Use of human milk beta-casein-derived peptides or derivatives thereof for inhibiting angiotensin converting enzyme

Abstract

The application discloses application of a human milk beta-casein source peptide or a derivative thereof to inhibition of angiotensin converting enzyme, and the human milk beta-casein source peptide and the pharmaceutically acceptable derivative thereof are characterized in containing a Val-Leu-Lys-Ser-Pro-Thr-Ile-Pro-Phe-Phe-Asp-Pro-Gln-Ile-Pro peptide segment. The human milk beta-casein source peptide, the derivative and the composition can effectively inhibit the activity and the protein level of the angiotensin converting enzyme, improve the function of vascular endothelial cells, and can be used as a human source and food source angiotensin converting enzyme inhibitor for prevention and treatment of related diseases. Experimental results show that the human milk beta-casein source peptide can effectively reduce the blood pressure of rats, and the antihypertensive effect in the rats is better than that of a commonly used clinical drug lisinopril under the same dose, and has excellent conversion application prospect.

Description

Technical Field

[0001] This invention relates to milk-derived polypeptides, and more particularly to the use of a human milk β-casein-derived peptide or its derivative in inhibiting angiotensin-converting enzyme. Background Technology

[0002] Angiotensin-converting enzyme (ACE) inhibitors are widely used in modern medicine and are one of the cornerstone drugs for treating cardiovascular diseases. ACE inhibitors improve vascular endothelial cell function on multiple levels by inhibiting the production of angiotensin II (Ang II) and increasing bradykinin levels: reducing oxidative stress and inflammation, promoting nitric oxide (NO) release, maintaining vasomotor balance and anticoagulation capacity, ultimately alleviating or reversing vascular endothelial damage. This endothelial protective effect is one of the key mechanisms by which they exert their therapeutic effects in diseases such as hypertension and coronary heart disease. Currently, commonly used ACE inhibitors include lisinopril and captopril; however, traditional chemically synthesized ACE inhibitors have certain side effects, including dry cough, rash, and angioedema. Developing safer and more effective ACE inhibitors remains essential.

[0003] In recent years, bioactive peptides derived from milk proteins have attracted widespread attention. Compared with traditional chemically synthesized drugs, milk-derived peptides exhibit unique advantages: these peptides not only have low molecular weight, high specificity, and are less prone to accumulation in the body, but their metabolic end products are also amino acids, resulting in higher safety, fewer side effects, and a wider range of applications. The bovine casein-derived peptides Ile-Pro-Pro and Val-Pro-Pro have been shown to lower blood pressure in humans and spontaneously hypertensive rats. In a rat model, oral administration of the bovine casein-derived peptide Met-Lys-Pro also reduced systolic blood pressure. These bovine casein-derived peptides primarily lower blood pressure by inhibiting ACE activity; however, human-derived ACE-inhibiting milk-derived peptides have not yet been reported. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a novel application of human milk β-casein-derived peptide clusters or derivatives thereof containing the amino acid sequence shown in SEQ ID NO: 1 in the inhibition of angiotensin-converting enzyme.

[0005] Technical solution: The present invention relates to the application of human milk β-casein-derived peptide clusters or derivatives thereof containing the amino acid sequence shown in SEQ ID NO: 1 in the inhibition of angiotensin-converting enzyme.

[0006] Preferably, the human milk β-casein-derived peptide is a peptide with an amino acid sequence as shown in any one of SEQ ID NO: 2-8.

[0007] Preferably, the derivative of the human milk β-casein-derived peptide is a pharmaceutically acceptable peptide modified with C-terminus and / or N-terminus and / or side chain and / or intermediate residues, or a D-type amino acid substitution peptide, or a PEG-modified peptide, or a cyclized peptide, or a membrane-penetrating peptide conjugate; more preferably, the modification of the C-terminus and / or N-terminus and / or side chain and / or intermediate residues is any one or more of acylation, amidation, alkylation, esterification, glycosylation, and phosphorylation.

