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PHARMACOLOGICAL EFFECTS AND ANTIDIABETIC CONSTITUENTS OF THE METHANOL LEAF EXTRACT OF Newbouldia laevis (P. BEAUV) Seeman ex Bureau
ABSTRACT
Pharmacological effects and Antidiabetic constituents of the methanol leaf extract of Newbouldia laevis (P.Beauv) Seeman ex Bureau. were carried out using standard in vivo and in vitro models in albino rats. The antidiabetic activities were evaluated using alloxan 150 mg/kg ip and various doses of NLE (62.5, 125.0 and 250.0 mg/kg), and glibenclamide, the reference drug 2.0 mg/kg. The yield of the methanol leaf extract of NLE was 5.27 w/w dry matter. Acute toxicity test of NLE did not show any sign of toxicity or death even at the highest dose of 6,000 mg/kg body weight after 48 h. The dose-response study showed that NLE caused significant (p < 0.05) time and dose-dependent reduction of the fasting blood sugar (FBS) of the treated rats when compared to the negative control, NLE (125.0 and 250.0 mg/kg) caused 25.0 and 33.6% at the 6th h, then 55.3 and 60.2% at the 24th h as compared to the reference drug glibenclamide, which caused 40.0 and 51.5% reductions in FBS at the same times of 6 and 24 h respectively. The extract and reference drug showed time-dependent decreases in FBS of normal glycaemic rats. The Oral glucose tolerance test (OGTT) showed that all the doses of NLE and the reference drug were able to inhibit the significant (p < 0.05) rise in the FBS within the 1st h after glucose load in both normal and diabetic rats. Antioxidant activity showed that NLE at concentration of 25 µg/ml, has 92.9 % and 94.2% at 400 µg/ml for the 1-1Diphenyl-2-picril hydroxyl (DPPH) and 1.45µM at 400µg/ml for the Ferric Reducing Antioxidant Power (FRAP) respectively. These effects were similar to Vitamin C, the positive control. In the sub-acute antidiabetic study, NLE caused various levels of significant (p < 0.05-0.001) dose and time-dependent reduction of FBS of the treated rats on day 21 as compared to the negative control. The various doses of the extract (62.5, 125.0 and 250.0 mg/kg) caused 40.1, 53.1 and 67.0 % decreases in FBS respectively as against 70.2% by glibenclamide. Furthermore, NLE at 125.0 and 250.0 mg/kg caused various levels of significant (p < 0.05-0.001) reductions in serum levels of bad lipids and the cardiovascular risk index of the diabetic rats when compared to the control. The effect of NLE at 250.0 mg/kg was comparable to glibenclamide in all the parameters tested. All levels of NLE also caused dose-dependent and significant (p < 0.05) increases in good lipids (HDL). The liver function parameters showed a dose-dependent and significant (p < 0.05) decrease in Alanine Aminotransferase (ALT), Alkaline Phosphate (ALP) and bilirubin but not Aspartate Aminotranferase (AST). NLE showed dose-dependent and significant (p < 0.05) decrease in serum urea and creatinine. All doses of NLE caused dose-dependent increases in superoxide and catalese. NLE (250.0 mg/kg) also significantly (p < 0.05) reduced the level of Malondialdehyde (MDA) when compared to the negative control. All doses of NLE showed significant (p < 0.05) increase in the WBC count, suggesting an immunomodulatory effect of NLE. Erythrocyte fragility test showed significant (p < 0.05) decrease in haemolysis between the NLE-treated groups, Vitamin C, glibenclamide and the negative control at the NaCl salt concentration of 0.85%. At this concentration, NLE at 62.5, 125.0 and 250.0 mg/kg caused 18.50, 24.55 and 11.08% haemolysis compared to 22.52% and 10.87% haemolysis for glibenclamide and vitamin C respectively. Histopathology showed vacuolation and decrease in pancreatic islet cells; fatty degeneration of hepatocytes in the liver and degenerative renal tubules in the kidney. These effects were reversed by glibenclamide and higher doses of NLE. Phytochemical analysis showed NLE to contain alkaloids, saponins, flavonoids, glycosides, tannins, sterols/terpenes, polyuronoids and carbohydrates. Elemental composition of NLE indicated the presence of lead, mercury, cadmium, zinc copper, calcium chromium, iron, magnesium selenium potassium and sodium. NLE also contains vitamins A, B1, B2, B3, C and E. Bioassay-guided fractionation of NLE yielded ten (10) fractions with fraction nine (F9) as the active fraction, which caused 66.0% reduction of FBS in alloxan-induced hyperglycaemic rats. Further purification using preparative thin layer chromatography (TLC), gave sub-fraction 9.2 as the active compound.. Sub-fraction 9.2 reduced the FBS by 61.4%. The characterization of F9:2 using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) confirmed it to be a polyunsaturated fatty acid 9-(4-Nonyl-phenyl)-non-8-enoic acid with the chemical formula C24H38O2 and molecular weight of 358.56. The sub-chronic treatment of the rats with graded doses (1.25, 2.5 and 5.0 g/kg) of NLE for 90 days caused no observable clinical signs of toxicity or death throughout the duration of the study, but a significant (p < 0.05) decrease in relative organ weight of the heart, lungs, kidneys and liver between day 30 and day 90 except in NLE 5.0 g/kg. It also caused a dose-dependent increase in insulin secretion on day 90. Generally NLE demonstrated significant antidiabetic activity which was comparable to glibenclamide, a standard sulfanylurea. This antidiabetic activity is likely due to its elemental, phytochemical and or vitamin composition. It also has potent antioxidant activity and demonstrated cardioprotective, hepatoprotective and nephroprotective properties. This implies that NLE can delay or protect from diabetic complications. It was well tolerated in the short term therapy, but caution should be employed in long term use. The present study justified the folkloric use of N. laevis in the management of diabetes mellitus by the natives.
