ACE inhibitory peptide with antihypertensive activity and application thereof

Abstract

The invention discloses an ACE (angiotensin converting enzyme) inhibitory peptide with antihypertensive activity and application thereof, and belongs to the technical field of active peptide biology, amino acid sequences of the ACE inhibitory peptide are EYPVK and SYPVK, the two peptides are obtained through module replacement design, the ACE inhibitory activity of the two peptides is remarkably improved compared with that of an original peptide LYPVK, IC50 values are respectively reduced to 14.82 + / -0.82 mu M and 12.83 + / -0.79 mu M from 117.14 + / -1.40 mu M, and the inhibitory activity is improved by 13.5% and 25.1%. Molecular simulation analysis shows that the interaction force between the two and ACE is stronger, and a potential modular effect exists between sequences. Besides, the polypeptide has remarkable blood pressure lowering activity on human umbilical vein endothelial cells, plays a role by regulating release of NO and ET-1, has the activity superior to that of original peptides, has the advantages of good digestive enzyme stability, small molecular weight and good water solubility, and is suitable for developing related products for lowering blood pressure.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and more particularly to an ACE inhibitory peptide with antihypertensive activity and its applications. Background Technology

[0002] Hypertension is a chronic cardiovascular syndrome characterized by persistently elevated systemic arterial blood pressure. According to data released by the World Health Organization (WHO) in 2024, the number of people with hypertension worldwide continues to rise, with approximately one-third of adults experiencing varying degrees of blood pressure abnormalities, and a significant trend towards affecting younger people. Hypertension and its related cardiovascular, cerebrovascular, and renal complications have become a major public health problem threatening human health and socio-economic development. Therefore, developing safe, effective, and suitable long-term intervention strategies for lowering blood pressure is a key focus in the cardiovascular and functional food industry.

[0003] Among the many neurohumoral factors involved in blood pressure homeostasis, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) together constitute an important regulatory network for maintaining vascular tone and fluid balance. Angiotensin-converting enzyme (ACE) is located at the key intersection of these two major blood pressure regulatory systems and plays a central role in the occurrence and progression of hypertension. ACE is widely distributed in the lungs, kidneys, vascular endothelium, and various tissues. Its upregulation and excessive activation of RAS are considered to be one of the typical characteristics of primary hypertension and some secondary hypertension. Based on the key role of ACE in the pathogenesis of hypertension, ACE inhibitors are widely used in clinical antihypertensive treatment. Currently marketed representative small-molecule ACE inhibitors effectively block the generation of AngII and slow down the degradation of bradykinin by binding to zinc ions or key amino acid residues in the active site of ACE, resulting in a significant and stable antihypertensive effect. However, long-term use of these drugs also has significant adverse reactions, such as irritating dry cough, hyperkalemia, hypoglycemia, angioedema, and increased liver and kidney burden in some patients.

[0004] In contrast, ACE inhibitory peptides derived from natural food protein raw materials are attracting widespread attention as a safe and highly effective functional component. Currently, ACE inhibitory peptides are mainly prepared by enzymatic hydrolysis of protein raw materials such as soybeans, oysters, and milk, followed by separation, purification, and characterization. Although a large number of food-derived ACE inhibitory peptides have been isolated and identified, their inhibitory activity is weak, and their blood pressure-lowering effect is limited, restricting their practical application. Against this background, developing ACE inhibitory peptides with strong ACE inhibitory activity is a problem that needs to be solved to apply ACE inhibitory peptides to the prevention and treatment of hypertension. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an ACE inhibitory peptide with antihypertensive activity and its application, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an ACE inhibitory peptide with antihypertensive activity, wherein the amino acid sequence of the ACE inhibitory peptide is X1YPVK, wherein X1 is selected from E or S.

[0007] Preferably, X1 is selected from S.

[0008] In this invention, the ACE inhibitory peptide is derived from the original ACE inhibitory peptide LYPVK using a module substitution strategy. By analyzing the dipeptide modules in the original LYPVK sequence that contribute less to activity, and replacing them with the identified dipeptide modules that contribute more to activity, the amino acid sequence of the ACE inhibitory peptide with enhanced ACE inhibitory activity is obtained. The two ACE inhibitory peptide amino acid sequences are then artificially synthesized to further verify their ACE inhibitory activity and inhibitory characteristics. The mechanism of enhanced ACE inhibitory activity is verified through molecular docking simulation and kinetic simulation. Furthermore, its antihypertensive activity is demonstrated through its action on human venous endothelial cells.