[0008] Preferably, the application is in the preparation of angiotensin-converting enzyme inhibitors.

[0009] Preferably, the application is in the preparation of drugs for the prevention and treatment of diseases related to elevated angiotensin-converting enzyme, or in the use of adjuvant drugs. It can be used alone or in combination with other drugs.

[0010] Preferably, the angiotensin-converting enzyme (ACE)-related diseases include cardiovascular and cerebrovascular diseases, kidney diseases, metabolic diseases, scleroderma hypertensive crisis, migraine, chronic obstructive pulmonary disease, and depression; more preferably, the cardiovascular and cerebrovascular diseases include hypertension, coronary heart disease, ventricular hypertrophy, left ventricular dysfunction or heart failure, myocardial infarction and ventricular remodeling, and stroke; the kidney diseases include diabetic nephropathy, chronic kidney disease, and proteinuria; and the metabolic diseases include diabetes and insulin resistance.

[0011] Preferably, the drug contains a human milk β-casein-derived peptide or a derivative thereof, or a pharmaceutically acceptable salt, solvate, or hydrate of either, as an active ingredient.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers. Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to propose and verify that human milk β-casein-derived peptide clusters with the Val-Leu-Lys-Ser-Pro-Thr-Ile-Pro-Phe-Phe-Asp-Pro-Gln-Ile-Pro peptide fragments can effectively inhibit the activity and protein level of angiotensin-converting enzyme and improve vascular endothelial cell function, thereby achieving the prevention and treatment of diseases related to elevated angiotensin-converting enzyme; 2. The human milk β-casein-derived peptide can effectively lower blood pressure. In vivo experiments in rats show that the antihypertensive effect at the same dose is superior to that of the commonly used clinical drug lisinopril, and it has excellent prospects for translational application. Attached Figure Description

[0014] Figure 1 A statistical diagram showing the results of an experiment on the inhibition of angiotensin-converting enzyme activity of human milk β-casein-derived peptides 1-7; Figure 2 Figure 1 shows the Western blot results of changes in angiotensin-converting enzyme protein levels in Huvec cells after treatment with human milk β-casein-derived peptide 3. Figure 3 A statistical result of human angiotensin II levels in the culture supernatant of Huvec cells after treatment with human milk β-casein-derived peptide 3; Figure 4 A statistical result of nitric oxide levels in the culture supernatant of Huvec cells after treatment with human milk β-casein-derived peptide 3; Figure 5 Figure showing the prediction results of the molecular docking model between human milk β-casein-derived peptide 3 and angiotensin-converting enzyme; Figure 6 This is a diagram showing the surface plasmon resonance results of human milk β-casein-derived peptide 3 and angiotensin-converting enzyme. Figure 7 The graph shows the blood pressure test results of normal rats treated with human milk β-casein peptide 3. Figure 8 The figure shows the blood pressure detection and statistical results of spontaneously hypertensive rats treated with human milk β-casein-derived peptide 3. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: Preparation of human milk β-casein-derived peptides Shanghai Ketai Biotechnology Co., Ltd. was commissioned to synthesize human milk β-casein-derived peptides 1-7 using chemical synthesis methods, with a purity >95%. In order to make the peptides more similar to the maternal protein and increase the stability of the peptides, human milk β-casein-derived peptides 1-7 were all subjected to N-terminal acetylation and C-terminal amidation. The synthesis information of human milk β-casein-derived peptides 1-7 is shown in Table 1.

