ACE inhibitory peptide and application thereof

Abstract

The invention provides an ACE inhibitory peptide and application thereof, and belongs to the technical field of bioactive peptide preparation. According to the invention, computer simulation guidance is utilized, six polypeptides with ACE inhibitory activity are efficiently screened from royal jelly protein, and the amino acid sequence of the ACE inhibitory peptide is shown as any one of SEQ ID NO.1-6. The ACE inhibitory peptide provided by the invention is clear in structure, clear in source and remarkable in ACE inhibitory activity, and is a novel food-borne ACE inhibitory peptide. Furthermore, the ACE inhibitory peptide derived from royal jelly provided by the invention is safe and free from toxic and side effects, and can be developed as a health food, a food additive, a drug synergist and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically relating to an ACE inhibitory peptide and its application. Background Technology

[0002] Hypertension is one of the most common chronic diseases worldwide and a significant modifiable risk factor for cardiovascular disease, causing numerous premature deaths each year and posing a serious threat to public health. Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS), regulating blood pressure by catalyzing the conversion of angiotensin I to angiotensin II. Therefore, ACE has become an important pharmacological target for antihypertensive therapy.

[0003] Currently, commonly used synthetic ACE inhibitors such as captopril and enalapril, while showing significant efficacy, often come with adverse reactions such as cough, kidney damage, rash, and taste disturbances. Therefore, discovering food-derived ACE-inhibiting peptides from natural food proteins that offer greater safety and fewer side effects has become a cutting-edge research area in functional food and antihypertensive drug development.

[0004] ACE is a zinc-containing carboxydipeptidase whose catalytic mechanism relies on the formation of a coordination bond between the zinc ion at its active site and the substrate. The mechanisms of action of ACE inhibitory peptides are mainly classified into three types: competitive inhibition, non-competitive inhibition, and mixed inhibition. Most ACE inhibitory peptides act as competitive inhibitors, meaning that specific amino acid residues at their N-terminus or C-terminus (such as tryptophan, tyrosine, phenylalanine, and proline) form hydrogen bonds, hydrophobic interactions, or electrostatic interactions with amino acid residues in the ACE active site, thereby preventing the substrate from entering the catalytic site.

[0005] From a structural perspective, the activity of ACE inhibitory peptides is closely related to their amino acid sequence. It is generally believed that peptides containing aromatic amino acids (Phe, Tyr, Trp) or proline at the C-terminus are more likely to bind to the S1 and S2 subsites of ACE; while hydrophobic amino acids at the N-terminus help enhance the stability of peptide-enzyme binding. Furthermore, peptide chain length is also a key factor. Dipeptides and tripeptides often exhibit higher inhibitory activity because shorter peptides can more flexibly adapt to the spatial conformation of the ACE active pocket. For example, dipeptides and tripeptides derived from bovine β-lactoglobulin have been confirmed in multiple clinical studies to have a definite antihypertensive effect, the mechanism of which is to exert a major inhibitory effect by blocking the C-domain of ACE.

[0006] ACE inhibitory peptides have a wide range of sources, covering almost all edible proteins. Milk protein was one of the earliest sources to be systematically studied; casein and whey protein, after enzymatic hydrolysis, can release a variety of highly active ACE inhibitory peptides. Fish protein, especially the muscle protein of marine fish such as skipjack tuna and sardines, has become an important source of highly active ACE inhibitory peptides due to its unique amino acid composition. Regarding plant proteins, soy protein, zein, and rice bran protein can also produce peptides with significant hypotensive activity after enzymatic hydrolysis. In recent years, novel protein resources such as insect protein and algal protein have also gradually entered the research field, providing new directions for expanding the raw materials for ACE inhibitory peptides.

[0007] Royal jelly is secreted by the hypopharyngeal and mandibular glands of worker bees, and its protein content accounts for 30% to 50% of its dry matter. Among them, the main protein in royal jelly is the main material basis for its biological activity. However, royal jelly protein still has certain limitations in practical applications: it is easily degraded and inactivated under room temperature conditions, requiring cold chain storage and transportation throughout the process, resulting in high industrialization costs; at the same time, the large protein molecules have a certain degree of allergenicity, and some people may experience mild allergic reactions after ingestion; and royal jelly itself has a sour and astringent taste and poor palatability.

[0008] To overcome these limitations, enzymatic hydrolysis technology has emerged as a promising strategy to degrade royal jelly proteins into small peptides, which not only helps reduce sensitization and improve stability but may also enhance their biological activity. Previous studies have reported the identification of several angiotensin-converting enzyme (ACE) inhibitory peptides from gastrointestinal mimic digestion products of royal jelly proteins, such as FRYR (IC). 50 = 608 μmol / L) and FFRNR (IC 50 = 684 μmol / L). These results indicate that the peptides released by enzymatic decomposition of royal jelly protein have ACE-inhibiting function, suggesting that they can serve as high-quality precursor proteins for dietary ACE-inhibiting peptides.

