淀粉是饮食中提供能量的主要碳水化合物,占人体摄入能量的40%～60%[1]。在淀粉消化吸收过程中,α-淀粉酶是其关键的水解酶之一[2-3]。α-淀粉酶通过作用于淀粉内部的α-1,4-糖苷键,将淀粉转化为小分子还原糖,如麦芽糖、葡萄糖[4],使餐后血糖水平迅速升高,此现象与某些代谢性疾病,如II型糖尿病和心血管疾病的患病风险呈正相关[5-7]。因此,通过抑制α-淀粉酶活性降低淀粉消化率,从而调节餐后血糖水平[8],对于缓解糖尿病、心血管疾病等非遗传性代谢疾病具有积极作用[9-10]。目前,虽然化学合成药物能抑制α-淀粉酶活性,但长期服用会有呕吐、胀气等副作用[11]。大量体外和体内实验的研究结果表明,酚酸对α-淀粉酶活性有抑制作用[12-13]。因此,具有多种生物活性的植物酚酸作为具有潜力的天然抑制剂成为研究热点,也更符合消费者对安全、绿色、多效的需求。

绿原酸(chlorogenic acid)是由咖啡酸和奎宁酸组成的一类缩合酚酸,广泛存在于咖啡、马铃薯、金银花等植物中[14],被原卫生部批准为保健食品原料[15-16]。绿原酸具有保护心血管、降脂和降糖、抗氧化、抗菌和抗癌等作用[17-19]。由于羟基位置不同,绿原酸的抗氧化能力强于咖啡酸、阿魏酸等多酚[20],更重要的是能有效清除高血糖引起的内源性活性氧,预防糖尿病并发症[21-22]。近年来,国内外相关文献主要研究了绿原酸及其衍生物对α-淀粉酶抑制效果的评估、酶反应动力学和抑制机制[23-25],但绿原酸含量、底物浓度、酶反应动力学模型的差异等因素导致抑制效果不同,结果相差较大。因此,为了明确绿原酸对α-淀粉酶的抑制作用与复杂的机制,需优化其作用条件,并通过直观数据由现象到机理系统性地阐述和揭示抑制机制。

本研究以玉米淀粉为底物,优化体外酶抑制实验条件,通过抑制动力学实验,探究绿原酸对α-淀粉酶的抑制作用与类型;采用分子对接模拟技术进一步分析绿原酸与α-淀粉酶相互作用过程中氨基酸残基的结合位点和模式。本研究拟从作用条件、抑制率、抑制动力学到抑制机理设计一整套系统研究框架,旨在为绿原酸应用于功能食品设计提供理论依据。

1 材料与方法

1.1 材料与试剂

猪胰腺α-淀粉酶(10 000 U/g),美国Sigma公司;绿原酸(纯度98%,金银花来源),西安泽邦生物科技有限公司;玉米淀粉,广州建达生物科技有限公司;葡萄糖标准溶液(分析纯),北京奥博星生物技术有限责任公司;3,5-二硝基水杨酸(DNS),北京索莱宝科技有限公司;其他试剂均为分析纯。

1.2 仪器与设备

UV-2550型紫外-可见分光光度计,岛津仪器(苏州)有限公司;AB104-S型分析天平,上海梅特勒-托利多仪器有限公司;DC-0506型电热恒温水浴锅,上海菁海仪器公司。

1.3 实验方法

1.3.1 还原糖含量的测定

称取适量玉米淀粉,加入pH值为6.9、浓度0.1 mol/L的磷酸盐缓冲液稀释,沸水浴15 min后冷却至室温,制备出不同质量浓度的糊化淀粉溶液,作为底物备用。玉米淀粉在α-淀粉酶的作用下水解,生成麦芽糖、葡萄糖等还原糖,还原糖的含量与α-淀粉酶的活性成正比,采用DNS法测定还原糖含量[26]。还原糖标准曲线方程为y=0.001 08x-0.035 83,x为吸光度,y为还原糖质量浓度(mg/mL),R2=0.999。