TABLE OF CONTENT
Title Page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Contents vi
List of Figures xv
List of Plates xvi
List of Tables xvii
Abstract xx
CHAPTER ONE
1.1 INTRODUCTION – – – – – – – – -1
1.2 STATEMENT OF THE PROBLEM – – – – – – -6
1.3 RESEARCH OBJECTIVE – – – – – – – -7
1.4 JUSTIFICATION OF THE STUDY – – – – – -7
CHAPTER TWO
2.1 LITERATURE REVIEW – – – – – – – -9
2.1.1 Definition and Prevalence of Diabetes Mellitus – – – – -9
2.1.2 Classification of Diabetes Mellitus – – – – – – -10
2.1.2.1 Type 1 Diabetes Mellitus – – – – – – – -11
2.1.2.2 Type 2 Diabetes Mellitus – – – – – – – -12
2.1.3.0 Other types of Diabetes Mellitus – – – – – – -13
2.1.3.1 Gestational Diabetes Mellitus (GDM) – – – – – -13
2.1.3.2 Diabetes Insipidus – – – – – – – – -13
2.1.3.3 Latent Immune Diabetes in Adults (LADA) – – – – – -14
2.1.3.4 Maturity Onset Diabetes of the Young (MODY) – – – – -14
2.1.3.5 Drugs or Chemical-Induced Diabetes – – – – – – -14
2.1.3.6 Brittle Diabetes – – – – – – – – -14
2.1.3.7 Malnutrition-Related Diabetes Mellitus (MRDM) – – – – -15
2.1.4 Aetiology and Predisposing Factors – – – – – -15
2.1.4.1 Autoimmune Mediated Destruction of β-cells of the Pancreas – – -15
2.1.4.2. Genetic Defect in β-cells Function – – – – – – -16
2.1.4.3 Environment – – – – – – – – – -16
2.1.4.4. Viral Infection – – – – – – – – -16
2.1.4.5. Chemicals and Drugs – – – – – – – -16
2.1.4.6. Diet – – – – – – – – – – -17
2.1.4.7 Exocrine Pancreatic Defects – – – – – – – -17
2.1.4.8. Endocrinopathies – – – – – – – – -17
2.1.4.9. Idiopathic Aetiology – – – – – – – – -17
2.1.5 Pathophysiology of Diabetes Mellitus – – – – – -19
2.1.6 Clinical Signs and Symptoms – – – – – – – -21
2.1.7 Complications of Diabetes Mellitus – – – – – – -22
2.1.7.1 Microvascular Complications – – – – – – – -22
2.1.7.1.1 Diabetic Retinopathy (Disease of the Retina) – – – – -22
2.1.7.1.2. Diabetic Nephropathy (Disease of the Kidney) – – – – -23
2.1.7.1.3. Diabetic Neuropathy (Disease of the nerves) – – – – -23
2.1.7.2 Macrovascular Complications – – – – – – -24
2.1.8 Diagnosis of Diabetes Mellitus – – – – – – -24
2.1.9 Treatment/Management of Diabetes Mellitus – – – – -25
2.1.9.1 Lifestyle Changes through diet Modification and Exercise – – – -26
2.1.9.2 Insulin Therapy – – – – – – – – -26
2.1.9.3 Oral Hypoglycaemic Agents – – – – – – – -27
2.1.9.4 Other Management Strategies in Diabetes Mellitus – – – – -31
2.1.10 Diabetes in Animals – – – – – – – – -32
2.1.11 Plants Products used in Diabetes Mellitus – – – – – -33
2.2. Newbouldia laevis – – – – – – – – –35
2.2.1 Botanical Profile of Newbouldia Laevis – – – – – -35
2.2.2 Common and Ethnics names of Newbouldia Laevis – – – – -35
2.2.3 Habitat and Geographic Distribution – – – – – – -36
2.2.4 Medicinal and Folkloric Uses – – – – – – – -36
CHAPTER THREE
3.0 Materials and Methods – – – – – – – -38
3.1 Materials – – – – – – – – – -38
3.1.1 Equipment/Instruments – – – – – – – -38
3.1.2 Chemicals and Reagents – – – – – – – -38
3.1.3 Consumables – – – – – – – – – -39
3.1.4 Glasswares – – – – – – – – – -39
3.1.5 Drugs – – – – – – – – – – -39
3.1.6 Animals – – – – – – – – – -39
3.2 Methods – – – – – – – – – -40
3.2.1 Collection and Identification of Plants – – – – – -40
3.2.2 Extraction of Selected Plant Material – – – – – – -40
3.2.3 Screening of the selected Plants – – – – – – -40
3.2.3.1 Introduction of Experimental Diabetes – – – – – -40
3.2.3.2 Screening of Extracts of the Seven Pilot Medicinal Plants for
