티로시나아제의 발현과 분해 조절을 통한 Gagunin D의 미백 효능 기전 연구

학위논문 (석사)-- 서울대학교 대학원 : 약학대학 약학과(천연물과학전공), 2017. 2. 이상국.Tyrosinase is the rate-limiting enzyme critical for melanin biosynthesis and controls pigmentation in the skin. Inhibition of tyrosinase has so far been the most common approach in the development of skin-whitening cosmetics. A number of tyrosinase inhibitors are used as cosmetic formulations. However, in this present study, a balance between tyrosinase expression and degradation is considered as a new target for skin whitening agent. Gagunin D (GD), a highly oxygenated diterpenoid isolated from the marine sponge Phorbas sp., has exhibited cytotoxicity toward human leukemia cells (K562). However, the effect of GD on normal cells and the molecular mechanisms of action remain to be elucidated. In this study, The anti-melanogenic activity of GD and its precise underlying mechanisms in mouse originated melan-a cells were identified. GD significantly inhibited melanin synthesis in the melan-a cells without cytotoxicity and had an effect on a reconstructed human skin model composed of human epidermis and melanocytes. Further analysis revealed that GD suppressed the expression of tyrosinase and increased the rate of tyrosinase degradation. GD also inhibited tyrosinase enzymatic activity. In addition, GD effectively suppressed the expression of proteins associated with melanosome-transfer. These findings suggest that GD is a potential candidate for cosmetic formulations due to its multi-functional properties which replaces tyrosinase inhibitor.I. Introduction 1 II. Materials and Methods 6 A. Materials 6 1. Reagents and antibodies 6 2. Compounds 6 3. Cell culture 7 B. Methods 9 1. Melanin content assay 9 2. MTT cell viability assay 9 3. Cell-free tyrosinase activity assay 9 4. Cellular tyrosinase activity assay 10 5. Western blotting 10 6. Real time polymerase chain reaction (PCR) 11 7. Immunocytochemistry 12 8. Transient transfection and dual luciferase assay 12 9. Reconstructed human skin model 13 10. Statistical analysis 13 III. Results 15 A. Inhibition of gagunin D (GD) on melanin biosynthesis in melan-a cells 15 B. Effects of GD on melanogenesis-related proteins 18 C. Inhibition of GD on the expression of PAX3 and SOX10 genes 21 D. Inhibition of GD on the expression of MITF, tyrosinase, TRP-1 genes 23 E. Inhibition of GD on both cell-free and cellular tyrosinase activity 28 F. Effects of GD on tyrosinase degradation 31 G. Effects of GD on melanosome-transfer-related proteins 34 H. Inhibition of GD on melanin biosynthesis in a reconstructed human skin model 39 IV. Discussion 42 References 46 Abstract in Korean 51Maste