Purification,Identification And Antihypertensive Effect Of ACE Inhibitory Peptides From Pine Nut Albumin (Pinus Koraiensis Sieb.et Zucc)

In 2016,hypertensive patients in China exceed 330 million,the number of deaths caused by hypertension accounted for 51% of the death toll.Long-term use of antihypertensive drugs will bring a variety of side effects,therefore,to seek more safe and effective antihypertensive drugs has practical significance.Pine nut(Pinus koraiensis Sieb.Et Zucc)is rich in nutrient elements.The pinealin protein was isolated by osborne method to obtain albumin,globulin and glutenin,among which albumin is water-soluble protein,and contains 8 kinds of essential amino acids human body needs,compared to other components of the protein,human body is more likely to absorb albumin.In this study,korean pine seed protein isolate was hydrolyzed with alcalase to prepare antihypertensive peptides.The ACE Inhibitory Peptides was isolated and identified.It was evaluated that antihypertensive activity of ACE peptides was evaluated by HUVECs.The stability of the antihypertensive peptides was evaluated in vitro,and the results of this study are demonstrated as follows:Pine nuts albumin as raw material,hydrolysate was prepared by Alcalase 2.4L.The optimized conditions for ACE inhibition ratio were determined through single-factor test and response surface(Box-Benhnken)optimization test: enzymolysis pH 9.0,temperature 55 ?,volume of enzyme 10000 U/g and concentration of substrate 2%.The ACE inhibition ratio of the hydrolysate under the conditions reached 75.2%,basically in line with model predictions.The substrate of albumin of red pine seed was purified through ultrafiltration separation,Sephadex G-25,Sephadex G-15 and RP-HPLC to conclude that F3 had the highest ACE inhibitory activity and its ACE inhibition ratio reached 93.7%.The structural identification of component F3 was conducted through ESI-MS/MS to get 19 polypeptides,verified by analysis to determine three polypeptides with high ACE inhibitory activity.Their amino acid sequences and relative molecular masses were respectively Tyr-Leu-Val-Pro-His(YLVPH),Tyr-Arg-Leu-Asp(YRLD)and Tyr-Leu-Leu-Lys(YLLK),and 627.34,565.29 and 535.34 Da.IC50 test result of three polypeptides with the purity of 98% produced through solid-phase synthesis were respectively 0.1510,0.1907 and 0.2825 ?mol/L and their molecular masses and sequential structures were all in line with theoretical values.The stability test in vitro is conducted to ACE inhibitory peptides with the result showingACE inhibitory peptides inhibition rate above 80% between 20 ?~100 ?,under the condition of pH is 2~10,indicating peptides with heat resistance,acid resistance and alkali resistance.More than 0.4 mol/L NaCl concentration,the inhibitory activity of peptides was significantly reduced(P<0.01)and the inhibition rate was about 50%.The inhibition rate of the peptides by different metal ions decreased and kept in the range of 50~80%.The simulation of gastroenteric environment in vitro proves that three kinds of ACE inhibitory peptides have resistance against gastrointestinal digestive enzymes,whose inhibition rate was decreased and stood at 50% above.It indicated that peptides intake of the human body and through the human digestive system can remain active and play a role in the function of organs or cells to make body blood pressure under control.With the HUVECs test,we discussed inhibition mechanism of ACE inhibitory peptides.The results showed that by observing the cell growth test,analysis and detection of intracellular ROS level,NO release quantity and SOD activity,further verifies the inhibition effect of YLVPH,YRLD and YLLK on the cellular level.These results suggest that the mechanism of lower blood pressure of ACE inhibitory peptides is related to improving SOD activity,increasing the release volume of NO and reducing the release of ROS.The experimental results layed an important theoretical foundation for further study of ACE inhibitory peptides for foodborne,improved the utilization rate of Changbai Mountain pine nut protein.It indicated that the pine nut albumin ACE inhibitory peptides can be further developed as a drop lowering blood pressure function products.