The Study Of Mechanism Of ACE Inhibitory Peptides From Foods And Their In Vivo Anti-hypertensive Activities

Angiotensin I-converting enzyme(ACE)plays a key role in the blood pressure homeostasis.It is the target for the treatment of hypertension.Antihypertensive peptides from foods can control blood pressure by inhibiting ACE with less side effects.In this work,the ACE inhibitory peptides from pistachios(Pistacia vera L.),sea-worm Phascolosoma esculenta and microalgae(Chlorella vulgaris)were studied in vitro and in vivo.The roles of C-terminal amino acids on ACE inhibitory activity were determined.The inhibitory pattern of the peptides were determined by Lineweaver-Burk plots.Moreover,the inhibitory mechanism was illustrated through molecular docking,and the determination of affinity,thermodynamics.The details are as follows.1.To study the effect of C-terminal amino acids on ACE inhibitory activity,the C-terminal amino acid of KAKP(IC50=7.23 ?M)was replaced by other hydrophobic amino acids,or deleted,or changed with the ortho-position residue,and 9 new peptides were obtained.The IC50 values of such peptides ranges from 2.02 to 6891 ?M,of which the most difference among the inhibitory activity is 3400 times.KAKW(IC50=2.02?M)is the most active one.KAKP and KAKW were non-competitive inhibitor and could keep stable activity on ACE during the reaction period of 24 h in vitro.The in vivo antihypertensive activity of the two peptides were evaluated by measuring the blood pressure of Spontaneously Hypertensive Rats(SHR).KAKP and KAKW could very significantly decrease the systolic blood pressure(SBP),and diastolic blood pressure(DBP)of SHRs.The docking results showed that KAKP and KAKW could bind with active pockets of ACE by hydrogen bonds,and KAKW formed more stable structure with ACE.RYDF,YASGR and GNGSGYVSR from Phascolosoma esculenta were non-competitive inhibitor.GNGSGYVSR might be hydrolyzed by ACE after reaction with ACE 4 h,and the hydrolysate of GNGSGYVSR could still inhibit ACE.GNGSGYVSR could significantly decreased the SBP and DBP of SHRs.2.Twelve pieces of ACE inhibitory peptide with C-terminal Trp from microalgae(Chlorella vulgaris)by virtual hydrolysis and screening method were selected for stufy.Non-competitive inhibitors,VHW(IC50=7.23 ?M)and TTW(IC50=7.23 ?M),are the most active peptides among the twelve.The docking results discovered the important role of C-terminal Trp on the ACE inhibitory activity.TTW and VHW could significantly decreased the SBP and DBP.3.ACE were purified by affinity chromatography using a Sepharose-NHS-Lisinopril affinity resin.The purification multiple of ACE is up to 6.1 with specific activity of 2 U/mg,and recovery of 65%.The purified ACE was used to study the interactions between ACE and ACE inhibitory peptides.The thermodynamic parameters of KAKP,KAKW,TTW and VHW showed that the interactions were spontaneous and entropy-driven.In terms of affinity,KAKP,KAKW,TTW and VHW could form a stable complex structure with ACE when the reaction reached equilibrium.