Almond peptide-polyphenol compound and application thereof

Abstract

The invention relates to the technical field of polypeptide compounds, and discloses an almond peptide-polyphenol compound and application thereof.The almond peptide-polyphenol compound is obtained by compounding almond peptide and polyphenol, the amino acid sequence of the almond peptide from the N terminal to the C terminal is FPWLQ, and the molecular weight is 689.80 Da; the polyphenol is chlorogenic acid. According to the almond peptide-polyphenol compound and the application thereof, the inhibitory activity of the almond peptide-polyphenol compound on angiotensin converting enzyme is measured, and the result shows that the enzyme inhibitory activity of the compound is greatly improved compared with that of single almond peptide and polyphenol substances, so that the compound is proved to have better enzyme inhibitory activity, and the application of the almond peptide-polyphenol compound is promoted. The method can be effectively applied to the development of blood pressure reducing products, provides an innovative solution for the creation of almond functional foods, realizes the high-added-value utilization of almond resources, and has important significance for promoting the sustainable development of the almond industry.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide complexes, and in particular to an almond peptide-polyphenol complex and applications thereof. Background Art

[0002] Hypertension is a major risk factor for cardiovascular disease, known as the "silent killer." It can easily cause myocardial infarction and stroke, and can be life-threatening in severe cases. Angiotensin-converting enzyme (ACE) regulates blood pressure through the renin-angiotensin system (RAS) and the bradykinin-releasing enzyme-bradykinin system (KKS). ACE inhibitors can reduce the conversion of ANG I and inactivate bradykinin, thereby lowering blood pressure. Common antihypertensive drugs include captopril and lisinopril, but finding natural angiotensin-converting enzyme inhibitors with fewer side effects is still important for lowering blood pressure.

[0003] As important functional ingredients, the synergistic mechanisms of polyphenols and bioactive peptides have become a research hotspot in modern food science. Polyphenols, due to their diverse phenolic hydroxyl structures and electron-donating properties, exhibit significant antioxidant and anti-inflammatory activity. Active peptides, with their high activity and low toxicity, offer unique advantages in the functional food sector. Polyphenol-peptide complexes not only improve the stability and bioavailability of individual components through non-covalent interactions such as intermolecular hydrogen bonding and hydrophobic interactions, but also produce synergistic effects. The natural polyphenol chlorogenic acid has been reported to exhibit ACE inhibitory activity. Polyphenols can form complexes with peptides through covalent interactions (such as binding of quinone oxide to thiol / amino groups on the peptide chain) or non-covalent interactions (hydrogen bonding and hydrophobic interactions), inducing conformational changes and modulating bioactivity. Synergistic complexation of polyphenols with peptides can enhance the ACE inhibitory activity and stability of peptides. If the inhibitory activity of almond peptides with ACE inhibitory activity can be enhanced by combining them with natural polyphenols, thereby leveraging the synergistic effects of almond peptides and polyphenols, this could provide new technical support for the development of functional almond foods and have important implications for the sustainable development of almond resources. Summary of the Invention

[0004] The purpose of the present invention is to provide an almond peptide-polyphenol complex and its application. The inhibitory activity of the almond peptide-polyphenol complex on angiotensin-converting enzyme was determined. The results showed that the enzyme inhibitory activity of the complex was greatly improved compared with that of single almond peptide and polyphenol substances, proving that the complex has good enzyme inhibitory activity and can be effectively used in the development of blood pressure lowering products. It not only provides an innovative solution for the creation of almond functional foods, but also realizes high added value utilization of almond resources, which is of great significance to promoting the sustainable development of the almond industry.

[0005] To achieve the above objectives, the present invention provides an almond peptide-polyphenol complex, which is obtained by compounding almond peptide and polyphenol. The amino acid sequence of almond peptide from N-terminus to C-terminus is FPWLQ, and the molecular weight is 689.80Da; the polyphenol is chlorogenic acid.

[0006] Furthermore, when almond peptide and polyphenol are compounded, the mass ratio of almond peptide and polyphenol is 1:1.

