Angiotensin converting enzyme inhibitory peptide and application thereof

Abstract

The invention provides an angiotensin converting enzyme inhibitory peptide and application thereof. The amino acid sequence of the angiotensin converting enzyme inhibitory peptide is PVKP. The ACE inhibitory activity of the inhibitory peptide PVKP provided by the invention is obviously improved compared with that of the original inhibitory peptide LYPVK, and the IC50 value is reduced from 10.6 mu g / mL to 9.6 mu g / mL; the digestive enzyme stability of the inhibitory peptide PVKP provided by the invention and the digestive enzyme stability of the original inhibitory peptide LYPVK under the same concentration are improved. Molecular simulation docking verifies that the interaction force between the ACE inhibitory peptide PVKP and ACE is enhanced compared with that of an original inhibitory peptide LYPVK, and the interaction force between alkyl groups and the interaction between the alkyl groups and Zn < 2 + > of an ACE active center are increased. The intracellular antihypertensive activity of the ACE inhibitory peptide PVKP is further verified through cell experiments, and compared with the original inhibitory peptide LYPVK, the intracellular NO release content is increased, and the ET-1 content is reduced. The ACE inhibitory peptide PVKP is small in molecular weight and good in water solubility, and application of the ACE inhibitory peptide PVKP in development of products with the blood pressure lowering effect is facilitated.

Description

Technical Field

[0001] The invention relates to the field of active peptide biotechnology, in particular to an angiotensin converting enzyme inhibitory peptide and its application. Background Art

[0002] Hypertension and the cardiovascular diseases it causes are one of the most common chronic diseases. According to data released by the World Health Organization, the number of deaths worldwide due to hypertension and related cardiovascular diseases has increased in the past 30 years, which is a major threat to human health. Angiotensin-converting enzyme (ACE) is a key conversion enzyme in the human blood pressure regulation system. It can participate in the regulation of human blood pressure, affect the content of key substances in the blood pressure-related pathways, and thus affect blood pressure. For example, ACE can promote the production of angiotensin II in the RAS system, and can also degrade bradykinin in the KKS system and reduce its content, thereby leading to increased blood pressure. Therefore, inhibiting the activity of ACE is an important target in the current blood pressure prevention and treatment strategy. Antihypertensive drugs such as captopril and enalapril exert their antihypertensive effects by targeting ACE and inhibiting the activity of ACE. This type of drug is currently the main treatment method for ACE inhibitors, but long-term use of drugs will bring certain side effects to the human body. The exploration of safe and green ACE inhibitors is a hot topic of current concern.

[0003] ACE inhibitory peptides are bioactive peptides widely derived from foods such as milk, soybeans, oysters, and fish, targeting ACE and having significant antihypertensive effects. They are the main targets of ACE inhibitory drug development in recent years. Although various ACE inhibitory peptides have been classified and identified from a wide range of food raw materials, the application of ACE inhibitory peptides is still restricted by problems such as weak ACE inhibitory activity, limited stability research, and unclear antihypertensive efficacy mechanism. The development of ACE inhibitory peptides with strong ACE inhibitory activity, strong stability, and significant antihypertensive effects is crucial for its application in the prevention and treatment of hypertension. LYPVK is an ACE inhibitory peptide derived from figs. It has been enzymatically hydrolyzed, separated, and identified from figs by researchers, but no stability analysis has been performed, and the inhibitory activity is weak. The components of the prepared product are complex, making it difficult to explain the antihypertensive effect of the peptide. It is of great significance to improve the inhibitory activity, study stability, explore the antihypertensive effect, and analyze the mechanism of LYPVK for its development as a food-borne ACE inhibitory peptide. Summary of the invention

[0004] The purpose of the present invention is to provide a new angiotensin converting enzyme inhibitory peptide with better inhibitory effect and its application.

