藏羊血红蛋白抗氧化肽模拟酶解筛选及活性验证  

Simulated enzymatic screening and activity validation of antioxidant peptides from Tibetan sheep hemoglobin

作　　者：刘鑫 罗唐君 韩丽娟 桂林生 侯生珍[2] 孙胜男 赵成周 LIU Xin;LUO Tangjun;HAN Lijuan;GUI Linsheng;HOU Shengzhen;SUN Shengnan;ZHAO Chengzhou(Medical Science Center,Qinghai University,Xining 810001,China;College of Agriculture and Animal Husbandry,Qinghai University,Xining 810016,China;College of Tibetan Medicine,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学医学部药学系,青海西宁810001 [2]青海大学农牧学院,青海西宁810016 [3]青海大学藏医学院,青海西宁810016

出　　处：《食品与发酵工业》2025年第7期64-73,共10页Food and Fermentation Industries

基　　金：青海省“帅才科学家负责制”项目(2022-NK-169-4)。

摘　　要：该文以生物信息学为理论基础,使用计算机模拟酶解藏羊血红蛋白,利用在线活性评分、活性预测和分子对接技术对藏羊血红蛋白抗氧化肽进行筛选,并通过DPPH自由基、ABTS阳离子自由基清除率实验对其抗氧化性进行验证。使用UniProt数据库筛选藏羊血红蛋白的氨基酸序列,对其潜在的生物活性进行评价,应用ExPASy Peptide Cutter在线工具对筛选的肽链进行酶解,应用Peptide Ranker对生物活性预测评分,利用在线工具Innovagen、ToxinPred、iBitter-SCM、A11erTOP和AdmetSAR对其溶解性、毒性、苦味、致敏性和ADMET进行筛选,使用Keap1受体进行分子对接,预测其活性,最后利用DPPH自由基、ABTS阳离子自由基清除率实验验证筛选得到的藏羊血红蛋白抗氧化肽。结果表明,5条藏羊血红蛋白链含有20种氨基酸,经酶解共得到258种多肽(485条),通过生物活性评分系统筛选出110种评分大于0.5的多肽(194条),经过活性预测,得到18种溶解性好、无毒、无苦味且非过敏原的多肽,对肠道吸收较好的10条多肽进行分子对接,将分子对接评分前3位的多肽KRM、FPL、FL进行体外抗氧化活性实验,结果显示,FPL和KRM的自由基清除能力均超过了阳性对照,其中KRM对DPPH自由基和ABTS阳离子自由基清除能力超过了阳性对照,IC_(50)分别为(0.125 0±0.012 3)和(0.339 1±0.031 4) mg/mL,FPL对DPPH自由基和ABTS阳离子自由基清除能力也超过了阳性对照,IC_(50)分别为(0.256 5±0.014 1)和(0.374 6±0.047 1) mg/mL。FL对DPPH自由基和ABTS阳离子自由基清除能力分别达到了阳性对照的88.90%和94.40%,IC_(50)分别为(1.106 1±0.279)和(1.601±0.296) mg/mL,均表现出较强的抗氧化活性。该方法与传统酶解实验相比,能快速、经济地筛选抗氧化肽,为藏羊血液资源的高值化利用与开发提供研究基础。Based on the theoretical basis of bioinformatics,this paper used the computer to simulate the enzymatic digestion of Tibetan sheep hemoglobin,using online activity scoring,activity prediction,and molecular docking techniques to screen their antioxidant peptides and verify their antioxidant properties by DPPH free radical and ABTS cationic free radical scavenging rate experiments.The amino acid sequences of Tibetan sheep hemoglobin were screened using the UniProt database to evaluate its potential bioactivities,ExPASy Peptide Cutter online tool was applied to enzymatically digest the screened peptide chains,Peptide Ranker was applied to score the bioactivity prediction,and the online tools Innovagen,ToxinPred,iBitter-SCM,A11erTOP,and AdmetSAR for solubility,toxicity,bitterness,sensitization and ADMET,molecular docking using Keap1 receptor for activity prediction,and finally validation of the screened antioxidant peptides of Tibetan sheep hemoglobin using DPPH free radical and ABTS cationic free radical scavenging rate assay.Results showed that five Tibetan sheep hemoglobin chains contained 20 amino acids,and a total of 258 kinds of peptide(485 peptides)were obtained by enzymatic digestion,and 110 kinds of peptide(194 peptides)with ratings greater than 0.5 were screened out by the bioactivity scoring system,and 18 kinds of peptide with good solubility,non-toxicity,non-bitterness,and non-allergenicity were obtained through the prediction of their activities,and the molecular docking was carried out for the 10 peptides that had a better intestinal absorption,and the top three peptides with molecular docking scores,KRM,FPL,and FL,were subjected to in vitro antioxidant activity assay,and the results showed that the free radical scavenging ability of FPL and KRM exceeded the positive control,with KRM exceeding the positive control for DPPH radicals and ABTS cationic radicals with IC_(50) of(0.1250±0.0123)and(0.3391±0.0314)mg/mL,respectively,and FPL exceeding the positive control for DPPH radicals and ABTS cationic rad

关 键 词：藏羊血红蛋白 虚拟酶解 模拟筛选 抗氧化肽 分子对接 

分 类 号：R28[医药卫生—中药学]