单环刺螠ACE抑制肽的分离、鉴定及活性评价
Isolation,identification and activity evaluation of ACE inhibitory peptides from Urechis unicinctus
聂鹏 1孟乐 1李针 1徐炫 1任春芝 2唐红军 3王玉梅 1孙坤来1
作者信息
- 1. 浙江海洋大学食品与药学学院,浙江舟山 316022
- 2. 舟山市妇幼保健院,浙江舟山 316000
- 3. 四川聚元药业集团有限公司,四川达州 635000
- 折叠
摘要
目的 以单环刺螠(Urechis unicinctus)为原材料,以血管紧张素转化酶(ACE)抑制活性为筛选指标,运用酶解技术和现代分离纯化技术,制备具有降压潜能的ACE抑制肽.方法 运用可控酶解技术,以ACE的抑制率为筛选指标,优化酶解条件.运用超滤、Sephadex G-15凝胶柱层析、反相高效液相色谱及质谱测序等一系列分离纯化和鉴定技术,制备具有ACE抑制活性的生物活性肽,并通过Lineweaver-Burk法确定其抑制剂类型.结果 最佳酶解条件为:胰蛋白酶,37 ℃、pH 8.0、加酶量4.0％、料液比1:30、酶解时间4.0 h.通过超滤、Sephadex G-15凝胶层析和反相高效液相色谱(RP-HPLC)进行分离纯化,得到5条活性肽,经质谱和氨基酸序列分析确定其分别为:Pro-Gln-Met-Thr-Phe(DHCY1,622.75 Da)、Pro-Tyr-Phe-Lys-His(DHCY2,690.8 Da)、Pro-Gly-Trp-Lys-Ala(DHCY3,557.66 Da)、Pro-Gln-Gly-Met-Ile-Val-Val(DHCY4,742.94 Da)、Val-Met-Arg-Ile-Ile(DHCY5,630.86 Da).其中 DHCY1、DHCY4、DHCY5 表现出较好的 ACE 抑制活性,其抑制率分别为(86.26±0.85)％、(92.11±1.13)％、(96.51±1.04)％.经判断,DHCY5 为竞争性 ACE 抑制剂.结论 胰蛋白酶酶解单环刺螠蛋白,可以产生具有显著ACE抑制活性的酶解肽,包括ACE竞争性抑制剂,具有潜在的降血压活性,可为单环刺螠酶解肽的开发利用和降血压产品的开发提供理论依据和参考.
Abstract
Objective Urechis unicinctus was used as raw material to prepare ACE inhibitory peptides with hypotensive potential by enzymatic hydrolysis and modern separation and purification techniques.ACE inhibitory activity was used as screening index.Methods The inhibition rate of ACE was used as the screening index,and controlled enzymatic hydrolysis technology was used to optimize the conditions of enzymatic hydrolysis.A series of separation,purification and identification techniques including ultrafiltration,Sephadex G-15 gel column chromatography,reversed phase high performance liquid chromatography(RP-HPLC)and mass spectrometry sequencing were used to prepare bioactive peptides with ACE inhibitory activity,and the inhibitor types were determined by Lineweaver-Burk method.Results The optimum conditions were trypsin,37 ℃,pH 8.0,4.0％enzyme dosage,1:30 feed-liquid ratio and 4.0 h enzymolysis time.Five active peptides were isolated,purified and identified as Pro-Gln-Met-Thr-Phe(DHCY1,622.75 Da),Pro-Tyr-Phe-Lys-His(DHCY2,690.8 Da),Pro-Gly-Trp-Lys-Ala(DHCY3,557.66 Da),Pro-Gln-Gly-Met-Ile-Val-Val(DHCY4,742.94 Da)and Val-Met-Arg-Ile-Ile(DHCY5,630.86 Da)by mass spectrometry and amino acid sequence analysis.Amongthem,DHCY1,DHCY4 and DHCY5 showed better ACE inhibitory activity,and the inhibitory rates were(86.26±0.85)％,(92.11±1.13)％and(96.51±1.04)％,respectively.DHCY5 were judged to be a competitive ACE inhibitor.Conclusion The trypsin hydrolyzes Urechis Unicinctus proteins could produce peptides with significant ACE inhibitory activity,including ACE competitive inhibition,which has potential antihypertensive activity,it could provide the theoretical basis and reference for the exploitation and utilization of U.unicinctus enzymatic peptides and the development of antihypertensive products.
关键词
单环刺螠/酶解肽/ACE抑制活性/分离纯化Key words
Urechis unicinctus/hydrolyzed peptides/ACE inhibitory activity/isolation and purification引用本文复制引用
基金项目
2023年浙江省中医药科技计划项目(2023ZR057)
舟山市医药卫生科技计划项目(2022YA10)
舟山市医药卫生科技计划项目(2022YA14)
达州市市校合作专项资()
出版年
2024
NETL
NSTL
万方数据