吳瑞碧臺灣大學：食品科技研究所吳寧容Wu, Ning-JungNing-JungWu2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/56357糖尿病是現今常見的一種慢性代謝疾病，隨著生活品質的提昇及物質充裕，糖尿病病患的比例也日益增加。根據世界衛生組織預測，到西元 2025 年全球罹患糖尿病的人數將達到三億人。番石榴（Psidium guajava Linn.）屬桃金孃科（Myrtaceae），本省民間常將野生番石榴果實或葉片曬乾熬湯飲用，作為降火氣、清血、降血糖之用，是坊間盛行的糖尿病藥草療法。本研究係以低劑量 Streptozotocin（65 mg/Kg BW）與胰臟保護劑 Nicotinamide（230 mg/Kg BW）注射 Sprague-Dawley 大白鼠，使產生類似第二型糖尿病的動物模式，探討番石榴（Psidium guajava Linn.）葉部之水與乙醇萃取物的降血糖效果，及其對肝臟碳水化合物代謝之影響。結果顯示，在急性試驗中，番石榴葉水或醇萃物皆具有顯著延緩血糖上升的效果，且高劑量組（400 mg/Kg BW）效果優於低劑量組（200 mg/Kg BW）。而在長期灌食實驗中，期間於第四、六週進行口服葡萄糖耐受性試驗，結果顯示番石榴葉（灌食劑量 400 mg/Kg BW）水萃物或醇萃物均能明顯改善糖尿病鼠的葡萄糖耐受性；試驗動物於第六週犧牲後測其肝臟中碳水化合物代謝酵素活性變化，結果顯示長期灌食番石榴葉水萃物能活化肝臟 hexokinase（HKase）、phosphofructokinase（PFKase）、glucose-6-phosphate dehydrogenas（G6PDHase）活性及降低 fructose-1,6-bisphosphatase（F1,6BPase）、Glucose -6-phosphatase（G6Pase）活性，顯示其能促進葡萄糖進入 glycolysis pathway 及 pentose monophosphate shunt 並減少肝臟葡萄糖的釋放而降低血糖；灌食番石榴葉乙醇萃取物之糖尿病大鼠只有 HKase 與 G6PDHase 活性較糖尿病組高，而 PFKase、 F1,6BPase 及 G6Pase 活性與糖尿病組並沒有顯著差異（p<0.05）。分析管餵六週後，糖尿病大鼠的骨骼肌及肝臟細胞葡萄糖攝入能力及肝醣合成能力的變化，結果顯示管餵葉粗萃物並不會促進骨骼肌對葡萄糖的攝入能力，但能增加肝臟細胞對葡萄糖的攝入能力並促進肝醣合成。綜合所得的結果得知，番石榴葉萃出物具顯著的降血糖效果，且其降血糖效果乃是藉由改變其肝臟碳水化合物代謝酵素活性，並增強糖尿病大白鼠肝臟對葡萄糖的攝入及肝醣合成能力，因而降低糖尿病大白鼠的高血糖現象。Diabetes mellitus (DM) is one of the major diseases that human being eagerly wants to overcome. In 2006, DM was the fourth of top ten causes of death in Taiwan and more than 95% are type 2 DM. Guava (Psidium guajava Linn.) is one of the most important economic fruit in Taiwan. Leaves and fruit of guava are believed to have hypoglycemic effects which have been popularly employed in traditional DM treatment in Taiwan. However, there is insufficient scientific evidence for guava to cure DM, especially type 2 DM. In the present study, we injected low-dose streptozotocin (STZ) plus nicotinamide (NA) into Sprague-Dawley (SD) rats to induce Type 2 diabetes and to evaluate the antihyperglycemic effect and the carbohydrate metabolism of water and ethanol extracts from guava leaves. The study contains two parts, the acute and the long term experiments. In the acute experiment, the water or ethanol extracts of guava leaves were prepared and in the oral glucose tolerance test (OGTT) was performed to evaluate the hypoglycemic effects. In the long term experiment, we investigated the effect of aqueous or ethanol extracts from guava leaves in STZ–NA induced diabetic rats daily administered for 6 weeks. Results of the acute experiment showed that plasma glucose levels of the diabetic rats administered with 200 or 400 mg/kg BW of aqueous or ethanol guava leaf extracts were lower than the diabetic control group (p<0.05). This indicated that aqueous or ethanol extracts from guava leaves could effectively delay the rise of plasma glucose after feeding in diabetic rats. Diabetic rats administered with 400 mg/kg BW of aqueous or ethanol guava leaf extracts significantly reduced plasma glucose than 200 mg/kg BW. Therefore, aqueous or ethanol extracts from guava leaves with dosage of 400 mg/kg BW were used to carry out the long term experiments. The result of the long term experiments, showed that the diabetes groups fed with 400 mg/kg BW of aqueous or ethanol