孫璐西臺灣大學:食品科技研究所黃潔妤Huang, Chieh-YuChieh-YuHuang2010-05-112018-06-292010-05-112018-06-292009U0001-1708200913552700http://ntur.lib.ntu.edu.tw//handle/246246/182216代謝症候群因與許多重大之慢性疾病(如:糖尿病及心血管疾病等) 間有密切相關性，為近年來日益受到重視之健康議題，其危險因子包含腹部肥胖、高血糖、血脂異常及高血壓等。本研究室於先前之研究已發現龍眼 (Dimocarpus longan Lour.) 花在體外試驗中具有很好的抗氧化活性，且在高果糖動物模式中證實龍眼花水萃物具有改善代謝症候群諸多症狀之功效。因此本研究之目的為評估龍眼花不同極性區分層對高果糖誘發代謝症候群之改善功效，以便日後尋找龍眼花中之有效成分。 本研究分為兩階段進行，第一階段為急性灌食試驗，用以觀察龍眼花水萃物及其區分層對大鼠葡萄糖耐受能力之影響。在此部分之試驗中，使用正常之Sprague-Dawley雄性大鼠，隨機分成五組，分別灌食龍眼花水萃物、乙酸乙酯區分層、正丁醇區分層、水區分層及去離子水後，進行口服葡萄糖耐受性試驗，觀察各組血糖變化情形。其結果顯示，乙酸乙酯區分層及水區分層皆具改善葡萄糖耐受能力的作用，因此同時選擇此二樣品進入第二階段之試驗。 第二階段則為長效期試驗，採高果糖飲食誘發大鼠代謝症候群之動物模式進行，用以觀察龍眼花水萃物及其區分層改善代謝症候群之功效。此部分試驗同樣選用Sprague-Dawley大鼠，並隨機分為五組：基礎值組 (B) 每日灌食去離子水並給予標準飼料；控制組 (C) 每日灌食去離子水並給予高果糖飼料；龍眼花水萃物組 (LFWE) 每日灌食龍眼花水萃物250 mg/ml/kgBW並給予高果糖飼料；龍眼花水萃物之乙酸乙酯區分層組 (EA) 每日灌食乙酸乙酯區分層36.3 mg/ml/kg BW並給予高果糖飼料；以及龍眼花水萃物之水區分層組 (W) 每日灌食水區分層147.5 mg/ml/kg BW並給予高果糖飼料。實驗期共16週。結果顯示，C組大鼠之禁食血漿葡萄糖、胰島素、三酸甘油酯、膽固醇濃度與血壓皆顯著上升，顯示長期餵飼高果糖飼料已顯著誘發出代謝症候群之症狀。 龍眼花水萃物於本實驗中，具有顯著改善腹部脂肪堆積、禁食血漿葡萄糖、胰島素、三酸甘油酯、總膽固醇濃度上升及高血壓等功效，有顯著改善胰島素阻抗之現象。而其效果可能來自於減少大鼠體內氧化壓力及提高IR、IRS-1、Akt、GLUT4等蛋白質之表現量。 在龍眼花水萃物之乙酸乙酯區分層部分，發現其與龍眼花水萃物具有許多相似之功效：顯著改善腹部脂肪堆積、禁食血漿葡萄糖、胰島素、三酸甘油酯濃度、高血壓及胰島素阻抗之現象。而在體內抗氧化壓力方面，也同樣具有改善血漿TBARS濃度，及增加肝臟抗氧化酵素Glutathione reductase之活性等功效，並且在胰島素訊息傳遞相關蛋白之表現量亦有上升的情形。 在龍眼花水萃物之水區分層部分，則具有顯著降低禁食血漿葡萄糖、總膽固醇濃度的作用，並同樣具有減少腹部脂肪堆積、降低肝中三酸甘油酯濃度等功效；但對胰島素阻抗與高三酸甘油酯血症並無顯著改善的功效。 綜合以上結果，乙酸乙酯區分層在改善代謝症候群之諸多症狀上皆類似於龍眼花水萃物，因此推測龍眼花水萃物中改善代謝症候群之有效成分應存在於乙酸乙酯區分層當中。Metabolic syndrome becomes an important issue recently. It has strong correlation with several chronic disease, for instance, diabetes and cardiovascular disease. The risk factors of metabolic syndrome contain abdominal obesity, hyperglycemia, dyslipidemia and hypertension. Previous study in our laboratory has shown that Longan (Dimocarpus Longan Lour.) flower had strong antioxidant activity in vitro. Furthermore, we also have demonstrated that Longan flower water extract (LFWE) can improve the symptoms of the metabolic syndrome in animal model. Therefore, the purpose of this study was to investigate the effect of each fraction of Longan flower water extract on metabolic syndrome. We expect to find effective compound in Longan flower in the future. he study was carried out into two parts. The first part was an acute study to examine the effects of LFWE and its fractions on glucose tolerance in rats. Male Sprague-Dawley rats were randomly divided into five groups, administered LFWE, ethyl acetate fraction, n-butanol fraction, water fraction and ddH2O by gavage respectly. Results of oral glucose tolerance test (OGTT) in rats showed that both EA and W fraction can improve glucose tolerance. Therefore, we choose these two fractions to do the following study.he following study was to investigate the effect of long-term treatment of EA and W fraction on rats with metabolic syndrome induced by high fructose diet. Male Sprague-Dawley rats were randomly divided into five groups: group B, fed with standard Purina chow; group C, fed with high fructose diet ; group LFWE, fed with high fructose diet plus LFWE 250 mg/ml/kg BW per day by gavage ; group EA, fed with high fructose diet plus EA 36.3 mg/ml/kg BW per day by gavage ; group W, fed with high fructose diet plus W 147.5 mg/ml/kg BW per day by gavage. The dietary manipulation lasted for 16 weeks. Results of this study showed that, high fructose feeding cause significant increase in abdominal fat mass, fasting plasma glucose, insulin, triglyceride, cholesterol levels and systolic blood pressure. These results indicated that fructose-rich diet could cause a cluster of disorders in metabolic syndrome. reatment with LFWE significantly improved abdominal fat accumulation, hypertriglyceridemia, hyperinsulinemia, hyperglycemia, hypercholesterolemia, hypertension and insulin resistance. To further evaluated the possible mechanism of LFWE, we found that the rats showed decreased oxidative stress and increased expression of insulin signaling pathway related protein, including insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt and glucose transporter Ⅳ(GLUT4).he supplement of EA fraction showed many similar efficacies as LFWE on abdominal fat, fasting glucose, insulin, triacylglyceride, hypertension and insulin resistance. EA fraction also showed the ability to decrease plasma TBARS and enhance glutathione reductase activity in fructose-fed rats. And the expressions of insulin signaling pathway related protein were also enhanced. fraction significantly decreased fasting glucose, total cholesterol, abdominal fat and liver triacylglycerol accumulation. But it shown no effects on insulin resistance and hypertriglyceridemia induced by high fructose diet instead. According to these finding, we suggested the effective components for ameliorating metabolic syndrome should be in the ethyl acetate fraction.摘要................................................................................................................................Ibstract........................................................................................................................Ⅲ錄..............................................................................................................................Ⅴ 次..............................................................................................................................ⅩⅠ表次.............................................................................................................................