臺灣博碩士論文加值系統

糖尿病患者中約有95%屬於第二型糖尿病，其主因多為胰島素阻抗而導致高血糖之情況。已有許多文獻證實桃金孃科 ( Myrtaceae family ) 植物具抗糖尿病之效用，本研究室先前以桃金孃科植物中的蓮霧幼果萃取物為材料，經分離、純化、鑑定與胰島素阻抗細胞模式試驗後，確認具有抗糖尿病潛力的活性化合物 Vescalagin 與 Gallic acid 。本研究進一步進行動物實驗，探討 Vescalagin與 Gallic acid 對以高果糖飼料誘導之高血糖、高胰島素血症糖尿病前期大鼠肝臟組織中胰島素阻抗與醣類代謝之影響，並探討其對改善糖尿病前期大鼠體內抗發炎與抗氧化之效果。
結果顯示，Vescalagin與 Gallic acid具有明顯降低高果糖飼料誘導糖尿病大鼠血糖之效果。西方墨點法分析結果顯示，在胰島素訊息傳遞部分， Vescalagin與 Gallic acid 可以增加胰島素受器( insulin receptor，IR )、胰島素受器受質( insulin receptor substrate-1，IRS-1 ) 、磷酸肌醇3激酶 ( Phosphatidylinositol-3 kinase，PI3K ) 、 AKT/蛋白激酶B ( AKT/Protein kinase B，AKT/PKB )、葡萄糖轉運蛋白2 ( glucose transporter-2，GLUT2 )等蛋白質之表現量。在醣類代謝部分， Vescalagin 與 Gallic acid 可以增加糖解作用酵素，包括 Hexokinase 、 Phosphofructokinase 與 Aldolase 之蛋白質表現量；肝醣合成酵素，如Glycogen synthase 之表現亦增加；然而糖質新生作用酵素，如 Frucotse-1,6-bisphosphatase 之表現會降低；而在磷酸五碳糖途徑酵素，如Glucose-6-phosphate dehydrogenase 之表現亦會增加。在抗發炎反應部分， Vescalagin 與 Gallic acid 降低了 NF-κB 路徑中的 NF-κB、COX-2、MCP-1 與 ICAM-1等發炎反應相關蛋白質之表現量。在抗氧化部分， Vescalagin 與 Gallic acid 則是增加了 SOD、Catalase 與 Glutathione peroxidase 酵素之活性，並且降低了體內脂質過氧化之程度。根據上述結果推測，蓮霧幼果分離物─ Vescalagin與 Gallic acid 可能藉由降低發炎反應及氧化壓力，因而改善高果糖飼料誘發之糖尿病前期大鼠胰島素阻抗現象，並調節醣類的正常代謝與利用。

Around 95% of the diabetic incidences belong to Type 2 diabetes mellitus (Type 2 DM). Type 2 DM is characterized as the insulin resistance and leaded to hyperglycemia. Previous studies have been confirmed the anti-diabetic effect of Myrtaceae family plants. Our laboratory previously found that vescalagin and gallic acid from wax apple, belongs to Maytaceae family plant and is one of important economic fruits in Taiwan and Oriental, fruit possessed potential on anti-diabetic in insulin resistance cell model. In the present study, we investigated the effect of vescalagin and gallic acid from pink wax apple on ameliorating carbohydrate metabolism, insulin resistance, anti-inflammation and antioxidation in vivo in high fructose diet (HFD)-induced prediabetic rats.
The results show that vescalagin and gallic acid from pink wax apple significantly decrease blood glucose level in HFD-induced prediabetic rats. The Western blot analysis reveals vescalagin and gallic acid promote the expression of insulin signaling-associated protein, including insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), AKT/Protein kinase B (AKT/ PKB) and glucose transporter-2 (GLUT2) in HFD rats. In the aspect of carbohydrate metabolism in liver, the results revealed that vescalagin and gallic acid increases the protein expression of glycolysis enzymes, including hexokinase, phosphofructokinase and aldolase; increases the protein expression of glycogen synthase; decreases the protein expressions of frucotse-1,6-bisphosphatase; increases the protein expression of glucose-6-phosphate dehydrogenase. Vescalagin and gallic acid decreased the expression of inflammatory factors, such as NF-κB, COX-2, MCP-1 and ICAM-1 I D in HFD rats. Vescalagin and gallic acid also increased the activity of anti-oxidative enzymes, including SOD, catalase and glutathione peroxidase, and decrease the production of MDA in lipid peroxidation.
In conclusion, vescalagin and gallic acid from pink wax apple fruit may decrease blood glucose level via their anti-inflammation and anti-oxidation stress abilities, therefore subsequently ameliorate the insulin resistance and carbohydrate metabolism in high fructose diet-induced prediabetic rats.

