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研究生:王延融
研究生(外文):Yen-Jung Wang
論文名稱:紅茶中茶黃素抗糖化效果
論文名稱(外文):Antiglycation effects of theaflavins in black tea
指導教授:羅至佑
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:食品科學系研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
中文關鍵詞:糖化反應進階糖化終產物茶黃素狗腎上皮細胞株紅茶
外文關鍵詞:glycationadvanced glycation endproducts (AGEs)theaflavinsMadin-Darby canine kidney (MDCK) epithelial cellsblack tea
相關次數:
  • 被引用被引用:3
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糖化反應是一種非酵素反應,為蛋白質與還原糖所進行的反應。蛋白質的胺基會與醣類的羰基反應產生席夫氏鹼 (schiff base),席夫氏鹼經安瑪多列重組產生安瑪多列產物 (amadori products),進一步反應形成進階糖化終產物 (advanced glycation endproducts, AGEs),其在身體組織累積而成為糖尿病併發症惡化的主要因素。本篇研究將探討紅茶初萃物,經由液、液萃取和 LH-20 管柱分離純化出的三種茶黃素 theaflavin (TF1)、theaflavin monogallate (TF2) 和 theaflavin digallate (TF3),再進一步以 flash column 進行分析得到純度分別為 98.4%、99.9% 和 99.9% 之 TF1、TF2 和 TF3,並探討此三種化合物抑制進階糖化終產物形成的效果,另以氨基胍 (aminoguanidine, AG) 作為對照組。將 TF1、TF2 和 TF3 分別添加於牛血清蛋白 (bovine serum albumin, BSA) 與果糖之中,在 50℃ 下反應 24 小時之反應混合物中,結果顯示能抑制螢光性進階糖化終產物並有劑量效應,其 IC50 為 513.57 ± 5.38、239.75 ± 1.83 和 162.24 ± 6.09 M,而 AG 為 5751.25 ± 142.75 M。進一步探討其抑制機制,發現所有的茶黃素都能夠降低雙羰基化合物、果糖胺 (安瑪多列產物) 、蛋白質聚合、蛋白質羰基的形成,其中以 TF3 之抗糖化效果最佳,因此將以 TF3 進行後續之細胞抗糖化實驗。細胞實驗選用Madin-Darby canine kidney (MDCK) epithelial cells (狗的腎上皮細胞株),探討在高濃度葡萄糖 (30 mM glucose) 下進行培養時,TF3 對腎臟細胞之保護效果,同樣以 AG 作為對照組。結果表示,TF3 能顯著提高細胞存活率並顯著減少細胞內活性氧分子 (reactive oxygen species, ROS) 含量,而抗氧化機制中具有關鍵影響力之蛋白質 nuclear factor (erythroid derived 2) like 2 (Nrf2),隨著 ROS 的減少亦隨之下降,再者除了 Nrf2 抗氧化機制的下游蛋白質 superoxide dismutase 1 (SOD-1) 之表現量顯著下降,heme oxygenase-1 (HO-1) 蛋白質表現量亦有下降之趨勢,而對於 catalase (Cat) 和 receptor for advanced glycation endproducts (RAGE) 則無明顯變化。
Glycation is a nonenzymatic reaction starting with the reaction of protein and reducing sugar. Schiff base is generated from an amino group in protein and carbonyl group in sugar. The Schiff base then undergoes amadori rearrangement to form amadori products. The amadori products can further undergo a series of reactions to form advanced glycation endproducts (AGEs). AGEs accumulation in tissues and cells will cause diabetic complications. In this study three kinds of theaflavins were purified through liquid phase extraction and LH-20 column chromatography of black tea extract, including theaflavin (TF1), theaflavin monogallate (TF2), and theaflavin digallate (TF3). Furthermore, we have purified these compounds by flash column and the purity of TF1-3 was 98.4%, 99.9% and 99.9%, respectively. The first step was to examine inhibitory effects of TF1, TF2, and TF3 on AGEs formation. TF1, TF2, and TF3 were individually added in the reaction mixture which included bovine serum albumin (BSA) and fructose. The mixtures were incubated at 50℃ for 24 h and aminoguanidine (AG) was treated as a control. The fluorescent AGEs inhibitory effect was concentration-dependently through the addition of TF1, TF2, and TF3. The half maximal inhibitory concentration (IC50) on fluorescent AGEs of TF1, TF2, TF3, and AG was 513.57 ± 5.38, 239.75 ± 1.83, 162.24 ± 6.09, and 5751.25 ± 142.75 M, respectively. Furthermore, we have illustrated the possible mechanisms. We discovered that theaflavin prepared from black tea extract decreased the formation of dicarbonyl compounds, fructosamine (an amadori product), and carbonyl content on protein associated with the reduction of protein aggregation. TF3 showed the best antiglycation performance among the three theaflavins. Therefore TF3 was further investigated in the following cellular model. In cellular experiments, we chose Madin-Darby canine kidney (MDCK) epithelial cells to evaluate the protective effect of TF3 on high level glucose (30 mM) incubation. AG was used as a control group. The results showed that TF3 could not only significantly increase the cell viability but also decrease the intracellular reactive oxygen species (ROS) content. With the decrease of ROS, significantly down-regulated expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a key factor in the intracellular antioxidant mechanism occurred, also the superoxide dismutase 1 (SOD-1), a downstream protein of Nrf2 antioxidant mechanism, decreased significantly. In addition, the protein expression of heme oxygenase-1 (HO-1) showed a tendency of decline, but there was no significant change in catalase (Cat) and receptor for advanced glycation endproducts (RAGE).
