Rice bran protein-derived ACE inhibitory peptide, its preparation method and application

Abstract

The present invention discloses a rice bran protein-derived ACE inhibitory peptide, its preparation method and application, belonging to the field of biotechnology. The amino acid sequence of the active peptide of the present invention is VFDGVLRPGQ, and its in vitro ACE inhibitory activity IC 50 value is 0.093 mg / mL. Molecular docking simulation visualization analysis shows that the high ACE inhibitory activity of this peptide is attributed to its effective interaction with the ACE receptor protein through hydrogen bonds, hydrophobic interactions, etc. Through the EA.hy926 cell model, it was found that VFDGVLRPGQ can promote the release of NO and reduce the content of ET-1 to achieve the effect of lowering blood pressure. This study provides a theoretical basis for the research and development of functional foods related to the prevention of cardiovascular diseases, drug development, and the high-value utilization of rice bran.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to rice bran protein-derived ACE inhibitory peptides, a preparation method thereof, and an application thereof. Background Art

[0002] Hypertension is one of the main causes of cardiovascular diseases (CVD) and premature death worldwide, and is the most common risk factor for myocardial infarction, stroke, heart failure, atrial fibrillation, and peripheral arterial disease. The prevention and treatment of hypertension have become an increasingly concerned issue. In the renin-angiotensin system (RAS), angiotensin I-converting enzyme (ACE) can cleave angiotensin I into angiotensin II, which can cause vasoconstriction and thus lead to hypertension. Currently, studies have proven that the RAS system is a key therapeutic target for hypertension, and ACE inhibitors can reduce the mortality rate of hypertensive patients. Clinically used chemically synthesized drugs for treating hypertension, such as captopril and lisinopril, although having good blood pressure-lowering effects, are accompanied by some side effects when taken. In contrast, bioactive peptides derived from nature are more green and safe, and have become a research hotspot in recent years.

[0003] Bioactive peptides are defined as specific protein fragments that have a positive impact on body functions or conditions and may affect health. They have hormone- or drug-like activities, such as antibacterial, antithrombotic, immunomodulatory, antioxidant, etc., and are widely used in disease prevention and are considered a new generation of bioactive regulators. Currently, a large number of studies have shown that food-derived bioactive peptides with ACE inhibitory activity have significant blood pressure-lowering effects, such as sesame peptides, mushroom peptides, moringa peptides, sorghum peptides, etc.

[0004] Rice (Oryza sativa L.) is a cereal widely cultivated worldwide, accounting for about 20% of the dietary intake of the global population. However, there is a serious phenomenon of resource waste in the rice processing process, and a large number of processing by-products have not been effectively developed. Rice bran is a by-product of polished rice after rice husking. When rice is processed into edible rice, about 10% of rice bran is produced, and the nutrients contained in this rice bran account for almost 65% of the whole rice. Rice bran contains about 12-20% protein, is rich in amino acids, and is considered a good source of low-allergenic protein. After hydrolysis of rice bran protein, various peptide segments with different biological activities can be produced, such as anti-cancer, antioxidant, blood pressure-lowering, blood sugar-lowering peptides, etc., which have great development potential. Summary of the Invention

[0005] To solve related problems, the primary object of the present invention is to provide rice bran protein-derived ACE inhibitory peptides.

[0006] Another object of the present invention is to provide a preparation method of the above-mentioned rice bran protein-derived ACE inhibitory peptides.

[0007] Another object of the present invention is to provide the application of the above-mentioned rice bran protein-derived ACE inhibitory peptide.

[0008] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0009] A rice bran protein-derived ACE inhibitory peptide, whose amino acid sequence is VFDGVLRPGQ (Val-Phe-Asp-Gly-Val-Leu-Arg-Pro-Gly-Gln).

[0010] The preparation method of the above-mentioned rice bran protein-derived ACE inhibitory peptide includes the following steps:

[0011] Step 1: Ultrafiltrate the rice bran protease hydrolyzate to obtain a <3KDa fraction;

[0012] Step 2: After the <3KDa fraction passes through a 0.22 μm filter, it is separated once by a C18 column in a preparative liquid phase LC-8 system;

[0013] Step 3: Take the peak with the best activity from the first separation, and after passing through a 0.22 μm filter, it is separated twice by a Shimadzu PRC-ODS(K) steel column in a preparative liquid phase LC-8 system;

[0014] Step 4: After separation and purification, it is further identified and analyzed by HPLC-MS / MS to obtain the above-mentioned rice bran protein-derived ACE inhibitory peptide.

