EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF AND FRUIT EXTRACTS OF Kigelia africana ON SOME OXIDATIVE AND BIOCHEMICAL PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

ABSTRACT

Globally, the estimated incidence of diabetes and projection for the year 2030 as given by the International Diabetes Federation (IDF) is 350 million. Kigelia africana is highly used for ethnomedicinal purposes although there is paucity of scientific information on its use. This work was therefore, aimed at evaluating the anti-diabetic and antioxidative potential of the plant. Ethanol, methanol and n- hexane extracts of the leaves of Kigelia africana were used for the study. Alloxan diabetes was induced in a total of 60 adult male albino rats weighing between 90 and 160 g. The alloxan was dissolved in cold normal saline. After 72 hr, diabetes was confirmed and the rats were divided into twelve (12) groups of five (5) rats each. Group 1 served as the normal control, group 2 was the diabetic untreated, group 3 received 2.5 mg /kg b.wt of glibenclamide, groups 4, 6 and 8 received ethanol, methanol and n-hexane leaves extract while group 5, 7 and 9 received ethanol, methanol and n-hexane fruit extract respectively of 500 mg/kg b.wt of the extracts. Groups 10-12 were administered equal combination of the leaves and fruits extracts. The rats were fed orally for 21 days after which some biochemical and oxidative parameters were statistically analysed. Phytochemical screening for different bioactive compounds was done using standard methods and indicated the presence of flavoniods, alkaloids, saponins, soluble carbohydrates, tannin, steroids, glycosides and reducing sugars. Proximate analysis revealed the presence of proteins (13.9%), carbohydrates (63.5%), fats and oil (11.4%) and crude fibre (2.2%). LD50 showed that the extracts were safe. The glucose level decreased while body weight increased in all the treated groups compared with the diabetic rats untreated. Oral administration of 500mg/kg b.w of K. africana extract significantly reduced (p<0.05), the sorbitol, glycohaemoglobin (HbA1c), total protein, and vitamin C concentrations in diabetic rats (groups 4-12) in comparison with the positive control. There were significant differences in glycohaemogolin, sobitol, total protein and vitamin C concentration in diabetic rats fed with a combination of the two parts of the plant extracts (groups 10-12) as against groups 4-9 administered single extracts. Malondiadehyde (MDA) concentration significantly decreased (p < 0.05) in all the test groups compared with the diabetic untreated rats. Low density lipoprotein, total cholesterol, and triacylgycerol levels decreased significantly (p < 0.05) in the treated groups in comparison with the positive control animals (group 3). However, administration of 500 mg/kg b.w of K. africana increased significantly (p<0.05) the high density lipoprotein (HDL) across the test groups as against the diabetic untreated group. Significant decreased (p<0.05) in the lipid profiles (except HDL) was recorded in groups 10, 11 and 12 treated with a combination of two parts (leaf and fruit) of K. africana in comparison with groups 4-9 orally fed with a single plant extract. Furthermore, the data recorded significantly increased (p < 0.05) antioxidant enzymes (SOD, CAT GPX) activities in diabetic treated groups (both combination and single) with reference to the positive control group. Similarly, significant increase (p > 0.05) of SOD and CAT activities and SOD percentage inhibition was observed in group 3 treated with 2.5 mg/kg b.wt of glibenclamide (standard) compared with all the test groups. Significant reduction (p < 0.05) in the activities of ALT, ALT and total bilirubin concentration were observed in the test groups treated with the extracts compared with the diabetic untreated rats. ALT activity and total bilirubin level decreased significantly (p < 0.05) in groups 10, 11 and 12 administered a combination of leaf and fruit extracts as against groups 4-9 treated with either leaf or fruits only. The results suggest that management and prevention of diabetic complications can be achieved by the use of K. africana.

TABLE OF CONTENTS

Title Page
Certification
Dedication
Acknowledgements
Abstract
Table of Contents
List of Figures
List of Tables
List of Abbreviations

CHAPTER ONE: INTRODUCTION

1.1 Kigelia africana
1.1.1 Description of Kigelia africana
1.1.2 Taxonomy of Kigelia africana
1.1.3 Traditional uses of Kigelia africana
1.1.4 Chemical constituents of Kigelia africana
1.1.5 Antibacterial and antifungi
1.2 Diabetes
1.2.1 Diabetes mellitus
1.2.2 Diabetes Type 1 and 2
1.2.3 Insulin resistance
1.2.4 Diabetic complications
1.3 Hyperglycemia and diabetic complication
1.4 Mechanism of tissue damage mediated by hyperglycemia
1.4.1 Aldose reductase pathway
1.4.2 Non-enzymatic glycation
1.4.3 Carbonyl stress in diabetes
1.4.4 Activation of protein kinase C isoforms
1.5 Oxidative stress
1.5.1 Mechanism of increased oxidative stress in diabetes mellitus
1.5.2 Glucose autoxidation
1.5.3 Free radicals
1.5.4 Reactive oxygen species and oxidative stress
1.6 Antioxidant system
1.6.1 Scavenging properties of antioxidants
1.6.2 Positive and negative effects of free radicals
1.7 Lipid peroxidation
1.8 Antioxidant supplementation in diabetes mellitus
1.9 Alloxan
1.9.1 Alloxan diabetes and streptozotocin diabetes
1.9.2 Alloxan: Mechanism of action
1.9.3 Beta cell toxicity and diabetogenicity of alloxan
1.9.4 Streptozotocin: Mechanism of action and beta cell selectivity
1.9.5 Beta cell toxicity of streptozotocin
1.9 Rationale for the study
1.10 Aim and objectives of the study
1.10.1 Aim of the study
1.10.2 Specific objectives of the study

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials
2.1.1 Chemicals
2.1.2 Instrument/Equipment
2.1.3 Drug
2.1.4 Plant material
2.2 Methods
2.2.1 Animal management
2.2.2 Preparation of plant extracts
2.2.3 Design of the experiment
2.2.4 Yield of extracts
2.2.5 Phytochemical analysis of the crude extracts
2.2.5.1 Test for the presence of alkaloids
2.2.5.2 Test for carbohydrates
2.2.5.3 Test for reducing sugar
2.2.5.4 Test for protein
2.2.5.5 Test for fats and oil
2.2.5.6 Test for glycosides
2.2.5.7 Test for acidic substances
2.2.5.8 Test for the presence of flavonoids
2.2.5.9 Test for the presence of steroids
2.2.5.10 Test for tannins
2.2.5.11 Test for resins
2.2.5.12 Test for saponins
2.2.3.13 Test for terpenoids and steroids
2.2.6 Proximate Analysis
2.2.6.1 Crude protein
2.2.6.2 Crude fat
2.2.6.3 Moisture
2.2.6.4 Ash /Mineral matter
2.2.6.5 Crude fibre
2.2.6.6 Carbohydrate or nitrogen free extract (NFE)
2.2.7 Acute toxicity test
2.2.7.1 Determination of LD50 of the extract
2.2.8 Induction of diabetes
2.2.9 Determination of fasting and random glucose concentrations
2.2.10 Determination of sorbitol concentration
2.2.11 Determination of total protein concentration
2.2.12 Determination of haemoglobin glycosylation
2.2.13 Determination of malondialdehyde concentration
2.2.14 Determination of vitamin C concentration
2.2.15 Assay of catalase activity
2.2.16 Assay of superoxide dismutase (SOD) activity
2.2.17 Assay of glutathione peroxidase activity
2.2.18 Determination of total cholesterol concentration
2.2.19 Determination of high density lipoprotein (HDL) cholesterol concentration
2.2.20 Determination of low density lipoprotein (LDL) cholesterol concentration
2.2.21 Determination of triacylglycerol concentration
2.2.22 Assay of aspartate aminotransferase (AST) activity
2.2.23 Assay of alanine aminotranferase (ALT) activity
2.2.24 Determination of total bilirubin concentration
2.3 Statistical analysis

