儿茶素单体EGC对胰脂肪酶的抑制作用及其机理研究

doi:10.13305/j.cnki.jts.2022.06.001

摘要/Abstract

摘要： 为研究儿茶素单体表没食子儿茶素（Epigallocatechin,EGC）对胰脂肪酶的抑制作用及机制,以干燥绿茶为原料,采用热水提取法、三氯甲烷脱色素和柱层析分离,得到EGC单体。采用扫描电子显微镜、傅里叶红外光谱、核磁共振氢谱对纯化的EGC单体进行结构表征,再通过滴定法研究EGC对胰脂肪酶的抑制作用及类型,然后通过荧光光谱法以及分子对接技术表征了EGC对胰脂肪酶结构的影响。结果表明,EGC以非竞争性方式对胰脂肪酶表现出抑制作用,并且抑制效果随EGC浓度的增加呈持续上升趋势,半抑制浓度为(1.62±0.085) mg·mL^-1。EGC对胰脂肪酶有荧光猝灭作用,可通过分子间氢键和疏水相互作用与酶中的氨基酸残基结合,导致酶的化学结构和空间构象发生变化,从而降低酶活性。EGC主要通过改变胰脂肪酶的化学结构和空间构象来抑制该酶活性,从而达到降血脂功效。

关键词: 表没食子儿茶素, 胰脂肪酶, 分子间氢键, 机制

Abstract: In order to study the inhibitory effect and mechanism of epigallocatechin (EGC) on pancreatic lipase, EGC monomer was obtained from dry green tea by hot water extraction, chloroform depigmentation and column chromatography. The structure of purified EGC monomer was characterized by scanning electron microscope, Fourier transform infrared spectroscopy and ¹H-NMR spectroscopy. The inhibitory effect and types of EGC on pancreatic lipase were studied by titration, and the effect of EGC on pancreatic lipase structure was characterized by fluorescence spectroscopy and molecular docking. The results show that EGC exhibited the inhibition on pancreatic lipase in a non-competitive manner. The inhibitory effect continued to rise with the increase of EGC concentration, and the half inhibitory concentration (IC₅₀) was (1.62±0.085) mg·mL^-1. There was a fluorescence quenching effect of EGC on pancreatic lipase. EGC could bind to amino acid residues in the enzyme through intermolecular hydrogen bonds and hydrophobic interactions, resulting in the changes in chemical structure and spatial conformation of the enzymes, thus decreasing the enzyme activity. The results show that EGC mainly inhibited the activity of pancreatic lipase by changing the chemical structure and spatial conformation of pancreatic lipase, so as to achieve the hypolipidemic effect.

Key words: epigallocatechin, pancreatic lipase, intermolecular hydrogen bonds, mechanism

中图分类号:

满子意, 凤怡, 吴祥庭. 儿茶素单体EGC对胰脂肪酶的抑制作用及其机理研究[J]. 茶叶科学, 2022, 42(6): 863-874. doi: 10.13305/j.cnki.jts.2022.06.001.

MAN Ziyi, FENG Yi, WU Xiangting. Inhibitory Effect of Catechin Monomer EGC on Pancreatic Lipase and Mechanism[J]. Journal of Tea Science, 2022, 42(6): 863-874. doi: 10.13305/j.cnki.jts.2022.06.001.