[0009] In a second aspect, the present invention provides the use of any of the above-described ACE inhibitory peptides in the preparation of ACE inhibitors and / or antihypertensive drugs.

[0010] In a third aspect, the present invention provides the use of the ACE inhibitory peptide described above in the preparation of products that help maintain healthy blood pressure levels.

[0011] Furthermore, the product is a health supplement or functional food.

[0012] In a fourth aspect, the present invention provides an angiotensin-converting enzyme inhibitor, comprising the ACE inhibitory peptide described above.

[0013] In a fifth aspect, the present invention provides an antihypertensive drug, the active ingredient of which includes the ACE inhibitory peptide described above.

[0014] Furthermore, the blood pressure-lowering drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the drug dosage form includes tablets, capsules, powders, pills, granules, oral liquids, injections, or emulsions.

[0016] In the sequence structure of ACE inhibitory peptides, their functional activity is highly dependent on the composition, sequence, and regional distribution of key amino acids. In particular, the dipeptide fragments, hydrophobic residues, and overall net charge of the sequence have a decisive influence on their binding ability to the ACE active site. In an ACE inhibitory peptide sequence, a combination of two or more amino acids that are structurally or functionally independent but synergistic can be considered a functional module. These modules exhibit strong intrinsic synergy in spatial configuration, physicochemical properties, and functional effects. Different modules can be combined to construct diverse functional peptide sequences, thus exhibiting varying degrees of functional activity. Therefore, adopting the above approach, based on a module replacement strategy, by analyzing the dipeptide modules in the original ACE inhibitory peptide LYPVK sequence that contribute less to activity, and replacing them with the analyzed dipeptide modules that contribute more to activity, a rationally designed novel ACE inhibitory peptide amino acid sequence was obtained, and its ACE inhibitory activity was effectively enhanced.

[0017] The beneficial effects of this invention include at least the following: The ACE inhibitory peptides EYPVK and SYPVK provided by this invention are obtained through a module substitution design strategy, and their inhibitory activity against ACE is significantly improved compared to the original inhibitory peptide LYPVK, with an IC50 value of [missing information]. 50 The values ​​decreased from 117.14 ± 1.40 μM to 14.82 ± 0.82 μM and 12.83 ± 0.79 μM, respectively, representing increases in inhibitory activity of 13.5% and 25.1%, respectively. Further molecular simulation docking and kinetic simulation analyses showed that the interaction forces between the ACE inhibitory peptides EYPVK and SYPVK and ACE were enhanced compared to the original inhibitory peptide LYPVK, and the interaction force analysis indicated a potential modular effect among the ACE inhibitory peptide sequences. Furthermore, the ACE inhibitory peptides EYPVK and SYPVK provided by this invention also exhibited significant antihypertensive activity against human umbilical vein endothelial cells, exerting their antihypertensive effect by regulating the release of NO and ET-1 in cells, and their activity was enhanced compared to the original inhibitory peptide LYPVK, consistent with in vitro activity results. The ACE inhibitory peptides EYPVK and SYPVK described in this invention also possess good digestive enzyme stability, small molecular weight, and good water solubility, which is beneficial for their application in the development of products with antihypertensive effects. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the contribution of the dipeptide module to the original ACE-inhibiting peptide LYPVK. (A) Predicted docking energy of the dipeptide module to ACE. (B) IC50 value of the dipeptide module for ACE inhibition. 50 Value determination.

[0019] Figure 2Schematic diagram of the contribution analysis of the “XY” dipeptide module: (A) Predicted docking energy values ​​of the entire combination of dipeptide modules to ACE; (B) IC50 of the “XY” sequence dipeptide module to ACE inhibition. 50 Value determination.

[0020] Figure 3 This is a comparison of the amino acids of the ACE inhibitory peptides EYPVK and SYPVK with the original ACE inhibitory peptide LYPVK.

[0021] Figure 4 The IC50 of the ACE inhibitory peptides EYPVK and SYPVK of this invention with the original ACE inhibitory peptide LYPVK for ACE inhibition is shown to be... 50 value.

