PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING PROPERTIES AND ANTIDIABETIC POTENTIALS OF METHANOL EXTRACT OF THE STEM (BARK) OF Alstonia boonei ON ALLOXAN-INDUCED DIABETIC ALBINO RATS

ABSTRACT

This study was carried out to investigate the phytochemical constituents of the methanol extract of the stem (bark) of Alstonia boonei (MEAB), in vitro antioxidant activity of the extract and its possible antidiabetic and antioxidant potential using alloxan- induced diabetic rats as model. The qualitative analysis of the extract showed a wide range of phytochemicals, which could be physiologically potent in ameliorating several diseases. Quantitative phytochemical analysis revealed that the extract contains relatively high concentration on tannin (7.375±0.005mg/100g), flavonoid (6.176±0.003 mg/100g) and phenols (5.867±0.003 mg/100g). The quantitative result of antioxidant vitamins shows that vitamin C was highest (24.91±0.005mg/100g) compared to vitamin A (1.314±0.002µg/g) and vitamin E (0.886±0.002mg/100g). The methanol extract of Alstonia boonei scavenged 1, 1- diphenyl-2-picrylhydrazyl radical (DPPH^.) in a concentration dependent manner with a correlation coefficient (R²) of 0.7066, indicating antioxidant activity with effective concentration that inhibits 50 percent of the radicals (EC₅₀) of 12.33 ± 0.2µg/ml compared to ascorbic acid standard EC₅₀  of 98 ± 0.02µg/ml. The superoxide radical scavenging activity was concentration-dependent with an EC₅₀ of 7.03±0.42µg/ml compared with ascorbic acid and rutin standards with EC₅₀ of 812.97±0.97µg/ml and 3.47 ± 0.6µg/ml respectively. The extract also showed hydroxyl radical scavenging activity with an EC₅₀ of 42.75±0.02µg/ml compared to α- tocophenol standard with EC₅₀ of 232.31 ± 6.97µg/ml. The nitric oxide radical scavenging activity shows that the extract scavenged nitric oxide radical in a concentration dependent manner with 500µg/ml being more effective than 500µg/ml of ascorbic acid standard. There was a significant increase (P > 0.05) in the serum glucose level in group 2 (diabetic untreated) compared to group 1 (normal control). Significant decrease (P < 0.05) in glucose serum concentration was recorded in all groups treated with the extract and the standard drug compared to group 2 (diabetic untreated) . There was a significant increase (P < 0.05) in urea, creatinine, sodium ion and chloride ion concentrations of group 2 (diabetic untreated) compared to group 1 (normal control). Significant decrease (P < 0.05) in urea, creatinine, sodium ion and chloride ion concentrations was recorded in all groups treated with the methanol extract and the standard drug(group 3 to 6) compared to group 2 (diabetic untreated). There was no significant increase (P > 0.05) in potassium ion concentration of group 2 when compared with group 1. There was a significant increase (P < 0.05) in the serum concentration of Aspartate aminotransferase, (AST), Alanine aminotransferase, (ALT) and Alkaline phosphatase, (ALP) of group 2 (diabetic untreated) when compared with group 1 and significantly reduced (P < 0.05) in all groups treated with the extract and standard drugs when compared to group 2. Serum superoxide and catalase activities were significantly (P < 0.5) reduced in group 2 (diabetic untreated) when compared to the normal control. Serum superoxide and catalase activities increased significantly (P < 0.05) in all groups treated with the extract and standard drugs when compared to group 2 (diabetic untreated). There was a significant increase (P < 0.05) in serum malondialdehyde (MDA) concentration of group 2 (diabetic untreated) and a significant reduction (P < 0.05) in all groups treated with the extract and the standard drug compare to group 2. These results suggest that methanol extract of the stem (bark) of Alstonia boonei (MEAB), possesses and antidiadetic and antioxidant potentials.