[0009] Chinese invention patent application CN117462649A, published on January 30, 2024, discloses a royal jelly active peptide composition with ACE inhibitory effect, its preparation method and application. Specifically, it discloses that the amino acid sequence composition of the main active peptides in the obtained ACE inhibitory peptide composition is HNSDDSFHRL, SFHRL, KNYPF, DVNFR and VEIPH.

[0010] However, current research on ACE inhibitory peptides derived from royal jelly proteins is still relatively limited, and most studies rely on empirical protease screening strategies. Further exploration and improvement are needed to establish a systematic, computer-aided pre-screening method for royal jelly proteins to accelerate the discovery and identification of ACE inhibitory peptides. Summary of the Invention

[0011] The first objective of this invention is to provide an ACE-inhibiting peptide, which provides a polypeptide derived from royal jelly and possessing good ACE-inhibiting activity, thus providing an alternative to the prior art.

[0012] The second objective of this invention is to provide an application of an ACE inhibitory peptide, offering a novel food-derived ACE inhibitory peptide with a well-defined structure, clear origin, and significant ACE inhibitory activity compared to existing technologies.

[0013] To achieve the above objectives, the technical solution adopted by the ACE inhibitory peptide in this invention is as follows: An ACE inhibitory peptide, wherein the amino acid sequence of the ACE inhibitory peptide is shown in any one of SEQ ID NO. 1 to 6.

[0014] The beneficial effects of the above scheme are as follows: This invention, which discloses an ACE-inhibiting peptide, is a pioneering invention. This invention utilizes computer simulation guidance to efficiently screen six polypeptides with ACE-inhibiting activity from royal jelly proteins. The ACE-inhibiting peptide provided by this invention has a well-defined structure, a clear origin, and significant ACE-inhibiting activity, making it a novel food-derived ACE-inhibiting peptide.

[0015] Furthermore, the ACE inhibitory peptide derived from royal jelly provided by this invention is safe and has no toxic side effects, and can be developed as a health food, food additive, drug synergist, etc.

[0016] As a further improvement, the amino acid sequence of the ACE inhibitory peptide is shown in SEQ ID NO.5.

[0017] The beneficial effects of the above-mentioned scheme are as follows: This invention provides an ACE inhibitory peptide derived from royal jelly protein, with the amino acid sequence Met-Tyr-Ile-Arg (abbreviated as MYIR). This ACE inhibitory peptide has a well-defined structure, binds tightly to ACE, exhibits significant inhibitory activity against ACE (significantly higher than the other five peptides), and demonstrates vascular endothelial protection. Furthermore, this ACE inhibitory peptide has good water solubility, high safety, no cytotoxicity, and no blood-brain barrier permeability.

[0018] As a further improvement, the ACE-inhibiting peptide is obtained by a method comprising the following steps: (1) Screen proteases based on computer simulation results of royal jelly protein hydrolysis, and use the screened proteases to hydrolyze royal jelly proteins to obtain hydrolysate; (2) The enzymatic hydrolysate obtained in step (1) was separated and purified, and ACE inhibitory peptides were obtained by liquid chromatography-tandem mass spectrometry analysis and peptidomics screening.

[0019] The beneficial effects of the above scheme are as follows: This invention establishes an integrated "computer simulation-guided - experimental verification" method for the efficient screening of ACE inhibitory peptides, using virtual enzymatic digestion with A... E The evaluation index guides protease screening, significantly improving the discovery efficiency of bioactive peptides and avoiding the drawbacks of long screening cycles and high costs associated with traditional enzyme screening experiments.

[0020] Preferably, the royal jelly protein obtained by computer-simulated enzymatic hydrolysis in step (1) is MRJP1-9, and its protein sequences are obtained from the NCBI database: MRJP1 (O18330), MRJP2 (O77061), MRJP3 (Q17060), MRJP4 (Q17061), MRJP5 (O97432), MRJP6 (A0A8U0WQ84), MRJP7 (A0A7M6W5F9), MRJP8 (A0A8U1C100), and MRJP9 (A0A7M6W880).

[0021] Preferably, the protease in step (1) includes animal protease, plant protease, microbial protease, and complex protease; the animal protease is selected from pepsin (EC 3.4.23.1), trypsin (EC 3.4.21.4), chymotrypsin C (EC 3.4.21.2), metridin (EC 3.4.21.3), leukocyte elastase (EC 3.4.21.37), cathepsin G (EC 3.4.21.20), chymotrypsin (EC 3.4.21.39), and plasmin (EC 3.4.21.7); the plant protease is selected from papain (EC 3.4.22.2) and bromelain (EC 3.4.22.32); the microbial protease is thermolysin (EC 3.4.24.27); and the complex protease is a combination of pepsin and trypsin.

[0022] As a further improvement, the protease obtained in step (1) is bromelain.

[0023] As a further improvement, the enzymatic hydrolysis conditions in step (1) are as follows: the amount of enzyme added is 5-6% of the substrate mass, and the enzymatic hydrolysis is carried out at pH 7.0-7.5 and 55-60℃ for 1-2 hours.

