic cells (Wei et al., 2006). Furthermore, low birth weight is commonly associated with a reduced beta cell mass which is a vital factor inthe pathogenesis of diabetes. Worldwide, an estimated 4.9 million people have type 1 diabetes (Gadsby, 2002). And approximately 35,100 children have type 1 diabetes in Africa (Beran et al., 2006).

1.5.5 Type II diabetes mellitus

Type II diabetes mellitus, formerly known as non-insulin dependent diabetes(NIDDM) is the most common type of diabetes mellitus accounting for 90 to 95% ofall diagnosed cases of diabetes (American Diabetes Association, 2004). Type IIdiabetes is a polygenic disease caused by diminished response of target cells to insulinaction, and/or insufficient production of insulin. Inadequate insulin production oftencharacterizes later stages of type II diabetes and is attributed

to reduced cell mass orbeta cell dysfunction (Jaime, 2013). Hepatic gluconeogenesis and renal gluconeogenesis significantly contribute to postprandial hyperglycaemia in type II diabetes (Meyer et al., 1998).

In recent times, interest has been generated into the possibility of involvement of the hypothalamus – pituitary – adrenal axis in the pathogenesis of type II diabetes (Koshiyama et al.,

2006). Risk factors for type 2 diabetes include genotype, age, previous history of gestational diabetes, behavioral factors including poor diet, and sedentary lifestyle. Genetic pre-disposition interacts with the environmental factors to influence disease prevalence (Zachary et al., 2012). In industrialized nations the increase in prevalence and incidences of diabetes are due in part to the increase in the age of the populations. Obesity is present in 50% and 70% of type II diabetic men and women, respectively (Etie et al., 2013). It leads to increased lipolysis which results in increased levels of circulating free fatty acids and triglycerides and their subsequent deposition in muscle bed (McGarry, 2001).

1.5.6 Gestational diabetes mellitus

Gestational diabetes, which occurs during pregnancy, is the third commonest type of diabetes. It is  derangement  in  carbohydrate metabolism  resulting  in  hyperglycaemia  with  onset  or first detection during pregnancy (ADA, 2016). Causative factors for gestational diabetes mellitus are unknown but autoimmune activities in the pancreatic b-cells of pregnant mothers are suggested (McEvoy, et al., 2004). It has high prevalence in developed countries at 3-19%, and is low in Africa, at 0 to1 % (Seyoum et al., 1999). The significance of gestational diabetes mellitus is that it poses 16 to 63 % risk factor to the mother for future development of type II diabetes within 5 to 16 years (Hyer and Shehata, 2005).

Gestational hyperglycaemia also exposes the foetus to risk of future diabetes,hypertension, and obesity (Maresh, 2005). In addition to the pre-natal morbidity, immediate dangers of gestational diabetes include pre-term birth, macrosomia, pre-eclampsia and caesarean section (Fan et al.,

2006). Excessive foetal exposure to glucose as a result of maternal hyperglycaemia results in permanent reduction in levels of GLUT-l and GLUT-4 transporters in the new born and adult offspring of a gestational diabetic mother (Boloker et al., 2002). Down regulation of these transporters may be an  adaptive mechanism by which the  foetus protects itself against the

adverse effects of hyperglycaemia. The foetus adapts to a changed environment in the uterus that may enhance its chance of short-term survival but at the expense of a long-term capacity for normal growth and development, resultbing in the development of diabetes (Boloker et al.,

2002). Foetal exposure to excessive levels of glucocorticoids in diabetic pregnancy is one of several factors that subject them to growth retardation, hypertension and diabetes mellitus later in life (Fujisawa et al., 2004). Also demonstrated that increased plasma cortisol levelswere strongly associated with hypertension, hyperglycaemia, fasting hypertriglyceridaemia and insulin resistance in adults.