[0022] Figure 5 The inhibition modes of the ACE-inhibiting peptides EYPVK and SYPVK of the present invention are shown in Figure 1. (A) ACE inhibition mode of EYPVK, (B) ACE inhibition mode of SYPVK.

[0023] Figure 6 For the digestive enzyme stability of the ACE inhibitory peptides EYPVK and SYPVK of the present invention, (A) the pepsin digestion stability of EYPVK and SYPVK, and (B) the trypsin digestion stability of EYPVK and SYPVK.

[0024] Figure 7 The diagram shows the molecular simulation docking of the ACE inhibitory peptides EYPVK and SYPVK of the present invention with the original ACE inhibitory peptide LYPVK and ACE. (A) Two-dimensional diagram of the interaction forces, (B) Diagram of the interacting residues and sites, and (C) Diagram of the interaction forces and their strength.

[0025] Figure 8 This is a schematic diagram of the kinetic simulation analysis of the ACE inhibitory peptides EYPVK and SYPVK of the present invention with the original ACE inhibitory peptide LYPVK and ACE. (A) RMSD, (B) RMSF, (C) Number of interacting hydrogen bonds, (D) SASA.

[0026] Figure 9 The diagram illustrates the hypotensive effects of the ACE inhibitory peptides EYPVK and SYPVK, and the original ACE inhibitory peptide LYPVK, on ​​human umbilical vein endothelial cells. (A) Cell activity of EYPVK, (B) Cell activity of SYPVK, (C) NO release content of EYPVK, (D) NO release content of SYPVK, (E) ET-1 secretion content of EYPVK, and (F) ET-1 secretion content of SYPVK. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Contribution ranking of dipeptide modules in the LYPVK sequence and verification of ACE inhibitory activity. LYPVK (a naturally derived ACE inhibitory peptide) was divided into different consecutive dipeptide modules, and their contributions to ACE binding and inhibitory activity were evaluated. Specifically, the amino acid sequence of LYPVK was modularized into four dipeptide modules: LY, YP, PV, and VK. The three-dimensional structures of each of the four dipeptide modules were then constructed, and molecular docking analysis was performed. By comparing the docking energy, conformational rationality, and interactions with key residues in the active site of different dipeptide modules, the potential contributions of each dipeptide module in the LYPVK sequence to overall ACE binding and inhibition were ranked, providing a theoretical basis for subsequent substitution design based on "high-contribution modules" and "low-contribution modules." Based on the molecular docking analysis, the in vitro ACE inhibitory activity of the four dipeptides LY, YP, PV, and VK was further determined. This yielded the inhibitory activity strength and contribution of each dipeptide module in the LYPVK sequence.

[0030] IC50 of ACE peptides 50The specific method for determining the ACE inhibitory activity of the ACE inhibitory peptide was as follows: ACE inhibitory activity was measured using a visible spectrophotometer in a 96-well plate. 1 mM N-[3-(2-furanyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) was used as the reaction substrate, dissolved in HEPES buffer (1.901 g HEPES reagent, 1.755 g NaCl reagent, pH 8.3), and the angiotensin-converting enzyme (ACE) concentration was set at 0.1 U / mL. During the assay, different concentrations of the sample, along with appropriate amounts of FAPPG substrate, ACE, and HEPES buffer, were added sequentially to the 96-well plate. The absorbance of the mixture at 340 nm was measured using a microplate reader. The system was then incubated at 37°C with shaking for 30 min, and 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 sample absorbance) / change in blank absorbance. 50 The value represents the concentration of ACE-inhibiting peptides when the inhibition rate of ACE is 50%.

[0031] Based on the results obtained from the CDOCKER molecular docking model, the docking energies of LY, YP, PV, and VK with ACE are -58.286, -72.736, -63.269, and -65.168 kcal / mol, respectively. Figure 1 A). It is generally believed that the lower the docking energy, the more stable the complex formed between the ligand and the target, and the stronger the binding affinity. Meanwhile, to functionally verify the above molecular docking predictions, the in vitro ACE inhibitory activity of four dipeptide modules was further measured, and their IC50 values ​​were calculated. 50 Values. The results show that the IC values ​​of LY, YP, PV, and VK are... 50 The concentrations were 38.50 ± 2.53, 16.24 ± 0.98, 32.15 ± 1.89, and 23.68 ± 2.06 μmol / L, respectively. Figure 1 B). From IC 50 In terms of size, YP exhibits the strongest inhibitory effect on ACE, followed by VK and PV, while LY shows the weakest inhibitory effect. Therefore, the binding affinity of the four dipeptide modules to ACE, from strongest to weakest, is YP, PV, VK, and LY.

