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목차
Part I. 카로티노이드 색소류의 광안정성, 광반응성 평가 및 빛 조사에 의한 생리활성 변화 평가 11
1. 서론 12
2. 재료 및 방법 17
2.1. 실험재료 17
2.2. 광조사 및 측정 기기 17
2.3. 광안정성 및 감광활성 평가 17
2.4. Reactive oxygen species (ROS) 생성량 측정 18
2.5. 용매별 광안정성 평가 및 HPLC분석 19
2.6. Annatto의 과산화물생성능 19
2.7. 산화방지활성 분석 20
2.8. Fenton reaction에 의한 산화방지활성 측정 20
2.9. Metal chelating activity 측정 21
2.10. 통계처리 21
3. 결과 및 고찰 22
3.1. 카로티노이드류 색소의 광안정성 평가 22
3.2. 카로티노이드류 색소의 감광활성 평가 23
3.3. 카로티노이드류 색소의 ROS 생성능 31
3.4. Annatto의 용매별 광안정성 평가 32
3.5. Annatto의 지질산패 및 ROS 생성 유도 32
3.6. 광 조사에 의한 annatto의 산화방지활성 변화 40
3.7. Annatto의 금속이온 제거 효과 41
Part II. 카로티노이드류 색소의 피부세포에 미치는 영향 및 면역조절 활성 평가 50
1. 서론 51
2. 재료 및 방법 54
2.1. 실험재료 및 세포주 54
2.2. 세포독성 평가 55
2.3. Colony formation assay (CFA) 55
2.4. Melanogenesis에 대한 영향 분석 56
2.5. Thiol기에 대한 lutein과 세포대사물의 반응성 분석 56
2.6. Pro-inflammatory cytokine 수준 측정 57
2.7. HaCaT세포의 과다증식에 대한 영향 평가 57
2.8. 분화 THP-1 배양액에 의한 피부세포의 영향 평가 58
2.9. Western blot 58
2.10. 통계처리 59
3. 결과 및 고찰 61
3.1. 카로티노이드의 피부세포에의 영향 61
3.2. 카로티노이드에 의한 면역조절 활성 75
참고문헌 96
ABSTRACT 102
Table 1. The maximum absorption and maximum absorptivity of different carotenoids calculated based on Fieser-Kuhn rule. 28
Table 2. Evaluation of photosensitizing properties of annatto and crocin under different light sources using the MTT formazan probe. 29
Table 3. The qRT-PCR primers used for detecting inflammatory marker. 60
Table 4. Evaluation of human keratinocyte (HaCaT) proliferation by different carotenoids using the colony formation assay (n=4). 65
Fig. 1. Structures of norbixin (A), bixin (B), crocin (C), and lutein (D) used in the present study. 15
Fig. 2. Emission spectrums of the light sources including fluorescent light (A), white LED (B), and blue, green, red LEDs (C) used in the present study. 16
Fig. 3. Evaluation of photo-stability of different carotenoids under fluorescent light and blue LED. 27
Fig. 4. Evaluation of photosensitizing activity of different carotenoids using the NBT probe. 30
Fig. 5. Detection of ROS formation by different carotenoids under light. 35
Fig. 6. Changes in chemical stability of annatto pigments under light. 38
Fig. 7. Lipid oxidation by photosensitivity of annatto under light or dark condition. 39
Fig. 8. Changes in antioxidant activities of annatto by irradiation. 46
Fig. 9. Effects of annatto on the Fenton reaction-induced lipid peroxidation and on metal ion chelation. 49
Fig. 10. Effects of carotenoid on the growth of different skin cells. 64
Fig. 11. Long-term effects on the growth of different skin cells by carotenoids. 67
Fig. 12. Effects of different carotenoids on the melanogenesis in the mouse melanoma. 72
Fig. 13. Reactivity evaluation of lutein and its cellular metabolites with thiol compounds. 74
Fig. 14. Effects of phorbol 12-myristate 13-acetate (PMA) on growth and cytokine mRNA levels of HaCaT. 78
Fig. 15. Changes in mRNA levels of pro-inflammatory cytokines in PMA-stimulated HaCaT by different carotenoids. 80
Fig. 16. Effects on PMA-induced keratinocyte proliferation by carotenoid. 82
Fig. 17. Changes in cell viability of THP-1 and differentiated THP-1 by different carotenoids. 86
Fig. 18. Effects of different carotenoids on PMA-induced differentiation of THP-1. 88
Fig. 19. Changes in cyclooxygenase-2 expression levels by carotenoid in differentiated THP-1. 93
Fig. 20. Effects of THP-1 media incubated with carotenoids on different skin cells. 95