It has been shown that diabetic pregnancy suppresses expression of 11 beta hydroxyl steroid dehydrogenase (llh-HSD) type 2 in the placenta and foetal kidney (Fujisawa et al., 2004). llh- HSD is a high affinity NAD+ -dependent enzyme that inactivates glucocorticoids. The role of glucocorticoids in insulin resistance is well established. Glucocorticoids mediate lipolysis resulting in increased availability of free fatty acids which culminates in reduced glucose oxidation and uptake in peripheral tissues. Moreover, the presence of glucocorticoids also compromises translocation of GLUT-4 transporters to the cell membranes (Mlinar et al., 2007). These substances promote blood vessel vasoconstriction, gluconeogenesis and glucose release and discourage glucose uptake in the peripheral tissues (Mlinar et al., 2007).

There are opposing  views  about  influence of birth  weight  on  future occurrence of  chronic diseases including diabetes mellitus. One view asserts that gestational diabetes leads to low birth weight which is a risk factor for type l diabetes (Boney et al., 2005).

1.5.7 Other types of diabetes mellitus

Other specific types of diabetes are: Diabetes due to genetic defect of beta cells, alsoknown as Maturity onset diabetes of the young, (MODy), diabetes due to geneticdefects in insulin action, diabetes due to diseases of the exocrine pancreas, diabetes due to endocrinopathies, and drug or chemical-induced diabetes (American Diabetes Association, 2004).

1.6 Diabetic complications

Complications  arising  from  hyperglycaemia  are  both  microvascular  and  macro  vascular  in nature. Microvascular complications, affecting the smaller blood vessels frequently result from

type 1 diabetes while macrovascular complications affecting large blood vessels, originate from type II (Girach and Vignati, 2006). The two are the leading cause of morbidity and death in people with diabetes.

1.6.1 Micro-vascular complications

Micro-vascular complications includes: (a) neuropathy, (b) retinopathy and (c) nephropathy.

1.6.2 Diabetic neuropathy

Diabetes mellitus, a common metabolic disease with a rising global prevalence, is associated with long-term complications of peripheral nervous system and the central nervous system (Chen et  al.,  2011).  Diabetic  neuropathy  is  a  chronic  micro-vascular  complication  affecting  both somatic and autonomic peripheral nerves. It may be defined as the presence of symptoms or signs of peripheral nerve dysfunction in people with diabetes, after the exclusion of other causes of neuropathy. Neuropathy is the common complication of diabetes and is due to high blood sugar, chemical changes that occur in the nerves (Heltianu and Guja, 2011). Generally it starts in the nerves of feet as they are the longest nerves and nourished with longest blood vessels of the body. This condition is called diabetic foot or diabetic peripheral neuropathy or distal symmetric neuropathy. Diabetes can reduce the blood supply to the foot and gradually damages the nerves which carry sensation. Diabetic neuropathy can cause foot ulcers and foot infections as advanced complications  in  diabetic  patients.  Signs  and  symptoms  of  Diabetic  Neuropathy  include, decrease or no sweating, numbness, or tingling, and some sort of burning sensation, weakness and loss of reflexes. Diabetic Polyneuropathy is a major complication of diabetes mellitus that frequently leads to foot ulceration (Viswanathan and Kumpatla, 2011).

1.6.3 Diabetic nephropathy

Diabetic  nephropathy  is  the  most  important  cause  of  renal  failure  in  developed  countries (Amador, et al., 2000). Patients with early renal damage manifest with micro-albuminuria which progresses into protein uria (Leiter, 2005). In renal tissue, advanced glycation end-products can induce activities of transforming growth factor and raise expressions of various extracellular matrix mRNAs. These mediate hypertrophy of the glomeruli, thickening of basal membrane, and

expansion of the mesangial extracellular matrix. Such structural changes are associated with albuminuria and protein-uria (Stitt, 2003).

