表没食子儿茶素没食子酸酯对葡萄糖淀粉酶的抑制机制

doi:10.3969/j.issn.1004-1524.20240700

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (9): 1951-1957.DOI: 10.3969/j.issn.1004-1524.20240700

表没食子儿茶素没食子酸酯对葡萄糖淀粉酶的抑制机制

黄伟红¹(), 刘一²^,^*(), 郑富明³

¹ 浙江省农业技术推广中心, 浙江杭州 310020
² 浙江农林大学食品与健康学院, 浙江杭州 311300
³ 浙江遂昌原食健康农业发展有限公司, 浙江遂昌 323300

收稿日期:2024-08-01 出版日期:2025-09-25 发布日期:2025-10-15
作者简介:*刘一,E-mail: liuyi@zafu.edu.cn
黄伟红(1970—),女,绍兴柯桥人,学士,高级农艺师,主要从事茶叶技术推广工作。E-mail: hnnytwh@163.com
通讯作者: 刘一
基金资助:
浙江农林大学人才启动项目(2024LFR006)

Inhibition mechanism of epigallocatechin gallate on glucoamylase

HUANG Weihong¹(), LIU Yi²^,^*(), ZHENG Fuming³

¹ Zhejiang Agricultural Technology Extension Center, Hangzhou 310020, China
² College of Food and Health, Zhejiang A & F University, Hangzhou 311300, China
³ Suichang Yuanshi Health Agriculture Development Co., Ltd., Suichang 323300, Zhejiang, China

Received:2024-08-01 Online:2025-09-25 Published:2025-10-15
Contact: LIU Yi

摘要/Abstract

摘要： 综合运用抑制动力学、荧光强度、表面疏水性分析和分子对接等方法研究表没食子儿茶素没食子酸酯(EGCG)对葡萄糖淀粉酶的抑制作用,及消化过程中葡萄糖淀粉酶结构的变化。结果表明,EGCG对葡萄糖淀粉酶的抑制属于混合竞争性抑制,以竞争抑制为主。随着EGCG质量浓度的升高,葡萄糖淀粉酶在425 nm处的荧光强度逐渐降低,荧光猝灭Stern-Volmer曲线的线性关系良好,荧光方式属于静态猝灭。随着EGCG质量浓度的增加,葡萄糖淀粉酶的表面疏水作用力逐渐降低。分子对接的结果表明,EGCG与葡萄糖淀粉酶非催化位点上的氨基酸结合。综上,EGCG与葡萄糖淀粉酶非催化位点的结合,影响催化位点与底物的结合,改变葡萄糖淀粉酶的结构,影响酶的表面疏水性,从而降低酶的催化效率。研究结果可为理解EGCG与葡萄糖淀粉酶的作用机制提供理论依据。

关键词: 表没食子儿茶素没食子酸酯, 葡萄糖淀粉酶, 抑制动力学

Abstract:

To investigate the inhibition mechanism of epigallocatechin gallate (EGCG) on glucoamylase and its effect on glucoamylase structure during the digestion process, the methods including inhibition kinetics, fluorescence intensity, surface hydrophobicity analysis and molecular docking were comprehensively adopted. It was shown that EGCG exhibited mixed competitive inhibition on glucoamylase with competitive inhibition as the main factor. As the mass concentration of EGCG increased, the fluorescence intensity of glucoamylase at 425 nm gradually decreased, and the linear relationship of the Stern-Volmer curve of fluorescence quenching was good, and the fluorescence quenching mode belonged to static quenching. With the increase in EGCG mass concentration, the surface hydrophobic interaction of glucoamylase gradually decreased. The molecular docking revealed that the EGCG was bound to amino acids on the non-catalytic site of glucoamylase. It was concluded that the binding of EGCG to the non-catalytic site of glucoamylase affected the binding of the catalytic site and substrate, changed the structure of glucoamylase, affected the hydrophobic interaction of glucoamylase, thus reduced the catalytic efficiency of glucoamylase. These findings provided theoretical basis for the understanding of the interaction mechanism between EGCG and glucoamylase.

