Title Page
Contents
LIST OF ABBREVIATIONS 17
ABSTRACT 21
I. Introduction 24
1. Seaweed and its application 24
2. Neurodegeneration in Alzheimer's disease 26
3. Oxidative stresss in Alzheimer's disease 32
4. Antioxidant defense system in Alzheimer's disease 34
5. BDNF/TrkB pathway in Alzheimer's disease 36
6. Seaweed and neuroprotection 39
7. Aim of Research 40
II. Materials and Methods 42
1. Chemicals 42
2. Preparation and extraction of Enteromorpha prolifera 43
2.1. Preparation of Enteromorpha prolifera 43
2.2. Recipe of macaron added with Enteromorpha prolifera 43
2.3. Extraction procedure from Enteromorpha prolifera 45
3. Quality characteristics assay 47
3.1. Proximate compostion analysis 47
3.2. Physicohemical properties 47
3.3. Measurement of carotenoid and chlorophyll 50
3.4. Measurement of phycocyanins 50
3.5. Measurement of flavonoid 51
3.6. Measurement of total phenolic compounds 51
3.7. Measurement of texture profiles 52
3.8. Antioxidant activity 53
3.9. Sensory evaluation - intensity test 55
3.10. Analysis of aromatic compounds using gas-chromatography mass spectrometry (GC/MS) 56
3.11. Comparison of aroma using electronic nose (E-nose) 57
3.12. Analysis of high performance liquid chromatography 58
4. In vitro assay 59
4.1. Cell viability assay 59
4.2. Measurement of intracellular ROS level 59
4.3. Flow cytometry analysis 60
4.4. Protein determination 60
4.5. Extraction of nuclear and cytosolic protein 60
4.6. Western blot analysis 61
5. In vivo assay 62
5.1. Animals and experimental induction-scopolamine 62
5.2. Tissue preparation and collection 64
5.3. Morris water maze test 64
5.4. Passive avoidance test 65
5.5. Protein Determination 65
5.6. Measurement of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity 66
5.7. Measurement of lipid peroxide concentration in brain 66
5.8. Measurement of antioxidant enzyme activities in brain 66
5.9. Histological examination 67
5.10. Western blot analysis 67
6. Statistical analysis 69
6.1. Quality characteristics assay 69
6.2. In vitro assay 69
6.3. In vivo assay 70
III. Results and Discussion 71
1. Comparison of Enteromporpha prolifera from Seosan and Muan in Korea 71
1.1. Proximate composition 71
1.2. Physicochemical properties 73
1.3. Contents of carotenoid and chlorophyll 77
1.4. Contents of phycocyanin 77
1.5. Contents of flavonoid and total phenolic compound 80
1.6. Antioxidant activity 81
2. Aroma analysis and application of Enteromorpha prolifera 84
2.1. Aromatic compounds in Enteromorpha prolifera using gas-chromatography mass spectrometry (GC/MS) 84
2.2. Comparison of aroma in Enteromorpha prolifera using electronic nose (E-nose) 89
2.3. Comparison of aroma Enteromorpha prolifera added with butter for removal of fishery smell 104
2.4. Quality characteristics of macaron added with Enteromorpha prolifera 109
2.5. Antioxidant activity of macaron 122
2.6. Sensory evaluation - intenstity test of macaron 124
3. Antioxidant activities and phytochemicals of Enteromorpha prolifera extract 127
3.1. Content of chlorophyll 127
3.2. Content of phcocyanin 127
3.3. Content of flavonoid 130
3.4. Content of total phenolic compound 130
3.5. Antioxidant activity 133
3.6. Biochemical compounds using high performance liquid chromatography 137
4. Neuroprotective effect of Enteromorpha prolifera extract against oxidative stress in glutamate-treated hippocampal neuronal cells 143
4.1. Cell viability 143
4.2. Intracellular ROS level in glutamate-treated HT-22 cells 144
4.3. Anti-apoptotic effect of EAEP in flow cytometry 146
4.4. Expression of Akt, Nrf2 and AREs in antioxidant defense system 148
4.5. Expression of BDNF, ERK, CREB, and TrkB in neurotrophic system 151
4.6. Neutralizing effect of K252a and MK2206 on the neuroprotection of EAEP 155
5. Neuroprotective effect of Enteromorpha prolifera extract against oxidative stress in scopolamine-treated mice 162
5.1. Weight of the body and brains of mice treated with EAEP 162
5.2. Spatial learning ability in the Morris Water Maze 164
5.3. Memory ability in the passive avoidance test 167
5.4. Acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity 169
5.5. Antioxidant enzyme activities and lipid peroxide contents 172
5.6. Hippocampal neurons in the CA1 and CA3 regions 175
5.7. β-Amyloid and tau expression in western blotting analysis 178
5.8. Protein expression via BDNF/TrkB/Akt pathway 180
IV. Summary and Conclusion 183
V. References 188
국문초록 212
Table 1. Ingredients of macarons added with different amount of Enteromorpha... 45
Table 2. Condition of texture analyser (two bite test) 52
