ACE inhibiting peptides and use thereof

Abstract

This invention belongs to the field of bioactive peptide technology and discloses an ACE-inhibiting peptide and its applications. The peptide amino acid sequence provided by this invention is shown in SEQ ID NO.1 or SEQ ID NO.2, and it exhibits significant ACE-inhibiting activity, IC50... 50 With a molecular weight as low as 87.82±8.58 μM, it belongs to a highly active food-derived ACE inhibitory peptide. This peptide has a simple structure, is easy to prepare industrially through solid-phase synthesis, and its purity is easy to control.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to an ACE inhibitory peptide and its applications. Background Technology

[0002] Hypertension is a significant risk factor for cardiovascular disease. Angiotensin-converting enzyme (ACE) plays a crucial role in the renin-angiotensin system, catalyzing the conversion of angiotensin I to angiotensin II, thus leading to elevated blood pressure. Therefore, inhibiting ACE activity is an important strategy for the prevention and treatment of hypertension. Compared with chemically synthesized ACE inhibitors, dietary ACE-inhibiting peptides have advantages such as high safety, fewer side effects, and easy absorption, and have become a research hotspot in the fields of functional foods and biomedicine.

[0003] Collagen is rich in characteristic amino acid sequences and is an important source of ACE inhibitory peptides. Bovine gelatin, as a food-grade raw material, is widely available and highly safe, making it a suitable raw material for preparing collagen-derived ACE inhibitory peptides. While there are numerous potential ACE inhibitory peptides in bovine collagen sequences, the currently reported ACE inhibitory peptide sequences are still relatively limited, leaving room for further exploration and research.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to solve at least one of the above technical problems, and provides an ACE inhibitory peptide and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An ACE inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] Preferably, the ACE inhibitory peptide is isolated and identified from the alkaline protease hydrolysate of bovine gelatin.

[0008] Preferably, the preparation method of the ACE inhibitory peptide includes: dispersing bovine gelatin in water, adjusting the pH to the optimal pH range for alkaline protease, adding alkaline protease for enzymatic hydrolysis, inactivating the enzyme after enzymatic hydrolysis, centrifuging to collect the supernatant, and obtaining the peptide through separation and purification.

[0009] Preferably, the ACE inhibitory peptide is obtained by chemical synthesis and has a purity of not less than 95%.

[0010] The present invention also provides an ACE inhibitor comprising the ACE inhibitory peptide described in any one of the above claims as an active component.

[0011] Preferably, the ACE inhibitor is in the form of an oral liquid, tablet, capsule, or lyophilized powder.

[0012] The present invention also provides the use of the ACE inhibitory peptide described in any of the above claims in the preparation of products for lowering blood pressure.

[0013] Preferably, the product is a health food or a food for special medical purposes.

[0014] Preferably, the product is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The peptide provided by this invention has significant ACE inhibitory activity, IC50. 50 With a molecular weight as low as 87.82±8.58 μM, it belongs to a highly active food-derived ACE inhibitory peptide. This peptide has a simple structure, is easy to prepare industrially through solid-phase synthesis, and its purity is easy to control. Attached Figure Description

[0016] Figure 1 The effect of alkaline protease on ACE inhibition rate at different time points in Example 1; Figure 2 MS / MS secondary mass spectra and fragment ion analysis of FOGP and GAOGFL identified in Example 2; Figure 3 The dose-response curves and IC50 values ​​of FOGP and GAOGFL on the ACE inhibitory activity in Example 3 are shown. 50 Fit analysis. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The first embodiment of the present invention provides an ACE inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0019] The ACE inhibitory peptides provided by this invention include peptides with amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2. The peptide shown in SEQ ID NO.1 consists of four amino acid residues with the sequence Phe-Hyp-Gly-Pro (phenylalanine-hydroxyproline-glycine-proline), i.e., FOGP; the peptide shown in SEQ ID NO.2 consists of six amino acid residues with the sequence Gly-Ala-Hyp-Gly-Phe-Leu (glycine-alanine-hydroxyproline-glycine-phenylalanine-leucine), i.e., GAOGFL.

[0020] According to the experimental results of this invention, both FOGP and GAOGFL exhibit ACE inhibitory activity. The half-maximal inhibitory concentration (IC50) of FOGP... 50 The concentration of GAOGFL was 87.82 ± 8.58 μM. 50 The concentration was 188.39 ± 6.03 μM. From a structure-activity relationship perspective, FOGP, as a tetrapeptide, has less steric hindrance, making it easier to penetrate the catalytic pocket of the target enzyme; its C-terminus is the characteristic strongly hydrophobic amino acid proline (Pro), with an embedded hydroxyproline (Hyp), and its N-terminus contains an aromatic phenylalanine (Phe). This classic "aromatic / hydrophobic residue-proline-rich" sequence pattern not only allows it to interact with the zinc ion (Zn) in the ACE active site... 2+ The peptide forms a strong bond with the surrounding hydrophobic subunits, which also endows the peptide with a strong ability to resist protease degradation, thus exhibiting excellent inhibitory activity.