CHAPTER ONE

INTRODUCTION

Diabetes is one of the most challenging health problems in the twenty first century (Rahman et al., 2009). Diabetes currently afflicts 171 million people worldwide (Boden and Taggart, 2009). Normal non-diabetic patients maintain plasma glucose <100 mg/dl in the fasting and <135 mg/dl in the post prandial period (Rossetti et al., 2008). Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both (American Diabetes Association, 2009a). Diabetes mellitus is classified as type 1, type 2, with other specific examples and gestational diabetes (American Diabetes Association, 2008). Type 1 diabetes is known as insulin dependent diabetes or Juvenile-onset diabetes and type 2 diabetes is known as non-insulin dependent or adult onset diabetes (American Diabetes Association, 2009b). A slowly progressive form of type 1 diabetes was acknowledged as latent autoimmune diabetes in adults (LADA) by the World Health Organization (WHO) and American Diabetes Association (ADA) (Van Deutekom et al., 2008). Classification schemes define type 1 diabetes as a state of absolute insulin deficiency and type 2 as a state of insulin resistance combined with inadequate insulin secretion (Greenbaum et al., 2009). Type 1 diabetes is an autoimmune disease where auto reactive immune cells attack insulin producing β-cells, destroying insulin reserve leading to hyperglycemia (Eldor et al., 2009). The rate of loss of β-cell function is affected by factors like age at diagnosis, degree of metabolic control, immune status, genetics and marked inter-individual variation (Palmer, 2009). The symptoms of type 1 diabetes are significant weight loss and ketoacidosis (Ludvigsson et al., 2008). Diabetes Type 2 diabetes is a progressive disease characterized by declining β-cell function that in concert with insulin resistance, leads to loss of glycemic control and eventual diabetes complications (Nauck et al., 2009). Type 1.5 diabetes also known as latent autoimmune diabetes in adults (LADA) is an important form of diabetes although it is frequently under estimated (Mayer et al., 2007). Type 1.5 diabetes is also known as slowly progressive type I diabetes, autoimmune diabetes in adults with slowly progressive β-cell failure, autoimmune diabetes not requiring insulin at diagnosis, autoimmune diabetes in adults and type 1.5 diabetes (Dunn et al., 2008). Type 1.5 diabetes has a later onset and slower progression towards an absolute insulin requirement (Cernea et al., 2009). Type 1.5 diabetes occurs in about 10% of patients classified as type 2 diabetes and not initially requiring insulin (Agardh et al., 2009). Diagnosis of type 1.5 diabetes is difficult due to lack of defining features (Jasem et al., 2010).

1.1 Alstonia boonei

There has been worldwide increase in realization of the use of medicinal plants in various traditional health systems of many countries. Recent estimates by the World Health Organization (WHO) revealed that about 80% of the population in Africa relies on traditional medicine of which the botanicals constituted greater components (Kayode and Kayode, 2008). These botanicals had over the years been subjected to wide and unsustainable use (Kayode, 2006). Many of these medicinal plants provide relief of symptoms comparable to that obtained from allopathic medicine (Choi and Hwang, 2003). One of these plants, Alstonia boonei is a herbal medicinal plant of West Africa origin. The plant parts have been traditionally used for its antimalarial, aphrodisiac, antidiabetic, antimicrobial, and antipyretic activities, which have also been proved scientifically (Kweifio-Okai, 1999). The plant parts are rich in various bioactive compounds such as echitamidine, Nα-formylechitamidine, boonein, loganin, lupeol, ursolic acid, and β-amyrin among which the alkaloids and triterpenoids form a major portion (Tepongning et al., 2011).

1.1.1 Morphology of Alstonia boonei

            Alstonia grows into a giant tree in most of the evergreen rain forests of tropical West Africa. The plant thrives very well in damp riverbanks. It is well known to all the traditional healers practicing along the west coast of Africa. Alstonia boonei De Wild is a deciduous tree up to 35 meters high. It buttresses deep-fluted high and narrow. Its white latexes are copious. The leaves are in whorls at nodes, oblanceolate, apex rounded to acuminate, lateral vein prominent almost at right angle to midrib. The flowers are white with lax terminal cymes. The fruits are paired with slender follicle up to 16cm long with brown floss at each end.