CHAPTER THREE: RESULTS

3.1 Qualitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.2 Quantitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.3 Percentage proximate compositions of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.4 Percentage yield of leaf and fruit samples of Kigelia Africana
3.5 Acute toxicity studies
3.6 Effect of ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana on sugar level of diabetic rats
3.7 Body weights of diabetic rats treated with ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana before and after experiment
3.8 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on sorbitol concentration in alloxan-induced diabetic rats
3.9 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigeria africana on total protein in alloxan-induced diabetic rats
3.10 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on glycosylated haemoglobin concentration in alloxin-induced diabetic rats
3.11 Effect of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on malondialdehyde (MDA) concentration in alloxan-induced diabetic rats
3.12 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on vitamin C concentration in alloxan-induced diabetic rats
3.13 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on catalase activity in alloxan-induced diabetic rats
3.14 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on superoxide dismutase (SOD) activity in alloxan- induced diabetic rats
3.15 Effect of ethanol methanol and n-hexane leaf and fruit extracts of Kigelia africana on percentage inhibition of superoxide dismutase in alloxan-induced diabetic rats
3.16 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on glutathione peroxidase activity in alloxan-induced diabetic rats
3.17 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on cholesterol concentration in alloxan-induced diabetic rat
3.18 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana high density lipoprotein in alloxan-induced diabetic rats
3.19 Effect of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana on low density lipoprotein concentration in alloxan-induced diabetic rats
3.20 Effect of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana on triacylglycerol (TAG) concentration in alloxan-induced diabetic rats
3.21 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on aspartate aminotranferase (AST) in alloxan-induced diabetic rats
3.22 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on alanine aminotransferase (ALT) in alloxan-induced diabetic rats
3.23 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on total bilirubin concentration in alloxan-induced diabetic rats

CHAPTER FOUR: DISCUSSION

4.1 Discussion
4.2 Conclusion
REFERENCES
APPENDICES

LIST OF ABBREVIATIONS

A Alloxan

AAS Atomic absorption spectrophotometer

AST Aspartate aminotransferase

ALT Alanine aminotransferase

ADP Adenosine diphosphate,

AGEs Advanced glycation end- product,

AVP Arginine vasopressin,

AQP-2 Aquaporin-2,

AVPR2 Arginine vasopressin 2 receptor,

AQP-2 Aquaporin-2,

ATP Adenosine triphosphate,

BH4 Tetrahydrobiopterin,

CAT Catalase,

Ca2+ Calmodulin.,

Cu2+ Copper (ii) Iron,

CUZnSOD Copper zinc superoxide dismutase,

DAG Diacylglycerol,

DHLA Dihydrolipoic acid,

DMSO Dimethyl sulphoxide,

DNA Deoxyribonucleic acid,

3-DG 3-Deoxyglucosone,

DM Diabetes mellitus,

DI Diabetes insipidus,

EDTA Ethylene diamine tetraacetate,

EDRF Endothelium derived relaxing factor,

eNOS Endothelial nitric oxide synthase,

FAD Flavin adenine dinucleotide,

Fe2+ Iron,

FMN Flavin mononucleotide,

GAPD Glyceraldehydes -3-Phosphate dehydrogenase,

GLO Glyoxal,

GR Glutathione reductase,

GSH Glutathione,

GSSG Oxidized glutathione,

GPx Glutathione peroxidase,

GLUT2 Glucose transporter,

HDL High density lipoprotein,

HOCL Hydrochlorous acid,

H2O2 Hydrogen peroxide,

HO. Hydroxyl radical,

HNE 4-Hydroxy-2-nonenal,

HK Hexokinase,

IDDM Insulin -dependent diabetes mellitus,

I.N.T 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-Phenyl Tetrazolium chloride,

1L-1 Interleukin -1,

LA Lipoic acid,

LD50 Lethal dose,

LDL Low density lipoprotein,

MNU N- methyl-N-nitrosourea,

MDA Malondialdehyde,

Mn-SOD Manganese superoxide dismutase,

MGD Methylglyoxal,

NADPH Nicotinamide adenine dinucleotide phosphate (Reduced form),

NAD+ Nicotinamide adenine dinucleotide (oxidized form),

NIDDM Non- insulin dependent diabetes mellitus,

NOS Nitrogen oxygen species,

NO Nitric oxide,

ONOO- Peroxynitrite,

1O2 Singlet oxygen,

O2 -.Superoxide anion,

PDGF Platelet- derived growth factor,

PKC Protein kinase C,

PS Phosphatidylserine,

PPP Pentose phosphate pathway,

PVS Polyvinyl sulphate,

PUFA Polyunsaturated fatty acid,

PVN Paraventricular nuclei,

RAGE Advanced glycation end- product,

RNS Reactive nitrogen species,

RO. Alkoxy radicals,

ROS Reactive oxygen species,

STZ Streptozotocin,

SON Supraoptic nuclei,

SOD Superoxide dismutase,

TG Triacylglycerol,

TLC Thin layer chromatography,

TNF Tumor necrosis factor,

TBA 2-Thiobarbituric acid,

TCA Trichloroacetic acid,

UKPDS United kingdom prospective diabetes study,

VEGF Vascular endothelial growth factor,

XOD Xanthine oxidase

CHAPTER ONE

INTRODUCTION

Diabetes mellitus is a metabolic disorder resulting from a defect in insulin secretion, insulin action or both. Insulin deficiency in turn leads to chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism (Kumar et al., 2011).

During diabetes, failure of insulin-stimulated glucose uptake by fat and muscle cause glucose concentration in the blood to remain high, consequently glucose uptake by insulin-independent tissue increases. Increased glucose flux both enhances oxidant production and impairs antioxidant defenses by multiple interacting non-enzymatic, enzymatic and mitochondrial pathways (Klotz 2002; Mehta et al., 2006). These include activation of protein kinase C isoforms (Inoguchi et al., 2000), increased hexosamine pathway (Kaneto et al., 2001), glucose autoxidation (Brownlee, 2001), increased methylglyoxal and advanced glycation end-product (AGEs) formation (Thornalley, 1998) as well as increased polyol pathway flux ( Lee and Chung, 1999). These seemingly different mechanisms are the results of a single process-that is, overproduction of superoxide by the mitochondrial electron transport system (Tushuizen et al., 2005). This hyperglycaemia-induced oxidative stress ultimately results in modification of intracellular proteins resulting in an altered function and DNA damage, activation of the cellular transcription (NFK B), causing abnormal changes in gene expression, decreased production of nitric oxide, and increased expression of cytokines, growth factors and pro-coagulant and pro-inflammatory molecules (Palmer et al., 1988; Evans et al., 2002; Klotz, 2002; Taniyama and Griendling, 2003). Oxidative stress is responsible for molecular and cellular tissue damage in a wide spectrum of human diseases (Halliwell, 1994), amongst which is diabetes mellitus. Diabetes produces disturbances of lipid profiles, especially an increased susceptibility to lipid peroxidation (Lyons, 1991), which is responsible for increased incidence of atherosclerosis (Guigliano et al., 1996), a major complication of diabetes mellitus . An enhanced oxidative stress has been observed in these patients as indicated by increased free radical production, lipid peroxidation and diminished antioxidant status (Baynes, 1991).

Globally, the estimated incidence of diabetes and projection for year 2030, as given by International Diabetes Federation is 350 million (Ananda et al., 2012). Currently available pharmacotherapies for the treatment of diabetes mellitus include oral hypoglycemic agents and insulin. However these drugs do not restore normal glucose homeostasis and they are not free from side effects (Bandawane et al., 2011). In view of the adverse effects associated with the synthetic drugs and as plants are safer, affordable and effective, conventional antidiabetic plants can be explored (Kumar et al., 2010). Over 400 traditional plants have been reported for the treatment of diabetes (Ramachandran et al., 2011). Furthermore, following World Health Organization recommendations, investigation of hypoglycemic agents from medicinal plants has become more important (Kumar et al., 2010). Also diabetes has been treated orally with several medicinal plants based on folklore medicine since ancient times.

Kigelia africana (Lam.) Benth (Family: Bignoniaceae) plant has many medicinal properties due to the presence of numerous secondary metabolites. These compounds include iridoids, flavonoids, naphthoquinones and volatile constituents (Houghton, 2002; Gorman, 2004; Asekun et al., 2006). Experimentally, the plant has shown antibacterial, antifungal, antineoplastic, analgesic, anti-inflammatory and antioxidant properties (Saini et al., 2009). Crude extracts of herbs and spices and other materials rich in phenolics are of increasing interest in the food industry because they retard oxidative degradation of lipids and thereby improving the quality and nutritional value of food. Flavonoids, are groups of polyphenolic compounds with known properties, which include free radical-scavenging and anti-inflammatory activities (Frankel, 1995).

