Exposure to environmental toxicant was known to cause neuronal disorders in humans. Hence, the present study evaluates the toxic effect of leachate (an environmental toxicant) in the synaptosomes of female rats and the reversal effect of phenolic-rich fraction from Croton zambesicus. Fifty animals were divided into five groups. Group I (Control) received 0.5ml of distilled water only, Group II (non-withdrawal) received 0.5ml of leachate for 14 weeks, Group III (withdrawal) received 0.5ml of leachate for 11 weeks and withdrawn for 3 weeks, Group IV (L+EXTRACT) received 0.5ml of leachate for 11 weeks and 400mg/kg extract for 3 weeks, Lastly, Group V (EXTRACT ONLY) received 400mg/kg extract only for 3 weeks. The experiment lasted for 14 weeks. Both non-withdrawal and withdrawal exposure of animals to leachate caused oxidative and neuronal damage. This study suggests that battery recycling site leachate elicits damage in female rats’ synaptosomes by increasing the malondialdehyde level, and decreasing Reduced glutathione level. The activities of catalase, superoxide dismutase, Lactate dehydrogenase and other enzymatic antioxidants were decreased, also the activities of aminerging catabolizing enzymes i.e Acetylcholinesterase, Butyrylcholinesterase and Monoamine oxidase were also elevated. The phenolic-rich fraction from Croton zambesicus significantly reversed the toxicity. The preventive and protective effect of phenolic compounds (Gallic acid, Caffeic acid, Quercetin, Luteolin and Apigenin) from phenolic-rich fraction from Croton zambesicus validates that they have therapeutic application in neuronal and oxidative damage of the brain.
TABLE OF CONTENT
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of contents v-ix
List of Figures x
List of Tables xi-xii
Abstract xiv
CHAPTER ONE
1.0 Introduction 1-3
1.1 Justification 3
1.2 Aims 3
1.3 Objectives 4
CHAPTER TWO
2.0 Literature review 5
2.1 Medicinal plants 5
2.1.1 Croton zambesicus 5-7
2.2 Antioxidants 8
2.2.1 Flavonoids and phenolic acids 8-9
2.2.1.1. Gallic acid 9
2.2.1.2 Luteolin 9
2.2.1.3 Quercitin 10
2.2.1.4 Caffeic acid 10
2.2.1.5 Apigenin 10-11
2.2.2. Tannins 11-12
2.2.3 Polyphenols 12
2.2.4 Saponins 12-13
2.3.1 Elewi odo battery recycling site 13
2.4 Neurotoxicity 13-15
2.4.1 Alzheimer’s disease 15-16
2.5.1 Free radicals 16
2.5.2 Reactive Oxygen Species 16-17
2.5.3 Oxidative stress 17-19
2.5.3.1 Chemical and Biological effects of oxidative stress 19
2.5.4 Oxidative stress and neurotoxicity 20
2.6.1 Endogenous antioxidants 20
2.6.2 Antioxidant enzymes 20-21
2.7 Lipid peroxidation 21-23
2.7.1 Malondialdehyde 23
2.7.1.1 Structure and Synthesis 23-24
2.7.1.2 Metabolism of Malondialdehyde 24
2.8 Acetylcholine, Butyrylcholine and Monoamine oxidase 25-26
2.9 Lactate dehydrogenase and 5’Nucleotidase 26
CHAPTER THREE
3.0 Materials and Methods 27
3.1 Materials 27
3.1.1 Plant Collection 27
3.1.2 Experimental Animal 27
3.1.3 Collection of the Battery recycling site leachate 27
3.1.4 Chemicals and Reagents 27-28
3.2 Methods 28
3.2.1 Preparation of phenolic extract 28
3.2.2 Method of HPLC-DAD 28-29
3.2.3 LOD and LOQ 29
3.2.4 Animal exposure to EBRSL 29-30
3.2.5 Preparation of the synaptosomal fraction of the brain 30
3.3 In-Vivo analysis 30
3.3.1 Estimation of Reduced Glutathione level 30-32
3.3.2 Assessment of Lipid peroxidation 32-34
3.3.3 Determination of Catalase activity 34-36
3.3 Determination of Tissue Lactate dehydrogenase 36
3.3.5 Determination of Superoxide dismutase activity 36-38
3.3.6 Estimation of Glutathione-S- transferase level 38-40
3.3.7 Neuronal 5’ Nucleotidase 40-43
3.3.8 Protein determination 44-45
3.3.9 Acetylcholinesterase inhibition 45-46
3.3.10 Butyrylcholinesterase inhibition 46-47
3.3.11 Monoamine Oxidase 47-48
3.4 Statistical Analysis 48
CHAPTER FOUR
4.0 Results and discussion 49
4.1 Results 49
4.1.2 Histopathology 65
4.2 Discussion 68-70
4.3 Conclusion 70
References 71-81
Appendix 82-91
LIST OF FIGURES
Figure 2.1: Picture showing Croton zambesicus 7
Figure 2.2: Structure Showing Oxidative stress and cellular responses 18
Figure 2.3: Structure of malondialdehyde 23
Figure 4.1: HPLC Profile of extracts 51
Figure 4.2: Catalase activity 54
Figure 4.3: Reduced glutathione level 55
Figure 4.4 MDA level 56
Figure 4.5: Superoxide dismutase activity 57
Figure 4.6a : Mono-amine oxidase activity for PMF 58
Figure 4.6b: Mono-amine oxidase activity for synaptosomes 59
Figure 4.7: Lactate dehydrogenase activity 60
Figure 4.8: Acetyl cholinesterase activity 61
Figure 4.9: Butryl cholinesterase activity 62
Figure 4.10: Neuronal-5’-Nucleotidase 63
Figure 4.11: Glutathione -S-tranferase activity 64
Figure 4.12: Histopathology; Control 65
Figure 4.13: Histopathology; Leachate-nonwithdrawal 65
Figure 4.14: Histopathology; Leachate-withdrawal 66
Figure 4.15: Histopathology; Leachate + Extract 66
Figure 4.16: Histopathology; Extract only 67
LIST OF TABLES
Table 1: Characterization of organic pollutants in EBRSL 49
Table 2: Quantitative phytochemical screening of Croton zambesicus 50
Table 3: Component of extract 52
Table 4: Effect of Croton zambesicus on weight of animals 53
Table 5: Protocol for the preparation of GSH standard curve 83
Table 6: Protocol for the preparation protein standard curve 84
Table 7: Protocol for the preparation of catalase standard curve 85
Table 8: Lactate dehydrogenase activity 86
Table 9: Reduced glutathione activity 86
Table 10: Glutathione- S- transferase activity 87
Table 11: Superoxide dismutase activity 87
Table 12: Catalase activity 88
Table 13: Lipid peroxidation level 88
Table 14: Neuronal- 51- nucleotidase activity 89
Table 15: Acetyl cholinesterase activity 89
Table 16: Butryl cholinesterase activity 90
Table 17a: Mono-amine oxidase activity for PMF 90
Table 17b: Mono-amine oxidase activity for synaptosomes 91
