CN115974969A - Selenium-enriched oyster-derived peptide with ACE (angiotensin converting enzyme) inhibitory activity and application thereof - Google Patents

Abstract

The invention relates to a peptide with ACE inhibitory activity derived from selenium-rich oysters and application thereof. The peptide with ACE inhibitory activity has the structure shown in SEQ ID NO:1, and the sulfur element in methionine M in the peptide having ACE inhibitory activity is substituted by selenium. The peptide with ACE inhibitory activity has high selenium content, can be combined with a plurality of amino acid residues in S1, S2 and S1' active pockets of ACE through hydrogen bonds and electrostatic action, and can effectively inhibit the ACE activity; and the release of EA.hy926 cell NO is promoted, the secretion of ET-1 is inhibited, and the preparation method has wide application in the aspects of preparing ACE inhibitors, functional foods, medicaments and the like.

Description

Selenium-enriched oyster-derived peptide with ACE (angiotensin converting enzyme) inhibitory activity and application thereof

Technical Field

The invention belongs to the technical field of active peptides, and particularly relates to a peptide with ACE inhibitory activity and derived from selenium-rich oysters and application thereof.

Background

Hypertension is a chronic disease characterized mainly by elevated systemic arterial blood pressure (systolic/diastolic pressure is 140mmHg/90 mmHg), and its prevalence rate is increasing year by year and the trend of younger patients is showing, and it is expected that the number of people suffering from hypertension worldwide will increase to nearly 15.6 hundred million by 2025. Hypertension complications such as stroke, myocardial infarction, heart failure, chronic kidney disease and the like have high disability and fatality rate, cause heavy burden to families and society, and become a great public health problem in the global scope. Therefore, how to effectively prevent and treat hypertension has become a focus of current research.

Angiotensin Converting Enzyme (ACE) is a key enzyme in two blood pressure regulating systems, the Renin-Angiotensin system (RAS) and the Kallikrein-kinin system (KKS). ACE hydrolyzes the carboxyl terminal of inactive decapeptide Angiotensin I (Ang I) into active octapeptide Angiotensin ii (Ang ii), resulting in an increase in systolic blood pressure, and in addition, it acts on Bradykinin (BK) having vasodilatory action and hydrolyzes it into inactive fragments, thereby causing an increase in blood pressure. Therefore, effective inhibition of ACE activity is considered to be a key approach for the prevention and treatment of hypertension. Since most of chemically synthesized ACE inhibitors (captopril, enalapril, lisinopril, benazepril and the like) have potential side effects such as renal function damage, dry cough, rash, angioneurotic edema and the like, ACE inhibitory peptides of natural sources, which are high in safety and easy to absorb, have become research hotspots. The source of food-derived ACE inhibitory peptides is wide, and a large number of researchers have obtained ACE inhibitory peptides with different compositions from a plurality of animal and plant protein resources such as milk sources, marine animals, grains, soybeans, nuts and the like.

Patent CN102311484A discloses a hexapeptide for inhibiting angiotensin transferase, which can effectively inhibit ACE activity. But the related studies are still less.

Therefore, the development of a natural active peptide with high ACE (angiotensin converting enzyme) inhibition activity has important research significance.

Disclosure of Invention

The invention aims to overcome the defect or deficiency of the lack of natural active peptide with high ACE inhibitory activity and provides the peptide with the ACE inhibitory activity, which is derived from selenium-enriched oysters. The peptide with ACE inhibitory activity has high selenium content, can be combined with a plurality of amino acid residues in S1, S2 and S1' active pockets of ACE through hydrogen bonds and electrostatic action, and can effectively inhibit the ACE activity; and the release of EA.hy926 cell NO is promoted, the secretion of ET-1 is inhibited, and the preparation method has wide application in the aspects of preparing ACE inhibitors, functional foods, medicaments and the like.

The invention also aims to provide application of the peptide with ACE inhibitory activity in preparing ACE inhibitors.

Another object of the present invention is to provide the use of the above-mentioned peptide having ACE inhibitory activity for the preparation of functional foods.

