臺灣博碩士論文加值系統

文獻指出微脂體包覆活性物質可增進其生物利用率，已知薑黃萃取 薑黃素為脂溶性多酚類物質，本研究以精鍊磷脂質搭配薄膜水合法，製 備搭載薑黃素的微脂體，利用加速性試驗、紫外光破壞試驗以及細胞培 養試驗，探討不同均質條件製備微脂體安定性以及抗氧化活性。結果顯 示，鐳射粒度分布儀測量微脂體粒徑與粒徑分布，在儲存 28天後，低轉 速及中轉速平均粒徑變動不大，又以高轉速呈現較不穩定，而在不同劑 量的UVB照射下，則因劑量提升造成粒徑變大趨勢。在細胞毒性試驗中， 細胞在不同濃度薑黃素經處理24、48 小時，IC50值濃度為 177.7ug/ml、 59.49ug/ml，在不同濃度薑黃素的微脂體，IC50 值濃度為 250.4ug/ml、 80.93ug/ml，兩者具有顯著差異，顯示出微脂體包覆薑黃素後有利於細 胞提高細胞存活率。在活性氧測試中，與對照組 H2O2 比較，添加 H2O2 分別與薑黃素及搭載薑黃素的微脂體共同處理，ROS 生成量明顯下降很多 ， 有 助 於 減 緩 H2O2 的氧化壓力達到保護細胞效果。

Research indicates that the liposomes systems as a carrier can improve the biological efficiency. It is known that curcumin extracted from turmeric is a fat-soluble polyphenolic substance, in this study, the thin-film hydration method was used to encapsulating curcumin by refined phospholipids to liposomes and the method was conducted to evaluate the stability of the liposomes and the activity of antioxidant activity by an accelerated test, UVB irradiation and cell culture assay. The instrument of laser diffraction particle size analyzer was used to measure the particles size and the distribution of the liposomes. After 28 days of storage, the average particle size showed insignificant differences between low spinning speed and medium spinning speed, while unstable particle size showed under high spinning speed. However, the particle size showed a dose dependent UVB irradiation that it will decrease in higher UVB irradiation. In cytotoxicity assay, the concentration of IC50 was 177.7 ug/ml and 59.49 ug/ml when the cells were treated with different concentrations of curcumin for 24 and 48 hours, and the concentrations of curcumin loading liposomes in different concentrations were 250.4 ug/ml and 80.93 ug/ml. As a result of the above, the significant differences between curcumin and curcumin-loaded liposomes elucidated that, it is beneficial for cells to increase cell viability after liposomes are coated with curcumin. In the reactive oxygen species assay, they were co-treated with curcumin and curcumin-loaded liposomes. The amount of ROS production was significantly reduced, which helped to reduce the oxidation pressure to protect cell. In the reactive oxygen species (ROS) assay, the co-treatment between H2O2 with curcumin and curcumin-loaded liposomes demonstrated a significant reduced on the amount of ROS production compare to the control. This result explained that curcumin and curcumin-loaded liposomes able to protect the cells from the oxidation stress.

中文摘要 1
Abstract II
誌謝 IV
目錄 V
第一章 前言 1
1.1 研究背景 1
1.2研究目的 1
1.3 研究架構 1
第二章 文獻回顧 4
2.1微脂體 4
2.1.1微脂體簡介 4
2.1.2 微脂體分類 5
2.1.3 微脂體配置方法 5
2.1.4 影響微脂體穩定度因子 7
2.1.5 微脂體參與細胞作用機制 7
2.2巨噬細胞及抗氧化能力 8
2.2.1 巨噬細胞(macrophage)功能 8
2.2.2 自由基(free radical) 9
2.2.3 抗氧化劑功效 10
2.3薑黃素 10
2.3.1 薑黃素簡介 10
2.3.2 多酚類化合物 11
2.3.3 薑黃醫療應用 11
第三章 材料與方法 13
3.1 實驗材料 13
3.1.1 儀器設備 13
3.1.2 試劑及藥品 14
3.1.3細胞品系 14
3.2實驗方法 15
3.2.1 微脂體製備 15
3.2.1.1薑黃素純化萃取 15
3.2.1.2磷脂質精煉 16
3.2.1.3 薑黃微脂體配製 17
3.2.2 微脂體裝載薑黃素安定性評估 17
3.2.2.1 加速性試驗 17
3.2.2.2紫外光(UVB)破壞試驗 18
3.2.3細胞活性試驗 18
3.2.3.1細胞培養基配製 18
3.2.3.2緩衝溶液的配製 (PBS) 18
3.2.3.3細胞及解凍活化與保存 19
3.2.3.4 RAW264.7小鼠巨噬細胞培養 19
3.2.3.5 RAW264.7巨噬細胞培養繼代 19
3.2.3.6細胞計數 19
3.2.3.7 MTT試劑溶液體配製 20
3.2.3.8 MTT試驗細胞前處理 20
3.2.3.9細胞存活率檢測 (MTT) 20
3.2.3.10 ROS試驗細胞前處理 21
3.2.3.11 RAW264.7細胞抗氧化能力 (ROS) 21
3.2.3.12 IC50濃度計算 22
第四章 結果 23
4.1薑黃素搭載微脂體製備方法之存放安定性影響 23
4.1.1加速性試驗評估 23
4.1.2紫外光破壞試驗 29
4.2 體外細胞試驗 35
4.2.1 RAW264.7細胞存活率分析 (MTT) 35
4.2.2 活性氧測試 (ROS) 36
第五章 討論 37
參考文獻 39
作者介紹 44