An enhanced oxidative stress has been observed in diabetic patients as indicated by increased free radical production, lipid peroxidation and diminished antioxidant status (Baynes, 1991). In diabetes mellitus, alterations in the endogenous free radical scavenging defense mechanisms may lead to ineffective scavenging of reactive oxygen species, resulting in oxidative damage and tissue injury. Oxidative stress is currently suggested as mechanism underlying diabetes and diabetic complications. Oxidative stress may cause oxidative damage of cellular membranes and changes in the structural and functional integrity of subcellular organelles and many produce effects that result in the various complications in diabetic disease (West, 2000). Recently, there has been an upsurge of interest in the therapeutic potentials of plants, as antioxidants in reducing free radical induced tissue injury. Although several synthetic antioxidants, such as ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene are commercially available, they are quite unsafe and toxic (Viny, et al.,2010). A survey of literature revealed that there is no experimental evidence of the antidiabetic effect of Kigelia africana. Therefore, the present work explores this and will in addition study its potential effect on the oxidative and biochemical parameters of alloxan-induced diabetic rats.

1.1 Kigelia africana

1.1.1 Description of Kigelia africana

Nature has been a source of medicinal agents including modern drugs for the thousands of years (Cordell, 2000). Plant based traditional medicine system continues to play an essential role in health care with about 80% of the world’s inhabitants relying mainly on traditional medicines for their primary health care .

Kigelia africana (Lam) Benth of Bignoniaceae family is widely distributed in South central and West Africa. It is known as the cucumber or sausage tree because of its huge fruits (average 0.6m in length and 4kg in weight) which hang from long fibrous stalks. It is also known as Balm Khene in Hindi and is distributed all over India but found in abundance in West Bengal. It is found mostly in wetter areas and spread abundantly across wet Savannah and riverine areas (Sofowora et al., 1980). The plant grows approximately 10m high, with odd pinnate, composite opposite leaves; leaflets are ovate- to- oblong in shape and 4-18 cm long. The flowers are found in the spring or summer season, hanging ancillary panicles up to 2 m long. It has a corolla with fused petals, irregularly bell shaped 9-13 cm long, yellowish outside and purple on inside . Fruits are oblong, 30-50 cm long, hanging on stalk for several months.

Taxonomy of Kigelia africana

Kingdom: Plantae

Order: Scrophulariales

Family: Bignoniaceae

Genus: Kigelia Dc-Sausage family

Species: Kigelia africana (Lam) Benth

Scientific name: Kigelia africana

Traditional Uses of Kigelia africana

The Kigelia plant has medicinal properties with characteristics of bitterness and astringent. It has long history of use by African countries particularly for medicinal properties. Several parts of the plant are employed for medicinal purposes by certain aboriginal people. In Mali during famine the seeds are roasted and eaten. The baked fruits of K.africana are used for fermentation of beer. Most commonly traditional healers use it to treat a wide range of skin ailments like fungal infections, boils, psoriasis, and eczema.

It has also internal applications including treatment of dysentery, ringworm, tapeworm, postpartum haemorrhage, malaria, diabetes, pneumonia and tooth care (Gill, 1992). The Tonga women of Zambezi valley regularly apply cosmetic preparations of kigelia fruits to their faces to ensure a blemish- free complexion (Pooleg, 1993). In folk medicine, the fruits of the plant are used as dressing for ulcers, purgative and for increasing the flow of milk in lactating women (Oliver-Bever, 1986). The roots are said to yield a bright yellow dye. The Shona people from India tend to use the bark or root as powder or infusion for application to ulcers, or in the treatment of pneumonia, as a gargle for toothache and backache (Maisiri and Gundidza, 1999).

In West Africa, the root and unripe fruit is used as vermifuge and as a treatment for haemorrhoids and rheumatism. The bark is traditionally used as remedy for syphilis and gonorrhea. The fruits and bark ground and boiled in water are taken orally or used as an enema in treating children’s stomach ailments – usually worms. The unripe fruit is used in Central Africa as a dressing for wounds, haemorrhoids and rheumatism. Palm wine extracts of leaf, bark and fruit extracts of the tree are used for treatment of venereal diseases (Walt el al., 1962).

Chemical Constituents of Kigelia africana

1. africana plant has many medicinal properties due to the presence of numerous secondary metabolites. These compounds include iridoids, flavonoids and naphthoquinones and volatile constituents (Houghton, 2002; Gorman et al., 2004; Asekun et al., 2006). Pinnatal and isopinatal were isolated from tropical trees that belong to the plant family of Bignoniaceae. Pinnata was found in a root bark extract of the plant. Thin layer chromatography (TLC) examination of the most active fractions of both stem bark and fruits showed the presence of some major components which were found to be rovibrational and B-sitosterol. Gouda et al.(2006) isolated a furanone derivative, 3- (2¹-hydroxyethyl)-5-(2^” – hydroxypropyl)- dihydrofuran -2(3H)- one and four iridoids, 7 hydroxy videoid II, 7 hydroxy eucommic acid, 1- hydroxy – 10- deoxycortisol and 10-deoxy eucomm oil together with seven known iridoids, jiofuran, jioglutolide, 1-dehydroxy-3, 4- dehydro lucigenin, des-p-hydroxybenzoyl kisasagenol B, ajugol, verminoside and 6-trans-caffeoyl jugol from the fruit (Gouda et al., 2003). They also isolated a phenyl propanoid derivatives identified as 6-p-coumaroyl-sucrose together with ten known phenylpropanoid and phenylethanoid derivatives and a flavonoid glycoside from fruits of K.africana (Gouda et al., 2006). The structures of the isolated compounds were characterized by different spectroscopic methods. Govindachari et al. (1971) isolated kingelin as the major constituent of the plant from the root heartwood.

Antibacterial and Antifungal

A biologically monitored fractionation of the methanolic extracts of the root and fruits led to the isolation of the naphthoquinones, kigeline, iso-pinnata l, dehydro-α- Lapachone, and lapachol and the phenylpropanoids, p-coumaric acid and ferulic acid as the compounds responsible for the observed antibacterial and antifungal activity (Binutu et al., 1996). The compounds isolated were tested for their activities against Staphylococcus aureus, Bacillus Subtilis, Corynebacterium diphtheriae, Aspergillus niger, A. flavus, Candida albicans and Pullularia pullulans (Aureobasidium sp). The steroids and flavonoids are hygroscopic and have fungicidal properties.

Chemical investigation showed that the aqueous extracts of the stem bark of the plant contain iridoids as major components. In the light of traditional uses of this plant, antimicrobial activities of the aqueous extracts and two major iridoids were tested against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albican. The crude aqueous extracts showed significant antimicrobial activity, which could be partially explained by the activity of the iridoids present (Akunyili et al., 1991). The fruits are popular sources of traditional medicine throughout Africa. The stem bark has been widely analyzed for pharmacological activity but fruit is limited despite more extensive use in traditional remedies.

In the microtitre plate bioassay, stem bark and fruit extracts of K.africana showed similar antibacterial activity against Gram negative and Gram positive bacteria. A mixture of free fatty acids exhibiting antibacterial effect was isolated from the ethyl acetate extract of the fruits using bioassay-guided fractionation. Palmitic acid, already known to possess antibacterial activity, was the major compound in this mixture. These results confirm antibacterial activity of K. africana fruits and stem bark, and support the traditional use of the plant in therapy of bacterial infections (Grace et al., 2002). A disc diffusion susceptibility test was used to screen concentrated extracts from the bark of 3 medicinal plants (Alstonia boonei de wild, Morinda lucida Benth and K. africana) for antimicrobial activity (Kwo and Craker, 1996). Solvents with different polarities were used for the extraction (methylene Chloride, ethyl acetate, 95% ethanol and acetonitrile), and the extracts were tested against Candida albicans, Staphylococcus aureus, Enterococcus   faecalis, Escherichia coli and Pseudomonas aeruginosa. The patterns of inhibition varied with the plant extract, the solvent used for extraction and the organism tested. The largest zone of inhibition was observed for ethanol extracts of K. africana against S. aureus and P. aeruginosa. S. aureus was the most inhibited new organism. No inhibitory effects were observed against C. albican. The extent of the inhibition of the bacteria was related to the concentration of the plant extract (Kwo and Craker, 1996).