Blocking activities of advanced glycation end products by a phenyl hydrazine compound, amino- guanidine, slows down mesangial expansion and proteinuria (Adler, 2003). In addition, hypertension associated with diabetes is one of the most important factors in the pathogenesis and progression of diabetic nephropathy (Bretzel, 1997). In turn, diabetic nephropathy is critical in the development of hypertension in diabetics (Parving et al., 2008). Diabetic nephropathy results  in  progressive  deterioration  of  glomerular  function.  The  decline  in  renal  function, however, can be attenuated by management of hypertension. Hyperglycaemia causes a redox imbalance.  This  is  as  a  result  of  excessive  reactive  oxygen  species  generation  as  well  as glycation of enzymes responsible for scavenging reactive oxygen species (Marcheva et al.,

2010). Enzymes involved in cellular defence against excessive reactive oxygen species build up include catalase, glutathione peroxidase and superoxide dismutase. Increased incidences of cardiovascular complications and renal function deterioration are partly attributed to reduced excretion of electrolytes such as sodium (Dahar and Gutkowska, 2000). This was supported by other trials that showed that reduction of hypertension in addition to managing glycaemia to acceptable  levels  did  reduce  incidences  of  macrovascular  complications  (Bate  and  Jerums,

2003).

1.6.4 Diabetic retinopathy

Diabetic retinopathy is the commonest cause of blindness in people afflicted with diabetes, aged between 20 and 74 years (AAO, 2016). Retinopathy is characterized by increased vascular permeability by vascular closure mediated by the formation of new blood vessels neovascularization, on the retina and posterior surface of the vitreous. Diabetic retinopathy is a micro-vascular disease, characterized by damage to the blood vessels and retina of the eyes. This condition occurs in both type 1 and type 2 diabetics (da Silva at al., 2010). It can be classified as non-prolifeative diabetic retinopathy and proliferative diabetic retinopathy or diabetic macular edema (Abràmoff, et al., 2013). In diabetic retinopathy, the micro vessel supplying blood to the retina of eye is affected and can cause blindness. Retinopathy is related to high blood sugar level and obstructs the flow of oxygen to the cells of the retina (Zhang et al., 2010). For the vision of eye, retina receives signals of light and sent them to the brain forming a three dimensional figure

which is identified. Finally it is sent back to the eye by which one can recognize the things around (Muaka et al., 2011). This working mechanism of passing light through the retina is hindered by the high glucose levels. The initial stage of this disease is known as Non- proliferate Diabetic retinopathy where as Proliferative diabetic retinopathy is the advanced form of diabetic retinopathy in which new as well as weak blood vessels break and leak blood into vitreous of the eye causing floating spots in the eye (Chakrabarti et al., 2012). Gradually, the swollen and scar nerve tissue of the retina is totally destroyed and leads to retinal detachment. The ground cause for blindness among diabetes is due to the retinal detachment (Raman et al., 2009).

1.6.5 Macro-vascular complications

Macro-vascular complications are largely responsible for mortality in diabetes, with cardiovascular diseases accounting for up to 80% morbidity (Akula et al., 2003). Macro-vascular complications include (a) atherosclerosis, (b) reproductive problems, (c) stroke and (d) coronary artery diseases. (Chakrabarti et al., 2012).

1.6.6 Atherosclerosis

Atherosclerosis is common in smokers and those with high blood pressure and abnormal fat levels in the blood. It is commonly fatty deposits in arteries or hardening of arteries. It accounts for virtually 80% of all deaths among diabetic patients (Soumya and Srilatha, 2011). Due to hyperglycaemia, fonnation of advanced glycation end products in arterial collagen results in trapping of low density lipoproteins. This process speeds up therate of atheroma fonnation in blood vessels (Ahmed, 2005). In addition advance dglycation end products promote smooth muscle  cell  proliferation  in  blood  vessels  (Basta  et  al.,  2004).  In  coronary  artery  disease, advanced glycation end products cause increased deposition of lipids in the cardiac cells, and compromise ventricular compliance (Sobel and Schneider, 2005).