1.3.2 α-淀粉酶最适水解条件的确定

取0.20 mL不同质量浓度(6.0、8.0、10.0、12.0、14.0 mg/mL)的糊化淀粉溶液,加入0.25 mL α-淀粉酶溶液在37 ℃进行水解。改变α-淀粉酶活力(0.5、1.0、1.5、2.0、3.0 U/mL)和水解时间(5、10、15、20、25、30 min),测定还原糖含量,以还原糖含量为指标,确定最适的底物浓度、α-淀粉酶活力和反应时间。

1.3.3 绿原酸对α-淀粉酶抑制率的测定

根据Bernfeld法[27-28]测定绿原酸对α-淀粉酶的抑制作用。准确移取0.25 mL的α-淀粉酶溶液(酶活力为2.0 U/mL),加入0.25 mL不同质量浓度(0、2.5、5.0、7.5、10.0、12.5、15.0、20.0 mg/mL)的绿原酸溶液,不添加绿原酸溶液的样品为空白组,其余为样品组。向各组溶液中加入0.2 mL糊化淀粉溶液,37 ℃水解20 min后,沸水浴5 min,使酶失活,加入0.5 mL的DNS试剂,充分混匀,避光冷却至室温,测定还原糖含量并计算酶活。根据式(1)计算绿原酸对α-淀粉酶的抑制率。

抑制率

(1)

式(1)中,A为空白组的α-淀粉酶酶活,B为样品组的α-淀粉酶酶活。

1.3.4 抑制动力学分析

参考1.3.3节的方法,研究在不同的底物浓度下,不同质量浓度绿原酸溶液对酶促反应速率的影响。取2.0 U/mL的α-淀粉酶溶液0.25 mL与0.25 mL的绿原酸溶液(质量浓度分别为0、3.45、5.00 mg/mL)混合,在不同绿原酸质量浓度的反应体系中分别加入6.0、7.5、10.0、12.5 mg/mL的淀粉溶液0.20 mL,充分混合后测定混合溶液的吸光度,计算得到还原糖生成量及不同底物浓度下酶促反应速率。根据Lineweaver-Burk方程,以底物浓度的倒数(1/[S])为横坐标,反应速率的倒数(1/v)为纵坐标,绘制不同质量浓度下绿原酸的双倒数曲线,判断抑制类型[29]。Lineweaver-Burk方程见式(2)。

(2)

式(2)中,v为酶促反应速率,vmax为酶促反应最大反应速率,Km为米氏常数,[S]为底物浓度。

1.3.5 分子对接模拟

α-淀粉酶(PDB ID:1BLI)的三维结构模型使用蛋白质结构数据库(Protein Data Bank,PDB)构建,绿原酸(ZINC ID:21238728)的分子结构模型使用ZINC数据库构建。利用PyMOL软件对α-淀粉酶分子模型进行预处理,包括去除水分子、移除配体。利用AutoDock对α-淀粉酶分子模型和绿原酸分子模型进行加氢处理,GridBox包裹住整个α-淀粉酶分子,以确保所有活性位点的对接准确性,然后进行分子对接,运算次数为100次。对接完成后,从所有运算结果中筛选出结合能最低且结合次数最多的最优对接结果,进行结果可视化及表面结构可视化分析。

1.4 数据处理

所有实验重复3次;所得数据利用SPSS 22.0统计软件进行显著性分析,P<0.05表示存在显著性差异;使用Origin 2018软件绘图。

2 结果与分析

2.1 α-淀粉酶最适水解条件分析

底物浓度对α-淀粉酶水解作用的影响如图1。由图1可知,当底物浓度小于10 mg/mL时,随着底物浓度的增加,α-淀粉酶水解生成的还原糖含量增加。根据活性中间络合物学说可知,底物的增加导致与酶形成的酶-底物络合物随之增加,生成的产物也增加[30-31]。当底物浓度超过10 mg/mL时,还原糖质量浓度并不随着底物浓度的增加而增加,此时酶-底物络合物的生成速度与分解速度达到平衡,酶促反应体系进入稳态。为了排除底物浓度这一影响因素,保证抑制动力学分析中反应处于零级反应,底物最适浓度为10 mg/mL。