Diabetes

Diabetes Mellitus

Diabetes mellitus (DM) in all its heterogeneity has taken the centre stage as one of the ultimate medical challenges. Diabetes complications are the major cause of morbidity and mortality in patients with Diabetes mellitus (Wolf, 1993). Diabetes mellitus is considered to be one of a rank free radical diseases which propagates complications with increased free radical formation (Baynes, 1991; Varvarovska et al., 2004).

One of the major hypotheses proposed to explain the hyperglycemia-induced onset of diabetic complications is an increase in oxidative stress (Brownlee, 2001; Sheetz and King, 2002, Creager et al., 2003). Similar to their proposed role in the onset of diabetic complications, reactive oxygen species (ROS) such as superoxide, (0₂^.-), hydroxyl radical, (OH^.) and hydrogen peroxide H₂O₂ have been linked to other diseases and injury states, including Alzheimer’s disease, (Yamagishi et al., 2001), Parkinson’s disease (Practico, 1999; Hyun et al., 2002), Chronic obstructive pulmonary disease (Practico et al; 1998), and Ischemia (Roberts and Morrow, 2000). Evidence suggests that ROS function not only as mediators of destruction, but also as intracellular second messengers that regulate signal transduction cascades and gene expression (Varvarovska, et al., 2004).

Diabetes Type 1 and 2

Diabetes is associated with a variety of metabolic abnormalities. The so-called metabolic syndromes include hyperglycaemia characterized by hypertriaglyceridemia, reduced High Density lipoprotein cholesterol (HDL) and abnormal postprandial lipidemia, atherosclerosis and pro-coagulant state. The metabolic syndrome represents a cycle whereby insulin resistance leads to compensatory hyperinsulinemia which maintain normal plasma glucose and may exacerbate insulin resistance.

Type 1 diabetes or insulin dependent diabetes mellitus (IDDM) is a complex multifactorial disease involving severe destruction of the insulin-producing pancreatic β-cells. Type 1 diabetes is generally associated with young juvenile onset. Type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM) typically occurs with older age and obesity. Although glycemic control, insulin treatment and other chemical therapies can control many aspects of diabetes, numerous complications are common and diverse. Diabetic patients have an increased risk of developing various clinical complications that are largely irreversible and due to microvascular or macro -vascular disease (Table 1)

Table 1: Vascular complications of diabetes mellitus

	Microvascular	Macrovascular
	Nephropathy	Ischemic heart disease
	Retinopathy	Stroke
	Neuropathy	Peripheral vascular disease
	Source: (Jakus, 2000).

 

The impact of microvascular disease in diabetes includes nephropathy, retinopathy and neuropathy (Table 1). Macrovascular disease is associated with the 2-4 fold increased risk for atherosclerosis and ischemic heart disease that occur in diabetic individuals. The complications of macrovascular disease are important causes of morbidity, mortality and disability in people with Type 2 diabetes mellitus. Although the increased death rate is mainly due to cardiovascular disease, deaths from noncardiovascular causes are also increased. In the pathogenesis of diabetic complications, important risk factors include not only duration of diabetes, but also dyslipidemia, hypertension and cigarette smoking. The results of the diabetic control and complications trails clearly establish hyperglycemia as the major causal factor for the development of diabetic microvascular complication (Jakus,2000).

The role of hyperglycaemia as an independent risk factor (not the major) for the development of cardiovascular disease is supported by United Kingdom prospective Diabetes Study. Improving glycaemic control delays the onset and reduces the severity of diabetic complication. However, even with intensive current antidiabetic agents, more than 50% of diabetic patient with type 2 suffer poor glycaemic control and 18% develop serious complications within six years of diagnosis (Jakus, 2000). Thus,there is need for new antidiabetic agents.

Insulin Resistance and Oxidative Stress

Several studies show that acute hyperglycemia can impair the physiological homeostasis of many systems in living organisms. Excessive hyperglycemia may impair insulin activity and sensitivity by the mechanism of “glucose toxicity” (Mooradian, 1999). Insulin stimulates the uptake and utilization of glucose in muscle and adipose tissue, inhibits glycogenolysis and gluconeogenesis in the liver and lipolysis in adipose tissue. Deficient action of insulin reverses the metabolism of carbohydrates. Thus, with increased lipolysis are enhanced level of free fatty acids and their oxidation in liver. In animal models, hyperglycemia increases fatty acid availability in muscle. Thus, both “glucotoxicity” and “lipotoxicity” could lead to insulin resistance and hyperinsulinemia (McGarry and Dobbis, 1999). It appears that insulin resistance must occur in both muscle and liver for Type 2 diabetes. Both hyperglycemia and insulin resistance are accompanied by reduced insulin action. Hyperglycemia and insulin resistance may also be accompanied by oxidative stress.

Ceriello (2001) hypothesized a model that oxidative stress represents the common pathway through which hyperglycaemia and insulin induce a depressed insulin action. This point of view is supported by studies with antioxidants, which are able to improve the activity of insulin.

Diabetic Complications

Diabetes is both a metabolic and vascular disease associated with numerous long-term clinical complications that contribute to increased morbidity of the disease(Schalkwijk and Stehouwer 2005). Vascular complications of diabetes can be divided into micro-and macro-vascular. Retinal and renal microangiopathy cause diabetic retinopathy and nephropathy respectively while microangiopathy of the vasa nervorum is important in the pathogenesis of neuropathy. Clinically, the complications are manifested as blindness, end stage renal failure, defective nerve conduction and impaired wound healing. Macroangiopathy in diabetes refers to a disease of larger vessels consisting mainly of an accelerated form of atherosclerosis that affects the coronary, carotid and peripheral arteries, thus increasing the risk of myocardial infarction, angina, congestive heart failure and stroke (Fantus, 2002).

Hypoglycemia has been identified as a risk factor for the development of diabetic complications. A number of equally tenable hypotheses have been put forward to account for the association of complications with this small molecule, glucose. These include but are not limited to increased aldose reductase pathway, advanced glycation end- products (AGES) formation, oxidative stress and increased protein kinase C (PKC) pathway. All these mechanisms have been extensively studied and reviewed over a number of years (Wolff et al., 1991; Guigliano et al., 1996; Brownlee, 2005). An abnormal activity of aldose reductase pathway by sustained hyperglycemia seems to trigger a number of cellular and molecular changes that are responsible for the micro- and macro- vascular complications.

Hyperglycemia and Diabetic Complications

The factors that strongly affect the risk of diabetic complications are disease duration and degree of glycemic control (Nathan, 1998 ; Semenkovich and Heinecke,1997). These observations have given rise to the “glucose hypoth esis” which suggests that glucose mediates many of the deleterious effects of diabetes. Although, this appears to be an over-simplification of a complex process, it has gained strong support from clinical trials in Type 1 and Type 2 diabetes. Both the Diabetes Control and Complication Trial and United Kingdom Prospective Diabetes Study found that strict glycemic control dramatically lowered the incidence of retinopathy, nephropathy and neuropathy (Nathan, 1998). This salutary finding suggests that hyperglycemia promotes or even initiates these complications. Therefore, glucose itself may be toxic to the micro vasculature. However, strict glycemic control alone does not prevent diabetic complications, suggesting the involvement of additional factors. Thus, factors other than glucose, such as abnormalities in lipoproteins, metabolic derangements (insulin resistance, elevated free fatty acid levels) and variations in gene controlling lipid metabolism might be important in macrovascular as well as microvascular disease (Semenkovich and Heinecke, 1997).

• Mechanism of Tissue Damage Mediated by Hyperglycemia

Proposed mechanisms for the pathogenesis of diabetic complications include formation of advanced glycosylation end-products (AGES) (Baynes and Thorpe, 2000; Stem, et al.,2002), oxidative stress ( Monnier, 2001), carbonyl stress (Baynes and Thorpe, 2000; Monnier, 2001), increased protein Kinase C activity (Ishie et al., 1998), altered growth factor or cytokine activities ( Sharma and Ziyadeh, 1997), reductive stress or pseudohypoxia (Ido et al., 1997), and mitochondrial dysfunction (Nishikawa et al., 2000; Brownlee, 2001). Some of these hypothesis overlap. For example, AGES might promote growth factor expression and oxidative stress, and oxidative stress might promote AGES formation ( Baynes and Thorpe, 2000; Monnier, 2001). All these hypotheses are supported by extensive data, but a unifying hypothesis remains elusive. The existence of several credible hypotheses might mean that different tissues are differentially vulnerable to various oxidative pathways.

