HPLC法测定胰蛋白酶抑制剂及其在中药木鳖子提取液研究中的应用

doi:10.5246/jcps.2011.04.050

HPLC法测定胰蛋白酶抑制剂及其在中药木鳖子提取液研究中的应用

林志燕, 余祝宏, 陈晨, 董琦鑫, 王銘章, 郁韵秋^*

1. 复旦大学药学院药物分析教研室, 上海 201203
2. 华东理工大学化学与分子工程学院, 上海 200237

收稿日期:2011-02-18 修回日期:2011-05-30 出版日期:2011-07-01 发布日期:2011-07-01
通讯作者: 郁韵秋*

An HPLC method for the assay of trypsin inhibitors and its application to the study of Momordica cochinchinensis extract

Zhi-Yan Lin, Zhu-Hong Yu, Chen Chen, Qi-Xin Dong, Ming-Zhang Wang, Yun-Qiu Yu^*

1. Department of Pharmaceutical Analysis, School of Pharmacy, Fudan University, Shanghai 201203, China
2. School of Chemistry and Molecule Engineering, East China University of Science and Technology, Shanghai 200237, China

Received:2011-02-18 Revised:2011-05-30 Online:2011-07-01 Published:2011-07-01
Contact: Yun-Qiu Yu*

摘要/Abstract

摘要： 本研究介绍了HPLC方法测定胰蛋白酶抑制剂活性及其在中药木鳖子胰蛋白酶抑制剂活性成分测定中的应用, 由于分析样品中的胰蛋白酶抑制剂可以减低胰蛋白酶的活性, 所以可以通过测定胰蛋白酶活性的下降来评价胰蛋白酶抑制剂的活性。测定原理是: 胰蛋白酶能水解N-α-苯甲酰-DL-精氨酰-4-硝基苯胺盐酸盐生成产物4-硝基苯胺, 采用HPLC方法对产物4-硝基苯胺进行分离和定量, 最终测定样品中胰蛋白酶抑制剂的活性。该方法与一般的紫外分光光度法相比选择性更好。

关键词: HPLC, 木鳖子, 胰蛋白酶抑制剂

Abstract: A reliable and validated HPLC method was established for the assay of trypsin inhibitors (TI) and it was used in the investigation of the active TI components in Momordica cochinchinensis (Cucurbitaceae family). The underlying principle of the assay is the measurement of the decrease in trypsin activity due to the presence of TI in analyzed samples, which was achieved by using HPLC separation and quantification of p-nitroanilide that was generated by tryptic hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide. The results showed that the HPLC method had higher selectivity than conventional spectrophotometric assay.

Key words: HPLC, Momordica cochinchinensis, Trypsin inhibitor

中图分类号:

R917.01

Supporting:

Foundation items: National Natural Science Foundation of China (Grant No. 30873405) and National Drug Innovation Program (Grant No. 2009ZX09301-011).

^*Corresponding author. Tel.: 13641691775; E-mail: yqyu@shmu.edu.cn

林志燕, 余祝宏, 陈晨, 董琦鑫, 王銘章, 郁韵秋^*. HPLC法测定胰蛋白酶抑制剂及其在中药木鳖子提取液研究中的应用[J]. , DOI: 10.5246/jcps.2011.04.050.

Zhi-Yan Lin, Zhu-Hong Yu, Chen Chen, Qi-Xin Dong, Ming-Zhang Wang, Yun-Qiu Yu^* . An HPLC method for the assay of trypsin inhibitors and its application to the study of Momordica cochinchinensis extract [J]. , DOI: 10.5246/jcps.2011.04.050.

参考文献

[1] Wong, R.C.H.; Fong, W.P.; Ng, T.B. Peptides. 2004, 25, 163-169.
[2] Huang, B.; Ng, T.B.; Fong, W.P.; Wan, C.C.; Yeung, H.W. Int. J. Biochem. Cell Biol. 1999, 31, 707-715.
[3] Chan, L.Y.; Wang, C.K.L.; Major, J.M.; Greenwood, K.P.; Lewis, R.J.; Craik, D.J.; Daly, N.L. J. Nat. Prod. 2009, 72, 1453-1458.
[4] Kobayashi, H.; Gotoh, J.; Hirashima, Y.; Terao, T. J. Biol. Chem. 1996, 271, 11362-11367.
[5] American Association of Cereal Chemists. Approved methods of the American Association of Cereal Chemists Method 71-10[S].
[6] ISO 14902: 2001 Animal feeding stuffs: Determination of trypsin inhibitor activity of soya products.
[7] Shi, Y.Y.; Liu, J.H.; Pang, X.Y.; Liu, J.H. Food Sci. 2009, 30, 237-240.
[8] Wang, G.D.; Wang, Z.; Yang, J.M.; Han, J.H.; Yang, Y.X. Chin. J. Food Hyg. 2009, 21, 232-235.
[9] Fu, Z.P.; Zhou, J.Y.; Wang, F.J. Food Drug. 2007, 9, 14-17.
[10] Deng, L.; Xiong, Z.D.; Li, P.G.; Mu, T.H. Food Sci. Technol. 2009, 34, 252-256.
[11] Smith, C.; Vanmegen, W.; Twaalfhoven, L.; Hitchcock, C. J. Sci. Food Agric. 1980, 31, 341-350.
[12] Kunitz, M. J. Gen. Phyical. 1946, 29, 149-154.
[13] Kreutzmann, P.; Schulz, A.; Standker, L; Forssmann, W.G.; Magert, H.E. J. Chromatogr. B. 2004, 803, 75-81.
[14] Sbrana, E.; Spinetti, M.; Secco, F.; Raspi, G. Polyhedron. 2002, 21, 1475-1479.
[15] Kowalska, J.; Pszczola, K.; Wilimowska-Pelc, A.; Lorenc-Kubis, I.; Zuziak, E.; Lugowski, M.; Legowska, A.; Kwiatkowska, A.; Sleszynska, M.; Lesner, A.; Walewska, A.; Zablotna, E.; Rolka, K.; Wilusz, T. Phytochemistry. 2007, 68, 1487-1496.
[16] Kubiak, A.; Jakimowicz, P.; Polanowski, A. Int. J. Biol. Macromol. 2009, 45, 194-199.
[17] Felizmenio-Quimio, M.E.; Daly, N.L.; Craik, D.J. J. Biol. Chem. 2001, 276, 22875-22882.
[18] Heitz, A.; Hernandez, J.F.; Gagnon, J.; Hong, T.T.; Pham, T.T.C.; Nguyen, T.M.; Le-Nguyen, D.; Chiche, L. Biochemistry. 2001, 40, 7973-7983.
[19] Pallaghy, P.K.; Nielsen, K.J.; Craik, D.J.; Norton, R.S. Protein Sci. 1994, 3, 1833-1839.
[20] Craik, D.J.; Daly, N.L.; Waine, C. Toxicon. 2001, 39, 43-60.