1.1.2 Classification of Alstonia boonei

Alstonia comprises about 40 species and has a pantropical distribution. There are about twelve species of the genus Alstonia. Alstonia boonei De Wild belongs to the family Apocynaceae. The species are scattered all over the world of which two are indigenous to Africa. The plant is known locally as Onyame dua, Egbu e.t.c . Elsewhere, Alstonia is known as Australian fever bush, Australian quinine, Devil tree, Dita bark, fever bark, or palimara (Gosse et al., 1999). The scientific classification of Alstonia boonei is presented thus;

Kingdom: Plantae Phylum: Tracheophyta Order: Gentianales Family: Apocynaceae Genus: Alstonia Species: boonei Botanical name: Alstonia boonei Common name: Devil tree.

Figure 1: Stem of Alstonia boonei

Figure 2: Leaves of Alstonia boonei

1.1.3 Uses of Alstonia boonei

            The bark of Alstonia tree is one of the most effective analgesic herbs available in nature. All the parts of the plant are very useful but the thick bark cut from the matured tree is the part that is most commonly used for therapeutic purposes. The bark of the tree is highly effective when it is used in its fresh form; however, the dried one could equally be used. Therapeutically, the bark has been found to possess antirheumatic, anti-inflammatory, analgesic/pain-killing, antimalaria/antipyretic, antidiabetic (mild hypoglycaemic), antihelminthic, antimicrobial and antibiotic properties (Abbiw, 1990). A decoction could be sweetened with pure honey and be taken up to 4 times daily as an effective painkiller for the relief of Painful menstruation (dysmenorrhoea), when associated with uterine fibroid or ovarian cysts in women; lower abdominal and pelvic congestion associated with gynaecological problems such as pelvic inflammatory diseases; to relieve the painful urethritis common with gonococcus or other microbial infections in men. Alstonia decoction also exerts a mild antibacterial effect in this case, relieving the aches and pains associated with malaria fever. Alstonia is taken in the form of preparations that exhibits antipyrexia and anti-malaria effects, to combat rheumatic and arthritic pains. The decoction of Alstonia bark could be taken alone as an effective pain-killing agent. A cold infusion made from the fresh or dried bark of Alstonia taken orally two to three times daily exerts a mild hypoglycaemic effect on diabetic patients. The cold infusion is also administered orally for the purpose of expelling round worms, threadworms and other intestinal parasites in children.The fresh bark of Alstonia could be used in preparing herbal tinctures; it is particularly useful as an effective antidote against snake, rat, or scorpion poison. It is also useful in expelling retained products of conception and afterbirth when given to women. Asthma can be treated with a drink prepared from parts of Trema orientalis and decoction of the bark of Alstonia boonei mixed with the roots and bark of cola and fruits of Xylopia parviflora with hard potash . The bark decoction of Alstonia boonei is used with other preparations in the treatment of fractures or dislocation, jaundice, and for inducing breast milk. Its latex is taken as a purgative. The hardened latex is used for the treatment of yaws. Alstonia booneiDe Wild is regarded as one of few herbs with potential anti-HIV indicators (Adotey et al., 2012). In some African countries Alstonia boonei is considered a sacred tree and worshiped in the forest and hence human beings in those countries do not eat its parts.

1.2 Reactive Oxygen Species (ROS)

            A free radical is defined as any molecule or atom which contains one or more unpaired electrons in its outer orbit (Halliwell and Gutteridge 1989). The term “reactive oxygen species” (ROS) is often used to refer to free radicals and other oxygen-related reactive compounds, such as singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), and hydroperoxides (ROOH) (Halliwell and Gutteridge 1989). All these compounds have the potential to cause free-radical reactions (Pryor, 1986). When a free radical reacts with a non-radical molecule, the target molecule is converted to a radical, which may further react with another molecule. Oxidative stress is a physiological condition that occurs when there is a significant imbalance between production of reactive oxygen species and antioxidant defenses. In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Parkinson’s disease and Alzheimer’s disease (Valko et al, 2005), but it may also be important in prevention of aging by induction of a process named mitohormesis. ROS can be beneficial, as they are used by the immune system as a way to attack and kill pathogens. ROS are also used in cell signaling. This is dubbed redox signaling. A particularly destructive aspect of oxidative stress is the production of ROS, which include free radicals and peroxides. Some of the less reactive of these species (such as superoxide) can be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage. (Valko et al., 2005).