Key words: epigallocatechin gallate, glucoamylase, inhibition kinetics

中图分类号:

黄伟红, 刘一, 郑富明. 表没食子儿茶素没食子酸酯对葡萄糖淀粉酶的抑制机制[J]. 浙江农业学报, 2025, 37(9): 1951-1957.

HUANG Weihong, LIU Yi, ZHENG Fuming. Inhibition mechanism of epigallocatechin gallate on glucoamylase[J]. Acta Agriculturae Zhejiangensis, 2025, 37(9): 1951-1957.

图/表 5

图1 表没食子儿茶素没食子酸酯(EGCG)、淀粉与葡萄糖淀粉酶相互作用的Lineweaver-Burk曲线 S为淀粉的质量浓度,V为反应速率,R2为决定系数。

Fig.1 Lineweaver-Burk curves of epigallocatechin gallate (EGCG), starch and glucoamylase S is the mass concentration of starch. V is the reaction rate. R2 is the determination coefficient.

图2 表没食子儿茶素没食子酸酯(EGCG)与葡萄糖淀粉酶相互作用的荧光强度柱上无相同字母的表示差异显著(p<0.05)。下同。

Fig.2 Fluorescence intensity of the interaction between epigallocatechin gallate (EGCG) and glucoamylase Bars marked without the same letters indicate significant difference at p<0.05.The same as below.

图3 表没食子儿茶素没食子酸酯(EGCG)与葡萄糖淀粉酶的Stern-Volmer曲线 F0、F分别为荧光猝灭前、后的荧光强度。

Fig.3 Stern-Volmer curve between epigallocatechin gallate (EGCG) and glucoamylase F0 and F are the fluorescence intensities before and after fluorescence quenching, respectively.

图4 表没食子儿茶素没食子酸酯(EGCG)与葡萄糖淀粉酶的表面疏水作用力

Fig.4 Surface hydrophobic interaction between epigallocatechin gallate (EGCG) and glucoamylase