[0024] Preferably, the enzymatic hydrolysis conditions in step (1) are: the amount of enzyme added is 6% of the substrate mass, and the enzymatic hydrolysis is carried out at pH 7.0 and 60°C for 1 h.

[0025] As a further improvement, the royal jelly protein in step (1) is prepared by the following method: royal jelly is dissolved in buffer solution, the supernatant is obtained by solid-liquid separation, the concentrate is collected by ultrafiltration, and then freeze-dried.

[0026] As a further improvement, the separation and purification in step (2) involves first separating the enzymatic hydrolysate using an ultrafiltration membrane, and then purifying the separated and collected components using a gel chromatography column.

[0027] As a further improvement, the molecular weight of the separated and collected components is less than 3 kDa.

[0028] As a further improvement, the gel chromatography column is a Sephadex G-15 gel chromatography column.

[0029] To achieve the above objectives, the technical solution adopted in the application of an ACE inhibitory peptide in this invention is as follows: Application of an ACE-inhibiting peptide, said application comprising any one of the following: (1) Preparation of formulations that inhibit ACE activity; (2) To prepare drugs for the treatment or prevention of hypertension; (3) Prepare health products or functional foods with the function of assisting in lowering blood pressure.

[0030] The beneficial effects of the above-mentioned solution are as follows: This invention also provides the application of the ACE inhibitory peptide derived from royal jelly protein in the preparation of foods, health products, or nutritional supplements with blood pressure regulation or cardiovascular protection functions. The ACE inhibitory peptide is derived from natural raw materials, has high safety, and possesses good ACE inhibitory activity. It can be used as a functional ingredient in foods or health products, providing basic angiotensin-converting enzyme regulation functions for related products and having potential cardiovascular health support effects. Attached Figure Description

[0031] Figure 1 The frequency of ACE inhibitory peptide release from royal jelly protein by different proteases in computer simulation of Example 1 of this invention (A) E (Value) Comparison Chart; Figure 2 This is a graph showing the verification results of the ACE inhibitory activity of the in vitro enzymatic hydrolysis product of the protease selected based on computer simulation results in Example 2 of the present invention; Figure 3 This is a graph showing the ACE inhibition activity of the hydrolysate obtained from bromelain at different enzymatic hydrolysis times in Example 3 of the present invention. Figure 4 This is a graph showing the ACE inhibitory activity of different molecular weight components obtained by ultrafiltration separation of the hydrolysis products of bromelain after 1 h of enzymatic hydrolysis in Example 4 of the present invention. Figure 5 This is a chromatography chromatogram of the royal jelly protein <3 kDa ultrafiltration fraction separated by Sephadex G-15 gel chromatography in Example 4 of the present invention; Figure 6This is a graph showing the ACE inhibition activity determination results of each separated component in Example 4 of the present invention using Sephadex G-15 gel chromatography. Figure 7 This is a determination of the ACE inhibitory activity of the synthesized candidate peptide in Example 7 of the present invention; Figure 8 This is a diagram illustrating the enzyme kinetic inhibition pattern of MYIR on ACE in Example 7 of the present invention; Figure 9 This is a three-dimensional structural model diagram of the predicted docking between MYIR and ACE molecules in Example 8 of the present invention; Figure 10 This is a two-dimensional schematic diagram of the interaction between MYIR and key residues in the ACE active pocket in Example 8 of the present invention; Figure 11 This is a graph showing the effect of MYIR on the viability of EA.hy926 endothelial cells in Example 9 of the present invention; Figure 12 This is a graph showing the effect of MYIR on Ang II-induced migration of EA.hy926 endothelial cells in Example 10 of the present invention. Figure 13 The figure shows the effect of MYIR on NO release in Ang II-induced EA.hy926 endothelial cells in Example 10 of this invention. Figure 14 The figure shows the effect of MYIR on the amount of ET-1 secreted by Ang II-induced EA.hy926 endothelial cells in Example 10 of the present invention. Detailed Implementation

[0032] Hypertension is a clinical syndrome characterized by elevated systemic arterial blood pressure (systolic and / or diastolic blood pressure), which can lead to diseases such as myocardial infarction, stroke, and coronary artery disease. The number of hypertensive patients worldwide is increasing annually, making hypertension a significant global public health issue. Angiotensin-converting enzyme (ACE) increases blood pressure by converting angiotensin I to angiotensin II, inactivating bradykinin (which has vasodilatory effects), and promoting aldosterone secretion. Therefore, inhibiting ACE activity can treat hypertension. Currently, commonly used ACE inhibitors include enalapril, captopril, benazepril, lamivudine, and lixinpril, which have significant therapeutic effects on hypertension. However, long-term use can easily lead to kidney damage, hyperkalemia, allergic reactions, and fetal maldevelopment.