[0032] Example 2: Screening of the contribution of the whole combination dipeptide modules and verification of ACE inhibitory activity Based on twenty standard amino acids, 400 theoretical dipeptide sequences were constructed. The contribution of each combination of dipeptide modules was ranked and verified using a combination of molecular docking and in vitro inhibitory activity assays. First, a virtual screening of the interactions between the 400 dipeptide molecules and ACE was performed. Based on docking energy and the abundance of interactions with key residues in the active site, the potential contributions of the 400 dipeptides were initially ranked. Based on low-contribution modules in LYPVK, and based on molecular docking predictions, XY-type dipeptide modules were selected as key targets for further in vitro ACE inhibitory activity verification. The IC50 was determined based on the calculated ACE inhibition rate of each dipeptide. 50 The value is used to characterize the actual ACE inhibitory activity of each combination of dipeptide modules.

[0033] Molecular docking results showed that the docking energies of the 400 dipeptides with ACE were distributed over a wide range, indicating that different amino acid combinations had significant differences in their interaction ability with ACE. Figure 2 A). Based on this, and combined with the aforementioned analysis results of the LYPVK internal modules, considering that the YP module showed the highest contribution while the LY module was a low-contribution module, a series of dipeptides with Tyr fixed at the C-terminus were screened and validated using "XY" as the template sequence. This approach preserved the beneficial role of Tyr as an aromatic hydrophobic residue for the ACE active site, while also comparing the effects of different amino acids at this position on ACE inhibitory activity by systematically replacing the N-terminal residue X. In in vitro activity validation, the inhibitory effect of different "XY" dipeptide modules on ACE was measured using FAPPGG as a substrate. With increasing dipeptide concentration, the ACE inhibition rate of each module gradually increased. The results showed that among the measured "XY" dipeptides, EY, SY, KY, and VY exhibited the strongest ACE inhibitory ability, with IC50 values ​​of [missing information]. 50 The concentrations were 2.68 ± 0.05, 11.70 ± 1.75, 7.70 ± 0.86, and 16.12 ± 1.04 μmol / L, respectively. Figure 2 Therefore, in the “XY” dipeptide, EY and SY can be identified as the dipeptide modules that contribute the most to ACE inhibitory activity.

[0034] Example 3: Rational Design of ACE Repressor Peptides Based on Module Replacement Strategy In the LYPVK sequence, LY is a dipeptide module with relatively low contribution, while EY and SY exhibit superior ACE inhibitory activity and more favorable binding characteristics in the overall "XY" dipeptide module. Based on this, using the N-terminal dipeptide module of LYPVK as the main modification site, LY in the sequence was replaced with the high-contribution modules EY and SY, respectively, to design two novel ACE inhibitory peptides, EYPVK and SYPVK (Figure 3). This rational design strategy not only preserves the core sequence framework of LYPVK related to ACE binding, avoiding excessive perturbation to the peptide chain length and overall conformation, but also, by introducing charged residues or residues more conducive to hydrogen bond formation at the N-terminus, it is expected to enhance the interaction strength between the peptide and the ACE active pocket and its surrounding key residues, thereby improving ACE inhibitory activity.

[0035] Example 4: Assay of ACE Inhibitory Activity In the LYPVK sequence, LY is a low-contribution dipeptide module, while EY and SY, obtained through full combinatorial screening, exhibit higher ACE inhibitory potential. Based on this, LY in LYPVK was replaced with EY and SY, respectively, constructing two new sequences, EYPVK and SYPVK, and their ACE inhibitory activities were verified. The results showed that the activities of both module-replaced peptides were improved compared to the parent peptides, with SYPVK exhibiting the strongest inhibitory activity (IC50, 100%). 50 The IC50 of EYPVK was 12.83 ± 0.79 μmol / L. 50 The concentration was 14.82 ± 0.82 μmol / L. Replacing LY with SY and EY increased the ACE inhibitory activity by approximately 25.1% and 13.5%, respectively (Figure 4), indicating that the two novel peptide sequences obtained based on the module replacement strategy have stronger ACE inhibitory activity levels compared to the parent peptide LYPVK.

