Neuroprotective Effect of Croton Zambesicus Phenolic Extract on Synaptosomes of Rats Exposed to Environmental Toxicant

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ABSTRACT

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received 0.5ml of leachate for 14 weeks, Group III (withdrawal) received 0.5ml of leachate for 11 weeks and withdrawn for 3 weeks, Group IV (L+EXTRACT) received 0.5ml of leachate for 11 weeks and 400mg/kg extract for 3 weeks, Lastly, Group V (EXTRACT ONLY) received 400mg/kg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

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Neuroprotective Effect of Croton Zambesicus Phenolic Extract on Synaptosomes of Rats Exposed to Environmental Toxicant

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