CN106554388B

CN106554388B - Polypeptide with ACE and DPP-IV inhibitory activity and application thereof

Info

Publication number: CN106554388B
Application number: CN201510622792.0A
Authority: CN
Inventors: 邹汉法; 靳艳; 于洋; 晏嘉泽
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2015-09-25
Filing date: 2015-09-25
Publication date: 2020-01-21
Anticipated expiration: 2035-09-25
Also published as: CN106554388A

Abstract

The invention relates to a polypeptide which is derived from casein and has angiotensin-converting enzyme (ACE) and DPP-IV double inhibition activity. The amino acid sequence is Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe. The polypeptide FVAPFPEVF has ACE inhibitory activity and DPP-IV inhibitory activity, and can be used as health products and drug lead compounds for lowering blood pressure and blood sugar.

Description

Polypeptide with ACE and DPP-IV inhibitory activity and application thereof

Technical Field

The invention relates to application of polypeptide FVAPFPEVF in preparation of drugs and health care products for inhibiting Angiotensin Converting Enzyme (ACE), inhibiting dipeptidyl peptidase-IV (DPP-IV) or lowering blood pressure and blood sugar.

Background

1. ACE inhibitory Activity

Hypertension is a common cardiovascular disease, has high morbidity, and is an important risk factor for causing various complications of heart, brain, kidney, blood vessels and the like, causing stroke, and promoting atherosclerosis and coronary heart disease. The average incidence rate of all countries in the world is 10% -20%, the number of hypertension patients in China exceeds 1.6 hundred million people, and the treatment and prevention of hypertension are very important subjects of the current society. Peptides are an important blood pressure lowering drug, and the blood pressure lowering effect target of peptide compounds is the inhibition of the activity of angiotensin-converting enzyme (ACE) (document 1: Vanessa Vermeissen, John Van Camp, Willy Verstraete, British Journal of Nutrition 2004, 92: 357) 366). Angiotensin converting enzyme can convert inactive decapeptide angiotensin I into octapeptide angiotensin II with strong vasoconstrictive effect, thereby increasing blood pressure, and inhibiting ACE activity can effectively control hypertension. The polypeptide is an important ACE inhibitor, and the zymolytic peptide of natural protein is the main source of the ACE inhibitor peptide (literature 2: Lieselot Vercruysse, John Van Camp, Guy Smagghe, J.Agric.food Chem 2005,53: 8106-. Angiotensin converting enzyme plays an important role in regulating the blood pressure and cardiovascular function of the body, so that drugs inhibiting the activity of ACE play an important role in the treatment of cardiovascular and heart failure and other diseases. The polypeptide ACE inhibitor can reduce blood pressure without causing side effects such as dry cough of common blood pressure lowering medicines.

2. DPP-IV inhibitory Activity

Dipeptidyl peptidase-IV (DPP-IV) (EC3.4.14.5) is a serine protease that is widely distributed in the human body. DPP-IV inactivates polypeptides through shearing, thereby achieving the effect of regulating physiological functions. DPP-IV preferentially hydrolyzes proteins having proline (Pro) or alanine (Ala) at the second position of the N-terminus. The action substrates comprise: glucagon-like peptide-1 (GLP217-36), gastric inhibitory peptide (GIP1-42), Neuropeptide (NPY), YY Peptide (PYY), pancreatic polypeptide family (PP-family), and the like. The common feature of these substances is that the amino acid residue at the second position of the N-terminus is either a proline or alanine residue. DPP-IV plays an important role in diseases related to diabetes, glucose tolerance, obesity, appetite regulation, hyperlipidemia, osteoporosis, neuropeptide metabolism, T-cell activation, and the like, by acting on the above polypeptide substrate. Therefore, in vivo administration of the DPP-IV inhibitor can prevent N-terminal degradation of related substrate peptide, thereby ensuring normal operation of human body functions.