guava leaf extracts presented a significantly reduction of blood sugar level comparing to diabetes and normal groups (both fed with saline) and could delay the elevation of plasma glucose during the OGTT test after the fourth and sixth weeks. The rats were sacrificed at the 6 weeks and the carbohydrate metabolism enzyme activities in liver were measured. The activities of hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenas in diabetic rats fed with aqueous guava leaf extracts were higher than those of diabetic control rats (p<0.05), while the activity of fructose-1, 6-bisphosphatase and glucose-6-phosphatase was markedly lower than diabetic control rats (p<0.05). In addition, the activities of hexokinase and glucose-6-phosphate dehydrogenase in diabetic rats fed with ethanol guava leaf extracts were higher than diabetic control rats, while the activities of phosphofructokinase, fructose-1, 6-bisphosphatase, and glucose-6-phosphatase were no different with each group. The blood glucose reduction ability of extracts from guava leaves in diabetic rats may be related with the activity of hepatic carbohydrate metabolic enzymes. In additional, the abilities of glucose uptake and glycogen synthesis are significantly promoted in hepatocytes of diabetic rats treated with aqueous or ethanol extracts from guava leaves. These results indicate that extracts of guava leaves may regulate the plasma glucose in STZ–NA induced type 2 diabetic rats.中文摘要..................................................i 英文摘要.................................................ii 目錄.....................................................iv 表目錄.................................................viii 圖目錄...................................................ix 第一章 前言...............................................1 第二章 文獻整理...........................................2 第一節 番石榴.............................................2 一、番石榴簡介............................................2 二、番石榴葉成分之研究....................................2 三、番石榴抗糖尿病或降血糖之研究..........................3 四、番石榴抗糖尿病成分之研究..............................3 第二節 糖尿病.............................................4 一、糖尿病簡介............................................4 二、糖尿病的典型症狀......................................5 三、糖尿病及血糖恆定失調的診斷標準........................5 四、糖尿病分類............................................8 五、糖尿病形成的原因.....................................11 六、糖尿病的併發症.......................................12 第三節 胰島素訊息傳遞與作用..............................14 一、胰島素...............................................14 二、胰島素受體（Insulin receptor, IR）...................14 三、胰島素受體基質（Insulin receptor substrate, IRS）....14 四、磷酸肌醇3激酶（phosphatidylinositol-3-OH-kinase）....15 五、葡萄糖轉運蛋白（Glucose transporter, GLUT）..........15 六、胰島素在細胞層次之作用...............................17 七、胰島素阻抗...........................................17 第四節 糖尿病研究之動物模式..............................18 一、研究糖尿病的動物模式.................................18 二、Streptozotocin (STZ)化學誘導糖尿病動物模式...........19 第三章 研究動機與目的及實驗架構..........................23 第一節 研究動機與目的....................................23 第二節 實驗架構..........................................25 第四章 材料與方法........................................27 第一節 番石榴(Psidium guajava Linn.)