Effect Of Ethanol Extract Of Euphorbia Kamerunicus On Potassium Bromate-Induced Toxicity In Albino Rats

ABSTRACT

This study investigated the modulatory effect of ethanol extract of Euphorbia kamerunicus on potassium bromate-induced toxicity in Albino rats. The crude ethanol extract was subjected to phytochemical and, GC-MS analyses and in vitro antioxidant and acute toxicity evaluations. Results obtained following phytochemical studies of the crude extract revealed the presence of alkaloids, saponins, tannins, terpenes, flavonoids, phenols, cardiac glycosides and steroids in various amounts. Alkaloids were the most abundant (21.63±0.15%) while cardiac glycosides were the least (4.60±0.26%). GC-MS analysis revealed the presence of 33 compounds in the crude extract with oleic acid as the most abundant (12.54%) and 2-Propenoic acid the least (0.20%). Other compounds with high abundance in the crude extract were 11-Octadecenoic acid (8.69%), Butyl 9-tetradecenoate (8.63%), n-Decanoic acid (6.98%), 1,3-Dioxolane (9.31%), 6-Octadecenoic acid (7.17%), Methyl stearate (4.27%), 2-Trifluoroacetoxypentadecane (4.54%) and Hexadecanoic acid (3.74%). The crude extract was also fractionated into five fractions. Investigation of the effects of the extract in potassium bromate-induced toxicity was carried out in two phases, first on the crude and then on the fractions. For the crude extract, 30 rats randomly assigned to 6 groups of five rats were used. The rats were treated according to the order: group 1 (normal control with no treatment), group 2 (potassium bromate, 100 mg/kg only), group 3 (200 mg/kg of crude extract + potassium bromate, 100 mg/kg), group 4 (400 mg/kg of crude extract + potassium bromate, 100 mg/kg), group 5 (800 mg/kg of crude extract + potassium bromate, 100 mg/kg) and group 6 (100 mg/kg vitamin C + potassium bromate, 100 mg/kg). Treatment lasted 28 days before animals were sacrificed for haematological and biochemical analyses. In the second phase using the fractions, treatments for groups 1-3 were repeated but groups 4-8 were treated with extract fractions 1-5 for 28 days before sacrifice and analyses of collected samples. Results of in vitro antioxidant activities showed significant nitric oxide and DPPH scavenging activities and mild ferric reducing antioxidant power activity. Acute toxicity value obtained for potassium bromate in rats was 346.41 mg/kg body weight while that of the crude extract was greater than 5000 mg/kg. Results of liver function parameters showed significantly higher AST, ALT and ALP activities in the group administered only potassium bromate when compared with those co-treated with the extract. The crude extract also significantly inhibited anomalies observed in total protein and serum bilirubin values due to potassium bromate intoxication. Higher levels of urea, uric acid and creatinine due to potassium bromate were also significantly lowered in the extract treated groups (p<0.05). Lipid profile values were not significantly altered following treatment with potassium bromate and treatment with the extract (p>0.05), but antioxidant parameters including GSH, GPx, SOD and catalase significantly depreciated with concurrent rise in the bromate only group but ameliorated in the groups treated with the extract (p<0.05). The fall in the values of haematological parameters were also significantly up regulated in the crude-extract-treated groups. Elevated values of cardiac parameters (lactate dehydrogenase, creatine phosphokinase and cardiac troponin) due to bromate intoxication were  significantly lowered in groups treated with the crude extract and fractions (p<0.05). Inflammatory markers (interleukin 1b, prostaglandin E2 and tumor necrosis factor) were also lowered significantly (P<0.05). Of all the fractions evaluated, fraction 4 had the higher activities than the other fractions and produced effects similar to that of the crude extract. Cactus plant extract may be of value in the management of potassium bromate-induced systemic toxicity and could be a potential source of control agent for oxidative stress-induced diseases caused by environmental oxidants.