[0007] Furthermore, the present invention also provides the use of the above-mentioned amygdaloid peptide-polyphenol complex in the preparation of angiotensin converting enzyme inhibitors.

[0008] Furthermore, the present invention also provides the use of the above-mentioned almond peptide-polyphenol complex in the preparation of a blood pressure lowering product.

[0009] Furthermore, the product includes medicine.

[0010] Furthermore, the medicine includes tablets, capsules, and powders.

[0011] Furthermore, the present invention also provides a blood pressure lowering medicine, the active ingredient of which includes the above-mentioned amygdaloid peptide-polyphenol complex.

[0012] The advantages and positive effects of the almond peptide-polyphenol complex and its application described in the present invention are:

[0013] 1. The present invention combines almond peptide FPWLQ and natural polyphenol chlorogenic acid in a mass ratio of 1:1. According to the in vitro activity test results (IC 50 =0.16 mg / mL), showing a significant decrease in the half-inhibitory concentration compared to single amygdaloid peptide FPWLQ and chlorogenic acid. The synergistic index CI value was <1, indicating a synergistic inhibitory effect of FPWLQ and chlorogenic acid on ACE.

[0014] 2. This study used molecular docking to investigate the interaction sites and forces between the FPWLQ-chlorogenic acid complex and ACE, laying a theoretical foundation for the further processing of almond protein. Molecular docking results showed that FPWLQ forms hydrogen bonds with amino acid residues located in the active region of ACE, while chlorogenic acid interacts with amino acid residues in the inactive pocket region of ACE (Asp358, Tyr360, Asn66, Arg124, and Asn70). This suggests that FPWLQ in the complex blocks substrate binding by occupying the active region, while chlorogenic acid alters the conformation of ACE, thereby inhibiting enzyme activity.

[0015] 3. The present invention prepares a complex of almond peptide FPWLQ and chlorogenic acid, which not only provides new technical support for almond functional food ingredients, but also opens up a new path for the comprehensive utilization of almonds, which is of great significance to the sustainable development of almond resources.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The in vitro ACE inhibitory activity of amygdaloid peptide FPWLQ, chlorogenic acid, and FPWLQ-chlorogenic acid complex;

[0018] Figure 2 This is a dose-effect diagram of the combined inhibitory effect of amygdaloid peptide FPWLQ and chlorogenic acid on ACE;

[0019] Figure 3 This is the combined index graph of the combined inhibitory effect of amygdaloid peptide FPWLQ and chlorogenic acid on ACE;

[0020] Figure 4 This is the isobologram of the combined inhibitory effect of amygdaloid peptide FPWLQ and chlorogenic acid on ACE;

[0021] Figure 5 This is the docking result diagram of FPWLQ-chlorogenic acid complex and ACE;

[0022] Figure 6 for Figure 5 Enlarge the image in the box. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0024] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0025] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0026] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0027] Example

[0028] (1) Preparation of almond peptide:

[0029] 4g of almond protein was dissolved in 100mL of distilled water and stirred in an 85°C water bath for 20 minutes before cooling to room temperature. The pH and temperature of the protein solution were adjusted to 6.9 and 45°C, respectively. 2000U / g of neutral protease was then added for enzymatic hydrolysis. After 2.5 hours of hydrolysis, the protein solution was placed in a 100°C water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, the protein solution was adjusted to pH 9.0 and heated to 55°C. 2000U / g of alkaline protease was then added for 2.5 hours. The enzymatic hydrolysis product was ultrafiltered using a 1kDa ultrafiltration membrane, and the sequence of the almond peptide was identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Bioinformatics and molecular docking were used to screen out the peptide FPWLQ with angiotensin-converting enzyme inhibitory activity.

[0030] (2) Synthesis of almond peptide:

[0031] FPWLQ (shown in SEQ ID NO. 1) was synthesized using the Fmoc solid phase method (purity ≥ 95%), and the purity and molecular weight of the synthesized peptide were identified by HPLC-MS / MS. The sample was stored at -20°C for further analysis.