1.2.1 Superoxide Radical

Superoxide (O₂^–) is generated by multiple enzymatic and non-enzymatic pathways and is often at the start of the oxidative stress cascade. A major source is via the cellular electron transport chains, such as those of mitochondria, chloroplasts and the endoplasmic reticulum (Halliwell and Gutteridge, 1990) where some electrons passing through the chain “leak” directly from the intermediate electron carriers onto O₂. Since oxygen accepts one electron at a time, O₂^– is formed (Halliwell, 1992). Superoxide anions are generated enzymatically by a number of oxidases, such as xanthine oxidase and the oxidase that is found in the plasmalemma of phagocytic cells (Reiter et al., 1995). Activated phagocytic cells (such as monocytes, neutrophils, eosinophils and macrophages including microglia) also produce superoxide, which plays an important part in the mechanism by which bacteria are engulfed and destroyed (Colton and Gilbert, 1987). Thus excessive activation of phagocytic cells (as in chronic inflammation) can lead to free radical damage. The toxicity of superoxide is seen in its ability to inhibit certain enzymes and thereby attenuate vital metabolic pathways, as well as in its effects on other major classes of biological molecules (McCord, 2000). E. coli deficient in superoxide dismutase (SOD) activity show increased rates of mutagenesis (Touati and Farr, 1990), illustrating the role of the radical in DNA damage.

1.2.2 Hydroxyl Radical

The OH^. radical is probably the most reactive of the ROS species (Poeggler et al., 1993; Dawson and Dawson, 1996) as it will react with almost all molecules in living cells (Fridovich, 1974). Hydroxyl radicals are short-lived and can be formed from O₂^– and H₂O₂ through the Haber-Weiss reaction or through the interaction of metals such as iron or copper and H₂O₂, through the Fenton reaction (Halliwell and Gutteridge, 1985) as shown in the equations below.

O₂^– + H₂O₂ → H₂O + OH ^– + OH^. Haber-Weiss Reaction (1)

Fe³⁺ + O₂^– → Fe²⁺ + O₂

Fe²⁺ + H₂O₂→ Fe³⁺ + OH^. + OH ^– Fenton Reaction (2)

The hydroxyl radical has been implicated in damage to proteins, carbohydrates, DNA, and lipids(Reiter et al., 1995; Dawson and Dawson, 1996; Volterra et al., 1994). Action on DNA results in strand breakage and chemical alterations of the deoxyribose and of the purine and pyrimidine bases. A main physiological target of free radicals is the polyunsaturated fatty acids of cell membranes and the resultant degradation causes alterations in membrane structure and function (Viani et al., 1991).

1.2.3 Nitric Oxide

Nitric oxide (NO) is a free radical released by several cell types, especially vascular endothelial cells and phagocytes (Moncada et al., 1991). Nitric oxide radical is formed by nitric oxide synthase (NOS). The process involves the conversion of L-arginine to L-citrulline (Knowles and Moncada, 1994). NO has been suggested to be involved in both the normal functioning of excitatory amino acids such as glutamate and in the damaging effects produced by their generation in excess (Dawson et al., 1991; Forstermann et al., 1991).

