Small Yellow Croaker ACE Inhibitory Peptide Separation And Purification, Identification And Structure-activity Relationships

In this paper, small yellow croaker was used as basic materials which hydrolyzed to prepared ACE inhibitory peptides. Then the product was introduced to purified and identified. Meanwhile, we made a QSAR reaserch of ACE inhibitory pentapeptides and screened a novel ACE inhibitory peptide, also used it in antihypertensive mechanism research at the cell level with the hope that we can provide a theoretical basis on the research and development of ACE inhibitory peptides.The effect of process conditions on the angiotensin-I converting enzyme (ACE) inhibitory of small yellow croaker hydrolysed with trypsin were investigated using response surface methodology (RSM). RP-HPLC method was also introduced to measure ACE inhibitory activity of hydrolysate. Results showed that the optimal hydrolysis conditions were as follows:pH7.0,50℃of hydrolysis temperature,16.5h of hydrolysis time, substrate at a concentration level of5.6%and enzyme/substrate ratio of9%o. The inhibitory activity of peptides obtained under the optimal conditions was up to87.36%.The crude ACE inhibitory obtained under the optimal conditions was purified step by step. First, we used ultrafiltration which molecular weight cutoff was3000Da, Then sieve chromatography of Sephadex G25, Sephadex G10were used to separation and purification peptides after ultrafiltration. Afterwards, LC-TOFMS was used to make an identification of the fraction of the highest activity between these fractions. Artificially synthesized and measured the ACE inhibitory activity of the peptides in vitro. Results showed that peptide with molecular weight smaller than3000Da could play potential ACE inhibitory activity, which reached98.1%. And the activity of fraction3-3could reach69.49%. The identifidition of small yellow croaker which obtained two peptides, HDHSE (IC50=38.741μmol/L) and GENSQ (IC50=65.377μ mol/L), respectively.In thisstudy, we collected63pentapeptides to establish a QSAR model of ACE inhibitory pentapeptides by the method of partial least-squares regression (PLS). The internal validation of the model was conducted by leave-one-out method, while at the same time, using identified small yellow croaker peptides to make external validation of the model. The results showed that the cross-validation correlation coefficient of the model was0.5752and the observed and calculated ACE inhibitory activities of small yellow croaker peptides had less difference, which revealed that the model have a certain potential predictive ability. Besides, the analysis of the QSAR model demonstated that the electrical parameter of the first amino acid and the hydrophobicity of the second amino acid from C-terminus had strong effects on ACE inhibitory activity of the pentapeptides (positive correlation). Besides, we screened a novel peptide high activity, which was Ile-Trp-His-Asp-Cys (IWHDC, IC50=19.179μmol/L).The effect of IWHDC on cell proliferation and the release of NO and MDA and the activity of NOS and SOD by oxidized human umbilical vein endothelial cells (HUVECs) was measured. Our results suggested that IWHDC could suppress cell proliferation and modulate NO and MDA release and influence the activity of NOS and SOD by HUVECS. These effects, among other mechanisms, like antioxidation, may exert a potential hypotensive effect via endothelium-dependent vasodilation.