[0021] In some preferred embodiments, FOGP and GAOGFL can be isolated and identified from the enzymatic hydrolysis products of food-grade bovine gelatin. Identification of FOGP and GAOGFL from complex enzymatic hydrolysates can be performed using conventional LC-MS / MS techniques in the art. Targeting the highly hydroxylated structural characteristics of collagen, this invention uses hydroxyproline (Hyp) as a key variable modification for database searching to more comprehensively identify the peptide composition in the enzymatic hydrolysate, providing data support for subsequent analysis of the contribution of specific modifications to ACE inhibitory activity.

[0022] In some preferred embodiments, to eliminate interference from other components in the enzymatic hydrolysate and accurately verify the ACE inhibitory activity of FOGP and GAOGFL, chemical synthesis can be performed using conventional solid-phase peptide synthesis techniques. The synthesis strategy, resin type, coupling reagents, lysis reagents, etc., can be conventionally selected by those skilled in the art based on the peptide sequence characteristics. After purification by reversed-phase high-performance liquid chromatography, the purity of the synthesized product can reach over 95%. After mass spectrometry confirmation of the molecular weight, it is used for activity evaluation experiments.

[0023] The second embodiment of the present invention provides the application of ACE inhibitory peptides. FOGP and GAOGFL have significant ACE inhibitory activity and can be further prepared as ACE inhibitors or used in the development of blood pressure-lowering related products.

[0024] In some embodiments, FOGP and / or GAOGFL can be used as active ingredients, combined with pharmaceutically or food-acceptable carriers, excipients, or diluents to prepare ACE inhibitors. The selection and dosage of the carriers and excipients can be routinely determined by those skilled in the art based on the dosage form and route of administration.

[0025] The dosage forms of the ACE inhibitors include, but are not limited to, oral solutions, tablets, capsules, or lyophilized powders. For example, oral solutions can be prepared by dissolving FOGP and / or GAOGFL in water or other suitable solvents and adding conventional excipients such as flavoring agents and stabilizers; tablets and capsules can be prepared by mixing FOGP and / or GAOGFL with conventional excipients such as fillers, binders, and disintegrants, followed by conventional processes such as wet granulation or dry granulation; lyophilized powders can be obtained by freeze-drying a solution containing FOGP and / or GAOGFL. The preparation methods for the above dosage forms are all conventional techniques in the art.

[0026] Based on the ACE-inhibiting activity of FOGP and GAOGFL, the peptides can be used to prepare products for lowering blood pressure. These products include, but are not limited to, health foods, foods for special medical purposes, or pharmaceutical compositions.

[0027] In health foods or foods for special medical purposes, FOGP and / or GAOGFL can be added as functional factors to ordinary food matrices, or prepared as dietary supplements such as oral liquids, tablets, and capsules. The amount added can be routinely determined by those skilled in the art based on the product type and expected efficacy.

[0028] In the pharmaceutical composition, FOGP and / or GAOGFL can be used as active ingredients, combined with pharmaceutically acceptable carriers (such as physiological saline, buffer solutions, fillers, etc.) to prepare a formulation suitable for oral or parenteral administration. The preparation method and dosage of the pharmaceutical composition can be determined by those skilled in the art based on routine pharmacological experiments.

[0029] The preparation and properties of ACE inhibitory peptides are described in detail below through examples.

[0030] The following is some information about the reagents used in the examples: The cowhide gelatin was purchased from Zhengzhou Haijia Food Co., Ltd., and is food grade. Alkaline protease was purchased from Vinoxin. Angiotensin-converting enzyme (ACE), hippuryl-histyl-leucine (HHL), and acetonitrile were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Hippuric acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Hydrochloric acid (HCl), sodium hydroxide (NaOH), trifluoroacetic acid, formic acid, and other reagents were purchased from Chengdu Jinshudu Laboratory Equipment Co., Ltd.