[0015] Further, in the first separation described in Step 2, the chromatographic conditions are as follows: Mobile phase A: distilled water + 0.1% (volume fraction, the same below) trifluoroacetic acid; Mobile phase B: methanol + 0.1% trifluoroacetic acid; Both mobile phases need to pass through a 0.2 μm filter membrane; Injection volume: 5 mL; Elution flow rate: 10 mL / min; Detection wavelengths: 214 nm and 280 nm; Gradient elution: 0 - 10.00 min: 8% - 15% mobile phase B; 10.00 - 30.00 min: 15% - 50% mobile phase B; 30.00 - 70.00 min: 50% - 70% mobile phase B; 70.00 - 80.00 min: 70% - 95% mobile phase B; 80.00 - 85.00 min: 95% - 8% mobile phase B.

[0016] Further, in the secondary separation described in Step 3, the chromatographic conditions are as follows: Mobile phase A: distilled water + 0.1% trifluoroacetic acid; Mobile phase B: methanol + 0.1% trifluoroacetic acid; both mobile phases need to pass through a 0.2 μm filter membrane; injection volume: 1 mL; elution flow rate: 10 mL / min; detection wavelengths: 214 nm and 280 nm; gradient elution: 0.00 - 30.00 min: 8% - 50% Mobile phase B; 30.00 - 60.00 min: 50% - 70% Mobile phase B; 60.00 - 65.00 min: 70% - 95% Mobile phase B; 65.00 - 75.00 min: 95% Mobile phase B; 75.00 - 80.00 min: 95% - 8% Mobile phase B; 80.00 - 95.00 min: 8% Mobile phase B.

[0017] Use of the above-mentioned rice bran protein-derived ACE inhibitory peptide in the preparation of an ACE inhibitor and / or an antihypertensive drug.

[0018] Further, in the above-mentioned use, the ACE inhibitor is an ACE non-competitive inhibitor.

[0019] Further, in the above-mentioned use, the antihypertensive drug has the effects of promoting the release of NO and reducing the release of ET-1.

[0020] Further, in the above-mentioned use, the ACE inhibitor and / or the antihypertensive drug uses the above-mentioned rice bran protein-derived ACE inhibitory peptide as an active ingredient and may be added with pharmaceutically acceptable carriers or excipients.

[0021] An ACE inhibitor comprising the above-mentioned rice bran protein-derived ACE inhibitory peptide as an active ingredient.

[0022] Further, the ACE inhibitor also contains pharmaceutically acceptable carriers or excipients.

[0023] An antihypertensive drug comprising the above-mentioned rice bran protein-derived ACE inhibitory peptide as an active ingredient.

[0024] Further, the antihypertensive drug also contains pharmaceutically acceptable carriers or excipients.

[0025] The present invention has the following advantages and effects compared with the prior art:

[0026] To develop new rice bran products and achieve the high-value utilization of rice by-products, the rice bran protease hydrolysate was separated and purified by ultrafiltration and reverse-phase high-performance liquid chromatography, and the peptides were identified by LC-MS / MS. A total of 40 peptides were identified. Through molecular docking simulation, a new and highly active ACE inhibitory peptide VFDGVLRPGQ (1086.582 Da) was screened out, and its in vitro ACE inhibitory activity IC 50 value was 0.093 mg / mL. Visual analysis of molecular docking simulation showed that the high ACE inhibitory activity of this peptide was attributed to its effective interaction with the ACE receptor protein through hydrogen bonds, hydrophobic interactions, etc. Through the EA.hy926 cell model, it was found that VFDGVLRPGQ could promote the release of NO and reduce the content of ET-1 to achieve the effect of lowering blood pressure. This study provides a theoretical basis for the research and development of drugs related to the prevention of cardiovascular diseases and the high-value utilization of rice bran. Description of the Drawings

[0027] Figure 1 Reverse-phase high-performance liquid chromatography chart of the ultrafiltrate with a molecular weight less than 3 kDa; among them, (a) chromatogram of the preparation liquid for separating the ultrafiltrate component with a molecular weight less than 3 kDa of the rice bran protease hydrolysate, (b) inhibition rate of each peak at a concentration of 0.2 mg / mL;

