CN112521446B

CN112521446B - ACE inhibitory peptide and application thereof

Info

Publication number: CN112521446B
Application number: CN202011231915.5A
Authority: CN
Inventors: 王斌; 迟长凤; 潘筱鸯; 赵玉勤
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Baiyimeiheng Beijing Technology Co ltd; Xi'an Huaqi Zhongxin Technology Development Co ltd
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2022-06-21
Anticipated expiration: 2040-11-06
Also published as: CN114835773A; CN112521446A

Abstract

The invention provides an ACE inhibitory peptide and application thereof, belonging to the technical field of bioactive peptides, wherein the amino acid sequence of the ACE inhibitory peptide is Met-Phe-Arg or Met-Phe-Val or Lys-Pro or Gln-Val-Lys or Ile-Lys or Tyr-Lys-Val or Ile-Arg-Lys or Met-Leu-Lys-Val or Asn-Phe-Arg-Pro-Gln or Tyr-Glu-Gly-Asp-Pro or Trp-Phe or Gly-Pro-Glu or Ser-Trp-Ile-Ser or Ser-Val-Glu-Trp-Lys or Phe-Lys-Trp-His. The ACE inhibitory peptide is used for preparing a blood pressure reducing product and/or a product for preventing and/or treating hypertension.

Description

ACE inhibitory peptide and application thereof

Technical Field

The invention belongs to the technical field of bioactive peptides, and particularly relates to ACE inhibitory peptide and application thereof.

Background

Hypertension is the major risk factor for stroke, myocardial infarction, heart failure, chronic kidney disease, and statistically, the number of deaths caused by hypertension accounts for about 12.8% of the total deaths worldwide each year. At present, common chemically synthesized antihypertensive drugs include captopril, lisinopril, enalapril and the like, but clinically, the drugs cause adverse reactions such as blood potassium level increase, taste disturbance, cough, anaphylactic reaction and the like. Food-derived bioactive peptides have many advantages over chemically synthesized ACE inhibitors, such as being relatively mild, safe, and easily absorbed. Meanwhile, the peptides also have the functions of oxidation resistance, aging resistance, tumor resistance and the like, so the food-borne ACE inhibitory peptide is more and more popular with people and has wide development and application prospects. Therefore, the search for a safe and effective antihypertensive drug to replace the chemical synthetic drug is a necessary trend. Nowadays, more and more researchers are focusing on finding ACE inhibiting peptides from food proteins.

The common mussel is also named as mussel and sea red, and has high nutritive value and medicinal health care effect. Mussels have been recorded in ancient times of China as having medical and health care functions, for example, mussels recorded in Bencao Shiyi (herbal medicine for enuresis) have deficiency-dominating strain, barren birth, blood-qi stagnation, abdominal cold, borborygmus, diarrhea, waist pain and leukorrhagia. Mussel contains rich protein and taurine, and the essential amino acids in the protein are complete in variety. Modern research shows that mussels contain polyunsaturated fatty acids and can be used for preventing atherosclerosis in the middle-aged and elderly people. The selenium also contains the trace element selenium which is necessary for human bodies, and related researches show that the selenium has the effects of cancer prevention, cancer resistance, aging resistance and the like. Mussel extract has anti-tumor, antioxidant, blood pressure lowering, antiinflammatory, antibacterial, and antiaging effects. The method researches the activity and action mechanism of the ACE inhibitory peptide of the common mussel, finds out novel ACE inhibitory peptide, has important significance for the development and development of peptide antihypertensive drugs, and provides theoretical basis for high-value utilization of the common mussel.

Disclosure of Invention

The invention aims to provide an ACE inhibitory peptide with high ACE inhibitory activity.

The technical scheme adopted by the invention for realizing the purpose is as follows:

an ACE inhibitory peptide having an amino acid sequence Met-Phe-Arg (MFR) or Met-Phe-Val (MFV) or Phe-Val (FV) or Lys-Pro (KP) or Gln-Pro (QP) or Gln-Val-Lys (QVK) or Ile-Lys (IK) or Tyr-Lys-Val (YKV) or Ile-Arg-Lys (IRK) or Met-Leu-Lys-Val (MLKV) or Asn-Phe-Arg-Pro-Gln (NFRPQ) or Tyr-Glu-Gly-Asp-Pro (YEGDP) or Trp-Phe (WF) or Gly-Pro-Glu (GPE) or Ser-Trp-Ile-Ser (SWISS) or Ser-Val-Glu-Trp-SVEWK) or Phe-Lys-Trp-FK (His).

The ACE inhibitory peptide has high ACE inhibitory activity, is mainly combined with ACE protein through hydrogen bonds, electrostatic force and hydrophobic effect, has NO cytotoxicity, can increase the content of endogenous relaxing factor NO in HUVEC cells, reduce the content of endogenous contraction factor ET-1, and inhibit H-formation₂O₂Induced apoptosis, the activity of SOD and GSH-Px and NO content in the HUVEC cells with oxidative damage are improved, MDA content and ROS level are reduced, and the HUVEC cells with oxidative damage have a certain protection effect. Therefore, the ACE inhibitory peptide can be used as a potential natural antihypertensive drug or functional food for research, and provides theoretical support for improving the commercial value of the Mytilus edulis.

Preferably, the ACE inhibitory peptide has an ACE semi-inhibitory concentration IC₅₀≤5.6mg/mL。

Preferably, the amino acid sequence of the ACE inhibiting peptide is IK or YEGDP or WF or SWISS.

Preferably, the amino acid sequence of the ACE inhibiting peptide is YEGDP or WF.

