세포 배양시스템에서 녹차 카테킨의 화학적 행태에 따른 생리활성 변화 및 항산화제와 일반의약품과의 상호작용

다국어 초록 (Multilingual Abstract)

Various physiological effects of tea catechins, has been studied extensively, including antioxidant, anti-inflammatory, and anti-cancer activities. Many of these activities and related mechanisms have been investigated using a cell culture system. Tea catechins are, however, unstable in cell culture conditions and are rapidly converted to oxidative products through the autoxidation process. In the present study, changes in bioactivities of a major tea catechin, epigallocatechin-3-gallate (EGCG), by chemical behaviors under cell culture systems and its interaction with different antioxidants or over-the-counter (OTC) drugs were explored.
First, fluorescent properties of EGCG during its autooxidation process were investigated. The fluorescence intensity of EGCG　was significantly enhanced after 4 h-incubation in phosphate-buffered saline or culture media showing a peak emission at 470 nm with an excitation at 380 nm. Enhancement of the fluorescence intensity was proportional to formation of EGCG oxidative products and was not observed in the presence of EGCG stabilizing agents including ascorbic acid and superoxide dismutase (SOD). The fluorescence signal from EGCG oxidative products can be a convenient marker for expecting EGCG stability and its oxidation process.
Uptake, efflux as well as cytotoxic properties of EGCG and its oxidative products were investigated in HepG2 cells. Cytotoxic effect of EGCG was mainly due to reactive oxygen species (ROS) generated from EGCG in media; intracellular ROS level decreased by EGCG. EGCG oxidative products rather stimulated cell proliferation slightly. Uptake form into cells was mainly free EGCG; its oxidation products was not detected in cells. Efflux transporter modulators including Ko143 and indomethacin increased the cellular levels of EGCG and (-)-epicatechin gallate (ECG) in HepG2 cells, suggesting that EGCG and ECG are substrates for MRP and/or BCRP. EGCG also increased the BCRP protein level in a concentration- and time-dependent manner without affecting its mRNA level. MRP-1 mRNA level decreased in cells treated with EGCG; EGCG, however, increased MRP-2 mRNA level. The effect of EGCG on mRNA level of MRP-2 was significantly less pronounced in the presence of SOD. The results suggest that EGCG could modulate activities and levels of these transporters and affect efflux of certain drugs that interact with these proteins. Among CYPs including 1A2, 3A4, and 2E1, levels of mRNA of CYP2E1 and CYP1A2 was decreased in HT-29 cells by EGCG; EGCG did not affect protein level of CYP2E1.
Changes of chemical stability and cytotoxic properties of EGCG in the presence of various antioxidants were investigated. The antioxidants used in the present study modulated chemical stability of EGCG. SOD, ascorbic acid, N-acetylcysteine (NAC), and glutathione (GSH) significantly increased EGCG stability. The H2O2 level generated from EGCG was decreased by NAC but not by GSH. The cytotoxic effects of EGCG also decreased in the presence of NAC, catalase, and SOD. GSH, however, showed a complicated modulatory pattern according to EGCG and GSH concentrations; ascorbic acid rather enhanced EGCG cytotoxicity. The results suggest that many antioxidants could modulate the cytotoxic properties of EGCG in a cell culture system not only by removing ROS but by modulating chemical stability and other factors, which should be considered carefully when studying ROS-dependent mechanisms of EGCG.
Tea catechins and commonly-consumed OTC drugs could be encountered and their interactions in body are expected. Acetylsalicylic acid (Asp) increased the chemical stability of EGCG. Non-toxic concentrations of acetaminophen (AAP) significantly enhanced the EGCG cytotoxicity. Non-toxic dose of EGCG (up to 20 µM) also enhanced ibuprofen cytotoxicity significantly. The isobologram analysis revealed the cytotoxic synergism by combination of ibuprofen with EGCG. There was, however, no dramatic change observed in cytotoxicity of Asp combined with EGCG. EGCG significantly decreased AAP toxicity in HepG2 cells, showing increased GSH levels. When consequently treated with AAP after EGCG incubation, cell viability was significantly improved as compared to cells treated with EGCG alone. Intracellular level of AAP were increased by Ko143; it was not changed significantly in the presence of EGCG.
The present results indicates that bioactivities of tea catechins, mainly EGCG, can be modulated dramatically by changes of their chemical behaviors (e.g. chemical stability, auto-oxidation, and generation of hydrogen peroxide etc.) under cell culture conditions. This study also suggests that interaction of EGCG with antioxidants, efflux modulators as well as OTC drugs could modify physiological actions of EGCG or its interacting compounds through modulating their chemical behaviors.