底物浓度为10 mg/mL时,排除了底物浓度的影响,酶促反应仅受酶本身催化性质和活力影响。酶活力对α-淀粉酶水解作用的影响如图2。由图2可知,随着α-淀粉酶活力升高,还原糖质量浓度也增加,当α-淀粉酶活力为2 U/mL时,还原糖质量浓度达到最高。但当生成的还原糖质量浓度过高时,反而对α-淀粉酶活力产生抑制作用[32]。因此,α-淀粉酶活力大于2 U/mL时,还原糖质量浓度不再增加而呈下降趋势。Liu等[33]采用相对较高活力的酶长时间水解淀粉时,反应速率呈降低趋势。因此,水解反应中α-淀粉酶的活力为2 U/mL时较适合。

不同底物浓度与反应时间对α-淀粉酶水解作用的影响如图3。由图3可知,随着反应时间的增加,不同底物浓度下α-淀粉酶催化产生的还原糖质量浓度逐渐增加;当反应时间达到20 min时,淀粉水解物中还原糖质量浓度达到最大值;水解时间超过20 min时,还原糖质量浓度几乎不变。因此,最适酶解时间为20 min。

2.2 绿原酸对α-淀粉酶的抑制作用分析

不同质量浓度绿原酸对α-淀粉酶的抑制率如图4。由图4可知,随着绿原酸质量浓度增加,其对α-淀粉酶的抑制率显著提高(P<0.05),IC50为4.2 mg/mL。绿原酸质量浓度为5 mg/mL时,抑制率达到66.1%;当质量浓度大于5 mg/mL时,绿原酸对α-淀粉酶的抑制效果趋于平稳。结果表明:绿原酸对α-淀粉酶具有显著的抑制作用,其中绿原酸质量浓度在0～5 mg/mL时对α-淀粉酶抑制率呈浓度依赖性关系;绿原酸质量浓度在5～20 mg/mL时,抑制率不随绿原酸质量浓度增大而增加。Zheng等[34]证明,阿魏酸对α-淀粉酶抑制率呈剂量依赖关系,IC50为0.622 mg/ml,对α-淀粉酶的抑制率能达到74.93%,并表明阿魏酸通过氢键与α-淀粉酶结合,改变淀粉酶的二级结构及某些氨基酸残基的微环境,从而达到对α-淀粉酶的抑制作用。徐冬兰等[35]发现天然提取物咖啡酰奎宁酸(CQA)的IC50为1.54 mg/mL,对α-淀粉酶有较强的抑制作用。当其衍生物3,5-双咖啡酰奎宁酸质量浓度为1.0 mg/mL时,抑制率接近100%,并发现CQA通过疏水作用力与α-淀粉酶结合成稳定的复合物,破坏酶蛋白的天然构象,表现为α-螺旋含量降低,β-转角含量增多,导致酶的稳定性下降,从而降低酶的催化活性。

多酚化合物对α-淀粉酶的抑制作用是酶与多酚化合物之间相互作用的结果[36-38]。阿魏酸、咖啡酰奎宁酸作为肉桂酸的衍生物,化学结构和性质与绿原酸有类似性,它们对α-淀粉酶的抑制作用与其化学结构有关。其中,共轭结构特征和多个羟基起到关键作用[39],多个羟基可以和酶的活性催化位点之间形成氢键[38,40];CO或CC双键和苯环之间形成的电子域能增强多酚化合物对酶的疏水作用,进而增强抑制作用[41]。正是酶与多酚类物质之间氢键和疏水力相互作用导致了酶的抑制作用。因此,作为酚酸的绿原酸对α-淀粉酶的抑制作用可能也是通过与酶相互作用来降低酶的活性。

通常用IC50表示多酚类化合物对α-淀粉酶活性的抑制程度[42]。对于大多数多酚类化合物,IC50取决于抑制剂和底物对酶活性位点的竞争,与抑制剂浓度以及底物浓度有关,这导致不同条件下测定得到的IC50难以直接比较[43]。因此,需要更详细的动力学分析来再现性地表征多酚化合物对酶活性的抑制作用。

2.3 绿原酸对α-淀粉酶的抑制动力学分析

抑制类型包括竞争性抑制、非竞争性抑制、反竞争性抑制和混合型抑制4种,根据Lineweaver-Burk曲线判断绿原酸对α-淀粉酶的抑制类型,结果如图5。由图5可知,随着绿原酸质量浓度升高,Lineweaver-Burk曲线斜率增大,与y轴的交点上移,即最大反应速率vmax减小,Km增大,且所有直线都相交于第三象限的一点,表明绿原酸对α-淀粉酶的抑制类型不属于竞争性抑制与非竞争性抑制。因此,需要借助Dixon和Cornish-Bowden方程进一步分析抑制类型。

