Title Page
Contents
국문초록 8
ABSTRACT 11
Ⅰ. INTRODUCTION 21
Ⅱ. PURPOSE OF STUDY 25
Ⅲ. MATERIALS AND METHODS 29
1. Materials 29
2. Preparation of O. japonicus extract and fraction 30
3. UPLC-Q-TOF-MS analysis 30
4. Cell culture 31
A. 3T3_L1 adipocyte 31
B. HaCaT keratinocyte and NIH3T3 fibroblast 32
5. Cytotoxicity assay 32
6. Cell differentiation and Oil Red O staining 32
7. Animals, diets, and experimental design 33
8. Plasma biochemistry in mice 35
9. Assay of mRNA expression by RT-PCR 36
A. Anti-obesity 36
B. Anti-oxidant and anti-inflammation 37
10. Assay of protein level by western blot 38
A. Anti-obesity 38
B. Anti-oxidant and anti-inflammation 39
11. Anti-oxidant activity assay 39
A. DPPH free radical scavenging activity 39
B. ABTS free radical scavenging activity 40
C. Ferric reducing anti-oxidant power (FRAP) assay 41
12. Statistical analysis 41
Ⅳ. RESULT 42
1. Measurement of cytotoxicity 42
A. 3T3_L1 adipocyte 42
B. HaCaT keratinocyte and NIH3T3 fibroblast 44
2. UPLC Q-TOF-MS analysis 46
3. Oil Red O staining in 3T3_L1 differentiation 52
4. Expression of PPAR-γ, C/EBP-α, FABP4, FAS and SCD-1 mRNA in 3T3_L1 differentiation 55
5. PPAR-γ, C/EBP-α, SCD-1, ACC, and Leptin protein levels in 3T3_L1 differentiation 59
6. AMPK phosphorylation levels in 3T3_L1 differentiation 62
7. Effects of O. japonicus extracts on body weight gain and food intake 64
8. Effects of O. japonicus extracts on liver and epididymal tissue weight 67
9. Effects of O. japonicus extracts on plasma biochemical parameters 70
10. Effects of O. japonicus extracts on mRNA expression of genes in adipose tissue 74
11. Effects of O. japonicus extracts on protein levels in adipose tissue 78
12. AMPK phosphorylation levels in adipose tissue 81
13. Measurement of anti-oxidant activity 83
14. mRNA expression of SOD-1, SOD-2, CAT, and Nrf-2 87
15. Protein levels of SOD-1, SOD-2, and Nrf-2 90
16. Protein level of NF-κB 93
17. Protein levles of MAPK signaling pathway 95
Ⅴ. DISCUSSION 98
A. Anti-obesity 98
B. Anti-oxidant and anti-inflammation 101
Ⅵ. CONCLUSION 105
Ⅶ. REFERENCES 106
Table 1. Composition of high-fat diets (D12492) and nomal diet (D12450B), total calories were 4,057 Kcal. 34
Table 2. UPLC Q-TOF MS analysis of O. japonicus fractions 51
Table 3. Anti-oxidant activities of O. japonicus fractions measured by DPPH, ABTS radicals, and FRAP 84
Fig. 1. Cytotoxicity of O. japonicus extracts. extrats were treated 24h, the viability of mouse preadipocyte 3T3_L1 cells were... 43
Fig. 2. Cytotoxicity of O. japonicus fractions. Fractions were treated for 24 h, the viability of (a) human skin keratinocyte... 45
Fig. 3. UPLC Q-TOF MS chromatograms of OJ_EtOH (a), OJ_Water, OJ_EtOAc (c), and OJ_DCM (d). 49
Fig. 4. Structure of compounds identifided in UPLC Q-TOF MS chromatograms of OJ_EtOH, OJ_Water, OJ_EtOAc, and OJ_DCM. 50
Fig. 5. Effect of OJ_EtOH and OJ_Water on lipid accumulation of 3T3_L1 cells. A) Lipid droplets were photographed after Oil Red O... 53
Fig. 6. Effect of OJ_EtOH and HCA on lipid accumulation of 3T3_L1 cells. A) Lipid droplets were photographed after Oil Red O... 54
Fig. 7. Effect of OJ_EtOH and OJ_Water on expression levels of adipogenic gene in 3T3_L1 cells. a) Adipocytes treated with various... 58
Fig. 8. Effect of OJ_EtOH and OJ_Water on expression levels of adipogenic protein in 3T3_L1 cells. a) Adipocytes treated with... 61
Fig. 9. Effect of OJ_EtOH and OJ_Water on AMPK phosphorylation in 3T3_L1 cells. a) Adipocytes treated with various concentration of... 63
Fig. 10. Effects of OJ_EtOH on a) sacrificed mouse b) body weight, c) body weight gain, d) food intake and e) food efficiency ratio... 66
Fig. 11. Effects of OJ_EtOH on a) liver tissue b) liver weight c) epididymal tissue, and d) epididymal weight in HFD-induced obese... 69
Fig. 12. Effects of OJ_EtOH on serum biomarker. a) TG b) TC c) HDL, d) LDL, e) leptin, f) adiponectin, g) ALT, and h) AST in... 73
Fig. 13. Effect of OJ_EtOH on expression levels of adipogenic gene in epididymal tissue. a) mRNA expression levels of adipogenesis... 77
Fig. 14. Effect of OJ_EtOH on levels of adipogenic protein in epididymal tissue. a) expression levels of adipogenesis related... 80
Fig. 15. Effect of OJ_EtOH on AMPK phosphorylation in epididymal tissue. a) expression levels of AMPK, p-AMPK proteins. b)... 82
Fig. 16. DPPH free radical scavenging activity of OJ_EtOH, OJ_EtOAC, OJ_DCM, and ascorbic acid. The percent inhibitions... 85
Fig. 17. ABTS free radical scavenging activity of OJ_EtOH, OJ_EtOAC, OJ_DCM, and ascorbic acid. The percent inhibitions... 86
Fig. 18. Measurement of effects of OJ_EtOH, OJ_EtOAc, and OJ_DCM on mRNA expressions of b) SOD-1, c) SOD-2, d) CAT,... 89
Fig. 19. Measurement of effects of OJ_EtOH, OJ_EtOAc, and OJ_DCM on protein levels of b) SOD-1, c) SOD-2, and d) Nrf-2.... 92
Fig. 20. Measurement of effects of OJ_EtOH, OJ_EtOAc, and OJ_DCM on protein levels of NF-κB. The results were expressed... 94
Fig. 21. Measurement of effects of OJ_EtOH, OJ_EtOAc, and OJ_DCM on protein levels of b) p-ERK, c) p-JNK and d) p-p38.... 97