Another object of the present invention is to provide the use of the above-mentioned peptide having ACE inhibitory activity for the preparation of a medicament for lowering blood pressure.

The invention also aims to provide the application of the peptide with ACE inhibitory activity in preparing health products.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a peptide with ACE inhibitory activity derived from selenium-rich oyster has the structure shown in SEQ ID NO:1, and the sulfur element in methionine M in the peptide having ACE inhibitory activity is substituted by selenium.

Specifically, seq id no: the peptide of the sequence shown in 1 is MFRTSSK (Met-Phe-Arg-Thr-Ser-Ser-Lys), the sequence of methionine M with selenium replaced is marked as SeMFRTSSK, and SeM represents that the sulfur element of methionine M is replaced by selenium.

The inventor obtains the peptide with ACE inhibitory activity of the specific sequence from oyster, and the molecular weight is 903.3717Da. Through determination, the peptide with the ACE inhibitory activity has higher selenium content, can be combined with a plurality of amino acid residues in S1, S2 and S1' active pockets of ACE through hydrogen bonds and electrostatic action, and can effectively inhibit the ACE activity; and the release of EA.hy926 cell NO is promoted, the secretion of ET-1 is inhibited, and the preparation method has wide application in the aspects of preparing ACE inhibitors, functional foods, medicaments and the like.

The use of the above-mentioned peptides with ACE inhibitory activity for the preparation of ACE inhibitors is also within the scope of the present invention.

Preferably, the use of said peptide having ACE inhibitory activity for the preparation of an ACE inhibitor which binds to the amino acid residues of ACE, ala354, glu384, tyr523, gln281, his353, lys511, his513 by hydrogen bonding.

Researches find that the peptide with ACE inhibitory activity can be combined with a plurality of amino acid residues in the active pockets of S1, S2 and S1' of ACE through hydrogen bonds and electrostatic action, and the ACE activity can be effectively inhibited.

On the one hand, semfrssk can form hydrogen bonds with amino acid residues Ala354, glu384, tyr523, gln281, his353, lys511, and His513 in ACE active pocket, and plays the most important role in stabilizing docking complexes and inhibiting ACE inhibitory activity.

Preferably, the use of said peptide having ACE inhibitory activity for the preparation of an ACE inhibitor hydrogen bonded to the amino acid residues Ala354, glu384, tyr523, gln281, his353, lys511, his513 of ACE.

On the other hand, seMFRTSSK can form electrostatic interaction with Glu162 amino acid residues in an ACE active pocket, thereby inhibiting ACE activity.

Preferably, the use of the peptide having ACE inhibitory activity for the preparation of an ACE inhibitor which binds electrostatically to the amino acid residue Glu162 of ACE.

The invention also claims the use of peptides with ACE inhibitory activity for the preparation of functional food products.

The application of the peptide with ACE inhibitory activity in preparing the medicine for reducing blood pressure is also within the protection scope of the invention.

Researches show that the peptide with ACE inhibitory activity can promote the release of EA.hy926 cell NO and inhibit the secretion of ET-1, and further can be used for preparing a medicine for reducing blood pressure.

Preferably, the use of the peptide having ACE inhibitory activity for the manufacture of a medicament for promoting the release of NO.

Preferably, the use of the peptide having ACE inhibitory activity for the manufacture of a medicament for inhibiting the secretion of ET-1.

The application of the peptide with ACE inhibitory activity in preparing health care products is also within the protection scope of the invention.

Preferably, the addition amount of the peptide with ACE inhibitory activity in an ACE inhibitor, a functional food, a medicine or a health care product is 0.005-0.025 mu g/mL.

Compared with the prior art, the invention has the following beneficial effects:

the peptide with ACE inhibitory activity has high selenium content, can be combined with a plurality of amino acid residues in S1, S2 and S1' active pockets of ACE through hydrogen bonds and electrostatic action, and can effectively inhibit the ACE activity; and the release of EA.hy926 cell NO is promoted, the secretion of ET-1 is inhibited, and the preparation method has wide application in the aspects of preparing ACE inhibitors, functional foods, medicaments and the like.