葉粗萃物之製備.......27 一、實驗材料.............................................27 （一）原料...............................................27 （二）實驗藥品與試劑.....................................27 （三）實驗儀器...........................................27 二、萃取方法和步驟.......................................28 （一）樣品製備...........................................28 （二）樣品蛋白質含量測定.................................28 （三）樣品總醣含量測定...................................29 （四）樣品總酚含量測定...................................29 第二節 急性實驗：探討急性灌食番石榴葉粗萃物對 STZ-NA 誘發之第二型糖尿病大鼠葡萄糖耐受性的影響.......................30 一、實驗流程.............................................30 二、實驗材料.............................................31 （一）樣品來源...........................................31 （二）實驗動物...........................................31 （三）動物飼料...........................................31 （四）實驗藥品與試劑.....................................31 （五）實驗儀器...........................................31 三、實驗步驟和方法.......................................32 （一）動物飼養...........................................32 （二）動物分組及誘導.....................................32 （三）急性灌食番石榴葉之水及乙醇萃出物對 STZ-NA 誘導糖尿病大鼠口服葡萄糖耐受性之影響...............................33 （四）血液的收集與處理...................................33 （五）血漿葡萄糖濃度之測定...............................34 第三節 長效性試驗：探討連續餵食番石榴葉萃出物對 STZ-NA 誘發之第二型糖尿病大鼠的影響.................................35 一、實驗流程.............................................35 二、實驗材料.............................................36 （一）樣品來源...........................................36 （二）實驗動物...........................................36 （三）動物飼料...........................................36 （四）實驗藥品與試劑.....................................36 （五）實驗儀器...........................................39 三、實驗步驟和方法.......................................40 （一）動物飼養...........................................40 （二）動物誘導及分組.....................................40 （三）長效性試驗.........................................40 （四）試驗方法...........................................41 1. 口服葡萄糖耐受性實驗..................................41 2. 血液處理..............................................41 3. 臟器的收集............................................41 4. 血漿葡萄糖濃度之測定..................................42 5. 血漿胰島素濃度之測定..................................42 6. 肝臟酵素活性測定......................................42 7. 血糖調控相關蛋白質含量之測定..........................44 8. 大鼠肝臟細胞葡萄糖攝入能力及及肝醣合成作用............48 9. 肝臟肝醣合成測定法....................................50 10. 大鼠骨骼肌對葡萄糖攝入測定...........................50 11. 統計分析.............................................51 第五章 結果..............................................52 第一節 番石榴(Psidium guajava Linn.)葉粗萃物之製備.......52 一、萃出率之測定.........................................52 二、樣品蛋白質、總醣及總酚含量...........................52 第二節 急性實驗：探討急性灌食番石榴葉粗萃物對 STZ-NA 誘發之第二型糖尿病大鼠葡萄糖耐受性的影響.......................53 一、番石榴葉粗萃物對大鼠口服葡萄糖耐受性影響.............53 第三節 長效性試驗：探討連續餵食番石榴葉粗萃物對 STZ-NA 誘發之第二型糖尿病大鼠的影響.................................54 一、葡萄糖耐受性實驗結果.................................54 （一）灌食番石榴葉粗萃物前之OGTT 血糖變化................54 （二）灌食番石榴葉粗萃物4週後 OGTT 血糖之變化............54 （三）灌食番石榴葉粗萃物6週後 OGTT 血糖之變化............55 （四）灌食番石榴葉粗萃物6週後 