DPP-IV cleaves 2N-terminal amino acid residues in complete GLP-1 and GIP reactions, inactivates them, and affects physiological reactions such as GLP-1 and GIP-related blood glucose regulation. In vivo administration of a DPP-IV inhibitor prevents N-terminal degradation of GLP-1 and GIP, increasing insulin secretion and thereby improving glucose tolerance. In view of the close relationship between DPP-IV inhibitor and blood sugar metabolism, DPP-IV inhibitor has become a hot research focus of novel type 2 diabetes mellitus drugs.

Disclosure of Invention

The invention aims to provide application of the polypeptide FVAPFPEVF in inhibiting ACE activity, inhibiting DPP-IV activity, lowering blood pressure and lowering blood sugar; the polypeptide FVAPFPEVF has ACE inhibitory activity, DPP-IV inhibitory activity, blood pressure lowering activity and blood sugar lowering activity, and has good application prospect as a health product and a medicine lead compound for diseases such as hypertension, heart disease, cardiovascular disease, diabetes, obesity, nephropathy, immune disorder and the like.

In order to achieve the purpose, the polypeptide FVAPFPEVF is used as an active ingredient for ACE inhibitory activity, DPP-IV inhibitory activity, blood pressure lowering activity and blood sugar lowering activity.

It has the sequence table of SEQ ID NO: 1, amino acid sequence; the polypeptide FVAPFPEVF is active ingredient of ACE inhibitory activity, DPP-IV inhibitory activity, blood pressure lowering, blood sugar lowering medicine and health product, and pharmaceutically acceptable carrier or adjuvant can be added.

The polypeptide FVAPFPEVF has ACE inhibitory activity, DPP-IV inhibitory activity, blood pressure lowering activity and blood sugar lowering activity, has an amino acid sequence of Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe, is a single-chain linear structure, is white powder, is easy to dissolve in water, and has the molecular weight of 1051.5 Da; has strong inhibiting effect on ACE activity, and IC50 is 35.76 μ M; has stronger inhibiting effect on DPP-IV activity, and the IC50 is 2.52 mM.

Polypeptide FVAPFPEVF possesses the characteristics required for ACE inhibitors:

1. ACE inhibiting active peptides mainly depend on C-terminal amino acids, which are aromatic (Trp, Phe, Tyr) or hydrophobic; and the peptide segment with hydrophobic amino acid at the N end has stronger ACE inhibitory activity. The C-terminal dipeptides of the polypeptide FVAPFPEVF are all the above amino acids, Val (hydrophobic) and Phe (aromatic, hydrophobic), respectively, and the N-terminal is the hydrophobic amino acid Phe, thus completely meeting the requirements.

2. The content of hydrophobic amino acids in peptides is an important factor affecting the inhibitory activity of peptides, and peptides with high inhibitory activity all contain a large amount of hydrophobic amino acids. The hydrophobic amino acids of the polypeptide FVAPFPEVF include Phe, Val, Ala, Pro, Phe, Pro, Val, and Phe, and many of these hydrophobic amino acids are present.

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

the invention obtains and determines the structure of the active compound from casein for the first time, and the compound has better activity of inhibiting ACE and DPP-IV, so that the compound has good potential and application prospect as a health product and a medicine lead compound for treating cardiovascular diseases such as hypertension and the like and diseases such as diabetes, heart disease, obesity, nephropathy, immune disorder and the like.

Detailed Description

EXAMPLE 1 preparation of polypeptide FVAPFPEVF

The method combining LC-MS/MS and Shotgun proteomics technology is adopted. The method comprises the steps of taking milk yoghourt as a raw material, carrying out protein enzymolysis, centrifuging, ultrafiltration and LC-MS/MS analysis, and screening peptide segments with inhibiting effects on ACE and DPP-IV by combining structure-activity relationship characteristics.