Drawings

FIG. 1 shows the effect of protease species on ACE inhibitory activity of selenium-enriched oyster protein hydrolysates;

FIG. 2 is the result of purification and enrichment of the peptide having ACE inhibitory activity in example 2, wherein FIG. 2A is a preparative liquid phase primary separation chromatogram; FIG. 2B shows ACE inhibitory activity at a concentration of 1mg/mL for each isolated fraction in a single separation; FIG. 2C is a preparative liquid phase secondary separation chromatogram; FIG. 2D shows ACE inhibitory activity at a concentration of 1mg/mL for two separate fractions;

FIG. 3 shows the molecular docking results of peptides with ACE inhibitory activity; wherein FIG. 3A is an ACE-SeMFRTSSK complex; FIG. 3B is a 2D schematic of the interaction of SeMFRTSSK with the amino acid residues of ACE.

Figure 4 is a graph of the effect of peptides with ACE inhibitory activity on ea.hy926 cell viability.

Fig. 5 is a graph of the effect of peptides with ACE inhibitory activity on NO content of ea.hy926 cells.

Figure 6 is a graph of the effect of peptides with ACE inhibitory activity on ET-1 content in ea.hy926 cells.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.

The main materials and reagents used in the examples are described below:

the selenium-rich oyster protein is provided by coastal selenium-rich functional agriculture research institute of northern gulf; alkaline protease (2X 10) ⁵ U/g), neutral protease (2X 10) ⁵ U/g), papain (2X 10) ⁵ U/g), trypsin (2500 Usp U/mg), nanning Pang Bo bioengineering, inc.; angiotensin converting enzyme (derived from rabbit lung), equacyl-histidyl-leucine (N-hippuryl-His-Leu tetrahydronate, HHL), sigma, usa; thiazoleave (MTT), shanghai Michelin Biochemical technology, inc.; DMEM high glucose medium, fetal bovine serum, diabase (penicillin/streptomycin), gibico, usa; nitric Oxide (NO) detection kit, bi yun tian biotechnology limited; human endothelin-1 (ET-1) enzyme linked immunosorbent assay kit, CUSABIO science and technology, inc.; other reagents are analytically pure.

Example 1 preparation of selenium-enriched oyster protein hydrolysate

This example utilizes a series of single enzymes: the selenium-rich oyster protein is subjected to enzymolysis by alkaline protease (55 ℃, pH8.5), neutral protease (45 ℃, pH7.0), papain (55 ℃, pH6.5) and trypsin (37 ℃, pH8.0) to obtain the ACE inhibitory peptide with high activity.

The specific process is as follows: dissolving the selenium-enriched oyster protein powder in deionized water according to the mass concentration of a substrate of 5g/100mL, and adjusting the pH value of the solution to an optimal value. Then, each type of protease with an enzyme-substrate ratio of 0.3% was added. After 3 hours of enzymolysis at the optimum temperature, the enzymolysis solution was heated at 95 ℃ for 10 minutes to terminate the reaction. Cooling to room temperature, centrifuging at 4000r/min for 20min, collecting supernatant, vacuum freeze drying, and storing at-20 deg.C.

Fig. 1 is a graph showing the influence of different proteases on ACE inhibitory activity of selenium-enriched oyster protein zymolyte, wherein the ACE inhibitory activity is determined according to the following method:

ACE inhibitory activity was determined with minor modifications to the existing methods. Firstly, freeze-drying a selenium-rich oyster protein zymolyte sample, and preparing an ACE inhibitor (ACEI) solution with the required concentration by using a 0.1mol/L boric acid-borax buffer solution (pH8.3, containing 0.3mol/L NaCl). The reaction loading sequence was as follows: adding 10 mu L of ACE solution (0.1U/mL), adding 10 mu L of ACEI into a sample tube, preheating in 37 ℃ water bath for 5min, adding 30 mu L of HHL solution (5.0 mmol/L), reacting in 37 ℃ water bath for 1h, adding 80 mu L of HCl solution (1.0 mol/L), shaking and mixing uniformly to stop the reaction, cooling to room temperature to obtain a reaction product, and using the reaction product for sample injection detection of the generation amount of hippuric acid. The control tube replaced the ACEI with 10. Mu.L of 0.1mol/L boric acid-borax buffer (pH 8.3, containing 0.3mol/L NaCl), and the blank tube inactivated the enzyme with 80. Mu.L of HCl solution (1.0 mol/L) before the addition of the ACE solution. The reaction solution was filtered through a 0.45 μm filter and analyzed by HPLC. High performance liquid chromatography conditions: welch Ultimate LC-C18 (HS) (250 mm. Times.4.6 mm,5 μm) chromatography column; the elution procedure was acetonitrile: ultrapure water =25 (containing 0.1% (v/v) TFA); flow rate: 1.0mL/min; detection wavelength: 228nm; column temperature: 30 ℃; sample injection amount: 20 μ L. The calculation formula is as follows:

in the formula: a is the chromatographic peak area of hippuric acid in the control group; b is the chromatographic peak area of hippuric acid in the sample group; a. The ₀ The area of the chromatographic peak of hippuric acid in the blank tube.

As can be seen from figure 1, the selenium-rich oyster protein is hydrolyzed for 3 hours under the optimal conditions of the proteases, the ACE inhibition rates of the four protease hydrolysates are different, and the specificity of the proteases is reflected. Wherein, the hydrolysate generated by the trypsin has the strongest ACE inhibitory rate activity which reaches 90.95 +/-0.28 percent, and the papain hydrolysate is the lowest (65.76 +/-1.13 percent). Therefore, trypsin is the most suitable enzyme for preparing the selenium-rich oyster protein zymolyte with ACE inhibitory activity in large scale for subsequent analysis.

Example 2 purification identification and molecular docking analysis of peptides having ACE inhibitory activity

(1) Ultrafiltration

Based on the molecular weight, separating the selenium-rich oyster protein enzymatic hydrolysate by using an ultrafiltration tube (Amicon Ultra-15, the molecular weight of a filter membrane is 10 kDa), and centrifuging for 30min at 4000 r/min. Fractions with a molecular weight of less than 10kDa were collected and stored at-20 ℃ for subsequent analysis.

(2) Preparative liquid chromatography separation

The ultrafiltrated fraction (< 10 kDa) was subjected to preparative liquid chromatography using a Shimadzu PRC-ODS (K) column (30 mm. Times.250mm, 15 μm). The preparation conditions are as follows: mobile phase (A: primary water +0.1% TFA, B: methanol +0.1% TFA); elution procedure: 0-45min, 5-10% of mobile phase B; 45-65min, 10-20% of mobile phase B; 65-75min, 20-50% of mobile phase B; 75-85min, 95-95% of mobile phase B; 85-90min, 95-5% of mobile phase B; the sample injection amount is 5mL; the elution flow rate is 10mL/min; the detection wavelengths were 214nm and 280nm. And (3) converging the same elution peaks, evaporating, concentrating, freeze-drying, detecting the ACE inhibitory activity of the mixture, and carrying out next separation and purification on the component with the strongest ACE inhibitory activity.

In the test of ACE inhibitory activity, a sample obtained by collecting elution peaks, evaporating and concentrating the elution peaks and then lyophilizing the collected elution peaks was used as a sample to be tested, and the rest conditions were the same as those in the test method in example 1.

As shown in FIG. 2A, 5 major peaks, designated as M1-M5 fractions, were isolated after purification of the ultrafiltrate fraction with the preparative liquid phase. When the concentration of the sample is 1mg/mL, the M1-M5 components show certain ACE inhibitory activity (figure 2B), wherein the ACE inhibitory activity of the M4 component is the highest and reaches 34.20 +/-0.41%, and the obvious difference is realized (p is less than 0.05). Therefore, M4 was selected for further isolation and analysis.