Preferably, the half inhibitory concentration of ACE IC of IK₅₀＝0.77±0.02mg/mL。

Preferably, the ACE half inhibitory concentration IC of YEGDP₅₀＝0.19±0.010mg/mL。

Preferably, the ACE half inhibitory concentration IC of WF₅₀＝0.40±0.015mg/mL。

Preferably, the ACE half inhibitory concentration IC of SWISS₅₀＝0.32±0.017mg/mL。

Preferably, the ACE inhibiting peptide is derived from perna canaliculus.

The invention also discloses application of the ACE inhibitory peptide in preparation of a product for reducing blood pressure and/or a product for preventing and/or treating hypertension.

Preferably, the product is a pharmaceutical or functional food.

Preferably, the use of an ACE inhibiting peptide for promoting NO release in HUVEC cells.

Preferably, the use of an ACE inhibitory peptide for inhibiting the production of ET-1.

Preferably, the use of ACE inhibiting peptides in the depressurization and regulation of HUVEC cells.

Preferably, the use of an ACE inhibiting peptide for protecting oxidatively damaged HUVEC cells.

The invention also discloses application of the ACE inhibitory peptide in preparation of an antioxidant product.

Preferably, the product is a pharmaceutical or functional food.

The invention also discloses a product for reducing blood pressure and/or a product for preventing and/or treating hypertension, which contains the ACE inhibitory peptide as an active ingredient.

Preferably, the ACE inhibiting drug comprises MFR and/or MFV and/or FV and/or KP and/or QP and/or QVK and/or IK and/or YKV and/or IRK and/or MLKV and/or NFRPQ and/or YEGDP and/or WF and/or GPE and/or SWISS and/or SVEWK and/or FKWH.

The invention also discloses a preparation method of the ACE inhibitory peptide, which comprises the following steps:

1) degreasing the common mussels by using ethyl acetate;

2) sequentially adopting pepsin and trypsin to carry out enzymolysis on the common mussels step by step;

3) and (3) purifying the enzymolysis liquid obtained in the step 2) by ultrafiltration, ion exchange chromatography, gel chromatography and reversed phase high performance liquid chromatography to obtain the ACE inhibitory peptide.

The preparation method takes the common mussel as a material, takes ACE inhibitory activity as an index, and 17 oligopeptides with ACE inhibitory activity are prepared from enzymolysis digestion products by a series of separation and purification technologies such as ultrafiltration, QFF anion exchange chromatography, G-15 gel chromatography, reversed phase high performance liquid chromatography (RP-HLPC) and the like, and the sequences of the oligopeptides are respectively MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK and WHFK.

Preferably, in the step 1), the feed liquid of the common mussel and the ethyl acetate is 1:4-6(w/v), and the degreasing time is 24-72 h.

Preferably, in the step 2), the pH value of the pepsin enzymolysis is 1.0-3.0, the pepsin-substrate (w/w) is 1:40-60, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 1-5 h.

Preferably, in the step 2), the pH value of the trypsin enzymolysis is 7.0-9.0, the ratio of the trypsin to the substrate (w/w) is 1:20-30, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 2-6 h.

Preferably, in step 2), after the enzymolysis is finished, the enzyme is inactivated at 100 ℃ for 5-20 min.

Preferably, in step 3), the ultrafiltration is specifically: ultrafiltering the Mytilus edulis enzymolysis liquid with an ultrafiltration membrane with molecular weight cutoff of 1kDa to obtain a component with MW <1kDa, and freeze-drying.

Preferably, in step 3), the ion exchange chromatography is specifically: partitioning the ultrafiltered components into 40-60mg/mL solution, filtering with 0.22 μ M microporous membrane, and loading onto Q Sepharose FF column; and eluting with Tris-HCl, 0.1M NaCl + Tris-HCl, 0.5M NaCl + Tris-HCl and 1M NaCl + Tris-HCl solutions step by step at a flow rate of 1-3mL/min, collecting one tube every 2-5min, detecting the absorbance value at 214nm absorbance, combining the solutions according to peaks, desalting, freeze-drying, measuring the ACE inhibitory activity of each component at a concentration of 0.5-2.0mg/mL, selecting the component with the highest ACE inhibitory activity, and purifying in the next step.

Preferably, in step 3), the gel chromatography is specifically: loading the component with the highest ACE inhibitory activity obtained by ion exchange chromatography at a concentration of 40-60mg/mL, eluting with ultrapure water at a flow rate of 0.5-0.8mL/min, collecting one tube every 2-5min, detecting at 214nm absorbance, respectively collecting different peaks, lyophilizing, respectively preparing into 0.5-2.0mg/mL for ACE inhibitory activity determination, selecting the component with the highest ACE inhibitory activity, and purifying.

Preferably, in the step 3), the purification by reversed-phase high performance liquid chromatography specifically comprises: dissolving the component with the highest ACE inhibitory activity obtained by gel chromatography in ultrapure water, passing through a 0.22 mu M microporous filter membrane, and loading the component on a Zorbax C18 column for separation; the eluent contained two kinds, one a liquid 0.05% TFA (trifluoroacetic acid) ultrapure water and one B liquid 0.05% TFA acetonitrile, using gradient elution: 0-5min, 10% B; 5-10min, 10% -60% B; 60-100% B for 10-35 min; 35-40min, 0% -100% B. Measuring absorbance value at 214nm, collecting each component, freeze-drying, collecting 17 components, and sequencing to obtain 17 oligopeptides with sequences of MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK, FKWH.