ⅩⅣ、 前言........................................................................................................................1、文獻回顧..................................................................................................................2一節、龍眼花..............................................................................................................2、植物簡介..............................................................................................................2 、龍眼花之成分與功效..........................................................................................3二節、代謝症候群......................................................................................................7、代謝症候群之定義及其診斷標準......................................................................7、代謝症候群與肥胖............................................................................................10、代謝症候群與第二型糖尿病............................................................................11、代謝症候群與心血管疾病................................................................................12、代謝症候群與高血壓........................................................................................12、代謝症候群與氧化壓力....................................................................................13七、高果糖誘發Sprague-Dawle大鼠代謝異常之動物模式..................................14三節、胰島素阻抗....................................................................................................15、胰島素................................................................................................................15、胰島素訊息傳遞之路徑....................................................................................15、胰島素阻抗........................................................................................................17四節、果糖代謝異常................................................................................................18 一、果糖與之質代謝異常........................................................................................18、 果糖與胰島素阻抗..........................................................................................19、果糖與高血壓…................................................................................................20、果糖與肥胖........................................................................................................20、果糖與氧化壓力................................................................................................20amp;#21442;、研究目的與實驗架構............................................................................................22、研究目的............................................................................................................22、實驗架構............................................................................................................23一) 樣品製備......................................................................................................23二) 急性試驗......................................................................................................24三) 長效性試驗..................................................................................................25、材料與方法...........................................................................................................26一節、實驗材料與儀器設備...................................................................................26、實驗樣品...........................................................................................................26、化學藥劑............................................................................................................26一) 化學藥品......................................................................................................26二) 溶劑..............................................................................................................27三) 酵素套組......................................................................................................27四) 動物飼料組成..............................................................................................28五) 西方點墨法..................................................................................................31、儀器設備...........................................................................................................31一) 用於原料前處理..........................................................................................31二) 用於動物飼料之配製..................................................................................32三) 用於動物實驗樣品之分析..........................................................................32二節、實驗方法.......................................................................................................33驗Ⅰ、龍眼花水萃物之區分方法...........................................................................33、樣品前處理.......................................................................................................33、製備龍眼花水萃物..........................................................................................33、製備龍眼花水萃物之各區分層.......................................................................33、各區分層樣品之萃出率....................................................................................33驗Ⅱ、急性期試驗－探討急性灌食龍眼花水萃物之各區分層對大鼠葡萄糖耐受性 (glucose tolerance) 的影響....................................................................................34、實驗動物…......................................................................................................34、口服葡萄糖耐受性試驗 (Oral Glucose Tolerance Test, OGTT) ..................34一)血液樣本之處理..........................................................................................34二)血漿葡萄糖濃度的測定..............................................................................34驗Ⅲ、長效期試驗－探討連續灌食龍眼花水萃物不同區分層 16 週對高果糖餵飼大鼠誘發代謝症候群的影響...................................................................................35、實驗動物...........................................................................................................35、動物分組與飼養...............................................................................................35、血壓之測量.......................................................................................................36、禁食狀態血液之採集與處理...........................................................................36一) 葡萄糖濃度之測定...................................................................................37二) 胰島素濃度之測定...................................................................................37三) 三酸甘油脂濃度之測定...........................................................................37四) 總膽固醇濃度之測定...............................................................................38五) 高密度脂蛋白膽固醇濃度之測定...........................................................38、口服葡萄糖耐受性試驗....................................................................................38、動物犧牲與檢體之收集與分析........................................................................39一) 動物犧牲當日檢體之收集與處理…......................................................39.血漿.......................................................................................................39.血清.......................................................................................................39.