[0028] Figure 2 Reverse-phase high-performance liquid chromatography chart of the ultrafiltrate of fraction F2; among them, (a) chromatogram of the preparation liquid for separating fraction F2, (b) ACE inhibition rate of each peak at a concentration of 0.2 mg / mL;

[0029] Figure 3 Total ion current chromatogram of the highly active component of rice bran protein;

[0030] Figure 4 Lineweaver-Burk plot of the inhibition of ACE by peptide VFDGVLRPGQ;

[0031] Figure 5 Molecular docking results of peptide VFDGVLRPGQ and ACE; among them, (a) 3D structural surface conformation of the VFDGVLRPGQ-ACE complex, (b) 2D detailed diagram of the interaction between VFDGVLRPGQ and ACE;

[0032] Figure 6 Results chart of the effect of peptide VFDGVLRPGQ at different concentrations on the viability of EA.hy926 cells;

[0033] Figure 7 Results chart of the effect of peptide VFDGVLRPGQ at different concentrations on the NO release of EA.hy926 cells;

[0034] Figure 8 The figure shows the research results of the effects of peptides VFDGVLRPGQ at different concentrations on the ET-1 content in EA.hy926 cells. Detailed implementation manners

[0035] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto.

[0036] Example 1

[0037] 1. Preparation of rice bran protease hydrolysate

[0038] Prepare rice bran protein hydrolysate according to the results of our previous research. Mix rice bran protein with distilled water at a substrate concentration of 8.2% evenly, add 0.3% neutral protease at pH 7.2, and carry out enzymatic hydrolysis in a water bath at 46 °C for 3 h. Then inactivate the enzyme at high temperature for 10 min. After the enzymatic hydrolysate is cooled to room temperature, centrifuge it at 4000 r / min for 20 min, and take the supernatant. Store it in a -20 °C refrigerator after collection.

[0039] 2. Detection method for in vitro ACE inhibitory activity and inhibition mode

[0040] Refer to Luo Lin's 96-well plate method to determine the ACE inhibition rate of the enzymatic hydrolysate. Using furanacryloyl tripeptide (FAPGG) as a simulated substrate, detect the ACE inhibitory activity of the sample solution by measuring the change in absorbance at 340 nm. Modify as follows: Use a 96-well plate, add 40 μL of the sample solution, 50 μL of FAPGG, and 10 μL of ACE enzyme to the sample group respectively; add 40 μL of HEPES buffer solution, 50 μL of FAPGG, and 10 μL of ACE enzyme to the blank group respectively. Use an enzyme-labeled instrument to detect the absorbance value at 340 nm and record it. After incubating at 37 °C for 30 min, detect the absorbance value at 340 nm again and record it. The calculation formula for the ACE inhibition rate is formula (1):

[0041] QUOTE (1)

[0042] In the formula:

[0043] a 1 —— The initial absorbance at 340 nm of the blank group;

[0044] a 2 —— The absorbance at 340 nm of the blank group after incubation at 37 °C for 30 min;

[0045] b 1 —— The initial absorbance at 340 nm of the sample group;

[0046] b 2 —— Absorbance at 340 nm of the sample group after incubation at 37°C for 30 min;

[0047] A—— Decrease value a of absorbance in the blank group 1 -a 2 ;

[0048] B—— Decrease value b of absorbance in the sample group 1 -b 2 .

[0049] Add ACE inhibitory peptides with different concentrations (0.25, 0.5 mg / mL) to each reaction system, and use substrates FAPGG with different concentrations (3, 1.5, 0.75, 0.375 mM) to detect the enzyme activity. According to the Michaelis-Menten kinetic equation, use the data of the Lineweaver-burke plot to calculate the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax), so as to determine the kinetics of ACE enzyme in the presence of inhibitors.

[0050] 3. Isolation and purification of ACE inhibitory peptides

[0051] 3.1 Ultrafiltration

[0052] Use an ultrafiltration device to pass the prepared enzymatic hydrolysate through a 3 kDa ultrafiltration membrane, and divide it into two parts with a molecular weight above 3 kDa (>3 kDa) and below 3 kDa (<3 kDa). After freeze-drying the partial components, measure their in vitro ACE inhibition rate.