The invention also discloses a preparation method of the ACE inhibitory peptide of the Mytilus edulis, the ACE inhibitory peptide containing the amino acid sequence of MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK and FKWH comprises the following steps:

1) degreasing the common mussels by using ethyl acetate;

3) filtering, concentrating and drying the enzymolysis liquid obtained in the step 2) to obtain the ACE inhibitory peptide of the common mussel.

Preferably, in the step 2), the pH value of the enzymolysis of the trypsin is 7.0-9.0, the ratio of the trypsin to a substrate (w/w) is 1:20-30, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 2-6 h.

Preferably, the ACE inhibitory peptide of Mytilus edulis has an ACE inhibitory rate of > 24%.

The invention has the following beneficial effects: the ACE inhibitory peptide has high ACE inhibitory activity, is mainly combined with ACE protein through hydrogen bonds, electrostatic force and hydrophobic effect, has NO cytotoxicity, can increase the content of endogenous relaxing factor NO in HUVEC cells, reduce the content of endogenous contraction factor ET-1, and inhibit H-formation₂O₂The induced apoptosis can improve the activity of SOD and GSH-Px and the content of NO in the HUVEC cells damaged by oxidation, reduce the MDA content and the ROS level, and have certain protection effect on the HUVEC cells damaged by oxidation. Therefore, the ACE inhibitory peptide can be used as a potential natural antihypertensive drug or functional food, and provides theoretical support for improving the commercial value of the Mytilus edulis.

Drawings

FIG. 1 is a graph showing the effect of ACE inhibitory peptides of Mytilus edulis according to the present invention on ACE inhibitory activity;

FIG. 2 is a QFF anion exchange step elution profile of the present invention;

FIG. 3 is a graph showing the effect of QFF anion exchange elution peaks on ACE inhibitory activity;

FIG. 4 is a Sephadex G-15 gel column chromatography elution curve of the present invention;

FIG. 5 shows the effect of various elution peaks of Sephadex G-15 gel column chromatography on ACE inhibitory activity;

FIG. 6 is a spectrum of a Zorbax C18 reversed phase high performance liquid phase of MH1-4-2 of the present invention;

FIG. 7 shows the molecular docking results of IK and ACE;

FIG. 8 shows the molecular docking results of YEGDP with ACE according to the present invention;

FIG. 9 shows the molecular docking results of WF and ACE according to the present invention;

FIG. 10 shows the molecular docking results of SWISS and ACE in accordance with the present invention;

FIG. 11 is a graph showing the effect of ACE inhibitory peptides of the present invention on HUVEC cell viability;

FIG. 12 is a graph showing the effect of ACE inhibitory peptides of the invention on NO levels in HUVEC cells;

FIG. 13 is a graph of the effect of ACE inhibitory peptides of the invention on ET-1 levels in HUVEC cells;

FIG. 14 shows the effect of hydrogen peroxide of different concentrations on HUVEC cell damage for 4 hours;

FIG. 15 shows HUVEC protective effects of ACE inhibitory peptides of the present invention on oxidative damage;

FIG. 16 is a graph showing the protective effect of YEGDP and WF according to various concentrations of the present invention on HUVEC oxidative damage;

FIG. 17 is a DCFH-DA staining protocol of the invention measuring ROS levels in HUVEC;

FIG. 18 shows the result of the AV-PI staining method for determining apoptosis;

FIG. 19 is a graph of the effect of different concentrations of YEGDP and WF according to the present invention on GSH-Px, SOD levels in HUVEC cells;

FIG. 20 is a graph of the effect of different concentrations of YEGDP and WF according to the invention on MDA levels in HUVEC cells;

FIG. 21 is a graph showing the effect of different concentrations of YEGDP and WF according to the present invention on NO levels in HUVEC cells.

Detailed Description

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It is not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

These examples are provided only for more specifically illustrating the present invention, and it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

The present invention is further described in detail with reference to the following examples:

example 1:

1 materials and instruments

1.1 materials

Purple mussel purchased from Zhoushan Sheng wolfberry island.

1.2 Primary reagents

TABLE 1 Main reagents

1.3 instruments

TABLE 2 Instrument

2 method

2.1A method for preparing an ACE inhibitory peptide of Mytilus edulis, which comprises the following steps:

1) firstly, shelling the common mussels, collecting meat, removing byssus, mincing, degreasing for 48 hours by using ethyl acetate according to the material-liquid ratio of 1:5(w/v), removing the ethyl acetate by vacuum filtration of the degreased common mussels, drying and crushing to obtain degreased common mussels;

2) adjusting the pH of a 20mg/mL defatted common mussel solution to 2.0 by using 1M HCl, adding pepsin according to the ratio of enzyme to substrate (w/w) being 1:50, uniformly mixing, and stirring at 37 ℃ for 2 hours; then saturated NaHCO₃Adjusting pH to 5.3 with 0.9M solution, further adjusting pH to 7.5 with 2M NaOH, adding trypsin at a ratio of enzyme to substrate (w/w) of 1:25, mixing, stirring at 37 deg.C for enzymolysis for 4h, heating in 100 deg.C boiling water to inactivate enzyme for 10min after reaction, and filtering to obtain enzymolysis solution;

2.2 determination of ACE inhibitory Activity

Preparation of a reagent: 80mM HEPES buffer (pH 8.3, Cl)^－Concentration 300 mM): HEPES 1.910g, NaCl 1.755g, dissolved in double distilled water, and adjusted with NaOHAdjusting the pH value to 8.3, supplementing water to 100mL, and placing at 4 ℃ for later use; fagg solution (1 mM): 3.994mg of FAPGG powder is taken and added with HEPES buffer solution, mixed and dissolved, the volume is determined to 10mL, and the mixture is stored in the dark at 4 ℃; ACE inhibitor (test peptide): weighing a proper amount of active peptide powder, and preparing a series of active peptide solutions with a series of concentrations by using HEPES buffer solution as a solvent; ACE solution (0.1U/mL): 0.25U ACE is dissolved in 2.5mL HEPES buffer solution to obtain 0.1U/mL ACE solution, and the solution is stored at-20 ℃ for later use.

