The Hypotensive Components And Mechanisms Of Small Peptides Derived From Laminaria Japonica

Hypertension is one of the most important risk factors triggering cardiovascular diseases(CVD).It can trigger series of chronic diseases,such as heart failure,atherosclerosis,myocardial infarction etc.,which is seriously harm to the human health and safety.At present,the common hypotensive drugs are chemical synthetics,which can lead to some adverse effects while play the antihypertensive role.Therefore,it would become a research focus in the world to extract the hypotensive functional components with little side-effect and high safety from the natural food.Laminaria japonica is a kind of large edible and medical marine algea,which was used as the folk hypotensive prescription in ancientry.The laminaria japonica production in China had ranked first in the world,however the processing level is still at a primary stage.The resource waste is severity and the utilization rate is extremely low.In this study,antihypertensive peptides which could have a significant inhibition on the activity of angiotensin converting enzyme(ACE)were prepared by the controlled enzymolysis of crude protein,and the preliminary inquiry of the action mechanism was carrid out;Meanwhile,the by-products,mannitol and fucoidan,were recycled efficiently,and the utilization and value-added of laminaria japonica were improved.This study was contributed to providing the basic data for enzymatic preparing antihypertensive peptides and investigating their mechanisms,and also giving the theoretical guidance for the high-value utilization of laminaria japonica.First,using ultrasonic-assisted method to extract the mannitol and fucoidan sequentially and response surface method to optimize the parameters,the optimum extraction conditions for the mannitol were as follows:95%ethanol as the extraction solvent,liquid-solid ratio 52:1(mL/g),ultrasonic treatment time 33 min,temperature 60?,power 300 w,and twice extraction.Under this condition,the highest extraction rate of mannitol was 16.36%;The optimum extraction conditions of fucoidan were as follows:liquid-solid ratio 20:1,temperature 49?,time 25 min,power 300 w.And the maximum yield of fucoidan was 3.18g/100g extracting by 0.1mol/L HCl.The optimization of the two-step extraction conditions were contributed to the comprehensive utilization of laminaria japonica,but also laid a foundation for the enzymatic preparation of antihypertensive peptides.Second,a simple and rapid reversed-phase high-performance liquid chromatography method(RP-HPLC)was established.Eight antihypertensive peptides(KY?GKY?SKTY?AKY?AKYSY?KKFY?FY?KFKY)were quantified simultaneously in laminaria japonica enzymatic hydrolysate.Furthermore,the HPLC/ESI-MS/MS method was used to analyze the structure and identification of the eight small antihypertensive peptides.The IC50 values which were carried out by the tests of ACE inhibitory activity in vitro,were 5.24 ?mol/L?7.94 ?mol/L?20.63?mol/L?7.52 ?mol/L?2.42 ?mol/L?15.33 ?mol/L?4.83 ?mol/L,respectively.Meanwhile,the ACE inhibition activities of different enzymatic hydrolysates were detected,and the results indicated that the strongest activity was prepared by complex enzyme(including alkaline protease,papain,trypsin),followed by alkaline protease hydrolysates,and the weakest one was by papains.Third,the response surface method was adopted to optimize the characteristic parameters of complex enzyme and the conditions of enzyme hydrolysis for the laminaria japonica crude protein.The optimal characteristic parameters of complex enzyme were enzyme ratio 1:2:1(alkaline protease:papain:trypsin),pH 8.0,temperature 60?.Based on the above optimal characteristic parameters,the optimal processing conditions were substrate concentration 1.7%,enzyme-substrate ratio 4.4%,enzymolysis time 5.1 h.Under these conditions,the IC50 values of ACE inhibitory activity was 5.64 mg/mL.The relative standard deviation was less than 0.95%between the real value of optimized parameters and the prediction ones.The degree of fitting was high,so the results could provide the reference value.Finally,Surflex-Dock model of software SYBYL8.1 was adopted to simulate molecular docking of the eight antihypertensive peptides and 3D structure of tACE crystalline,for acquiring the eight stable combining conformation of equipotential surface figure.The result showed that all of the eight antihypertensive peptides had a strong ability to combine with tACE in many active sites,especially His383,Glu384,Glu411 and His387.The binding energy was mainly provided by hydrogen bonding force.Thus,the correlation between the ACE inhibitory activity and molecular conformation was preliminarily discussed.