江珧柱抗氧化肽的制备及其对血管内皮细胞氧化损伤的保护作用

Preparation of antioxidant peptides from Pinna(Atrina)pectinata and their protective effects on oxidative damage of human umbilical vein endothelial cells

以江珧[Pinna(Atrina)pectinate]柱为原材料,探究水解肽的抗氧化水平及对H_(2)O_(2)诱导的氧化损伤细胞的保护作用。采用可控酶解技术、以DPPH自由基清除率和水解度(hydrolysis degree,DH)作为评价指标,筛选最佳水解酶,并优化酶解条件;运用超滤、Q强阴离子交换快速流层析柱(Q sepharose fast flow,QFF)、反相高效液相色谱(reversed phase high-performance liquid chromatography,RP-HPLC)、氨基酸N端测序和质谱分析,分离、鉴定活性酶解肽;以H_(2)O_(2)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)为氧化损伤模型,研究活性肽对氧化损伤细胞的保护作用。研究发现,最佳酶解条件为:木瓜蛋白酶,37℃、pH 8.0、加酶量3.0%、料液比1∶20(g∶mL)、酶解时间3.0 h。从该条件酶解产物中分离、鉴定得到5条寡肽,经质谱和氨基酸序列分析确定其分别为Val-Ala-His(JY1,325.18 Da)、Glu-Pro(JY2,244.11 Da)、Ala-Val(JY3,188.12 Da)、Trp-Tyr(JY4,367.15 Da)、Trp-Val(JY5,303.16 Da)。其中JY1、JY4表现出较好的抗氧化活性,其对DPPH自由基、ABTS阳离子自由基、超氧阴离子自由基3种自由基清除活性的EC_(50)分别为(2.102±0.01)、(4.215±0.024)、(3.019±0.021)mg/mL,(2.981±0.014)、(5.385±0.027)、(4.204±0.031)mg/mL;并可减少细胞氧化损伤,显著提升H_(2)O_(2)诱导的HUVEC损伤细胞存活率。由此可见,江珧柱蛋白可以酶解产生具有抗氧化作用的活性的肽,并对H_(2)O_(2)诱导的HUVEC细胞氧化损伤起保护作用,为心血管疾病的预防和治疗、江珧柱的开发利用和抗氧化产品的开发提供理论依据和参考。

Pinna pectinata was used as raw material to extract and the antioxidant levels of the hydrolyzed peptides and the protective effects against H_(2)O_(2)-induced oxidative damage were explored.The controlled enzymatic hydrolysis technology was used to screen the best hydrolase and optimize the enzymatic hydrolysis conditions,DPPH radical scavenging rates and the degrees of hydrolysis were used as the main detection indexes.Ultrafiltration,Q sepharose fast flow(QFF)anion column,reversed phase high-performance liquid chromatography(RP-HPLC),amino acid N-terminal sequencing,and mass spectrometry were used to isolate and identify active enzymatic hydrolyzed peptides.H_(2)O_(2)-induced human umbilical vein endothelial cells(HUVECs)were used as an oxidative damage model to study the protective effects of active peptides against oxidative damage.The optimal enzymolysis conditions were:papain,37℃,pH 8.0,enzyme dosage of 3.0%,solid-liquid ratio of 1∶20,enzymolysis time of 3.0 h.Five oligopeptides were isolated and identified from the enzymolysis products,which were identified by mass spectrometry and amino acid sequence analysis as Val-Ala-His(JY1,325.18 Da),Glu-Pro(JY2,244.11 Da),Ala-Val(JY3,188.12 Da),Trp-Tyr(JY4,367.15 Da),and Trp-Val(JY5,303.16 Da).Of which,JY1 and JY4 showed better antioxidant activities,and the EC_(50)values against DPPH radicals,ABTS cationic radicals,·O 2-were(2.102±0.01),(4.215±0.024),(3.019±0.021)mg/mL,(2.981±0.014),(5.385±0.027),(4.204±0.031)mg/mL,respectively.They both had significant protective effects on H_(2)O_(2)-induced HUVECs damage,and can significantly improve cell survival rates and reduce cell damage.The enzymatic hydrolysis of P.pectinata protein have antioxidant activities and can protect HUVECs from H_(2)O_(2)induced oxidative damage.The results of this study can provide theoretical basis and reference for the prevention and treatment of cardiovascular diseases,the development of antioxidant products,and utilization of P.pectinata.