臺灣博碩士論文加值系統

攝取過多食物常會造成肥胖、糖尿病、高血壓等慢性疾病，其發生之主要原因是因為醣類的羰基和蛋白質的胺基在體內進行糖化反應，最後形成進階糖化終產物 (advanced glycation end-products, AGEs)。AGEs 在體內累積會造成氧化壓力、發炎反應等現象，並伴隨著細胞或組織的受損，進而引發上述疾病發生。因此，找尋天然抗糖化食品來抵禦慢性疾病的發生是一個值得討論的議題。
綠茶是現今茶飲市場中消費量最大的飲品之一，綠茶是經過萎凋、殺菁、乾燥等步驟製成，因為未經發酵處理，所以又被稱作不發酵茶，保留了茶葉多種茶多酚和維生素。綠茶之兒茶素已被證實具有抗氧化、抗自由基等功效，然而其是否具備抗糖化能力則尚待闡明，因此本篇的研究目的即為探討綠茶之兒茶素對於糖化反應的抑制效果。
在兒茶素抑制螢光性AGEs活性實驗中，ECG 和 EGCG 之 IC50 值分別為446.60 ± 74.2 μM及315.79 ± 6.0 μM，其結果明顯比正對照組aminoguanidine (AG)(IC50值 6760.36 ± 72.3μM) 具有較佳抑制AGEs形成的能力。由 β-amyloid、Carbonyl、Fructosamine 結果來看，同樣是以ECG和 EGCG 對於上述糖化指標具有較佳抑制能力，降低的比率分別達到 66%、69%、47% 和 76%、70%、43%，由以上結果可得知 EGCG 的抗糖化能力是四種兒茶素中較為突出的，推測可能與其結構之沒食子酸酯和羥基數量有關。
經由先前的研究和本實驗結果可以推測綠茶不只具備抗氧化功效外，也具備了抑制糖化指標的能力，其中又以 EGCG 之抗糖化能力最為顯著。

Taking excess food often causes obesity, diabetes mellitus, hypertension and other chronic diseases. The main reason for the above is carbonhydrate’s carbonyl group and protein’s amino group occur glycation reaction and form advanced glycation end-products (AGEs). Accumulation of AGEs in the body would cause oxidative stress, inflammation and other phenomena accompanied by cell or tissues damage, and then trigger these diseases.Therefore, it’s worth to find natural anti-glycation foods against chronic diseases.
Green tea is one of the most consumed beverage in today's tea market.
Green tea was made through withering, blanching, drying and other steps. Because it has not been fermented so it is also called non-fermented tea, which preserves a variety of tea polyphenols and vitamins. Green tea’s catechins have been proved having anti-oxidation, anti-free radical and other benefits. However, whether catechins have anti-glycation ability remains to be elucidated. Thus, the aim of this study was to investigate catechins’s anti-glycation ability.
In AGEs inhibition test, the IC50 values for ECG and EGCG were 446.60 ± 74.2 μM and 315.79 ± 6.0 μM, respectively. It showed that these two were much better than postive-control aminoguanidine (AG) (IC50 value 6760.36 ± 72.3 μM) in suppression of AGEs formation. From the results, ECG and EGCG also demonstrated higher inhibitory effect on glycation marker β-amyloid, carbonyl and fructosamine, with the decreasing rates of 66%, 69%, 47% (ECG), and 76% 70% 43% (EGCG), respectively. Based on this results EGCG has the highest anti-glycation ability among the four catechins, presumably related to the structure of gallate and the number of hydroxyl group.
Based on the previous reports and the results of this study, we can speculate that green tea possesses not only the anti-oxidant effect but also the ability to inhibit the glycation index. Among them, EGCG has the best anti-glycation ability.