[0005] Specifically, the present invention is based on the original ACE inhibitory peptide LYPVK amino acid sequence, and adopts the idea that molecular weight, hydrophobic residues, amino acid sequence distribution, arrangement and the type of amino acid residues at the end of the sequence will affect the inhibitory activity of the ACE peptide, and rationally designs its original amino acid sequence to obtain the amino acid sequence of the ACE inhibitory peptide with improved ACE inhibitory activity, and artificially synthesizes two ACE inhibitory peptide amino acid sequences to further verify their ACE inhibitory activity, inhibitory characteristics, and gastrointestinal stability, and uses molecular simulation docking to verify the mechanism of improved ACE inhibitory activity, and verifies and compares the intracellular hypotensive effects of the two ACE inhibitory peptides, and analyzes the changes in the levels of intracellular blood pressure-related products after the action of the two ACE inhibitory peptides to explore the intracellular hypotensive efficacy.

[0006] The larger the molecular weight of the ACE inhibitory peptide, the more difficult it is to enter the active center of ACE, and the lower the ACE inhibitory activity; and according to the amino acid composition of the ACE active center, the hydrophobic amino acids, lysine and proline at the end of the ACE inhibitory peptide are easy to interact with ACE, which is more conducive to inhibiting the activity of ACE. Therefore, the above ideas are used to rationally design ACE inhibitory peptides and develop new ACE inhibitory peptides, which is conducive to improving their ACE inhibitory activity.

[0007] To achieve the above object, the present invention proposes the following technical solution: an angiotensin converting enzyme inhibitory peptide, wherein the amino acid sequence of the angiotensin converting enzyme inhibitory peptide is PVKP, Pro-Val-Lys-Pro.

[0008] The use of the above-mentioned angiotensin converting enzyme inhibitory peptide in the preparation of angiotensin converting enzyme inhibitors.

[0009] Such as the use of angiotensin converting enzyme inhibitory peptide in the preparation of antihypertensive drugs.

[0010] An angiotensin converting enzyme inhibitor comprises the above angiotensin converting enzyme inhibitory peptide as an active ingredient.

[0011] A blood pressure lowering pharmaceutical composition comprises the above-mentioned angiotensin converting enzyme inhibitory peptide.

[0012] A blood pressure lowering pharmaceutical preparation comprises the above angiotensin converting enzyme inhibitory peptide or the above blood pressure lowering pharmaceutical composition, and also comprises a pharmaceutically acceptable carrier.

[0013] Furthermore, in the present invention, the pharmaceutically acceptable carrier is suitable for tablets, capsules, powders, pills, granules, oral liquids, injections or emulsions.

[0014] Beneficial effects: The technical solution of this application has the following technical effects:

[0015] The ACE inhibitory peptide PVKP provided by the present invention has an improved inhibitory activity on ACE compared with the original inhibitory peptide LYPVK. 50 The value was reduced from 10.6 μg / mL to 9.6 μg / mL, and at the same concentration, the digestive enzyme stability of the ACE inhibitory peptide PVKP provided by the present invention was stronger than that of the original inhibitory peptide LYPVK. Molecular simulation docking verified that the interaction between the ACE inhibitory peptide YPLKP and ACE was stronger than that of the original inhibitory peptide LYPVK, and the interaction between the alkyl groups and the Zn 2+ The action force is increased. The intracellular hypotensive effect of the ACE inhibitory peptide PVKP of the present invention was further verified by cell experiments. The original inhibitory peptide LYPVK was improved, the NO content was increased, and the ET-1 content was reduced. The ACE inhibitory peptide PVKP of the present invention has a small molecular weight and good water solubility, which is conducive to its application in the development of products with hypotensive effects.

[0016] It should be appreciated that all combinations of the foregoing concepts, as well as additional concepts described in greater detail below, may be considered to be part of the inventive subject matter of the present disclosure, provided such concepts are not mutually inconsistent.