Preparation of liquid phase System, reversed phase column SunAire Prep C18 OBD, using Prep 150 ^TM (19 mm. Times.250mm, 5 μ M, waters), and further purifying the fraction M4 having the strongest antioxidant activity. The preparation conditions are as follows: mobile phase (A: double distilled water +0.1% TFA B: methanol +0.1% TFA); gradient elution: 0-15min, 6% -8% of mobile phase B; 15-20min, 8-40% of mobile phase B; 20-40min, 40-50% of mobile phase B; 40-50min, 95-95% of mobile phase B; 50-55min, 95-6% of mobile phase B; the sample injection amount is 1mL; the elution flow rate was 10mL/min, and the peak was monitored at a UV wavelength of 214 nm. Collecting active components, performing rotary evaporation concentration, performing freeze-drying, detecting the ACE inhibitory activity of the active components, and screening the components with the strongest ACE inhibitory activity for mass spectrum identification.

In the ACE inhibitory activity test, a sample obtained by rotary evaporation concentration and freeze drying of an active component after M4 separation is used as a sample to be tested, and the rest conditions are the same as the test method in example 1.

The secondary chromatogram of 2C, M4 fraction is further divided into 3 peaks (M4-1-M4-3), and each fraction is collected, concentrated, lyophilized, and evaluated for ACE inhibitory activity. As shown in fig. 2D, the ACE inhibition of the M4-2 component increased to 56.96 ± 0.11% significantly higher than the remaining two components (p < 0.05) when the sample concentration was 1 mg/mL. However, the M4-1 and M4-3 components do not have ACE inhibitory activity. Thus, the M4-2 fraction was collected in large quantities for subsequent analysis.

(3) Determination of selenium content

This example measured the selenium content of the active fraction during the purification and enrichment of peptides with ACE inhibitory activity, as shown in table 1. The test procedure was as follows: the selenium content of the sample is determined by the method of GB5009.93-2017 hydride atomic fluorescence spectrometry for determining selenium in food. The sample digestion adopts wet digestion, a proper amount of sample is weighed and placed in a conical flask, 10mL of nitric acid-perchloric acid mixed acid (v/v, 9:1) and a plurality of glass beads are added, and the surface dish is covered for cold digestion overnight. Heating on an electric heating plate the next day, and adding nitric acid in time. When the solution became clear and colorless with white smoke, the solution was further heated to a residual volume of about 2mL and cut into pieces which could not be evaporated to dryness. After cooling, 5mL of hydrochloric acid solution (6.0 mol/L) was added and heating was continued until the solution became clear and colorless with the appearance of white smoke. After cooling, the mixture is transferred to a 10mL volumetric flask, 2.5mL ferric potassium oxide solution (100 g/L) is added, water is used for constant volume, and the mixture is uniformly mixed for testing. And simultaneously, carrying out a reagent blank test.

The instrument conditions were: negative high voltage 280V; selenium lamp current 80mA; the carrier gas flow is 300mL/min; the shielding gas flow is 800mL/min; the reading time is 12s; a delay time of 3s; measurement mode standard curve method.

TABLE 1 selenium content and IC of ACE inhibitory Activity ₅₀ Value analysis

As can be seen from Table 1, after optimization of trypsin enzymolysis, the selenium content of the obtained zymolyte is obviously increased to 2.44 +/-0.71 mg/kg. After two times of separation and purification, the selenium content of the component M4-2 with the highest ACE inhibitory activity reaches 37.00 +/-0.56 mg/kg, and is increased by 15.4 times compared with the selenium content of an zymolyte, which shows that the selenium is excellently enriched in the separation and purification process.

IC ₅₀ The value representing the concentration of ACE inhibiting peptide required to reduce ACE activity by 50% is a key indicator for assessing the inhibitory potency of the peptide, i.e. IC ₅₀ The lower the value, the stronger the ACE inhibitory effect of the peptide. Interestingly, the IC of ACE inhibitory Activity of the M4-2 component ₅₀ The value is reduced to 0.774mg/mL, which is obviously lower than that of zymolyte (IC) ₅₀ =2.801 mg/mL), indicating that enrichment of elemental selenium may contribute significantly to an increase in its ACE inhibitory activity. (4) Identification of peptides having ACE inhibitory Activity