antihyperglycaemic activity – – – – – – – -41
3.2.4 Acute toxicity effect of the Methanol leaf extract of
Newbouldia Laevis in rats – – – – – – – -42
3.2.5 Antidiabetic Study Using Newbouldia Laevis Extract (NLE) – – -42
3.2.5.1 Effect of NLE on normal glycaemic rats – – – – – -42
3.2.5.2 Effect of graded-doses of NLE on
Alloxan-Induced hyperglycaemia in rats – – – – – -43
3.2.5.3 Effect of NLE on Oral Glucose Tolerance Test (OGTT) in normal rats – -43
3.2.5.4 Effects of NLE on Oral glucose Tolerance Test (OGTT) in
Diabetic Rats – – – – – – – – – -43
3.2.6 Evaluation of the in vitro antioxidant activity of NLE – – – -44
3.2.6.1 1.1 Diphenyl-2-Picrilhydrazyl (DPPH) Photometric Assay – – – -44
3.2.6.2 Ferric Reducing Antioxidant Power (FRAP) Assay of NLE – – – -44
3.2.7 Effects of 21 days Oral Administration of NLE on Body
Weight, FBS, Serum Biochemistry, Haematology and Lipid,
Peroxidation in Alloxan-induced diabetic rats – – – – -45
3.2.7.1 Lipid profile Assay – – – – – – – – -46
3.2.7.1.1 Total Cholesterol – – – – – – – – -46
3.2.7.1.2 Triglycerides (TG) – – – – – – – – -46
3.2.7.1.3 High Density Lipids (HDL) – – – – – – -47
3.2.7.1.4. Low Density Lipids (LDL) – – – – – – – -48
3.2.7.1.5. Very Low Density Lipids (VLDL) – – – – – -48
3.2.7.2 Effects of NLE on Liver Markers Enzymes – – – – – -49
3.2.7.2.1 Serum Aspartate Aminotransferase (AST) – – – – – -49
3.2.7.2.2. Serum Alanine Aminotransferase (ALT) – – – – – -50
3.2.7.2.3. Serum Alkaline Phosphatase (ALP) – – – – – -50
3.2.7.2.4. Total Protein Estimation – – – – – – – -51
3.3.7.2.5. Total Bilirubin Estimation – – – – – – – -51
3.2.7.3 Effects of NLE on Kidney Functions – – – – – – -52
3.2.7.3.1. Estimation of serum Urea – – – – – – – -52
3.2.7.3.2. Estimation of Serum Creatinine – – – – – – -53
3.2.7.4 In vivo Antioxidant activities/lipid peroxidation – – – – -54
3.2.7.4.1 Malondialdehyde (MDA) – – – – – – – -54
3.2.7.4.2. Estimation of Superoxide Dismutase (SOD) – – – – -54
3.2.7.4.3 Catalase Determination – – – – – – – -54
3.2.7.5 Haematology – – – – – – – – – -55
3.2.7.5.1. Packed Cell Volume (PCV) Determination – – – – -55
3.2.7.5.2. Total White Blood Cell (WBC) Count – – – – – -55
3.2.7.5.3. Red Blood Cell (RBC) Count – – – – – – -56
3.2.7.5.4. Haemoglobin (Hb) Concentration Determination – – – – -56
3.2.7.5.5. Mean Corpuscular Volume (MCV) Determination – – – -56
3.2.7.5.6. Mean Corpuscular Haemoglobin (MCH) Determination. – – – -56
3.2.7.5.7. Mean Corpuscular Haemoglobin Concentration (MCHC) – – – -57
3.2.7.6 Effects of NLE on Erythrocytes Osmotic-Fragility in
alloxan-induced diabetic rats – – – – – – – -57
3.2.7.7. Histopathology – – – – – – – – -58
3.2.8 Phytochemical, Elemental and Vitamin Analysis of NLE – – – -58
3.2.8.1. Phytochemical Content – – – – – – – -58
3.2.8.2. Elemental Analysis – – – – – – – – -60
3.2.8.3. Vitamin Composition – – – – – – – -60
3.2.8.4. Proximate Analysis – – – – – – – – -61
3.2.9 Bioassay guided Fractionation/Determination of active
Compound – – – – – – – – – -63
3.2.9.1 Column Chromatography – – – – – – – -63
3.2.9.2 Packing of the glass Column – – – – – – – -63
3.2.9.3 Preparation and Elution of NLE – – – – – – -64
3.2.9.4 Selection of Solvent System for the Thin layer
Chromatography – – – – – – – – -66
3.2.9.5 Thin-layer Chromatography (TLC) – – – – – – -66
3.2.10 Bioassay Guided Isolation of Active Compound – – – – -67
3.2.10.1 Screening of fractions of NLE for bioactivity/
