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Title: | 探討中國橄欖水萃殘渣之甲醇萃取物乙酸乙酯區分層中的α-葡萄糖苷酶抑制成分 Investigation of α-glucosidase inhibitory compounds in ethyl acetate fraction of methanol extract from water extract residues of Chinese olive (Canarium album L.) |
Authors: | Chung-Ching Lee 李重慶 |
Advisor: | 謝淑貞(Shu-Chen Hsieh) |
Co-Advisor: | 李宗徽(Tzong-Huei Lee) |
Keyword: | 糖尿病,α-葡萄糖??,中國橄欖,可水解型單寧, diabetes,α-glucosidase,Chinese olive (Canarium album (Lour.) Raeusch.),hydrolysable tannins, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 目前治療糖尿病的藥物中,有一類藥物為α-葡萄糖苷酶(α-glucosidase)抑制劑,可抑制α-葡萄糖苷酶將澱粉、寡糖或雙糖分解成單糖的活性,進而延緩糖類吸收,達到穩定餐後血糖的效果,而在天然物中也存在著各種不同形式的α-葡萄糖苷酶抑制劑,本實驗室先前已發現將中國橄欖(Canarium album (Lour.) Raeusch.)水萃物殘渣經甲醇萃取後之乙酸乙酯分層(COE)作為樣品,在小鼠巨噬細胞Raw 264.7具有抗發炎效果,在高飲食糖尿病大鼠可以降低血糖及改善脂肪代謝,所以本實驗繼續探討此萃取物的抗糖尿病機制。
本實驗以體外酵素抑制實驗作為篩選平台,發現中國橄欖萃取物具有抑制酵母α-葡萄糖苷酶的效果,於是以層析的方式純化萃取物中所含有的功效成分,並鑑定其結構。最後純化到2個可水解型單寧化合物(hydrolysable tannins) 1,2,3,6-tetra-O-galloyl-β-glucopyranose和chebulagic acid。在本實驗的條件下測得抑制酵母菌α-葡萄糖苷酶之IC50由低到高分別為1,2,3,6-tetra-O-galloyl-β-glucopyranose (0.44±0.07 μg/ml)、chebulagic acid (0.94±0.13 μg/ml)、COE (1.25±0.12 μg/ml)、acarbose (214.80±15.45 μg/ml)。再以Caco-2細胞酵素抑制實驗發現COE和所分離到的單寧化合物的確對人類α-葡萄糖苷酶有抑制效果,但在5 μg/ml濃度下只有COE效果跟acarbose效果相當,而2個單寧化合物則比acarbose效果差,代表COE中可能有其他活性成分。COE水解的產物主要為gallic acid、ellagic acid與其他兩個UV吸收最大值在262 nm和272 nm的未知化合物(可能為酚類化合物),表示COE含有大量的可水解型單寧化合物,而因為經強酸加熱水解後的COE不再具有抑制α-葡萄糖苷酶的活性,我們推測COE中其它具有活性的成分也是可水解型單寧。另一方面又發現添加牛血清蛋白後即減弱了COE和單寧化合物抑制α-葡萄糖苷酶之效果,顯示COE可能與其他蛋白有非專一性的結合。綜合以上實驗推測COE含有的可水解型單寧為抑制α-葡萄糖苷酶功效的主要來源。 α-glucosidase inhibitor, one of the interference used for diabetes treatment, can inhibit the activity of α-glucosidase to decompose starch, oligosaccharide or disaccharide into monosaccharides, thereby delaying the absorption of sugar and stabilizing postprandial blood glucose. Various α-glucosidase inhibitors in nature exhibit the ability for modulating blood sugar. Our previous studies display that ethyl acetate fraction of methanol extract from water extract residues of Chinese olive (Canarium album (Lour.) Raeusch.) (COE) has anti-inflammatory effect on mouse macrophage Raw 264.7 cell, and can reduce blood sugar and attenuate metabolic dysfunction in diabetic rats under high fat diet challenge. We thus continue to explore the anti-diabetic mechanism of COE in this study. Firstly, in vitro enzyme inhibition experiment was used as a screening platform to identify compounds in COE with the inhibitory effect on yeast α-glucosidase. Based on the results from platform screening, we separated compounds from COE by chromatography, and purified two hydrolysable tannins, 1,2,3,6-tetra-O-galloyl-β-glucopyranose and chebulagic acid. Our in vitro enzyme screening revealed that the IC50 to yeast α-glucosidase from low to high is 1,2,3,6-tetra-O-galloyl-β-glucopyranose (0.44±0.07 μg/ml), chebulagic acid (0.94±0.13 μg/ml), COE (1.25±0.12 μg/ml) and acarbose (214.80±15.45 μg/ml). We further used Caco-2 cell enzyme inhibition assay to investigate whether COE and the compounds we found have an inhibitory effect on human α-glucosidase, and result demonstrated that the inhibitory effect of COE was almost equivalent to acarbose at 5 μg/ml. However, the effect of two compounds were lower than the effect of acarbose, indicating there are other active compounds in COE. The hydrolyzed products are mainly gallic acid, ellagic acid and two other unknown compounds (UV absorption λmax are 262 nm and 272 nm, may be phenolic compounds), indicated that COE contains a large amount of hydrolysable tannins. Since strong acid with heating hydrolyzed COE no longer exhibited inhibitory activities, we propose that hydrolysable tannin is also the major contributor in other active fractions from COE. On the other hand, the addition of bovine serum albumin reduces the effect of COE and tannin compounds on inhibiting α-glucosidase, indicating that the active ingredients in COE may non-specifically bind other proteins. In conclusion, we declared that the hydrolysable tannins of COE may contribute to the inhibitory effect on α-glucosidase. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21242 |
DOI: | 10.6342/NTU201903489 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 食品科技研究所 |
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