The peptide component with ACE inhibitory activity after desalting is dried by centrifugation and then re-dissolved in deionized water containing 0.1% formic acid for on-line LC/MS analysis. The liquid phase was an Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), and the samples were applied to a nanobipe C18 pre-column (3 μm,

) After desalination and retentionThen passing through a C18 reverse phase chromatographic column (Acclaim PepMap RSLC,75 μm × 25cm C18-2 μm->

) The separation was carried out with a gradient of mobile phase B (80% acetonitrile, 0.1% formic acid) rising from 5% to 38% within 60 min. Mass Spectrometry A ThermoFisher Q active system (ThermoFisher, USA) was used in combination with a nanoliter nebulizing Nano Flex ion source (ThermoFisher, USA), the nebulizing voltage was 1.9kV, and the ion transfer tube heating temperature was 275 ℃. The mass spectrum scanning mode is under the information-Dependent acquisition working mode (DDA, data Dependent Analysis), the primary mass spectrum scanning resolution is 70000, the scanning range is set to be 100-1500m/z, and the AGC target is set to be 3 multiplied by 10 ⁶ Maximum implant time 100ms. At most 20 secondary spectra with charge of 1+ to 3+ are collected under each DDA cycle, the AGC target of the secondary mass spectrum is set to 8000, and the maximum injection time is 50ms. High energy collision induced dissociation (HCD) was set to 28eV and dynamic exclusion was set to 6s. Data processing and search analysis was performed using de novo software PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, canada). Searching the crosssearch gigas database, the local false discovery rate of PSM was 1.0%, and two cleavages were missing at maximum. The retrieval parameters are set as: oxidation (M), acetylation (Protein N-term), deamination (NQ), pyro-glu from E, pyro-glu from Q, carbamidomethylation (C) and Selenium reproduction (MC) are variably modified. The precursor and fragment mass tolerances were 10ppm and 0.05Da respectively.

91 ALC (%). Gtoreq.60 selenium peptide sequences are identified in the active component M4-2, and the peptide sequences are composed of 4-13 amino acid residues.

(5) Molecular docking screening of peptide monomers with ACE inhibitory activity

The molecular docking technology is a method for intuitively and effectively researching the interaction between a small molecular ligand and a receptor, the binding energy is the most intuitive embodiment of the docking result, and the lower the binding energy of the ligand and the receptor is, the higher the stability of the ligand-receptor complex conformation structure is.

This example screens peptides for potential ACE inhibitory activity based on molecular docking techniques. The process is as follows: receptor molecule 1O8AThe crystal structure of the Protein was downloaded from RCSB Protein Data Bank (www.rcsb.org). Removing water molecules and original ligand contained in receptor molecules, zn in ACE, by PyMol software ²⁺ Plus a positive charge of 0.95e, saved as a PDB format file. And then, the receptor molecules after PyMol treatment are opened by using Autodock Tools 1.5.6 software, and are subjected to hydrotreatment and stored as pdbqt format files for later use. The two-dimensional structure of the ligand micromolecules is drawn by MarvinSketch software, energy minimization structure optimization is carried out, the three-dimensional structure of the ligand micromolecules is output to be a mol2 format file, the mol2 format file is opened by using Autodock Tools software, the ligand micromolecules are subjected to hydrotreating, gasteiger is calculated, a rotary key is arranged, and the pdbqt file is stored. The center position of the Grid Box (43.821, 38.24, 46.712), the Box size (100.125 × 100.125 × 100.125), exhaustiverse =20 are set. Molecular docking was performed using Autodock Vina 1.1.2, with default values for all other parameters, except as otherwise noted. The docking results are expressed as the binding energy values, and the conformation with the smallest binding energy is selected as the optimal binding site. Finally, the molecular docking results were visually analyzed using Discovery studio 4.5 software.

The results showed that SeMFRTSSK (903.3717 Da) among 91 peptides had the lowest binding affinity for ACE, at-9.8 kcal/mol, which is presumed to have potential ACE inhibitory activity. SeMFRTSSK has selenium in the form of SeMet (the sulfur element of methionine M in the peptide is replaced by selenium), and the results of structural identification and molecular docking screening are shown in Table 2.

