Almond peptide-polyphenol complex and application thereof

Abstract

The present application relates to the technical field of polypeptide complex, and discloses an almond peptide-polyphenol complex and application thereof, wherein the almond peptide-polyphenol complex is obtained by compounding almond peptide and polyphenol, the amino acid sequence of the almond peptide from N-terminal to C-terminal is FPWLQ, and the molecular weight is 689.80 Da; the polyphenol is chlorogenic acid.The present application determines the inhibitory activity of the almond peptide-polyphenol complex on angiotensin converting enzyme, and the results show that the enzyme inhibitory activity of the complex is greatly improved compared with single almond peptide and polyphenol substance, which proves that the complex has good enzyme inhibitory activity and can be effectively applied to the development of antihypertensive drugs, realizes high value-added utilization of almond resources, and has important significance for promoting the sustainable development of the almond industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide complex, and particularly relates to an almond peptide-polyphenol complex and application thereof. BACKGROUND

[0002] Hypertension is a major risk factor for cardiovascular disease, known as "silent killer", which is easy to cause myocardial infarction and stroke, and even life-threatening. Angiotensin converting enzyme (ACE) is involved in blood pressure regulation 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 reducing blood pressure. Common antihypertensive drugs include captopril and lisinopril, etc., but it is still of great significance to find natural ACE inhibitors with small side effects.

[0003] As important functional ingredients, polyphenols and bioactive peptides have become a research hotspot in the field of modern food science. Polyphenols exhibit significant activity in antioxidant and anti-inflammatory due to their diverse phenolic hydroxyl structures and electron donor properties. Bioactive peptides show unique advantages in the field of functional foods due to their high activity and low toxicity. The polyphenol-polypeptide complex system not only improves the stability and bioavailability of single components through non-covalent binding such as intermolecular hydrogen bonding and hydrophobic interaction, but also produces synergistic effects. Natural polyphenol chlorogenic acid has been reported to have ACE inhibitory activity. Polyphenols can form complexes with polypeptides through covalent (such as the combination of oxidized quinone and peptide thiol / amino groups) or non-covalent interactions (hydrogen bonding and hydrophobic interaction), induce conformational changes, and regulate biological activity. The synergistic complex of polyphenols and polypeptides can improve the ACE inhibitory activity and stability of polypeptides. If the ACE inhibitory activity of almond peptides can be improved by complexing with natural polyphenols, the synergistic effect of almond peptides and polyphenols can be exerted, which is of great significance for the sustainable development of almond resources. SUMMARY

[0004] The purpose of the present application is to provide an almond peptide-polyphenol complex and application thereof. The ACE inhibitory activity of the almond peptide-polyphenol complex is determined, and the results show that the enzyme inhibitory activity of the complex is greatly improved compared with single almond peptide and polyphenol substances, proving that the complex has good enzyme inhibitory activity and can be effectively applied to the development of antihypertensive drugs, realizing the high-value utilization of almond resources and having important significance for promoting the sustainable development of the almond industry.

[0005] To achieve the above purpose, the present application provides an almond peptide-polyphenol complex, which is obtained by complexing almond peptides and polyphenols. The amino acid sequence of the almond peptide from N-terminal to C-terminal is FPWLQ, and the molecular weight is 689.80 Da. The polyphenol is chlorogenic acid.

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

[0007] Further, the application further provides application of the almond peptide-polyphenol complex in preparation of an angiotensin converting enzyme inhibitor.

[0008] Further, the application further provides application of the almond peptide-polyphenol complex in preparation of an angiotensin converting enzyme inhibitor.

[0009] Further, the medicine includes a tablet, a capsule and a powder.

[0010] Further, the application further provides a medicine for reducing blood pressure, and the effective component of the medicine includes the almond peptide-polyphenol complex.

[0011] The almond peptide-polyphenol complex and the application thereof have the following advantages and positive effects:

[0012] 1. The almond peptide FPWLQ and the natural polyphenol chlorogenic acid are compounded in a mass ratio of 1:1, and according to the in-vitro activity experiment result (IC 50 = 0.16 mg / mL), the half-inhibition concentration is significantly reduced compared with the single almond peptide FPWLQ and the chlorogenic acid. The synergistic index CI value is less than 1, which indicates that the FPWLQ and the chlorogenic acid have a synergistic inhibitory effect on ACE.

[0013] 2. The molecular docking technology is used to explore the interaction site and the interaction force of the FPWLQ-chlorogenic acid complex and ACE, which lays a theoretical foundation for deep processing of almond protein. The molecular docking result shows that the FPWLQ forms a hydrogen bond with the amino acid residues located in the active region of ACE, and the chlorogenic acid interacts with the amino acid residues (Asp358, Tyr360, Asn66, Arg124 and Asn70) in the non-active pocket region of ACE, which indicates that the FPWLQ in the complex can occupy the active region to prevent the substrate from being combined, and the chlorogenic acid can change the conformation of ACE, so as to inhibit the activity of the enzyme.