[0036] Example 5: ACE Inhibition Pattern Analysis Lineweaver–Burk plotting results show that as the concentration of EYPVK increases, the reaction K... m It remained basically stable, while V max The decline is consistent with the non-competitive inhibition pattern. Figure 5A) indicates that, like LYPVK, EYPVK primarily achieves inhibition by simultaneously binding to free ACE and the ACE-substrate complex, thereby reducing the enzyme's catalytic activity. This also explains that while replacing LY with the high-contribution module EY can enhance ACE inhibitory activity, it does not alter the overall inhibitory mechanism, maintaining a non-competitive level. In contrast, the enzyme kinetics of SYPVK are more complex. With increasing SYPVK concentration, the Kt of the reaction... m With V max The values ​​all showed a decreasing trend (Figure 5B), a result consistent with the characteristics of a mixed inhibition mode, where the inhibitor can bind to both the enzyme's active site and its allosteric site, and exhibits different affinities for the enzyme-substrate complex and the free enzyme. This suggests that SYPVK may simultaneously interfere with the regulation of substrate entry into the catalytic center and the conformational state of ACE, thereby weakening the catalytic activity of ACE at multiple levels. This characteristic reflects, to some extent, that the module substitution strategy not only enhances the activity of the ACE inhibitory peptide but also alters its inhibition mode, evolving it from a single binding mode into a more diverse and complex regulatory mode.

[0037] Example 6: Gastrointestinal Digestive Tolerance Analysis In simulated gastric digestion, the ACE inhibition rates of both the parent peptide and the designed peptide remained largely unchanged after 120 min of pepsin treatment, showing no significant decrease, indicating good tolerance to the gastric environment. The ACE inhibition rate of the parent peptide LYPVK decreased only slightly from 50.59 ± 1.64% to 48.50 ± 3.68%, a small change, suggesting that its structure remained relatively stable under acidic conditions and pepsin activity. Similarly, EYPVK and SYPVK, obtained based on a module replacement strategy, showed a similar trend, with ACE inhibition rates of 48.02 ± 0.94% and 47.42 ± 1.26% respectively after simulated gastric digestion, similar to the changes observed in the parent peptide LYPVK. Figure 6 A). In the subsequent simulated intestinal digestion, after 180 min of combined action of trypsin and chymotrypsin, the ACE inhibition rate showed a slight decrease overall, but remained at a high level, indicating good intestinal digestion tolerance. The ACE inhibition rate of LYPVK further decreased slightly from 48.50 ± 3.68% at the end of gastric digestion to 47.20 ± 1.74%, indicating that it maintained relatively stable activity throughout the simulated gastrointestinal digestion process. In contrast, the ACE inhibition rates of EYPVK and SYPVK after 180 min of intestinal digestion were 47.64 ± 0.96% and 46.54 ± 1.90%, respectively, which were similar to those of the parent peptide ( Figure 6 B). Overall, the decrease in ACE inhibition rate after simulated intestinal digestion was relatively small for all peptides, indicating that although the sequence modification may have altered some cleavage sites, it did not significantly affect their stability.

[0038] Example 7 Molecular Simulation Docking and Interaction Force Analysis Molecular docking results showed that both EYPVK and SYPVK, in their lowest energy conformations, could form a wide variety of non-covalent interactions with the ACE active site, including electrostatic interactions, conventional hydrogen and C-H bonds, π-cation and π-anion interactions, π-π T-shaped stacking, alkyl-π-alkyl interactions, and metal-acceptor interactions (Figure 7A). Compared with LYPVK, EYPVK and SYPVK exhibited more diverse interaction types and a significantly increased total number of interactions, suggesting a tighter and more stable binding to ACE. Docking analysis indicated that EYPVK and SYPVK formed more hydrogen bonds and hydrophobic interactions with ACE than LYPVK. This enhanced force network is likely an important structural basis for their improved ACE inhibitory activity. After module replacement, SYPVK could interact with 18 residues, EYPVK with 17 residues, while the parent peptide LYPVK interacted with only about 15 residues (Figure 7A). The enlarged interface means that the peptides after module replacement have better spatial complementarity with the ACE active pocket, which is conducive to the formation of a more stable enzyme-peptide complex.