[0017] The foregoing and other aspects, embodiments and features of the present invention can be more fully understood from the following description in conjunction with the accompanying drawings. Other additional aspects of the present invention, such as the features and / or beneficial effects of the exemplary embodiments, will be apparent from the following description or learned from the practice of the specific embodiments according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures may be represented by the same reference numeral. For clarity, not every component is labeled in every figure. Embodiments of various aspects of the present invention will now be described by way of example and with reference to the accompanying drawings, in which:

[0019] Figure 1 This is an amino acid comparison chart of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK;

[0020] Figure 2 The IC of ACE inhibition by the ACE inhibitory peptide PVKP of the present invention and the original ACE inhibitory peptide LYPVK 50 value;

[0021] Figure 3 The digestive enzyme stability of the ACE inhibitory peptide PVKP of the present invention and the original ACE inhibitory peptide LYPVK;

[0022] Figure 4It is a schematic diagram of the molecular simulation docking interaction force between the ACE inhibitory peptide PVKP of the present invention and ACE;

[0023] Figure 5 Schematic diagram of the molecular simulation docking interaction force between the original ACE inhibitory peptide LYPVK and ACE;

[0024] Figure 6 The cytotoxicity of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK;

[0025] Figure 7 The intracellular NO content of ACE inhibitory peptide PVKP and original ACE inhibitory peptide LYPVK;

[0026] Figure 8 It is the intracellular ET-1 content of ACE inhibitory peptide PVKP and original ACE inhibitory peptide LYPVK. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are cited and described as follows in conjunction with the accompanying drawings. Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily defined to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways, because the concepts and embodiments disclosed in the present invention are not limited to any implementation. In addition, some aspects disclosed in the present invention can be used alone, or in any appropriate combination with other aspects disclosed in the present invention.

[0028] Example 1

[0029] Rational design of ACE inhibitory peptides

[0030] The original ACE inhibitory peptide LYPVK was rationally designed to obtain the amino acid sequences of two inhibitory peptides: Figure 1 As shown, the ACE inhibitory peptide sequence of the present invention is Pro-Val-Lys-Pro, and the original ACE inhibitory peptide LYPVK and the ACE inhibitory peptide PVKP of the present invention are artificially synthesized to obtain two inhibitory peptide samples for subsequent activity detection.

[0031] Example 2

[0032] ACE inhibitory activity assay

[0033] The ACE inhibitory activity of ACE inhibitory peptide PVKP and original ACE inhibitory peptide LYPVK was determined using a 96-well plate using a visible spectrophotometer. 1 mM N-[3-(2-furyl) acryloyl]-L-phenylalanylglycylglycine (FAPPG), HEPES buffer (1.901 g HEPES reagent, 1.755 g NaCl reagent, pH 8.3), and 0.1 U / mL angiotensin converting enzyme (ACE) were prepared respectively. Different concentrations of ACE inhibitory peptide PVKP and original ACE inhibitory peptide LYPVK samples (2.5, 5, 10, 20 μg / mL), as well as appropriate amounts of FAPPG substrate, ACE and HEPES buffer, were added to a 96-well plate; the absorbance of the mixed system at 340 nm was measured using an enzyme reader; the system was placed in a 37°C shaker for 30 minutes, and the absorbance at 340 nm was measured again using an enzyme reader after the reaction; the ACE inhibition rate of the sample was determined by calculating the change in absorbance before and after the reaction. The calculation formula is: ACE inhibition rate % = 1-change in sample absorbance value / change in blank absorbance value. IC of ACE inhibitory peptide 50 The value is the ACE inhibitory peptide concentration when the inhibition rate of ACE is 50%.

[0034] The results are as follows Figure 2 As shown, the IC of ACE inhibition by the ACE inhibitory peptide PVKP is 50 The value was 9.6 μg / mL, while the IC of the original ACE inhibitory peptide LYPVK for ACE inhibition was 50 The value was 10.6 μg / mL, and the ACE inhibitory activity of the ACE inhibitory peptide PVKP was improved.

[0035] Example 3

[0036] Prediction and analysis of the physicochemical properties of ACE inhibitory peptide PVKP

[0037] The physicochemical properties of ACE inhibitory peptide YPLKP were analyzed using software or online websites. The isoelectric point was analyzed using the online website (https: / / www.innovagen.com / proteomics-tools); the toxicity was analyzed using the ToxinPred website (https: / / crdd.osdd.net / raghava / toxinpred / ) and the water solubility was analyzed using the online website (http: / / www.innovagen.com / proteomics-tools).