OGTT 胰島素之變化..........55 二、實驗期間大鼠體重變化.................................55 三、臟器相對重量.........................................56 四、肝臟碳水化合物代謝酵素活性變化.......................56 （一）Hexokinase (HKase) ................................56 （二）Phosphofructokinase（PFKase）......................56 （三）G-6-phosphate dehydrogenase（G6PDase）.............56 （四）Fructose-1,6-biphosphatase（F1,6BPase）............57 （五）Glucose-6-phosphatase (G-6-Pase) ..................57 五、血糖調控相關蛋白質含量之測定.........................57 六、番石榴葉萃出物對第二型糖尿病大鼠骨骼肌葡萄糖攝入能力的影響.....................................................58 七、番石榴葉萃出物對第二型糖尿病大鼠肝細胞葡萄糖攝入能力及肝醣合成作用的影響.......................................58 第六章 討論..............................................60 一、急性灌食番石榴葉粗萃物對大鼠葡萄糖耐受性的影響.......60 二、連續餵食番石榴葉粗萃物對 STZ-NA 誘發之第二型糖尿病大鼠葡萄糖耐受性及胰島素濃度的影響...........................60 三、連續餵食番石榴葉粗萃物對第二型糖尿病大鼠體重及臟器重量變化的影響...............................................61 四、連續餵食番石榴葉粗萃物對第二型糖尿病大鼠肝臟醣類代謝的影響.....................................................61 五、連續餵食番石榴葉粗萃物對第二型糖尿病大鼠肝臟細胞葡萄糖轉運蛋白Ⅱ表現量的影響...................................63 六、連續餵食番石榴葉粗萃物對第二型糖尿病大鼠第二型糖尿病大鼠骨骼肌葡萄糖攝入能力的影響.............................64 七、連續餵食番石榴葉粗萃物對第二型糖尿病大鼠肝細胞葡萄糖攝入能力及肝醣合成作用的影響...............................64 第七章 結論..............................................65 參考文獻.................................................83 表目錄 表 一、糖尿病診斷標準.....................................7 表 二、人類的葡萄糖轉運蛋白..............................16 表 三、番石榴葉水萃出物與乙醇萃出物蛋白質、總醣及總酚含量.......................................................66 表 四、餵食番石榴葉萃出物六週後對第二型糖尿病鼠器官相對重量之影響...................................................67 圖目錄 圖 一、糖尿病分類.......................................10 圖 二、Streptozotocin 之化學結構........................21 圖 三、STZ造成大鼠胰臟β細胞死亡之機制...................21 圖 四、實驗架構.........................................24 圖 五、急性灌食番石榴葉萃出物並進行口服葡萄糖耐受性試驗對糖尿病大鼠血漿葡萄糖濃度變化之影響.......................68 圖 六、糖尿病大鼠長期灌食番石榴葉萃出物前進行口服葡萄糖耐受性試驗時血漿葡萄糖濃度之變化...........................69 圖 七、灌食番石榴葉萃出物四週後進行口服葡萄糖耐受性試驗時糖尿病大鼠血漿葡萄糖濃度之變化...........................70 圖 八、灌食番石榴葉萃出物六週後進行口服葡萄糖耐受性試驗時糖尿病大鼠血漿葡萄糖濃度之變化...........................71 圖 九、灌食番石榴葉萃出物六週後進行口服葡萄糖耐受性試驗時糖尿病大鼠血漿胰島素濃度之變化...........................72 圖 十、長期灌食番石榴葉萃出物六週期間大鼠體重變化.......73 圖十一、灌食番石榴葉萃出物六週後大鼠肝臟 hexokinase活性..74 圖十二、灌食番石榴葉萃出物六週後大鼠肝臟 phosphofructokinase活性..................................75 圖十三、灌食番石榴葉萃出物六週後大鼠肝臟glucose-6-phosphate dehydrogenase活性..............................76 圖十四、灌食番石榴葉萃出物六週後大鼠肝臟fructose-1,6-bisphosphatase活性.......................................77 圖十五、灌食番石榴葉萃出物六週後大鼠肝臟 Glucose-6-phosphatase 活性.........................................78 圖十六、灌食番石榴葉萃出物六週後大鼠肝臟細胞葡萄糖轉運蛋白Ⅱ之含量.................................................79 圖十七、灌食番石榴葉萃出物六週對糖尿病大鼠骨骼肌葡萄糖攝入作用的影響...............................................80 圖十八、灌食番石榴葉萃出物六週後對大鼠肝臟細胞葡萄糖攝入作用的影響.................................................81 圖十九、灌食番石榴葉萃出物六週後對大鼠肝臟細胞肝醣合成之影響.......................................................821326831 bytesapplication/pdfen-US番石榴糖尿病降血糖作用口服葡萄糖耐受性試驗碳水化合物代謝Psidium guajava Linn.diabetesantihyperglycemic effectOGTTcarbohydrate metabolism[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56357/1/ntu-96-R94641004-1.pdf