Aldose Reductase Pathway

An increased flux of glucose via the polyol pathway leads to the intracellular accumulation of sorbitol. Accumulation of this non-permeable sugar in cells especially the lens and nerves results in osmotic stress, cellular edema, redox imbalance, depletion of water soluble antioxidants and susceptibility to oxidative insult (Cameron et al., 1999). Under normoglycemia, most of the cellular glucose is phosphorylated to glucose 6-phosphate by hexokinase. A minute part of non-phosphorylated glucose (approximately 8%) enters the so-called polyol pathway, the alternative route of glucose metabolism (Maria et al., 2007), implicating the enzyme aldose reductase. Aldose reductase normally reduces toxic aldehydes in the cell to inactive alcohols, but when the glucose concentration in the cell becomes too high, glucose reduces to sorbitol in the presence of aldose reductase and NADPH which is later oxidized to fructose by the sorbitol dehydrogenase at the cost of NAD⁺ (Fig. 2)

Fig. 2: Pathway leading to AGE formation. AGE formations arise from decomposition of Amadori products, fragmentation products of polyol pathway lipid peroxidation products which react with amino group of protein. HK = hexokinase; Glo = glyoxal; MGO = methylglyoxal; 3-DG = 3-deoxy glucosone; PPP = Pentose Phosphate Pathway.

Source: (Maria et al., 2007)

Under hyperglycemia, there is an increase in the use of glucose through the pentose phosphate pathway together with increased conversion of glucose via the polyol pathway (more than 30% of glucose.

The sorbitol pathway increases in activity in diabetes in those tissues that do not require insulin for cellular glucose uptake, such as the retina, kidney, peripheral nerves and blood vessels. This pathway may impair endothelial function through some mechanisms. First, sorbitol does not diffuse through cell membranes easily and accumulates, causing osmotic damages. Sorbitol accumulation decreases other osmolytes such as myo-inositol and taurine . However, the relatively low expressions of aldose reductase in the endothelial cells may not be sufficient to cause significant sorbitol accumulation. Secondly, hyperglycemia leads to over flow of the products of the polyol pathway along with depletion in the reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is an essential reducing equivalent for the regeneration of reduced glutathione (GSH) by glutathione reductase (Fig 2) and for the activity of the NADPH- dependent thioredoxin system, two important cell antioxidants against oxidative damage. Cells have several sources of NADPH, including the two dehydrogenases of the pentose–phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase; both insulin- induced enzymes), the malic enzyme and the NADPH-dependent isocitrate dehydrogenase. The impairment of the hexose monophosphate shunt leads to a reduced NADPH availability, and negatively influences other enzymes and systems involved in defensive process against oxidative agents, such as the catalase and glutathione systems. Several papers have been published that underline the role of glucose 6- phosphate dehydrogenase deficiency in the pathogenesis of diabetes (West, 2002,)

NADPH is a cofactor of important enzymes of reactive nitrogen species (RNS) and reactive oxygen species (ROS) metabolism, nitrogen oxygen species (NOS) (Nishikawa et al., 2000) and NADPH- Oxidase respectively. Intracellular depletion of NADPH leads to a decreased NO synthesis since NADPH is cofactor of NO synthesis, which synthesizes NO from L-arginine. All isoforms of NOS contain a reductase domain and an oxygenase domain separated by a calmodulin-binding region. NOS requires five cofactors/ prosthetic groups such as flavin adenine dinucleotide, FAD, flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and Ca2+- calmodulin.

1.4.2 Non-Enzymatic Glycation

The non-enzymatic reaction of glucose with free amino groups, a variety of the long -lived proteins represents another important mechanism of diabetic pathology that ultimately leads to accumulation of advanced alycation end products (AGES). These products can form covalent cross-linkages with proteins such as collagen and laminin and increase the stiffness of the extracellular matrix (Tsilibary et al., 1988). Apart from this direct protein modification, circulating advanced glycation end-products (AGEs) have been found to bind receptors for AGEs, called receptor for advanced glycation end-products (RAGE) on various cells (Endothelial cells, macrophages and mesangial cells) ( Li at al., 1996).

Binding of these receptors, stimulate the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF -α) and interleukin – 1 (1L-1), growth factor such as vascular endothelial growth factor (VEGF) and platelet – de rived growth factor (PDGF) and transcription factor (Yamagishi et al,. 1998). RAGE activation increases the vascular permeability that generates ischemia and microaneurysm in the eye and hemorrhage in the brain (Paolino and Garner, 2005).

Cataract occurs as a result of the precipitation of glycated lens crystalline proteins and glycation of retinal vessels leading to inflammation and microhemorrhage. Accumulation of AGE products such as carboxymethyl lysine, pentosidine, and imidazolone in the aqueous humor has been implicated in the diabetic retinopathy (Franke et al, 2003)

Carbonyl Stress in Diabetes

Carbonyl stress explains increased modification of protein in diabetes, uremia and other diseases by a generalized increase in the concentration of reactive carbonyl precursors of AGEs, glycoxidation and lipoxidation products (Lyons and Jenkins, 1997). These carbonyls may damage not only proteins but phospholipids and nucleotide base DNA. Carbonyl stress may result from an increase in the deficiency of detoxification of carbonyl compounds.

Carbonyl stress refers to the intracellular generation and accumulation of reactive compounds with carbonyl groups. These include both six- carbon derivative of glucose such as 3-deoxyglucosone, and 3–carbon fragmentation products of glycolytic intermediate, glyceraldehyde–3-phosphate called glyoxal and methylglyoxal (Begenbardt et al., 1998). These reactive species form covalent linkages with the amino groups of proteins both intra – and extracellularly resulting in altered structure and function.

Activation of Protein Kinase C isoforms

Excess glucose may activate protein kinase C (PKC) directly by several mechanisms, including through de novo synthesis of diacylglycerol (DAG), by activation of phospholipase C, and by inhibition of DAG kinase (Keogh et al., 1997) or indirectly (via ligation of AGE receptors or increased activity of the polyol pathway). Increased activity of protein kinase C results in functional changes to vascular cells via activation of phospholipase A₂ (the enzyme supplying the substrates arachidonic acid for prostaglandin production), the expression of growth factors e.g. transforming growth factor– β, endothelial, and vascular endothelial growth factor) and alterations in the expression of certain basement membrane proteins (Koya, et al., 1997). Glycation may be responsible for the increased deformability of granulocytes observed in diabetes (Sulochana et al., 2001).

Glycated products can be oxidized by several ROS, including HO^. and ONOO^– to give AGEs shown in the Fig.3 below (Yim et al., 2002; Ahmed et al., 2005). Glycoxidation products such as pentosidine and N^E carboxyl methyl lysine are the best chemically characterized AGEs compounds found in humans. The non- enzymatic glycation reaction proceeds slowly through different stages leading to alteration of protein structure and molecular surface topology that profoundly change the biochemical properties of the affected molecule. The major biological effects of excessive glycation include inhibition of regulatory molecule binding , cross linking of glycated proteins, trapping of soluble protein by glycated extracellular matrix, decreased susceptibility to proteolysis, inactivation of enzymes and transformation factors and abnormalities in relation to immune complex formation (Turk, 2001;Yim et al., 2002; Ahmed et al., 2005; Halliwell and Gutteridge, 2007). Glycation is faster at elevated glucose in diabetic patients. Some tissues such as the liver, kidneys, and erythrocytes are more susceptible to AGEs formation than others (Bohlender et al., 2005). Glycated haemoglobin (Hb_A1c) contains a glucose amadori product attached to the N– terminal valine of the β-chain. Glucose also glycates CuZnSOD in the erythrocyte, decreasing its activity; this may account for the lower SOD activity reported in the blood of some diabetics (Aral et al., 1987). Both CuZnSOD and ceruloplasmin can fragment after glycation to release pro- oxidant copper ions (Islam et al., 1995). Fig. 3 shows the formation of advanced glycation end-products (AGEs) by combination of glycation and oxidation.

Fig. 3: Formation of advanced glycation end products (AGEs) by combination of glycation and oxidation.

Source: (Maria et al., 2007).