劉得任. (2000). 微卡計於液膜交互作用力量測之應用----微脂粒系統穩定度之探討. 國立中央大學圖書館,
林名薇. (2013). 探討 Eupafolin 抑制脂多醣體誘導 RAW 264.7 巨噬細胞發炎反應之效用與作用機制. 臺灣大學解剖學暨細胞生物學研究所學位論文, 1-64.
林欣榜. (2002). 植物多酚類的機能性及其利用. 食品工業, 34(8), 39-47.
王偉丞. (2001). DPPC/DHDP 混合微脂粒的物理穩定性之研究. 國立成功大學化學工程學系碩 士論文.
邱智信. (2005). 不同磷脂質包覆維生素 E 之微脂粒系統特性研究.
陳美鈴. (2003). 局部用維他命 A 酸微脂立劑型之組成配方及穿皮動態之體外研究. National Taiwan University

Allen, T. M. (1998). Liposomal drug formulations. Rationale for development and what we can expect for the future. Drugs, 56(5), 747-756.
Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007). Bioavailability of curcumin: problems and promises. Molecular pharmaceutics, 4(6), 807-818.
Burhans, W. C., & Heintz, N. H. (2009). The cell cycle is a redox cycle: linking phase-specific targets to cell fate. Free Radical Biology and Medicine, 47(9), 1282-1293.
Castile, J. D., & Taylor, K. M. G. (1999). Factors affecting the size distribution of liposomes produced by freeze–thaw extrusion. International Journal of Pharmaceutics, 188(1), 87-95. Chen, Y.-R., & Tan, T.-H. (1998). Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway by curcumin. Oncogene, 17(2), 173.
Curtin, J. F., Donovan, M., & Cotter, T. G. (2002). Regulation and measurement of oxidative stress in apoptosis. Journal of immunological methods, 265(1-2), 49-72.
Deshpande, P. P., Biswas, S., & Torchilin, V. P. (2013). Current trends in the use of liposomes for tumor targeting. Nanomedicine, 8(9), 1509-1528.
Dhillon, N., Aggarwal, B. B., Newman, R. A., Wolff, R. A., Kunnumakkara, A. B., Abbruzzese, J. L., Kurzrock, R. (2008). Phase II trial of curcumin in patients with advanced pancreatic cancer. Clinical Cancer Research, 14(14), 4491-4499.
Fernandes-Alnemri, T., Armstrong, R. C., Krebs, J., Srinivasula, S. M., Wang, L., Bullrich, F., Litwack, G. (1996). In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proceedings of the National Academy of Sciences, 93(15), 7464-7469.
Forman, H. J., & Torres, M. (2002). Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. American journal of respiratory and critical care medicine, 166(supplement_1), S4-S8.
Gupta, S. C., Kismali, G., & Aggarwal, B. B. (2013). Curcumin, a component of turmeric: from farm to pharmacy. Biofactors, 39(1), 2-13.
Hollman, P., & Katan, M. (1997). Absorption, metabolism and health effects of dietary flavonoids in man. Biomedicine & Pharmacotherapy, 51(8), 305-310.
Hong, R.-L., Spohn, W. H., & Hung, M.-C. (1999). Curcumin Inhibits Tyrosine Kinase Activity of p185 neu and Also Depletes p185 neu. Clinical Cancer Research, 5(7), 1884-1891.
Korutla, L., Cheung, J. Y., Medelsohn, J., & Kumar, R. (1995). Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin. Carcinogenesis, 16(8), 1741-1745.
Kwon, Y., Malik, M., & Magnuson, B. A. (2004). Inhibition of colonic aberrant crypt foci by curcumin in rats is affected by age. Nutrition and cancer, 48(1), 37-43.
Larrauri, J. A., Rupérez, P., & Saura-Calixto, F. (1997). Effect of drying temperature on the stability of polyphenols and antioxidant activity of red grape pomace peels. Journal of agricultural and food chemistry, 45(4), 1390-1393.
Makino, K., Yamada, T., Kimura, M., Oka, T., Ohshima, H., & Kondo, T. (1991). Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data. Biophysical Chemistry, 41(2), 175-183.
Montaner, L. J., da Silva, R. P., Sun, J., Sutterwala, S., Hollinshead, M., Vaux, D., & Gordon, S. (1999). Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-γ or IL-10. The Journal of Immunology, 162(8), 4606-4613.