[0031] Example 1: Preparation of peptides Weigh an appropriate amount of bovine gelatin raw material and place it in an Erlenmeyer flask. Add distilled water at a material-to-liquid ratio of 1:20 (w / v) and stir to disperse evenly. Then adjust the pH of the system to 9.0 and stabilize for 10 min. Next, add alkaline protease at 0.5% (w / w) of the substrate content, mix well, and place in a 50 ℃ constant temperature water bath shaker for enzymatic hydrolysis. Immediately after the enzymatic hydrolysis is completed, remove the sample and heat in a 90 ℃ oil bath for 15 min to terminate the enzyme reaction. After cooling to room temperature, centrifuge the sample at 4 ℃ and 8000 rpm for 15 min. Collect the supernatant, aliquot and lyophilize, and store at 4 ℃.

[0032] To investigate the changes in ACE inhibitory activity during the enzymatic hydrolysis of bovine gelatin, the ACE inhibitory activity of the hydrolysates obtained at different hydrolysis times (0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h) was examined under the above conditions.

[0033] The ACE inhibition rate was determined as follows: 30 μL of 2.5 mM HCl solution (prepared with pH 8.3 borate buffer) was mixed with 10 μL of sample solution (1 mg / mL), and reacted at 37 ℃ for 5 min. Then, 20 μL of 0.1 U / mL ACE solution was added, and the reaction was continued at 37 ℃ for 60 min. Finally, 60 μL of 1.0 M HCl was added to terminate the reaction, and the mixture was filtered through a 0.22 μm filter after adding 200 μL of UP water. The standard hippuric acid concentration was 10 mg / mL. The detection conditions were: mobile phase A (0.1% formic acid) and mobile phase B (100% acetonitrile) were eluted isocratically at a ratio of 82:18, the flow rate was 0.8 mL / min, and the detection wavelength was 220 nm. The ACE inhibition rate was calculated according to formula (1).

[0034] (1); ΔAc and ΔAs represent the peak areas of HA in the control group (excluding samples) and the hydrolysate group, respectively.

[0035] The results are as follows Figure 1As shown, enzymatic hydrolysis time has a significant impact on the ACE inhibition rate of protein hydrolysates. The alkaline protease-mediated hydrolysis reaction exhibits a clear time-dependent characteristic, with the ACE inhibitory activity of the products showing an inverted "U"-shaped trend of first increasing and then decreasing with prolonged hydrolysis time. At 6 h of hydrolysis, the hydrolysates showed the strongest inhibitory effect, reaching a peak of 63.54% ± 2.58%. When the hydrolysis time was extended to 8 h, the inhibition rate significantly decreased to 51.42% ± 2.00%, which may be attributed to excessive hydrolysis leading to the further degradation of highly active target peptides into less active small peptides or free amino acids.

[0036] Example 2 Identification of Peptides The sample from Example 1, after 6 h of enzymatic hydrolysis, was identified by LC-MS / MS. The identification method was as follows: The sample was prepared as a 0.5 mg / mL solution, filtered through a 0.22 μm filter membrane, and 2 μL was injected into a UPLC system equipped with a Waters HSS T3 column (2.1 mm × 100 mm, 1.8 μm). Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–10 min, 5%–40% B; 10–12 min, 40%–90% B; 12–14 min, 90% B; 14–15 min, 90%–5% B; 15–18 min, 5% B. The flow rate was 0.2 mL / min, and the column temperature was 30 °C. The obtained mass spectrometry data were converted from raw data to centroidal MGF and mzML formats suitable for peptidomics analysis using MSConvert software. Subsequently, the data were compared and identified using a PepOSX (v4.1.0) workstation with a reviewed bovine collagen sequence from the UniProt database as a template. Based on the highly hydroxylated structural characteristics of collagen, this invention sets hydroxyproline ([Hyp], O) as a key variable modification.

[0037] Mass spectrometry data were automatically de-weighted and convolved, and database searches were performed to successfully identify several high-confidence sequences, including FOGP (Phe-Hyp-Gly-Pro) and GAOGFL (Gly-Ala-Hyp-Gly-Phe-Leu).

[0038] like Figure 2As shown, the two-stage mass spectrometry (MS / MS) spectrum reveals a clear and continuous series of b and y ions, covering the key amino acid residues of the target peptide. In the FOGP (m / z 433.209) secondary mass spectrometry, characteristic fragment peaks at m / z 286.139 and m / z 173.091 were detected, corresponding to the y3 and y2 ions after the loss of C-terminal residues, respectively, directly confirming the presence of the Phe-Hyp group in the sequence. Furthermore, a residue increment of 113.048 Da was observed in all identified peptides (instead of the 97.053 Da of proline), confirming the successful localization of hydroxylation modification at the mass spectrometry level.