中文摘要 I
Abstract II
謝誌 III
目錄 IV
圖次 VI
表次 VII
壹、 前言 1
貳、 文獻回顧 2
一、 茶類概論 2
二、 茶多酚 (Tea polyphenols) 3
三、 糖化反應 (Glycation) 5
四、 糖尿病 (Diabetes mellitus) 10
五、 抗糖化作用 14
六、 天然物之抗糖化作用 15
參、 研究目的 16
肆、 實驗架構 17
伍、 材料與方法 18
一、 實驗原料 18
二、 化學藥品及溶劑 18
2.1 以下藥品購買於Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan) 18
2.2 以下藥品購買於Shimakyu Co., Ltd. (Osaka, Japan) 18
2.3 以下藥品購買於Sigma公司 (St. Louis, MO, USA) 18
2.4 以下藥品購買於Merck公司 (Darmstadt, Germany) 18
2.5 以下藥品購買於Santoku Chemical Industries Co., Ltd. (Tokyo, Japan) 19
2.6 以下藥品購買於聯工化學股份有限公司 (Hsinchu, Taiwan) 19
三、 儀器設備與耗材 20
3.1 儀器設備 20
3.2 耗材 20
四、 實驗方法 21
4.1 四種兒茶素樣品 (EC、EGC、ECG和EGCG) 體外抗糖化效果評估 …………………………………………………………………….21
4.2 統計分析 26
陸、 結果與討論 27
一、 四種兒茶素樣品 (EC、EGC、ECG和EGCG) 之體外抗糖化能力評估 ……………………………………………………………………….27
1. 抑制進階糖化終產物AGEs之活性 27
2. 抑制β-amyloid交聯結構之活性 30
3. 抑制羰基 (carbonyl) 化合物生成之效果 32
4. 抑制果糖胺 (fructosamine) 生成之效果 34
5. 保護硫醇基 (thiol) 之效果 36
6. 抑制雙羰基化合物 (dicarbonyl) 之效果 38
柒、 結論 42
捌、 參考文獻 43
圖次
圖一、兒茶素之結構。………..………………………….……………………..4
圖二、糖化反應路徑圖。………………………………………………………5
圖三、安瑪多立產物之降解作用。……………………………………………8
圖四、兩種AGEs之結構 (A) 螢光且交聯型AGEs； (B) 非螢光且非交聯型AGEs。……..……………………………….………………………………..9
圖五、高血糖引起之蛋白質糖化反應所造成的影響。…….…………….….12
圖六、AGEs與其受體RAGE相互作用導致糖尿病併發症之反應路徑。.…13
圖七、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制進階糖化終產物 (AGEs) 之活性比較。……...…………………..…………28
圖八、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制β-amyloid生成之影響。……...……………………………………………….31
圖九、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制羰基生成之影響。………………………………………………….…………33
圖十、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制果糖胺生成之影響。……...………………………………………….……….35
圖十一、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對硫醇基之保護效果。…….………………………………………………….…...37
圖十二、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制乙二醛生成之影響。…..………………………………………….………..39
圖十三、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制甲基乙二醛生成之影響。…………………………………….……………40
圖十四、EC、EGC、ECG和EGCG與對照組Aminoguanidine (AG) 對抑制3-葡萄糖醛酮生成之影響。……………………………………….………41
 
表次
表一、HPLC沖提梯度。….....……………………………………….………25
表二、四種兒茶素 (EC、EGC、ECG和EGCG) 與對照組Aminoguanidine (AG) 抑制進階糖化終產物 (AGEs) 之IC50。…………...…………………29

Abdallah, H. M., El-Bassossy, H., Mohamed, G. A., El-Halawany, A. M., Alshali, K. Z., & Banjar, Z. M. (2016). Phenolics from Garcinia mangostana inhibit advanced glycation endproducts formation: effect on amadori products, cross-linked structures and protein thiols. Molecules, 21(2), 251-265.
Ahmad, M. S. and N. Ahmed (2006). Antiglycation properties of aged garlic extract: possible role in prevention of diabetic complications. J Nutr 136(3), 796-799.
Ahmed, M. U., S. R. Thorpe and J. W. Baynes (1986). Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J Biol Chem 261(11), 4889-4894.
Ahmed, N. (2005). Advanced glycation endproducts--role in pathology of diabetic complications. Diabetes Res Clin Pract, 67(1), 3-21.
Ahmed, S., Rahman, A., Hasnain, A., Lalonde, M., Goldberg, V. M., & Haqqi, T. M. (2002). Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes. Free Radic Biol Med, 33(8), 1097-1105.
Alberti, K. G., & Zimmet, P. Z. (1998). Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med, 15(7), 539-553.
Azizan, N., Suter, M. A., Liu, Y., & Logsdon, C. D. (2017). RAGE maintains high levels of NFκB and oncogenic Kras activity in pancreatic cancer. Biochem Biophys Res Commun, 493(1), 592-597.
Basta, G., Schmidt, A. M., & De Caterina, R. (2004). Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes. Cardiovasc Res, 63(4), 582-592.