1.6.7 Reproductive complicationscaused by diabetes mellitus

Hyperglycaemia due to diabetes is a significant underlying cause of a number of sexual disorders associated  with  diabetes  mellitus.  About  90  %  of  diabetics  manifest  sexual  problems characterized by impotence, infertility and low sexual drive (Amaral et al., 2006). These are attributed to hyperglycaemia-induced reactive oxygen species generation. Erectile dysfunction in

diabetes  is  caused  by  lack  of  smooth  muscle  relaxation  mediated  by  nitric  oxide  due  to endothelial dysfunction and autonomic neuropathy (Price, 2006). Moreover, interaction of reactive oxygen species generated as a consequence of diabetes mellitus, with DNA of spermatozoa, results in reduced motility and fertility of human sperm (Amaral et al., 2006).

1.6.8 Pathogenesis of diabetic complications

Development and progression of diabetic complications are mediated by four major pathways which includes increased sorbitol (polyol) pathway flux, increased formation of advanced glycosylation end products, increased activities of protein kinase C, and increased hexosamine pathway flux (Geraldesand King, 2010 ).

Generation of reactive oxygen species have been implicated as the main pathogenic factor as well as the result, in these processes. In endothelial cells, glucose enters into cells via plasma membrane transported by insulin-independent GLUT-1 transporter. Its subsequent accumulation in  the  cells  causes  generation  of  superoxide  which  is  considered  a  key  component  in  the initiation of all other pathways (Lapolla, 2005). Excessive formation of reactive oxygen species results in oxidative damage to DNA, proteins and lipids (Dandona et al., 1996). The most affected organs appear to be those whose glucose uptake is independent of insulin influence including the nervous system, kidney, heart and small blood vessels (Ahmed, 2005).

1.7 Diagnosis and clinical manifestations

The diagnosis of diabetes depends on the demonstration of carbohydrate intolerance; the symptoms are characterized by elevated fasting or post-prandial blood glucose level.

Several assays are used in diagnosing diabetes, these includes; oral glucose tolerance assay (OGTA), fasting blood sugar assay (FBS), random blood sugar assay (RBS), urine assay and glycosylated haemoglobin assay (Yehuda et al., 20011).

1.7.1 The fasting plasma glucose test: Is done in the morning before eating. Normal fasting plasma glucose levels are less than 110 milligrams per decilitre (mg/dl). Fasting plasma glucose levels of more than 126 mg/dl on two or more tests on different days indicate diabetes.

1.7.2 Random plasma glucose test: Differs from situation to situation and is done the same way as the fasting glucose test but without fasting. It shows different levels depending on the time difference of food-intake. Because of more and more problems of low-level glucose by diabetics in the morning, medical doctors change from tests after fasting to this kind of testing.

1.7.3 Oral glucose tolerance test (OGTT): For the oral glucose tolerance test (or Impaired Glucose Tolerance Test, IGT), you must fast overnight (at least 8 but not more than 16 hours) and go to your doctor’s office or the laboratory in the morning. First, your fasting plasma glucose is tested. After this test, you receive 75 grams of glucose. Blood samples are taken up to four times during the following hours to measure the blood glucose (Saxena et al., 2010). Other glucose level assay includes urine assay and glycosylated haemoglobin assay (Saxena et al.,

2010).

1.8 Pharmacological intervention

1.8.1 Insulin

Insulin  is  one  of  the  major  hypoglycaemic  agents  presently  used  in  diabetes  mellitus management.  It  was  discovered  in  1921  and  is  the  mainstay  antidiabetic  agent  for  the management of type 1 diabetes and is also used in late-stage type II diabetes (Emilien, et al.,

1999). There are several types of insulin classified based on their duration of action. These are rapid (ultra short) acting (e.g. lispro, aspart), short-acting (e.g regular insulin), intermediate – acting (e.g. lente) and long-acting (e.g. glargine, detemir) (Bethel and Feinglos, 2005). Insulin is currently administered subcutaneously using multiple daily injections or external pump for continuous delivery (Hirsch, et al.,2005). Other delivery routes include oral, inhaled, nasal, rectal, ocular, intravaginal and transdermal (William,2011). Intensive insulin therapy to maintain optimal glucose levels in type 1 diabetes can reduce incidences and exacerbation of micro- vascular complications by 50 to 70% (Elizabeth et al., 2014). Shortfalls of insulin therapy include ineffectiveness on oral application due to first pass hepatic metabolism, short shelf life, and severe hypo-glycaemia on over dosage, non-compliance to painful injections and weight gain (Shilpa et al., 2012).