摘要 I
謝誌 III
圖次 VIII
表次 IX
壹、前言 1
貳、文獻回顧 2
一、茶 2
1.緣起 2
2.加工方法 2
3.多酚化合物 3
4.生物活性 7
二、糖尿病 10
1.簡介 10
2.分類 10
3.糖尿病併發症 14
三、糖化反應 16
1.反應途徑 18
2.糖化反應產物-進階糖化終產物 21
3.食品中進階糖化終產物 23
4.進階糖化終產物接受器 25
四、抗糖化 26
1.體內抗糖化機轉 26
2.抗糖化物質 29
參、實驗目的 36
肆、實驗架構 37
伍、材料與方法 38
一、實驗材料 38
1.樣品來源 38
2.細胞株來源 38
二、化學藥品、溶劑與抗體 38
1.以下抗體購買自Abbiotec公司 (San Diego, USA) 38
2.以下抗體購買自Abcam公司 (Cambridge, UK) 38
3.以下藥品購買自Alfa Aesar公司 (Ward Hill, USA) 38
4.以下藥品購買自AppliChem公司 (New Taipei, Taiwan) 38
5.以下溶劑購買自Duksan Pure Chemicals公司 (Gyeonggi-do, South Korea) 38
6.以下藥品購買自G-Biosciences公司 (St. Louis, USA) 38
7.以下藥品購買自GE Healthcare公司 (Chicago, USA) 39
8.以下藥品購買自Hayashi Pure Chemical公司 (OSAKA, JP) 39
9.以下抗體購買自Invitrogen公司 (Carlsbad, USA) 39
10.以下藥品購買自J.T Baker公司 (Center Valley, USA) 39
11.以下藥品購買自Jackson ImmunoResearch公司 (West Grove, USA) 39
12.以下藥品購買自Merck公司 (Kenilworth, USA) 39
13.以下溶劑購買自MP biomedicals公司 (Santa Ana, USA) 39
14.以下抗體購買自OriGene公司 (Rockville, USA) 39
15.以下藥品購買自Santaku Chemical公司 (OSAKA, JP) 39
16.以下藥品購買自Shimakyu Chemical公司 (OSAKA, JP) 39
17.以下藥品購買自Sigma公司 (St. Louis, USA) 40
18.以下藥品購買自Toronto Research Chemicals公司 (Toronto, Canada) 40
19.以下藥品購買自ThermoFisher公司 (Waltham, USA) 40
20.以下溶劑購買自友和貿易股份有限公司 (New Taipei, Taiwan) 41
21.以下溶劑購買自虹御生技有限公司 (Chiayi, Taiwan) 41
三、儀器設備與耗材 41
1.以下儀器購買自Clubio公司 (Berlin, Germany) 41
2.以下耗材購買自Corning公司 (NY, USA) 41
3.以下儀器購買自Delta公司 (New Taipei, Taiwan) 41
4.以下儀器購買自DENG YNG Instruments公司 (New Taipei, Taiwan) 41
5.以下儀器購買自Firstek公司 (New Taipei, Taiwan) 41
6.以下儀器購買自GAU JIE公司 (Chiayi, Taiwan) 41
7.以下耗材購買自GE Healthcare公司 (Chicago, USA) 41
8.以下儀器購買自GE Healthcare公司 (Chicago, USA) 42
9.以下耗材購買自Gunster Biotech公司 (New Taipei, Taiwan) 42
10.以下儀器購買自Heidolph公司 (Schwabach, Germany) 42
11.以下儀器購買自HIPOINT公司 (Taichung, Taiwan) 42
12.以下儀器購買自HSIANGTAI公司 (New Taipei, Taiwan) 42
13.以下儀器購買自KINGMECH公司 (Taipei, Taiwan) 42
14.以下儀器購買自Major Science公司 (Saratoga, USA) 42
15.以下儀器購買自Marienfeld-Superior公司 (Lauda-Königshofen, Germany) 42
16.以下儀器購買自Panasonic Healthcare公司 (Tokyo, JP) 42
17.以下儀器購買自PANCHUM公司 (Kaohsiung, Taiwan) 42
18.以下儀器購買自Sartorius公司 (Goettingen, Germany) 42
19.以下儀器購買自TOMIN公司 (New Taipei, Taiwan) 43
20.以下儀器購買自ThermoFisher公司 (Waltham, USA) 43
21.以下儀器購買自Millpore公司 (MA, USA) 43
四、實驗方法 44
1.茶黃素樣品之製備 44
2.抗糖化活性評估 45
3.MDCK細胞株之培養方式 47
4.MDCK細胞株細胞存活率測定 48
5.MDCK細胞株活性氧分子含量測定 48
6.MDCK細胞株西方墨點法之操作 49
7.統計分析 51
陸、結果與討論 52
一、茶黃素樣品之純化與鑑定 52
1.紅茶初萃物分析 52
2.茶黃素分離 54
3.茶黃素鑑定 54
二、抗糖化活性 57
1.抑制螢光性進階糖化終產物之活性 57
2.安瑪多列產物 (果糖胺) 含量 57
3.蛋白質硫醇基含量 58
4.抑制第二型澱粉狀蛋白之活性 58
5.蛋白質羰基含量 58
6.活性雙羰基化合物含量 59
三、茶黃素對腎臟細胞保護效果評估 70
1.細胞存活率測定 70
2.細胞內氧化壓力測定 70
3.茶黃素對腎臟細胞蛋白質表現的影響 73
柒、結論 80
捌、參考文獻 81
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