杂色蛤酶解物的降糖活性初步评价及其物质基础研究

Preliminary Evaluation of Hypoglycemic Activity and Its Material Basis of Ruditapes philippinarum Hydrolysate

目的:明确杂色蛤酶解肽对降糖活性相关指标的作用,研究其肽类成分组成。方法:利用酸性蛋白酶制备杂色蛤酶解物样品,通过60%乙醇沉淀得到上清部分,采用Sep-Pak C18固相萃取小柱在不同浓度乙腈洗脱条件下对分离上清部分,以二肽基肽酶-Ⅳ(DDP-Ⅳ)体外抑制模型与人肝癌细胞(HepG-2)胰岛素抵抗模型筛选杂色蛤酶解物的最佳活性部分,采用Nano LC-MS/MS串联质谱对活性部分进行分析鉴定。结果:经10%乙腈洗脱部分具有最佳的抑制DPP-Ⅳ活性作用,可促进胰岛素抵抗细胞的葡萄糖摄取量,杂色蛤酶解物的10%乙腈洗脱部分中共鉴定114个多肽,其中83.3%为亲水性多肽,90.4%的肽分子量低于2000 Da,且N末端疏水性氨基酸较多。结论:杂色蛤酶解物中肽类成分可通过抑制DPP-Ⅳ的活性及减少胰岛素抵抗发挥降糖作用。

Objective:To clarify the effect of enzymatic hydrolyzed peptides on related indexes of hypoglycemic activity and its peptide composition.Methods:Hydrolysate of R.philippinarum were prepared by acid protease treatment and separated by Sep-Pak C18 solid phase extraction column under different concentrations of acetonitrile.The human dipeptidyl peptidase Ⅳ(DDP-Ⅳ)inhibition model in vitro and the HepG-2 insulin resistance(IR)model were screened to determine the active peptide fraction.Then the fraction with best activities was identified by nano liquid chromatography Q-exactive-orbitraptandem mass spectrometry(Nano LC-Orbitrap MS/MS).Results:10% acetonitrile eluting fraction had the best inhibitory effect on DPP-Ⅳ,which increased glucose uptake.Finally,a total of 114 peptides were identified from 10% acetonitrile eluting fraction by nano LC-MS/MS.Among those peptides,83.3% of which was hydrophilic polypeptides,and 90.4%with the molecular weight was lower than 2000 Da,and most of them contained hydrophobic amino acid was at their N-terminal.Conclusion:These results suggest that the peptides from hydrolysate of R.philippinarum may exert hypoglycemic effects by inhibiting DPP-Ⅳ activity and reducing insulin resistance.