[0053] 3.2 Separation and purification of ACE inhibitory peptides by reversed-phase high performance liquid chromatography

[0054] The ultrafiltration fraction with strong ACE inhibitory activity was selected. After passing through a 0.22 μm filter membrane, it was separated once using a C18 column (20 mm×450 mm, 10 μm) in a preparative liquid chromatography (LC-8, Shimadzu, Japan) system. The chromatographic conditions were as follows: mobile phase A: distilled water + 0.1% (volume fraction, the same below) trifluoroacetic acid; mobile phase B: methanol + 0.1% trifluoroacetic acid; both mobile phases needed to pass through a 0.2 μm filter membrane; injection volume: 5 mL; elution flow rate: 10 mL / min; detection wavelengths: 214 nm and 280 nm. Gradient elution: 0 - 10.00 min: 8% - 15% mobile phase B; 10.00 - 30.00 min: 15% - 50% mobile phase B; 30.00 - 70.00 min: 50% - 70% mobile phase B; 70.00 - 80.00 min: 70% - 95% mobile phase B; 80.00 - 85.00 min: 95% - 8% mobile phase B. Each elution peak was collected, and after concentration, a part was taken for lyophilization to determine the ACE inhibitory activity among different peak components.

[0055] The fraction with the best activity from the first separation was taken. After passing through a 0.22 μm filter, it was separated twice using a Shimadzu PRC-ODS(K) steel column (30 mm×250 mm, 15 μm) in a preparative liquid chromatography (LC-8, Shimadzu, Japan) system. Mobile phase A: distilled water + 0.1% trifluoroacetic acid; mobile phase B: methanol + 0.1% trifluoroacetic acid; both mobile phases needed to pass through a 0.2 μm filter membrane; injection volume: 1 mL; elution flow rate: 10 mL / min; detection wavelengths: 214 nm and 280 nm. Gradient elution: 0.00 - 30.00 min: 8% - 50% mobile phase B; 30.00 - 60.00 min: 50% - 70% mobile phase B; 60.00 - 65.00 min: 70% - 95% mobile phase B; 65.00 - 75.00 min: 95% mobile phase B; 75.00 - 80.00 min: 95% - 8% mobile phase B; 80.00 - 95.00 min: 8% mobile phase B. Each elution peak was collected, and after concentration, a part was taken for lyophilization to determine the ACE inhibitory activity among different peak components.

[0056] 4. Identification of Amino Acid Sequence of Purified Peptide

[0057] Select the purified component with the highest activity, collect it after freeze-drying, and perform identification of the amino acid sequence. Take an appropriate amount of the sample and desalt it using a C18 desalting column, and analyze the sample by LC-MS / MS equipped with an online nanoelectrospray ion source. The entire system is a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with EASY-nanoLC1200. The chromatographic conditions are as follows: analytical column: Acclaim PepMap C18, 75 μm x 25 cm); sample loading volume: 3 μL of the sample; column flow rate: 300 nL / min; column temperature: 40°C; electrospray voltage: 2 kV; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: ACN solution containing 0.1% formic acid; chromatographic gradient is as follows: 0 - 3.00 min: 2% - 6% mobile phase B; 3.00 - 42.00 min: 6% - 20% mobile phase B; 42.00 - 47.00 min: 20% - 35% mobile phase B; 47.00 - 48.00 min: 35% - 100% mobile phase B; 48.00 - 60.00 min: 100% mobile phase B. The tandem mass spectra are analyzed by PEAKS Studio version X+ (Bioinformatics Solutions Inc., Waterloo, Canada). The PEAKS DB is used to search the uniprot - Oryza_sativa (version 201907, 37344 entries) database, and no enzyme digestion is set. The search parameters are as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 7 ppm, maximum number of missed cleavages: 2, variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98. The protein score value is: -10lgP≥0, with at least 1 unique peptide; the peptide score value is: -10lgP≥15.

[0058] 5. Molecular docking study

[0059] The structure of the bioactive peptide was drawn using Marvin Sketch software and optimized by energy minimization to generate a mol2 format file for subsequent docking. The crystal structure of the receptor molecule, angiotensin-converting enzyme (ACE) protein (1O8A), was downloaded from the PDB database (http: / / www.rcsb.org / ). Ligands and water molecules in the receptor were removed using Pymol software, and the final file was saved in pdb format. The receptor molecule and peptide monomer were processed for hydrogen addition and charge calculation using AutodockTools 1.5.6 software and output as pdbqt format files for docking with Vina. The result with the lowest binding energy from Vina docking was visually analyzed using Pymol and Discovery Studio 4.5 software. The specific parameter settings were as follows: the center coordinates were x: 43.821, y: 38.240, z: 46.712, and a reaction constraint box with a spacing of 0.375 Å and a size of 100 Å × 100 Å × 100 Å was established.

