苦丁冬青苦丁茶咖啡酰奎尼酸类物质与α-淀粉酶的相互作用特性

doi:10.7506/spkx1002-6630-201613002

摘要/Abstract

摘要：

探讨苦丁冬青苦丁茶中咖啡酰奎尼酸（caffeoylquinic acids，CQA）：3-CQA、4-CQA、5-CQA、3,4-diCQA、3,5-diCQA和4,5-diCQA对α-淀粉酶的体外抑制活性，并采用荧光光谱法和圆二色谱法分析CQA与α-淀粉酶的相互作用特性，使用修正后的Stern-Volmer方程与van’t Hoff方程探讨CQA-酶之间的结合常数、结合位点数及热力学参数。结果表明：3-CQA、4-CQA、5-CQA、3,4-diCQA、3,5-diCQA和4,5-diCQA对α-淀粉酶均有较强的抑制作用，半抑制浓度（IC50）分别为1.54、1.05、1.28、0.96、0.33、0.64 mg/mL；CQA与α-淀粉酶发生结合反应，生成稳定的复合物，从而造成酶分子内部荧光发生猝灭；热力学参数分析表明CQA与α-淀粉酶主要靠疏水作用力结合，反应自发进行。此外，圆二色谱结果显示CQA的结合引起α-淀粉酶二级结构的变化，破坏了酶蛋白的天然构象，从而降低了酶的催化活性。

关键词: &alpha, -淀粉酶, 咖啡酰奎尼酸, 荧光光谱, 圆二色谱, 相互作用

Abstract:

The inhibitory effects of six caffeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA,
3,5-diCQA and 4,5-diCQA) against α-amylase were studied comparatively by inhibitory activity assay. Furthermore, the
potential interaction mechanisms between CQA derivative and α-amylase were investigated by fluorescence quenching and
circular dichroism (CD) spectroscopy. The binding parameters were calculated according to modified Stern-Volmer equation,
and the thermodynamic parameters were determined by the van’t Hoff equation. The results showed that all CQA derivatives
exhibited inhibitory effects on α-amylase and the half inhibitory concentrations (IC50) were 1.54, 1.05, 1.28, 0.96, 0.33 and
0.64 mg/mL, respectively. CQA derivatives interacted with α-amylase, forming stable complexes and leading to fluorescence
quenching. Thermodynamic analysis indicated that the interaction process was spontaneous, and hydrophobic force might
be primarily responsible for the interaction. In addition, the CD spectra suggested that the binding of CQA derivatives to the
enzyme induced the change of protein structure, thus destabilizing the enzyme and reducing its activity.

Key words: α-amylase, caffeoylquinic acid, fluorescence spectroscopy, circular dichroism spectrum, interaction

中图分类号:

徐冬兰，王晴川，曾晓雄，孙怡. 苦丁冬青苦丁茶咖啡酰奎尼酸类物质与α-淀粉酶的相互作用特性[J]. 食品科学, doi: 10.7506/spkx1002-6630-201613002.

XU Donglan, WANG Qingchuan, ZENG Xiaoxiong, SUN Yi. Interaction Properties of Caffeoylquinic Acid Derivatives from Ilex kudingcha C. J. Tseng with α-Amylase[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201613002.

参考文献

[1] HOLMAN R R, SOURIJ H, CALIFF R M. Cardiovascular outcome

trials of glucose-lowering drugs or strategies in type 2 diabetes[J]. The

Lancet, 2014, 383: 2008-2017. DOI:10.1016/S0140-6736(14)60794-7.

[2] CE R I E L L O A. Postprandial hyperglycemia and diabetes

complications[J]. Diabetes, 2005, 54(1): 1-7. DOI:10.2337/

diabetes.54.1.1.

[3] CAI Xin, YU Jianan, XU Liman. The mechanism study in the

interactions of sorghum procyanidins trimer with porcine pancreatic

α-amylase[J]. Food Chemistry, 2015, 174: 291-298. DOI:10.1016/

j.foodchem.2014.10.131.

[4] 康明丽. 淀粉酶及其作用方式[J]. 食品工程, 2008(3): 11-13.

DOI:10.3969/j.issn.1673-6044.2008.03.004.

[5] DALAR A, KONCZAK I. Phenolic contents, antioxidant capacities

and inhibitory activities against key metabolic syndrome relevant

enzymes of herbal teas from Eastern Anatolia[J]. Industrial Crops and

Products, 2013, 44: 383-390. DOI:10.1016/j.indcrop.2012.11.037.

[6] HE Qiang, LÜ Yuanping, YAO Kai. Effects of tea polyphenols on the

activities of α-amylase, pepsin, trypsin and lipase[J]. Food Chemistry,

2007, 101(3): 1178-1182. DOI:10.1016/j.foodchem.2006.03.020.

[7] FEI Qunqin, GAO Yun, ZHANG Xin, 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. DOI:10.1021/jf5032907.

[8] MIAO Ming, JIANG Huan, JIANG Bo, et al. Phytonutrients

for controlling starch digestion: evaluation of grape skin

extract[J]. Food Chemistry, 2014, 145: 205-211. DOI:10.1016/

j.foodchem.2013.08.056.

[9] 阮妙芸, 张根义. 茶多酚对淀粉酶抑制作用的研究[J]. 安徽农业科学,

2008, 36(11): 4371-4373. DOI:10.3969/j.issn.0517-6611.2008.11.004.

[10] THUONG P T, SU N D, NGOC T M, et al. Antioxidant activity and

principles of Vietnam bitter tea Ilex kudingcha[J]. Food Chemistry,

2009, 113(1): 139-145. DOI:10.1016/j.foodchem.2008.07.041.