图5 分子对接结果

Fig.5 Results of molecular docking

参考文献 37

[1]	WANG S J, LI C L, COPELAND L, et al. Starch retrogradation: a comprehensive review[J]. Comprehensive Reviews in Food Science and Food Safety, 2015, 14(5): 568-585.
[2]	REAVEN G. Insulin resistance type 2 diabetes mellitus, and cardiovascular disease: the end of the beginning[J]. Circulation, 2005, 112(20): 3030-3032.
[3]	PATEL T P, RAWAL K, BAGCHI A K, et al. Insulin resistance: an additional risk factor in the pathogenesis of cardiovascular disease in type 2 diabetes[J]. Heart Failure Reviews, 2016, 21(1): 11-23.
[4]	REAVEN G, ABBASI F, MCLAUGHLIN T. Obesity, insulin resistance, and cardiovascular disease[J]. Recent Progress in Hormone Research, 2004, 59: 207-223.
[5]	CHIBA S. Molecular mechanism in α-glucosidase and glucoamylase[J]. Bioscience, Biotechnology, and Biochemistry, 1997, 61(8): 1233-1239.
[6]	ZONG X Y, WEN L, WANG Y T, et al. Research progress of glucoamylase with industrial potential[J]. Journal of Food Biochemistry, 2022, 46(7): e14099.
[7]	CHANDRASEKARAN M. Enzymes in food and beverage production: an overview[M]// Enzymes in food and beverage Processing. Boca Raton: CRC Press, 2015: 133-154.
[8]	SVIHUS B, HERVIK A K. Digestion and metabolic fates of starch, and its relation to major nutrition-related health problems: a review[J]. Starch-Stärke, 2016, 68(3/4): 302-313.
[9]	SUN L, MIAO M. Dietary polyphenols modulate starch digestion and glycaemic level: a review[J]. Critical Reviews in Food Science and Nutrition, 2020, 60(4): 541-555.
[10]	SUN L J, WARREN F J, GIDLEY M J. Natural products for glycaemic control: polyphenols as inhibitors of alpha-amylase[J]. Trends in Food Science & Technology, 2019, 91: 262-273.
[11]	HE Q, LV Y P, YAO K. Effects of tea polyphenols on the activities of α-amylase, pepsin, trypsin and lipase[J]. Food Chemistry, 2007, 101(3): 1178-1182.
[12]	WILLIAMSON G. Possible effects of dietary polyphenols on sugar absorption and digestion[J]. Molecular Nutrition & Food Research, 2013, 57(1): 48-57.
[13]	CIANCIOSI D, FORBES-HERNÁNDEZ T Y, REGOLO L, et al. The reciprocal interaction between polyphenols and other dietary compounds: impact on bioavailability, antioxidant capacity and other physico-chemical and nutritional parameters[J]. Food Chemistry, 2022, 375: 131904.
[14]	WANG L, LI P H, FENG K. EGCG adjuvant chemotherapy: current status and future perspectives[J]. European Journal of Medicinal Chemistry, 2023, 250: 115197.
[15]	ZHONG Y, SHAHIDI F. Lipophilised epigallocatechin gallate (EGCG) derivatives and their antioxidant potential in food and biological systems[J]. Food Chemistry, 2012, 131(1): 22-30.
[16]	ZHONG Y, CHIOU Y S, PAN M H, et al. Anti-inflammatory activity of lipophilic epigallocatechin gallate (EGCG) derivatives in LPS-stimulated murine macrophages[J]. Food Chemistry, 2012, 134(2): 742-748.
[17]	KUMAZOE M, TACHIBANA H. Anti-cancer effect of EGCG and its mechanisms[J]. Functional Foods in Health and Disease, 2016, 6(2): 70.
[18]	SIMSEK M, QUEZADA-CALVILLO R, NICHOLS B, et al. Inhibition of individual subunits of maltase-glucoamylase and sucrase-isomaltase by polyphenols (1045.26)[J]. The FASEB Journal, 2014, 28(S1): 1045.26.
[19]	SIMSEK M, QUEZADA-CALVILLO R, FERRUZZI M G, et al. Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release[J]. Journal of Agricultural and Food Chemistry, 2015, 63(15): 3873-3879.
[20]	NGUYEN T T H, JUNG S H, LEE S, et al. Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition[J]. Biotechnology and Bioprocess Engineering, 2012, 17(5): 966-971.
[21]	陈南, 高浩祥, 何强, 等. 植物多酚与淀粉的分子相互作用研究进展[J]. 食品工业科技, 2023, 44(2): 497-505.
	CHEN N, GAO H X, HE Q, et al. A review of the molecular interaction between plant polyphenols and starch[J]. Science and Technology of Food Industry, 2023, 44(2): 497-505. (in Chinese with English abstract)