[0060] 6. Peptide synthesis

[0061] The monomeric peptide was synthesized by solid-phase synthesis with a peptide purity ≥ 98%. The monomeric peptides in this study were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

[0062] 7. Detection of ACE inhibitory rate of monomeric peptide on EA.hy 926 cells

[0063] 7.1 Cytotoxicity assay

[0064] It was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. EA.hy926 cells were seeded at a density of 1×10 5 cells / well into fresh medium in a 96-well plate and placed at 37 °C with 5% CO 2Incubate in an incubator for 24 h. After incubation for 24 h, aspirate all the culture medium, add 100 μL of peptide monomers diluted with fresh culture medium at gradient concentrations (0.1, 0.25, 0.5, 1, 1.5, 2 mg / mL), and continue to culture the cells for 24 h (add normal culture medium to the blank sample). After culturing for 24 h, aspirate the culture medium containing the sample, and then add a quantitative MTT solution to each well. Place the 96-well plate in the incubator and incubate for 4 h. Then, discard the treated culture solution from the wells, and add a certain amount of dimethyl sulfoxide (DMSO). After shaking for 20 min, use a microplate reader to measure the optical density of the sample at a wavelength of 490 nm. Use captopril as a positive control.

[0065] 7.2 Effect of antihypertensive peptides on NO release from cells

[0066] Seed EA.hy 926 cells into fresh culture medium in a 96-well plate at a density of 1×10 5 cells / well, and place it in an incubator at 37 °C with 5% CO 2 Incubate for 24 h. After incubation for 24 h, aspirate all the culture medium, add 100 μL of peptide monomers diluted with fresh culture medium at gradient concentrations (0.1, 0.25, 0.5 mg / mL), and continue to culture the cells for 24 h (add normal culture medium to the blank sample). After incubation for 24 h, detect the NO release from the cells using a NO detection kit. Use captopril as a positive control.

[0067] 7.3 Effect of antihypertensive peptides on the content of endothelin (ET-1) in cells

[0068] Seed EA.hy 926 cells into fresh culture medium in a 96-well plate at a density of 1×10 5 cells / well, and place it in an incubator at 37 °C with 5% CO 2 Incubate for 24 h. After incubation for 24 h, aspirate all the culture medium, add 100 μL of peptide monomers diluted with fresh culture medium at gradient concentrations (0.1, 0.25, 0.5 mg / mL), and continue to culture the cells for 24 h (add normal culture medium to the blank sample). After incubation for 24 h, detect the ET-1 content in the cells using an ET-1 detection kit (ELISA Kit). Use captopril as a positive control.

[0069] 8. Data analysis

[0070] The data graphs were plotted using Origin 8.6, and the IC 50 values were calculated using SPSS Statistics 21 software, and the differences between the data were analyzed using one-way ANOVA and Duncan's method. P < 0.05 indicates a significant difference.

[0071] 9. Results and Analysis

[0072] 9.1 Isolation and Purification of ACE Inhibitory Peptides from Rice Bran Protein Hydrolysate

[0073] The enzymatic hydrolysate was separated into two fractions, <3 kDa and >3 kDa, using a 3 kDa ultrafiltration membrane. Subsequently, the two ultrafiltration fractions and the enzymatic hydrolysate were lyophilized and their ACE inhibitory activities were measured. The results are shown in Table 1. The <3 kDa fraction (IC 50 value: 0.284 mg / mL) showed better ACE inhibitory activity than the enzymatic hydrolysate (IC 50 value: 0.390 mg / mL) and the >3 kDa fraction (IC 50 value: 0.693 mg / mL), indicating that molecular weight is one of the factors affecting the ACE inhibition rate.