[0038] The results showed that the isoelectric point of the ACE inhibitory peptide PVKP was 9.46, it had good water solubility, and was non-toxic at normal concentrations.

[0039] Example 4

[0040] Gastrointestinal digestive enzyme stability of ACE inhibitory peptides

[0041] The ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK samples were subjected to gastrointestinal digestive enzyme stability test. The pH of the system was adjusted to 2 with a 1M concentration of HCl solution, and pepsin was added at a concentration of 2% to prepare a simulated gastric digestion solution. The ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK samples (at a concentration of 10 μg / mL) were mixed with the pepsin solution and incubated at 37°C. After the reaction, the pH was adjusted to 7.5 with a 1M concentration of NaOH to inactivate the enzymatic hydrolysis product, and the gastric hydrolyzate was obtained for trypsin treatment. After trypsinization, the sample was inactivated at 95°C for 10 minutes, cooled to 37°C, and the in vitro ACE inhibition rate was determined.

[0042] The results are as follows Figure 3 As shown, at the same concentration, after the ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK were treated with the above-mentioned gastrointestinal digestive enzymes, the inhibition rate of the digested ACE inhibitory peptide PVKP on ACE was 52.3%, and it still had good ACE inhibitory activity; after the same concentration of the original inhibitory peptide LYPVK was treated with the same gastrointestinal digestive enzymes, the inhibition rate of ACE was 43.5%, indicating that the ACE inhibitory peptide PVKP of the present invention has gastrointestinal digestive enzyme tolerance and improved gastrointestinal stability compared with the original inhibitory peptide LYPVK.

[0043] Example 5

[0044] Molecular simulation and docking analysis

[0045] The ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK were docked with ACE by molecular simulation, and the mechanism of the activity enhancement of the ACE inhibitory peptide YPLKP was elucidated from the perspective of the interaction force of amino acid residues. The three-dimensional conformational diagrams of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK were drawn using Discovery studio software, and molecular simulation docking was performed with the catalytic active center of ACE (PDB: 1o8a), and the interaction force between the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK and the key amino acid residues in the ACE active center was analyzed based on the docking results.

[0046] The results are as follows Figure 4As shown in the figure, the ACE inhibitory peptide PVKP can be docked with the ACE active center and can be simulated and docked within the normal analysis time. The ACE inhibitory peptide PVKP interacts with various amino acid residues in the ACE active center. The main forces are hydrogen bonds, salt bridges, mutual attraction, metal ion receptors, interactions between alkyl groups, and interactions between π-alkyl groups. Compared with the analysis results of the interaction force between the original ACE inhibitory peptide LYPVK and the amino acid residues in the ACE active center, as shown in the figure, Figure 4 and Figure 5 Compared with the original ACE inhibitory peptide LYPVK, the ACE inhibitory peptide PVKP of the present invention has a stronger interaction force with the key amino acid residue alkyl group of the ACE active center, and has an increased metal ion receptor, which indicates that the inhibitory peptide PVKP of the present invention is easier to combine with the ACE active center, thereby enhancing the ACE inhibitory activity.

[0047] Example 6

[0048] Study on the intracellular hypotensive effect

[0049] Human umbilical vein endothelial cells EA.hy926 were used as the research object to explore the intracellular hypotensive effect of PVKP and the original ACE inhibitory peptide LYPVK described in the present invention. First, the toxicity of the two inhibitory peptide samples to cells was determined. Specifically: dilute the cells to make a cell suspension. The cells to be used were washed with PBS and digested with trypsin from the culture bottle, and then dissolved with complete culture medium to make a cell suspension, and counted with a hemocytometer. Dilute the cells to an appropriate concentration with complete culture medium, cover the 96-well plate, and culture in an incubator for about 24 hours to adhere to the wall. Use a gun to suck away the supernatant culture fluid from each well of the cell, add 100 μL PBS solution to each well for washing, and then suck it away with a gun. Dilute the sample with a culture medium that does not contain FBS (i.e., DMEM-H+1% P / S). Weigh the sample in advance, dilute it to different concentrations in the clean bench, and sterilize it with a membrane. Add 100 μL of diluted sample to each well and place it in an incubator for about 24 hours. Use a gun to suck away the supernatant culture medium of each well of the cells, add 100 μL PBS solution to each well for washing, and then suck it away with a gun. Use a culture medium without FBS (i.e. DMEM-H + 1% P / S) to prepare CCK (the ratio is: 90 μL culture medium + 10 μL CCK). Add 100 μL of culture medium containing CCK to each well, place it in an incubator for incubation, and measure the absorbance at 450nm with an enzyme marker. Calculate the cell survival rate after treatment with the inhibitory peptide sample according to the cell viability formula.