Determination of ACE inhibitory activity: reactants were added to 96-well plates as designed in Table 3 and the initial absorbance was measured at 340nm for blank and sample wells, respectively (A)₁，B₁) Reacting at 37 deg.C for 30min, and measuring its absorbance (A)₂，B₂). The ACE inhibitory activity is calculated as follows:

wherein: bi (B)₁-B₂) Change of absorbance at 30min for sample wells: ai (A)₁-A₂) The absorbance of the blank wells was changed at 30 min.

TABLE 3 ACE inhibitory Activity assay

2.3 data processing

The results are expressed as mean ± standard deviation (n ═ 3) and plotted and data analyzed using Origin and SPSS 22.0 software.

Example 2:

the difference from example 1 is that: a preparation method of Mytilus edulis ACE inhibitory peptide comprises the step 2) that the concentration of degreased Mytilus edulis solution is 5 mg/mL.

Example 3:

the difference from example 1 is that: a preparation method of a common mussel ACE inhibitory peptide comprises the step 2) that the concentration of a degreased common mussel solution is 10 mg/mL.

Example 4:

the difference from example 1 is that: a preparation method of a common mussel ACE inhibitory peptide comprises the step 2) that a defatted common mussel solution is used, and the concentration of the defatted common mussel solution is 40 mg/mL.

Examples 1-4 effects of the ACE inhibitory peptides of Mytilus edulis on ACE inhibitory activity As shown in FIG. 1, it can be seen that the ACE inhibitory rate of the ACE inhibitory peptides of examples 1-4 Mytilus edulis is > 24%.

Example 5:

1 materials and instruments

The same as in example 1.

2 method

2.1A process for the preparation of an ACE inhibitory peptide which comprises:

1) the same as example 1;

2) the same as example 1;

Wherein, the step 3) is specifically as follows:

ultrafiltering the Mytilus edulis enzymatic hydrolysate with ultrafiltration membrane with molecular weight cutoff of 1kDa to obtain MH1 component with MW <1kDa, the ACE inhibitory activity of MH1 is (54.80 + -1.63%), and freeze drying;

partitioning the ultrafiltered components into 50mg/mL solution, filtering with 0.22 μ M microporous membrane, and loading onto Q Sepharose FF column; then eluting with Tris-HCl, 0.1M NaCl + Tris-HCl, 0.5M NaCl + Tris-HCl and 1M NaCl + Tris-HCl solutions step by step at a flow rate of 2mL/min, collecting one tube every 3min, detecting absorbance values at 214nm absorbance, merging the solutions according to the peaks to obtain 5 peaks (as shown in figure 2), desalting, freeze-drying, and performing ACE inhibitory activity determination on five components of MH1-1, MH1-2, MH1-3, MH1-4 and MH1-5 after freeze-drying at a concentration of 1.0mg/mL, wherein the result is shown in figure 3, the ACE inhibitory activity (67.18 +/-2.87%) of MH1-4 is obviously higher than that of the other four components, so that the MH1-4 component is selected for next purification;

loading MH1-4 component at a concentration of 50mg/mL onto Sephadex G-15(3.6 × 150cm) column, eluting with ultrapure water at a flow rate of 0.6mL/min, collecting one tube every 3min, detecting at 214nm absorbance, collecting different peaks respectively to obtain 3 peaks (as shown in FIG. 4), lyophilizing, preparing into 1.0mg/mL respectively, and measuring ACE inhibitory activity, with the results shown in FIG. 5: MH1-4-2 has ACE inhibitory activity (75.79 + -1.56%) higher than MH1-4-1(63.30 + -1.05%), so MH1-4-2 component is selected for freeze-drying for further purification;

dissolving MH1-4-2 component in ultrapure water, passing through a 0.22 mu M microporous filter membrane, and loading on a Zorbax C18 column for separation; the eluent contained two kinds, one a liquid 0.05% TFA (trifluoroacetic acid) ultrapure water and one B liquid 0.05% TFA acetonitrile, using gradient elution: 0-5min, 10% B; 5-10min, 10% -60% B; 60-100% B for 10-35 min; 35-40min, 0% -100% B. Measuring absorbance at 214nm, collecting each component, lyophilizing, collecting 17 components (shown in FIG. 6), and sequencing by Edman degradation at detection center. The amino acid sequencing was entrusted to Shanghai Beta Biotechnology Co., Ltd, and was synthesized using a Shimadzu PSSM-8 type polypeptide automatic synthesizer, and the purity was 95% or more. The sequence of the obtained 17 oligopeptides is MFR, MFV, FV, KP, QP, QVK, IK, YKV, IRK, MLKV, NFRPQ, YEGDP, WF, GPE, SWISS, SVEWK and FKWH respectively.

Determining ACE inhibition rate of 17 oligopeptides under different concentrations, performing Statistics by using SPSS Statistics 22 software, and calculating IC of 17 oligopeptides₅₀The results are shown in Table 4.

Table 417 oligopeptide amino acid sequences and activities

Example 5:

the oligopeptides IK, YEGDP, WF and SWISS were submitted to Shanghai Nempu Biotech Co., Ltd for mock molecular docking.