[0074] Table 1. IC 50 Values of in vitro ACE inhibition rates of each fraction

[0075]

[0076] Reverse-phase high-performance liquid chromatography has the advantages of high sensitivity, high resolution, and high column efficiency, and is widely used in the separation and purification of polypeptides. The <3 kDa ultrafiltrate was separated by preparative liquid chromatography using a reverse C18 column. The chromatogram is shown in Figure 1 (a). Peaks F1, F2, and F3 were collected, concentrated, and lyophilized, and their ACE inhibition rates were measured at a concentration of 0.2 mg / mL. The results are shown in Figure 1 (b). Fraction F2 showed the best ACE inhibitory activity, with an IC 50 value of 0.223 mg / mL (Table 1). Fraction F2 was further separated by preparative liquid chromatography using a reverse C18 column. The chromatogram is shown in Figure 2 (a). Each peak was collected, concentrated, and lyophilized, and their ACE inhibition rates were measured at a concentration of 0.2 mg / mL. The results are shown in Figure 2 (b).

[0077] 9.2 Peptide Identification and Molecular Docking Screening

[0078] The highly active fractions F2-5 and F2-6 were mixed, concentrated, and lyophilized, and then further analyzed and identified by LC / MS-MS. The total ion chromatogram is shown in Figure 3 .

[0079] Set -10lgP ≥ 45, peptide chain length ≤ 10, and a total of 40 peptides were identified. The binding energy generated in molecular docking can to a certain extent show the tightness of binding to the receptor. Therefore, the molecular simulation docking technology was used to dock and analyze the peptide segments with the crystal structure of the receptor molecule ACE protein (1O8A). According to the binding energy results, a potential highly active peptide monomer VFDGVLRPGQ (1086.582 Da) (Table 2) was screened out, and its ACE inhibition rate IC 50 value was 0.093 mg / mL, showing better activity than the ACE inhibitory peptides Pro–Gln–Phe–Tyr–Trp (IC 50 value of 0.104 mg / mL) and Val–Val–Leu–Tyr–Arg (IC 50 value of 0.244 mg / mL) derived from coconut meal.

[0080] Table 2. Peptide sequence identification and molecular docking analysis

[0081] Peptide sequence Mass m / z -10lgP Binding energy (kcal / mol) VFDGVLRPGQ 1086.582 544.3055 56.06 -9.4

[0082] Studies have shown that the activity of bioactive peptides is related to their amino acid types, sequences, etc., and most peptide segments with ACE activity are generally composed of 2 - 12 amino acids. Hydrophobic amino acids are often considered to play a very important role in the ACE inhibitory activity of peptides. The peptide VFDGVLRPGQ contains hydrophobic amino acids Pro, Leu, Val, and Phe, which may be one of the reasons for the high ACE inhibitory activity of VFDGVLRPGQ. In addition, acidic amino acids are also closely related to ACE inhibitory activity. The acidic amino acids Gln and Asp in VFDGVLRPGQ may also be beneficial to its ACE inhibitory activity.

[0083] 9.3 Analysis of the ACE inhibition mode of active peptides

[0084] Enzyme kinetics studies provide information on the affinity of substrates and products for the enzyme. Understanding the dynamic characteristics of enzyme catalysis is a prerequisite for designing ACE inhibitors. The ACE kinetic constants of active peptides are as Figure 4 shown. As the concentration of the peptide VFDGVLRPGQ increases, the Michaelis - Menten constant (Km) remains unchanged, while the maximum reaction rate (Vmax) decreases. This is a non - competitive inhibition mode. The results show that VFDGVLRPGQ cannot bind to the active site of ACE, but at other exclusive sites of the ACE active site.

[0085] 9.4 Analysis of the binding site and interaction force between the active peptide and the receptor protein

[0086] Molecular docking is used to study the behavior of small molecules at the binding sites of target proteins, simulate molecular interactions and predict the binding mode and affinity between receptors and ligands, and is widely used in the research of ACE inhibitory peptides. The binding pockets related to ACE activity are mainly distributed in three regions: S1, S2 and S1'. The S1 region is located at the upper end of ACE and includes key amino acid residues such as Ala 353, Lys 384, Tyr 523, etc. The S2 region is located in the middle of ACE and includes key amino acid residues such as Gln 281, His 353, Lys 511, His513, Tyr 520, etc. The S1' region is located at the lower end of ACE and includes key amino acid residues such as Glu 162. At the same time, the interaction between ACE inhibitors and the Zn 2+ between usually also plays an important role. Zn 2+ combines with the key amino acid residues His 383, His 387 and Glu 411 to form a tetrahedral coordination structure.