Nathan, C., & Shiloh, M. U. (2000). Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proceedings of the National Academy of Sciences, 97(16), 8841-8848.
Parsegian, V. A. (2005). Van der Waals forces: a handbook for biologists, chemists, engineers, and physicists: Cambridge University Press.
Pillai, S., Oresajo, C., & Hayward, J. (2005). Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation‐induced matrix degradation–a review. International journal of cosmetic science, 27(1), 17-34.
Polasa, K., Naidu, A., Ravindranath, I., & Krishnaswamy, K. (2004). Inhibition of B (a) P induced strand breaks in presence of curcumin. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 557(2), 203-213.
Rajagopalan, S., Meng, X. P., Ramasamy, S., Harrison, D. G., & Galis, Z. S. (1996). Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. The Journal of clinical investigation, 98(11), 2572-2579.
Rashmi, R., Santhosh Kumar, T., & Karunagaran, D. (2003). Human colon cancer cells differ in their sensitivity to curcumin‐induced apoptosis and heat shock protects them by inhibiting the release of apoptosis‐inducing factor and caspases. FEBS letters, 538(1-3), 19-24.
Samad, A., Sultana, Y., & Aqil, M. (2007). Liposomal drug delivery systems: an update review. Current drug delivery, 4(4), 297-305.
Sessa, G., & Weissmann, G. (1968). Phospholipid spherules (liposomes) as a model for biological membranes. J Lipid Res, 9(3), 310-318.
Singhal, P. C., Bhaskaran, M., Patel, J., Patel, K., Kasinath, B. S., Duraisamy, S., Kapasi, A. A. (2002). Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis. The Journal of Immunology, 168(8), 4025-4033.
Singhal, P. C., Reddy, K., Ding, G., Kapasi, A., Franki, N., Ranjan, R., . . . Gibbons, N. (1999). Ethanol-induced macrophage apoptosis: the role of TGF-β. The Journal of Immunology, 162(5), 3031-3036.
Smith, A. M., Rahman, F. Z., Hayee, B. H., Graham, S. J., Marks, D. J., Sewell, G. W., Gloger, I. S. (2009). Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease. Journal of Experimental Medicine, 206(9), 1883-1897.
Torchilin, V., & Weissig, V. (2003). Liposomes: a practical approach: Oxford University Press.
Tu, C. X., Lin, M., Lu, S. S., Qi, X. Y., Zhang, R. X., & Zhang, Y. Y. (2012). Curcumin inhibits melanogenesis in human melanocytes. Phytotherapy Research, 26(2), 174-179.
Villanueva, J., Lee, S., Giannini, E. H., Graham, T. B., Passo, M. H., Filipovich, A., & Grom, A. A. (2004). Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome. Arthritis Res Ther, 7(1), R30.
Walde, P., & Ichikawa, S. (2001). Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomolecular Engineering, 18(4), 143-177.
Webb, M., Harasym, T., Masin, D., Bally, M., & Mayer, L. (1995). Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models. British journal of cancer, 72(4), 896.
Witschi, H.(1986). Enhanced tumour development by butylated hydroxytoluene (BHT) in the liver, lung and gastro-intestinal tract. Food and chemical toxicology, 24(10-11), 1127-1130.
Woo, J.-H., Kim, Y.-H., Choi, Y.-J., Kim, D.-G., Lee, K.-S., Bae, J. H., Lee, Y. H. (2003). Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-X L and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis, 24(7), 1199-1208.
Yin, M., Faustman, C., Riesen, J., & Williams, S. (1993). α‐Tocopherol and ascorbate delay oxymyoglobin and phospholipid oxidation in vitro. Journal of Food Science, 58(6), 1273-1276.