Selection of Active Compound- – – – – – – -67
3.2.10.2 Phytochemical Composition of Fractions – – – – – -68
3.2.11 Purification of the active Compound – – – – – – -68
3.2.11.1 Preparative Thin Layer Chromatography – – – – – -68
3.2.11.1.1 Preparation of Thin Layer Chromatographic Plates – – – -68
3.2.11.1.2 Purification of the Active Fraction – – – – – -68
3.2.11.1.3 Elution of the Separated Spot/zones – – – – – -69
3.2.12 Screening of the Active Compound – – – – – – -69
3.2.12.1 Determination of the Active Compound – – – – – -69
3.2.12.2 Thin Layer Chromatography of the Active Compound – – – -69
3.2.13 Characterization and Structural Elucidation of Pure Compound Using
Nuclear Magnetic Resonance and Mass spectroscopy – – – -70
3.2.14 Sub-chronic Toxicity Study- – – – – – – – -70
3.2.14.1 Preparation of Experimental Feed – – – – – – -70
3.2.14.2 Experimental Procedure – – – – – – – -71
3.2.14.3 Determination of Parameters – – – – – – -71
3.2.14.3. 1. Body Weights and Relative Organ Weights – – – – -71
3.2.14.3.2. Haematological Parameters – – – – – – -72
3.2.14.3.3. Lipid Profile Assays – – – – – – – -72
3.2.14.3.4. Liver Function Tests – – – – – – – -72
3.2.14.3.5. Kidney Function Tests – – – – – – – -72
3.2.14.3.6. Lipid Peroxidation/ In vivo Antioxidant Activities – – – -72
3.2.14.3.7. Histopathology – – – – – – – – -72
3.2.15 Effect of NLE on Insulin Secretion – – – – – – -73
3.3 Statistical Analysis – – – – – – – – -74
CHAPTER FOUR
4.0 Results – – – – – – – – – – -75
4.1 Screening of Pilot Plants for Antidiabetic Activity – – – – -75
4.2 Yield of Newbouldia Laeves Leaf Extract (NLE) – – – – -75
4.3 Acute Toxicity Test in rats – – – – – – – -75
4.4 Effect of NLE on Normal Glycaemia of rats – – – – – -77
4.5 Effect of Graded-Doses of NLE on FBS Level of
Alloxan- Induced Hyperglycaemic rats – – – – – -76
4.6.1 Effect of NLE Oral Glucose Tolerance (OGT) of
Normal Glycaemic rats – – – – – – – -80
4.6.2 Effect of NLE on Oral Glucose Tolerance (OGT)
of alloxan- Induced Diabetic rats – – – – – – -79
4.7 In vitro Antioxidant Activities of NLE – – – – – -83
4.7.1 1-1 Diphenyl – 2- – Picril hydrazyl (DPPH) Photometric Assay – – -83
4.7.2 Ferric Reducing Antioxidant Power (FRAP) Assay – – – – -83
4.8 Effect of Subacute Administration of NLE on Alloxan- Induced
Diabetic Rats for 21 Days – – – – – – – -83
4.8.1 Effect of NLE on Fasting Blood Sugar (FBS) – – – – -83
4.8.2 Effect of NLE on Mean Body Weight – – – – – -87
4.8.3 Effect of NLE on Lipid Profile of Alloxan Diabetic rats – – – -87
4.8.4 Effect of NLE on Liver Marker Enzymes of Alloxan- Induced Diabetic rats -90
4.8.5 Effect of NLE on Kidney Function of Alloxan -Induced Diabetic rats – -90
4.8.6 Effect of NLE on Lipid Peroxidation (In vivo antioxidant activity) in
Alloxan- Induced Diabetic rats – – – – – – -93
4.8.7 Effect of NLE on Haematological Parameters of Alloxan- Induced
Diabetic rats – – – – – – – – – -95
4.8.8 Effect of NLE on Erythrocyte Fragility in Alloxan- Induced Diabetic rats – -97
4.8.9 Histopathology – – – – – – – – -99
4.9 Phytochemical, Elemental, Vitamin and Proximate Analyses of NLE – -103
4.9.1 Phytochemical Analysis of NLE – – – – – – -103
4.9.2 Elemental/Mineral Composition of NLE – – – – – -103
4.9.3 Vitamin Analysis of NLE – – – – – – – -103
4.9.4 Proximate Analysis of NLE – – – – – – – -103
4.10 Bioassay Guided Fractionation of NLE and Isolation of Active Compound – -108
4.10.1 Column and Thin Layer Chromatography – – – – – -108