[0087] Figure 5 It shows that VFDGVLRPGQ generates 13 hydrogen bonds with the ACE amino acid residues Lys 118, Glu 123, Asp 121, Trp 59, Tyr51, Thr 92, Arg 522, Ala 356, Asn 66, including 12 conventional hydrogen bonds and 1 non-conventional hydrogen bond. It also forms 10 hydrophobic interactions with the amino acid residues Ala 63, Trp 357, Val 351, Phe 512, Pro 407, His 410, Pro 519, Met223 and 3 electrostatic interactions with the amino acid residues Glu 403, Asp358, Glu 143. Hydrogen bonds are crucial for the binding of ACE and oligopeptides. These 13 hydrogen bond forces may be one of the reasons for the good ACE inhibitory activity of the decapeptide VFDGVLRPGQ. Moreover, this peptide generates a hydrogen bond at a site very close to the key amino acid residue Ala 354 in the S1 of the ACE active pocket, and has hydrophobic interactions and electrostatic forces at sites close to the key amino acid residues His 383, His 387 and Glu 411 in the Zn 2+ active site, which may affect its ACE inhibitory activity.

[0088] 9.5 Evaluation of the antihypertensive peptide activity based on the EA.hy926 cell model

[0089] 9.5.1 Detection of the cytotoxicity of antihypertensive peptides on EA.hy926 cells

[0090] Vascular endothelial cells cover the inner surface of blood vessels and maintain blood pressure balance by producing key regulatory factors of vascular tone (vasodilators and vasoconstrictors). Therefore, vascular endothelial cells are an ideal cell model for exploring the intracellular mechanism of antihypertensive peptides.

[0091] The cytotoxicity of VFDGVLRPGQ on EA.hy926 cells was determined by MTT colorimetric assay to detect the cell viability of EA.hy926, and captopril was used as a positive control. The results are as Figure 6 shown. Compared with the blank group, there were no significant differences in the range of 0.1 - 2.0 mg / mL dose of VFDGVLRPGQ, and the viability was above 95%, without obvious cytotoxicity, indicating that the peptide is safe and non-toxic to EA.hy926 cells within an appropriate dose range. The positive drug captopril had a certain impact on cell viability with the increase of dose. When the dose was 2.0 mg / mL, there was a significant difference compared with the blank group, and the cell viability was 87.95 ± 3.11%.

[0092] 9.5.2 Effect of antihypertensive peptide on NO release in EA.hy 926 cells

[0093] NO is an endogenous vasodilator gas molecule that can relax the surrounding vascular smooth muscle cells and dilate blood vessels, playing a direct role in reducing blood pressure. EA.hy926 cells were treated with peptides at different dose groups (0.1, 0.25, 0.5 mg / mL) and cultured for 24 h, and then their NO release was measured. At the same time, captopril was used as a positive control.

[0094] The results are as Figure 7 shown. Compared with the blank group, the NO release in cells treated with VFDGVLRPGQ and captopril increased significantly, and there was a certain dose-dependence. Even at a low dose of 0.1 mg / mL, the NO release in the VFDGVLRPGQ treatment group was more than twice that of the blank group. It can be seen that the peptide VFDGVLRPGQ can significantly promote the release of NO in EA.hy 926 cells, thereby dilating blood vessels and achieving the effect of reducing blood pressure.

[0095] 9.5.3 Effect of antihypertensive peptide on the content of endothelin (ET-1) in EA.hy 926 cells

[0096] ET-1 is a 21-amino acid peptide. As a known vasoconstrictor factor, its overexpression is one of the causes of hypertension-related vascular dysfunction. EA.hy926 cells were treated with peptides at different dose groups (0.1, 0.25, 0.5 mg / mL) and cultured for 24 h, and then their ET-1 content was measured. At the same time, captopril was used as a positive control.

[0097] The results are asFigure 8 As shown, compared with the blank group, the intracellular ET-1 content decreased significantly after VFDGVLRPGQ treatment, and showed a dose-dependent manner. At the low dose of 0.1 mg / mL, compared with the blank group, the ET-1 content in the VFDGVLRPGQ treatment group decreased significantly, by 30.64%. At the high dose of 0.5 mg / mL, the VFDGVLRPGQ treatment group showed a good inhibitory effect on ET-1 production. Compared with the blank group, the ET-1 content decreased from 24.64 ± 0.055 pg / mL to 15.78 ± 0.70 pg / mL, a decrease of 35.97%, and there was no significant difference from the positive drug captopril treatment group. The results showed that the peptide VFDGVLRPGQ could inhibit the production of ET-1, thus achieving the effect of lowering blood pressure.