第一章、 前言 …………………………………………………………………1
第二章、 文獻回顧 ……………………………………………………………3
第一節 糖尿病 ………………………………………………………………3
一、 糖尿病流行病學………………………………………………………3
二、 糖尿病之定義…………………………………………………………3
三、 糖尿病分類……………………………………………………………6
第二節 胰島素 ………………………………………………………………10
一、 胰島素簡介…………………………………………………………10
二、 胰島素之作用………………………………………………………10
三、 胰島素於細胞層次之作用…………………………………………11
四、 胰島素訊息傳遞路徑………………………………………………12
五、 胰島素與三大營養素代謝之作用 ………………………………18
第三節 胰島素阻抗………………………………………………………20
一、 胰島素阻抗 ……………………………………………………20
二、 高果糖飲食與胰島素阻抗 ……………………………………20
三、 細胞內氧化壓力與胰島素阻抗………………………………22
四、 發炎反應與胰島素阻抗………………………………………24
第四節 體內抗氧化系統………………………………………………27
一、自由基與活性氧之簡介…………………………………………27
二、自由基對生物體之傷害…………………………………………28
三、體內的抗氧化系統………………………………………………29
第五節 抗糖尿病物質與酚類化合物………………………………33
一、 常見之抗糖尿病藥物 ……………………………………33
二、 酚酸化合物與糖尿病 ……………………………………35
第六節 蓮霧幼果分離物─Vescalagin與Gallic acid……………39
一、 蓮霧幼果分離物─Vescalagin與Gallic acid……………39
二、 Vescalagin …………………………………………………39
三、 Gallic acid…………………………………………………40
第三章、 研究動機與實驗架構…………………………………………42
第一節 研究動機與目的………………………………………………42
第二節 實驗架構…………………………………………………………43
第四章、 材料與方法……………………………………………………44
第一節 蓮霧幼果分離物─Vescalagin與Gallic acid之製備……44
第二節 實驗材料………………………………………………………46
第三節 實驗步驟與方法………………………………………………51
第五章、 結果……………………………………………………………63
第一節 蓮霧幼果分離物─ Vescalagin 對高果糖飼料誘導糖尿病前期大鼠
之影響……………………………………………………………63
一、 蓮霧幼果分離物─ Vescalagin 對高果糖飼料誘導糖尿病前期大鼠生理、生化之影響………………………………………………63
二、 蓮霧幼果分離物─ Vescalagin對高果糖飼料誘導糖尿病前期大鼠肝臟醣類代謝、胰島素訊息傳遞與發炎反應之影響…………70
第二節 蓮霧幼果分離物─ Gallic acid 對高果糖飼料誘導糖尿病前期大鼠之影響…………………………………………………………76
一、 蓮霧幼果分離物─ Gallic acid對高果糖飼料誘導糖尿病前期大鼠生理、生化之影響………………………………………………76
二、 蓮霧幼果分離物─ Gallic acid對高果糖飼料誘導糖尿病前期大鼠肝臟醣類代謝、胰島素訊息傳遞與發炎反應之影響…………83