Baynes, J. W. (1991). Role of oxidative stress in development of complications in diabetes. Diabetes, 40(4), 405-412.
Bode, A. M., & Dong, Z. (2009). Epigallocatechin 3-gallate and green tea catechins: united they work, divided they fail. Cancer Prev Res, 2(6), 514-517.
Bokuchava, M. A., & Skobeleva, N. I. (1980). The biochemistry and technology of tea manufacture. Crit Rev Food Sci Nutr, 12(4), 303-370.
Bouma, B., L. M. Kroon-Batenburg, Y. P. Wu, B. Brunjes, G. Posthuma, O. Kranenburg, P. G. de Groot, E. E. Voest and M. F. Gebbink. (2003). Glycation induces formation of amyloid cross-beta structure in albumin. J Biol Chem, 278(43), 41810-41819.
Chen, H. J., Chen, Y. C., Hsiao, C. F., & Chen, P. F. (2015). Mass spectrometric analysis of glyoxal and methylglyoxal-induced modifications in human hemoglobin from poorly controlled type 2 diabetes mellitus patients. Chem Res Toxicol, 28(12), 2377-2389.
Chen, X.-Y., Huang, I. M., Hwang, L. S., Ho, C.-T., Li, S., & Lo, C.-Y. (2014). Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal. J Funct Foods, 8, 259-268.
Clark, A., Charge, S. B. P., Badman, M. K., MacArthur, D. A., & de Koning, E. J. P. (1996). Islet amyloid polypeptide: actions and role in the pathogenesis of diabetes. Biochem Soc Trans, 24(2), 594-599.
Coughlan, M. T., Cooper, M. E., & Thomas, M. C. (2007). Can you reduce your AGE?:Strategies to prevent AGE accumulation in diabetes. Drug Discovery Today: Ther Strategies, 4(1), 85-92.
Dalluge, J. J., & Nelson, B. C. (2000). Determination of tea catechins. J Chromatogr A, 881(1-2), 411-424.
Filipović, N., Stojević, Z., Mašek, T., Mikulec, Ž., & Prvanović, N. (2011). Relationship between fructosamine with serum protein, albumin and glucose concentrations in dairy ewes. Small Rumi Res, 96(1), 46-48.
Gkogkolou, P., & Bohm, M. (2012). Advanced glycation end products: Key players in skin aging? Dermatoendocrinol, 4(3), 259-270.
Golde, T. E., Eckman, C. B., & Younkin, S. G. (2000). Biochemical detection of Abeta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer's disease. Biochim Biophys Acta, 1502(1), 172-187.
Gonta, M., Duca, G., & Porubina, D. (2008). Establishment of the antioxidant/antiradical activity of the inhibitors using the dpph – radical. CJM ASM MD, 3(1), 118-126.
Han, M. K. (2003). Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic beta-cell damage. Exp Mol Med, 35(2), 136-139.
Henle, T. (2005). Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods. Amino Acids, 29(4), 313-322.
Hofmann, M. A., Drury, S., Fu, C., Qu, W., Taguchi, A., Lu, Y., Avila, C., Kambham, N., Bierhaus, A., Nawroth, P., Neurath, M. F., Slattery, T., Beach, D., McClary, J., Nagashima, M., Morser, J., Stern, D., Schmidt, A. M. (1999). RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell, 97(7), 889-901.
Huang, S. M., C. L. Hsu, H. C. Chuang, P. H. Shih, C. H. Wu and G. C. Yen (2008). Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells. Neurotoxicology, 29(6), 1016-1022.
Johnson, R. N., Metcalf, P. A., & Baker, J. R. (1983). Fructosamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin Chim Acta, 127(1), 87-95.
Kumar, S., & Pandey, A. K. (2013). Chemistry and biological activities of flavonoids: an overview. SCI WORLD J, 2013, 162750.
Li, N., Alam, J., Venkatesan, M. I., Eiguren-Fernandez, A., Schmitz, D., Di Stefano, E., Slaughter, N., Killeen, E., Wang, X., Huang, A., Wang, M., Miguel, A.H., Cho, A., Sioutas, C., Nel, A. E. (2004). Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. J Immunol, 173(5), 3467-3481.
Lo, C. Y., Li, S., Tan, D., Pan, M. H., Sang, S., & Ho, C. T. (2006). Trapping reactions of reactive carbonyl species with tea polyphenols in simulated physiological conditions. Mol Nutr Food Res, 50(12), 1118-1128.