Fig. 3 depicts another way of making AGEs by first oxidizing the glucose and then allowing the oxidation product to react with protein. In the presence of transition metals, glucose can be oxidized to produce O^–₂, H₂O₂, HO^. and toxic dicarbonyls which can damage proteins (Halliwell and Gutteridge, 2007).The above reactions occur under hyperglycemic condition with production of radicals which can attack biomelecules such as lipids, proteins and DNA. Under these conditions depletion of antioxidant enzymes such as SOD, CAT GPx could be attributed to increased levels of the ROS produced.The disequilibrium between free radical and antioxidant in favour of the former contributes to AGE formation and the word glycoxidation is often used to describe the pathway involved. Once formed, AGE – modified protein causes more oxidative stress. Glycation of protein in the electron transport chain can impair normal electron flow and promote “leakage” t o form O₂^-. Binding of glucose to amino group on both Apo B and on lipids in LDL facilities LDL oxidation. Thus AGEs formation is probably a significant contributor to the onset of diabetes complication mainly atherosclerosis (Mehta et al., 2006).

Oxidative Stresses

An alteration in the level of oxidant and antioxidants, called oxidative stress initiated by hyperglycemia contributes to tissue pathology. Early studies showed that glucose autoxidation occurs in the presence of metal ions generating superoxide radical (O₂^–) and hydrogen peroxide (H₂O₂) and if the scavenging enzymes superoxide dismutase and catalase are impaired this may result in the formation of hydroxyl radicals (HO^.) that react rapidly with and damage lipids, protein and DNA (Baynes, 1991). So many data (clinical and experimental) have clearly documented the depletion of extra and intracellular antioxidants in the diabetic state and the prevention of complications by antioxidant supplementation (Baynes and Therpe, 2000; Cederberg et al., 2001; Yamagishi et al., 2001). Browlee (2001) speculates that in the setting of hyperglycemia, over- production of superoxide by mitochondrial electron transport chain and the resultant oxidative stress is the unifying mechanism linking the major biochemical pathways triggered by hyperglycaemia. Oxidative stress may be important in diabetes not just because of its role in the development of complication but because persistent hyperglycaemia, secondary to insulin resistance may induce oxidative stress and contribute to beta cell destruction in Type 2 diabetes (Maria et al., 2007). Protein kinase C activation induced by glycemia may also represent a common pathway by which oxidants and glycation products may mediate their advanced effects.

1.5.1 Mechanism of Increased Oxidative Stress in Diabetes Mellitus

Many studies have shown that increased lipid peroxides and/or oxidative stress are present in diabetic subjects. Oxidative stress can be increased before clinical signs of diabetic complications. In diabetes, oxidative stress is caused by both increased production of reactive oxygen species (ROS), sharp reduction in antioxidant defenses and altered cellular redox status (West, 2000). Hyperglycaemia may lead to increased generation of free radicals via multiple mechanisms. Patients with diabetes may be prone to acute and chronic oxidative stress which enhances the development of late diabetic complications. Enhanced oxidative stress in hyperglycemia is indicated by urinary excretion of 8 -iso –prostaglandin F ₂ alpha. Oxidative stress measured as index an of lipid peroxidation and protein oxidation has been shown to increase in both insulin-dependent diabetes and non-insulin dependent diabetes (Nishikawa et al., 2000; Cerieillo et al., 2001; Mohora et al., 2006; Stephens et al., 2006). Although, the source of oxidative stress remains unclear, it has been suggested that the chronic hyperglycaemia in diabetes enhances the production of ROS from glucose autoxidation, protein glycation and glycoxidation, which leads to tissue damage. Also, cumulative episode of acute glycemia can be source of acute oxidative stress.

During diabetes or insulin resistance, increased oxidative glucose metabolism itself increases mitochondria production of O₂^–, which will be then converted to OH^., and H₂O₂ (Nishikawa et al, 2000). Beyond glucose, ROS formation is also increased by free fatty acids, through direct effects on mitochondria (Evans et al., 2002). Enhanced oxidative stress in diabetes (Type 2) has further a variety of important effects in atherogenesis, including lipoprotein oxidation, particularly low density lipoprotein (LDL) oxidation. Lipid peroxidation of polyunsaturated fatty acid (PUFA), one of the radical reactions in vivo, can adequately reflect increased oxidative stress in diabetes (Slatter et al., 2002). The overproduction of O^._2, in particular by mitochondria, causes inhibition of the glyceraldehyde -3-phosphate dehydrogenase (GAPDH) and of cytochrome enzyme of the electron transport system responsible for oxidative phosphorylation associated with the Krebs cycle (Nishikawa et al., 2000). Hyperglycaemia- induced GAPDH inhibition was a consequence of poly (ADP-ribosylation) of GAPDH by poly (ADP-ribose) polymerase], which was activated by DNA strand breaks produced by mitochondrial superoxide over production (Du et al., 2003).

As a result, glycolytic intermediates upstream of GAPDH accumulate, leading to increased substrate- directed activity of the diacylglycerol synthetic pathway , which further activates PKC (protein kinase C isoforms) and NADPH oxidase , as well as the hexosamine and polyol biosynthetic pathways ( Fig 4).

1.5.2 Glucose Autoxidation

Hyperglycaemia –induced oxidative stress also occur in non-nucleated cells lacking mitochondria and the NADPH oxidase (i.e. erythrocytes) (Jain, 1989). There must therefore be another mechanism of ROS formation in these cells. One hypothesis is glucose autoxidation. Glucose and many of its metabolites can react with hydrogen peroxide in the presence of transition metals, such as Fe2+ and Cu2+, to form hydroxyl radical (OH.), the most reactive ROS (Robertson et al., 2003).

1.5.3 Free Radicals

Free radicals are defined as atoms or molecules that contain one or more unpaired electrons, making them unstable and highly reactive. The most important ROS are superoxide anion (O2-) hydroxyl radical (OH.) hydrogen peroxide (H2O2), alkoxyl (RO.), peroxyl (ROO.) and hydrochlorous acid (HOCl). Other non-oxygen species existing as reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite have also important bioactivity. ROS are

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continuously generated in physiological conditions and effectively eliminated by several intracellular and extracellular antioxidant systems (Halliwell and Guttreridge, 1999). Free radicals may be electrically neutral or either positively or negatively charged. They attack sites of increased electron density such as the nitrogen atom present in proteins and DNA predominantly and carbon-carbon double bonds present in polyunsaturated fatty acids and phospholipids to produce additional free radicals which are often reactive intermediates (Knight, 1999). Uncontrolled production of ROS often leads to damage of cellular macromolecules,DNA, lipids and proteins and compromise cell function leading to cell death by necrosis or apoptosis.

1.5.4 Reactive Oxygen Species and Oxidative Stress

Oxidative stress is defined as a disturbance in the proxidant- antioxidant balance in favor of the former leading to potential damage and disruption of redox signaling and control. Cellular metabolism generates reactive oxygen species (ROS). Molecular or ground state oxygen can be activated to a ROS by means of energy transfer (e.g. under the influence of ultraviolet radiation), forming singlet oxygen (1O2), or by electron transfer, forming incomplete reduction products i.e. the superoxide anion radical (O2-). Small amount of oxygen (between 0.4 and 4% of all oxygen consumed) are reduced to O-2 by the mitochondria electron transport chain during the course of normal respiration which is essential for generating ATP (Boveris, 1984). Subsequently, O-2 can be converted into other ROS and RNS as shown in Fig.5.

Under normal conditions, O2 – molecules are quickly converted to H2O2 by the key mitochondrial enzyme, manganese superoxide dismutase (Mn-SOD) within the mitochondria and by copper and zinc (CuZn-SOD) in the cytosol (Mendez et al., 2006). H2O2 is then either detoxified to H2O and O2 by glutathione peroxidase (in the mitochondria) in conjunction with glutathione reductase, or diffuse into the cytosol and detoxified by catalase in peroxisomes. H2O2 can also be converted to the highly reactive hydroxyl radicals (HO-) in the presence of reduced transition metals such as Cu or Fe (Fenton reaction). Further reactive oxygen species may be derived from H2O2 such as the hypochlorite (OCL-) peroxyl radicals (ROO.) and alkoxyl radicals (RO.) or from peroxidation of polyunsaturated fatty acids (PUFA) such as conjugated dienes, lipid hydroperoxides and malondialdehyde (MDA) (Taniyama and Griendling, 2003).

