龍眼花水萃物對高果糖誘發代謝症候群大鼠之影響

Abstract

代謝症候群是近年來受到重視的健康議題，其症狀包括高胰島素血症、胰島素阻抗、高三酸甘油酯血症和高血壓等，此些危險因子的叢聚現象與第二型糖尿病和心血管疾病有密切的相關性。前人研究也證實，於飲食中多攝取抗氧化物質有助於預防代謝症候群因而降低心血管疾病的發生。本研究室於先前的研究發現龍眼 (Dimocarpus longan Lour.) 花在體外具有很好的抗氧化活性，因此本研究目的在探討龍眼花粗萃物的高抗氧化活性是否能改善代謝症候群之諸多症狀。 實驗第一階段為體外抗氧化性試驗，先將龍眼花粉末分別以沸水萃取及室溫下以95%乙醇或甲醇溶劑攪拌萃取。再以兩種抗氧化活性評估方法，包括清除DPPH自由基能力試驗和抑制銅離子誘導人類低密度脂蛋白氧化檢測此三種不同溶劑之龍眼花粗萃物。實驗結果顯示，龍眼花水萃物於此二種抗氧化實驗中皆展現較高之抗氧化力，其清除DPPH自由基之EC50值為3.75±0.61 μg/mL，且延緩銅離子誘導LDL氧化效果為同濃度下正控制組trolox之1.57倍。整體而言抗氧化活性以水萃物最佳，其次為乙醇和甲醇萃取物。 第二階段為對大鼠進行急性灌食試驗，觀察龍眼花粗萃物對其耐糖能力的影響。急性試驗中，以龍眼花粗萃物125或250 mg/kg body weight (BW) 的劑量灌食正常Sprague-Dawley大鼠，同時進行口服葡萄糖耐受性試驗。結果顯示，大鼠灌食龍眼花水萃物250 mg/kg BW，在第30分鐘的血漿葡萄糖值顯著低於對照組，但胰島素濃度在各時間點則無顯著差異。此外，灌食龍眼花乙醇萃物125或250 mg/kg BW的劑量，不論在血漿葡萄糖或胰島素濃度與對照組比較皆無顯著差異。顯示大鼠急性灌食龍眼花水萃物可以減緩血糖上升的速度，其作用不在於改善胰島素敏感性，可能與延緩或干擾腸道對糖分的吸收有關。 第三階段為長效性試驗，以高果糖飼料誘發大鼠產生代謝症候群，觀察龍眼花水萃物是否能改善其症狀。將體重約250克Sprague-Dawley大鼠隨機分為4組：C組給予標準飼料；F組給予高果糖飼料；L組除了高果糖飼料外，每隻每天灌食龍眼花水萃物 125 mg/kg BW，為低劑量組；H組除了高果糖飼料外，每隻每天灌食龍眼花水萃物 250 mg/kg BW，為高劑量組，C組與F組則以灌食去離子水作為對照。實驗期為14週。F組自第2週起，收縮壓、禁食狀態血漿三酸甘油酯和胰島素皆明顯開始上升，但血漿葡萄糖濃度仍維持正常；於第4週進行口服葡萄糖耐受性試驗，已有胰島素阻抗之情況發生，顯示長期餵飼高果糖飼料已明顯誘導大鼠產生代謝症候群之症狀。在體內抗氧化能力試驗中，餵飼高果糖飼料14週後，會造成體內氧化壓力增加，血漿脂質過氧化物TBARS濃度明顯上升且肝臟抗氧化酵素活性下降。 給予低劑量125 mg/kg BW龍眼花水萃物的組別，可降低高血壓，於實驗11和12週血壓已明顯低於F組。在胰島素敏感性方面，於4、8和12週的口服葡萄糖耐受性試驗中，有改善胰島素阻抗的現象，但效果並不明顯。對於禁食血漿三酸甘油酯濃度仍不具有調節作用。在體內抗氧化能力試驗中可提升肝臟抗氧化酵素Glutathione reductase的活性，而在血漿脂質過氧化物TBARS濃度則無差異。 給予高劑量250 mg/kg BW龍眼花水萃物的組別，降血壓效果顯著，於實驗第7週開始已明顯低於F組。在胰島素敏感性方面，於第4週口服葡萄糖耐受性試驗中即開始有改善胰島素阻抗之現象。而給予高劑量的龍眼花水萃物仍無法降低禁食血漿三酸甘油酯濃度。在體內抗氧化能力試驗中可提升肝臟抗氧化酵素Glutathione reductase的活性，且可降低血漿脂質過氧化物TBARS的濃度。 分析脂肪細胞胰島素訊息傳遞相關蛋白質表現量的結果顯示，給予高果糖飼料會造成insulin receptor substrate (IRS-1)、Akt和glucose transporter 4 (GLUT 4)表現量降低，導致胰島素受體對胰島素之敏感性降低。而給予低劑量龍眼花水萃物14週後有增加IRS-1的表現量，而給予高劑量龍眼花水萃物14週後更提升了IRS-1和GLUT 4的表現量，此結果顯示龍眼花水萃物可藉由增加胰島素訊息傳遞相關蛋白質之表現量，使胰島素敏感性增加。 本研究結果顯示，龍眼花水萃物具有很好的抗氧化活性，且可改善高血壓、高胰島素血症、胰島素阻抗和降低體內氧化壓力。