[0033] Compared with chemically synthesized ACE inhibitors, food-derived peptide ACE inhibitors have advantages such as high safety, easy absorption, and fewer side effects, providing a new approach for the prevention and treatment of hypertension. Currently, there are numerous reports on the enzymatic preparation of ACE-inhibiting peptides from food-derived proteins. Royal jelly, secreted by the hypopharyngeal and mandibular glands of worker bees, is milky white or pale yellow, translucent, slightly viscous, with a distinctive aroma and a sour, astringent, pungent, and slightly sweet taste. It possesses various physiological activities, including anti-aging, antibacterial, anti-fatigue, anti-inflammatory, antioxidant, blood pressure regulation, blood sugar reduction, and cell growth promotion. However, fresh royal jelly has poor stability, requiring a complete cold chain for storage and transportation, resulting in extremely high logistics and warehousing costs. It also has poor water solubility and an unpleasant sour, astringent, and spicy taste. Furthermore, sensitive individuals may be allergic to royal jelly; research suggests that the allergens are the main proteins 1 and 2 in royal jelly. These drawbacks significantly impact the commercial value of royal jelly and limit its application in various fields.

[0034] To overcome these limitations, enzymatic hydrolysis technology has emerged as a promising strategy, capable of degrading royal jelly proteins into small peptide fragments. This not only helps reduce sensitization and improve stability but may also enhance their biological activity. However, research on ACE inhibitory peptides derived from royal jelly proteins is still in its early stages, with few reports available. Most studies focus on exploring simple hydrolysis conditions, and no commercially available products with blood pressure-lowering effects have yet been developed.

[0035] Based on this, the present invention provides an ACE-inhibiting peptide and its applications. This invention utilizes computer simulation guidance to efficiently screen six new peptides with well-defined structures, good activity, and mechanisms of action from royal jelly proteins, providing a feasible solution for hypertension management and the development of functional foods or health products.

[0036] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and comparative examples are commercially available.

[0037] Specific embodiments of an ACE inhibitory peptide and its application according to the present invention: This invention identifies an ACE-inhibiting peptide screened from royal jelly. The amino acid sequence of the ACE-inhibiting peptide is shown in any one of SEQ ID NO. 1-6. The ACE-inhibiting peptide provided by this invention is derived from natural royal jelly protein, exhibits good water solubility, high safety, no cytotoxicity, and no blood-brain barrier permeability. It has good potential for oral development and can be used to prepare antihypertensive drugs, health products, or functional foods. Specific implementation steps are as follows: Example 1: Computer-simulated enzymatic digestion-guided protease screening This embodiment uses royal jelly protein as raw material. Based on computer simulation enzymatic hydrolysis results, proteases are screened. The specific implementation steps are as follows: Retrieved Italian honeybees from the UniProt database ( Apis mellifera The amino acid sequences of the nine major royal jelly proteins (MRJP1-9) are as follows, with accession numbers of: MRJP1 (O18330), MRJP2 (O77061), MRJP3 (Q17060), MRJP4 (Q17061), MRJP5 (O97432), MRJP6 (A0A8U0WQ84), MRJP7 (A0A7M6W5F9), MRJP8 (A0A8U1C100), and MRJP9 (A0A7M6W880).

[0038] Using the "Enzyme Action" function of the BIOPEP-UWM database, virtual enzyme digestion was performed on 12 proteases from different sources based on known enzyme digestion specificities. These included: animal proteases (pepsin EC3.4.23.1, trypsin EC3.4.21.4, chymotrypsin C EC 3.4.21.2, metridin EC 3.4.21.3, leukocyte elastase EC 3.4.21.37, cathepsin G EC 3.4.21.20, chymotrypsin EC 3.4.21.39, plasmin EC 3.4.21.7), plant proteases (papain EC 3.4.22.2, bromelain EC 3.4.22.32), microbial proteases (thermolysin EC 3.4.24.27), and a pepsin-trypsin complex. The A value of ACE inhibitory peptides released by each protease was calculated. E Value, A E The higher the value, the greater the theoretical potential for the enzyme to produce ACE inhibitory peptides, as shown in the results. Figure 1 As shown.

[0039] As shown in the figure, bromelain A E The value was highest, followed by papain, indicating that bromelain has the best theoretical potential in releasing ACE inhibitory peptides.

[0040] Example 2: Extraction and in vitro enzymatic hydrolysis verification of royal jelly proteins Based on the screening results of Example 1, this embodiment verifies in vitro the effects of bromelain, papain, and gastropancreatic complex protease on the enzymatic hydrolysis of royal jelly proteins. The specific implementation procedures are as follows: 1. Extraction of royal jelly protein: Fresh royal jelly was dissolved in 0.1 mol / L phosphate buffer (PBS, pH 7.0) at a ratio of 1:8 (g / mL), and magnetically stirred at 4°C for 4 h. The solution was then centrifuged at 10,000 rpm / min for 30 min, and the supernatant was collected. The supernatant was ultrafiltered using a 10 kDa molecular weight cutoff membrane at 4°C for 24 h. The concentrate was collected, centrifuged at 10,000 rpm / min for 30 min, and the supernatant was freeze-dried for 48 h to obtain lyophilized royal jelly protein powder.