IK. Molecular docking results for YEGDP, WF and SWISS with ACE are shown in fig. 7-10. As can be seen in fig. 7, IK hydrogen bonds with ACE amino acid residue His 387; forms hydrophobic interaction with Phe457, Tyr523 and Tyr 520; and Asp415, Glu384, His353 and Glu411 are acted by electrostatic force. As can be seen from fig. 8, YEGDP forms hydrogen bonds with ACE amino acid residues Asp453, Glu376, Ala354, Tyr520, wherein hydrogen bonds with the S1 active pocket (Ala354) and with the S2 active pocket (Tyr 520); forms hydrophobic interaction with Val380, His353 and Tyr523, wherein His353 and Tyr523 react with each other; asp415 is reacted by electrostatic forces. As can be seen from fig. 9, WF forms a hydrophobic interaction with ACE amino acid residues Phe457, Phe527, His353, His383, Val 380; asp377, Glu376 and Lys454 are acted by electrostatic force. As can be seen from fig. 10, SWISS hydrogen bonds with ACE amino acid residues Asp377, Gys352, Ala354, Tyr523, His383, His353, His387, wherein the active pocket with S1 (Ala354, Tyr523) and the active pocket with S2 (His353) form; forms hydrophobic interaction with Val380 and Trp 279; the electrostatic force acts on Glu384 and Glu 411. This suggests that ACE inhibitory peptides exert inhibitory effects by binding to ACE proteins mainly through hydrogen bonds, electrostatic forces, hydrophobic effects, and that ACE inhibitory activity may be closely related to active pocket interactions and to ACE protein binding strength. Meanwhile, the affinity of SWISS, IK, YEGDP and WF for ACE is-8.6 Kcal/mol, -6.1Kcal/mol, -8.8Kcal/mol and-7.6 Kcal/mol, respectively.

Example 6:

materials and instruments

1.1 materials

Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Shanghai Cell bank of Chinese academy of sciences.

1.2 reagents

TABLE 5 reagents

1.3 instruments

TABLE 6 Instrument

2 method

2.1 culture of HUVEC cells

HUVEC cells were cultured using DMEM + 10% Fetal Bovine Serum (FBS) + 1% diabody (penicillin-streptomycin) medium. The culture process is as follows: the recovered cells were placed in a medium containing 5% CO₂Culturing at 37 deg.C in incubator, changing liquid after 12 hr, digesting with pancreatin and subculturing when cell grows to 80% -90% of the bottom of the bottle. HUVEC cells in logarithmic growth phase were taken as experimental material.

2.2 HUVEC cytotoxicity assay (MTT method)

HUVECs in logarithmic growth phase were inoculated into several 96-well culture plates. 180. mu.L per well, cell count 0.9X 10⁴One cell/well, at 37 ℃ in 5% CO₂And (4) incubating for 24 h. Adding 20 mu L of oligopeptide into the sample group, wherein the final concentration of the oligopeptide is respectively 100, 200, 300, 400 and 500 mu M, adding 20 mu L of PBS into the blank group, incubating for 24h, adding 20 mu L of MTT, incubating for 4h, removing the culture solution, adding 150 mu L of DMSO, incubating for 10min at 37 ℃, measuring the absorbance value at 570nm by using an enzyme-labeling instrument, and calculating the cell survival rate.

The cell survival rate calculation formula is as follows: cell survival (%) (experimental/control) x 100

2.3 Experimental grouping and processing

HUVECs in logarithmic growth phase were inoculated into several 6-well culture plates. 1.8mL per well, 3.5X 10 cell number⁵One cell/well, at 37 ℃ in 5% CO₂And (4) incubating for 24 h. The random groupings were as follows:

(1) blank control group: treating the cells without adding any reagent;

(2) captopril (Cap) group: adding Cap with the final concentration of 1 mu M;

(3) noradrenaline (NE) group: adding NE to the solution to a final concentration of 0.5 μ M;

(4) ACE inhibitory peptide low dose group: adding peptide to a final concentration of 100. mu.M;

(5) ACE inhibitory peptides medium dose groups: adding peptide to a final concentration of 200. mu.M;

(6) ACE inhibitory peptide high dose group: peptide was added to a final concentration of 400. mu.M;

(7) ACE inhibitory peptides + NE group: peptide and NE were added to final concentrations of 400. mu.M, 0.5. mu.M, respectively.

2.4 measurement of the intracellular NO content of HUVEC

A nitrate reductase kit is adopted. The determination principle is as follows: NO is active chemically and is metabolized in vivo to Nitrate (NO)₃ ^-) And Nitrite (NO)₂ ^-) And Nitrite (NO)₂ ^-) Yet further converted into Nitrate (NO)₃ ^-) The method utilizes the specificity of nitrate reductase to specifically react Nitrate (NO)₃ ^-) Reduction to Nitrate (NO)₂ ^-) The NO content was calculated by measuring the absorbance at 550 nm. After 24h of action, the culture medium was discarded, washed three times with PBS, each group of cells was scraped off using a cell scraper and collected in an EP tube, the cells were disrupted with a cell disruptor in an ice water bath, 300. mu.L of PBS was added, and the procedure was followed on the NO kit.