[0039] Similarly, in another representative hexapeptide GAOGFL secondary mass spectrum, abundant and continuous fragment ions were also observed. Notably, the base peak (strongest signal) of this spectrum was precisely located at m / z 449.240, perfectly matching the y4 ion (Hyp-Gly-Phe-Leu) containing the characteristic hydroxyproline. This high-abundance characteristic peak not only strongly confirms the arrangement of the C-terminal core sequence of this peptide but also indirectly reflects the structural stability of the Hyp residue-containing fragment during mass spectrometry fragmentation. Furthermore, combined with the clearly detected y5 ion (m / z 520.276) and b5 ion (m / z 448.200) in the spectrum, and the abundant continuous fragments in the low-mass region, key evidence of fragmentation further enables unambiguous identification of this sequence.

[0040] Example 3: Synthesis of peptides and evaluation of ACE inhibitory activity The candidate peptides FOGP and GAOGFL obtained in Example 2 were commissioned to Nanjing Jietai Biotechnology Co., Ltd. for custom synthesis. The experiment employed an Fmoc / tBu solid-phase synthesis strategy, using 2-Cl resin as the matrix, and coupling amino acids sequentially from the C-terminus to the N-terminus via a DMF system. Under the catalysis of DIEA, the first amino acid was immobilized on the activated resin, followed by deprotection with 20% piperidine and washing cycles to extend the peptide chain. The coupling efficiency was monitored in real time using the Kaiser method. After sequence extension, the ends were modified with azidoacetic acid. Subsequently, the protecting group was removed by cleavage using a TFA system, and the crude product was collected by cold ether precipitation and centrifugation. Desalting and purification were performed by RP-HPLC, and the molecular weight of the final product was determined by ESI-MS, yielding a 100 mg lyophilized powder with a purity higher than 95%.

[0041] Through computer-aided simulation screening, the present invention chemically synthesized the selected key peptides and achieved a half-inhibitory concentration (IC50). 50FOGP and GAOGFL were used as the core indicator for evaluating their ACE inhibitory activity. Following the method described in Example 1, the inhibition rates of FOGP and GAOGFL at concentration gradients of 62.5–2000 μg / mL were determined. The IC50 values ​​of the two candidate peptides were calculated using nonlinear regression fitting of the concentration-inhibition rate curves with Origin software. 50 value.

[0042] The results are as follows Figure 3 As shown, both peptides exhibited significant dose-dependent inhibitory effects with excellent goodness of fit (correlation coefficient R0). 2 The values ​​were as high as 0.990 and 0.998, respectively. Based on model calculations and conversions, the half-maximal inhibitory concentration (IC50) of FOGP was... 50 The concentration was only 87.82 ± 8.58 μM (corresponding to 37.98 ± 3.71 μg / mL), while GAOGFL's IC50 was much higher. 50 The value was 188.39 ± 6.03 μM (corresponding to 107.98 ± 3.43 μg / mL). This shows that FOGP exhibits a lower IC50 value. 50 It has the most outstanding ACE inhibition potential.

[0043] In summary, the ACE-inhibiting peptide FOGP (Phe-Hyp-Gly-Pro) provided by this invention has a well-defined sequence characteristic and excellent ACE-inhibiting activity (IC50). 50 = 87.82 ± 8.58 μM), and GAOGFL (Gly-Ala-Hyp-Gly-Phe-Leu) also showed certain ACE inhibitory activity. Both are derived from safe food raw materials, are easy to synthesize industrially, and have broad application prospects in the fields of antihypertensive functional foods and medicines.

[0044] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. An ACE-inhibiting peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The ACE-inhibiting peptide as described in claim 1, characterized in that, The ACE inhibitory peptide was isolated and identified from the alkaline protease hydrolysate of bovine gelatin.

3. The ACE-inhibiting peptide as described in claim 1, characterized in that, The method for preparing the ACE inhibitory peptide includes: Bovine gelatin was dispersed in water, and the pH was adjusted to the optimal pH range for alkaline protease. Alkaline protease was added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated, the supernatant was collected by centrifugation, and then purified by separation.

4. The ACE-inhibiting peptide as described in claim 1, characterized in that, The ACE inhibitory peptide was obtained by chemical synthesis and had a purity of not less than 95%.

5. An ACE inhibitor, characterized in that, It contains the ACE inhibitory peptide as described in any one of claims 1-4 as an active component.

6. The ACE inhibitor as described in claim 5, characterized in that, The ACE inhibitor is in the form of oral liquid, tablet, capsule or lyophilized powder.

7. The use of the ACE inhibitory peptide according to any one of claims 1-4 in the preparation of a product for lowering blood pressure.

8. The application as described in claim 7, characterized in that, The product is a health food or a food for special medical purposes.

9. The application as described in claim 7, characterized in that, The product is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

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