1.8.2 Oral blood glucose lowering agents

There are five classes of antidiabetic agents presently in use, including biguanides, sulphonylureas, thiazolidinediones, benzoic acid derivatives and a-glucosidase inhibitors. Highlighted below are some of their modes of actions, advantages and limitations (Richard et al., 2012).

1.8.3 Sulphonylurea

Sulphonylureas act by stimulating insulin secretion. They bind to beta cell receptors that are linked to ATP-dependent potassium gated channels. Interaction with these receptors inhibits opening of potassium channels. Potassium builds up in the cell causes cellular depolarization evoking opening of calcium channels, and subsequently, influx of calcium ions which mediate exocytosis of secretory granules containing insulin (Sim and Hernandez, 2002). Sulphonylureas usage is restricted to type II diabetes mellitus because it requires a functioning pancreatic beta cell mass. Examples of agents presently in use include Gilbenclamide, glipizide, tolbutamide and gliclazide.  Hypoglycaemia  is  the  main  side  effect  of  sulphonylureas  (porksen,  2006).  The advance effect of sulphonylurea class of antidiabetic agents is that they stimulate insulin release by the pancreatic beta cells independent of glucose loading thereby subjecting the beta cells to exhaustion. Furthermore, they cause allergy and weight gain as a result of increased blood levels of insulin.

1.8.4 Biguanides

Biguanides  were introduced  for use between  1957  and  1960  (Sim6 and  Hernandez,  2002). Biguanides (e.g. metformin) mediate antihyperglycaemic effects by sensitizing target tissues cells  to  insulin  action  and  blocking  hepatic  gluconeogenesis  (pari  and  Satheesh,  2006). Metformin (Dianben~ is the principal drug in this class. The drug has been proved effective when used either in mono-therapy or in combination with other groups of drugs such as sulphonylureas or insulin (Chadwick et al., 2007). Its mechanisms of actions are still unclear. Current studies, however, show that on a subcellular level, metformin mediates hypoglycaemic effects by stimulating AMP-activated protein kinase (AMPK). Stimulation of AMPK results in fatty acid catabolism and enhanced muscle glucose uptake (Zhou et al., 20011). Therefore, usage of metformin prevents the occurrence of diabetic complications in addition to controlling glucose

levels (Julio, at al., 2010). Other examples of drugs in this class include fenformin and buformin (Kiho et al., 2005).

1.8.5 Thiazolidinediones

Thiazolidinediones (TZDs) mediate their hypoglycaemic effects by enhancing insulin sensitivity, and reducing gluconeogenesis by the liver (Emilien et al., 1999). This group of drugs targets the perixosome proliferator gamma receptors (pPAR-y) which are nuclear and expressed mainly in the adipose tissues. Other tissues expressing PP AR-y include liver, skeletal muscle, kidney, heart, large and small intestine and colon (Song et al., 2004). Interaction TZDs with the receptors leads to modification of expression of major genes responsible for glucose metabolism (Hevener et al., 2001). When administered at high doses TZDs suppress hepatic gluconeogenesis (Fiordelllis et al., 2005). A major shortfall of thiazolidinediones is that they lead to increased plasma levels of cholesterol (Bailey, 2000).