[0032] (3) Preparation of amygdaloid peptide-polyphenol complex:

[0033] Accurately weigh 10 mg of chlorogenic acid and dissolve it in 0.5 mL of anhydrous ethanol (a small amount of DMSO can be added to aid solubilization). Take an appropriate amount of polyphenol mother solution and dilute it to the target concentration (0.0625-16 mg / mL) with 0.01 M pH 8.3 (containing 0.3 M NaCl) borate buffer solution. Accurately weigh 10 mg of almond peptide and dissolve it in 0.01 M borate buffer solution to prepare a 16 mg / mL peptide solution, and then dilute it to the target concentration (0.0625-4 mg / mL) by gradient dilution. Take an equal volume of 200 μL of peptide and polyphenol solutions of different concentrations and mix them in a 0.5 mL centrifuge tube and vortex at room temperature for 10 minutes.

[0034] (4) In vitro determination of the inhibitory effect of ACE inhibitors on ACE activity:

[0035] The sample, hippuryl-histidyl-leucine hydrate (HHL), and angiotensin-converting enzyme (ACE) were dissolved in 0.01M borate buffer (pH 8.3) (containing 0.3M NaCl). 25 μL of 4.7mM HHL and 12.5 μL of sample were mixed and incubated at 37°C for 5 minutes. 25 μL of ACE was added and reacted at 37°C for 30 minutes. 375 μL of 0.3M NaOH solution was used to terminate the reaction and maintain the alkalinity of the reaction system. 25 μL of 2% o-phthalaldehyde methanol solution was added. The reaction was incubated at room temperature in the dark for 10 minutes, and then 50 μL of 6M HCl was added to terminate the reaction. Finally, 200 μL of the liquid was transferred to a black 96-well plate, and the fluorescence intensity was measured at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The ACE inhibition rate was calculated as shown in formula (1):

[0036]

[0037] Among them, F a is the fluorescence intensity of the control group (without peptide), F b is the fluorescence intensity of the sample group, F c is the fluorescence intensity of the blank group (without peptide and ACE).

[0038] In vitro activity test results Figure 1 As shown, Figure 1 It showed that the IC values of FPWLQ and chlorogenic acid 50 Compared with the single almond peptide FPWLQ, the IC 50 It was 0.16 mg / mL, a decrease of 3.6 times.

[0039] (5) Calculation of the combined index of amygdaloid peptide-polyphenol complex:

[0040] The combined index method was used to evaluate the synergistic ACE inhibition effect of the amygdaloid peptide-polyphenol complex. The combined index calculation formula is as follows:

[0041]

[0042] Wherein, (D)1, (D)2, (Dx)1, and (Dx)2 are the concentrations at which ACE inhibition is 50% when drug 1 and drug 2 are used together; and the concentrations at which ACE inhibition is 50% when drug 1 and drug 2 are used alone. A CI value < 1 indicates synergistic effect.

[0043] The combined index (CI) of the amygdaloid peptide-polyphenol complex was calculated by ComboSyn software, with a CI value of 0.39, indicating that FPWLQ and chlorogenic acid had a synergistic inhibitory effect on ACE.

[0044] Dose-effect Figure 2 It can be seen that when amygdaloid peptide FPWLQ and chlorogenic acid are used alone to inhibit ACE, there is a good dose-effect relationship, and the inhibitory effect of FPWLQ-chlorogenic acid complex (mass ratio of 1:1) on ACE is stronger than that of FPWLQ and chlorogenic acid. Figure 3 The combined inhibitory effect of FPWLQ-chlorogenic acid complex on angiotensin-converting enzyme was not concentration-dependent, but at all concentrations (0.0125-3.2 mg / mL), the CI values were less than 1, indicating that the FPWLQ-chlorogenic acid complex (mass ratio of 1:1) had a synergistic inhibitory effect on ACE. Figure 4 It shows that the data points of Fa=0.5, Fa=0.75, and Fa=0.9 all fall below the corresponding hypotenuse, indicating that in ACE inhibition, the FPWLQ-chlorogenic acid complex exhibits stable synergistic enhancement characteristics at different effect levels.

