Title Page
Contents
ABSTRACT 10
국문초록 12
I. INTRODUCTION 14
A. Orostachys japonicus and cynaroside 14
B. Anti-oxidant activity 16
C. Anti-inflammatory activity 17
D. Anti-cancer activity 19
II. PURPOSE 22
III. Materials and methods 24
A. Cell lines and reagents 24
B. HPLC analysis 25
C. Cell culture 26
D. DPPH radical scavenging assay 27
E. Cell cytotoxicity assay 28
F. NO assay 29
G. Cell viability assay 30
H. 4, 6-diamidino-2-phenylindole (DAPI) assay 31
I. Cell cycle analysis 32
J. Western blot analysis 33
K. Statistical Analysis 34
IV. Results 35
A. HPLC analysis of OJB 35
B. Effects of cytotoxicity of OJB and cynaroside on RAW 264.7 macrophage cells 37
C. DPPH Radical Scavenging Activity 39
D. HO-1protein level expression by OJB and cynaroside in LPS-induced RAW 264.7 cells 41
E. OJB and cynaroside inhibition of NO formation 43
F. iNOS protein level expression by OJB and cynaroside in LPS-induced RAW 264.7 cells 45
G. OJB and cynaroside protein expression of PGE2 in LPS-induced RAW 264.7 cells 47
H. OJB and cynaroside protein expression of COX-2 in LPS-induced RAW 264.7 cells 49
I. OJB and cynaroside protein expression of NF-κB in LPS-induced RAW 264.7 51
J. Inhibition of proliferation of pancreatic cancer cells 54
K. Effects of OJB and cynaroside-induced apoptosis 57
L. Effects of OJB protein expression of caspase in PANC-1 cells 59
M. Cell cycle arrest 65
V. Discussion 70
VI. CONCLUSION 73
VII. REFERENCES 74
Table. 1. Analysis of cell cycle distribution using flow cytometry of OJB-treated PANC-1 cells at various concentrations 69
Figure 1. Constituents of n-butanol fracion of s Orostachys japonicus EtOH extracts and structures of cynaroside 15
Figure 2. Model for inhibitory mechanism of OJB and cynaroside on LPS-treated inflammation 18
Figure 3. Intrinsic and Extrinsic pathway of apoptosis 21
Figure 4. Correlation of oxidative stress, inflammation, and cancer 23
Figure 5. HPLC chromatograms of cynaroside and n-butanol fraction from Orostachys japonicus 36
Figure 6. Cytotoxicity effect of OJB on proliferation of RAW 264.7 38
Figure 7. DPPH assay 40
Figure 8. Raw 264.7 cells treated with cynaroside and OJB and inflamed with LPS (10 µg/mL) expressed heme oxygenase 1 (HO-1) 42
Figure 9. Effects of cynaroside and OJB on LPS-induced NO production 44
Figure 10. Expression of iNOS in RAW 264.7 cells inflamed with LPS (10 µg/mL) and treated with cynaroside and OJB 46
Figure 11. Expression of prostaglandin E2 (PGE₂) in RAW 264.7 cells inflamed with LPS (10 µg/mL) and treated with cynaroside and OJB 48
Figure 12. Expression of COX-2 in RAW 264.7 cells inflamed with LPS (10 µg/mL) and treated with cynaroside and OJB 50
Figure 13. Expression of NF-κB and IκB in RAW 264.7 cells inflamed with LPS (10 µg/mL) and treated with cynaroside and OJB 53
Figure 14. Inhibitory effect of cynaroside on proliferation of PANC-1 55
Figure 15. Inhibitory effect of OJB on proliferation of PANC-1 56
Figure 16. PANC-1 cells Nuclear morphological changed, displaying signs of apoptosis 58
Figure 17. Effect of OJB on the pro-caspase 3 protein levels in PANC-1 pancreatic cancer cells 60
Figure 18. Effect of OJB on the pro-caspase 8 and cleaved caspase-8 protein levels in PANC-1 pancreatic cancer cells 62
Figure 19. Effect of OJB on pro-caspase 9 and cleaved caspase-9 protein levels in PANC-1 pancreatic cancer cells 64
Figure 20. Analysis of the cell cycle distribution using flow cytometry of OJB-treated PANC-1 cells at various concentration 66
Figure 21. Effect of OJB on the protein levels of CDK2 and CDK4 in PANC-1 pancreatic cancer cells 68