Study On Preparation And Antihypertensive Mechanism Of Antihypertensive Peptides From Lepidotrigla Microptera

Hypertension is a common cardiovascular disease（CVD）characterized by elevated blood pressure.Every year,the number of people who died from CVD accounts for 31%of the global deaths,of which more than 9.4 million people died from hypertension.Because drugs for treating hypertension were usually accompanied by serious side effects（eg,rash,oliguria dizziness,etc.）,finding safe and effective substitutes for treating hypertension has become the focus of current research,and natural antihypertensive peptides with low toxicity and strong specificity have come into people’s sight.As a low-value marine fish,Lepidotrigla microptera has the characteristics of small size,strong reproductive ability,rapid generation renewal and high protein content,and was mainly used as raw materials for fish meal or surimi processing,resulting in great waste of resources.Therefore,in this study,the protein peptide with high angiotensin convening enzyme（ACE）inhibitory activity was prepared by screening protease types and optimizing enzymatic hydrolysis conditions.Ultrafiltration-gel chromatography-liquid chromatography was used to separate and identify the protein peptide of Lepidotrigla microptera.The interaction between antihypertensive peptide and ACE was analyzed by molecular docking combined with chromatography.Using hypertensive cell model,the hypotensive mechanism of antihypertensive peptides was studied at the cellular level.Taking spontaneously hypertensive rats（SHR）of hypertensive rats as animal model,the hypotensive effect of antihypertensive peptides in vivo and its effect on intestinal flora of rats were studied.The main research contents and results are as follows:（1）To optimize the process conditions for preparing antihypertensive peptides.Combined with different chromatographic techniques,the molecular weight and structural changes of the protein of the Lepidotrigla microptera were determined under the action of different proteases.The results showed that the alcalase hydrolysate had the highest ACE inhibitory activity.In addition,the content of hydrophobic amino acids in alcalase hydrolysate was the highest,which was 200.5 mg/g;In structural characterization,theβ-turn angle,random curl and low molecular weight（<1000 Da）of alcalase hydrolysate were also higher than those of other groups,accounting for 69.68%,23.00%and 88.20%respectively.Taking the ACE inhibition rate and degree of hydrolysis（DH）of the enzymatic hydrolysates as an index,the parameters of enzymatic hydrolysis of Lepidotrigla microptera surimi protein were optimized using response surface analysis.The results showed that the optimal enzymatic hydrolysis conditions for the preparation of antihypertensive peptide by response surface method were obtained as follows:enzyme-substrate ratio enzymatic 1.4%,hydrolysis temperature 54℃,p H 9 and enzymatic hydrolysis time 2 h.（2）Isolation and identification of antihypertensive peptides.Taking the Angiotensin converting enzyme（ACE）inhibition activity as an index,the protein peptide of Lepidotrigla microptera was separated by ultrafiltration,and the LMF1 component with the highest ACE activity was collected and purified by gel chromatography,then five components（F1,F2,F3,F4 and F5）were obtained,among which F4 component had the highest ACE activity.Therefore,F4 component was collected and further separated and purified by semi-preparative liquid chromatography.The results showed that F4-12 was the highest ACE active component.The amino acid sequences of F4-12 components were analyzed and identified.A novel blood pressure reducing peptide（DLTAGLLE）with molecular weight of 830.43 was obtained,and its IC₅₀value was 0.13 mg/m L.Subsequently,sensory evaluation,electronic tongue and GC-IMS were used to detect the flavor changes of the antihypertensive peptides during the purification process.The results showed that the bitterness response value decreased gradually with the purification process.（3）Interaction mechanism between ACE and antihypertensive peptide.The interaction mechanism between ACE and DLTAGLLE was analyzed by Lineweaver-Burk mapping,ultraviolet chromatography,fluorescence chromatography,circular binary chromatography and molecular docking.The results showed that the inhibition type of DLTAGLLE peptide on ACE was non-competitive.DLTAGLLE peptide was linked to 12 amino acid residues at the inactive site of ACE through hydrogen bonding,which showed high binding affinity,and the DLTAGLLE-ACE complex was very stable.The stability test shows that DLTAGLLE has good digestion stability and temperature,p H and ion stability.（4）Based on hypertensive cell model,the regulatory mechanism of antihypertensive peptides on hypertension was studied.angiotensin II（AngⅡ）was used to induce human umbilical vein endothelial cells（HUVECs）to establish a hypertensive cell model,and the effect of DLTAGLLE peptide on HUVECs induced by AngⅡwas observed.The results showed that DLTAGLLE peptide inhibited ROS,decreased ET-1 in cell culture supernatant,and increased the contents of SOD and vascular relaxing factor NO,which inhibited cell apoptosis.The results showed that DLTAGLLE peptide could effectively promote the proliferation of HUVEC induced by AngⅡ,inhibit apoptosis and oxidative stress,and protect HUVEC dysfunction induced by Ang II.The results of RT-PCR showed that the relative expression of ACE,ACE2 and ET-1 genes in HUVECs was decreased by DLTAGLLE peptide,while the relative expression of ACE2 gene was increased in a concentration-dependent manner.（5）Effect of antihypertensive peptides on blood pressure in vivo.Taking SHR rats as disease model,the effects of antihypertensive peptides（DLTAGLLE）on diastolic blood pressure,systolic blood pressure,heart rate,cardiac ultrasonic indexes and pathological detection in SHR rats were observed.To study the efficacy of DLTAGLLE peptide in the treatment of hypertension.the short-term and long-term antihypertensive results showed that after 2 hours of administration,the blood pressure of rats in each group was the lowest,and the systolic blood pressure in the high dose SHR-H group and the low dose SHR-L group were 152±6 mm Hg and 182±11 mm Hg,respectively,and the diastolic blood pressure were116±8 mm Hg and 128±10 mm Hg respectively.In the process of drug intervention for 28 d,the blood pressure of rats in high dose SHR-H group decreased slowly.Compared with the SHR group,the results of color Doppler ultrasound showed that LVEDV,LVSV,LVPWd and IVSs in SHR-H and SHR-L groups decreased,while EF,FS,LVIDd,LVIDs and LVPWs increased.At the same time,the effects of antihypertensive peptides on RAS system were evaluated with the activities of AngⅡ,bradykinin and ACE in rat blood as indicators.The results showed that after the intervention of antihypertensive peptides,the activity of ACE in serum increased,the content of AngⅡin plasma decreased,and the content of bradykinin increased,which led to the decrease of blood pressure,thus affecting renin-angiotensin system（RAS）system.The results of blood biochemistry and routine blood tests showed that after the intervention of antihypertensive peptides,MCH and RBC increased,while WBC,MCV,PDW,MCHC and UA decreased.In addition,the pathological examination of heart and kidney showed that compared with SHR group,the inflammatory reaction of heart tissue in SHR-H and SHR-L groups was reduced,the shape of myocardial cells was regular and the gap was moderate,and the collagen deposition in the gap between myocardial fibers was reduced.In SHR-H and SHR-L groups,the glomerular morphology was normal,the renal tubules were arranged in order,the renal interstitial inflammation was alleviated,and the glomerular fibrosis area was reduced.（6）To study the effect of antihypertensive peptides on intestinal flora in rats.The effects of DLTAGLLE peptides intervention on intestinal flora and short-chain fatty acids were investigated by gas chromatography combined with 16S r DNA sequencing.The results showed that DLTAGLLE peptide significantly improved the diversity and richness of intestinal flora in hypertensive rats,and also improved the intestinal flora and short-chain fatty acid disorder caused by hypertension.