Production of one ROS may lead to the production of the other through radical chain reaction. As summarized in Fig.5, O-2 is produced by one electron reduction of oxygen by several different oxidases including NAD(P)H oxidase , xanthine oxidase, cyclooxygenase and even endothelial nitric oxide synthase (eNOS) under certain conditions (Guzik et al., 2002; Mehta et al., 2006).

Reactive nitrogen species (RNS) include free radicals like nitric oxide (NO.) and nitrogen dioxide (NO2), as well as non-radicals such as peroxylnitrite (ONOO-). NO., also known as endothelium-derived relaxation factor (EDRF) produced from L-arginine by eNOS in the vasculature is considered vasculo protective. However, NO. can easily react with O2-, generating the highly reactive molecule ONOO-. Thus, variation in the production of and O-2 by endothelium might provide one mechanism for the regulation of vascular tone and NO. hence of blood pressure.

Although these ROS and RNS differ with regard to their stability, reactivity and molecular targets, a common denominator is that the generation of ROS exceeding the antioxidant capacity of the cells results in damage and oxidation of biomolecules such as lipids, proteins and nucleic acids (Maria et al., 2007).

1.6 Antioxidant System

The reactive oxygen intermediates produced in mitochondria, peroxisomes, and the cytosol, are scavenged by cellular defense systems including enzymatic (e.g. super oxide dismutase, glutathione peroxidase, glutathione reductase, and catalase) and non-enzymatic antioxidants (e.g. glutathione (GSH), thioredoxin, lipoic acid, ubiquinol, albumin, uric acid, flavonoids, vitamins A, C and E, ).These antioxidants are located in the cell membranes, the cytosol or blood plasma (Maritim et al., 2003). A major cellular thiol antioxidant and redox buffer of the cell is reduced glutathione (GSH) which is regenerated most efficiently from oxidized form (GSSG) by glutathione reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH). Glutathione is highly abundant in cytosol (1 – 11 mM) nuclei (3 – 15mM); and mitochondria (5 – 11mM) and is the major soluble antioxidant in these compartments (Masella et al., 2005; Valko et al., 2007). GSH in the nucleus maintains the redox state of critical protein sulfhydryl that are necessary for DNA repair and expression. Oxidized glutathione is accumulated inside the cell and the ratio of GSH/GSSG is a good measure of oxidative stress of an organism (Valko et al., 2007). There are many other redox couples in the cell, examples include NAD+ /NADH, ascorbate/dehydroascorbate, NADP+/NADPH, and α – lipoic acid (LA)/dihydrolipoic acid (DHLA).

The main protective roles of glutathione against oxidative stress are (i) glutathione is a cofactor of several detoxifying enzymes against oxidative stress, e.g. glutathione peroxidase, (GPX), glutathione reductase, glyoxalases and enzymes involved in leucotriene synthesis.

(ii) GSH can react with ONOO- leading to formation of some nitrosothiol (GSNO), which can decompose to regenerate NO., hence GSH can to some extent, recycle ONOO- to NO.

(iii) GSH scavenges hydroxyl radicals and singlet oxygen directly; detoxifying hydrogen peroxides by the catalytic action of glutathione peroxidase (iv) Glutathione is able to regenerate the most important antioxidants lipoic acid, vitamin C and E, back to their active forms. Glutathione can reduce the tocopherol radicals of vitamin E directly or indirectly via reduction of semi dehydroascorbate to ascorbate (Masella et al., 2005). The capacity of glutathione to regenerate the most important antioxidant is linked with the redox state of glutathione disulphide – glutathione couple (GSSG/2 GSH) (Pastore et al., 2003)

1.6.1 Scavenging Properties of Antioxidants

A number of major cellular antioxidant defense mechanisms exist to neutralize the damaging effects of free radicals. Enzymatic antioxidant system (Cu, Zn and Mn- superoxide dismutase (SOD), catalase,glutathione (GSH), glutathione peroxidase (GPX), and glutathione reductase (GR) function by indirect or sequential removal of ROS, thereby terminating their activities (Jakus, 2007). The biological roles of these antioxidants are shown in following equations.

These antioxidant enzymes and vitamins catalyze the reactions that neutralize free radicals and reactive oxygen species. They form the body’s endogenous mechanisms to help protect against free radical-induced cell damage. The antioxidants enzymes glutathione peroxidase, catalase, and superoxide dismutase metabolism oxidative toxic intermediates. Vitamin C and E molecules can interrupt free radical chain reactions by capturing the free radical. The free hydroxyl group on the aromatic ring is responsible for the antioxidant properties. The hydrogen from this group is donated to the free radical, resulting in a relatively stable free radical form of vitamin E.

To minimize transition metal – induced catalysis of Fenton and Haber Weiss reaction which generate the most reactive hydroxyl radical, Several specific metal binding proteins such as ceruloplasmin, ferritin, transferrin, haptoglobin, lactoferrin, and albumin ensures that these metals (copper and iron) are cryptic (Jacus, 2007).Non-enzymatic antioxidant systems consist of scavenging molecules that are endogenously produced (GSH,ubiquinol,uric acid) or those derived from the diet (vitamin C and E, carotenoids,α-lipoic acid and selenium).These molecules donates electrons, and themselves become free radicals that can either initiate chain reactions, or conversely be regenerated.

Regeneration of endogenous antioxidants occurs through a cooperative set of reactions. The hydroxyl radicals as shown in Fig. 6 can abstract an electron from polyunsaturated fatty acid.(LH) to give rise to carbon-centered lipid radicals (L ). The lipid radical (L ) can further interact with molecular oxygen to give a lipid peroxyl radical (LOO.), the lipid peroxyl radical (LOO.) is reduced within the membrane by the reduced form of vitamin E resulting in the formation of a lipid hydroperoxide and a radical of vitamin E. The vitamin E radical is reduced back to vitamin E by vitamin C leaving behind the vitamin C radical. The oxidized vitamin E radical can also be reduced by GSH. The oxidized glutathione (GSSG) and the vitamin C radicals are reduced back to GSH and vitamin C, respectively by dihydrolipoic acid (DHLA) which itself is converted to α-lipoic acid (LA). Α –lipoic acid (LA) after reduction by nicotinamide adenine dinucleotide phosphate (NADPH) to dihydrolipoic acid (DHLA) is able to facilitate the non-enzymatic regeneration of vitamin C and GSH, both of which are able to regenerate vitamin E (Maria et al., 2007).

1.6.2 Positive and Negative Effects of Free Radicals

Respiratory burst is a remarkable property of the neutrophils, macrophages, β- cells and other phagocytic cells (Kerrigan et al., 2009; Sluauch, 2011). These cells after activation increase their oxygen uptake,which may raise up to 50 folds. This increased oxygen uptake is then followed by the breakdown of the oxygen by respiratory burst oxidase enzyme as shown below:

The presence of low concentrations of free radicals is important for normal cellular redox status, immune function and intracellular signaling. Free radicals can serve as second messengers or modify oxidation- reduction (redox) states. They are involved in some enzymes activation, drugs detoxification and play an essential role in muscle contraction (Finaud et al., 2006).

Lipid Peroxidation

Lipid peroxidation refers to the oxidative deterioration of lipid. It is the process in which free radicals ‘steal’ electrons from the lipids in cell membranes resulting in cell damage. Lipid peroxidation proceeds by free radical chain reaction. Polyunsaturated fatty acids are most often being affected because of the presence of multiple double bonds in between which lie methylene bridges (-CH₂-) that possess reactive hydrogens.When the radical removes hydrogen atom, it leaves behind an unpaired electron in the lipid (Niki, 2009). This in turn leads to chain reaction. L-H + OH → H₂O + L

The lipid radicals formed lead to cell damage. Three mechanisms are able to induce lipid peroxidation: autoxidation (by free radicals reaction), photooxidation and enzyme action. Autoxidation is a radical-chain process involving three stages: initiation, propagation and termination. The general process of lipid peroxidation consists of three stages: initiation, propagation and termination. Initiation occurs when oxygen is partly reduced by Fe²⁺ to species able to abstract a hydrogen atom from a methylene carbon .The resulting alkyl radical reacts with oxygen to form a peroxy radical (LOO ), which itself can liberate LOOH via hydrogen abstraction from a neighbouring alkyl bonds.

In propagation, fatty acid radicals react with molecular oxygen forming a peroxyl-fatty acid radical. This radical is also an unstable species that reacts with another free radical acid, producing a different fatty radical and a lipid peroxide or acyclic peroxide if it had reacted with itself. The cycle continues as the new fatty acid radical react in the same way.

