An angiotensin-converting enzyme inhibitory peptide and its application

Abstract

This invention discloses an angiotensin-converting enzyme (ACE) inhibitory peptide and its applications. The amino acid sequence of the ACE inhibitory peptide is PVKP. The PVKP inhibitory peptide provided by this invention exhibits significantly enhanced ACE inhibitory activity compared to the original inhibitory peptide LYPVK, with an IC50 value of [missing information]. 50 The concentration decreased from 10.6 μg / mL to 9.6 μg / mL; and the digestive enzyme stability of the ACE inhibitor PVKP provided by this invention at the same concentration is improved compared to the original inhibitor LYPVK. Molecular simulation docking verified that the interaction between the ACE inhibitor PVKP and ACE is enhanced compared to the original inhibitor LYPVK, increasing the interaction between alkyl groups and the Zn group at the ACE active site. 2+ Interactions. Further cell experiments verified the intracellular antihypertensive activity of the ACE inhibitory peptide PVKP described in this invention. Compared to the original inhibitory peptide LYPVK, it showed increased intracellular NO release and decreased ET-1 content. The ACE inhibitory peptide PVKP described in this invention has a small molecular weight and good water solubility, which is beneficial for its application in the development of products with antihypertensive effects.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and in particular to an angiotensin-converting enzyme inhibitory peptide and its applications. Background Technology

[0002] Hypertension and its related cardiovascular diseases are among the most common chronic diseases. According to publicly released data from the World Health Organization, the number of deaths worldwide due to hypertension and related cardiovascular diseases has increased in the past 30 years, posing a significant threat to human health. Angiotensin-converting enzyme (ACE) is a key enzyme in the human blood pressure regulation system, participating in blood pressure regulation and affecting the levels of key substances in blood pressure-related pathways, thereby influencing blood pressure. For example, ACE can promote the production of angiotensin II in the RAS system and degrade bradykinin in the KKS system, reducing its levels and leading to elevated blood pressure. Therefore, inhibiting ACE activity is an important target in current strategies for lowering and preventing blood pressure. Antihypertensive drugs such as captopril and enalapril target ACE, inhibiting its activity to lower blood pressure. These drugs are currently the main treatment method for ACE inhibitors, but long-term use can cause certain side effects. Developing safe and environmentally friendly ACE inhibitors is a current focus.

[0003] ACE inhibitory peptides are bioactive peptides widely derived from foods such as milk, soybeans, oysters, and fish. They target the ACE pathway and possess significant antihypertensive effects, making them a primary focus of ACE inhibitory drug development in recent years. Although various ACE inhibitory peptides have been classified and identified from a wide range of food ingredients, their application is still limited by issues such as weak ACE inhibitory activity, limited stability studies, and unclear mechanisms of action for lowering blood pressure. Developing ACE inhibitory peptides with strong ACE inhibitory activity, high stability, and significant antihypertensive effects is crucial for their application in hypertension prevention and treatment. LYPVK is an ACE inhibitory peptide derived from figs. It has been enzymatically digested, isolated, and identified by researchers, but stability analyses have not been performed, and its inhibitory activity is weak. The resulting product has a complex composition, making it difficult to explain the peptide's antihypertensive effect. Therefore, improving the inhibitory activity, studying its stability, exploring its antihypertensive effects, and analyzing its mechanisms are of great significance for the development of LYPVK as a food-derived ACE inhibitory peptide. Summary of the Invention

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

[0005] Specifically, this invention, based on the original amino acid sequence of the ACE inhibitory peptide LYPVK, rationally designs the original amino acid sequence by considering factors such as molecular weight, hydrophobic residues, amino acid sequence distribution, arrangement, and the types of terminal amino acid residues that affect the inhibitory activity of the ACE peptide. This results in the amino acid sequence of the ACE inhibitory peptide with enhanced ACE inhibitory activity. Furthermore, the two ACE inhibitory peptide amino acid sequences are artificially synthesized to further verify their ACE inhibitory activity, inhibitory characteristics, and gastrointestinal stability. Molecular simulation docking is used to verify the mechanism of enhanced ACE inhibitory activity. The intracellular blood pressure-lowering effects of the two ACE inhibitory peptides are verified and compared, and the changes in intracellular blood pressure-related product levels after the action of the two ACE inhibitory peptides are analyzed to explore the intracellular blood pressure-lowering efficacy.