[0039] Using dipeptide modules as units, the Leu residue in the LY module is a typical aliphatic hydrophobic residue. While its side chain helps provide a hydrophobic interface, its polar interactions are relatively limited. When Leu in the LY module is replaced by Glu or Ser, the physicochemical properties of the module's side chain change significantly. The Glu residue in EYPVK is a negatively charged polar residue, and its carboxyl side chain can participate in salt bridging, electrostatic attraction, C-H bonds, and various hydrogen bonds. Figure 7(B) Compared to Leu in the original LY module, the EY module shows a significant increase in the effective interaction sites formed by Glu and ACE residues, thus significantly improving local binding capacity and enhancing the peptide's ability to occupy multiple active pockets simultaneously, which is beneficial for improving overall inhibitory activity. The Ser in SYPVK introduces a polar side chain with hydroxyl groups, which also possesses the potential to form hydrogen bonds and some electrostatic interactions. Docking results show that Ser can form specific interactions with residues such as Glu162 and Glu376, which to some extent helps stabilize the peptide's conformation in the catalytic trench (Figure 7C). Although, from the perspective of single-residue comparison, the number of specific interactions formed by Ser is less than all the interactions involved by Leu in LYPVK, the overall binding interface and interaction network of SYPVK are actually enhanced after module replacement, indicating that its activity enhancement is not merely due to the simple superposition of single-residue interactions, but is closely related to the synergistic effect between residues within the module.

[0040] Example 8: Dynamic Simulation Analysis Molecular dynamics simulations are an important computational tool for predicting molecular structure and dynamic behavior. They can be used to assess the conformational stability of ligand-receptor complexes and analyze their binding process and key binding sites over time. Using RMSD, RMSF, hydrogen bond count, and SASA as indicators, the structural stability and dynamic characteristics of peptide-ACE complexes under different design strategies were compared and analyzed, further elucidating the molecular basis for their enhanced ACE inhibitory activity. The average RMSD values ​​of the SYPVK-ACE and EYPVK-ACE complexes obtained by module replacement were 0.215 nm and 0.197 nm, respectively, both slightly higher than the 0.196 nm of the parent peptide LYPVK-ACE complex (Figure 8A). This indicates that the peptides after module replacement can also induce more significant conformational adjustments in ACE. Furthermore, after binding to ACE, EYPVK and SYPVK can maintain the overall conformational stability of the complex and guide ACE to a more favorable conformational state in local regions, consistent with the trend of enhanced in vitro ACE inhibitory activity. The RMSF fluctuation range of the SYPVK–ACE and EYPVK–ACE complexes was generally lower than that of the LYPVK–ACE complex (Figure 8B), indicating that the introduction of the high-contribution module reduced the local flexibility of the complexes and made the structure more compact, which is conducive to the formation of a durable and stable binding interface. This supports the high inhibitory activity of the module-replaced peptides from a kinetic perspective. The average SASA values ​​of the SYPVK–ACE and EYPVK–ACE complexes were slightly higher than those of LYPVK–ACE (Figure 8C), indicating that these two peptides could enter the active cavity of ACE more deeply or fully after binding to ACE, forming a larger contact interface with the active site. The number of hydrogen bonds formed by the SYPVK–ACE and EYPVK–ACE complexes was generally higher than that of LYPVK–ACE throughout the simulation (Figure 8D), indicating that these two peptides anchored the ACE active site and its surrounding residues through more hydrogen bonds, thereby significantly improving the stability and binding affinity of the complexes. By combining multiple indicators such as RMSD, RMSF, SASA, and the number of hydrogen bonds, it can be seen that the binding affinity of EYPVK and SYPVK to ACE is significantly improved, that is, the docking energy is lower and the number of interaction sites is more, while the stability and durability of their complexes with ACE are also improved at the dynamic level.