Termination occurs when new radicals reacts and produce a non-radical species. Antioxidant vitamin E and antioxidant enzymes play a major role in the termination process (Marnett, 2002).

Photo-oxidation occurs when singlet oxygen of highly electrophilic reacts with unsaturated lipids. In the presence of sensitizers (chlorophyll, porphyrins, myoglobin,riboflavin, bilirubin), a double bond interacts with singlet oxygen produced from O₂ by light. The oxygen is added at either end carbon of a double bond which takes the trans-configuration. Thus, the possible reaction of singlet oxygen with double bond produces hydroperoxides (Hossam and Mohamed, 2013).

Malondialdehyde (MDA) is a late–stage lipid oxidation by-product that can be formed non enzymatically as a by-product of cyclooxygenase activity (Slatter et al., 2002). MDA is a highly toxic product formed in part by lipid oxidation-derived free radicals. Many studies have shown that its concentration is considerably high in diabetes mellitus correlating with poor glycemic control (Slatter et al., 2002, Hoff et al., 2003). MDA is a volatile molecule that reacts, via Schiff base formation, with free amine groups of proteins, lipids and DNA. It is estimated that up to 80% of MDA is protein-bound (Slatter et al., 2002). In addition, accumulation of MDA affects membrane organization by increasing phosphatidylserine (PS) externalization. Accumulation of MDA and MDA adducts were correlated with many disease state, such as hepatitis C, Down syndrome (Muchova et al., 2001), cancer (Marneth et al., 2002), liver injury (Tuma, 2002), neurodegenerative disease and diabetes mellitus (Slatter et al., 2002).

4-Hydroxy–nonenal is another lipid oxidation by-product which can form non-enzymatically by-product. 4-Hydroxynonenal is formed from scission of precursor lipid hydroperoxide and degradation of cyclic intermediates in lipid oxidation (Hoff et al., 2003). Compared with MDA, 4-HNE is more reactive with proteins, potentiated by the ability for Michael addition as well as Schiff based information. 4-HNE reacts with lysine from proteins and forms pyrroles, cysteine and histidine. Few studies have demonstrated the accumulation of 4-HNE in diabetes mellitus. 4-HNE is increased in microsomes and mitochondria of the IDDM model mice (Traverso et al., 2002) and in the kidney of STZ-induced diabetic rats.

Lipid hydroperoxides are intermediates of lipid oxidation and are also formed enzymatically through the action of lipoxygenases. Both forms of lipid peroxidation by-products are structurally similar and can be further modified to hydroxyl fatty acid, leukotrienes, and lipids by lipoxygenases and glutathione peroxidase. These molecules are ultimately important in regulation of inflammation and atherosclerosis (Funk and Cyrus, 2001 and Natarajan and Nadle, 2003). They initiate apoptosis in vascular smooth muscle cells (Dandona and Mjada, 2002).There is evidence that both non-enzymatically and enzymatically formed lipid hydroperoxides and their derivatives are elevated in diabetic state and are associated with a few of the diabetic complications.

Non-enzymatically produced lipid hydroperoxide increased in the retina of STZ- induced diabetic rats (Kowluru, 2003) and in the plasma of NIDDM (Nourooz-zadeh et al., 1997) and IDDM (Davison et al., 2002) patients.

Isoprostanes are nonenzymatic products of arachidonic acid oxidation that are formed in situ in the cell membrane and are released through the action of phospholipases. Isoprostanes metabolites, such as epoxy isoprostanes and epoxy cyclopentenones, stimulate endothelial cell protein expression and synthesis (Subbanagounder et al., 2002). In addition isoprostane have been used extensively as indicators of oxidative stress in cigarette smoking – induced oxidation, pesticide exposure, chronic obstructive pulmonary disease, athero-thrombotic disease, heart disease, atherosclerosis and diabetic mellitus (Subbanagounder et al., 2002). They offer specificity and sensitivity advantages over MDA, the classic oxidant indicator.

Antioxidant Supplementation in Diabetes Mellitus

Given the involvement of oxidative stress in diabetic complications, supplementation with antioxidants could be of interest since they delay the appearance or the development of vascular complications. Some information is available on the effects of treatments with classical antioxidants such as vitamin E, vitamin C or lipoic acid. Specifically, vitamin E normalizes retinal blood flow and PKC activity in vascular tissue of diabetic rats (Kunisaki et al., 1995). Lipoic acid has been suggested but not proven, to decrease the severity of diabetic neuropathy by maintaining G-SH level and / or by its direct antioxidant properties (Vincent et al., 2004). Vitamin E decreases the extent of diabetic complications, including renal damage, and embryo pathway, nerve damage and vascular dysfunction. It equally delays lipid peroxidation.

Alloxan

Alloxan and streptozotocin are the most prominent diabetogenic chemicals in diabetes research. Both are cytotoxic glucose analogues. Although their cytotoxicity is achieved via different pathways, their mechanisms of beta cell selective action are identical (Lenzen, 2007). In 1938 Wohler and Liebig synthesized a pyrmidine derivative, which they later called alloxan (Lenzen et al., 1996). In 1943, interest in alloxan increased when Dunn and Mc letchie reported that it could induce diabetes in animals as a result of the specific necrosis of the pancreatic beta cells (Peschke et al., 2000). The resulting insulinopenia causes a state of experimental diabetes mellitus called alloxan diabetes. The reduction product of alloxan, dialuric acid, has been shown to be diabetogenic in animals and to cause ultrastructural changes identical to those observed in response to alloxan (Jorns et al., 1997). It was reported that streptozotocin is diabetogenic and could cause diabetes by specific necrosis of the pancreatic beta cell. Research has provided a unifying explanation for selective toxicity of these most prominent diabetogenic agents.

1.9.1 Alloxan Diabetes and Streptozotocin Diabetes

Fig. 7 shows a schematic diagram of the tetraphasic and triphasic glucose responses to alloxan and streptozotocin respectively, when injected (Lenzen, 2007). The responses are accompanied by corresponding inverse changes in plasma insulin and sequential ultra structural changes resulting in necrotic beta cell death. A first transient hypoglycemia phase of up to 30 minutes starts within minutes of alloxan injection. This short lived hypoglycemic response is the result of a transient stimulation of insulin secretion as documented by an increase in the plasma insulin concentration.

As demonstrated in the diagram above, the initial transient hypoglycemic phase is not observed in response to streptozotocin injection, since streptozotocin does not inhibit glucokinase. Morphological alterations are minimal during this phase. The second phase starts with an increase in the blood glucose concentration 1h after administration of the toxins, and a decrease in plasma insulin. This first hyperglycemic phase, which usually lasts 2-4h, is caused by inhibition of insulin secretion leading to hypoinsulinemia. The third phase, again a hypoglycemic phase typically occurs 4-8h after the injection of the toxins and last several hours. It may be so severe that it causes convulsions, and may even be fatal without glucose administration, in particular when liver glycogen stores are depleted through starvation. This severe transitional hypoglycemia is produced by the flooding of the circulation with insulin as a result of toxin – ind uced secretory granule and cell membrane rupture (Lenzen., 2008). The fourth phase is the permanent diabetic hyperglycemic phase. Morphologically, complete degranulation and loss of beta cell integrity is seen within 12-48h. Non–beta cells remain intact, demonstrating the bet a cell- selective character of the toxin. Thus, injection of alloxan and streptozotocin principally induce the same blood glucose and plasma insulin response and cause an insulin-dependent type 1- like diabetic syndrome. All of the described morphological features of beta cell destruction are characteristic of necrotic cell death (Lenzen, 2007).

Alloxan: Mechanism of Action

 Alloxan has two distinct pathological effects: it selectively inhibits glucose-induced insulin secretion through specific inhibition of glucokinase, the glucose sensor of the beta cell, and causes a state of insulin-dependent diabetes through its ability to induce ROS formation, resulting in the selective necrosis of beta cells (Lenzen, 2008). Due to its chemical properties, in particular the greater stability (Table 2), streptozotocin is the agent of choice for reproducible induction of a diabetic metabolic state in experimental animals (Lenzen et al., 1996). Alloxan on the other hand, as a model compound of ROS-mediated beta cell toxicity, is the agent with the greater impact upon the understanding of ROS mediated mechanisms of beta cell death in type 1 and type 2 diabetes mellitus.

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