4.10.2 Screening of Fractions of NLE for Bioactivity/Selection of Active Fraction -108
4.10.3 Phytochemical Analysis of the Fractions – – – – – -108
4.11 Purification of Active Fraction/Isolation of Pure Compound – – – -112
4.12. Characterization and Structural Elucidation of active Fraction – – -112
4.13 Chronic Toxicity Result – – – – – – – -119
4.13.1 Clinical Observation – – – – – – – – -119
4.13.2 Body Weight Changes – – – – – – – -119
4.13.3 Relative Organ Weight (ROW) – – – – – – -119
4.13.3.1 Heart – – – – – – – – – – -119
413.3.2 Lungs – – – – – – – – – -121
413.3.3 Kidney – – – – – – – – – -121
413.3.4 Liver – – – – – – – – – – -121
4.13.4 Haematology – – – – – – – – – -126
4.13.4.1 Packed Cell Volume (PCV) – – – – – – – -126
4.13.4.2 Mean Haemoglobin (HB) Concentration – – – – – -126
4.13.4.3 Total Red Blood Cell (RBC) Count – – – – – – -126
4.13.4.4 Total White Blood Cell (WBC) Count – – – – – -127
4.13.4.5 Mean Corpuscular Volume (MCV) – – – – – – -127
4.13.4.6 Mean Corpuscular Haemoglobin (MCH) – – – – – -127
4.13.4.7 Mean Corpuscular Haemoglobin Concentration ( MCHC) – – – -127
4.13.5 Lipid Profile – – – – – – – – – -135
4.13.5.1 Total Cholesterol – – – – – – – – -135
4.13.5.2 Triglycerides – – – – – – – – -135
4.13.5.3 High Density Lipids (HDL) – – – – – – – -135
4.13.5.4 Low Density Lipids (LDL) – – – – – – – -135
4.13.5.5 Very Low Density Lipids (VLDL) – – – – – – -136
4.13.6 Liver Enzyme Markers – – – – – – – -142
4.13.6.1 Aspartate Aminotransferase (AST) – – – – – – -142
4.13.6.2 Alanine Aminotransferase (ALT) – – – – – – -142
4.13.6.3 Alkaline Phosphate (ALP) – – – – – – – -142
4.13.6.4 Total Protein – – – – – – – – -142
4.13.6.5 Total Bilirubin – – – – – – – – -143
4.13.6.6 Total Albumin – – – – – – – – -143
4.13.7 Kidney Fuction Test – – – – – – – – -150
4.13.7.1 Serum Urea – – – – – – – – – -150
4.13.7.2 Serum Creatinine – – – – – – – – -150
4.13.8 Lipid Peroxidation – – – – – – – – -153
4.13.8.1 Malondialdehyde (MDA) – – – – – – – -153
4.13.8.2 Catalase – – – – – – – – – -153
4.13.8.3 Superoxide Dismutase (SOD) – – – – – – -153
4.13.9 Effect of NLE on Insulin Secretion – – – – – – -157
4.13.10 Histopathology – – – – – – – – -159
CHAPTER FIVE
5.0 Discussion and Conclusion – – – – – – – -163
5.1 Discussion – – – – – – – – – -163
5.2 Conclusion – – – – – – – – – -178
REFERENCES – – – – – – – – -179
APPENDIXES – – – – – – – – -205
CHAPTER ONE
1.1 Introduction
The use of natural products by humans as medicine is as old as human civilization (Evans, 2009). Plants, animals and mineral products were the main sources of drugs (Pamplona-Rodgers, 2004). Farnsworth, (1994) defined ethnobotanic medicine as the use of plants by humans as medicine. According to Schneider, (2004), all civilizations took advantage of herbs in order to ease suffering and, many times, in order to cure diseases as well. Maybe the creator gave humans this vegetal world, with all its curative power, to make our life easier. Despite the great advances in modern medicine observed in recent decades, plants still make an important contribution to primary health care delivery system as an important source of drugs. Pamplona-Rogers (2004) stated that medicinal herbs, ignored during some ages and even dismissed in others, have been waiting quietly and patiently for several thousand years for humanity to turn their eyes to them in order to know, to study, to use and to love them.