1.3 Diabetes: Definition and Types

            Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both

(American Diabetes Association, 2009a). Diabetes mellitus is classified as type 1, type 2, other specific types and gestational diabetes (American Diabetes Association, 2008). Type 1 diabetes is known as insulin dependent diabetes or Juvenile-onset diabetes and type 2 diabetes is known as non-insulin dependent or adult onset diabetes (American Diabetes Association, 2009b). A slowly progressive form of type 1 diabetes was acknowledged as latent autoimmune diabetes in adults (LADA) by the World Health Organization (WHO) and American Diabetes Association (ADA) (Van Deutekom et al., 2008). Classification schemes define type 1diabetes as a state of absolute insulin deficiency and type 2 as a state of insulin resistance combined with inadequate insulin secretion (Greenbaum et al., 2009). Type 1 diabetes is an autoimmune disease where auto reactive immune cells attack insulin producing β-cells, destroying insulin reserve leading to hyperglycemia (Eldor et al., 2009). The rate of loss of β-cell function is affected by factors like age at diagnosis, degree of metabolic control, immune status, genetics and marked inter-individual variation (Palmer, 2009). The symptoms of type 1 diabetes are significant weight loss and ketoacidosis (Ludvigsson et al., 2008). Type 2 diabetes is a progressive disease characterized by declining β-cell function that in concert with insulin resistance, leads to loss of glycemic control and eventual diabetes complications (Nauck et al., 2009). The symptoms of type 2 diabetes are polyuria, polyphagia, blurred vision and general malaise (Goldfine, 2008).     The criteria for the diagnosis of diabetes mellitus in clinical practice is fasting plasma glucose that is equal or greater than 126 mg/dL or two-hours post prandial plasma glucose greater than 200 mg/dL (Nwankwo et al., 2008). With good glycemic control, several long-term, life-threatening complications of diabetes can be prevented (Shabbidar et al., 2006).

1.3.1 Complications of Diabetes

            Severe long term abnormalities can result from diabetes. Complications already identified are eye complications, heart disease, kidney and foot problems if blood sugar levels are poorly controlled (Brophy et al., 2007). These complications are of two types, microvascular complications that include retinopathy, nephropathy, neuropathy and peripheral vascular disorders and macrovascular complications that include cardiovascular and cerebrovascular disorders. The complications of diabetes can involve multiple systems throughout the body that are susceptible to the detrimental effects of oxidative stress and apoptotic cell injury (Maiese et al., 2010). Diabetes also leads to long-term complications throughout the body involving cardiovascular, renal and nervous disorders (Daneman, 2006). The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of various organs especially the eyes, kidneys, nerves, heart and blood vessels (Chandramohan et al., 2009). The importance of protecting the body from hyperglycemia cannot be overstated; the direct and indirect effects on the human vascular tree are the major source of morbidity and mortality in both type 1 and type 2 diabetes. Generally, the injurious effects of hyperglycemia are separated into macrovascular complications (coronary artery disease, peripheral arterial disease and stroke) and microvascular complications (diabetic nephropathy, neuropathy, and retinopathy) (Fowler, 2008).