Metabolic syndrome is a cluster of disorders, including hyperinsulinemia, insulin resistance, hypertriglyceridemia and hypertension that increase one's risk for type 2 diabetes and cardiovascular disease . Natural antioxidants were reported to ameliorate metabolic syndrome. Previous study in our laboratory has shown that Longan (Dimocarpus longan Lour.) flower had strong antioxidant activity in vitro. Therefore, the objective of this study was to evaluate the effect of the supplemention of Longan flower extract on metabolic syndrome. The first stage of this study was to analyze the antioxidative activity of Longan flower in vitro, the crude extracts were prepared by extracting Longan flower with boiling water and two solvents (95% ethanol, methanol) at room temperature. Then, the three different solvent extracts of Longan flower were tested for two different antioxidant assays, including DPPH free radical scavenging effect and the inhibition of Cu2+-induced oxidation of human LDL. The results of antioxidant assays revealed that the best effect was exhibited by the water extract, followed by ethanol and methanol. The EC50 value of water extract in scavenging DPPH radicals was 3.75±0.61 µg/mL, and its effect on delaying LDL oxidation is 1.57 times better than trolox at the same concentration level (1 µg/mL). The second stage of the study was to observe the glucose tolerance of rats after the acute treatment with Longan flower crude extract (125 mg/kg or 250 mg/kg) and glucose. Results of oral glucose tolerance test (OGTT) in rats showed that the plasma glucose levels of the Sprague-Dawley rats administered 250 mg/kg BW of Longan flower water extract (LFWE) were lower than the control group after 30 minutes of ingestion but insulin concentrations showed no difference with the control group at each time intervals . However, both the plasma glucose and insulin levels of the rats ingested Longan flower ethanol extract showed no significant difference with control group. These findings suggest that Longan flower water extract may delay or interference with the sugar absorption in the gastrointestinal tract without changing insulin secretion and its action. The third stage is to investigate the effect of long-term treatment of LFWE on rats with metabolic syndrome induced by high fructose diet. Male Sprague-Dawley rats of body weight around 250 g were randomly divided into four groups: group C, fed with standard Purina chow; group F, fed with high fructose diet alone; group L, fed with high fructose diet plus LFWE 125 mg/kg BW per day by gavage (a low dose group) and group H, fed high fructose diet plus LFWE 250 mg/kg BW per day by gavage (a high dose group). The dietary manipulation lasted for 14 weeks. Results of our study showed that, high fructose feeding for 2 weeks cause significant increase in sytosolic blood pressure, fasting plasma triglyceride and insulin levels without elevating fasting plasma glucose. And insulin resistance was demonstrated after the 4th week by OGTT. These results indicated that fructose-rich diet could cause a cluster of disorders in metabolic syndrome. In antioxidative capacity analyses, at the end of the 12-week experiment high fructose diet increased plasma TBARS and significantly decreased liver antioxidant enzyme activity . The supplementation of LFWE ameliorated insulin resistance by enhancing the expression of insulin signaling pathway related proteins, including GLUT 4 and insulin receptor substrate-1. LFWE supplementation was also found to decrease SBP and ameliorated oxidative stress. These findings indicate that Longan flower water extract may improve the symptoms of the metabolic syndrome in fructose-fed rats.