TABLE OF CONTENTS

 

Title Page                                                                                                        i

Declaration                                                                                                      ii

Certification                                                                                                    iii

Dedication                                                                                                      iv

Acknowledgements                                                                                        v

Table of Contents                                                                                           vi

List of Tables                                                                                                  xii

List of Figures                                                                                                 xiv

Abstract                                                                                                          xv       

CHAPTER ONE

INTRODUCTION                                                                                        1

1.1              Background of the study                                                                    1

1.2              Statement of the problem                                                                   4

1.3              Justification for the study                                                                   6

1.4              Aim of the study                                                                                 6

 

1.5              Objectives of the study                                                                       6

CHAPTER TWO

LITERATURE REVIEW                                                                           8

2.1       Potassium bromate: an overview                                                        8

2.1.1    Physicochemical properties                                                                 9

2.1.2    Uses and action                                                                                   10

2.1.2.1 Toxicity and Safety                                                                            11

2.1.3.   Potassium bromated in bread making                                                 16

2.2       Systemic Targets For Potassium Bromate Induced Toxicity              19

2.2.1    Potassium bromate nephrotoxicity                                                      20

2.2.1.1 Pathophysiologic effect of KBrO₃ in the Liver                                  23

2.2.2    Potassium bromate induced-oxidative stress                                      24

2.2.3    Potassium bromate-induced hematological alterations                       26       

2.2.4    Potassium bromate-induced cardiac hypertrophy                               27

2.2.5    Pathophysiologic effect of KBrO₃ in Other Tissues                           27

2.3       Mechanism Action of Potassium Bromate Toxicity                           29

2.4       Effects of Plant-Derived Antioxidants                                              31

2.4.1    Antioxidants                                                                                       33

2.4.2    Potassium Bromate and Inflammatory Bowel Disease                      33

2.5       Biology of Euphorbia kamerunicus                                                    35

2.5.1    Medicinal benefits                                                                              36

CHAPTER THREE

MATERIALS AND METHODS                                                                37

3.1    Materials (Equipment and Reagents)                                                     37

3.2.   Collection and Drying of Plant Materials                                             37

3.2.1 Preparation of Extract                                                                            38

3.3.   Qualitative Phytochemical Study of the Extract                                   38

3.4.   Quantitative Phytochemical Tests                                                         40

3.5.   In Vitro Antioxidant Evaluation of the Extract                                                44

3.6.   Determination of Phytochemical Composition of crude cactus

  Extract by Gas Chromatography – Mass Spectrometry (Gc-Ms)        46

3.7.   Animals                                                                                                 48

3.8    Acute Toxicity Studies                                                                          48

3.8.1 Acute Toxicity Evaluation of Potassium Bromate                                49

3.8.2 Acute Toxicity Evaluation of Cactus Plant Extract                              49

3.9. Experimental Design                                                                                50

3.10. Assessment of Haematological Parameters                                           51

3.11. Assessment of Liver Function Parameters                                             52       

3.12 Assessment of Renal Function Parameters                                             56

3.13  Lipid Profile Parameters                                                                        60

3.14  Assessment of Antioxidant Parameters in Liver Tissues                       63

3.15. Estimation of cardiac parameters (LDH, CPK and cardiac

         troponin)                                                                                                            66

 

3.16. Estimation of inflammatory cytokines (tumor necrotic

         factor and interleukin-1b)                                                                      70

3.17  Liver And Kidney Histopathology                                                        74

3.18  Bioassay-Guided Fractionation of Cactus Plant Extract                      

        Using Chromatographic Techniques                                                       75