参考文献

[1] Dechakhamphu A, Wongchum N.Investigation of the kinetic properties of phyllanthus chamaepeuce ridl. Extracts for the inhibition of pancreatic lipase activity[J]. Journal of Herbal Medicine, 2022, 32: 100508. doi: 10.1016/j.hermed.2021.100508.
[2] Druso J, Fischbach C.Biophysical properties of extracellular matrix: linking obesity and cancer[J]. Trends in Cancer, 2018, 4(4): 271-273.
[3] Phyu M, Kongkatitham V, Mekboonsonglarp W, et al.Phenanthrenes from dendrobium senile and their pancreatic lipase inhibitory activity[J]. Journal of Asian Natural Products Research, 2022, 24(7): 697-702.
[4] Zhou J F, Wang W J, Yin Z P, et al.Quercetin is a promising pancreatic lipase inhibitor in reducing fat absorptionin vivo[J]. Food Bioscience, 2021, 43: 101248. doi: 10.1016/j.fbio.2021.101248.
[5] Etxeberria U, Garza A, Campión J, et al.Antidiabetic effects of natural plant extracts via inhibition of carbohydrate hydrolysis enzymes with emphasis on pancreatic alpha amylase[J]. Expert Opinion on Therapeutic Targets, 2012, 16(3): 269-297.
[6] Liu T T, Liu X T, Huang G L, et al.Theophylline extracted from Fu brick tea affects the metabolism of preadipocytes and body fat in mice as a pancreatic lipase inhibitor[J]. International Journal of Molecular Sciences, 2022, 23(5): 2525. doi: 10.3390/ijms23052525.
[7] Siegień J, Buchholz T, Popowski D, et al.Pancreatic lipase andα-amylase inhibitory activity of extracts from selected plant materials after gastrointestinal digestionin vitro[J]. Food Chemistry, 2021, 355: 12941. doi: 10.1016/j.foodchem.2021.129414.
[8] Negi H, Gupta M, Walia R, et al.Medicinal plants and natural products: more effective and safer pharmacological treatment for the management of obesity[J]. Current Drug Metabolism, 2021, 22(12): 918-930.
[9] Sang S, Lambert J D, Ho C T, et al.The chemistry and biotransformation of tea constituents[J]. Pharmacological Research, 2022, 64(2): 87-99.
[10] Moldoveanu S, Oden R.Antioxidant character and levels of polyphenols in several tea samples[J]. ACS Omega, 2021, 6(15): 9982-9988.
[11] 陈旭, 徐楚炎, 范露, 等. 山楂叶多酚提取工艺及其降糖降脂应用研究[J]. 饲料研究, 2022, 45(5): 78-83.
Chen X, Xu C Y, Fan L, et al.Study on extraction technology of polyphenols from hawthorn leaves and its application in reducing glucose and lipid[J]. Feed Research, 2022, 45(5): 78-83.
[12] 李锋, 王贤波. 茶叶中EGC对高脂大鼠的降血脂作用[J]. 浙江农业科学, 2012(1): 32-34.
Li F, Wang X B.The hypolipidemic effect of EGC in tea on hyperlipidemia rats[J]. Zhejiang Agricultural Science, 2012(1): 32-34.
[13] 王诗卉, 刘云. 茶叶儿茶素抑制剂对胰脂肪酶构效关系的影响[J]. 食品科学, 2013, 34(9): 104-107.
Wang S H, Liu Y.Effect of (-)-epigallocatechin-3-gallate as an inhibitor on the structure-activity relationship of pancreatic lipase[J]. Food Science, 2013, 34(9): 104-107.
[14] 魏志文. 绿茶中四种儿茶素单体(EC, EGC, ECG, EGCG)和槲皮素单体分离制备[D]. 合肥: 安徽农业大学, 2009.
Wei Z W.Separation and preparation of four individual catechins (EC, EGC, ECG, EGCG) and qurecetin monomer from green tea[D]. Hefei: Anhui Agricultural University, 2009.
[15] 顾峰. 一种茶叶中EGC儿茶素的提取方法: CN201811536009.9[P].2019-2-19.
Gu F. A kind of extraction method of EGC catechin in tea: CN201811536009.9[P].2019-2-19.