[11] SONG Chengwu, XIE Chao, ZHOU Zhiwen, et al. Antidiabetic effect

of an active components group from Ilex kudingcha and its chemical

composition[J]. Evidence-Based Complementary and Alternative

Medicine, 2012: 1-12. DOI:10.1155/2012/423690.

[12] 刘瑾, 丁平. 冬青属药用植物资源、化学成分及药理作用研究进

展[J]. 广州中医药大学学报, 2008, 25(3): 277-280. DOI:10.3969/

j.issn.1007-3213.2008.03.026.

[13] SUN Yi, XU Wenqing, ZHANG Wenqin, et al. Optimizing the

extraction of phenolic antioxidants from kudingcha made frrom Ilex

kudingcha C.J. Tseng by using response surface methodology[J].

Separation and Purification Technology, 2011, 78(3): 311-320.

DOI:10.1016/j.seppur.2011.01.038.

[14] 孙怡, 张鑫, 张文芹, 等. 苦丁茶冬青苦丁茶中多酚类物质的分离纯

化与结构解析[J]. 食品科学, 2011, 32(11): 60-63.

[15] 王晴川, 张鑫, 张文芹, 等. 苦丁冬青苦丁茶中咖啡酰奎尼酸类物

质的分离纯化和高效液相色谱法分析[J]. 食品科学, 2013, 34(22):

119-122. DOI:10.7506/spkx1002-6630-201322024.

[16] NARITA Y, INOUYE K. Inhibitory effects of chlorogenic acids from

green coffee beans and cinnamate derivatives on the activity of porcine

pancreas α-amylase isozyme I[J]. Food Chemistry, 2011, 127(4):

1532-1539. DOI:10.1016/j.foodchem.2011.02.013.

[17] HANSAWASDI C, KAWABATA J, KASAI T. α-Amylase

inhibitors from roselle (Hibiscus sabdariffa Linn.) tea[J]. Bioscience,

Biotechnology, and Biochemistry, 2000, 64(5): 1041-1043.

DOI:10.1271/bbb.64.1041.

[18] SKRT M, BENEDIK E, PODLIPNIK ?, et al. Interactions of different

polyphenols with bovine serum albumin using fluorescence quenching

and molecular docking[J]. Food Chemistry, 2012, 135(4): 2418-2424.

DOI:10.1016/j.foodchem.2012.06.114.

[19] BUISSON G, DUEE E, HASER R, et al. Three dimensional structure of

porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in

structure and activity[J]. EMBO Journal, 1987, 6(13): 3909-3916.

[20] RAWEL H M, FREY S K, MEIDTNER K, et al. Determining the

binding affinities of phenolic compounds to proteins by quenching of

the intrinsic tryptophan fluorescence[J]. Molecular Nutrition & Food

Research, 2006, 50(8): 705-713. DOI:10.1002/mnfr.200600013.

[21] WU Xuli, WANG Wenpu, ZHU T, et al. Phenylpropanoid glycoside

inhibition of pepsin, trypsin and α-chymotrypsin enzyme activity

in Kudingcha leaves from Ligustrum purpurascens[J]. Food

Research International, 2013, 54(2): 1376-1382. DOI:10.1016/

j.foodres.2013.10.020.

[22] LAKOWICZ J R. Principles of fluorescence spectroscopy[M]. New

York: Springer, 2006: 8-12.

[23] WU Xuli, DEY R, WU Hui, et al. Studies on the interaction

of (-)-epigallocatechin-3-gallate from green tea with bovine

β-lactoglobulin by spectroscopic methods and docking[J]. International

Journal of Dairy Technology, 2013, 66(1): 7-13. DOI:10.1111/j.1471-

0307.2012.00873.x.

[24] ROSS P D, SUBRAMANIAN S. Thermodynamics of protein

association reactions: forces contributing to stability[J]. Biochemistry

1981, 20(11): 3096-3102. DOI:10.1021/bi00514a017.

[25] SANDHYA B, HEGDE A H, SEETHARAMAPPA J. Elucidation

of binding mechanism and identification of binding site for an anti

HIV drug, stavudine on human blood proteins[J]. Molecular Biology

Reports, 2013, 40(5): 3817-3827. DOI:10.1007/s11033-012-2460-8.

[26] SHEN Haibo, GU Zhengqin, JIAN Kang, et al. In vitro study on

the binding of gemcitabine to bovine serum albumin[J]. Journal

of Pharmaceutical and Biomedical Analysis, 2013, 75: 86-93.

DOI:10.1016/j.jpba.2012.11.021.

[27] KANAKIS C D, HASNI I, BOURASSA P, et al. Milk β-lactoglobulin

complexes with tea polyphenols[J]. Food Chemistry, 2011, 127(3):

1046-1055. DOI:10.1016/j.foodchem.2011.01.079.

[28] LI Daojin, WANG Ye, CHEN Jianjun, et al. Characterization of the

interaction between farrerol and bovine serum albumin by fluorescence

and circular dichroism[J]. Spectrochimica Acta Part A: Molecular

and Biomolecular Spectroscopy, 2011, 79(3): 680-686. DOI:10.1016/

j.saa.2011.04.005.

[29] BESLEY N A, HIRST J D. Theoretical studies toward quantitative

protein circular dichroism calculations[J]. Journal of the American

Chemical Society, 1999, 121(41): 9636-9644. DOI:10.1021/ja990627l.

[30] TENG Yue, JI Fanying, LI Chao, et al. Interaction mechanism

between 4-aminoantipyrine and the enzyme lysozyme[J]. Journal

of Luminescence, 2011, 131(12): 2661-2667. DOI:10.1016/

j.jlumin.2011.07.005.