白芍熱水萃取物醣解酵素抑制成分之純化及對小鼠肝臟FL83B細胞之葡萄糖恆定調節

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题名:	白芍熱水萃取物醣解酵素抑制成分之純化及對小鼠肝臟FL83B細胞之葡萄糖恆定調節
其它题名:	Purification of Glycosidases Inhibitory Components from Hot Water Extract of Paeonia lactiflora Roots and Their Regulating Glucose Homeostasis on Murine FL83B Hepatocytes
作者:	李怡慧 Li, Yi-Huei
贡献者:	蘇正德 Su, Jeng-De 東海大學食品科學系
关键词:	降血糖活性;α-葡萄糖苷酶;α-澱粉酶;胰島素阻抗;albiflorin;白芍;抗氧化性 α-glucosidase;α-amylase;insulin-resistant;albiflorin;Paeonia lactiflora Roots;hypoglycemic activity;antioxidant activity
日期:	2011
上传时间:	2012-12-13T09:01:07Z (UTC)
摘要:	本實驗擬自中草藥中尋找具調節血糖功效之保健食材，首先選定的是降血糖驗方甘芍降糖片之白芍；白芍為毛莨科植物芍藥(Paeonia lactiflora Pall.)的乾燥根，已知具有抗憂鬱、抗發炎、調節免疫力、抗菌、抗病毒、抗過敏、降低血中膽固醇、三酸甘油脂、低密度脂蛋白的濃度及降血糖等功效。本研究先以熱水及乙醇進行萃取，並以抑制醣解酵素活性為篩選平台，進行活性成分之分離、純化及結構鑑定。結果得知白芍萃取物中以熱水萃取物對α-amylase及α-glucosidase抑制能力為最佳，此萃取物利用層析膠體( Amberlite XAD-7及 Cosmosil 75C18-OPN )進行區分，再以HPLC純化出具有醣解酵素抑制活性之5種純化物質，並利用MS、UV及1H、13C-NMR鑑定結構。目前已鑑定出methyl gallate (1)、gallic acid (2)、albiflorin (3)、 lactiflorin (4)、paeniflorin (5)。上述純化物質中以albiflorin抑制α-amylase及α-glucosidase醣解酵素的能力最佳，其濃度為1mg/mL時抑制率分別為56.0%及90.9%。Albiflorin及HWEP同時進行正常小鼠肝臟FL83B細胞之細胞XTT毒性試驗，其在250及500ppm以下與正常細胞相比，並無顯著差異。進一步探討albiflorin及HWEP於正常及阻抗性細胞調節血糖機制，以高糖誘導FL83B細胞產生胰島素阻抗性，分別針對葡萄糖攝入量及肝醣合成量進行觀察。流式細胞分析顯示添加albiflorin可提高正常及阻抗性細胞葡萄糖擬似物2-NBDG帶入量；肝醣含量分析顯示albiflorin隨作用濃度增加能促進正常細胞及阻抗性細胞肝醣合成。本研究證實，albiflorin具有抑制α-amylase及α-glucosidase醣解酵素的活性，可能藉由影響肝細胞葡萄糖利用，降低胰島素阻抗，增加葡萄糖帶入能力，而顯現降血糖作用。 Paeonia lactiflora roots, a compositional herbal medicine of Chinese hypoglycemic prescription Gan-Shaur hypoglycemic tablets, were selected to investigate the regulation of blood sugar. It have long been known for their antidepressant, anti-nflammatory, immune modulatory, anti-bacterial, anti-virus, anti-allergy, hypolipidemic and hypoglycemic effects. In this study, active components were isolated and identified from hot water and ethanol extract of P. lactiflora roots by inhibitory glycosicase activities, and were tested for their hypoglycemic activities in vitro. Hot water extract of P. lactiflora roots (HWEP) showed strong inhibitory properties against both α-amylase and α-glucosidase. The extract was further separated successively by Amberlite XAD-7, Cosmosil 75C18-OPN and reversed HPLC chromatographies to obtain five components. The isolated components were identified as methyl gallate (1), gallic acid (2), albiflorin (3), lactiflorin (4), and paeniflorin (5) by MS, UV and 1H, 13C-NMR analyses. Among them, albiflorin showed the strongest inhibitory activities toward α-amylase and α-glucosidase with inhibition rates of 56.0% and 90.9%, respectively, at the concentration of 1mg/mL. Hypoglycemic activities of albiflorin and HWEP were then further verified in a murine FL83B hepatocyte line. Both albiflorin and HWEP had no inhibitory effects on the growth of FL83B cells at the concentration of 250 and 500 ppm, respectively, by the XTT assay. To further investigate the antiglycemic mechanisms of HWEP and albiflorin under series of incubation concentrations, glucose uptake and glycogen content were observed on murine FL83B hepatocytes with or without the induction of insulin resistance by incubation with high glucose. In the presence of albiflorin, uptake of glucose analogue 2-NBDG was significantly enhanced when FL83B cells were rendered resistant to insulin by a prior incubation with high concentration of glucose. Glycogen content both in normal and in insulin resistant cells was also increased by adding albiflorin in a dose-dependent manner. Our results demonstrated that albiflorin from P. lactiflora roots may inhibit α-amylase and α-glucosidase, acted possibly through glucose utilization in hepatocytes, reduce the insulin resistant, and promote glucose uptake and storage in hepatocytes, implying a hypoglycemic activity in vivo.
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