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鹰嘴豆豆芽提取物降糖抗炎作用及机制研究
刘丛丛
学位类型硕士
导师阿吉艾克拜尔·艾萨 ; 窦君
2015-06-01
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词鹰嘴豆豆芽提取物 纯化物 降血糖作用 四氧嘧啶 抗氧化 抗炎
摘要

鹰嘴豆在维吾尔医药中有着悠久的应用历史,常被用作降血糖药物。研究发现鹰嘴豆豆芽可以富集鹰嘴豆中降血糖有效成分,但对鹰嘴豆豆芽的降糖机制还未有研究报道。鹰嘴豆按照专利(授权号:ZL 200710180020.1)中方法发芽,本课题组前期已经从鹰嘴豆豆芽中提取得到鹰嘴豆豆芽提取物及纯化物。前期已按照保健食品药效验证的规定,完成了鹰嘴豆豆芽提取物作用于四氧嘧啶诱导的糖尿病小鼠,验证其具有降血糖作用。本文分别在动物和细胞水平研究了鹰嘴豆芽提取物纯化物的降血糖及纯化物的抗炎作用机制。具体工作如下: 1.初步解释鹰嘴豆豆芽提取物的降糖功能及其作用机制。 前期研究已表明:提取物给予四氧嘧啶小鼠后,与模型对照组相比:给药组血糖显著下降(结果未显示在本文),为了解其可能作用机制,首先在体外观查了可能的作用靶标,观察到鹰嘴豆豆芽提取物对于α-GAA具有较好的抑制作用,并且可以增加L6细胞对葡萄糖的消耗。其次,对动物组织的分析表明:低剂量组己糖激酶及肝糖原含量升高;IRS1、AKT、GSK3β、AMPK蛋白磷酸化水平上升;TSOD、CAT酶活增加,MDA,iNOS的酶活及NO的含量降低。以上结果说明鹰嘴豆豆芽或许是通过改善糖尿病小鼠肝脏胰岛素信号通路,同时降低肝脏的氧化损伤,从而增加糖原合成的方式降低糖尿病小鼠血糖。 2.在细胞水平研究了鹰嘴豆纯化物的降糖作用及机制 首先以MTT及葡萄糖消耗确定鹰嘴豆豆芽纯化物作用于L6细胞的浓度为40μg/mL以内。Western Blot检测结果显示纯化物可以增加AMPK及GSK3β蛋白的磷酸化水平。上述结果显示,纯化物可能通过上调AMPK及GSK3β蛋白的磷酸化水平增加L6肌管细胞糖原的消耗。 3.首次确定了鹰嘴豆纯化物具有抗炎作用,并阐明其可能的作用途径 以LPS诱导小鼠RAW264.7作为炎症细胞模型,采用MTT方法确定纯化物对RAW264.7安全作用浓度为100μg/mL以内。ELISA结果显示纯化物可以抑制LPS模型细胞的TNF-α、IL-6、TGFβ1、MCP-1、PGE2蛋白表达,并且可以降低其NO的含量。Western Blot检测结果显示纯化物可以降低COX-2、iNOS及下调p38磷酸化水平,且呈剂量依赖性。上述结果表明纯化物是通过抑制COX-2、iNOS及p38磷酸化蛋白的表达,进而下调NO、TNF-α、IL-6、TGFβ1、MCP-1、PGE2的表达而发挥抗炎作用。本论文研究了鹰嘴豆豆芽提取物降血糖作用机制,同时在细胞水平上首次证明了鹰嘴豆豆芽纯化物具有抗炎作用,并在体外研究了其降糖抗炎机制,为进一步开发鹰嘴豆豆芽奠定了理论基础。

其他摘要

Chickpea, which is widely used as a kind of glucose-regulating medicine has been used in traditional Uygur medicine for a long time. Growing evidences show that the sprouts of Chickpea could represent the effective glucose-regulatingfraction. Followed by the method of the patent(ZL 200710180020.1), we get the extract of chickpea sprouts and purify it furthermore for the purpose of enriching effective components.In the following researches, the chickpea sprout extract has been tested in ALX-induced mice as a kind of glucose-regulating treatment. Its mechanisms about glucose-regulating and anti-inflammation in both animal and cell level were explored respectively. Our work show as follows: 1. Confirmation hypoglycemic effect of chickpea sprouts extracts and explanation of the related mechanism.The chickpea sprout extract as an inhibitor of α-GAAcould increase the glucose consumption in L6 cells in vitro. After the treatment of the extract to alloxan-induced diabetic mice model, the hexokinase and hepatic glycogen of low-dose group is increased in comparison to the model group; and the phosphorylation level of IRS1, AKT, GSK3β, AMPK were same as hexokinase and hepatic glycogen; the enzyme activity of TSOD, CAT were increased and MDA, iNOS, NO were decreased by compared with the model group. Above all, the chickpea sprout extract can decrease blood glucose level by reducing the oxidative damage, increasing the process of glycogen-synthesis and improving insulin signaling pathway in the liver of diabetic mice. 2. The determination of hypoglycemic activity and mechanisms of chickpea sprouts purification in cell level.The vitro study was used to determine the hypoglycemic effect and mechanism of purification. The MTT and glucose consumption assaysin L6 cells revealed the proper concentration of purification. Western Blot showed that the purification could increase the expression levels of AMPK and phosphorylated-GSK3β. The results indicated that the purification of chickpea sprouts can increase glycogen consumption of L6 cells by up-regulating the expression of AMPK and phosphorylated-GSK3β. 3. The determination anti-inflammation activity of chickpea sprouts purification and related mechanisms. LPS-induced RAW264.7 cells were used as inflammatory model to confirm the purification’s anti-inflammatory effects. The concentration of purification was determined within 100μg/mL by MTT method. The ELISA results showed that the purification could inhibit the expression of TNF-α, IL - 6, TGFβ1, MCP-1, PGE2 and reduce the level of NO in LPS-stimulated RAW264.7 cells. Western Blot results revealed that the purification could reduce the expression levels of COX-2, iNOS and phosphorylated-p38 in a dose-dependent manner. The results also showed that the purification could exert anti-inflammatory effect by inhibiting the expression of COX-2, iNOS and P-p38 and thendown-regulate the expression levels of NO and TNF-α, IL - 6, TGFβ1, MCP-1, PGE2. This study explored the hypoglycemicand anti-inflammatory effectsof chickpea sprouts extract, and demonstrated related mechanismsin vitro. In conclusion, our study provided a theoretical basis for chickpea sprouts’ exploitation.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4246
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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刘丛丛. 鹰嘴豆豆芽提取物降糖抗炎作用及机制研究[D]. 北京. 中国科学院大学,2015.
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