[0006] The larger the molecular weight of the ACE inhibitory peptide, the less likely it is to enter the active site of ACE, resulting in lower ACE inhibitory activity. Furthermore, based on the amino acid composition of the ACE active site, the hydrophobic amino acids, lysine, and proline at the terminal end of the ACE inhibitory peptide readily interact with ACE, thus further enhancing its inhibitory activity. Therefore, rationally designing ACE inhibitory peptides using the above approach to develop new ACE inhibitory peptides is beneficial for improving their ACE inhibitory activity.

[0007] To achieve the above objectives, 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] Such as the application of the angiotensin-converting enzyme inhibitory peptide mentioned above in the preparation of angiotensin-converting enzyme inhibitors.

[0009] Such as the application of the angiotensin-converting enzyme inhibitory peptide mentioned above in the preparation of antihypertensive drugs.

[0010] An angiotensin-converting enzyme inhibitor comprising the aforementioned angiotensin-converting enzyme inhibitory peptide as an active ingredient.

[0011] A pharmaceutical composition for lowering blood pressure includes the aforementioned angiotensin-converting enzyme inhibitory peptide.

[0012] A blood pressure-lowering pharmaceutical preparation comprising the aforementioned angiotensin-converting enzyme inhibitory peptide or the aforementioned blood pressure-lowering pharmaceutical composition, and further comprising a pharmaceutically acceptable carrier.

[0013] Furthermore, in this 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 this invention exhibits improved ACE inhibitory activity compared to the original inhibitory peptide LYPVK, with an IC50 value of [missing value]. 50 The concentration decreased 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 this invention was stronger than that of the original inhibitory peptide LYPVK. Molecular simulation docking verified that the interaction force between the ACE inhibitory peptide YPLKP and ACE was enhanced compared to the original inhibitory peptide LYPVK, as were the interactions between alkyl groups and with Zn. 2+ Increased potency. Further cell experiments verified that the intracellular antihypertensive efficacy of the ACE inhibitory peptide PVKP described in this invention was enhanced by an increase in the original inhibitory peptide LYPVK, with increased NO content and decreased ET-1 content. The ACE inhibitory peptide PVKP described in this invention has a small molecular weight and good water solubility, which is beneficial for its application in the development of products with antihypertensive effects.

[0016] It should be understood that all combinations of the foregoing concepts and the additional concepts described in more detail below can be considered part of the inventive subject matter of this disclosure, provided that such concepts do not contradict each other.

[0017] The foregoing and other aspects, embodiments, and features of the teachings of the present invention will be more fully understood from the following description in conjunction with the accompanying drawings. Other additional aspects of the invention, such as features and / or beneficial effects of exemplary embodiments, will become apparent from the following description or may be learned through practice of specific embodiments according to the teachings of the present invention. Attached Figure Description

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

[0019] Figure 1 A comparison of the amino acids of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK;

[0020] Figure 2 The IC50 of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK in this invention for ACE inhibition 50 value;

[0021] Figure 3 To assess the digestive enzyme stability of the ACE inhibitory peptide PVKP of this invention and the original ACE inhibitory peptide LYPVK;

[0022] Figure 4This is a schematic diagram of the molecular simulated docking interaction between the ACE inhibitory peptide PVKP and ACE in this invention.

[0023] Figure 5 A schematic diagram of the molecular docking interaction 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 the original ACE inhibitory peptide LYPVK;

[0026] Figure 8 The intracellular ET-1 content of ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK. Detailed Implementation

[0027] To better understand the technical content of this invention, specific embodiments are described below in conjunction with the accompanying drawings. Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, which illustrate numerous illustrative embodiments. The embodiments of this disclosure are not necessarily defined to include all aspects of the invention. It should be understood that the various concepts and embodiments described 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 this invention are not limited to any particular implementation. Furthermore, some aspects of this invention can be used alone or in any suitable combination with other aspects of this invention.

