论文摘要
α-淀粉酶抑制剂(α-amylase inhibitor, α-AI)属于一种糖苷水解酶抑制剂,广泛存在于植物种子的胚乳和微生物的代谢产物中,α-AI作为一种新型糖尿病治疗药物,以人体糖代谢的关键酶淀粉酶为作用靶点,抑制其生物活性,进而减少人体对食物中碳水化合物的消化吸收以及脂肪生成,起到降糖、降脂的作用。目前α-AI在医药和农业等领域有广泛的用途,特别是在临床上对糖尿病,高血糖,高血脂,脂肪过多症等疾病的治疗具有重要价值。另外,淀粉酶抑制剂提取物的疗效及安全性取决于加工和提取工艺,得到的抑制剂可被添加到食品中,用来增加糖尿病人的碳水化合物耐受量,降低摄取的能量,从而避免出现肥胖症。本文首次利用硫酸铵分级沉淀、阳离子柱交换以及Sephadex G-75凝胶过滤技术从鹰嘴豆(Cicer arietinum L.)种子中分离纯化得到α-淀粉酶抑制剂,并进行部分性质研究。鹰嘴豆种子经脱皮、碾磨、脱脂后,利用正交试验法对提取温度(A)、pH值(B)、提取时间(C)、溶剂用量(D)4个因素进行优化,确定α-AI粗提条件为在pH4.0,提取温度50℃,提取液体积比为1:35条件下浸提4.0h,其抑制活力最高。粗提液经35%65%饱和硫酸铵分步沉淀,进一步利用CM-52纤维素离子交换和Sephadex G-75分子筛层析,分离纯化得到其中的一种α-淀粉酶抑制剂(α-AI)。经SDS-聚丙烯酰胺凝胶电泳,计算出该α-AI的分子量约为40.4kDa,对淀粉酶的抑制常数Ki为3.14×10-6mol·L-1(淀粉为底物时),最大半抑制剂浓度为1.03×10-6mol·L-1,是一种抑制活力很强的非竞争性抑制剂。鹰嘴豆α-AI的活性最佳孵化时间10min,最佳浓度为1.0×102μg·mL-1,在80℃加热30min,其抑制活性仍维持在85%左右,表明α-AI具有一定的耐热性。对pH值稳定性的研究表明,α-AI在pH值6.3附近时抑制活性最大,在pH3.011.0之间抑制活性相对稳定。
论文目录
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