[16] Xie H J, Liu C Z, Jian G, et al.Fabrication of zein-lecithin-EGCG complex nanoparticles: characterization, controlled release in simulated gastrointestinal digestion[J]. Food Chemistry, 2021, 365: 130542. doi: 10.1016/j.foodchem.2021.130542.
[17] Liu Y, Ye Y, Hu X, et al.Structural characterization and anti-inflammatory activity of a polysaccharide from the lignified okra[J]. Carbohydrate Polymers, 2021, 265(6): 118081. doi: 10.1016/j.carbpol.2021.118081.
[18] Sza B, Gh A, Gc B.Extraction, structural analysis, derivatization and antioxidant activity of polysaccharide from chinese yam[J]. Food Chemistry, 2021, 361: 130089. doi: 10.1016/j.foodchem.2021.130089.
[19] 王菲菲. 具有降脂功能的山楂原粉及其复合制剂的开发[D]. 石家庄: 河北科技大学, 2020.
Wang F F.Development of hawthorn powder and its compound preparation with lipid-lowering function[D]. Shijiazhuang: Hebei University of Science and Technology, 2020.
[20] 范金波, 李鑫芮, 葛春辉, 等. 荧光光谱法研究绿原酸与胰脂肪酶相互作用[J]. 食品与发酵科技, 2017, 53(6): 106-110.
Fan J B, Li X R, Ge C H, et al.Study on the interaction between chlorogenic acid and pancreatic lipase by fluorescence spectroscopy[J]. Food and Fermentation Sciences & Technology, 2017, 53(6): 106-110.
[21] Huang Y M, Peng W, Jian Y, et al.Mechanistic study on inhibition of porcine pancreaticα-amylase using the flavonoids from dandelion[J]. Food Chemistry, 2021, 344: 128610. doi: 10.1016/j.foodchem.2021.130089.
[22] 林心健, 夏旭东, 戚向阳, 等. 体外消化对油茶蒲提取物抑制α-葡萄糖苷酶活性的影响[J]. 核农学报, 2022, 36(2): 376-383.
Lin X J, Xia X D, Qi X Y, et al.Effect of vitro digestion on the inhibitory activity of oil tea camellia extract onα-glucosidase[J]. Journal of Nuclear Agricultural Sciences, 2022, 36(2): 376-383.
[23] 张宏图, 董伟进, 陈南, 等. 茶多酚对α-淀粉酶的抑制作用及分子机理[J]. 食品工业科技, 2022, 43(4): 90-96.
Zhang H T, Dong J W, Chen N, et al.The inhibition effort and molecular mechanism of tea polyphenols on theα-amylase[J]. Science and Technology of Food Industry, 2022, 43(4): 90-96.
[24] 张国文, 黎沙, 朱苗. 白杨素对胰脂肪酶的抑制作用及机制[J]. 南昌大学学报(理科版), 2021, 45(6): 545-552.
Zhang G W, Li S, Zhu M.Inhibitoryan mechanism of chrysin on pancreatic lipase[J]. Journal of Nanchang University (Natural Science), 2021, 45(6): 545-552.
[25] 董燕茹, 曹妍彦, 卢佳纯, 等. 儿茶素与咖啡碱络合物的晶体学研究[J]. 茶叶科学, 2021, 41(1): 80-89.
Dong Y R, Cao Y Y, Lu J C, et al.Crystallographic study on the complex of catechins and caffeine[J]. Journal of Tea Science, 2021, 41(1): 80-89.
[26] Wu G, Fan G, Zhou J, et al.Structure and main polyphenols in the haze of blackberry wine[J]. LWT-Food Science and Technology, 2021, 149(1): 111821. doi: 10.1016/j.lwt.2021.111821.
[27] Liu Y, Ye Y, Hu X, et al.Structural characterization and anti-inflammatory activity of a polysaccharide from the lignified okra[J]. Carbohydrate Polymers, 2021, 265(6): 118081. doi: 10.1016/j.carbpol.2021.118081.
[28] Xin C, Zhao M Y, Wang J H, et al.Hawthorn polyphenols, D-chiro-inositol, and epigallocatechin gallate exert a synergistic hypoglycemic effect[J]. Journal of Food Biochemistry, 2021, 45(7): e1377. doi: 10.1111/jfbc.13771.
[29] 黎沙. 基于光谱法结合分子模拟技术研究黄酮化合物对胰脂肪酶的抑制作用机制[D]. 南昌: 南昌大学, 2021.
Li S.Study on the inhibitory effect of flavonoids on pancreatic lipase by spectroscopy combined with molecular simulation technology[D]. Nanchang: Nanchang University, 2021.