[0098] Conclusion:

[0099] A novel ACE inhibitory peptide - VFDGVLRPGQ (1086.582 Da) was isolated, purified and identified from rice bran protease hydrolysate by means of ultrafiltration, reverse-phase high performance liquid chromatography, LC-MS / MS, etc. Its IC 50 value was 0.093 mg / mL, showing a non-competitive inhibition mode. Molecular simulation results showed that VFDGVLRPGQ exhibited strong binding force with ACE protein through a large number of hydrogen bonds and hydrophobic interactions, which might be the reason for its good ACE inhibitory activity. Through the EA.hy926 cell model, it was found that VFDGVLRPGQ could achieve the effect of lowering blood pressure by promoting the production of NO and inhibiting the secretion of ET-1.

[0100] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A rice bran protein-derived ACE inhibitory peptide, characterized in that: its amino acid sequence is Val-Phe-Asp-Gly-Val-Leu-Arg-Pro-Gly-Gln.

2. The preparation method of the rice bran protein-derived ACE inhibitory peptide according to claim 1, characterized in that: it includes the following steps: Step 1: Ultrafilter the rice bran protease hydrolyzate to obtain a fraction <3KDa; Step 2: After the <3KDa fraction passes through a 0.22 μm filter, it is separated once by a C18 column in a preparative liquid chromatography LC-8 system; Step 3: Take the peak with the best activity from the first separation, pass it through a 0.22 μm filter, and then separate it twice by a Shimadzu PRC-ODS(K) steel column in a preparative liquid chromatography LC-8 system; Step 4: After separation and purification, it is further identified and analyzed by HPLC-MS / MS to obtain the described rice bran protein-derived ACE inhibitory peptide.

3. The preparation method of the rice bran protein-derived ACE inhibitory peptide according to claim 2, characterized in that: In the first separation described in Step 2, the chromatographic conditions are as follows: Mobile phase A: distilled water + 0.1% trifluoroacetic acid; Mobile phase B: methanol + 0.1% trifluoroacetic acid; Both mobile phases need to pass through a 0.2 μm filter membrane; Injection volume: 5 mL; Elution flow rate: 10 mL / min; Detection wavelengths: 214 nm and 280 nm; Gradient elution: 0 - 10.00 min: 8% - 15% mobile phase B; 10.00 - 30.00 min: 15% - 50% mobile phase B; 30.00 - 70.00 min: 50% - 70% mobile phase B; 70.00 - 80.00 min: 70% - 95% mobile phase B; 80.00 - 85.00 min: 95% - 8% mobile phase B; In the second separation described in Step 3, the chromatographic conditions are as follows: Mobile phase A: distilled water + 0.1% trifluoroacetic acid; Mobile phase B: methanol + 0.1% trifluoroacetic acid; Both mobile phases need to pass through a 0.2 μm filter membrane; Injection volume: 1 mL; Elution flow rate: 10 mL / min; Detection wavelengths: 214 nm and 280 nm; Gradient elution: 0.00 - 30.00 min: 8% - 50% mobile phase B; 30.00 - 60.00 min: 50% - 70% mobile phase B; 60.00 - 65.00 min: 70% - 95% mobile phase B; 65.00 - 75.00 min: 95% mobile phase B; 75.00 - 80.00 min: 95% - 8% mobile phase B; 80.00 - 95.00 min: 8% mobile phase B.

4. The application of the rice bran protein-derived ACE inhibitory peptide according to claim 1 in the preparation of ACE inhibitors and / or antihypertensive drugs.

5. The application according to claim 4, characterized in that: in the described application, the ACE inhibitor is an ACE non-competitive inhibitor.

6. The application according to claim 4, characterized in that: In the application described above, the antihypertensive drug has the effects of promoting the release of NO and reducing the release of ET-1.

7. The application according to claim 4, wherein: In the application described above, the ACE inhibitor and / or the antihypertensive drug uses the furoprotein-derived ACE inhibitory peptide as an active ingredient, and also contains a pharmaceutically acceptable carrier or excipient.

8. An ACE inhibitor, wherein: It contains the furoprotein-derived ACE inhibitory peptide described in claim 1 as an active ingredient.

9. An antihypertensive drug, wherein: It contains the furoprotein-derived ACE inhibitory peptide described in claim 1 as an active ingredient.

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