목차 (Table of Contents)

• I. 서론 1
• 1.1 녹차의 일반적 특성 1
• 1.2 차 카테킨의 화학적 특성 3
• 1.3 차 카테킨들의 생리활성 및 관련 기작 3
• 1.4 일반의약품 4
• 1.5 식품과 약물의 상호작용 8
• 1.5.1 화학반응을 통한 직접 상호작용 8
• 1.5.2 Efflux transporter에 의한 상호작용 10
• 1.5.3 CYP450에 의한 상호작용 11
• 1.6 연구 목적 13
• II. 실험재료 및 방법 15
• 2.1 실험재료 15
• 2.2 세포주 및 세포 배양 15
• 2.3 형광 특성 변화 분석 16
• 2.4 Hydrogen peroxide (H2O2) 생성량 및 DPPH radical 소거능 측정 16
• 2.5 세포 독성 및 증식 측정 17
• 2.6 세포 내 H2O2 및 GSH 정량 18
• 2.7 세포 주기 분석 19
• 2.8 차 카테킨 성분의 cellular uptake 20
• 2.9 HPLC 분석 20
• 2.10 RNA 추출 및 역전사효소 반응 24
• 2.11 Western blot 24
• 2.12 통계분석 26
• III. 결과 및 고찰 28
• 3.1 차 카테킨의 행태 및 생리활성의 변화 28
• 3.1.1. 차 카테킨의 형광도 분석 28
• 3.1.2. 세포배양계 상에서의 ECGC 형광특성 변화 35
• 3.1.3. EGCG의 행태 변화에 따른 세포독성에의 영향 40
• 3.1.4. EGCG 자동산화 및 ROS 생성에 미치는 각종 항산화제의 영향 43
• 3.1.5. EGCG 행태 변화에 따른 세포 내 분포 45
• 3.2 차 카테킨 성분이 efflux transporter와 CYP450에 미치는 영향 51
• 3.2.1 Efflux transporter 저해를 통한 EGCG, ECG의 세포 내 수준 변화 51
• 3.2.2 차 카테킨 성분의 efflux transporter 발현 변화 56
• 3.2.3 차 카테킨 성분이 CYP450에 미치는 영향 58
• 3.3 차 카테킨과 항산화제 간의 상호작용 68
• 3.3.1 각종 항산화제들에 의한 EGCG의 화학 안정성 변화 68
• 3.3.2 각종 항산화제들에 의한 차 카테킨의 세포독성 변화 71
• 3.4 차 카테킨과 일반의약품 성분과의 상호작용 80
• 3.4.1 일반의약품 성분이 EGCG의 화학안정성에 미치는 영향 80
• 3.4.2 일반의약품 성분에 의한 EGCG의 세포독성 변화 80
• 3.4.3 EGCG에 의한 일반의약품 성분의 세포독성 변화 87
• 3.4.4 EGCG와 일반의약품 성분의 다양한 처리 조건에 따른 세포독성 변화 97
• 3.4.5 EGCG가 AAP의 세포 내 수준에 미치는 영향 101
• IV. 참고 문헌 103
• ABSTRACT 117