1.3.2 Oxidative Stress and Diabetic Complications

            Oxidative stress plays both microvascular and cardiovascular pivotal roles in the development of diabetes complications. The metabolic abnormalities of diabetes cause mitochondrial superoxide overproduction in endothelial cells of both large and small vessels, as well as in the myocardium (Du et al., 2000; Nishikawa et al., 2000). This increased superoxide production causes the activation of 5 major pathways involved in the pathogenesis of complications: polyol pathway flux, increased formation of advanced glycation end products (AGEs), increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C isoforms, and overactivity of the hexosamine pathway. It also directly inactivates 2 critical antiatherosclerotic enzymes, endothelial nitric oxide synthase and prostacyclin synthase (Nascimento et al. 2006 ; Xie et al., 2008). Through these pathways, increased intracellular reactive oxygen species (ROS) cause defective angiogenesis in response to ischemia, activate a number of proinflammatory pathways, and cause long-lasting epigenetic changes that drive persistent expression of proinflammatory genes after glycemia is normalized (“hyperglycemic memory”) (Yip et al., 1998; Du et al., 2006). Atherosclerosis and cardiomyopathy in type 2 diabetes are caused in part by pathway-selective insulin resistance, which increases mitochondrial ROS production from free fatty acids and by inactivation of antiatherosclerosis enzymes by ROS (Brown and Goldstein, 2008; Li et al., 2010). Oxidative stress may promote the onset of diabetes by decreasing insulin sensitivity and destroying the insulin-producing cells (Wallace, 1992). ROS can penetrate through cell membranes and cause damage to β-cells of pancreas (Chen et al., 2005). A high fat diet or free fatty acids also has been shown to release ROS and contribute to mitochondrial DNA damage and impaired pancreatic β-cell function (Rachek et al., 2006). Oxidant stress and ROS exposure can result in the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial NAD+ stores and result in apoptotic cell injury (Chong et al., 2005). Free fatty acids also can lead to ROS release, mitochondrial DNA damage and impaired pancreatic β-cell function (Li et al., 2008). The development of diabetes has been associated with a decrease in the levels of mitochondrial proteins and mitochondrial DNA (Choo et al., 2006). Long standing diabetes mellitus is associated with an increased prevalence of microvascular and macrovascular diseases (Mehta et al.; 2009a, Mehta et al., 2009b). Cellular pathways in diabetes are closely associated to cellular energy maintenance and intact mitochondrial function (Newsholme et al., 2007). Figure 1 represents the protein kinase C (PKC) pathway and polyol pathway in the pathogenesis of diabetic complications.

Figure 3: schematic representation of protein kinase C and polyol pathways in the pathogenesis     of diabetic complications

Figure 4: Increased production of AGE precursors and their pathologic consequences

The pathologic consequences of increased advanced glycation end products (AGE) precursors and increased flux through hexosamine pathway are depicted in Figure 3 and Figure 4 respectively.

Figure 5: Increased flux by hyperglycemia through hexosamine pathway

 The DNA damage induced by ROS through Poly (ADP-ribose) polymerase (PARP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is depicted in figure 5.

 Figure 6: Activation of PARP and modification of GAPDH by ROS-induced DNA damage

1.4 Phytochemicals

Phytochemicals are large group of plant-derived compounds responsible for much of the disease protection conferred on plants (Arts and Hollman, 2005). Medicinal plants contain some organic compounds which produce definite physiological action on the human body and these bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids (Edoga et al., 2005). Phenolics have been known to possess a capacity to scavenge free radicals. The antioxidant activity of phenolics is principally due to their redox properties, which allow them to act as reducing agents, hydrogen donors. Phenolics are especially common in leaves, flowering tissues and woody parts,such as stems and barks. Studies have shown that they play an important preventive role in the development of cancer, heart diseases and ageing related diseases (Larson, 1988). Flavonoids are potent water-soluble antioxidants and free radical scavengers which prevent oxidative cell damage and have strong anticancer activity (Salah et al.,1995; Del-Rio et al., 1997; Okwu, 2004). Flavonoids also lower the risk of heart diseases. Saponins are capable of neutralizing some enzymes in the intestine that can become harmful, building the immune system and promoting wound healing. Alkaloids have been documented to possess analgesic, antispasmodic and bactericidal effects. Tannins hasten the healing of wounds and inflamed mucous membrane (Okwu and Okwu, 2004). Cardiac steroids are widely used in the treatment of congestive heart failure. They help in increasing the force of contraction of the heart (positive inotropic activity) in heart failure patients.

 Aim and Objectives

• • Aim

The complications of diabetes can involve multiple systems throughout the body that are susceptible to the detrimental effects of oxidative stress and apoptotic cell injury. The main aim of this study is to investigate in vitro antioxidant activity of the methanol extract of A. boonei, its antidiadetic and in vivo antioxidant potential using alloxan induced diabetic rats as model.

1.5.2    Specific Objectives

The study was designed to achieve the following specific objectives:

• To determine the phytochemical constituents of Alstonia boonei
• To determine possible acute toxicity(LD₅₀) concentration of Alstonia boonei
• To determine the anti-oxidant potential of the methanol extract of Alstonia boonei in-vitro and in-vivo
• To determine the hypoglyceamic potentials of the methanol extract

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