2.5 assay of ET-1 content in HUVEC cells

Enzyme-linked immunosorbent assay (ELISA) using double antibody one-step sandwich method is adopted. The principle is as follows: and adding the specimen, the standard substance and the detection antibody marked by the HRP into the coated micropores previously coated with the endothelin 1(ET-1) antibody, incubating and thoroughly washing. Color development with the substrate TMB, TMB conversion to blue catalyzed by catalase and finally to yellow by acid. The shade of the color is positively correlated with endothelin 1(ET-1) in the sample. And measuring the light absorption value of the sample at the wavelength of 450nm by using a microplate reader, and calculating the concentration of the sample. The specific steps of ET-1 content determination are carried out according to the kit instructions.

3 results of the experiment

The effect of ACE inhibitory peptides on cell survival of HUVEC As shown in FIG. 11, the cell survival of the IK, YEGDP and WF groups was above 90% in the 100-400 μ M concentration range compared to the blank group.

The effect of ACE inhibitory peptides on NO levels in HUVEC cells as shown in figure 12, the NO content in captopril (Cap) group was significantly increased to 56.27 ± 2.63 μ M (P <0.01) compared to the blank control group. In addition, oligopeptides IK, YEGDP and WF in the sample group were applied to cells at concentrations of 100, 200 and 400 μ M, respectively, and the results showed that NO content exhibited dose dependence. IK. YEGDP and WF the NO content in cells at a concentration of 400. mu.M was 44.23. + -. 0.29, 47.61. + -. 0.64 and 45.39. + -. 0.99. mu.M, respectively. Meanwhile, Norepinephrine (NE) inhibits the release of NO (19.47 +/-0.60 mu M) in cells, and the NO content is obviously increased (P <0.01) after the ACE inhibitory peptide is added, which indicates that the NO release effect of NE in cells can be antagonized by the ACE inhibitory peptide.

The effect of ACE inhibitory peptides on ET-1 levels in HUVEC cells is shown in FIG. 13, with the ET-1 content in captopril (Cap) group being significantly reduced to 25.79. + -. 1.10. mu.M (P <0.01) compared to the blank control group. In addition, oligopeptides IK, YEGDP and WF in the sample group act on cells at concentrations of 100, 200 and 400. mu.M, respectively, and ET-1 content appears dose-dependent. IK. The content of ET-1 in the cells at a concentration of 400. mu.M for YEGDP and WF was 50.51. + -. 1.44, 42.85. + -. 0.73 and 44.89. + -. 0.49. mu.M, respectively. Meanwhile, Norepinephrine (NE) inhibits the release of ET-1 in cells (71.30 +/-1.50 mu M), and the content of ET-1 is remarkably reduced (P is less than 0.01) after the ACE inhibitory peptide is added, which indicates that the ACE inhibitory peptide can inhibit the release of ET-1 in cells.

The compound action result of the three oligopeptides of IK, YEGDP and WF on two factors of NO and ET-1 shows that the IK, YEGDP and WF have NO obvious toxicity to HUVEC, can promote the release of endogenous relaxing factor Nitric Oxide (NO) in HUVEC cells and inhibit the generation of endogenous contraction factor endothelin (ET-1), and shows that the three oligopeptides have certain functions of reducing blood pressure and regulating HUVEC cells. Thus, under in vivo conditions, IK, YEGDP and WF can exert their hypotensive effects by affecting the function of vascular endothelial cells.

Example 7:

materials and instruments

1.1 reagents

TABLE 7 reagents

1.2 instruments

TABLE 8 Instrument

2 method

2.1 culture of HUVEC cells

The same as in example 6.

2.2 with H₂O₂Induction of HUVEC cells

HUVECs in logarithmic growth phase were inoculated into 96-well culture plates. 180. mu.L per well, cell count 0.9X 10⁴One/well, at 37 ℃ and 5% CO₂And (4) incubating for 24 h. The model group is added with the final concentration of 500, 600, 700, 800 and 900 mu M H respectively at the concentration of 20 mu L₂O₂The blank group was incubated with 20. mu.L of PBS for 4 hours, and the cell viability was measured by MTT method.

2.3 screening of ACE inhibitory peptides on oxidative stress of HUVEC cells

HUVECs in logarithmic growth phase were inoculated into several 96-well culture plates. 160. mu.L per well, cell count 0.9X 10⁴One cell/well, at 37 ℃ in 5% CO₂And incubating for 24 h. Adding oligopeptide with final concentration of 400 μ M in 20 μ L into sample group, adding Glutathione (GSH) with final concentration of 1mM in 20 μ L into positive drug group, adding PBS in 20 μ L into blank group and model group, incubating for 3 hr, adding oligopeptide with final concentration of 600 μ M H in 20 μ L into sample group, model group and positive drug group₂O₂The blank group was incubated with 20. mu.L of PBS for 4 hours, and the cell viability was measured by MTT method. Selecting oligopeptides with better protection effect on HUVEC cell oxidative stress by 400 mu M active peptides, and then respectively acting on cells at the concentrations of 100, 200 and 400 mu M to observe whether ACE inhibitory peptides have protection effect on HUVEC cell oxidative stress.

2.4 measurement of ROS levels in HUVEC cells

HUVECs in logarithmic growth phase were inoculated into several 96-well culture plates. 160. mu.L per well, cell count 0.9X 10⁴One/well, standing at 37 deg.C and 5%CO₂And (4) incubating for 24 h. Adding 20 μ L active peptide with final concentration of 100, 200, 400 μ M into sample group, adding 20 μ L GSH with final concentration of 1mM into positive drug group, adding 20 μ L PBS into blank group and model group, incubating for 3 hr, adding 20 μ L active peptide with final concentration of 600 μ M H into sample group, model group and positive drug group₂O₂Adding 20 mu L of PBS into the blank group, incubating for 4h, discarding the culture solution, washing for 2 times by using the PBS, adding 100 mu L of 10 mu m DCFH-DA fluorescent probe solution, incubating for 40min at 37 ℃, washing for 3 times by using the PBS, and detecting the intracellular reactive oxygen level by using a multifunctional microplate reader under the conditions of excitation wavelength of 500 +/-15 nm and emission wavelength of 530 +/-20 nm.