肝臟...................................................................................................... 39.心臟..................................................................................................40.腎臟..................................................................................................40.腎周脂及腸繫膜..............................................................................40.副睪脂肪組織..................................................................................40.脂肪細胞之製備..............................................................................40 (二) 動物犧牲後之生化分析項目............................................................42. 血漿之生化值分析.........................................................................421) 血漿中葡萄糖之濃度測定.....................................................422) 血漿中胰島素之濃度測定.....................................................423) 血漿中三酸甘油脂之濃度測定.............................................424) 血漿中總膽固醇之濃度測定.................................................425) 血漿中高密度脂蛋白膽固醇之濃度測定.............................436) 血清中低密度脂蛋白膽固醇之濃度測定.............................437) 血漿TBARS之測定................................................................43. 肝臟脂質之分析............................................................................441) 肝臟脂質萃取.........................................................................442) 肝臟中三酸甘油酯之測定.....................................................443) 肝臟中總膽固醇之測定.........................................................44. 肝臟氧化壓力之分析....................................................................441) 肝臟抗氧化酵素樣品製備.....................................................442) 麩胱甘肽過氧化酶(glutathione peroxidase, GPx)之活性.....453) 麩胱甘肽還原酶 (glutathione reductase, GRd) 之活性.......45三) 血糖調控相關蛋白質含量之測定...................................................46. 脂肪細胞蛋白質定量分析............................................................46. 西方轉漬法 (Western blot) 分析蛋白質含量.............................46三節、統計分析......................................................................................................49、結果與討論..........................................................................................................50驗Ⅰ、龍眼花水萃物各區分物之萃出率及灌食劑量計算..................................50驗Ⅱ、急性口服葡萄糖耐受性實驗.......................................................................51、結果...................................................................................................................51、討論...................................................................................................................54驗Ⅲ、長效性試驗－探討連續灌食龍眼花水萃物之不同區分層16週對高果糖誘發大鼠代謝症候群之影響......................................................................................55 一、結果..................................................................................................................55一) 生長狀況.....................................................................................................54 (二) 飼養期間禁食血漿葡萄糖、胰島素濃度及HOMA-R數值之變化.......55 2.1血漿葡萄糖濃度之變化................................................................................55 2.2.血漿胰島素濃度之變化................................................................................55 2.3胰島素阻抗現象(HOMA-IR) .......................................................................56 (三) 飼養期間之血壓變化..................................................................................60四) 飼養期間血漿三酸甘油酯及膽固醇濃度之變化......................................61 4.1血漿三酸甘油脂.............................................................................................61 4.2血漿總膽固醇.................................................................................................61五) 口服葡萄糖耐受性試驗 (Oral Glucose Tolerance Test, OGTT) ...............62.1大鼠飼養4週後之OGTT結果.....................................................................62.2大鼠飼養8週後之OGTT結果.....................................................................66.3大鼠飼養15週後之OGTT結果...................................................................69 (六) 組織臟器重量...............................................................................................73 6.1腹部脂肪 (Abdominal fat) 重量....................................................................73 6.2肝臟重量..........................................................................................................73 6.3腎臟重量..........................................................................................................74七) 禁食血液之分析...........................................................................................77.1禁食血漿葡萄糖及胰島素濃度.....................................................................77.2禁食血漿三酸甘油酯、總膽固醇濃度及高/低密度脂蛋白膽固醇與總膽 固醇之比例....................................................................................................77 (八) 肝臟脂質分析.............................................................................................78 8.1肝臟三酸甘油脂濃度....................................................................................78 8.2肝臟總膽固醇濃度........................................................................................78九) 氧化壓力分析..............................................................................................82十) 脂肪細胞胰島素訊息傳遞相關蛋白質表現量..........................................84、討論..................................................................................................................88一) 高果糖動物模式..........................................................................................88二) 生長狀況與組織臟器重量..........................................................................88三) 胰島素阻抗與高胰島素血症......................................................................89四) 高血壓..........................................................................................................90五) 血脂異常....................................90六) 氧化壓力.......................................91七) 胰島素訊息傳遞相關蛋白質表現量及其磷酸化情形.........92、結論......................................94、參考文獻...............................................95錄.....................................................110application/pdf1940803 bytesapplication/pdfen-US龍眼花代謝症候群乙酸乙酯區分層胰島素阻抗Longan flowermetabolic syndromeethyl acetate fractioninsulin resistance[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182216/1/ntu-98-R96641033-1.pdf