Zhongguo youzhi (Feb 2023)

南瓜籽ACE抑制肽的Plastein反应修饰及分离鉴定Plastein reaction modification of pumpkin seed ACE inhibitory peptide and its isolation and identification

  • 胡田媛1,杨晨1，陈哲2,3，郑竟成1,3，何东平1,3，杨永2,3，雷芬芬1,3 HU Tianyuan1, YANG Chen1, CHEN Zhe2,3, ZHENG Jingcheng1,3, HE Dongping1,3, YANG Yong2,3, LEI Fenfen1,3

DOI
https://doi.org/10.19902/j.cnki.zgyz.1003-7969.210722
Journal volume & issue
Vol. 48, no. 2
pp. 29 – 35

Abstract

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为进一步提升南瓜籽多肽的ACE抑制活性，利用Plastein反应对南瓜籽ACE抑制肽进行修饰，探究底物质量分数、反应温度、反应时间以及3种外源氨基酸添加量对ACE抑制率的影响。采用超滤和Sephadex G-25柱层析等分离修饰产物，并利用LC-MS/MS鉴定肽序列。结果表明：Plastein反应修饰的最佳条件为底物质量分数45%，反应温度20 ℃，反应时间3 h；分别添加亮氨酸、苯丙氨酸或甘氨酸均能显著提升修饰产物的ACE抑制率，其中添加0.5 mmol/g亮氨酸时，修饰产物的ACE抑制率最高，比修饰前提高了24.50百分点；经超滤和Sephadex G-25柱层析分离，获得ACE抑制率达89.61%的组分，经LC-MS/MS鉴定出76个肽段，固相合成其中4种多肽IFH、IFF、LAAF、DFHPR，其抑制ACE的IC50分别为1.55、2.24、3.79 mmol/L和7.86 mmol/L。综上，Plastein反应修饰可显著改善南瓜籽ACE抑制肽的ACE抑制活性，经超滤和Sephadex G-25柱层析分离，可获得高ACE抑制活性的南瓜籽ACE抑制肽。 Plastein reaction was applied to modify pumpkin seed polypeptide to further enhance its ACE inhibitory activity. The effects of substrate mass fraction, reaction temperature, reaction time, and the addition of three exogenous amino acids on the inhibition rate of ACE inhibitory peptides were studied. The modified products were separated by ultrafiltration and Sephadex G-25 column chromatography, and were identified by LC-MS/MS. The results showed that the optimal conditions for Plastein reaction modification were substrate mass fraction 45%, reaction temperature 20 ℃, and reaction time 3 h. Adding leucine, phenylalanine or glycine respectively could significantly increase the ACE inhibition rate of the modified products. When 0.5 mmol/g leucine was added, the ACE inhibition rate of the modified product was up to 24.50 percentage points higher than that before the modification. After ultrafiltration and Sephadex G-25 column chromatography separation, the fraction with 89.61% ACE inhibition rate was obtained. A total of 76 peptides was identified by LC-MS/MS, and 4 of them with IFH, IFF, LAAF and DFHPR were synthesized by solid phase, and their IC50 for ACE inhibition were 155, 2.24, 3.79 mmol/L and 7.86 mmol/L, respectively. In summary, Plastein reaction can significantly improve the ACE inhibitory activity of pumpkin seed ACE inhibitory peptide, and the pumpkin seed ACE inhibitory peptide with high ACE inhibitory activity can be separated by ultrafiltration and Sephadex G-25 column chromatography.

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