根据Dixon方程,竞争性抑制方程见式(3)。

(3)

根据Cornish-Bowden方程,非竞争性抑制方程见式(4)。

(4)

式(3)和式(4)中, v为酶促反应速率,vmax为酶促反应最大反应速率,Km为米氏常数,[S]为底物浓度,Kic为竞争性抑制常数,Kiu为非竞争性抑制常数。

绿原酸对α-淀粉酶的抑制动力学分析结果如图6、图7。由图6和图7可知,不同质量浓度下绿原酸的Dixon曲线和Cornish-Bowden曲线均相交于一点,确定绿原酸对α-淀粉酶的抑制类型属于竞争性抑制与非竞争性抑制相结合的混合型抑制,即绿原酸不仅与淀粉竞争α-淀粉酶结合位点,并且与淀粉酶-淀粉复合物结合,以此来影响α-淀粉酶的水解能力,这与Aleixandre等[44]的结论一致。

2.4 绿原酸对α-淀粉酶抑制机制的分子对接模拟分析

根据2.3节抑制动力学的结果可知,绿原酸对α-淀粉酶的抑制类型属于混合型抑制。但是,对绿原酸与α-淀粉酶之间的这种分子相互作用以及抑制机理的分子基础缺少直观数据。分子对接模拟技术作为研究分子间相互作用的重要手段,通过分子间的空间匹配和能量匹配,展现最佳结合能等直观实验数据[45],预测其结合位点、结合模式和亲和力等信息,从而阐明分子间的相互作用机制[46]。

为了使绿原酸与α-淀粉酶的分子间相互作用可视化,通过氨基酸残基结合位点的分析预测抑制类型,利用AutoDock对绿原酸和α-淀粉酶的结构变化进行表征,见图8。选取最优对接结果,即为绿原酸与α-淀粉酶的最佳结合区域,此区域结合次数最多,结合能最低,为-33.1 kJ/mol。

图8绿原酸与α-淀粉酶结构模型的具体对接结果表明:绿原酸被α-淀粉酶的氨基酸残基Tyr193、Asp328、Glu261、His327和Ala232包围,并通过氢键与这些催化活性关键位点的氨基酸残基结合。绿原酸和α-淀粉酶活性部位通过氢键和疏水作用力相互作用,影响α-淀粉酶催化位点与底物的结合,从而抑制α-淀粉酶对底物的水解效果。

其中,Tyr193、Asp328和Glu261是α-淀粉酶发挥催化活性的关键位点[47-48]。Tyr193苯环端氢原子与绿原酸苯环上羟基形成氢键,保守区内的Asp328与绿原酸的酚羟基形成氢键,Glu261作为水解反应的质子供体,是α-淀粉酶发挥催化活性的关键位点,其通过氢键与绿原酸结合。这些α-淀粉酶的经典催化活性位点与绿原酸结合,阻碍底物进入或占据淀粉的结合位点,从而进一步抑制α-淀粉酶的活性,因此,呈现出竞争性抑制[48]。Zaynullin等[47]研究表明:二氢槲皮素通过氢键与α-淀粉酶催化活性位点的Tyr193相互作用,对α-淀粉酶活性产生竞争性抑制,与本研究的分子对接预测结果吻合。

此外,His327是催化反应中的质子供体,也可以作为质子受体在酶反应中发挥催化作用,其咪唑基是催化中最有效最活泼的催化功能基,绿原酸六元环上的羟基可与咪唑基相结合;作为α-淀粉酶基本组成的氨基酸残基,Ala232也通过氢键与绿原酸结合。相对于Tyr193、Asp328和Glu261,His327、Ala232远离α-淀粉酶的活性位点,绿原酸与二者的结合表现出非竞争性结合抑制[47]。

另外,前人利用分子对接技术研究了不同抑制剂对α-淀粉酶的抑制作用,发现α-淀粉酶结构中的Glu233、Asp300和His305在抑制作用中起到关键作用[49-51],与本研究的结果不一致,这可能是由于运行软件不同以及不同分子间结合能不同造成的。

