국립강릉원주대학교

목차 도움말

Abstract i
Table of contents iii
List of tables viii
List of figures ix
Abbreviations xii
Chapter 1. Introduction 1
1.1 Dementia 1
1.2 Alzheimer’s disease 2
1.2.1 Stages 3
1.2.2 Diagnosis 4
1.2.3 Genetics and risk factors 6
1.3 Pathology of AD 8
1.3.1 Amyloid β 9
1.3.2 Tauopathies 12
1.3.3 Synaptic dysfunction and cholinergic pathology 13
1.3.4 Oxidative stress and neuroinflammation 15
1.4 Therapeutics of AD 17
1.4.1 Drug treatments 17
1.4.2 Non-drug treatments 18
1.4.3 Current clinical pipelines 19
1.5 Ginsenosides 22
1.5.1 Structural properties 25
1.5.2 Structural conversion 26
1.5.3 Adaptogenic effects 28
1.6 Effect of ginsenosides on the AD pathogenesis 30
1.6.1 Inhibition of Aβ formation 31
1.6.2 Inhibition of NFTs formation 33
1.6.3 Antioxidative effect 34
1.6.4 Anti-inflammatory effect 35
1.6.5 Anti-apoptotic effect 37
1.7 Study overview and objectives 39
Chapter 2. Pharmacological activities of ginsenosides derived from ginseng berry extract 41
2.1 Introduction 41
2.2 Materials & methods 43
2.2.1 Ginseng berry bioactive extract 43
2.2.2 Preparation of ginsenosides 44
2.2.2.1 HPLC analysis and preparative HPLC isolation 44
2.2.2.2 LC-MS analysis 44
2.2.3 ADMET prediction 45
2.2.4 Evaluation of BBB penetration 45
2.2.4.1 Animal and administration 45
2.2.4.2 LC-MS/MS analysis 46
2.2.5 Antioxidant activities 47
2.2.5.1 DPPH radical scavenging activity 47
2.2.5.2 Hydroxyl radical scavenging activity 47
2.2.6 Cell culture 48
2.2.7 Cell viability assay 49
2.2.8 NO measurement 49
2.2.8.1 Non-cellular NO scavenging activity 49
2.2.8.2 Measurement of cellular NO production 50
2.2.9 RT-qPCR analysis 50
2.2.10 Western blot analysis 51
2.2.11 Statistical analysis 52
2.3 Results 53
2.3.1 Quantification and isolation of ginsenosides 53
2.3.2 ADMET prediction summary in ginsenosides 60
2.3.2.1 Physicochemical properties 60
2.3.2.2 Medicinal chemistry 62
2.3.2.3 Pharmacokinetics 64
2.3.2.4 Toxicity 68
2.3.3 Evaluation of BBB penetration 71
2.3.4 Antioxidant activities 73
2.3.5 Reduction of cellular NO concentration 76
2.3.6 Physiological activities in brain cells 79
2.3.6.1 Cell viability assessment 79
2.3.6.2 Neuronal marker expression in N2a cells 81
2.3.6.3 Neuronal marker expression in SK-N-MC cells 86
2.3.6.4 Activation of ERK and Akt pathways in HMO6 cells 88
2.4 Discussion 90
Chapter 3. Activation of M2 microglia by ginsenoside Rg3 via selective CB2 receptor agonism 94
3.1 Introduction 94
3.2 Materials & methods 96
3.2.1 Sample preparation 96
3.2.2 GPCR profiling 96
3.2.3 Molecular docking simulation 98
3.2.4 Cell culture 98
3.2.5 Preparation of M1 and M2 polarized microglial cells 99
3.2.6 Cell viability and cytotoxicity assay 99
3.2.7 RT-qPCR analysis 100
3.2.8 Western blot analysis 101
3.2.9 Confocal microscopy analysis 102
3.2.10 Statistical analysis 102
3.3 Results 103
3.3.1 Profiling of GPCR responsiveness relevant to Rg3 103
3.3.2 Binding affinity of Rg3 to CB2 and Vina score 107
3.3.3 Cell viability and cytotoxicity in HMO6 cells 117
3.3.4 CB2 receptor agonism of Rg3 119
3.3.5 Contribution of Rg3 in M1 and M2 polarization 124
3.3.6 Rg3-induced acceleration of phagocytosis 130
3.4 Discussion 134
Chapter 4. Regulation of Aβ pathogenesis by ginsenoside Rg3 via modulation of Aβ production and clearance 140
4.1 Introduction 140
4.2 Materials & methods 142
4.2.1 Preparation of samples 142
4.2.2 Aβ peptides 142
4.2.3 Aβ42 aggregates PICUP assay 142
4.2.4 Cell culture 143
4.2.5 Immunoprecipitation 144
4.2.6 Western blot analysis 144
4.2.7 Confocal microscopy analysis 146
4.2.8 Statistical analysis 146
4.3 Result 147
4.3.1 Inhibition of Aβ fibril formation 147
4.3.2 Promotion of non-amyloidogenesis 149
4.3.3 Inhibition of tau phosphorylation 152
4.3.4 Accelerating Aβ uptake and degradation 154
4.3.4.1 Assessment of protein expression levels 154
4.3.4.2 Investigation of the relationship between SRA distribution and Aβ42 uptake 157
4.3.4.3 Distribution analysis of endocytosis-associated proteins 159
4.3.4.4 Assessment of Aβ-degrading enzyme distribution 161
4.4 Discussion 163
Chapter 5. Enhancing cognitive function in APPswe/PS1dE9 mice with a pharmaceutical composition derived from ginseng berries 167
5.1 Introduction 167
5.2 Materials & methods 170
5.2.1 Sample Preparation 170
5.2.2 HPLC analysis 170
5.2.3 GC-MS analysis 170
5.2.4 Animal model 171
5.2.5 Passive avoidance test 173
5.2.6 Morris water maze test 173
5.2.7 Acetylcholine concentration 174
5.2.8 Aβ concentration 174
5.2.9 Western blot analysis 175
5.2.10 Immunohistochemistry 176
5.2.11 Statistical analysis 176
5.3 Result 177
5.3.1 Biologically active compound analysis 177
5.3.2 Restoration of cognitive function 181
5.3.3 Acetylcholine concentration in brain tissue 184
5.3.4 Protein expression profiles in brain tissue 186
5.3.4.1 Expression of choline acetyltransferase and neurotrophins 186
5.3.4.2 Expression of Aβ clearance-related proteins 189
5.3.4.3 Analysis of Aβ42 and GFAP levels 191
5.3.5 Assessment of Aβ deposition in the brain 193
5.3.6 Examination of GFAP activation in the brain 195
5.4 Discussion 197
Chapter 6. Conclusion and future directions 202
6.1 Conclusion 202
6.2 Future directions 204
Bibliography 207
국문초록 237