[0017] Table 1. Information on the synthesis of peptides derived from human milk β-casein

[0018] Example 2: Human milk β-casein-derived peptides inhibit angiotensin-converting enzyme (ACE) activity (1) Mix 10 μL of ACE aqueous solution (Sigma A6778, final activity 12.5 U / L) with 40 μL of human milk β-casein-derived peptide 1-7 aqueous solution (final concentration 4 mg / mL) or ultrapure water in the wells of a 96-well plate at room temperature, with 6 sub-wells for each treatment; (2) N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG, CAS 64967-39-1) was dissolved in 50 mM Tris-HCl buffer containing 0.3 M NaCl at pH 7.5 to obtain FAPGG solution. 150 μL of 0.88 mM FAPGG solution was added to each well of a 96-well plate, and the absorbance was immediately measured at 340 nm using a microplate reader. After incubation at 37℃ for 30 min, the absorbance was measured again. The difference in absorbance before and after incubation was recorded as ΔA. The ACE inhibition rate was calculated using the following formula:

[0019] Where, ΔA a The absorbance change after incubation with the added peptide for 30 min; ΔA b The value represents the change in absorbance 30 minutes after the addition of water.

[0020] The results are as follows Figure 1 As shown, human milk β-casein-derived peptides 1-7 can significantly inhibit the enzyme activity of ACE, and the inhibitory effect of human milk β-casein-derived peptide 3, whose sequence is shown in SEQ ID NO: 4, is the most obvious.

[0021] Example 3: Human milk β-casein-derived peptides inhibit ACE protein expression levels (1) Human umbilical vein endothelial Huvec cells were cultured in 6-well plates in endothelial cell ECM medium (Sciencell, 1001) until the confluence was greater than 80%. Fresh ECM medium was then added and human milk β-casein-derived peptide 3 (polypeptide) was added to a final concentration of 100 μM. An equal volume of the polypeptide solvent, i.e., sterile enzyme-free water, was used as a control. The treatment was carried out for 1, 3 or 6 h. (2) Cells were collected after treatment, and total protein was extracted using RIPA lysis buffer containing protease inhibitors. The protein was quantified using the BCA method. The quantified protein samples were subjected to electrophoresis, transfer to a membrane, and blocking. After blocking, the samples were incubated overnight at 4°C with 1:1000 diluted ACE antibody (Invitrogen, PA5-86568). The next day, after washing, the samples were incubated at room temperature for 1 h with 1:5000 diluted anti-rabbit secondary antibody (Biosharp, BL003A) and then developed using chemiluminescence.

[0022] The results are as follows Figure 2As shown, treatment with human milk β-casein-derived peptides can significantly reduce the expression level of ACE protein in cells.

[0023] Example 4: Human milk β-casein-derived peptides improve endothelial cell function 1. Human milk β-casein-derived peptides reduce the level of angiotensin II in cell culture supernatant. (1) Human umbilical vein endothelial Huvec cells were cultured in 6-well plates in endothelial cell ECM medium (Sciencell, 1001) until the confluence was greater than 80%. Fresh ECM medium was replaced and human milk β-casein-derived peptide 3 (polypeptide) with a final concentration of 100 μM was added. An equal volume of peptide solvent, i.e., sterile enzyme-free water, was used as a control and the cells were treated for 1 h. (2) Collect cell culture supernatant and use human angiotensin II (Ang II) enzyme-linked immunosorbent assay kit (purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number CSB-E04493h) to detect the Ang II level in the supernatant.

[0024] The results are as follows Figure 3 As shown, treatment with human milk β-casein-derived peptides significantly reduced the level of Ang II in cell culture supernatant.

[0025] 2. Human milk β-casein-derived peptides reduce the level of nitric oxide in cell culture supernatant. (1) Huvec cells were cultured in 6-well plates in endothelial cell ECM medium (Sciencell, 1001) until the confluence was greater than 80%. Fresh ECM medium was then added and human milk β-casein-derived peptide 3 (polypeptide) with a final concentration of 100 μM was added. An equal volume of peptide solvent, i.e., sterile enzyme-free water, was used as a control and the cells were treated for 3 h. (2) Collect cell culture supernatant and use a nitric oxide (NO) detection kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0021S) to detect the NO level in the supernatant.

[0026] The results are as follows Figure 4 As shown, treatment with human milk β-casein-derived peptides significantly increased the level of NO in cell culture supernatant.