The specific method comprises the following steps: yogurt samples were mixed with 35mM NaCl solution at 1:1, regulating the pH value to 2.0 by using 1M hydrochloric acid, measuring the protein content in the yoghourt, adding pepsin, wherein the adding amount is that the pepsin: and (3) carrying out enzymolysis on the yoghourt sample at 37 ℃ for 1h, wherein the mass ratio of the protein in the yoghourt sample is 1: 100. The reaction was terminated by adjusting the pH to 7.0 with 1M NaOH. Centrifuging the enzymolysis solution at 4 deg.C and 10000g (3000-10000 g) for 40 min (3000-10000 g), ultrafiltering the supernatant with ultrafiltration membrane (3000-10000 g) with cut-off molecular weight of 10000, and desalting the filtrate with C18-SPE column. After desalting, the enzymolysis sample is freeze-dried and dissolved in formic acid water with mass concentration of 0.1%, and LTQ Orbitrap XL (ion trap cyclotron resonance combination mass spectrometer) is used for mass spectrum analysis of the sample.

Peptide sequence library search: and (3) carrying out mass spectrum analysis on the sample by using LTQ Orbitrap XL with an ion source of ESI, retrieving the obtained data in a bone.

Example 2 detection of ACE inhibitory Activity of polypeptide FVAPFPEVF

Principle of

ACE catalyzes the simulated substrate Hippuryl-L-Histidyl-L-Leucine (HHL) of angiotensin I to generate hippuric acid at 37 ℃ and pH8.3, and the substance has a characteristic ultraviolet absorption peak at 228 nm. When an ACE inhibitor is added, the catalytic action of ACE on HHL is inhibited and the production of hippuric acid is reduced. The magnitude of the inhibitory activity can be calculated by measuring the ultraviolet absorbance of hippuric acid before and after the addition of the inhibitor.

Reaction system

The buffer solution is 0.05M, pH8.3 borate buffer solution; the substrate is Hippuryl-L-Histidyl-L-Leucine (HHL) and MW 429.47, and the buffer solution is used for preparing 5 mM; ACE (angiotensin-converting enzyme) was prepared at 0.1U/ml with the above buffer.

OD_A(control, absorbance in the absence of inhibitor but in the presence of enzyme): 50 mul buffer solution +50ul HHL +50 mul buffer solution is put in water bath at 37 ℃ for 5min, then 50 mul ACE is added, water bath at 37 ℃ is carried out for 30min, 200 mul HCl with the concentration of 1M is added to stop the reaction, 1ml ethyl acetate extraction product hippuric acid is added, oscillation is carried out for 15S, 3500r/min centrifugation is carried out for 5min, 0.8ml supernatant is taken, water bath at 90 ℃ is dried for 15min, redissolved in 0.8ml distilled water, and the absorbance value detected at 228nm is OD_A。

OD_B(sample group, absorbance value in the presence of inhibitor and enzyme) 50. mu.l of sample + 50. mu.l of HHL + 50. mu.l of buffer solution in 37 ℃ water bath for 5min, then 50. mu.l of ACE is added, the 37 ℃ water bath is carried out for 30min, 200. mu.l of 1M HCl is added to terminate the reaction, 1ml of ethyl acetate extraction product hippuric acid is added, 15S is shaken, 3500r/min is centrifuged for 5min, 0.8ml of supernatant is taken, the 90 ℃ water bath is dried for 15min, the supernatant is redissolved in 0.8ml of distilled water, and the absorbance value detected at 228nm is OD_B。

The operation was carried out using the polypeptide FVAPFPEVF obtained in example 1 as a sample.

OD_C(blank, absorbance in the absence of inhibitor and enzyme): 50 mul buffer solution +50 mul HHL +50 mul buffer solution are put in water bath at 37 ℃ for 5min, then 50 mul buffer solution is added, water bath at 37 ℃ is carried out for 30min, 200 mul and 1M HCl are added to stop the reaction, 1ml ethyl acetate extraction product hippuric acid is added, oscillation is carried out for 15S, 3500r/min centrifugation is carried out for 5min, 0.8ml supernatant is taken, water bath at 90 ℃ is dried for 15min, redissolved in 0.8ml distilled water, and the absorbance value detected at 228nm is OD_C。

ACE inhibition ratio (%) ═ OD_A-OD_B)/(OD_A-OD_C)×100％

The ACE inhibitory activity of polypeptide FVAPFPEVF was determined as described above at different concentrations. The results are as follows:

EXAMPLE 3 detection of DPP-IV inhibitory Activity of polypeptide FVAPFPEVF

Principle of

A color development substrate method with glycylproline p-nitroaniline (Gly-Pro-PNA) as a substrate is adopted to screen the DPP-IV inhibitor, the detection principle of the method is that the DPP-IV catalyzes the substrate Gly-Pro-p-nitroanilide to hydrolyze under an alkaline condition to generate yellow p-nitroaniline, the yellow p-nitroaniline has a characteristic absorption peak at the wavelength of 405nm, and the absorbance measured at the wavelength of 405nm by a microplate reader reflects the activity of enzyme.

Method of producing a composite material

Sample preparation: the sample was dissolved in 100mM Tris-HCl buffer (pH 8.0) to prepare a stock solution of 40mg/mL, and the stock solution was diluted to different concentrations to prepare sample solutions.

Substrate: gly-pro-p-nitroanalide was dissolved in 100mM Tris-HCl buffer (pH 8.0) to prepare 1.59mM Gly-pro-p-nitroanalide solution. Positive drug: diprotin a solution, Diprotin a was prepared as a 2mM stock solution in 100mM Tris-HCl buffer (pH 8.0) and diluted to 800uM before use. The rest stock solution is divided into a plurality of tubes and stored at-20 ℃.

Enzyme: the DPP-IV solution was prepared in 0.01U/mL of Tris-HCl buffer (pH 8.0) at 100 mM.

Stop solution: 1M acetate-sodium acetate buffer (pH 4.0).

The assay was performed in 96-well plates and absorbance was measured at 405nm using a microplate reader. Firstly, enzyme, buffer solution and medicine are respectively subjected to water bath for 30min at the temperature of 37 ℃, then a sample (or the buffer solution) and a substrate are sequentially added into a 96-well plate and incubated for 10 min at the temperature of 37 ℃, DPP-IV enzyme solution is added, the mixture is uniformly mixed and incubated for 60 min at the temperature of 37 ℃, and 100 mu L of 1M acetic acid-sodium acetate buffer solution (pH 4.0) is added to terminate the reaction. Absorbance at 405nm was measured with a microplate reader. The total volume of the reaction was 100. mu.l. The experiment was divided into 4 groups, each group being provided with 3 multiple wells. The groups are respectively as follows:

sample set (S set): sample + enzyme + substrate.

Sample blank (SB group): sample + substrate.

Negative control group (group C): enzyme + substrate.

Blank group (group B): a substrate.

Specific groups and samples are shown in table 1.

TABLE 1 DPP-VI inhibitory Activity assay Components and sample amounts

Note: (1) the numbers in the table are all in. mu.l.

(2) The total volume of the reaction was 100. mu.l, and the final volume was made up to 100. mu.l with buffer after addition of the reactants for each group as listed in the table.

(3) Calculation of inhibition Rate

Inhibition ratio of S group (1-). times.100%

(4) The result of the detection

DPP-IV inhibitory activity assay was performed as described above using polypeptide concentrations of 300. mu.g/ml, 600. mu.g/ml, 1200. mu.g/ml, respectively. The results are given in the following table:

warp IC₅₀The calculator calculates the IC of the sequence₅₀It was 2.65mg/mL, i.e., 2.52 mM.

Claims

1. The application of a polypeptide with ACE and DPP-IV inhibitory activity in the process of preparing DPP-IV inhibitors is characterized in that: the polypeptide is FVAPFPEVF, and has a sequence table SEQ ID NO: 1, amino acid sequence; the amino acid sequence of the polypeptide is Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe.

2. Use of the polypeptide of claim 1 in the manufacture of a medicament for lowering blood glucose.

3. Use according to claim 2, characterized in that: one or two of the DPP-IV inhibitor and the hypoglycemic drug take the polypeptide FVAPFPEVF as an active ingredient.

4. Use according to claim 2 or 3, characterized in that: one or two of the DPP-IV inhibitor and the hypoglycemic drug can be added with a pharmaceutically acceptable carrier or auxiliary material.