The universal role of plants in the treatment of disease is exemplified by their employment in all the major branches of medicine irrespective of the underlying philosophical premise throughout history and continues to serve as the basis for many pharmaceuticals today. For example, the Western medicine with origins in Mesopotamia and Egypt, the Unani (Islamic) and Ayurvedic (Hindu) in Western Asia and the Indian subcontinent and those of orient in China, Japan, Tibet etc. How and when such medicinal plants were first used is in many cases lost in prehistory (Patwardhan, 2000; Kong et al, 2009; Evans, 2009; Wagner and Ulrich, 2009). Following the oral transmission of medicinal information came the use of writing. For example, the Egyptian Papyrus Ebers 1600 BC, baked clay tablets 650 BC from Ashurbanipal’s library at Nineve, now in British museum all refer to drugs well known today, Parchments and manuscript herbals after invention of printing in 1440 AD, pharmacopoeias and other works of reference (first London pharmacopoeia in 1618) and most recently electronic storage of data. Similar records exist for Chinese medicinal plants from the 4th century BC, Ayurvedic medicine (Ayurveda 2500-600 BC) and Unani medicine (Kitab-Al-Shifa, the magnum opus of Avicenna 980-1037 AD (Evans 2009). An extensive list of medicinal herbs and their virtues was compiled by the Greek physician Dioscorides in 70 AD. It was originally written in Greek and later translated into Latin as De Materia Medica and remained the authority in medicinal plants for a period of over 1500 years (Mendonca-filho, 2006). Indeed other animals apart from man appear to have their own material medica (Evans, 2009). Another Greek physician, Galen (AD 129-200), developed the pharmacopoeia which described the appearance, properties and use of many plants of his time. In addition to the above recorded information, there is a great wealth of knowledge concerning the medicinal, narcotic and other properties of plants that is still transmitted orally from generation to generation by tribal societies, particularly those of Africa, North and South America and the pacific countries. These are areas containing the world’s greatest number of plant species not found elsewhere, and with the westernization of so many of the people of these zones there is a pressing need to record local knowledge before it is lost forever.
Important drugs were discovered from plants in the course of time which sparked the interest in the study of plants as sources of drugs. Some of the important drugs include: quinine from Chinchona ledgeriana, vincristine and vinblastine from Catharanthus roseus, atropine and hyosoyamine from Atropa belladonna, morphine from Papaver somniferum, digitalis from Digitalis purpera, cocaine from Erythroxylon coca (Fabricant and Farnsworth, 2001; Pamplona-Rogers, 2004). Compounds such as yohimbine, physostigmine, nicotine, muscarine, colchicines, caffeine, emetine etc all obtained from plants are important substances used in pharmacological, physiological, biochemical and pathological studies (Williamson et al, 1996). Many drugs are derived from natural products as well. For example metformin, an important biguanide used in treating type 2 diabetes is derived from an active natural product galegine, a guanidine isolated from the plant Galega officinalis L. which was used in the medieval times to relieve the intense urination in diabetic people (Evans, 2009).
Despite the discovery of active natural products from higher plants, there was a disconnect in the 19th century between the historical link of plants and medicine, and the synthetic combinatorial chemistry and high throughput screening (HTS) of potential drug targets. The interest of Chemists, pharmaceutical scientists and pharmacologists turned towards the production of synthetic products or compounds and research was focused mainly on modification of natural products in an attempt to enhance biological activity, increase selectivity and decrease toxicity and side effects and above all, to increase the profit margins of pharmaceutical companies who feel it was cheaper getting a drug hit through this way (Burks, 2012).
Other reasons for these actions were that pure compounds were easily obtained from a pool of compounds available; structural modifications to obtain potentially more active and safer compounds could be easily performed with little or no cost. All these will ultimately enhance the profit margin and increase the economic powers of the pharmaceutical companies. On the other side, the use of natural products has been associated with magico-religious significance in the development of human cultures concerning health and disease (Sofowora, 2009). This association as against the beliefs of the industrialized western societies who owned most of these pharmaceutical companies and consider drugs from natural sources as options for the poorly educated or low income people or simply as religious superstition of no pharmacological value (Rates, 2001).