[0027] Example 5: Peptides derived from human milk β-casein can bind to ACE. 1. Molecular docking model prediction Molecular docking model predictions were performed using Pymol software (v1.7), based on the findings of Natesh R, Schwager SL, Sturrock ED, and Acharya KR. Crystal structure of the human angiotensin-converting enzyme-lisinopril complex. Nature The active sites of the structure-localized molecules reported in .2003;421(6922):551-554.

[0028] The results are as follows Figure 5 As shown, similar to the ACE inhibitor lisinopril, human milk β-casein-derived peptide 3 (peptide) can bind to the groove of the active site of ACE proteins.

[0029] 2. Surface Plasmon Resonance (SPR) Detection (1) Human ACE protein (purchased from MedChemExpress, catalog number HY-P7992) was prepared into a solution of 50 μg / mL with 10 mM sodium acetate at pH=4.0 and injected into the experimental channel of the CM5 chip (Cytiva BR-1005-30) at a flow rate of 10 μL / min. The fixed amount was about 5000 RU. At the same time, a reference channel without ACE protein was set up. (2) Dilute 0.1% Tween 20-phosphate buffer solution (0.1% PBST) to human milk β-casein-derived peptide 3 solutions with concentrations of 0, 3.125, 6.25, 12.5, 25, 50 or 100 μM and inject them into the experimental channel or reference channel at a flow rate of 30 μL / min to obtain the detection results.

[0030] The results are as follows Figure 6 As shown, peptides derived from human milk β-casein can bind to ACE proteins.

[0031] Example 6: Human milk β-casein-derived peptides effectively reduce blood pressure in rats. 1. Human milk β-casein-derived peptides effectively lower blood pressure in normal rats. (1) Eight-week-old male SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and randomly divided into human milk β-casein-derived peptide (polypeptide) treatment group, lisinopril treatment group and control group, with 3 rats in each group; (2) For rats in the polypeptide treatment group, human milk β-casein peptide 3 was injected into the tail vein at a dose of 10 mg / kg. For rats in the lisinopril treatment group, lisinopril (purchased from MedChemExpress, catalog number HY-18206) was injected into the tail vein at a dose of 20 mg / kg. For rats in the control group, an equal volume of physiological saline was injected into the tail vein. (3) Anesthetize the rats, cut open the right side of the neck, bluntly separate the muscles, find the triangle area and cut it, find the common carotid artery and make a small incision, insert the probe catheter of the biological function test system (Chengdu Taimeng BL-420S) into the common carotid artery, and monitor the blood pressure of the rats at 0 min and 30 min after treatment.

[0032] The results are as follows Figure 7 As shown, human milk β-casein-derived peptides can lower blood pressure in normal rats, and at the same dosage, they are more effective than lisinopril.

[0033] 2. Human milk β-casein-derived peptides effectively lower blood pressure in hypertensive rats. (1) Three 11-week-old male spontaneously hypertensive rats (SHR) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (2) Anesthetize the rats, cut open the right side of the neck, bluntly separate the muscles, find the triangle area and cut it, find the common carotid artery and make a small incision, insert the probe catheter of the biological function test system (Chengdu Taimeng BL-420S) into the common carotid artery, inject each rat with a dose of 10 mg / kg human milk β-casein peptide 3 via the tail vein, and monitor the blood pressure of the rats at 0 min and 30 min after treatment.

[0034] The results are as follows Figure 8 As shown, human milk β-casein-derived peptides can significantly reduce systolic blood pressure, diastolic blood pressure, and mean blood pressure in hypertensive rats.

Claims

1. The application of a human milk β-casein-derived peptide in the preparation of therapeutic or adjuvant therapeutic drugs for diseases related to elevated angiotensin-converting enzyme, characterized in that, The amino acid sequence of the human milk β-casein-derived peptide is shown in SEQ ID NO:

4. The angiotensin-converting enzyme-related disease is hypertension, and the human milk β-casein-derived peptide inhibits angiotensin-converting enzyme.

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