第六章、 討論……………………………………………………89
第一節、 粉紅種蓮霧幼果分離物─Vescalagin對高果糖飼料誘導糖尿病前期大鼠之影響…………………………………………………………89
一、 蓮霧幼果分離物─ Vescalagin 對高果糖飼料誘導糖尿病前期大鼠生理、生化之響……………………………………………………89
二、 蓮霧幼果分離物─ Vescalagin對高果糖飼料誘導糖尿病前期大鼠肝臟蛋白質表現量之影響…………………………………………98
第二節、 粉紅種蓮霧幼果分離物─ Gallic acid 對高果糖飼料誘導糖尿病前期大鼠之影響……………………………………………………109
一、 蓮霧幼果分離物─ Gallic acid 對高果糖尿病前期大鼠生理、生化之影響……………………………………………………………109
二、 蓮霧幼果分離物─ Gallic acid對高果糖飼料誘導糖尿病前期大鼠肝臟蛋白質表現量之影響…………………………………………118
第七章、 結論…………………………….………………………129
第八章、 參考文獻………………………………………………162
第九章、 附件……………………………………………………180


圖次

圖 2-1、胰島素調節之代謝作用………………………………………………11
圖 2-2、胰島素訊息傳遞路徑…………………………………………………12
圖 2-3、葡萄糖轉運蛋白之基本構型…………………………………………15
圖 2-4、小腸上皮細胞葡萄糖運送方式………………………………………15
圖 2-5、氧化壓力活化絲胺酸激酶誘導胰島素阻抗…………………………22
圖 2-6、肝臟組織中番木鱉甘抗氧化之機制…………………………………26
圖 2-7、自由基與活性氧的生成途徑…………………………………………28
圖 2-8、Hydroxycinnamic acid 與 Hydroxybenzoic acid 之化學結構……...................................................36
圖 2-9、Vescalagin 結構圖……………………………………………….41
圖 2-10、Gallic acid 結構圖……………………………………………41
圖 3-1、實驗架構……………………………………………………………43
圖 5-1、蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導
糖尿病前期大鼠攝食量之影響…………………………131
圖 5-2、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠體重之變化………………………………132
圖 5-3、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠空腹血液葡萄糖濃度之變化……………133
圖 5-4、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠血中胰島素濃度之變化…………………134
圖 5-5、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠血中 C-胜肽濃度之變...………………135
圖 5-6、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠 HOMA-IR Index之變化…………………136
圖 5-7、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠血中果糖胺之變化…………………………137
圖 5-8、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠口服葡萄糖耐受性之變化…………………138
圖 5-9、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠口服葡萄糖耐受性曲線下面積之變化……139
圖5-10、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠血中游離脂肪酸之變化……………………140
圖5-11、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠血中三酸甘油酯濃度之變化………………141
圖5-12、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠肝臟體重之變化……………………………142
圖5-13、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠脂質過氧化物之變化………………………143
圖5-14、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠超氧歧化酶之變化…………………………144
圖5-15、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠觸媒之變化…………………………………145
圖5-16、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠麩胱甘肽過氧化酶之變化……………..…146
圖5-17、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠六碳醣激酶之表現…………………………147
圖5-18、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠磷酸果糖激酶之表現………………………148
圖5-19、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠醛縮酶之表現………………………………149
圖5-20、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠肝醣合成酶之表現…………………………150
圖5-21、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠果糖-1,6-二磷酸酶之表現………………151
圖5-22、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠葡萄糖-6-磷酸脫氫酶之表現……………152
圖5-23、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠胰島素受器之表現……………………………153
圖5-24、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠胰島素受器受質之表現………………………154
圖5-25、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠磷酸肌醇3激酶之表現………………………155
圖5-26、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠AKT/ PKB之表現……………………………156
圖5-27、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠葡萄糖轉運蛋白-2之表現………………….157
圖5-28、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠細胞核轉錄因子之表現………………………158
圖5-29、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠環氧化酶-2之表現……………………………159
圖5-30、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠單核球趨化蛋白-1之表現……………………160
圖5-31、管餵蓮霧幼果分離物─ Vescalagin 與 Gallic acid 對高果糖飼料誘導糖尿病前期大鼠細胞間黏附分子-1之表現……………………161
圖 7-1、蓮霧幼果分離物─ Vescalagin 與 Gallic aicd 改善高果糖飼料誘導糖尿病前期大鼠胰島素阻抗、醣類代謝、抗發炎可能之機制…162



表次

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表2-2、輔助性葡萄糖轉運蛋白種類………………………………………17

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