Table 3. GC/MS operating condition for aromatic compounds in Enteromorpha prolifera 57
Table 4. E-nose condition for aromatic compounds in Enteromorpha prolifera 58
Table 5. Condition of mass spectrometry. 59
Table 6. Experimental design. 63
Table 7. Proximate composition of Enteromorpha Prolifera from Seosan and Muan... 72
Table 8. Contents of soluble soild and reducing sugar of Enteromorpha Prolifera... 74
Table 9. Quality characteristics of Enteromorpha Prolifera from Seosan and Muan... 76
Table 10. Carotenoid, chlorophyll and phycocyanin contents of Enteromorpha... 79
Table 11. Total phenol, flavonoid contents, DPPH radical scavenging, SOD-like... 83
Table 12. Aromatic compounds in Enteromorpha prolifera by gas-chromatography... 87
Table 13. Classification of aromatic compound in Enteromorpha prolifera according to sensory descriptors 91
Table 14. Comparison of aromatic compounds in Enteromorpha prolifera by E-nose 100
Table 15. Sensory evaluation of aroma in Enteromorpha prolifera (EP) 103
Table 16. Comparison of aromatic compounds in Enteromorpha prolifera added with butter by E-nose 108
Table 17. Moisture content, pH, soluble solid content, and reducing sugar... 112
Table 18. Hunter's color values of macarons added with Enteromorpha prolifera powder 114
Table 19. Chlorophyll, carotenoid, and phycocyanin, contents of macarons added... 117
Table 20. TP A (textrure profile analysis) of macarons added with Enteromorpha... 121
Table 21. Score of intensity test of macarons added with Enteromorpha prolifera powder 126
Table 22. Phycocyanin and chlorophyll content of Enteromorpha prolifera (EP) extracts 129
Table 23. Body and brain weights of both normal and scopolamine-treated mice. 163
Fig. 1. Pathology diagram in AD. (A) Processing APP according to type of secretase. 29
Fig. 2. Cholinergic nervous system related with acetylcholine. 32
Fig. 3. Mechanisms in Nrf2/ARE pathway under normal or abnormal condition.... 35
Fig. 4. Mechanisms in BDNF/CREB/TrkB pathway. Under physiological conditions,... 38
Fig. 5. Defense systems via brain-activation of neurotrophic (BDNF/CREB)... 38
Fig. 6. Photograph of Enteromorpha prolifera 41
Fig. 7. Procedures to prepare different solvent extracts (DWEP, ETEP, EAEP) of... 46
Fig. 8. The diagram of schedule in the experiment. 63
Fig. 9. Chromatogram of aromatic compounds by E-nose. (A) The peak of... 98
Fig. 10. Comparison of aromatic compounds in Enteromorpha prolifera by E-nose.... 99
Fig. 11. Chromatogram of aromatic compounds in Enteromorpha prolifem added... 106
Fig. 12. Comparison of aromatic compounds in Enteromorpha' prolifera added... 107
Fig. 13. Flavonoid (A) and total phenolic compound contents of macarons added... 119
Fig. 14. DPPH radical scavenging activities (A) and SOD-like activities (B) contents... 123
Fig. 15. Photograph of macaron added with 4.5 g Enteromorpha prolifera powder 126
Fig. 16. Flavonoid (A) and total phenolic compounds (B) content of Enteromorpha... 132
Fig. 17. DPPH radical scavenging (A), Hydroxyl radical scavenging (B), and (C)... 136
Fig. 18. TIC (total ion chromatogram) of the extract of Enteromorpha prolifera with ethyl acetate by UHPLC-Q-Exactive... 141
Fig. 19. Mass spectrum graph and intensity of dihydroactinidiolide (A),... 142
Fig. 20. Neuroprotective effect of Enteromorpha prolifera extracted with ethyl... 145
Fig. 21. Neuroprotective effect of Enteromorpha prolifera extracted with ethyl... 147
Fig. 22. Effect of Enteromorpha prolifera extracted with ethyl acetate (EAEP)... 149
Fig. 23. Expression of BDNF, ERK, CREB, and TikB in neurotrophic system... 153
Fig. 24. Inhibitive effect of K252a or MK-2206 neutralize neuroprotection of... 157
Fig. 25. Inhibitive effect of K252a or MK-2206 neutralize neuroprotection of... 160
Fig. 26. Inhibitive effect of K252a or MK-2206 inhibits neuroprotection of... 161
Fig. 27. Memory-improving effect of Entemmorpha pmlifem extracted with ethyl... 166
Fig. 28. Cognitive ability-enhacing effect of Enteromorpha prolifera extracted with... 168
Fig. 29. Activities of cholinergic nervous system by treatment with Enteromorpha... 171
Fig. 30. Activities of antioxidant defense system by treatment with Enteromorpha... 173
Fig. 31. Neuroprotective effect of Enteromorpha prolifera extracted with ethyl acetate (EAEP) on hippocampal neurons in... 176
Fig. 32. Effect of Enteromorpha prolifera extracted with ethyl acetate (EAEP) on... 179
Fig. 33. Effect of Enteromorpha prolifera extracted with ethyl acetate (EAEP) on... 181
Fig. 34. Neuroprective effect of Enteromorpha prolifera extracted with ethyl acetate (EAEP) via TrkB/Akt pathway. 182