ROS level calculation formula: ROS level (%) (experimental/control) x 100

2.5 determination of the apoptosis Rate of HUVEC cells

HUVECs in logarithmic growth phase were inoculated into several 6-well culture plates. 1.6mL per well, 3.5X 10 cell number⁵One cell/well, at 37 ℃ in 5% CO₂And (4) incubating for 24 h. Dividing the 6-well plate into a blank control group, a model group, a positive drug group and a sample group, adding 200 mu L PBS into the blank control group and the model group, adding 200 mu L GSH into the positive drug group, adding 200 mu L oligopeptide into the sample group (the final concentration of the sample is respectively 100, 200 and 400 mu M), and culturing for 3h in a constant-temperature cell culture box. The blank group was added with 200. mu.L of PBS, and each of the model group, the positive drug group and the sample group was added with 200. mu.L of PBS to a final concentration of 600. mu. M H₂O₂And culturing for 4 hours in a constant-temperature cell culture box.

(1) Sucking out the cell culture solution in the 6-well plate, washing adherent cells twice by using PBS, and digesting the cells for 1-3 min by using pancreatin cell digestive juice without EDTA.

(2) After cell digestion and blowing, the mixture was mixed with pre-cooled PBS and transferred to a centrifuge tube. Centrifuging at 1000rpm at 4 deg.C for 5min, discarding the supernatant, collecting the lower layer of precipitated cells, repeating twice, and completely absorbing PBS.

(3) Cells were suspended with 400. mu.L of 1 × Annexin-V binding solution to a resuspended cell concentration of 3 × 10⁵One per mL.

(4) Add 5. mu.L Annexin V-FITC staining solution to the cell suspension, mix gently, protect from light with aluminum foil, incubate at 2 ℃ for 10 min.

(5) Then 10. mu.L of propidium iodide was added, mixed gently, protected from light with aluminum foil, and incubated at 2 ℃ for 10 min.

(6) The cell sap to be detected is detected by a flow cytometer after passing through a cell sieve, and a green fluorescent signal of Annexin V-FITC can be detected by an FL1(FITC receiver) channel; PI is red fluorescent and can be detected by FL2(Propidium iodide receiver) channel.

Wherein, the upper left corner (Q1-UL) is the mechanically dead cell region, the upper right corner (Q1-UR) is the late apoptotic cell region, the lower left corner (Q1-LL) is the viable cell region, the lower right corner (Q1-LR) is the early apoptotic cell region, and the total apoptotic cells are the late apoptotic cell + early apoptotic cell region.

2.6 measurement of GSH-Px, SOD Activity and NO and MDA content in HUVEC cells

HUVECs in logarithmic growth phase were inoculated into several 6-well culture plates. 1.6mL per well, 3.5X 10 cell number⁵One cell/well, at 37 ℃ in 5% CO₂And (4) incubating for 24 h. Adding 200 μ L of oligopeptide with final concentration of 100, 200, 400 μ M into sample group, adding 200 μ L of GSH with final concentration of 1mM into positive drug group, adding 200 μ L of PBS into blank group and model group, incubating for 3h, adding 200 μ L of oligopeptide with final concentration of 600 μ M H into sample group, model group and positive drug group₂O₂Adding 200 mu L PBS into the blank group, incubating for 4h, then removing the culture solution, washing three times by PBS, scraping each group of cells by a cell scraper and collecting the cells into an EP tube, crushing the cells by a cell crusher in an ice water bath, and then measuring the protein concentration in each group of cells, the activity of GSH-Px and SOD and the content of NO and MDA according to the steps of a BCA protein kit, GSH-Px, SOD, NO and MDA kit.

3 results of the experiment

The damage effect of hydrogen peroxide with different concentrations on HUVEC cells for 4H is shown in figure 14, and is within a certain concentration range along with H₂O₂The survival of HUVEC cells decreased significantly with increasing concentration of (a). According to the report of relevant documents, when the survival rate of HUVEC cells is about 50 percent, the HUVEC cells are the best model, and therefore the HUVEC cells are established by selecting the hydrogen peroxide with the concentration of 600 mu M (51.33 +/-1.37 percent) to act for 4 hoursThe conditions of the damage model were studied.

The protective effect of ACE inhibitory peptide on oxidative damage HUVEC is shown in FIG. 15, and compared with blank group, the cell survival rate of model group is significantly different (P)<0.01) and 52.30 +/-2.70 percent, which indicates that the established oxidative damage model is successful. The cell viability of the oligopeptide sample groups (IK, YEGDP, WF) differed compared to the model group, where YEGDP and WF were vs. H₂O₂Injured HUVEC cells have significant protective effects (P)<0.01) to improve the cell survival rate from 52.30 + -2.70% to 61.56 + -1.80% and 60.43 + -1.16% respectively. Protection of HUVEC oxidative damage by different concentrations of YEGDP and WF fig. 16 shows that oligopeptides YEGDP and WF act on cells at concentrations of 100, 200 and 400 μ M, respectively, and as the concentrations of these oligopeptides increase, the oligopeptides gradually increase in protection of HUVEC cells from oxidative damage, i.e., YEGDP and WF are dose-dependent.