[0041] Example 9: Evaluation of the antihypertensive activity of human umbilical vein endothelial cells In the vascular system, endothelial cells, located in the innermost layer of the vascular lumen, finely regulate vascular tone and maintain vascular homeostasis by synthesizing and secreting various regulatory factors, serving as a crucial hub for blood pressure homeostasis. Therefore, endothelial cells are frequently used to evaluate the cellular efficacy of antihypertensive peptides and to elucidate their intracellular mechanisms of action. The human umbilical vein endothelial fusion cell line EA.hy926 was selected, possessing typical phenotypic and functional characteristics of endothelial cells, and has been widely used in in vitro studies of endothelial function and cardiovascular protective effects.

[0042] First, the cytotoxicity and safety of ACE inhibitory peptides were evaluated. Using the CCK-8 assay, EA.hy926 cells were treated with different concentrations of the ACE inhibitory peptides EYPVK and SYPVK, with the untreated group (0 µg / mL) serving as a blank control, and captopril as a positive control. Results showed that under all treatment concentrations, cell viability remained above 90% in all ACE inhibitory peptide treatment groups, with no significant difference compared to the control group. p >0.05) Figure 9 (A, 9B) indicates that the ACE inhibitory peptides EYPVK and SYPVK have no significant cytotoxicity to endothelial cells within the experimental concentration range and have little effect on cell proliferation and survival.

[0043] Using EA.hy926 endothelial cells as a model, different concentrations of the ACE inhibitory peptides EYPVK and SYPVK were treated for 24 h, with captopril as a positive control. The intracellular hypotensive function of the peptides was evaluated by detecting the NO content in the cell supernatant. The results showed that compared with the blank control group, EYPVK and SYPVK significantly increased the NO production level of EA.hy926 cells, and the NO release was similar to that of the captopril treatment group, exhibiting a concentration-dependent characteristic. Figure 9 (C, 9D). This indicates that the ACE inhibitory peptides EYPVK and SYPVK also exhibit a strong NO-promoting effect at the endothelial cell level, demonstrating outstanding cellular hypotensive potential.

[0044] ACE inhibitory peptides EYPVK and SYPVK were added at different concentrations, with captopril as a positive control. Changes in ET-1 levels in cell culture supernatants were detected to evaluate their intracellular hypotensive effects. Results showed that, compared with the blank control group, treatment with captopril and ACE inhibitory peptides EYPVK and SYPVK significantly decreased the ET-1 levels in the culture supernatant of EA.hy926 cells. p <0.05 indicates that these peptides can effectively inhibit the production of ET-1 at the cellular level, and have certain potential for intracellular blood pressure reduction. Figure 9 (E, 9F). Meanwhile, the decrease in ET-1 levels induced by all samples showed a certain dose-dependent effect; the ET-1 levels in the high-concentration treatment group were generally lower than those in the low-concentration group, indicating that within a certain concentration range, the inhibitory effect of peptides on ET-1 production increases with increasing dose.

[0045] In summary, analysis of NO and ET-1 levels in EA.hy926 endothelial cells revealed that the ACE inhibitory peptides EYPVK and SYPVK exhibited significant antihypertensive activity and have potential for further application.

[0046] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0047] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An ACE inhibitory peptide with antihypertensive activity, characterized in that, The amino acid sequence of the ACE inhibitory peptide is X1YPVK, where X1 is selected from E or S.

2. The ACE-inhibiting peptide according to claim 1, characterized in that, X1 is selected from S.

3. The use of the ACE inhibitory peptide according to claim 1 or 2 in the preparation of ACE inhibitors and / or antihypertensive drugs.

4. The use of the ACE inhibitory peptide as described in claim 1 or 2 in the preparation of products that help maintain healthy blood pressure levels.

5. The application according to claim 4, characterized in that, The product is a health supplement or functional food.

6. An angiotensin-converting enzyme inhibitor, characterized in that, Includes the ACE inhibitory peptide as described in claim 1 or 2.

7. A blood pressure-lowering drug, characterized in that, Its active ingredient includes the ACE inhibitory peptide as described in claim 1 or 2.

8. The antihypertensive drug according to claim 7, characterized in that, The antihypertensive drugs also include pharmaceutically acceptable excipients.

9. The antihypertensive drug according to claim 7, characterized in that, The drug dosage forms include tablets, capsules, powders, pills, granules, oral liquids, injections, or emulsions.

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