TABLE 2 structural identification and molecular docking screening of peptides with ACE inhibitory activity

(6) Molecular docking analysis of interaction of peptide with ACE inhibitory activity with ACE

To further explore the activity mode of action of the peptide semfrssk with ACE inhibitory activity, this example used molecular docking techniques to explore the binding mode and site of action between semfrssk and the receptor ACE. ACE has three major active site regions (S1, S2 and S1'). The S1 active pocket contains Ala354, glu384 and Tyr523 amino acid residues, the S2 active pocket contains Gln281, his353, lys511, his513 and Tyr520 amino acid residues, and S1' contains a Glu162 amino acid residue. Studies report that hydrogen bonding and hydrophobic interactions are the two major interactions that influence the stability and inhibitory effect of ACE and inhibitory drug complexes. FIG. 3A shows the best docking conformation of SeMFRTSSK with ACE, which peptide forms a stable complex with ACE, and FIG. 3B is a 2D schematic of the interaction of SeMFRTSSK with the amino acid residues of ACE. As shown in table 3, hydrogen bonding, hydrophobic interactions and electrostatic interactions are the main modes of action of SeMFRTSSK with ACE. SeMFRTSSK binds to amino acid residues in the active pockets of S1, S2 and S1', consistent with reported findings. SeMFRTSSK forms hydrogen bonds with Ala354, glu384, tyr523, gln281, his353, lys511, and His513 amino acid residues in the ACE active pocket, while the antihypertensive drug captopril also forms hydrogen bonds with Gln281, his353, his513, and Lys511 amino acid residues of ACE, suggesting that SeMFRTSSK may have some similar sites of action and mechanisms of activity as captopril. Furthermore, seMet of SeMFRTSSK forms hydrophobic interactions with the amino acid residues Val379 and Phe527 of ACE, and the results show that the presence of selenium may promote the binding between the peptide with ACE inhibitory activity and ACE, but the specific role of selenium is under intensive study.

(7) Synthesis of peptides with ACE inhibitory activity

The screened peptide SeMFRTSSK with ACE inhibitory activity is synthesized by Nanjing Jettin peptide biotechnology limited, and the purity of the peptide SeMFRTSSK is more than 98 percent after the analysis of High Performance Liquid Chromatography (HPLC), and the peptide SeMFRTSSK is stored at the temperature of minus 20 ℃.

Example 3 cellular hypotensive Activity of peptides with ACE inhibitory Activity

(1) Detection of cytotoxic Effect (MTT) of peptides having ACE inhibitory Activity on HepG2

Culture of hy926 cells was performed according to the prior art with minor modifications. Inoculating EA.hy926 cells in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin, and adding 5% CO ₂ And culturing in an incubator at 37 ℃. Mixing 100 μ L of EA.hy926 cell suspension (1×10 ⁵ cells/mL) in a 96-well plate, culturing for 24h in an adherent manner, removing the culture solution, adding 100 μ L of peptide solution (0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 mg/mL) with ACE inhibitory activity to the sample group, replacing the blank control group with the culture medium, and culturing for 24h with captopril (0.5 mg/mL) as the positive control group. After the grouping, cell viability was determined using the MTT method described by Mosmann. The culture medium was discarded, washed with PBS, and then 100. Mu.L of MTT solution (0.5 mg/mL) was added thereto to continue the incubation for 4 hours. Discarding the MTT solution, adding 100 mu L DMSO, shaking for 10min to completely dissolve the blue-violet crystal, and detecting the OD value at 490nm by using an enzyme-labeling instrument.

As shown in fig. 4, when the concentration was less than 0.5mg/mL, the cell viability of the SeMFRTSSK treated group was above 90%, and there was no significant toxic effect on ea.hy926 cells. SeMFRTSSK also promotes cell proliferation at concentrations ranging from 0.01 to 0.05 mg/mL. Thus, seMFRTSSK can be used as a safe ACE inhibitor at concentrations ranging from 0.001-0.25mg/mL, and is not cytotoxic to EA.hy926 cells.

