高分辨液相色谱-串联质谱法分析冻干西洋参抗氧化多肽

Analysis of Antioxidant Peptides Extracted from Freeze-dried American Ginseng by High Resolution Liquid Chromatography Tandem Mass Spectrometry

本研究利用超高效液相色谱-四极杆/静电场轨道阱串联质谱(UHPLC-Q-Orbitrap MS/MS)技术快速鉴定冻干西洋参多肽的氨基酸组成。采用碱溶酸沉法提取冻干西洋参粗蛋白,以1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率为评价指标,筛选酶解冻干西洋参粗蛋白的最佳蛋白酶,并用最佳蛋白酶将粗蛋白酶解,经3 ku截留分子质量的透析袋透析得到不同组分,采用蛋白液相分析系统分离纯化抗氧化活性较好的组分。结果表明,选择胃蛋白酶酶解1 h时,DPPH自由基清除活性较好,清除率为(56.02±2.01)%,且透析得到<3 ku组分的抗氧化活性优于>3 ku组分和内源性肽组分。用蛋白液相分析系统对抗氧化活性较好的组分(<3 ku)进行分离,评价所得各组分的DPPH自由基和羟自由基清除能力。结果表明,组分F1和F2具有较强的抗氧化活性。经UHPLC-QOrbitrap MS/MS法鉴定,这2个组分均为由2~4个氨基酸组成的多肽,包括疏水氨基酸甘氨酸(Gly)、缬氨酸(Val)和亮氨酸(Leu),这可能是其具有较强抗氧化活性的原因。该方法可快速有效地鉴定冻干西洋参抗氧化多肽的组成,为揭示其组成与抗氧化活性的关系提供了依据。

American ginseng contains a variety of active ingredients.Bioactive peptides with biological functions can be obtained by protease hydrolysis,which has physiological activities such as lowering blood pressure,anti-oxidation and antibacterial.The inorganic salts,polysaccharides and free amino acids contained in the hydrolysate of protease will affect the biological activity of peptides to some extent,so it is necessary to further separate and purify the hydrolysate.At present,the commonly used peptides separation methods are time-consuming and labor-intensive,and the yield of target peptide components is low.In this study,large-scale separation and purification of proteins in a short time can be achieved by the protein purification system,which improves work efficiency and reduces costs.Biological activity and functional properties of peptides are stronger than those of protein.Therefore,it is of great significance to study the antioxidant peptides in freeze-dried American ginseng for nutritional evaluation and pharmacological effects.The amino acid composition of the peptides extracted from freeze-dried American ginseng was investigated by ultra-high performance liquid chromatography-quadrupole electrostatic field-Orbitrap tandem mass spectrometry(UHPLC-Q-Orbitrap MS/MS).The crude protein in freeze-dried American ginseng was extracted by alkali-soluble acid precipitation method,and hydrolyzed by various proteases.The optimal protease for enzymatic defrosting of the crude protein was screened out using DPPH free radical clearance as an evaluation index.The crude protein was enzymolized with the best protease,and different components were obtained by 3 ku dialysis bags.The components with better anti-oxidation activity were separated by protein liquid phase analysis system.The results showed that after pepsin hydrolysis for 1 h,DPPH free radical scavenging activity is the best,and the clearance rate is(56.02±2.01)%.Further,the antioxidant activity of<3 ku components is better than that of>3 ku components or endogenous peptides.The components with good antioxidant activity(<3 ku)were separated byÄKTATMpure protein liquid phase analysis system to evaluate the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of each fraction,among which F1 and F2 have strong antioxidant activities.The amino acid composition was identified by UHPLC-Q-Orbitrap MS/MS.The hydrophobic amino acids such as Gly,Val and Leu are identified in F1 and F2,which may make them have good antioxidant activities.This method can quickly and effectively identify the antioxidant peptides composition in freeze-dried American ginseng,and provides a basis for revealing the relationship between antioxidant peptides composition and antioxidant activity.