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Discovery of Potential Neuroprotective Compounds from Marine Sponges and Algae

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Discovery of Potential Neuroprotective Compounds from Marine Sponges and Algae Discovery of potential neuroprotective compounds from marine sponges and algae by Mousa Alghazwi Thesis Submitted to Flinders University for the degree of Doctor of philosophy College of Medicine and Public Health 25th of July, 2018 Contents LIST OF FIGURES ...................................................................................................... vi LIST OF TABLES ..................................................................................................... viii SUMMARY .................................................................................................................. ix DECLARATION .......................................................................................................... xi ACKNOWLEDGEMENTS ......................................................................................... xii LIST OF PUBLICATIONS ........................................................................................ xiv 1. CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ...................... 1 1.1 Neurodegenerative diseases ............................................................................ 2 1.1.1 Introduction to Alzheimer’s disease (AD) ............................................... 2 1.1.2 Diagnosis of AD ...................................................................................... 4 1.1.3 Risk Factors for AD ................................................................................. 6 1.1.4 Current issue of treating AD .................................................................... 6 1.1.5 Beta Amyloid: features, folding and neurotoxicity .................................. 7 1.2 Current treatment for AD ................................................................................ 9 1.3 Natural products: experimental treatments with clinical potential................ 10 1.3.1 (−)-epigallocatechin-3-gallate (EGCG) ................................................. 11 1.3.2 Curcumin................................................................................................ 12 1.4 Marine sources of natural compounds for AD treatment and prevention ..... 13 1.4.1 Marine macroalgae................................................................................. 14 1.4.2 Marine sponges ...................................................................................... 15 1.5 Prospective Therapy ...................................................................................... 16 1.6 Hypotheses .................................................................................................... 17 1.7 Aims and scope of this thesis ........................................................................ 17 1.8 Significance ................................................................................................... 19 1.9 References ..................................................................................................... 20 2. CHAPTER 2: IN VITRO PROTECTIVE ACTIVITY OF SOUTH AUSTRALIAN MARINE SPONGE AND MACROALGAE EXTRACTS AGAINST AMYLOID BETA (AΒ1-42) INDUCED NEUROTOXICITY IN PC-12 CELLS ....... 28 2.1 Introduction ................................................................................................... 29 2.2 Materials and methods .................................................................................. 31 2.2.1 Samples collection ................................................................................. 31 2.2.2 Extract preparation ................................................................................. 36 2.2.3 Sample Preparation for the Queensland Compound Library (QCL) /Compounds Australia ......................................................................................... 36 2.2.4 Preparation of sponge and macroalgae extracts samples for biological assays 36 i 2.2.5 Reagents ................................................................................................. 36 2.2.6 Cell lines and cell culture ....................................................................... 37 2.2.7 Cell viability assessment using MTT assay ........................................... 37 2.2.8 Preparing Aβ for treating cells ............................................................... 38 2.2.9 Neuroprotection study ............................................................................ 38 2.2.10 Statistical analysis .................................................................................. 38 2.3 Results ........................................................................................................... 39 2.3.1 Cytotoxicity of extracts of marine sponges and macroalgae from South Australia ............................................................................................................... 39 2.3.2 Neuroprotective activity of extracts of marine sponges and macroalgae from South Australia ............................................................................................ 43 2.4 Discussion ..................................................................................................... 47 2.5 Conclusion ..................................................................................................... 53 2.6 References ..................................................................................................... 54 3. CHAPTER 3: IN SILICO SCREENING OF NEUROPROTECTIVE COMPOUNDS REPORTED FROM MARINE SPONGE AND ALGAE AGAINST AMYLOID BETA (AΒ) AGGREGATION ................................................................ 59 3.1 Introduction ................................................................................................... 60 3.2 Materials and methods .................................................................................. 61 3.2.1 Compounds selected from marine sponges and macroalgae ................. 61 3.2.2 Ligand preparation ................................................................................. 62 3.2.3 Molecular docking ................................................................................. 62 3.2.4 Screening for Drug-likeness .................................................................. 62 3.3 Results and discussion ................................................................................... 64 3.3.1 Molecular docking results ...................................................................... 64 3.3.2 Drug likeness and likelihood of BBB penetration ................................. 96 3.4 Conclusion ..................................................................................................... 98 3.5 References ................................................................................................... 100 4. CHAPTER4: IMPACT OF EXTRACTION PROCESSES ON THE NEUROPROTECTIVE ACTIVITIES OF ECKLONIA RADIATA AGAINST AMYLOID BETA (AΒ1-42) TOXICITY AND AGGREGATION ........................... 107 4.1 Introduction ................................................................................................. 108 4.2 Materials and Methods ................................................................................ 109 4.2.1 Reagents and materials ........................................................................ 109 4.2.2 Seaweed extraction and fractionation process ..................................... 109 4.2.3 PC12 cell culture .................................................................................. 111 4.2.4 Aβ preparation and treatment in PC12 cells ........................................ 111 4.2.5 MTT assay for neuronal cell viability .................................................. 111 ii 4.2.6 Thioflavin T assay of Aβ fibril formation............................................ 112 4.2.7 MTT assay for protecting PC-12 cells against H2O2-induced cytotoxicity 112 4.2.8 Nuclear staining for assessment of apoptosis ...................................... 113 4.2.9 Neurite outgrowth assay ...................................................................... 113 4.2.10 Statistical analysis ................................................................................ 114 4.3 Results and discussion ................................................................................. 114 4.3.1 Neurotoxicity and neuroprotective activity of E. radiata fractions against Aβ1-42 ..................................................................................................... 114 4.3.2 Effects of E. radiata fractions against Aβ1-42 fibril and aggregate formation ............................................................................................................ 118 4.3.3 Bioactivity of E. radiata fractions against H2O2 ................................ 121 4.3.4 Neuroprotective activities of E. radiata fractions in reducing the PC12 cell apoptosis induced by Aβ1-42 ........................................................................ 124 4.3.5 Neuroprotective activities of E. radiata fractions in enhancing neurite outgrowth ........................................................................................................... 126 4.4 Conclusion ................................................................................................... 129 4.5 References ................................................................................................... 130 5. CHAPTER 5: COMPARATIVE STUDY ON NEUROPROTECTIVE ACTIVITIES OF FUCOIDANS FROM FUCUS VESICULOSUS AND UNDARIA PINNATIFIDA ..........................................................................................................
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