[30] Winkler F K, D Arcy A, Hunziker W. Structure of human pancreatic lipase[J]. Nature, 1990, 343(6260): 771-774
[31] 杜沁岭, 杨芳, 徐文, 等. 银耳多糖对淀粉消化酶的抑制作用及其机理研究[J]. 食品工业科技, 2022, 43(2): 120-125.
Du Q L, Yang F, Xu W, et al.Inhibitory effect of tremella fuciformis polysaccharide on starch digestive enzymes and its action mechanism[J]. Science and Technology of Food Industry, 2022, 43(2): 120-125.
[32] 刘天囡, 徐梦佳, 胡冰, 等. 茯砖茶多酚类物质对胰脂肪酶活性的抑制作用[J]. 食品科学, 2015, 36(21): 46-49.
Liu T N, Xu M J, Hu B, et al.Inhibitory activity of polyphenols from fuzhuan brick tea on pancreatic lipase[J]. Food Science, 2015, 36(21): 46-49.
[33] 廖家乐, 方甜, 范艳丽. 枸杞叶黄酮对胰脂肪酶活性的抑制作用[J]. 中国食品学报, 2022, 22(5): 43-53.
Liao J L, Fang T, Fan Y L.Inhibitory effect ofLycium barbarumleaf flavonoids on pancreatic lipase activity[J]. Journal of Chinese Institute of Food Science and Technology, 2022, 22(5): 43-53.
[34] Zhang L, Zheng J, Ma M, et al.Drug-guided screening for pancreatic lipase inhibitors in functional foods[J]. Food & Function, 2021, 12(10): 4644-4653.
[35] 褚盼盼, 侯冬敏, 杨卫民. 苦荞麦多糖对胰脂肪酶抑制作用的研究[J]. 中国食品添加剂, 2015(9): 78-83.
Chu P P, Hou D M, Yang W M.Bitter buckwheat polysaccharide the study of pancreatic lipase inhibition[J]. China Food Additives, 2015(9): 78-83.
[36] 张静, 米佳, 禄璐, 等. 黑果枸杞花色苷提取物对胰脂肪酶活性的影响[J]. 食品科学, 2020, 41(5): 8-14.
Zhang J, Mi J, Lu L, et al.Effect of anthocyanins extract fromLycium ruthenicummurr. fruit on pancreatic lipase activity[J]. Food Science, 2020, 41(5): 8-14.
[37] 安欢, 叶云, 丁华杰, 等. 苦瓜多糖对胰脂肪酶抑制作用的研究[J]. 中国调味品, 2020, 45(2): 27-31.
An H, Ye Y, Ding H J, et al.Study on the inhibitory effect ofMomordica charantiapolysaccharide on pancreatic lipase[J]. China Condiment, 2020, 45(2): 27-31.
[38] 赵瑜, 周家春, 张靖伟, 等. 紫娟茶提取物对血管紧张素转换酶、α-淀粉酶和胰脂肪酶的体外抑制作用[J]. 食品工业科技, 2017, 38(19): 11-20.
Zhao Y, Zhou J C, Zhang J W, et al.Inhibitory effects of zijuan tea (Camellia sinensisvar. Kitamura) extracts on angiotensin converting enzyme,α-amylase and pancreatic lipase in vitro[J]. Science and Technology of Food Industry, 2017, 38(19): 11-20.
[39] 杨龙佳, 王进, 杨庆雄. 红茶提取物对胰脂肪酶抑制活性的研究[J]. 食品科技, 2015, 40(8): 212-222.
Yang L J, Wang J, Yang Q X.Inhibitory effects of black tea on pancreatic lipases[J]. Food Science and Technology, 2015, 40(8): 212-222.
[40] Sergent T, Vanderstraeten J, Winand J, et al.Phenolic compounds and plant extracts as potential natural anti-obesity substances[J]. Food Chemistry, 2012, 135: 68-73.
[41] Grove K A, Sae-Tan S, Kennett M J, et al.(-)-Epigallocatechin-3-gallate inhibits pancreatic lipase and reduces body weight gain in high fat-fed obese mice[J]. Obesity, 2013, 20(11): 2311-2313.
[42] Rahim A T, Takahashi Y, Yamaki K.Mode of pancreatic lipase inhibition activityin vitroby some flavonoids and non-flavonoid polyphenols[J]. Food Research International, 2015, 75(9): 289-294.
[43] 黄琳翔, 施乐乐, 蔡志英, 等. 黑木耳中抑制胰脂肪酶活性物质的提取工艺及体外抑制效果[J]. 菌物学报, 2020, 39(2): 441-451.
Huang L X, Shi L L, Cai Z Y, et al.Extraction technology and inhibitory effects in vitro of substances inhibiting pancreatic lipase activities from fruiting body ofAuricaria heimuer[J]. Mycosystema, 2020, 39(2): 441-451.
[44] 刘帅, 曹立民, 林洪, 等. 胰脂肪酶特异性卵黄抗体对胰脂肪酶活性的抑制作用研究[J]. 食品工业科技, 2016, 37(11): 350-361.
