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received 0.5ml of leachate for 14 weeks, Group III (withdrawal) received 0.5ml of leachate for 11 weeks and withdrawn for 3 weeks, Group IV (L+EXTRACT) received 0.5ml of leachate for 11 weeks and 400mg/kg extract for 3 weeks, Lastly, Group V (EXTRACT ONLY) received 400mg/kg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 Tablesxi-xii
Abstractxiv
CHAPTER ONE
1.0 Introduction 1-3
1.1 Justification3
1.2 Aims3
1.3 Objectives4
CHAPTER TWO
2.0 Literature review5
2.1 Medicinal plants5
2.1.1 Croton zambesicus5-7
2.2 Antioxidants8
2.2.1 Flavonoids and phenolic acids8-9
2.2.1.1. Gallic acid9
2.2.1.2 Luteolin9
2.2.1.3 Quercitin10
2.2.1.4 Caffeic acid 10
2.2.1.5 Apigenin10-11
2.2.2. Tannins11-12
2.2.3 Polyphenols12
2.2.4 Saponins12-13
2.3.1 Elewi odo battery recycling site 13
2.4 Neurotoxicity13-15
2.4.1 Alzheimer’s disease15-16
2.5.1 Free radicals16
2.5.2 Reactive Oxygen Species16-17
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 antioxidants20
2.6.2 Antioxidant enzymes20-21
2.7 Lipid peroxidation21-23
2.7.1 Malondialdehyde23
2.7.1.1 Structure and Synthesis23-24
2.7.1.2 Metabolism of Malondialdehyde24
2.8 Acetylcholine, Butyrylcholine and Monoamine oxidase25-26
2.9 Lactate dehydrogenase and 5’Nucleotidase26
CHAPTER THREE
3.0 Materials and Methods27
3.1 Materials27
3.1.1 Plant Collection27
3.1.2 Experimental Animal27
3.1.3 Collection of the Battery recycling site leachate 27
3.1.4 Chemicals and Reagents27-28
3.2 Methods28
3.2.1 Preparation of phenolic extract28
3.2.2 Method of HPLC-DAD28-29
3.2.3 LOD and LOQ29
3.2.4 Animal exposure to EBRSL29-30
3.2.5 Preparation of the synaptosomal fraction of the brain30
3.3 In-Vivo analysis30
3.3.1 Estimation of Reduced Glutathione level30-32
3.3.2 Assessment of Lipid peroxidation32-34
3.3.3 Determination of Catalase activity34-36
3.3 Determination of Tissue Lactate dehydrogenase36
3.3.5 Determination of Superoxide dismutase activity36-38
3.3.6 Estimation of Glutathione-S- transferase level38-40
3.3.7 Neuronal 5’ Nucleotidase40-43
3.3.8 Protein determination44-45
3.3.9 Acetylcholinesterase inhibition45-46
3.3.10 Butyrylcholinesterase inhibition46-47
3.3.11 Monoamine Oxidase47-48
3.4 Statistical Analysis48
CHAPTER FOUR
4.0 Results and discussion49
4.1Results49
4.1.2Histopathology65
4.2Discussion68-70
4.3Conclusion70
References71-81
Appendix82-91
LIST OF FIGURES
Figure 2.1: Picture showing Croton zambesicus7
Figure 2.2: Structure Showing Oxidative stress and cellular responses18
Figure 2.3: Structure of malondialdehyde 23
Figure 4.1: HPLC Profile of extracts51
Figure 4.2: Catalase activity54
Figure 4.3: Reduced glutathione level55
Figure 4.4 MDA level56
Figure 4.5: Superoxide dismutase activity57
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 activity60
Figure 4.8: Acetyl cholinesterase activity61
Figure 4.9: Butryl cholinesterase activity62
Figure 4.10: Neuronal-5’-Nucleotidase63
Figure 4.11: Glutathione -S-tranferase activity64
Figure 4.12: Histopathology; Control65
Figure 4.13: Histopathology; Leachate-nonwithdrawal65
Figure 4.14: Histopathology; Leachate-withdrawal66
Figure 4.15: Histopathology; Leachate + Extract66
Figure 4.16: Histopathology; Extract only67
LIST OF TABLES
Table 1: Characterization of organic pollutants in EBRSL49
Table 2: Quantitative phytochemical screening of Croton zambesicus50
Table 3: Component of extract52
Table 4: Effect of Croton zambesicus on weight of animals53
Table 5: Protocol for the preparation of GSH standard curve83
Table 6: Protocol for the preparation protein standard curve84
Table 7: Protocol for the preparation of catalase standard curve85
Table 8: Lactate dehydrogenase activity86
Table 9: Reduced glutathione activity86
Table 10: Glutathione- S- transferase activity87
Table 11: Superoxide dismutase activity87
Table 12: Catalase activity88
Table 13: Lipid peroxidation level88
Table 14: Neuronal- 51- nucleotidase activity89
Table 15: Acetyl cholinesterase activity89
Table 16: Butryl cholinesterase activity90
Table 17a: Mono-amine oxidase activity for PMF90
Table 17b: Mono-amine oxidase activity for synaptosomes91