[0041] 2. In vitro enzymatic digestion verification: Royal jelly protein lyophilized powder was dissolved in 0.1 mol / L phosphate-buffered saline (PBS, pH 7.0) at a substrate concentration of 1% (w / v). Bromelain, papain, and gastropancreatic complex protease were added for enzymatic hydrolysis. The enzyme addition amount was 6% (w / w) of the substrate mass for each enzyme, and hydrolysis was carried out for 4 h. The optimal reaction conditions for each enzyme are shown in Table 1. After hydrolysis, the reaction was terminated by heating in a boiling water bath for 10 min, followed by centrifugation at 10,000 rpm / min for 10 min. The supernatant was collected and freeze-dried for 48 h to obtain the royal jelly protein hydrolysate.

[0042] Table 1 Optimal reaction conditions for proteases Types of proteases Optimal temperature (°C) Optimal pH value Bromelain 60 7.0 Papain 55 7.0 pepsin 37 2.0 trypsin 37 7.5 Note: When hydrolyzing with gastric and pancreatic complex proteases, first hydrolyze with pepsin (pH 2.0, 37℃) for 2 hours, adjust the pH to 7.5, and then add trypsin (37℃) for 2 hours.

[0043] 3. Determination of ACE inhibitory activity: The hydrolysate was prepared to a concentration of 1 mg / mL, and the ACE inhibition rate was determined by high-performance liquid chromatography (HPLC). ACE (0.1 U / mL), hippuryl-histyl-leucine hydrate (HHL) (5.82 mmol / L), and hydrolysate (1 mg / mL) were prepared using 0.1 mol / L borate buffer (pH 8.3) containing 0.3 mol / L sodium chloride. 50 μL of sample solution (with an equal volume of buffer for the control group) was mixed with 100 μL of HHL and incubated at 37°C for 5 min. 15 μL of ACE solution was added to initiate the reaction, and incubation was continued at 37°C for 30 min. The reaction was terminated by adding 150 μL of 1 mol / L HCl. Captopril was used as a positive control.

[0044] The generated hippuric acid (HA) was detected using an Elite P3200 HPLC system under the following chromatographic conditions: Column: C18 (4.6 × 250 mm, 5 μm); Mobile phase: A (0.05% TFA aqueous solution): B (acetonitrile) = 75:25; Flow rate: 0.5 mL / min; Detection wavelength: 228 nm; Injection volume: 20 μL; The sample was filtered through a 0.22 μm membrane.

[0045] ACE inhibitory activity is calculated using the following formula: ; In the formula: A0, the peak area of ​​HA in the blank control; A1, peak area of ​​sample HA.

[0046] The results are as follows Figure 2 As shown (different lowercase letters indicate significant differences between groups (P<0.05)), the ACE inhibition rate of the bromelain hydrolysate (64.53%) was significantly higher than that of the papain group (48.38%) and the gastropancreatic complex protease group (37.21%). The experimental results were highly consistent with the calculated predictions, fully demonstrating the efficiency and rationality of computer-simulated enzymatic hydrolysis as a protease screening tool. Therefore, bromelain was selected as the hydrolytic enzyme for subsequent experiments.

[0047] Example 3: Optimization of Enzymatic Hydrolysis Time This embodiment optimizes the time for enzymatic hydrolysis of royal jelly protein by bromelain. The specific implementation steps are as follows: Using bromelain, royal jelly protein was enzymatically hydrolyzed for 0, 1, 2, 4, 6, and 8 hours, respectively, according to the method in Example 2, and the ACE inhibitory activity of the hydrolysate at each time point was measured.

[0048] The results are as follows Figure 3 As shown, the inhibitory activity significantly increased from 0 to 1 h, reaching a peak (79.81%) at 1 h. Thereafter, the activity gradually decreased with increasing hydrolysis time, and stabilized after 6 hours. Therefore, the optimal enzymatic hydrolysis time was determined to be 1 h.

[0049] Example 4: Isolation and purification of royal jelly protein hydrolysate This embodiment describes the separation and purification of royal jelly protein hydrolysate obtained by hydrolysis with bromelain. The specific implementation steps are as follows: 1. Ultrafiltration separation: The enzymatic hydrolysate from Example 3 was sequentially passed through ultrafiltration centrifuge tubes with molecular weight cutoffs of 5 kDa and 3 kDa, and centrifuged at 4,000 rpm / min for 20 min to collect three fractions: >5 kDa, 3-5 kDa, and <3 kDa. The ACE inhibitory activity of each fraction at 1 mg / mL was determined.

[0050] The results are as follows Figure 4 As shown, the <3 kDa fraction exhibited the highest ACE inhibition rate (95.39%), significantly higher than the 3-5 kDa fraction (83.14%) and the >5 kDa fraction (21.81%). Therefore, the <3 kDa fraction was collected for subsequent purification.