3.19 Statistical analysis                                                                                   80

CHAPTER FOUR

RESULTS                                                                                                     82

4.1       Results of Phytochemical Evaluation                                                 82

4.1.1    Qualitative phytochemical result                                                        82

4.1.2    Quantitative phytochemical result                                                      84

4.2       Result of Gc-Ms Analysis of Cactus Plant Crude Extract                 86

4.3       Antioxidant Activity Assay                                                                90

4.3.1    DPPH free radical scavenging activity                                               90

4.3.2    Ferric reducing antioxidant potential (FRAP) assay                          92

4.3.3    Nitric oxide (NO) radical scavenging activity                                    94

4.4       Results of Acute Toxicity Evaluation                                                 96

4.4.1    Result of acute toxicity evaluation of potassium bromate                  96       

4.4.2    Acute toxicity report of cactus plant extract                                      98

4.5       Liver Function Profile                                                                         100

4.6       Serum Renal Profile                                                                            103

4.7       Serum Lipid Profile Result                                                                 107

4.8       Antioxidant Assay                                                                              109

4.9       Haematological Profile                                                                       112

4.10     Evaluation of Fractions for Cactus                                                     115

4.10.1 Effects of fractions on haematological parameters                              115

4.10.2  Effects of fractions on liver function parameters                               119

4.10.3  Effects of fractions on renal function parameters                              122

4.10.4  Effects of fractions on lipid profile parameters                                  125

4.10.5  Effects of fractions on antioxidation  enzymes                                  130

4.10.6  Effects of fractions on cardiac biomarkers                                         134

4.10.7  Effects of fractions on carcinoma biomarkers                                    136

CHAPTER FIVE

DISCUSSION                                                                                               140

5.1       Phytochemicals                                                                                   140

5.2       Antioxidant Potential                                                                         142

5.3       Liver Function Profile                                                                         145

5.4       Lipid Profile                                                                                        147

5.5       Renal Function                                                                                    149

5.6       Hematological Evaluation                                                                   151

5.7       Acute Toxicity                                                                                    153

5.8       Antioxidants                                                                                       154

5.9       Cardiac Markers                                                                                  156

5.91     Evaluation of Fractions                                                                       157

5.92     Inflammatory Markers                                                                        158

5.93     Conclusion                                                                                          160

5.94     Recommendations                                                                              160

5.95     Suggestion for Further Study                                                             161

References                                                                                                     162

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES

 

Table 4.1a: Qualitative phytochemical components of ethanol extract

       of cactus                                                                                       83

 

Table 4.1b: Qualitative phytochemical components of ethanol extract

                   of cactus                                                                                       85

 

Table 4.2: GC-MS crude extract                                                                     88

Table 4.3a: Acute toxicity evaluation of potassium bromate phase 1 97

Table 4.3b: Acute toxicity evaluation of potassium bromate phase 2            97

Table 4.4a: Acute toxicity evaluation of ethanol extract of cactus phase 1   99

           

Table 4.4b: Acute toxicity evaluation of ethanol extract of cactus phase 2   99

 

Table 4.5: Effects of the ethanol extract of cactus on

                 some serum biochemical parameters treated exposed

                 to potassium bromate                                                                     102

Table 4.6: Effects of the ethanol extracts of cactus  on plasma urea,                                       creatinine, uric acid and electrolyte levels in potassium bromate                                      intoxicated Rats.                                                                             105

 

Table 4.7: Effects of the ethanol extract of cactus on some lipid

              profile of potassium bromate intoxicated Rats.                                108

 

Table 4.8: Effects of ethanol extract of cactus  on protective/antioxidant     parameters.                                                                                          111

Table 4.9: The effects of the extract cactus  on some

    haematological parameters induced with potassium bromate

     intoxicated Rats.                                                                            114

 

Table 4.10: Effect of 5 different fractions and crude extract of cactus

             on concentration of some haematological indices.                            117

 

Table 4.11: Effects of five different fractions and crude extract of cactus on          concentration of selected liver function parameters.                           120

 

Table 4.12: Effects of five different fractions and crude extract of

            cactus on selected renal function parameters.                                     125

 

 

Table 4.13: Effect of 5 different fractions and crude extract of cactus 

            on selected lipid parameters.                                                               128

 

Table 4.14: Effects of five different fractions and crude extract of cactus

            on redox status of potassium bromate intoxicated Rats                     132

 

Table 4.15: Effects of five different fractions and crude extract of cactus

            on some cardiac biomarkers of potassium bromate intoxicated Rats.136

 

Table 4. 16: Effects of five different fractions and crude extract of cactus

            plant on the concentration levels some carcinoma biomarkers exposed

            to potassium bromate.                                                                         139

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

Figure 2.1 Structure of the Potassium Bromate Molecule                              10       

Figure  4.1: GC-MS chromatogram showing 33 compounds in cactus                     

     plant crude extract.                                                                                    87

Figure 4.2a: DPPH free radical scavenging antioxidant activity.                   91

Figure 4.2b: Ferric reducing antioxidant power (FRAP) assay.                     93

Figure 4.2c: Nitric oxide radical scavenging activity.                                    95