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목차
Part Ⅰ. 다양한 광원 하에서 riboflavin 및 생체전환 isoalloxazine 유도체들의 광반응성 및 화학적 특성 변화와 세포 배양 시스템에서의 광독성 평가 14
1. 서론 15
2. 재료 및 방법 18
2.1. 실험재료 18
2.2. 세포주 및 세포 배양 18
2.3. 광 조사 및 측정 기기 19
2.4. 광안정성 평가 19
2.5. High-performance liquid chromatography (HPLC) 분석 20
2.6. 감광활성 평가 20
2.7. Reactive oxygen species (ROS) 생성량 측정 21
2.8. 지질과산화물 생성량 측정 22
2.9. 산화방지활성 분석 23
2.10. 세포독성 및 광독성 평가 24
2.11. 세포 내·외 ROS 측정 24
2.12. 세포 내 glutathione 수준 측정 25
2.13. 통계처리 25
3. 결과 및 고찰 31
3.1. Riboflavin 및 생체전환 isoalloxazine 유도체들의 광안정성 분석 31
3.2. Riboflavin 및 생체전환 isoalloxazine 유도체들의 광반응성 분석 56
3.3. Riboflavin 및 생체전환 isoalloxazine 유도체들의 산화방지활성 평가 71
3.4. 안구와 피부 세포에 대한 광독성 평가 78
4. 결론 92
Part Ⅱ. 빛 조사 하에서 riboflavin 및 생체전환 isoalloxazine 유도체들과 생체분자 및 식품 성분 간의 화학적 상호작용 분석 94
1. 서론 95
2. 재료 및 방법 98
2.1. 실험재료 98
2.2. 세포주 및 세포 배양배지 98
2.3. 광 조사 및 측정 기기 99
2.4. 세포독성 및 광독성 평가 99
2.5. Hydrogen peroxide (H₂O₂) 생성량 측정 100
2.6. NBT 환원활성 측정 101
2.7. Reactive oxygen species (ROS) 생성량 측정 101
2.8. 지질과산화물 생성량 측정 102
2.9. 세포 내 glutathione 수준 측정 103
2.10. 통계처리 103
3. 결과 및 고찰 104
3.1. Riboflavin과 아미노산 조합에 따른 광반응성 및 화학적 특성 변화 104
3.2. Oil in water (O/W) 반응계에서 Rb isoalloxazine 유도체와 철 이온과의 화학적 상호작용 129
4. 결론 134
참고문헌 135
ABSTRACT 142
Table 1. Conversion of the photometric units (illuminance, lx) of different lights used in the present study to its radiometric equivalent 29
Table 2. HPLC operating conditions for Rb and its derivatives analysis in the present study 30
Table 3. Comparison of photosensitizing property of different concentrations of Rb and its derivatives under different light sources based on the MTT formazan decolorization method 60
Table 4. Amino acid compositions in different culture media used in the present study. 108
Table 5. NBT reduction potential (△ abs. at 560 nm) from the interaction of Rb derivatives (Rb, FMN, LC, and LF, each 10 μM) combined with different... 112
Fig. 1. Structures of alloxazine (A), isoalloxazine (B), lumichrome (LC, C), lumiflavin (LF, D), riboflavin (Rb, E), flavin mononucleotide (FMN, F), and... 26
Fig. 2. Emission spectra of different visible light sources including fluorescent light (A), white (B), red (C), green (D), and blue (E) LED used in the... 28
Fig. 3. Correlation between photometric units (illuminance, lx) and radiometric units (irradiance, W/m²) of different light sources including fluorescent light,... 29
Fig. 4. Analysis of absorbance and fluorescence emission spectra of Rb, FMN, and FAD (each 50 μM) dissolved in distilled water. 34
Fig. 5. Comparison of photostability of Rb, FMN, and FAD under different light conditions. 37
Fig. 6. The kinetic property of Rb photodegradation under blue LED. 43
Fig. 7. The kinetic property of FMN photodegradation under blue LED. 46
Fig. 8. The kinetic property of FAD photodegradation under blue LED. 49
Fig. 9. The kinetic property of LC photodegradation under blue LED. 52
Fig. 10. The kinetic property of LF photodegradation under blue LED. 55
Fig. 11. Comparison of photosensitizing activities of Rb, FMN, and FAD under different light conditions using the MTT formazan probe. 59
Fig. 12. Evaluation of ROS generation by Rb, FMN, and FAD under different light sources using the DCFH fluorescent probe. 62
Fig. 13. Generation of H₂O₂ by Rb, FMN, and FAD under irradiation of fluorescence light and blue LED. 63
Fig. 14. Evaluation of NBT reduction potential by Rb, FMN, and FAD under irradiation of fluorescence light and blue LED. 65
Fig. 15. Effects of Rb, FMN, and FAD on lipid peroxidation induced by light irradiation. 70
Fig. 16. Evaluation of antioxidant property of photodegradation products of Rb, FMN, and FAD. 73
Fig. 17. Evaluation of antioxidant potential of Rb, FMN, and FAD under light irradiation. 77
Fig. 18. Evaluation of HLE B-3 and HaCaT phototoxicity induced by riboflavin and its derivatives under blue LED. 85
Fig. 19. Changes in cell viability of HLE B-3 and HaCaT by post-incubation period after blue LED irradiation and by blue LED irradiation time in the... 87
Fig. 20. Changes in intracellular ROS levels of HLE B-3 and HaCaT by riboflavin and its derivatives under blue LED. 88
Fig. 21. Changes in H₂O₂ levels in the culture media of cells treated with Rb, FMN, and FAD after blue LED irradiation. 89
Fig. 22. Changes in glutathione levels in HLE B-3 and HaCaT treated with Rb, FMN, and FAD after blue LED irradiation and effect of NAC on HLE... 91
Fig. 23. Evaluation of HaCaT phototoxicity induced by Rb in different media under blue LED. 106
Fig. 24. Evaluation of H₂O₂ generation by Rb in different solvent systems under blue LED irradiation. 107
Fig. 25. Effects of different amino acids on Rb derivatives-induced NBT reduction under light. 114
Fig. 26. Effects of SOD on NBT reduction by the interaction of Rb derivatives with different amino acids under light. 116
Fig. 27. Effects of Met, His, and Cys on Rb-induced ROS generation under blue LED irradiation. 120
Fig. 28. Effects of combination of Rb with Met and His on H₂O₂ generation under blue LED. 123
Fig. 29. Induction of HaCaT phototoxicity by the interaction of Rb with Met or His under blue LED. 127
Fig. 30. Changes in glutathione levels in HaCaT cells treated with combination of Rb with Met or His under blue LED irradiation. 128
Fig. 31. Induction of photo-oxidation by Rb in different lipid peroxidation system. 131
Fig. 32. Induction of photo-oxidation by FMN in different lipid peroxidation system. 132
Fig. 33. Induction of photo-oxidation by FAD in different lipid peroxidation system. 133