[0028] Example 1

[0029] Rational design of ACE inhibitory peptides

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

[0031] Example 2

[0032] ACE inhibitory activity assay

[0033] The ACE inhibitory activities of the ACE inhibitory peptide PVKP and the original ACE inhibitory peptide LYPVK were determined using a 96-well plate with visible spectrophotometry. 1 mM N-[3-(2-furanyl)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 the original ACE inhibitory peptide LYPVK (2.5, 5, 10, 20 μg / mL) were added sequentially to 96-well plates, along with appropriate amounts of FAPPG substrate, ACE, and HEPES buffer. The absorbance of the mixture at 340 nm was measured using a microplate reader. The mixture was then incubated at 37°C with shaking for 30 min. The absorbance at 340 nm was measured again 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 absorbance of sample / change in absorbance of blank. The IC50 of the ACE inhibitory peptide... 50 The value represents the concentration of ACE-inhibiting peptides when the inhibition rate of ACE is 50%.

[0034] The results are as follows Figure 2 As shown, the ACE-inhibiting peptide PVKP has an IC50 inhibitory effect on ACE. 50 The value was 9.6 μg / mL, while the IC50 of the original ACE-inhibiting peptide LYPVK for ACE inhibition was... 50 The value was 10.6 μg / mL, indicating that the ACE inhibitory activity of the ACE inhibitory peptide PVKP was enhanced.

[0035] Example 3

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

[0037] The physicochemical properties of the ACE-inhibiting peptide YPLKP were analyzed using software or online websites. The isoelectric point was analyzed using the online website (https: / / www.innovagen.com / proteomics-tools); toxicity was analyzed using the ToxinPred website (https: / / crdd.osdd.net / raghava / toxinpred / ); and 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 it was not toxic at normal concentrations.

[0039] Example 4

[0040] Stability of ACE inhibitory peptides to gastrointestinal digestive enzymes

[0041] The stability of ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK was determined by gastrointestinal digestive enzymes. The pH of the system was adjusted to 2 with 1M HCl solution, and pepsin was added at a concentration of 2% to prepare a simulated gastric digestive fluid. The ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK (concentration 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 1M NaOH to inactivate the enzymatic hydrolysate, yielding a gastric hydrolysate for trypsin treatment. After trypsin treatment, the sample was inactivated at 95°C for 10 min, cooled to 37°C, and the in vitro ACE inhibition rate was measured.

[0042] The results are as follows Figure 3 As shown, after treatment with the above-mentioned gastrointestinal digestive enzymes at the same concentration, the ACE inhibitory peptide PVKP showed an ACE inhibition rate of 52.3%, still exhibiting good ACE inhibitory activity. The original ACE inhibitory peptide LYPVK, at the same concentration, showed an ACE inhibition rate of 43.5% after treatment with the same gastrointestinal digestive enzymes. This indicates that the ACE inhibitory peptide PVKP described in this invention has gastrointestinal digestive enzyme tolerance and exhibits improved gastrointestinal stability compared to the original ACE inhibitory peptide LYPVK.

[0043] Example 5

[0044] Molecular simulation docking analysis

[0045] The ACE repressor peptide PVKP and the original ACE repressor peptide LYPVK were subjected to molecular simulation docking with ACE to elucidate the mechanism of ACE repressor peptide LYPVK activity enhancement from the perspective of amino acid residue interaction forces. Three-dimensional conformational diagrams of ACE repressor peptide PVKP and the original ACE repressor peptide LYPVK were constructed using Discovery Studio software, and molecular simulation docking was performed with the catalytic active site of ACE (PDB: 1o8a). Based on the docking results, the interaction forces between ACE repressor peptide PVKP and the original ACE repressor peptide LYPVK and key amino acid residues of the ACE active site were analyzed.

[0046] The results are as follows Figure 4As shown, the ACE inhibitory peptide PVKP can dock with the ACE active site, and simulated docking can be performed within the normal analysis time. Furthermore, PVKP interacts with various amino acid residues in the ACE active site, with the main interactions including hydrogen bonds, salt bridges, mutual attraction, metal ion acceptors, alkyl group interactions, and π-alkyl group interactions. Compared with the interaction analysis results of the original ACE inhibitory peptide LYPVK with the amino acid residues of the ACE active site, as shown... Figure 4 and Figure 5 Compared with the original ACE inhibitory peptide LYPVK, the ACE inhibitory peptide PVKP described in this invention has increased interaction forces between the key amino acid residues alkyl groups in the ACE active site and has added metal ion acceptors, indicating that the inhibitory peptide PVKP described in this invention is more likely to bind to the ACE active site, thereby enhancing ACE inhibitory activity.