The ROS level in HUVEC measured by DCFH-DA staining method is shown in figure 17, and the ROS content in the model group cells is 205.30 +/-7.04% of that in the control group, which is obviously higher than that in the control group (P < 0.01). The YEGDP and WF peptides in the sample groups were found to reduce intracellular ROS and to be dose-dependent when applied to cells at 100, 200 and 400. mu.M, respectively. YEGDP and WF acted on cells at a concentration of 400. mu.M, the ROS content in the cells was significantly reduced to 158.67 + -6.45% and 160.97 + -4.06%, respectively, of the control group (P < 0.01).

The apoptosis result measured by the AV-PI staining method is shown in figure 18, and compared with the blank group, the apoptosis rate of the model group is 49.6 +/-2.1 percent, which indicates that the apoptosis model is successfully established. The sample group inhibited H to a different extent than the model group₂O₂The induced apoptosis of HUVEC cells, YEGDP and WF can obviously reduce the apoptosis rate (P) under the concentration of 100, 200 and 400 mu M<0.05 or P<0.01), and all exhibit dose dependence. YEGDP and WF acted on the cells at a concentration of 400. mu.M, the apoptosis rates were 30.5. + -. 1.2% and 30.7. + -. 1.1%, respectively. The results show that: ACE inhibitory peptides YEGDP and WF can reduce H₂O₂Resulting in apoptosis of HUVEC cells and is dose-dependent.

The effects of different concentrations of YEGDP and WF on GSH-Px and SOD levels in HUVEC cells are shown in FIG. 19, the activities of SOD and GSH-Px in blank cells were 241.06 + -14.83U/mgprot and 62.18 + -1.94U/mgprot, respectively, the activities of SOD and GSH-Px in model cells were 119.31 + -9.48U/mgprot and 27.01 + -1.37U/mgprot, respectively, and the oligopeptides YEGDP and WF in sample cells were applied to the cells at concentrations of 100, 200 and 400. mu.M, respectively, and it was found that the activities of SOD and GSH-Px in cells increased with the increase in the oligopeptide concentration. Compared with the model group, when YEGDP and WF act on cells at a concentration of 400 mu M, the activity of SOD in the cells is remarkably improved to 174.32 +/-6.54U/mgprot and 171.97 +/-10.94U/mgprot respectively (P < 0.01). Meanwhile, the content of GSH-Px in the cells is obviously improved to 41.16 +/-2.31U/mgprot and 38.92 +/-1.31U/mgprot respectively (P is less than 0.01).

The effect of different concentrations of YEGDP and WF on the MDA level in HUVEC cells is shown in FIG. 20, the MDA content in blank cells is 13.03 + -0.58 nmoL/mgprot, the MDA content in model cells is 28.38 + -0.78 nmoL/mgprot, and the oligopeptides YEGDP and WF in the sample group act on the cells at concentrations of 100, 200 and 400. mu.M, respectively, and the MDA content in the cells is found to decrease with the increase of the oligopeptide concentration. YEGDP and WF the MDA content in the cells at 400. mu.M was significantly reduced to 20.73. + -. 1.20nmoL/mgprot and 21.46. + -. 1.03nmoL/mgprot, respectively (P < 0.01).

The effect of different concentrations of YEGDP and WF on the NO levels in HUVEC cells is shown in FIG. 21, the NO content in the blank cells is 35.69 + -1.50 μmol/L, the NO content in the model cells is 15.54 + -0.62 μmol/L, and the oligopeptides YEGDP and WF in the sample groups act on the cells at concentrations of 100, 200 and 400 μ M, respectively, and the NO content in the cells is found to be dose-dependent. YEGDP and WF the levels of NO in the cells increased significantly at 400. mu.M to 21.26. + -. 0.78. mu. mol/L and 20.74. + -. 0.78. mu. mol/L, respectively (P < 0.01).

The above results indicate that the oligopeptides YEGDP and WF can inhibit the inhibition by H₂O₂Induced apoptosis, improved SOD and GSH-Px activity and NO content in HUVEC cells with oxidative damage, reduced MDA content and ROS level, certain protection effect on HUVEC cells with oxidative damage, and can be used as natural antihypertensive peptide in medicine or functional food, and as novel antihypertensive peptide for further research。

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. An ACE inhibitory peptide, the amino acid sequence of which is Tyr-Glu-Gly-Asp-Pro.

2. An ACE inhibiting peptide according to claim 1, wherein: ACE half inhibitory concentration IC of the ACE inhibitory peptide₅₀=0.19±0.010mg/mL。

3. An ACE inhibiting peptide according to claim 1, wherein: the ACE inhibiting peptide is derived from Mytilus edulis.

4. Use of the ACE inhibiting peptide according to claim 1 for the preparation of a product for lowering blood pressure and/or a product for the prevention and/or treatment of hypertension.

5. Use according to claim 4, characterized in that: the use of the ACE inhibitory peptide in the hypotension and regulation of HUVEC cells.

6. Use according to claim 4, characterized in that: use of the ACE inhibitory peptide for protecting oxidatively damaged HUVEC cells.

7. Use of the ACE inhibiting peptide of claim 1 in the preparation of an antioxidant product.

8. A hypotensive product and/or a product for preventing and/or treating hypertension, comprising the ACE inhibitory peptide according to claim 1 as an active ingredient.

9. A method for preparing an ACE inhibiting peptide from a common mussel, comprising the ACE inhibiting peptide of claim 1, comprising the steps of:

1) degreasing the common mussels by using ethyl acetate;

10. The method of claim 9, wherein the method comprises the steps of: the ACE inhibitory rate of the ACE inhibitory peptide of the Mytilus edulis is more than 24%.