Over the past few decades, interest in drugs derived from higher plants has increased considerably. Pharmaceutical companies have once again turned their interest to natural products research especially those derived from higher plants (Phillipson, 2001; Pistorius and Oberer, 2012). It has been estimated that about 25% of all modern medicines prescribed worldwide are derived from higher plants and out of the 252 list of basic and essential drugs considered by the World Health Organization (WHO), 11% are exclusively of plant origin and a significant number is made up of synthetic drugs obtained from natural products (Farnsworth and Morris, 1976; De Smet, 1997; Shu, 1998). Some of the reasons that rekindle the interest in drugs of plant origin include the fact that synthetic drugs have many adverse effects; there is a phenomenal emergence of drug resistant microorganisms; there are emerging diseases with no drug available to treat them; a large population of the world cannot afford these conventional drugs (Petrovick, et al., 1997). Poverty and lack of access to modern drugs has forced about 80% of over 7 billion people of the world’s population living in developing countries to depend essentially on plants for their primary health care (Farnsworth et al, 1985; WHO, 2014). Till today, herbs are still found in 40 % of prescribed drugs in many developed countries of the world. (Smith and Winder, 1996; Andrede-cetto and Heinrich, 2005). There is also a worldwide green revolution which is reflected in the belief that herbal remedies are safer and less damaging to the human body than synthetic drugs (Williamson et al, 1996). They are readily available and cheaper (Gefu et al, 2000). They have advantage in toxicity considerations based on their long term use and their bioactive compounds are reported to have low animal and human toxicity (Fabricant and Farnsworth, 2001).
In view of the above facts, the World Health Organization (WHO) has recognized the role of herbal, traditional, alternative/complementary medicine in primary health care, especially in developing countries and has encouraged member nations to develop national policies for proper identification, sustainable exploitation, scientific development and appropriate utilization of herbal medicines appropriate for their situations, considering the fact that the use of medicinal plants is the most common form of traditional medicine world wide (WHO, 2005). World Health Organization (WHO) also considers phytotherapy in its health programmes and suggests basic procedures for the validation of drugs of plant origin (WHO, 1980; Vulto and Smeti, 1988). A variety of procedures have been adopted for the investigation of pharmacological properties of medicinal herb and those that are yet to be explored (Vogel, 2002; Verspohl, 2002).
Despite the long duration of herbal medical contribution to the control and treatment of diseases, only a few of the plants have been properly identified and documented (Sofowora, 2009). Out of the estimated 400,000 plant species on the earth surface only about 5% has been investigated scientifically for medical purposes (Rabo and Sanusi, 2001; Pamplona – Rogers, 2004). Hence there is serious need to assess all the discovered medicinal plants (Mendonca-Filho, 2006; Bnouham et al., 2006; Neelish et al., 2010). Herbal medicine will continue to grow in popularity and use as alternative or complementary to conventional medicine and has great potential to prevent, heal and cure many of the chronic diseases (Dharmananda, 1991; Evans, 2009; Singh, 2011). Controversial criticisms has trailed traditional medical practice due to lack of early documentation of research works for references as well as suspicion due to lack of knowledge of active ingredients in the concoctions that are used which may result in serious toxicity (Evans, 2009). Presently research is being carried out on the efficacy, toxicity and safety of some of the herbs used traditionally for treatment of diseases and information is beginning to emerge on Nigerian and African medicinal plants (Abdu-Aguye, 1977; Sofowora, 2009; Ezurike and Perieto, 2014). Traditional medicine therefore needs to be scientifically evaluated with a view to giving it due recognition and proper development in order to improve its safety, efficacy, availability and acceptability at minimal cost of production (Gefu et al., 2000; Sofowora, 2009).
Recently, the recognition of research on plants used in folk or herbal medicine as a suitable approach for the development of new drugs has led to an increase in the volume of research and publications in this area. Governmental and private institutions are now actively supporting such research programmes world wide (Calixto, 1996; Pistorius and Oberer, 2012). In 1993, the international programme of cooperation for biodiversity (IPCB) was formed to promote natural products in Latin America and Africa, with an objective to link universities, research institutes, industries and governments in a multi-disciplinary programme for the sustainable development and preservation of environment and its biodiversity. Also large pharmaceutical companies such as Merck, Ciba, Glaxo, Novartis among others have specific departments dedicated to the study of new drugs from natural sources (Rates, 2001; Pistorius and Oberer, 2012; Burks, 2012). Novartis particularly has been very active in Africa with centres in Ghana, Mali and Kenya. They train young African scientist in natural products and upgrade facilities in universities in developing countries (Burks, 2012)
In Africa, Latin America and Asia, people have a long standing history of using herbal drugs for treatment of various diseases. In some of these countries up to 90 % of the population depend exclusively on herbs for their primary healthcare (Hosteffman et al, 2004; Singh, 2011). Many of these plants have been documented and published. The Scientific Technical and Research Commission of the Organization of African Unity (OAU/STRC) a scientific arm of the OAU now African union (AU) has published an African Pharmacopia and carried out ethnobotanical surveys, in several African countries including Nigeria as at 2004 (Adjanohoun, 1991, 1993, 1996). In 1968, it organized a conference in Dakar Senegal which was dedicated to the efficacy of herbs used by traditional medical practitioners in areas of anticancer, anti-malarial antihelmintic, antimicrobial, antihypertensive, antisickling and antiviral (Sofowora, 2009). A summary of research as indicated by publications on African medicinal plant collated by Natural Products Alert (NAPRALERT) database for natural products indicated only 36 % of the publication dealt with bioassay–guided isolation of plant constituents along with their pharmacological and toxicological testing. The rest dealt with purely phytochimical research.
