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

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received 0.5ml of leachate for 14 weeks, Group III (withdrawal) received 0.5ml of leachate for 11 weeks and withdrawn for 3 weeks, Group IV (L+EXTRACT) received 0.5ml of leachate for 11 weeks and 400mg/kg extract for 3 weeks, Lastly, Group V (EXTRACT ONLY) received 400mg/kg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 CONTENTTitle page                                                                                                                                 iCertification                                                                                                                             iiDedication                                                                                                                               iiiAcknowledgement                                                                                                                  iv                                                                                                                                  Table of contents                                                                                                                    v-ixList of Figures                                                                                                                         xList of Tablesxi-xiiAbstractxivCHAPTER ONE1.0       Introduction               1-31.1       Justification31.2       Aims31.3       Objectives4CHAPTER TWO2.0       Literature review52.1          Medicinal plants52.1.1       Croton zambesicus5-72.2          Antioxidants82.2.1       Flavonoids and phenolic acids8-92.2.1.1.   Gallic acid92.2.1.2   Luteolin92.2.1.3  Quercitin102.2.1.4   Caffeic acid  102.2.1.5   Apigenin10-112.2.2.     Tannins11-122.2.3      Polyphenols122.2.4      Saponins12-132.3.1      Elewi odo battery recycling site                                                                                132.4         Neurotoxicity13-152.4.1     Alzheimer’s disease15-162.5.1      Free radicals162.5.2      Reactive Oxygen Species16-172.5.3      Oxidative stress            17-192.5.3.1   Chemical and Biological effects of oxidative stress           192.5.4     Oxidative stress and neurotoxicity            202.6.1     Endogenous antioxidants202.6.2     Antioxidant enzymes20-212.7         Lipid peroxidation21-232.7.1      Malondialdehyde232.7.1.1   Structure and Synthesis23-242.7.1.2    Metabolism of Malondialdehyde242.8         Acetylcholine, Butyrylcholine and Monoamine oxidase25-262.9         Lactate dehydrogenase and 5’Nucleotidase26CHAPTER THREE3.0         Materials and Methods273.1         Materials273.1.1      Plant Collection273.1.2      Experimental Animal273.1.3      Collection of the Battery recycling site leachate 273.1.4      Chemicals and Reagents27-283.2         Methods283.2.1      Preparation of phenolic extract283.2.2      Method of HPLC-DAD28-293.2.3     LOD and LOQ293.2.4     Animal exposure to EBRSL29-303.2.5     Preparation of the synaptosomal fraction of the brain303.3        In-Vivo analysis303.3.1     Estimation of Reduced Glutathione level30-323.3.2     Assessment of Lipid peroxidation32-343.3.3     Determination of Catalase activity34-363.3 Determination of Tissue Lactate dehydrogenase363.3.5     Determination of Superoxide dismutase activity36-383.3.6     Estimation of Glutathione-S- transferase level38-403.3.7     Neuronal 5’ Nucleotidase40-433.3.8     Protein determination44-453.3.9     Acetylcholinesterase inhibition45-463.3.10   Butyrylcholinesterase inhibition46-473.3.11    Monoamine Oxidase47-483.4        Statistical Analysis48CHAPTER FOUR4.0       Results and discussion494.1Results494.1.2Histopathology654.2Discussion68-704.3Conclusion70References71-81Appendix82-91





LIST OF FIGURESFigure 2.1:  Picture showing Croton zambesicus7Figure 2.2:  Structure Showing Oxidative stress and cellular responses18Figure 2.3:  Structure of malondialdehyde 23Figure 4.1: HPLC Profile of extracts51Figure 4.2: Catalase activity54Figure 4.3: Reduced glutathione level55Figure 4.4 MDA level56Figure 4.5: Superoxide dismutase activity57Figure 4.6a : Mono-amine oxidase activity for PMF                                    58Figure 4.6b: Mono-amine oxidase activity for synaptosomes       59Figure 4.7: Lactate dehydrogenase activity60Figure 4.8: Acetyl cholinesterase activity61Figure 4.9: Butryl cholinesterase activity62Figure 4.10: Neuronal-5’-Nucleotidase63Figure 4.11: Glutathione -S-tranferase activity64Figure 4.12: Histopathology; Control65Figure 4.13: Histopathology; Leachate-nonwithdrawal65Figure 4.14: Histopathology; Leachate-withdrawal66Figure 4.15: Histopathology; Leachate + Extract66Figure 4.16: Histopathology; Extract only67












LIST OF TABLESTable 1:  Characterization of organic pollutants in EBRSL49Table 2: Quantitative phytochemical screening of Croton zambesicus50Table 3:  Component of extract52Table 4: Effect of Croton zambesicus on weight of animals53Table 5: Protocol for the preparation of GSH standard curve83Table 6: Protocol for the preparation protein standard curve84Table 7: Protocol for the preparation of catalase standard curve85Table 8: Lactate dehydrogenase activity86Table 9: Reduced glutathione activity86Table 10: Glutathione- S- transferase activity87Table 11: Superoxide dismutase activity87Table 12: Catalase activity88Table 13: Lipid peroxidation level88Table 14: Neuronal- 51- nucleotidase activity89Table 15: Acetyl cholinesterase activity89Table 16: Butryl cholinesterase activity90Table 17a: Mono-amine oxidase activity for PMF90Table 17b: Mono-amine oxidase activity for synaptosomes91