(2) Effect of peptides having ACE inhibitory Activity on NO secretion from cells

Hy926 cells are a classical cell evaluation model commonly used in studies of hypotensive activity. NO is an important mediator for regulating endothelial cell function, has a relaxing effect on vascular smooth muscle, and has very important functions in balancing human blood pressure and maintaining constant vascular tension. Ea.hy926 cells were treated with different concentrations of peptide with ACE inhibitory activity (SeMFRTSSK) with captopril as a positive control and the results are shown in fig. 5. Compared with the blank control group, the NO content of the SeMFRTSSK treatment group is gradually increased along with the increase of the treatment concentration, and when the peptide concentration reaches 0.025mg/mL, the NO content of the SeMFRTSSK treatment group is increased by 202.65% and is remarkably higher than Yu Katuo pril positive control group (p is less than 0.05). Therefore, the peptide with ACE inhibitory activity can promote EA.hy926 cells to generate NO, thereby playing an antihypertensive effect and having a dose effect.

(3) Effect of peptides having ACE inhibitory Activity on cellular secretion of ET-1

In addition to the renin angiotensin system, the role of the endothelin system in blood pressure regulation is increasingly recognized, with ET-1 having potent vasoconstricting and pressure-raising properties. This example treats ea.hy926 cells with a peptide having ACE inhibitory activity (semfrssk), and the hypotensive activity of semfrssk can be measured by detecting changes in ET-1 levels. As can be seen from FIG. 6, after 24h of treatment, the SeMFRTSSK treatment group had a significant inhibitory effect on the production of cellular ET-1 (p < 0.05) when the sample concentration was greater than or equal to 0.01mg/mL, compared to the blank control group (109.72. + -. 1.66 pg/mL), which was close to the captopril positive control group (88.67. + -. 0.91 pg/mL). The experimental result shows that the peptide with ACE inhibitory activity has a good blood pressure lowering effect on the cellular level. In conclusion, seMFRTSSK has NO obvious toxic effect on EA.hy926 cells under a certain concentration, and can promote the generation of NO and inhibit the secretion of ET-1. It is shown that the peptide with ACE inhibitory activity can play a role in lowering blood pressure by increasing the release amount of NO and reducing the secretion of ET-1.

Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the present disclosure are to be considered within the scope of the claims of the present invention.

Claims (10)

1. A peptide with ACE inhibitory activity, which is derived from selenium-rich oysters, is characterized in that the peptide with ACE inhibitory activity has the amino acid sequence shown as SEQ ID NO:1, and the sulfur element in methionine M in the peptide having ACE inhibitory activity is substituted by selenium.

2. Use of a peptide having ACE inhibitory activity according to claim 1 in the preparation of an ACE inhibitor.

3. Use according to claim 2, characterized in that the peptide having ACE inhibitory activity is used for the preparation of ACE inhibitors that hydrogen bond to the amino acid residues of ACE, ala354, glu384, tyr523, gln281, his353, lys511, his 513.

4. Use according to claim 2, characterized in that the peptide having ACE inhibitory activity is used for the preparation of an ACE inhibitor which binds electrostatically to the amino acid residue Glu162 of ACE.

5. Use of a peptide having ACE inhibitory activity according to claim 1 for the preparation of a functional food.

6. Use of a peptide having ACE inhibiting activity according to claim 1 for the preparation of a medicament for lowering blood pressure.

7. Use of a peptide having ACE inhibitory activity according to claim 6 for the manufacture of a medicament for promoting the release of NO.

8. Use of a peptide having ACE inhibitory activity according to claim 6 in the manufacture of a medicament for inhibiting the secretion of ET-1.

9. Use of a peptide having ACE inhibitory activity according to claim 1 for the preparation of a health product.

10. The use according to any one of claims 2 to 9, wherein the peptide having ACE inhibitory activity is added to an ACE inhibitor, a functional food, a pharmaceutical or a health product in an amount of 0.005 to 0.025 μ g/mL.

Images