[0047] Example 6

[0048] Intracellular antihypertensive effect study

[0049] Using human umbilical vein endothelial cells EA.hy926 as the research object, this study investigated the intracellular hypotensive effects of PVKP and the original ACE inhibitory peptide LYPVK described in this invention. First, the toxicity of the two inhibitory peptide samples to the cells was determined. Specifically, cells were diluted to prepare a cell suspension. The cells to be used were washed with PBS and digested with trypsin from the culture flask, then resuspended in complete culture medium to prepare a cell suspension, and counted using a hemocytometer. The cells were diluted to an appropriate concentration with complete culture medium, filled into 96-well plates, and incubated in an incubator for approximately 24 hours until confluence. The supernatant culture medium from each well was aspirated with a pipette, and 100 μL of PBS solution was added to each well for washing, followed by aspiration. The samples were diluted with FBS-free culture medium (i.e., DMEM-H + 1% P / S). The samples were weighed in advance, diluted to different concentrations in a clean bench, and sterilized by membrane filtration. 100 μL of the diluted sample was added to each well, and the plates were incubated in an incubator for approximately 24 hours. The supernatant culture medium was aspirated from each well using a pipette. Each well was washed with 100 μL of PBS solution, which was then aspirated. CCK was prepared using FBS-free medium (DMEM-H + 1% P / S) at a ratio of 90 μL medium + 10 μL CCK. 100 μL of CCK-containing medium was added to each well, and the wells were incubated. The absorbance at 450 nm was measured using a microplate reader. The cell viability after treatment with the inhibitory peptide sample was calculated using the cell viability formula.

[0050] The results are as follows Figure 6As shown, neither the ACE-inhibiting peptide PVKP nor the original inhibitory peptide LYPVK described in this invention is cytotoxic. At concentrations between 2.5 and 40 μg / mL, cell viability was greater than 90%, indicating that at concentrations where the two inhibitory peptides effectively exert their ACE-inhibiting effect, they are not toxic to endothelial cells.

[0051] Intracellular NO and ET-1 levels are important biomarkers related to blood pressure. Whether the ACE inhibitory peptide can cause changes in NO and ET-1 levels is an important indicator for evaluating the intracellular antihypertensive efficacy. This study investigated the antihypertensive efficacy of human umbilical vein endothelial cells EA.hy926 after treatment with the ACE inhibitory peptide PVKP and the original inhibitory peptide LYPVK described in this invention by measuring the NO and ET-1 levels released. Captopril was used as a positive control. Results are as follows... Figure 7 As shown, after treatment with the two inhibitory peptides, both peptides increased the intracellular NO release compared to the negative control. At the same concentration of the two inhibitory peptides, the NO release from cells treated with the ACE inhibitory peptide PVKP described in this invention was higher than that of the original inhibitory peptide LYPVK, indicating that the ACE inhibitory peptide PVKP described in this invention has a stronger cellular blood pressure-lowering effect. Intracellular ET-1 content results are as follows... Figure 8 As shown, compared to the negative control, both peptides reduced the amount of NO released from cells. Under the same concentration of the two inhibitory peptides, the ET-1 content in cells treated with the ACE inhibitory peptide PVKP of this invention was lower than that of the original inhibitory peptide LYPVK, indicating that the ACE inhibitory peptide PVKP of this invention has a stronger cellular blood pressure-lowering effect.

[0052] In summary, based on the results of cell experiments, through rational design, the ACE inhibitory peptide PVKP described in this invention significantly enhances the intracellular blood pressure-lowering effect compared to the original inhibitory peptide LYPVK.

[0053] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. The application of an angiotensin-converting enzyme inhibitory peptide in the preparation of antihypertensive drugs, wherein the amino acid sequence of the angiotensin-converting enzyme inhibitory peptide is PVKP.

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