- 學位論文
芝麻中sesaminol triglucoside萃出物在大鼠吸收代謝及抗氧化活性之探討
Studies on the Metabolism of Sesaminol Triglucoside Extract and its Impact on Antioxidant Status in Rats
摘要
芝麻中含獨特之lignan化合物，其中sesamol及sesaminol等分子因在結構上有多酚OH官能基，具抗氧化活性，被認為與維持芝麻油安定性及提供生理活性相關。在脫脂芝麻粕中，所含lignans多以lignan glucosides型式存在，其中約80-90 % 為sesaminol triglucoside (ST)。本研究室曾以脫脂芝麻粕為試驗材料，對其所含lignan glucosides成分進行體外及體內試驗。結果顯示sesaminol glucosides在體外DPPH自由基清除效果不佳，但若經水解去醣基後，抗氧化能力顯著增高許多；在動物試驗中發現，長期餵予lignan glucosides具有降低高果糖餵飼大鼠體內之氧化壓力及增加胰島素敏感性等功效，推測其活性成分可能來自於lignan glucosides在動物體內經水解代謝釋出之lignans成分。故本研究之目的在探討芝麻粕中ST於大鼠體內可能之代謝、分佈及排除情形，並以維生素E不足之大鼠為動物模式，觀察ST對大鼠體內氧化壓力的影響。 試驗材料為未經炒焙之泰國黑芝麻，將脫油後之芝麻粕先以80 %甲醇溶液進行萃取，經濃縮後再以XAD-2膠體進行管柱層析，以不同濃度之甲醇水溶液沖提後，收集ST含量較高之60 % 甲醇水溶液區分層，行冷凍乾燥可得ST萃出物粉末。動物試驗中，將SD大鼠分為三組: B組給予正常飼料，C組及ST組給予維生素E缺乏飼料。預養10天之後，進行灌食試驗，每日以管餵方式給予ST組大鼠300 mg/kg body weight劑量之ST萃出物，B組與C組則灌食去離子水以供對照，實驗期為兩週。 結果顯示，以維生素E缺乏飼料預養10天後，C組及ST組大鼠血漿中α-tocopherol濃度已顯著低於B組，並隨灌食試驗天數增加而逐漸減少。飼養三週後犧牲，於體內抗氧化能力試驗中，可觀察到C組之LDL氧化遲滯期縮短，並在血漿、肝臟及腎臟中MDA含量均顯著增加，顯示三週維生素E的缺乏造成大鼠體內氧化壓力增加。連續灌食ST萃出物兩週後，能增加血漿中γ-tocopherol含量，延緩LDL的氧化，以及降低大鼠血漿、肝臟與腎臟中MDA生成量，顯示ST萃出物能改善因維生素E缺乏所造成之氧化傷害。 以HPLC對大鼠體內ST及其代謝物進行分析。結果顯示，於灌食ST萃出物6小時後，於小腸黏膜及盲腸中分別測得約5 % 及40 % 未被吸收之ST原型態。而在血漿以及各組織臟器中，皆測得ST及其代謝物sesaminol的存在；進一步將大鼠尿液檢體以LC/MS/MS進行分析，結果證實尿液中確實含有ST及sesaminol分子。以上結果顯示，ST能以帶醣基型式直接被吸收，且伴隨有少部分ST能在大鼠體內水解為sesaminol後被吸收。 Sesaminol在各組織及尿液中主要是以sulfate型式存在；但在糞便中則多以aglycone型式存在，顯示腸道中微生物可能具有水解ST醣基酵素 (如: β-glucosidase)，能切除ST之醣基以利腸道吸收。 綜合言之，ST經大鼠攝食後，部分能被水解為sesaminol，並經吸收後，分佈至血液及各組織中。Sesaminol在體外試驗中具有很強的抗氧化活性，推測其可能為降低維生素E攝取不足之大鼠體內氧化壓力原因之ㄧ。
並列摘要
Sesame seeds contain abundant lignans, like sesamol and sesaminol, exhibiting excellent antioxidative activity in vitro. Some of them possess phenolic OH group to react with free radicals, thus protect sesame oil from oxidation and are considered beneficial to health. In defatted sesame seed, lignans are usually present as lignan glucosides, and sesaminol triglucoside (ST) is the major lignan glucoside. Previous studies reported that sesame lignan glucosides have poor antioxidative properties in DPPH scavenging activity in vitro. But in high fructose-fed rats model, supplementation with sesame lignan glucosides extract could lower oxidative stress and increase insulin sensitivity. These results indicated that lignan glucosides may be converted to lignans after oral administration and express biological effects. Therefore, the aim of this work was to investigate the metabolites, distribution and elimination of ST in vivo and to determine the impact of a ST-enriched diet on the antioxidant status in rats. Unroasted black sesame seeds were pressed and defatted with n-hexane, extracted with 80 % methanol, and separated by XAD-2 gel open column chromatography eluting with a serious of concentrations of aqueous methanol solutions (20 %, 40 % and 60 %). The 60 % methanol eluate, which contained higher content of ST compound, were collected and freeze-dried to obtain the ST extract powder. In animal experiment, we used vitamin E deficient rat model to increase oxidative stress. First, SD rats were divided into three groups, eight rats per group, and fed different diets: group B, normal diet；group C and group ST, vitamin E deficient diet. After 10 days, animals in group ST were tube fed with 300 mg kg-1 body weight of ST extract daily for 2 weeks. Group B and group C animals were tube fed with water to replace ST extract. Results showed that feeding with a vitamin E-deficient diet for 10 days, the plasma α-tocopherol levels of group C and group ST rats were significantly lower than group B and decreasing with time. In antioxidative capacity analyses, rats fed with a vitaminE-deficient diet for 3 weeks, the lag time of plasma LDL oxidation was shortened, and the TBARS in plasma, liver and kidney were increased in group C. This indicated that the oxidative stress in vitaminE-deficient diet-fed rats is increased. On the other hand, a ST-enriched diet significantly enhanced antioxidant capacity in plasma LDL oxidation and lowered the TBARS in plasma, liver and kidney. HPLC system was used to analyze ST and its metabolites in rats. Results showed that there was 5 % and 40 % of tube-fed ST found in the small-intestinal mucosa and cecum, respectively, indicating that the absorption of ST may be low. However, ST and its aglycone were detected in all tissues, urine and feces. With the aid of LC/MS/MS system to analyze urine sample obtained from a ST tube-fed rat, we confirmed that ST and sesaminol existed in urine. The metabolite-sesaminol existed mainly in sulfate form in rats tissues and urine, but it existed as aglycone in cecum, indicating that intestinal microorganism have the ability in hydrolyzing ST to sesaminol aglycone. Our results show that ST can be absorbed as glucoside directly, and some of them could be converted to aglycone then absorbed into blood and tissues, finally eliminated in urine. Based on these results, we suggest that the antioxidative abilities of ST may come from its aglycone found in vivo.