[22]	DENG N, DENG Z, TANG C, et al. Formation, structure and properties of the starch-polyphenol inclusion complex: a review[J]. Trends in Food Science & Technology, 2021, 112: 667-675.
[23]	WANG C C, SHENG Z, ZHANG Y H, et al. Effects of epigallocatechin-3-gallate on the structural hierarchy of the gluten network in dough[J]. Food Hydrocolloids, 2023, 142: 108803.
[24]	郝飞龙, 梁骁, 范莹, 等. 黑豆皮多酚提取物体外对α-淀粉酶和α-葡萄糖苷酶活性的影响[J]. 食品安全质量检测学报, 2017, 8(9): 3359-3364.
	HAO F L, LIANG X, FAN Y, et al. Effects of polyphenol extracts from black soybean seed coat on the activity of α-amylase and α-glucosidase in vitro[J]. Journal of Food Safety & Quality, 2017, 8(9): 3359-3364. (in Chinese with English abstract)
[25]	汤夕瑶, 刘宇佳, 朱杰, 等. 酒糟结合多酚的抗氧化活性及对α-淀粉酶荧光特性的影响[J]. 食品安全质量检测学报, 2023, 14(17): 36-44.
	TANG X Y, LIU Y J, ZHU J, et al. Antioxidant activity of bound polyphenols from distiller's grains and the effects on fluorescence characteristics of α-amylase[J]. Journal of Food Safety & Quality, 2023, 14(17): 36-44. (in Chinese with English abstract)
[26]	FEI Q Q, GAO Y, ZHANG X, et al. Effects of oolong tea polyphenols, EGCG, and EGCG3″Me on pancreatic α-amylase activity in vitro[J]. Journal of Agricultural and Food Chemistry, 2014, 62(39): 9507-9514.
[27]	盛政, 杜文凯, 王崇崇, 等. 茶多酚对茶食品中还原糖检测方法的影响[J]. 茶叶科学, 2023, 43(4): 567-575.
	SHENG Z, DU W K, WANG C C, et al. Effect of tea polyphenols on the determination of reducing sugar in tea food[J]. Journal of Tea Science, 2023, 43(4): 567-575. (in Chinese with English abstract)
[28]	JIANG C, CHEN Y, YE X, et al. Three flavanols delay starch digestion by inhibiting α-amylase and binding with starch[J]. International Journal of Biological Macromolecules, 2021, 172: 503-514.
[29]	GEHLEN M H. The centenary of the Stern-Volmer equation of fluorescence quenching: from the single line plot to the SV quenching map[J]. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 2020, 42: 100338.
[30]	DIAS F F G, DE MOURA BELL J M L N. Understanding the impact of enzyme-assisted aqueous extraction on the structural, physicochemical, and functional properties of protein extracts from full-fat almond flour[J]. Food Hydrocolloids, 2022, 127: 107534.
[31]	SUN L J, CHEN W Q, MENG Y H, et al. Interactions between polyphenols in thinned young apples and porcine pancreatic α-amylase: inhibition, detailed kinetics and fluorescence quenching[J]. Food Chemistry, 2016, 208: 51-60.
[32]	ZHAO T, SUN L J, WANG Z C, et al. The antioxidant property and α-amylase inhibition activity of young apple polyphenols are related with apple varieties[J]. LWT, 2019, 111: 252-259.
[33]	MUNISHKINA L A, FINK A L. Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins[J]. Biochimica et Biophysica Acta, 2007, 1768(8): 1862-1885.
[34]	ZHENG Y X, YANG W H, SUN W X, et al. Inhibition of porcine pancreatic α-amylase activity by chlorogenic acid[J]. Journal of Functional Foods, 2020, 64: 103587.
[35]	马丽娜, 陈喜文, 甘睿, 等. 葡萄糖淀粉酶的结构和功能研究进展[J]. 生物技术通讯, 2005, 16(6): 677-680.
	MA L N, CHEN X W, GAN R, et al. Advances on structure and function of glucoamylase[J]. Letters in Biotechnology, 2005, 16(6): 677-680. (in Chinese with English abstract)
[36]	SIERKS M R, SVENSSON B. Functional roles of the invariant aspartic acid 55, tyrosine 306, and aspartic acid 309 in glucoamylase from Aspergillus awamori studied by mutagenesis[J]. Biochemistry, 1993, 32(4): 1113-1117.
[37]	TONG L G, ZHENG J, WANG X, et al. Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications[J]. Biotechnology for Biofuels, 2021, 14(1): 202.

表没食子儿茶素没食子酸酯对葡萄糖淀粉酶的抑制机制

Inhibition mechanism of epigallocatechin gallate on glucoamylase

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 37

相关文章 3

编辑推荐

Metrics

本文评价

[1]	杨春, 梁思慧, 汪安然, 陈娟, 李燕, 林开勤, 密孝增, 乔大河, 陈正武, 郭燕. 五十四份茶树种质的特征性代谢产物含量与耐寒性[J]. 浙江农业学报, 2025, 37(9): 1860-1871.
[2]	方明雅, 余宏伟, 武雅娴, 韩文炎, 李鑫, 刘海河. 外源表没食子儿茶素没食子酸酯对甜瓜幼苗白粉病抗性的影响[J]. 浙江农业学报, 2023, 35(1): 138-145.
[3]	李洋, 刘凯, 魏吉鹏, 张兰, 李鑫, 韩文炎, 李青云. 不同浓度EGCG对NaCl胁迫下黄瓜种子萌发及其抗性的影响[J]. 浙江农业学报, 2018, 30(7): 1160-1167.