Martin, D., Rojo, A. I., Salinas, M., Diaz, R., Gallardo, G., Alam, J., De Galarreta, C.M., Cuadrado, A. (2003). Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3 kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Bio Chem, 279(10), 8919-8929.
Martins, S. I. F. S., Jongen, W. M. F., & Boekel, M. A. J. S. v. (2001). A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci Technol, 11, 364-373.
Matsuda, H., Wang, T., Managi, H., & Yoshikawa, M. (2003). Structural requirements of flavonoids for inhibition of protein glycation and radical scavenging activities. Bioorg Med Chem, 11(24), 5317-5323.
McCance, D. R., D. G. Dyer, J. A. Dunn, K. E. Bailie, S. R. Thorpe, J. W. Baynes and T. J. Lyons (1993). Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J Clin Invest, 91(6), 2470-2478.
McLellan, A. C., Thornalley, P. J., Benn, J., & Sonksen, P. H. (1994). Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin Sci (Lond), 87(1), 21-29.
Montagnani, M. (2008). Diabetic cardiomyopathy: how much does it depend on AGE? Br J Pharmacol, 154(4), 725-726.
Münch, G., S. Mayer, J. Michaelis, A. R. Hipkiss, P. Riederer, R. Müller, A. Neumann, R. Schinzel and A. M. Cunningham. (1997). Influence of advanced glycation end-products and AGE-inhibitors on nucleation-dependent polymerization of β-amyloid peptide. Biochim Biophys Acta, Mol Basis Dis, 1360(1), 17-29.
Nakagawa, T., Yokozawa, T., Terasawa, K., Shu, S., & Juneja, L. R. (2002). Protective activity of green tea against free radical- and glucose-mediated protein damage. J Agric Food Chem, 50(8), 2418-2422.
Naponelli, V., Ramazzina, I., Lenzi, C., Bettuzzi, S., Rizzi F. (2017). Green tea catechins for prostate cancer prevention: present achievements and future challenges. Antioxidants, 6(2).
Navarrete Santos, A., Jacobs, K., Simm, A., Glaubitz, N., Horstkorte, R., & Hofmann, B. (2017). Dicarbonyls induce senescence of human vascular endothelial cells. Mech Ageing Dev, 166, 24-32.
Nowotny, K., Jung, T., Hohn, A., Weber, D., & Grune, T. (2015). Advanced glycation end products and oxidative stress in type 2 diabetes mellitus. Biomolecules, 5(1), 194-222.
Odani, H., Shinzato, T., Matsumoto, Y., Usami, J., & Maeda, K. (1999). Increase in three α,β-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced maillard reaction. Biochem Biophys Res Commun, 256(1), 89-93.
Olas, B., B. Wachowicz, A. Stochmal, P. Nowak, W. Oleszek, M. Kedzierska, A. Jeziorski, A. Tomczak and J. Piekarski (2008). Studies on antioxidant properties of polyphenol-rich extract from berries of aronia melanocarpa in blood platelets. J Physiol Pharmacol, 59(4), 823-835.
Pansarasa, O., Bertorelli, L., Vecchiet, J., Felzani, G., & Marzatico, F. (1999). Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle. Free Radic Biol Med, 27(5-6), 617-622.
Parlak, E. S., Alisik, M., Karalezli, A., Sayilir, A. G., Bastug, S., Er, M., Hasanoglu, H. C, Erel, O. (2017). Are the thiol/disulfide redox status and HDL cholesterol levels associated with pulmonary embolism?: Thiol/disulfide redox status in pulmonary embolism. Clin Biochem, 50(18), 1020-1024.
Poulsen, M. W., Hedegaard, R. V., Andersen, J. M., de Courten, B., Bugel, S., Nielsen, J., Skibsted, L.H., Dragsted, L. O. (2013). Advanced glycation endproducts in food and their effects on health. Food Chem Toxicol, 60, 10-37.
Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J. (2005). Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. J Neurosci, 25(38), 8807-8814.
Rodríguez-Segade, S., Rodríguez, J., & Camiña, F. (2017). Corrected fructosamine improves both correlation with HbA1C and diagnostic performance. Clin. Biochem, 50(3), 110-115.