In Nigeria traditional medicine could be of great value than importing sophisticated and expensive drugs which cannot be afforded by many and sometimes inefficacious. Therefore this urgent need to find cheaper and pharmacologically active substances from natural products has redefined research on medicinal plants in many research institutions and universities. For instance at the National Institute for Pharmaceutical Research and Development (NIPRD) Abuja, traditional remedies such as dopravil® and Cornavil® for HIV/AIDS, Niprisan® for sickle cell anaemia and Nipradials® for diabetes management were standardized and are now registered by the National Agency for Food and Drug Administration (NAFDAC) for public use (Shingu, 2003). The Science and Technical Education Post Basic (STEP-B) a World Bank assisted project in Nigeria was introduced with the aim of encouraging research in science and technical education. The centre at the University of Nigeria Nsukka was coordinated by Prof. I. U. Asuzu and aimed at discovering active principles from medicinal plants against diabetes mellitus, malaria and trypanosomosis.
Newbouldia laevis (P. Beauv) Seeman ex Bureau also known as “Border” or “Boundary tree” belongs to the family Bignoniaciae and has a genus of one species (Keay, 1989). It is widely used in traditional medicine in Nigeria and other west African countries to treat fever, ear aches, chest pain, convulsion and epilepsy in children (Keay, 1989; Burkil, 1994; Tanko et al., 2008). The stem bark is used for treating skin infections while the roots are used to treat arthritis, general malady and diarrhoea (NNMDA, 2006; Owolabi et al., 2011). Its major phytochemicals include alkaloids, tannins and saponins (NNMDA, 2006). However, Tiv traditional medical practitioners in Central Nigeria use the leaves of Newbouldia laevis to treat disease conditions that result in frequent urination and sweet urine. The high prevalence rate of diabetes in the developing world and its attendant high cost on healthcare have necessitated search for cheaper, effective and readily available altrenatives. Hence the evaluation of the pharmacological effects, isolation and structural elucidation of the antidiabetic principle(s) of the methanol leaf extract of Newbouldia laevis in rats so as to authenticate the pharmacological basis for its folkloric use.
1.2 Statement of the Problem
- Most ethnomedical practices lack scientific procedures and their effects are sometimes over exaggerated or based on trial and error (Wambebe, 1999).
- World Health Organisation (WHO) has mandated the research in medicinal plants on diabetes mellitus especially in developing countries (WHO, 1980).
- Despite the high morbidity and mortality associated with diabetes mellitus, there are no effective remedies which can delay or limit the cause of disease progression.
- The available synthetic hypoglycemic agents used in clinical management of diabetes mellitus have serious adverse effects (Wadker, et al, 2008), therefore the search for more effective, safer, less expensive and readily available oral antidiabetic agents of plant origin is expedient.
- The synthetic drugs used presently are given for a long duration and have complicated mode of intake (Blum et al, 1986; Zimmet, 2007). There is no single agent that yields optimal glucose-lowering effect in all treated patients (Bozkurt et al, 2011; ADA, 2012).
1.3 Research Objective
- To screen some selected medicinal plants for antihyperglycaemic effect using rodents models.
- To evaluate the antidiabetic effects of Newbouldia laevis using normaglycaemic and alloxan-induced hyperglycaemic rats.
- To determine the toxicity and or safety of the plant extract using both acute and subchronic toxicity models.
- To evaluate the antioxidant potential of the extract both in vitro and in vivo.
- To analyse the phytochemical, proximate, elemental and vitamin composition of the leaves of Newbouldia leavis.
- To determine the effect of subacute and sub-chronic oral administration of the extract on blood glucose, haematology and serum biochemistry of the alloxan induced diabetic rats.
- To isolate, characterize and identify the active compound(s) responsible for the antidiabetic activity of the plant.
1.4 Justification of the Study
- Mainly to establish the pharmacological basis for the use of Newbouldia laevis leaves in the treatment of diabetes mellitus.
- Adequate knowledge about the ethnomedical properties of the plant will help in producing a cheap, safe efficacious and readily available antidiabetic drug.
- The finding of this work (the active pure compound) will be relevant in the pharmaceutical industry for drug development both for human and animal needs.
- The results from this study will also show the level of safety of the plant and provide basis for dose regimen for its use.
PHARMACOLOGICAL EFFECTS AND ANTIDIABETIC CONSTITUENTS OF THE METHANOL LEAF EXTRACT OF Newbouldia laevis (P. BEAUV) Seeman ex Bureau
