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  • 學位論文

以α-葡萄糖苷酶之活性抑制為主軸探討五倍子之調節血糖功能與成分

Anti-hyperglycemia activity of Galla chinensis based on the inhibition of α- glucosidase activity

指導教授 : 蔣丙煌

摘要


糖尿病(diabetes mellitus;DM)為目前全球流行之慢性代謝疾病,在台灣,糖尿病多年來皆位於十大死因前五名之列,由於糖尿病患者體內長期處於高血糖之狀態,會進一步造成其病症惡化以及引發多種併發症,而飯後高血糖為造成糖尿病患許多併發症之主因之一。目前在治療糖尿病之主要藥物中,只有α-葡萄糖苷酶 (α-glucosidase) 抑制劑具有有效降低飯後血糖之效果。本實驗以抑制α-葡萄糖苷酶之活性為篩選平台,由十一種中草藥中篩選出具有抑制α-葡萄糖苷酶活性之材料,結果發現五倍子之抑制活性最為顯著,故選用五倍子做進一步的分離純化,希望從中找尋出具有調節血糖功效之活性成分,並期望能由多方面達到調節血糖以及改善糖尿病之效果。 在抑制α-葡萄糖苷酶活性試驗中,五倍子之乙酸乙酯萃取層,具有最高之抑制活性,且在抑制DPP-4酵素、抑制糖化終產物(AGEs)生成以及抑制α-澱粉酶 (α-amylase) 之實驗中,乙酸乙酯萃取層亦具有最佳之活性。進一步以 Sephadex LH-20 膠體將乙酸乙酯萃取物進行管柱層析分離為EA-A ~ EA-I九個區分層,將此九個區分層進行抑制α-葡萄糖苷酶活性試驗,實驗結果發現EA-G 區分層對酵母來源及大鼠來源之α-葡萄糖苷酶抑制性率分別為93.59 % 及42.91 %,與其他區分層相較有最佳之抑制活性。另外EA-G 區分層在清除 DPPH 自由基、抑制糖化終產物(AGEs)生成、抑制α-澱粉酶 (α-amylase) 以及促進小鼠肝細胞FL83B葡萄糖攝入之實驗中亦具有良好之活性。 由上述實驗結果選出 EA-G 區分層做進一步之純化,在經過高效液相層析儀分離純化後,得到之七個區分物G1 ~ G7,當中G4及G5兩個區分物對 α-葡萄糖苷酶之抑制活性分別為97.7 %以及99.93 %,其抑制能力顯著高於 EA-G 之其他區分物。進一步收集此兩區分物做 LC-MS 質譜分析並與文獻比對後,推知此兩個區分物為1,2,3,6-tetra-O-galloyl-β-d-glucose 及 1,2,3,4,6-penta-O-galloyl-β-d-glucose。 綜合以上實驗結果可知,五倍子具有良好之 α-葡萄糖苷酶抑制活性,並推測其主要活性成分為 1,2,3,6-tetra-O-galloyl-β-d-glucose 及 1,2,3,4,6-penta-O-galloyl -β-d-glucose。五倍子除了具有良好之 α-葡萄糖苷酶抑制活性外,尚有 α-澱粉酶抑制活性、DPP-4 抑制活性、清除 DPPH 自由基、抑制 AGEs 形成、刺激細胞吸收葡萄糖等各種生理活性,故五倍子具有由多方面性達到調節血糖之效果,以及發展為抗糖尿病活性產品之潛力。

並列摘要


Diabetes mellitus (DM) is a common metabolism disease worldwide. DM is also on the top five list of the ten major causes of death in Taiwan for many years. Hyperglycemia is the main symptom to the DM patients. It obten inducesvarious complications, and postprandial hyperglycemia is the principal factor of DM complications. Among many kinds of diabetic medicines, only α-glucosidase inhibitors can significantly lower postprandial blood glucose. This research used α-glucosidase inhibition assay to find the plant materials which has ability to inhibit α-glucosidase. It was found that among the eleven plant materials tested, Galla chinensis has the most potent inhibition activity. Therefor, we futher investigated the active component in Galla chinensis usingα-glucosidase inhibition assay. Various solvents were used to prepare Galla chinensis extracts, and ethyl acetate extract (EA) of Galla chinensis was found to have the highest ability to inhibit α-glucosidase, and it’s also had the highest activity in the DPP-4 inhibition assay, advanced glycation end products inhibition assay and α-amylase inhibition assay. We futher used Sephadex LH-20 gel to separate that EA-A ~ EA-I fractions from the Galla chinensis ethyl acetate extract, and found EA-G had the best ability to inhibit α-glucosidase among the nine fractions, the inhibition ratio of yeast α-glucosidase and rat α-glucosidase were 93.59 % and 42.91 %, respectively. The EA-G fraction also had well activity in the DPP-4 inhibition assay, DPPH free radical scavenge assay, advanced glycation end products inhibition assay and α-amylase inhibition assay, and it also increased glucose uptake of insulin resistant cells. According to the above experimental result, the EA-G fraction was futher purified using High Performance Liquid Chromatography and G1~ G7 fractions were obtained. The G4 and G5 fractions were found to have highest α-glucosidase inhibition activity among the seven fractions. These two fractions were futher analyzed by Mass Spectrometry, and found that they are 1, 2, 3, 6-tetra-O-galloyl-β-d-glucose and 1, 2, 3, 4, 6-penta-O-galloyl-β-d-glucose. This research has proved that Galla chinensis not only has high α-glucosidase inhibition activity, but also can inhibit DPP-4, scavenge free radicals, inhibit advanced glycation end products formation, inhibit α-amylase activity, and increase glucose uptake of insulin resistant cells. So, Galla chinensis can modulate blood sugar through various ways and has potential to be developed as an anti-diabetes product.

參考文獻


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