[0014] 3. The complex of the almond peptide FPWLQ and the chlorogenic acid is prepared, which opens up a new way for comprehensive utilization of almond and has important significance for sustainable development of almond resources.

[0015] The technical solutions of the application are further described in detail below with reference to the drawings and the embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the in-vitro ACE inhibitory activity of the almond peptide FPWLQ, the chlorogenic acid and the FPWLQ-chlorogenic acid complex;

[0017] Figure 2 is a dose-effect plot of the combined inhibition of ACE by almond peptide FPWLQ and chlorogenic acid;

[0018] Figure 3 is a combination index plot of the combined inhibition of ACE by almond peptide FPWLQ and chlorogenic acid;

[0019] Figure 4 is an isobologram plot of the combined inhibition of ACE by almond peptide FPWLQ and chlorogenic acid;

[0020] Figure 5 is a plot of the docking results of FPWLQ-chlorogenic acid complex with ACE;

[0021] Figure 6 is a zoom-in view of the box of Figure 5 . DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further illustrated by the accompanying drawings and examples.

[0023] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application pertains.

[0024] Based on the examples in the present application, all other examples obtained by those with ordinary skills in the art without making creative efforts shall fall within the scope of protection of the present application. The experimental methods not specified in the following examples shall be determined according to the national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0025] Unless otherwise defined or explained, all professional and scientific terms used in the present application shall have the same meanings as understood by those with ordinary skills in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0026] EXAMPLES

[0027] (1) Preparation of almond peptide:

[0028] The 4 g of almond protein was dissolved in 100 mL of distilled water, and after stirring in a water bath at 85°C for 20 min, it was cooled to room temperature. The pH and temperature of the protein solution were adjusted to 6.9 and 45°C, respectively, and then 2000 U / g of neutral protease was added for enzymolysis. After 2.5 h of enzymolysis, the protein solution was placed in a 100°C water bath for 10 min to inactivate the enzyme. After cooling to room temperature, the pH of the protein solution was adjusted to 9.0 and the temperature was raised to 55°C, and then 2000 U / g of alkaline protease was added for enzymolysis for 2.5 h. After the enzymolysis product was ultrafiltered using a 1 kDa ultrafiltration membrane, the sequence of the almond peptide was identified using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Through bioinformatics and molecular docking, the polypeptide FPWLQ with angiotensin-converting enzyme inhibitory activity was screened.

[0029] (2) Synthesis of almond peptide:

[0030] FPWLQ (SEQ ID NO. 1) was synthesized by Fmoc solid-phase method (purity ≥ 95%). 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.

[0031] (3) Preparation of almond peptide-polyphenol complex:

[0032] 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 assist dissolution). Take an appropriate amount of polyphenol stock 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 equal volumes 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 min.

[0033] (4) In vitro determination of the inhibitory effect of ACE inhibitors on ACE activity:

[0034] The sample, hippuric- histidyl-leucine hydrate (HHL) and angiotensin converting enzyme (ACE) were dissolved in 0.01M pH 8.3 (containing 0.3M NaCl) borate buffer solution, 4.7mM 25μL HHL and 12.5μL sample were mixed and incubated at 37℃ for 5min, 25μL ACE was added and reacted at 37℃ for 30min, 375μL 0.3M NaOH solution was used to terminate the reaction and keep the reaction system alkaline, 25μL 2% o-phthaldehyde methanol solution was added, after reaction at room temperature for 10min in the dark, 50μL 6M HCl was added to terminate the reaction, finally, 200μL liquid was taken to a full black 96-well plate, and the fluorescence intensity was determined at an excitation wavelength of 340nm and an emission wavelength of 455nm. The ACE inhibition rate calculation formula is shown in formula (1):

[0035]

[0036] Wherein, 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).

[0037] The in vitro activity experiment results are shown in Table 1. Figure 1 Figure 1 Table 1 shows that the IC 50 of FPWLQ and chlorogenic acid is 0.29mg / mL and 0.36mg / mL respectively. Compared with the almond peptide FPWLQ alone, the IC 50 of the FPWLQ-chlorogenic acid complex (mass ratio of 1:1) is 0.16mg / mL, which is reduced by 3.6 times.

[0038] (5) Calculation of the combination index of almond peptide-polyphenol complex:

[0039] The combination index method was used to evaluate the synergistic inhibition of ACE of the almond peptide-polyphenol complex. The combination index calculation formula is as follows:

[0040]

[0041] Wherein, (D)1, (D)2, (Dx)1, (Dx)2 are the concentrations of drug 1 and drug 2 when the ACE inhibition rate is 50%; the concentrations of drug 1 alone and drug 2 alone when the ACE inhibition rate is 50%. CI value <1, synergistic effect.