[0051] 2. Gel filtration chromatography purification: The <3 kDa lyophilized powder was reconstituted with ultrapure water to a 100 mg / mL solution and separated using a Sephadex G-15 gel chromatography column (1.6 × 70 cm). Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min, with one collection tube every 3 min. The detection wavelength was 220 nm. The elution peaks were combined, lyophilized, and the ACE inhibition rate was determined at a concentration of 0.5 mg / mL.

[0052] The results are as follows Figure 5 , Figure 6 As shown, eight elution peaks (F1-F8) were obtained using a Sephadex G-15 gel chromatography column, with fraction 5 exhibiting the highest ACE inhibitory activity (59.71%). Fraction F5 was collected for subsequent peptide identification.

[0053] Example 5: Identification of polypeptide sequences in royal jelly protein hydrolysate This embodiment identifies the F5 component collected in Example 4. The specific implementation steps are as follows: The polypeptide sequences in the F5 fraction were analyzed by LC-MS / MS (liquid chromatography-tandem mass spectrometry). The specific method is as follows: the sample was subjected to reductive alkylation and then analyzed by LC-MS / MS under the following conditions: Pre-column: 300 μm id × 5 mm, packed with Acclaim PepMap RPLCC18, 5 μm, 100 Å; Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLCC18, 1.9 μm, 100 Å; Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN; Gradient: 0 min, 4% mobile phase B; 0-2 min, 4%-8% mobile phase B; 2-45 min, 8%-28% mobile phase B; 45-55 min, 28%-40% mobile phase B; 55-56 min, 40%-95% mobile phase B; 56-66 min, 95% mobile phase B.

[0054] Flow rate: 600 nL / min.

[0055] Mass spectrometry data were used to identify peptides by searching royal jelly protein databases using Byonic software.

[0056] A total of 1467 peptides were identified, with abundance >10 being the most common. 7 Furthermore, a score > 100 was used as a high-confidence screening criterion to eliminate sequences containing post-translational modifications, ultimately yielding 58 highly reliable candidate peptides.

[0057] Example 6: Peptidomics Analysis of Candidate Polypeptides This embodiment performs peptidomics analysis on 58 high-confidence candidate peptides obtained in Example 5. The specific implementation steps are as follows: 1. Bioactivity evaluation: The 58 polypeptide sequences obtained in Example 5 were input into the PeptideRanker program for activity prediction. Polypeptides with a score >0.5 were selected as potential bioactive candidate peptides, resulting in 21 candidate sequences.

[0058] 2. Water solubility evaluation: The Peptide Property Calculator was used to analyze the water solubility of peptides, and six candidate peptides with good water solubility were screened out.

[0059] 3. Toxicity and ADMET evaluation: The toxicity of the six selected peptides was predicted using the ToxinPred program, and the absorption distribution and metabolic characteristics were predicted using the admetSAR 2.0 platform.

[0060] The results showed that all candidate peptides were predicted to be non-toxic and had no blood-brain barrier permeability (BBB-), indicating a low risk of central nervous system side effects (Table 2); among them, four peptides (IAIDKFDRL, TFDYDPRY, MYIR, YPFDVDR) were predicted to have human intestinal absorption characteristics (HIA+), and have the potential for oral development; all candidate peptides were predicted to be CYP450 2D6 non-substrate and non-inhibitor, indicating a low risk of drug metabolism interaction.

[0061] Table 2. PeptideRanker scores, toxicity, and ADMET evaluation of candidate ACE inhibitory peptides. polypeptide sequence PeptideRanker score toxicity <![CDATA[BBB 1 ]]> <![CDATA[HIA 2 ]]> <![CDATA[CYP450 2D6 Interaction 3 > FDYDPRY 0.83 Non-toxic - - - PPSLR 0.69 Non-toxic - - - IAIDKFDRL 0.65 Non-toxic - + - TFDYDPRY 0.62 Non-toxic - + - MYIR 0.59 Non-toxic - + - YPFDVDR 0.56 Non-toxic - + - Note: 1 BBB (Blood-Brain Barrier): "-" indicates a negative prediction result, meaning the peptide is unlikely to cross the blood-brain barrier; "+" indicates a positive prediction result, meaning the peptide may cross the blood-brain barrier. 2HIA (Human Intestinal Absorption): "-" indicates a negative prediction result, meaning the peptide is poorly absorbed by the human intestinal tract; "+" indicates a positive prediction result, meaning the peptide has good potential for absorption by the human intestinal tract. 3 CYP450 2D6 interaction: Cytochrome P450 2D6 enzyme interaction; "-" indicates that the peptide is predicted to be neither a CYP2D6 substrate nor an inhibitor; "+" indicates that the peptide is predicted to be a CYP2D6 substrate or an inhibitor.