Russell, P., Garland, D., Zigler, J. S., Jr., Meakin, S. O., Tsui, L. C., & Breitman, M. L. (1987). Aging effects of vitamin C on a human lens protein produced in vitro. FASEB J, 1(1), 32-35.
Sang, S., Shao, X., Bai, N., Lo, C.-Y., Yang, C. S., & Ho, C.-T. (2007). Tea polyphenol (−)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol, 20(12), 1862-1870.
Schonberg, A., & Moubacher, R. (1952). The strecker degradation of α-amino acids. Chemical Reviews, 50(2), 261-277.
Senthil Kumaran, V., Arulmathi, K., Srividhya, R., & Kalaiselvi, P. (2008). Repletion of antioxidant status by EGCG and retardation of oxidative damage induced macromolecular anomalies in aged rats. Exp Gerontol, 43(3), 176-183.
Singh, V. P., Bali, A., Singh, N., & Jaggi, A. S. (2014). Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol, 18(1), 1-14.
Sonoda, J. I., Ikeda, R., Baba, Y., Narumi, K., Kawachi, A., Tomishige, E., Nishihara, K., Takeda, Y., Yamada, K., Sato, K., Motoya, T. (2014). Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression. Exp Ther Med, 8(1), 59-63.
Soulis-Liparota, T., Cooper, M., Papazoglou, D., Clarke, B., & Jerums, G. (1991). Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes, 40(10), 1328-1334.
Stadtman, E. R., & Berlett, B. S. (1998). Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev, 30(2), 225-243.
Thilavech, T., Ngamukote, S., Abeywardena, M., & Adisakwattana, S. (2015). Protective effects of cyanidin-3-rutinoside against monosaccharides-induced protein glycation and oxidation. Int J Biol Macromol, 75, 515-520.
Uribarri, J., & Tuttle, K. R. (2006). Advanced glycation end products and nephrotoxicity of high-protein diets. Clin J Am Soc Nephrol, 1(6), 1293-1299.
Vassar, R. (2004). BACE1: the beta-secretase enzyme in Alzheimer's disease. J Mol Neurosci, 23(1-2), 105-114.
Wang, S. H., Chang, J. C., Pokkaew, R., Lee, J. F., & Chiou, R. Y. (2011). Modified fast procedure for the detection and screening of antiglycative phytochemicals. J Agric Food Chem, 59(13), 6906-6912.
Wang, Y., & Ho, C. T. (2012). Flavour chemistry of methylglyoxal and glyoxal. Chem Soc Rev, 41(11), 4140-4149.
Wu, C. H., Huang, S. M., Lin, J. A., & Yen, G. C. (2011). Inhibition of advanced glycation endproduct formation by foodstuffs. Food Funct, 2(5), 224-234.
Wu, C. H. and Yen, G. C. (2005). Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts. J Agric Food Chem, 53(8), 3167-3173.
Wu, L., & Juurlink, B. H. (2002). Increased methylglyoxal and oxidative stress in hypertensive rat vascular smooth muscle cells. Hypertension, 39(3), 809-814.
Yamabe, N., K. S. Kang, J. M. Hur and T. Yokozawa (2009). Matcha, a powdered green tea, ameliorates the progression of renal and hepatic damage in type 2 diabetic OLETF rats. J Med Food, 12(4), 714-721.
Yan, S. D., Zhu, H., Zhu, A., Golabek, A., Du, H., Roher, A., Yu, J., Soto, C., Schmidt, A. M., Stern, D., Kindy, M. (2000). Receptor-dependent cell stress and amyloid accumulation in systemic amyloidosis. Nat Med, 6(6), 643-651.
Yang, C. S., Wang, X., Lu, G., & Picinich, S. C. (2009). Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. Nat Rev Cancer, 9(6), 429-439.
Yao, D., & Brownlee, M. (2010). Hyperglycemia-induced reactive oxygen species increase expression of the receptor for advanced glycation end products (RAGE) and RAGE ligands. Diabetes, 59(1), 249-255.
阮逸明、張如華、張連發、Juan, I. M.、張如華、張連發。1988。不同烘焙溫度與時間對包種茶化學成分與品質之影響。台灣茶葉研究彙報8： 71-82。
林木連、蔡右任、張清寬、陳國任、楊盛勳、陳英玲、賴正南、陳玄、張如華。2003。各種茶類的製造方法。台灣的茶葉。頁92-113。
蘇桂玉。2014。利用高效能液相層析儀分析不同浸泡溫度下茶湯的成分變