1.8.6 alpha-glucosidase inhibitors

Alpha-glucosidase   inhibitors   mediate   their   hypoglycaemic   effects   by   lowering   glucose absorption into blood by suppressing digestion of polysaccharides and disaccharides into monosaccharides, thus decreasing post-prandial blood glucose (Fujisawa et al., 2005). This is primarily mediated by competitively and reversibly inhibiting the actions of alpha-glucosidase, a small intestine epithelial membrane bound enzyme which catalyses break down of polysaccharides into monosaccharides. Examples of a-glucosidase are acarbose and voglibose. Side effects include diarrhoea, flatulence abdominal bloating. These adverse effects are primarily due to malabsorption of glucose (Salvatore et al., 2013).

1.8.7 Non-pharmacological intervention

Weight loss as a non-pharmacological intervention for diabetes is significant in that reduced adipose tissue results in lesser production of proinflammatory cytokine tumour necrosis factor (TNF-a) which is thought among others to mediate insulin resistance (Mlinar et al., 2007). Consumption of food with low glycaemic index is beneficial in that it requires less insulin secretion and improves target tissues to insulin effects. Foods with low glycaemic index allow slow absorption of glucose in the gut. Regular exercise reduces levels of circulating cholesterol.

Furthermore, exercise is known to stimulate glucose uptake by the muscles and liver and its conversion to glycogen (Alan et al., 20013).

1.9 Experimental animal models of diabetes

Animal models of diabetes have allowed studies and provided understanding of pathogenesis and progression of diabetes mellitus in humans. Furthermore, animal models have allowed the evaluation of various therapeutic interventions which may be potentially used in man. Uses of animal models have made possible studies of disease characteristics in humans by providing genetic and immunological modifications that are not possible in humans (Bach, 1994).

1.10 Alloxan-induced diabetes

Alloxan, 2,4,5,6 (IH,3H)-pyrimidinetetrone, is probably the longest known and most  potent diabetogenic agent (Piotrowski, 2003). Uptake of alloxan by the pancreatic cells is via GLUT-2 transporters, due similarity in molecular shape to glucose (Elsner et al., 2006). Alloxan then undergoes  reduction  intracellulary  in  the  presence  of  tripeptide  thiol,  glutathione,  to  yield dialuric acid whose re-oxidation, regenerates alloxan. Hence a cyclic reaction (redox cycle) is created that ultimately results in reactive oxygen species generation (Elsner et al., 2006). Alloxan toxicity, however, affects other organs including the liver, kidney, gonads and lungs. Alloxan causes mortality in 37% of animals within a few days (Piotrowski 2003).

1.10.1 Mechanism of Alloxan

Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species. This causes an insulin-dependent diabetes mellitus (called “alloxan diabetes”) in these animals, with characteristics similar to type 1diabetes in humans. Alloxan is selectively toxic to insulin- producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT-2 glucose transporter. Alloxan, in the presence of intracellular thiols, generates reactive oxygen species (ROS) in a cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is initiated by free radicals formed in this redox reaction. One study suggests that alloxan does not cause diabetes in humans. Others found a significant difference in alloxan plasma levels in children with and without diabetes Type 1 (Lenzen, 2008).

1.10.2 Chemical structures of alloxan

The original preparation for alloxan was by oxidation of uric acid by nitric acid. In another method the monohydrate is prepared by oxidation of barbituric acid by chromium trioxide. Alloxan is a strong oxidizing agent and it forms a hemiacetal with its reduced reaction product dialuric acid (in which a carbonyl group is reduced to a hydroxyl group) which is called alloxantin.

Figure 4.Alloxan monohydrate structure.Holmgren, A. V.; Wenner, W. (1952), “Alloxan monohydrate”, Org. Synth. 32: 6; Coll. Vol. 4: 23.