[0062] Example 7: Activity verification and enzyme kinetic analysis of ACE inhibitory peptides This embodiment verifies the activity and performs enzyme kinetic analysis on the six candidate peptides screened in Example 6. The specific implementation steps are as follows: 1. Synthesis of ACE-inhibiting peptides: Candidate peptides FDYDPRY (SEQ ID NO.1), PPSLR (SEQ ID NO.2), IAIDKFDRL (SEQ ID NO.3), TFDYDPRY (SEQ ID NO.4), MYIR (SEQ ID NO.5), and YPFDVDR (SEQ ID NO.6) were synthesized by Hefei Kesheng Jingtai Biotechnology Co., Ltd. using solid-phase synthesis, with a purity of ≥98%. Different concentrations of the peptides were reacted with ACE and the substrate HHL, and the ACE inhibitory activity was determined by HPLC.

[0063] The results show that... Figure 7 As shown, MYIR exhibited the strongest inhibitory activity, with an inhibition rate of 75.64% at 1 mg / mL, and its IC50 value was [missing information]. 50 The concentration was 446.38 μmol / L. Therefore, MYIR was selected as the target active peptide for further validation.

[0064] 2. The inhibition mode of MYIR was analyzed using the Lineweaver-Burk double reciprocal plot method. Different concentrations of MYIR solutions (0, 233, and 875 μmol / L) were co-incubated with ACE (0.1 U / mL) and different concentrations of HHL (0.5, 1.0, 2.0, and 5.0 mmol / L). The Michaelis constant (Km) was determined. m ) and maximum reaction rate (V max The type of inhibition is determined by the change in ( ).

[0065] The results are as follows Figure 8 As shown, with the increase of MYIR concentration, V max Decrease and K m The change indicates that MYIR inhibits ACE in a mixed manner.

[0066] Example 8: Molecular docking analysis of MYIR and ACE This embodiment involves molecular docking analysis of the ACE inhibitory peptide MYIR with ACE. The specific implementation steps are as follows: Using ACE as the receptor (PDB ID: 1O86), its crystal structure was obtained from the RCSB protein database. The ACE inhibitory peptide MYIR was docked to the active pocket region of ACE using AutoDockVina software, with captopril as a positive control. Prior to docking, the protein structure underwent dehydration, hydrogenation, and energy minimization treatments.

[0067] The results showed that the binding energy of MYIR to ACE was -9.0 kcal / mol, significantly lower than that of captopril (-5.6 kcal / mol), indicating that MYIR has a stronger binding affinity to ACE and can form a stable complex. Figure 9 Interaction analysis shows that () Figure 10 MYIR forms hydrogen bonds with key amino acid residues such as the S1 (Ala354), S2 (Gln281) pockets and zinc ion coordinating residue (His383) at the ACE active site, stabilizing the MYIR-ACE complex. This result is consistent with mixed inhibition kinetics, suggesting that MYIR can both competitively bind to the active site and induce conformational changes in the enzyme by binding to the peripheral region.

[0068] Example 9: Cell viability assay of MYIR In this embodiment, the MTT assay was used to detect the cytotoxicity of MYIR on human umbilical vein fusion cells EA.hy926 to assess the safety of this ACE inhibitory peptide. The specific implementation procedure is as follows: Cells were seeded in 96-well plates (1×10⁶ cells per well). 5 Cells / mL were added to the culture medium after adhesion, and then treated for 24 h with different concentrations of MYIR (0.0625, 0.125, 0.25, 0.5, 1 mg / mL). MTT solution (20 μL) was added to each well and incubated for 4 h. After discarding the supernatant, formazan crystals were dissolved in dimethyl sulfoxide (DMSO), shaken for 10 min, and the absorbance was measured at 490 nm to calculate cell viability. Cell viability was calculated using the following formula: ; In the formula: A b The absorbance is for the blank control (cell-free culture medium); A c The absorbance is for the control group (cells cultured only in complete culture medium). A s This indicates the absorbance of cells treated with peptides.

[0069] The results are as follows Figure 11As shown, within the concentration range of 0.0625-1 mg / mL, the cell survival rate of the MYIR treatment group was 96%-108%, and no dose-dependent decreasing trend was observed. This indicates that the ACE inhibitory peptide MYIR of the present invention has no obvious cytotoxicity to EA.hy926 cells within this concentration range, has good cell compatibility, and can be used for subsequent cell function detection.

[0070] Example 10: Protective effect of MYIR against Ang II-induced endothelial cell dysfunction This embodiment explores the protective effect of the ACE inhibitory peptide MYIR on human umbilical vein fusion cells EA.hy926. The specific implementation is as follows: 1. Effects on cell migration: EA.hy926 cells were seeded in 6-well plates and cultured for 24 h. Except for the control group, cells in all other groups were treated with angiotensin II (Ang II, 1 μmol / L) for 12 h to establish an endothelial dysfunction model. Scratches were made on the cell monolayer using a 200 μL sterile pipette tip, and detached cells were removed by washing with PBS. 1% FBS medium containing different concentrations of MYIR (0.125, 0.25, 0.5 mg / mL) or captopril (0.25 mg / mL) was added. Scratches were photographed under a microscope at 0 h, 6 h, and 24 h to record healing progress. The scratch area and cell migration rate were calculated using ImageJ software.

