绿原酸对α-淀粉酶的抑制作用及其分子对接模拟

Inhibition of Chlorogenic Acid onα-Amylase and Its Molecular Docking Simulation

作为淀粉消化关键的水解酶之一,α-淀粉酶的水解作用使淀粉快速消化,引发餐后血糖水平迅速升高,持续性的餐后血糖升高与高血糖、高血脂等代谢性疾病发生呈正相关。利用天然化合物抑制α-淀粉酶活性对降低这些代谢性疾病的发生率具有重要意义。以绿原酸和α-淀粉酶为研究对象,通过体外模拟消化实验发现,当底物浓度为10 mg/mL时,α-淀粉酶的活力为2 U/mL;水解时间为20 min时,其抑制率为67.3%,IC_(50)为4.2 mg/mL,证实了绿原酸对α-淀粉酶活性的抑制作用。基于酶反应动力学,利用Lineweaver-Burk双倒数绘图法分析发现,酶的抑制类型为混合型抑制。利用分子对接技术揭示了绿原酸与α-淀粉酶的分子间作用,预测绿原酸对α-淀粉酶的抑制机制为混合型抑制,与酶反应动力学结果相符。分子对接模拟结果表明:绿原酸既能够与α-淀粉酶催化活性部位的Glu261、Asp328和Tyr193形成氢键,实现与淀粉的竞争性抑制;同时也能够与α-淀粉酶的His327、Ala232氨基酸残基形成氢键,实现与淀粉酶-底物复合物的非竞争性抑制。基于绿原酸对α-淀粉酶的抑制作用,利用绿原酸抑制淀粉酶活性从而降低淀粉消化率,延缓餐后由碳水化合物引起的血糖快速升高,以期为应用绿原酸开发适合Ⅱ型糖尿病患者的功能食品提供理论依据和有益参考。

As one of the key hydrolytic enzymes for starch digestion,the hydrolysis ofα-amylase enables starch to digest quickly,leading to a rapid increase of postprandial blood glucose levels.Persistent postprandial blood glucose increase is positively correlated with occurrence of metabolic diseases such as diabetes,hyperglycemia and hyperlipidemia,and etc..Using natural compounds to inhibit the activity ofα-amylase is of great significance in reducing the incidence of these metabolic diseases.Chlorogenic acid andα-amylase taken as the research object,the in vitro simulated digestion experiment was carried out.When the substrate concentration was 10 mg/mL,the activity ofα-amylase was 2 U/mL.When the hydrolysis time was 20 min,the inhibition rate was 67.3%,and the IC_(50)was 4.2 mg/mL,which confirmed that chlorogenic acid inhibited the activity ofα-amylase.Based on enzyme reaction kinetics,the results of Lineweaver-Burk double reciprocal mapping analysis showed that the inhibition type of the enzyme was mixed inhibition.Molecular docking technique was used to reveal the intermolecular interaction between chlorogenic acid andα-amylase.It was predicted that the inhibition mechanism of chlorogenic acid onα-amylase was a mixed inhibition,which was consistent with the results of enzyme reaction kinetics.Molecular docking simulation results showed that chlorogenic acid could not only form hydrogen bonds with Glu261,Asp328 and Tyr193 at the catalytic active site ofα-amylase to achieve competitive inhibition with starch,but also form hydrogen bonds with His327 and Ala232 amino acid residues ofα-amylase to achieve non-competitive inhibition combined with amylase-substrate complex.Based on the inhibitory effect of chlorogenic acid onα-amylase,chlorogenic acid could be used to inhibit the activity ofα-amylase to reduce the digestibility of starch and delay the rapid increase of postprandial blood glucose caused by carbohydrates.It was hoped to provide a theoretical basis and useful reference for the application of chlorogenic acid in food suitable for typeⅡdiabetes patients.