Liu S, Cao L M, Lin H, et al.Inhibitory effect of the specific chicken egg yolk antibody on pancreatic lipasein vitro[J]. Science and Technology of Food Industry, 2016, 37(11): 350-361.
[45] Lan W Q, Liu J L, Wang M, et al.Effects of apple polyphenols and chitosan-based coatings on quality and shelf life of large yellow croaker (Pseudosciaena crocea) as determined by low field nuclear magnetic resonance and fluorescence spectroscopy[J]. Journal of Food Safety, 2021, 41(3): e12887. doi: 10.1111/jfs.12887.
[46] Gong T, Yang X, Bai F T, et al.Young apple polyphenols as naturalα-glucosidase inhibitors:in vitroand in silico studies[J]. Bioorganic Chemistry, 2020, 96: 103625. doi: 10.1016/j.bioorg.2020.103625.
[47] Waner M J, Hiznay J M, Mustovich A T, et al.Streptavidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence[J]. Biochemistry and Biophysics Reports, 2019, 17: 127-131.
[48] Hua F, Zhou P, Wu H Y, et al.Inhibition ofα-glucosidase andα-amylase by flavonoid glycosides from Lu'an Guapian tea: molecular docking and interaction mechanism[J]. Food & Function, 2018, 9(8): 4173-4183.
[49] Martinez-Gonzalez A I, Alvarez-Parrilla E, Díaz-Sánchez Á G, et al.In vitroinhibition of pancreatic lipase by polyphenols: a kinetic, fluorescence spectroscopy and molecular docking study[J]. Food Technology & Biotechnology, 2017, 55(4): 519-530.
[50] 李勤, 黄建安, 傅冬和, 等. 茶叶减肥及对人体代谢综合征的预防功效[J]. 中国茶叶, 2019, 41(5): 7-13.
Li Q, Huang J A, Fu D H, et al.Tea for weight loss and its preventive effect on human metabolic syndrome[J]. Chinese Tea, 2019, 41(5): 7-13.
[51] Li Z, Liu Y, Zhao W, et al. Pathogenic effects and potential regulatory mechanisms of tea polyphenols on obesity[J]. BioMed Research International, 2019(14): 579734. dio: 10.1155/2019/2579734.
[52] 黄玉凤, 肖安风, 倪辉, 等. 单宁酶处理提高茶梗儿茶素含量及茶梗提取液生物活性[J]. 农业工程学报, 2013, 29(13): 277-285.
Huang Y F, Xiao A F, Ni H, et al.Tannase treatment improves contents of catechins and bioactivity of tea stalk infusion[J]. Transactions of the Chinese Society of Agricultural Engineering, 2013, 29(13): 277-285.
[53] 张忠. 茶多酚对胰脂肪酶活性的抑制作用[J]. 食品工业, 2013, 34(8): 168-170.
Zhang Z.The inhibition of tea polyphenol on pancreatic lipase[J]. The Food Industry, 2013, 34(8): 168-170.
[54] 李鑫磊. 不同茶类代谢产物差异及其水提物、差异代谢物对神经细胞保护作用与机制[D]. 福州: 福建农林大学, 2020.
Li X L.Study of the different metabolites of different tea types and their water extracts and differential metabolites neuronal cell protective effects and mechanism[D]. Fuzhou: Fujian Agriculture and Forestry University, 2020.
[55] Kamesh V, Chiu H F, Ju C C, et al.Comparative studies on the hypolipidemic, antioxidant and hepatoprotective activities of catechin-enriched green and oolong tea in a double-blind clinical trial[J]. Food & Function, 2018, 9(2): 1205-1213.
[56] 马小灵. 富硒茶油的品质特性及其降血脂与抗氧化功能研究[D]. 长沙: 中南林业科技大学, 2016.
Ma X L.Study on the quality characteristics, hypolipidemic and antioxidant functions of selenium-enrichedcamellia oil[D]. Changsha: Central South University of Forestry and Technology, 2016
[57] 周斌星, 孔令波, 李发志. 普洱茶(熟茶)不同发酵阶段在制品降血脂效果的比较研究[J]. 中国农学通报, 2010, 26(18): 231-236.
Zhou B X, Kong L B, Li F Z.Study on blood lipid-depressing function in the different stages of fermented Pu-erh tea[J]. Chinese Agricultural Science Bulletin, 2010, 26(18): 231-236.