3 结 论

本研究通过体外消化实验、抑制动力学分析和分子对接技术系统性研究了绿原酸对α-淀粉酶的抑制作用。结果发现,当底物浓度为10 mg/mL,α-淀粉酶活力为2 U/mL,水解时间为20 min时,抑制率为67.3%,IC50为4.2 mg/mL,绿原酸能够显著抑制α-淀粉酶活性。分子对接结果推断出绿原酸的抑制机制主要是通过氢键与α-淀粉酶催化活性位点的氨基酸残基和距活性位点一定距离的某些氨基酸残基结合,即绿原酸既能够优先与α-淀粉酶催化活性中心的氨基酸残基Glu261、Asp328和Tyr193结合,实现与淀粉的竞争性抑制,也能够优先与α-淀粉酶的氨基酸残基His327、Ala232结合,实现淀粉酶-底物复合物的非竞争性抑制。因此,绿原酸对α-淀粉酶的抑制类型为混合型抑制。本研究表明:绿原酸可作为一种具有明显抑制效果的α-淀粉酶天然抑制剂,基于酶反应动力学和分子对接技术,预测绿原酸通过竞争性抑制与非竞争性抑制的方式与α-淀粉酶的催化活性部位的氨基酸残基结合,从而抑制α-淀粉酶的活性,降低淀粉消化率并延缓餐后血糖的快速升高。

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Inhibition of Chlorogenic Acid on α-Amylase and Its Molecular Docking Simulation

SHI Mingwei1, ZHANG Mengyue1, CHU Hong1, LIU Li2, QU Wenxuan1, LIU Biqi1, HAN Jing1, SUN Wen1, XU Ning1,*, GUO Ling1,*

(1.College of Food Science,Northeast Agricultural University,Harbin 150030,China;2.Pharmaceutical Research Institute, Heilongjiang Provincial Institute for Drug Control and Research, Harbin 150001, China)

Abstract：As one of the key hydrolytic enzymes for starch digestion, the hydrolysis of α-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 of α-amylase is of great significance in reducing the incidence of these metabolic diseases. Chlorogenic acid and α-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 of α-amylase was 2 U/mL. When the hydrolysis time was 20 min, the inhibition rate was 67.3%, and the IC50 was 4.2 mg/mL, which confirmed that chlorogenic acid inhibited the activity of α-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 and α-amylase. It was predicted that the inhibition mechanism of chlorogenic acid on α-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 of α-amylase to achieve competitive inhibition with starch, but also form hydrogen bonds with His327 and Ala232 amino acid residues of α-amylase to achieve non-competitive inhibition combined with amylase-substrate complex. Based on the inhibitory effect of chlorogenic acid on α-amylase, chlorogenic acid could be used to inhibit the activity of α-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 type Ⅱ diabetes patients.

Keywords：chlorogenic acid; α-amylase; inhibition; in vitro digestion; molecular docking

中图分类号：TS201.2

文献标志码：A

doi：10.12301/spxb202200742

文章编号：2095-6002(2023)04-0135-09

引用格式：史明威, 张梦玥, 初鸿, 等. 绿原酸对α-淀粉酶的抑制作用及其分子对接模拟[J]. 食品科学技术学报,2023,41(4):135-143.

SHI Mingwei, ZHANG Mengyue, CHU Hong, et al. Inhibition of chlorogenic acid on α-amylase and its molecular docking simulation[J]. Journal of Food Science and Technology, 2023,41(4):135-143.

收稿日期：2022-07-18

基金项目：黑龙江省自然科学基金项目(LH2022C045);东农学者“学术骨干”计划项目(20XG14)。

Foundation: Natural Science Foundation of Heilongjiang Province of China(LH2022C045);Northeast Agricultural University Scholars Academic Backbone Program Project(20XG14).

第一作者：史明威,男,硕士研究生,研究方向为天然植物提取物的综合利用。

*通信作者：徐 宁,女,副教授,博士,主要从事植物大分子功能调控方面的研究;郭 鸰,女,教授,博士,主要从事食源性致病菌生物防控及应用方面的研究。

(责任编辑：张逸群)