1.10.3 Streptozotocin (STZ)-induced diabetes.

The streptozotocin-induced diabetic model is an extensively used animal model in studies of human diabetes mellitus. It is a well-described model and the toxicity of STZ is relatively low compared with other diabetogenic agents (Piotrowski, 2003). Streptozotocin (N- [methylnitrocarbamoyl]-D-glucosamine), STZ, is an antibiotic synthesised by Streptomycetes achromogenes and is commonly used in induction of experimental diabetes. STZ selectively destroys p-cells by alkylation of DNA through its nitrosourea moiety (Vessal et al., 2003). Other mechanisms of beta cell damage occur via reactive oxygen species and nitric oxide production. Pancreatic beta cells take up STZ via GLUT -2 transporters (Szkudelski, 2001).

1.11 Concepts associated with diabetes mellitus

1.11.1 Free radicals

A free radical is any molecule that contains one or more unpaired electrons. Free radicals are normal products of many metabolic pathways (Christen et al., 2014). They interact with various tissue components, such interactions can cause both acute and chronic dysfunction, but can also provide essential control of redox regulated signaling  pathways (Roberts et al. 2010). It should also be clear that, while free radicals may initiate a series of damaging biochemical events, they are not necessarily directly responsible for the final tissue dysfunction. Conversely, free radicals may arise subsequent to some earlier events that are the proximate cause of tissue injury, but these radicals may have either no effect or ameliorate or aggravate the final damage.

1.11.2 Reactive oxygen species.

Reactive oxygen species (ROS) is a term which encompasses all highly reactive, oxygen- containing molecules, including free radicals. Reactive Oxygen Species (ROS) have long been known to be a component of the killing response of immune cells to microbial invasion (Kops et al., 2002). The production of oxygen based radicals is the bane to all aerobic species. ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades (Hancock et al., 2001). The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including: superoxide; hydrogen peroxide; hydroxyl radical; hydroxyl ion; and nitric oxide (Sunil, 2014).

Figure 5. Electron structures of common reactive oxygen species. Each structure is provided with its name and chemical formula.

All are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes, and other small molecules, resulting in cellular damage. ROS are generated by a number of pathways (Sunil, 2014).

1.11.3 The roles of ROS in metabolic syndrome (eg diabetes).

The role of ROS in the pathogenesis of the metabolic syndrome, as well as both Type 1 and Type

2 diabetes, has been studied extensively (Rochette et al., 2014). Both an enhanced generation of ROS  and  a  dysfunctional  cellular  antioxidant  response  appear  to  be  factors.  For  example, elevated blood glucose and saturated fatty acid levels are linked to an enhanced production of superoxide, hydrogen peroxide through stimulated mitochondrial metabolism, as well as through activation of NADPH oxidases (Rochette et al., 2014). In addition, the hyperglycemia associated with poorly controlled diabetes can induce oxidative stress and may play an important role in diabetic complications, especially b-cell damage (Ogugua, 2000).

1.11.4 Oxidative stress

Reactive oxygen species percentage increases during infections, exercise, stress, exposure to pollutants, UV light, ionizing radiation, etc., thus increasing the degree of oxidative stress in the internal biological system (Manda et al., 2009).

Oxidative stress is a term used to refer to the shift towards the pro-oxidants in the prooxidants antioxidants balance that can occur as a result of an increase in oxidative metabolism (Manda, et al., 2009). ROS reactions with biomolecules such as lipid, protein and DNA, produce different types of secondary radicals like lipid radicals, sugar and base derived radicals, amino acid radicals depending upon the nature of the ROS (Niki et al., 2005). These radicals in the presence of oxygen are converted to peroxyl radicals. Peroxyl radicals are critical in biosystems, as they often induce chain reactions. These reactions exert oxidative stress on the cells, tissues and organs of the body. The biological implications of such reactions depends on several factors like site of generation, nature of the substrate, activation of repair mechanisms, redox status among many others (Goldstein et al., 1993). Oxidative stress is increased in diabetes mellitus owing to an increase in the production of oxygen free radicals, such as  superoxide (O2•-), hydrogen peroxide  (H2O2)  and  hydroxide  (OH•-)  radicals  which  overwhelm  the  natural  antioxidant defence mechanisms (Soliman, 2008).

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