高粱3-脱氧花青素对糖苷水解酶活性的抑制机制

Inhibitory Activity and Mechanism of 3-Deoxyanthocyanidins in Sorghum on Glycoside Hydrolase

摘要:

高粱3-脱氧花青素(3-DAS)对α-淀粉酶与α-葡萄糖苷酶活性具有抑制作用,但其作用机制尚不明确。考察了不同高粱品种间3-DAS的酶抑制活性差异,以确定对α-淀粉酶和α-葡萄糖苷酶抑制作用最强的高粱3-DAS提取物；采用多种光谱学技术及酶动力学分析法,进一步探究 3-DAS 与两种糖苷水解酶的相互作用机制。结果表明,红茅粱6号提取物中的3-DAS含量最高且对酶的活性抑制作用最强,但其对α-淀粉酶和α-葡萄糖苷酶的作用特性略有不同,3-DAS以非竞争性方式抑制α-淀粉酶,而以竞争性为主的混合型方式抑制α-葡萄糖苷酶。高粱3-DAS与酶结合后,可导致α-淀粉酶和α-葡萄糖苷酶在200nm附近的蛋白质骨架特征吸收峰发生红移,同时引起两种酶的内在荧光分别发生静态猝灭和静态-动态混合猝灭,并使酶的色氨酸和酪氨酸残基周围环境的疏水性升高。高粱3-DAS能减少α-淀粉酶和α-葡萄糖苷酶的α-螺旋结构含量,从而改变酶构象。高粱3-DAS与两种糖苷水解酶间的相互作用力均以静电作用力为主,结合过程为自发进行的放热反应。研究结果表明,高粱3-DAS具有作为α-淀粉酶和α-葡萄糖苷酶抑制剂的潜在价值,研究结果旨在为高粱及其副产物的高值化应用提供基础数据支撑。

Abstract:

Sorghum 3-deoxyanthocyanins (3-DAS) can inhibit the activities of α-amylase and α-glucosidase, but the mechanism is still unclear. The differences in the enzyme inhibitory activities of 3-DAS among different sorghum varieties were investigated to determine the sorghum 3-DAS extract with the strongest inhibitory effect on α-amylase and α-glucosidase. A variety of spectroscopic techniques and enzyme kinetic analysis were used to further explore the interaction mechanism between 3-DAS and the two glycoside hydrolases. The results showed that 3-DAS from Hong Mao Grain No.6 extract had the highest content and the strongest enzyme inhibitory activity, but its action characteristics for α-amylase and α-glucosidase were slightly different. 3-DAS inhibited α-amylase in a non-competitive manner, but inhibited α-glucosidase in a competitive mixed manner. The binding of sorghum 3-DAS to the enzyme resulted in a red shift of the characteristic absorption peaks of the protein cytoskeleton around 200nm for α-amylase and α-glucosidase, a static quenching and a static-dynamic mixed quenching of the intrinsic fluorescence of the two enzymes, respectively, and an increase in the hydrophobicity of the environment around the tryptophan and tyrosine residues of the enzymes. Sorghum 3-DAS could reduce the content of α-helix structure of α-amylase and α-glucosidase, thereby changing the conformation of the enzyme. The interaction between sorghum 3-DAS and the two glycoside hydrolases was dominated by electrostatic force, and the binding process was a spontaneous exothermic reaction. The results showed that sorghum 3-DAS had potential value as an inhibitor of α-amylase and α-glucosidase, and the results aimed to provide basic data support for high-value application of sorghum and its by-products.