[0050] The results are as follows Figure 6As shown, the ACE inhibitory peptide PVKP of the present invention and the original inhibitory peptide LYPVK have no cytotoxicity. At a concentration of 2.5-40 μg / mL, the cell survival rate is greater than 90%, indicating that at the concentration where the two inhibitory peptides effectively exert ACE inhibitory effects, they have no toxicity to endothelial cells.

[0051] The content of NO and ET-1 in cells is an important marker substance related to blood pressure. Whether the content of NO and ET-1 can be changed under the action of ACE inhibitory peptide is an important indicator for evaluating the antihypertensive effect of cells. The antihypertensive effect of the two inhibitory peptides was explored by measuring the NO content and ET-1 content released by human umbilical vein endothelial cells EA.hy926 after treatment with the ACE inhibitory peptide PVKP described in the present invention and the original inhibitory peptide LYPVK. Captopril, a blood pressure-lowering drug, was used as a positive control. The results are as follows. Figure 7 As shown in the figure, after the two inhibitory peptides acted, compared with the negative control, both peptides could increase the NO content released in cells. Under the same concentration treatment of the two inhibitory peptides, the NO content released by cells after the ACE inhibitory peptide PVKP of the present invention was higher than that of the original inhibitory peptide LYPVK, indicating that the ACE inhibitory peptide PVKP of the present invention has a stronger cell hypotensive effect. The results of intracellular ET-1 content are shown in the figure. Figure 8 As shown, compared with the negative control, both peptides can reduce the amount of NO released into cells. Under the same concentration treatment of the two inhibitory peptides, the ET-1 content of cells after treatment with the ACE inhibitory peptide PVKP of the present invention is lower than that of the original inhibitory peptide LYPVK, indicating that the ACE inhibitory peptide PVKP of the present invention has a stronger effect of lowering blood pressure in cells.

[0052] In summary, based on the results of cell experiments, after rational design, the ACE inhibitory peptide PVKP described in the present invention has a significantly improved intracellular blood pressure lowering effect compared to the original inhibitory peptide LYPVK.

[0053] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. An angiotensin converting enzyme inhibitory peptide, characterized in that: The amino acid sequence of the angiotensin converting enzyme inhibitory peptide is PVKP.

2. Use of the angiotensin converting enzyme inhibitor peptide according to claim 1 in the preparation of angiotensin converting enzyme inhibitors.

3. Use of the angiotensin converting enzyme inhibitory peptide according to claim 1 in the preparation of antihypertensive drugs.

4. An angiotensin converting enzyme inhibitor, characterized in that: The invention comprises the angiotensin converting enzyme inhibitory peptide according to claim 1 as an active ingredient.

5. A pharmaceutical composition for lowering blood pressure, characterized in that: The invention comprises the angiotensin converting enzyme inhibitory peptide according to claim 1.

6. A blood pressure lowering pharmaceutical preparation, characterized in that: A pharmaceutical composition comprising the angiotensin converting enzyme inhibitory peptide according to claim 1 or the blood pressure lowering pharmaceutical composition according to claim 5, and also comprising a pharmaceutically acceptable carrier.

7. A blood pressure lowering pharmaceutical preparation according to claim 6, characterized in that: The pharmaceutically acceptable carrier is suitable for tablets, capsules, powders, pills, granules, oral liquids, injections or emulsions.

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