[0045] (6) Docking of the amygdaloid peptide-polyphenol complex with ACE molecules:

[0046] The crystal structure of angiotensin-converting enzyme (PDB: 1O8A) was downloaded from the RCSB protein database (https: / / www.rcsb.org / ), and the ligands and water molecules in the protein structure were removed using PyMOL software. Polar hydrogen was added using AutoDock Vina, and the molecular structure file was saved in pdbqt format. The 3D structure of chlorogenic acid was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the structural formula of the ligand peptide was drawn using ChemDraw 20.0 software. The geometry of chlorogenic acid and peptide was optimized (energy minimized) using Chem 3D 20.0 software and saved as a PDB format file. The docking box was set at coordinates (x: 43.439, y: 34.009, z: 47.652), and the active center was covered by an 80×70×74 grid box with a grid distance of 0.375. The search parameters for molecular docking were set to 20. The docking model with the lowest binding energy value in each docking was selected as the most favorable combination. Finally, the interaction between the protein and ligand was analyzed using PyMOL.

[0047] The molecular docking results are as follows Figure 5 and Figure 6 As shown, Figure 5 and Figure 6The results showed that FPWLQ formed hydrogen bonds with Glu384 and Tyr523 in the active pocket S1 of ACE, His513 in the S2 pocket, and Arg522 and Ala356 near the active site, while chlorogenic acid interacted with amino acid residues in the inactive pocket region of ACE (Asp358, Tyr360, Asn66, Arg124, and Asn70). This suggests that FPWLQ in the complex can block substrate binding by occupying the active region, while chlorogenic acid can alter the structure of ACE and inhibit the catalytic activity of the enzyme.

[0048] The present invention compounds almond peptide FPWLQ and natural polyphenol chlorogenic acid in a mass ratio of 1:1. Through in vitro activity experiment verification and synergistic index calculation, the obtained complex has stronger angiotensin converting enzyme inhibitory activity than the single compound, IC 50 The concentration of FPWLQ and chlorogenic acid is 0.16 mg / mL, and the CI value is 0.39, indicating that FPWLQ and chlorogenic acid have a synergistic inhibitory effect on ACE. Molecular docking shows that FPWLQ in the complex occupies the active area, preventing substrate binding, and chlorogenic acid binds to the inactive area to change the structure of ACE and inhibit the catalytic activity of ACE. The present invention combines almond peptide FPWLQ and natural polyphenol chlorogenic acid, so that the two enhance the biological activity of almond peptide through synergistic synergy, which not only provides new technical support for almond functional food ingredients, but also opens up a new path for the comprehensive utilization of almonds, and has important practical value and application prospects.

[0049] Therefore, the present invention adopts the above-mentioned almond peptide-polyphenol complex and its application to determine the inhibitory activity of the almond peptide-polyphenol complex on angiotensin-converting enzyme. The results show that the enzyme inhibitory activity of the complex is greatly improved compared with that of single almond peptide and polyphenol, proving that the complex has good enzyme inhibitory activity and can be effectively used in the development of blood pressure lowering products. It not only provides an innovative solution for the creation of almond functional foods, but also realizes high added value utilization of almond resources, which is of great significance to promoting the sustainable development of the almond industry.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An almond peptide-polyphenol complex, characterized by: It is obtained by compounding almond peptide and polyphenol. The amino acid sequence of almond peptide from N-terminus to C-terminus is FPWLQ, with a molecular weight of 689.80Da; the polyphenol is chlorogenic acid.

2. The almond peptide-polyphenol complex according to claim 1, characterized in that: When almond peptide and polyphenols are compounded, the mass ratio of almond peptide and polyphenols is 1:

1.

3. Use of the almond peptide-polyphenol complex according to claim 1 or 2 in the preparation of angiotensin-converting enzyme inhibitors.

4. Use of the almond peptide-polyphenol complex according to claim 1 or 2 in the preparation of a blood pressure lowering product.

5. The use according to claim 4, characterized in that: The products include pharmaceuticals.

6. The use according to claim 5, characterized in that: The medicines include tablets, capsules and powders.

7. A blood pressure lowering drug, characterized in that: The effective ingredients include the almond peptide-polyphenol complex according to claim 1.

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