[0042] The combination index CI of the almond peptide-polyphenol complex was calculated by ComboSyn software, and the CI value was 0.39, indicating that FPWLQ and chlorogenic acid had synergistic inhibition effect on ACE. ​

[0043] From the dose-effect Figure 2 It can be seen that there is a good dose-effect relationship when apricot peptide FPWLQ and chlorogenic acid are used alone to inhibit ACE, and the inhibitory effect of FPWLQ-chlorogenic acid complex (mass ratio 1:1) on ACE is stronger than that of FPWLQ and chlorogenic acid. Figure 3 In the present application, the combined inhibitory effect of FPWLQ-chlorogenic acid complex on angiotensin converting enzyme is not concentration-dependent, but the CI value is less than 1 at all concentrations (0.0125-3.2 mg / mL), indicating that the FPWLQ-chlorogenic acid complex (mass ratio 1:1) has a synergistic inhibitory effect on ACE. Figure 4 It is shown that the data points of Fa=0.5, Fa=0.75, and Fa=0.9 all fall below the corresponding hypotenuse, indicating that the FPWLQ-chlorogenic acid complex exhibits stable synergistic characteristics at different effect levels in ACE inhibition.

[0044] (6) Molecular docking of apricot peptide-polyphenol complex and ACE:

[0045] The crystal structure of angiotensin converting enzyme (PDB: 1O8A) was downloaded from the RCSB protein database (https: / / www.rcsb.org / ), and the ligand and water molecules in the protein structure were removed using PyMOL software, AutoDockVina added polar hydrogen, 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 structure of the ligand polypeptide was drawn using ChemDraw20.0 software, and the geometric optimization (energy minimization) of chlorogenic acid and polypeptide was performed 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 80x70x74 grid box with a grid distance of The search parameters for molecular docking were set to 20 times. The docking model with the lowest binding energy in each docking was selected to represent the most favorable combination mode. Finally, the interactions between the protein and the ligand were analyzed using PyMOL.

[0046] The results of molecular docking are shown in Figure 5 and Figure 6 Figure 5 and Figure 6 ​It is shown that FPWLQ forms hydrogen bonds with Glu384, Tyr523 in the active pocket S1, His513 in the S2 pocket, and Arg522, Ala356 near the active site of ACE, and chlorogenic acid interacts with amino acid residues (Asp358, Tyr360, Asn66, Arg124, Asn70) in the non-active pocket region of ACE. It is indicated that FPWLQ in the complex can prevent the binding of the substrate by occupying the active region, and chlorogenic acid can change the structure of ACE and inhibit the catalytic activity of the enzyme.

[0047] The almond peptide FPWLQ and the natural polyphenol chlorogenic acid are compounded in a mass ratio of 1:1 in the present application, and the obtained complex has stronger angiotensin converting enzyme inhibitory activity than single compound through in vitro activity experiment verification and synergistic index calculation, IC 50 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 region to prevent the binding of the substrate, and chlorogenic acid binds to the non-active region to change the structure of ACE and inhibit the catalytic activity of ACE. The present application compounds the almond peptide FPWLQ and the natural polyphenol chlorogenic acid, so that the two can enhance the biological activity of the almond peptide through synergistic effect, opens up a new way for the comprehensive utilization of almond, and has important practical value and application prospect.

[0048] Therefore, the present application adopts the above-mentioned almond peptide-polyphenol complex and its application, and determines the angiotensin converting enzyme inhibitory activity of the almond peptide-polyphenol complex, and the results show that the enzyme inhibitory activity of the complex is greatly improved compared with single almond peptide and polyphenol, proving that the complex has good enzyme inhibitory activity and can be effectively applied to the development of antihypertensive drugs, realizing the high value-added utilization of almond resources, and having important significance for promoting the sustainable development of the almond industry.

[0049] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. An almond peptide-polyphenol complex, characterized by: The almond peptide and the polyphenol are compounded to obtain the almond peptide-polyphenol complex, the amino acid sequence of the almond peptide from N-terminal to C-terminal is FPWLQ, and the molecular weight is 689.80 Da; the polyphenol is chlorogenic acid; When the almond peptide and the chlorogenic acid are compounded, the mass ratio of the almond peptide to the chlorogenic acid is 1:1; The IC50of the compound of almond peptide and chlorogenic acid is 0.16 mg / mL, and the synergistic index of the compound is 0.

39. 50 =0.16mg / mL, the synergistic index of the compound is 0.39; After the almond peptide and the chlorogenic acid are compounded, the almond peptide in the complex occupies the active region by forming a hydrogen bond with the amino acid residues located in the active region of the ACE, prevents the substrate from being combined, and the chlorogenic acid interacts with the amino acid residues in the non-active pocket region of the ACE to change the conformation of the ACE, so that the activity of the ACE enzyme is synergistically inhibited.

2. Application of the almond peptide-polyphenol complex in claim 1 in the preparation of a blood pressure lowering drug.

3. Use according to claim 2, characterized in that: The drug includes tablets, capsules and powders.

4. A pharmaceutical product for lowering blood pressure, characterized in that: The effective component includes the almond peptide-polyphenol complex in claim 1.

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