[0071] The results are as follows Figure 12 As shown, compared with the control group, Ang II treatment significantly promoted cell migration (6 h migration rate: 34.06% vs. 26.77%; 24 h: 72.94% vs. 62.75%). Compared with the Ang II model group, MYIR inhibited cell migration in a concentration-dependent manner. At a concentration of 0.5 mg / mL, the migration rates at 6 h and 24 h decreased to 24.22% and 56.16%, respectively, with effects similar to those in the captopril group (21.88% and 47.60%).

[0072] This fully demonstrates that the ACE inhibitory peptide MYIR can effectively inhibit Ang II-induced abnormal endothelial cell migration and has the potential to improve endothelial dysfunction and vascular remodeling.

[0073] 2. Effects on NO and ET-1 secretion: EA.hy926 cells were seeded in 6-well plates (4 × 10⁻⁶ cells per well). 5Cells were divided into four groups (cells / mL): control group, Ang II model group, MYIR (0.125, 0.25, 0.5 mg / mL) + Ang II group, and captopril (0.25 mg / mL) + Ang II group. The treatment groups were pre-incubated with the corresponding concentration of MYIR or captopril for 24 h, the supernatant was discarded, and Ang II (1 μmol / L) was added for further culture for 12 h. Cell culture medium was collected, and NO and ET-1 levels were detected using the Griess method and ELISA kits, respectively.

[0074] The results are as follows Figure 13 and Figure 14 As shown, compared with the control group, the Ang II model group showed a significant decrease in NO level (38.65 μmol / g prot vs. 74.41 μmol / g prot) and an increase in ET-1 level (15.69 pg / mg prot vs. 14.29 pg / mg prot). Compared with the model group, MYIR increased NO level and decreased ET-1 level in a concentration-dependent manner; at a concentration of 0.5 mg / mL, NO recovered to 63.01 μmol / g prot, and ET-1 decreased to 12.27 pg / mg prot, with effects similar to those in the captopril group (NO 71.36 μmol / g prot, ET-1 11.86 pg / mg prot).

[0075] This fully demonstrates that the ACE inhibitory peptide MYIR can improve Ang II-induced endothelial dysfunction and exert an angiprotective effect by upregulating NO and downregulating ET-1.

[0076] In summary, this invention successfully obtained a novel royal jelly protein-derived ACE-inhibiting peptide, MYIR. Its preparation method is efficient and targeted, and the peptide exhibits significant in vitro ACE-inhibiting activity and vascular endothelial cell protection function, showing promising application prospects.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ACE-inhibiting peptide, characterized in that: The amino acid sequence of the ACE inhibitory peptide is shown in any one of SEQ ID NO. 1 to 6.

2. The ACE inhibitory peptide according to claim 1, characterized in that: The amino acid sequence of the ACE inhibitory peptide is shown in SEQ ID NO.

5.

3. The ACE-inhibiting peptide according to claim 1 or 2, characterized in that: The ACE inhibitory peptide is obtained by a method comprising the following steps: (1) Screen proteases based on computer simulation results of royal jelly protein hydrolysis, and use the screened proteases to hydrolyze royal jelly proteins to obtain hydrolysate; (2) The enzymatic hydrolysate obtained in step (1) was separated and purified, and ACE inhibitory peptides were obtained by liquid chromatography-tandem mass spectrometry analysis and peptidomics screening.

4. The ACE inhibitory peptide according to claim 3, characterized in that: The protease obtained in step (1) is bromelain.

5. The ACE inhibitory peptide according to claim 4, characterized in that: The enzymatic hydrolysis conditions described in step (1) are as follows: the amount of enzyme added is 5-6% of the substrate mass, and the enzymatic hydrolysis is carried out at pH 7.0-7.5 and 55-60℃ for 1-2 hours.

6. The ACE-inhibiting peptide according to claim 5, characterized in that: The royal jelly protein described in step (1) is prepared by the following method: royal jelly is dissolved in buffer solution, the supernatant is obtained by solid-liquid separation, the concentrate is collected by ultrafiltration, and then freeze-dried.

7. The ACE-inhibiting peptide according to claim 3, characterized in that: The separation and purification described in step (2) involves first separating the enzymatic hydrolysate using an ultrafiltration membrane, and then purifying the separated and collected components using a gel chromatography column.

8. The ACE-inhibiting peptide according to claim 7, characterized in that: The molecular weight of the separated and collected components is less than 3 kDa.

9. The ACE-inhibiting peptide according to claim 7, characterized in that: The gel chromatography column is a Sephadex G-15 gel chromatography column.

10. The application of the ACE-inhibiting peptide as described in any one of claims 1 to 9, characterized in that: The application includes any of the following: (1) Preparation of formulations that inhibit ACE activity; (2) To prepare drugs for the treatment or prevention of hypertension; (3) Prepare health products or functional foods with the function of assisting in lowering blood pressure.

Images