Grape seed protein peptide having ACE inhibitory activity and preparation method thereof

Abstract

This invention discloses a grape seed protein peptide with ACE inhibitory activity and its preparation method, belonging to the field of comprehensive utilization of wine fermentation by-products. This invention provides a grape seed protein peptide with ACE inhibitory activity, wherein the grape seed protein peptide is FFYPL, SGMFPFP, or WFFPGPR. The grape seed protein peptide of this invention can be used in pharmaceuticals; it has broad application prospects in the pharmaceutical field as a preventive or adjunctive treatment for patients with hypertension, or those with obesity, cardiovascular and cerebrovascular diseases, and other chronic metabolic disorders.

Description

Technical Field

[0001] This invention relates to grape seed protein peptides with ACE inhibitory activity and their preparation method, belonging to the field of comprehensive utilization of wine fermentation by-products. Background Technology

[0002] Hypertension is one of the leading global cardiovascular diseases. Patients often also have other metabolic disorders such as obesity, prediabetes, and atherosclerosis, which are often asymptomatic in their early stages. Related studies have shown that elevated blood pressure is related to angiotensin II. The renin-angiotensin system (RAS) is a key hormonal regulator of angiotensin II synthesis, primarily occurring in the circulatory system and organs such as the aorta, kidneys, lungs, and brain. Angiotensinogen is cleaved into angiotensin I by renin, which is then converted to angiotensin II by angiotensin-converting enzyme (ACE), thus leading to elevated blood pressure. The main mechanism of action of the angiotensin-converting enzyme (ACE) inhibitory peptide in this invention is to inhibit the catalytic conversion of angiotensin I to angiotensin II by ACE, thereby preventing a sustained increase in blood pressure in patients.

[0003] Grapes are recognized worldwide as one of the most valuable fruits, with 75-80% of the world's grapes used in the wine fermentation industry. This results in a large amount of grape byproducts that have attracted widespread attention. Therefore, to improve the rational resource utilization of wine processing waste, this invention proposes that defatted grape seeds produced during wine fermentation, after being pressed for oil extraction, are also a high-quality source of ACE inhibitory functional peptides.

[0004] Currently, the development of naturally derived antihypertensive functional peptides includes: Chinese patent application CN119264219A discloses two ACE inhibitory peptides extracted, isolated, and purified from rice wine residue, and explores their pathway mechanism through cell model experiments; Chinese patent application CN117756886A discloses a method for preparing a sea buckthorn leaf antihypertensive peptide, and elucidates the potential of the inhibitory peptide from a molecular perspective; commercially available angiotensin-converting enzyme inhibitors were initially isolated from snake venom, and drugs such as captopril, lisinopril, and enalapril have been developed. However, long-term use can lead to serious side effects such as dry cough, rash, and taste disturbance. Therefore, naturally derived ACE inhibitory peptides, due to their advantages of no side effects, high safety, and easy absorption, have become one of the main research directions in this field. Thus, we are committed to promoting the research and development of wine by-products while exploring plant-derived functional peptides with safe and efficient ACE inhibitory activity. Summary of the Invention

[0005] To address the existing problems mentioned above and to further improve the reuse of grape seeds, a byproduct of wine fermentation, this invention evaluates the potential of grape seed protein as a source of ACE (angiotensin-converting enzyme) inhibitory peptides and verifies that grape seed protein hydrolysate has a good ACE inhibitory effect, providing a grape seed protein peptide with ACE inhibitory activity and its preparation method.

[0006] This invention provides a grape seed protein peptide with ACE inhibitory activity, wherein the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR), as shown in SEQ ID NO. 1~3;

[0007] SEQ ID NO.1: FFYPL;

[0008] SEQ ID NO.2: SGMFPFP;

[0009] SEQ ID NO.3: WFFPGPR.

[0010] In one embodiment of the present invention, the grape seed protein peptide has good ACE inhibitory activity, with an ACE inhibitory activity (Ic50 value) of less than 0.5 μg / mL.

[0011] In one embodiment of the present invention, the grape seed protein peptide is prepared from defatted grape seeds.

[0012] In one embodiment of the present invention, the grape seed protein peptide can be used as a medicine; it is used to inhibit ACE and lower blood pressure, and can be used as a preventive or adjunctive treatment for patients with hypertension or chronic metabolic syndrome with obesity, low immunity, cardiovascular and cerebrovascular diseases.

[0013] This invention provides a method for preparing grape seed protein peptides with ACE inhibitory activity, the method comprising:

[0014] 1) Select defatted grape seeds from wine fermentation by-products as raw materials, grind the grape seeds into powder to obtain defatted grape seed powder; mix the obtained defatted grape seed powder with 50% ethanol at a solid-liquid ratio of 1:4~6, let stand and filter to obtain defatted and phenol-free grape seed powder; dissolve the defatted and phenol-free grape seed powder with sodium hydroxide at a solid-liquid ratio of 1:15~18, adjust the pH to 10~12, and extract with ultrasonication while stirring at a frequency of 30~50 kHz for 15~30 min, filter, add cross-linked polyvinylpyrrolidone at a mass-volume ratio of 4~6% (w / v) to the obtained filtrate, mix and react for 20~30 min, remove the lower solid phase to obtain grape seed protein alkaline solution;

[0015] 2) Adjust the pH of the alkaline solution of grape seed protein obtained in step 1) to the isoelectric point of 3.5-4, centrifuge to obtain acidic precipitated protein; finally, add distilled water to reconstitute the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0016] 3) Dissolve the grape seed protein powder obtained in step 2) in a phosphate buffer system, and add alkaline protease at 40-45 °C, pH=9.5-10.5, stirring speed 100-150 r / min, with an enzyme / protein concentration ratio of 3800-4200 U / g for the next enzymatic hydrolysis. After the reaction is stopped by boiling water bath after 3.5-4 h, add trichloroacetic acid solution to precipitate and centrifuge to remove excess protein, and freeze dry to obtain a mixture of grape seed protein peptides.

[0017] In one embodiment of the present invention, the method for preparing grape seed protein peptides includes the following steps:

[0018] This invention employs an ultrasound-assisted alkali-dissolving and acid-precipitating method for protein extraction, and improves the extraction rate and effect by adding cross-linked polyvinylpyrrolidone to adsorb phenols in the protein solution. On the other hand, it selects alkaline protease for enzymatic hydrolysis to release grape seed protein peptides with ACE inhibitory activity.

[0019] In one embodiment of the present invention, the method for preparing grape seed protein peptides includes the following steps:

[0020] 1) Grape seed powder undergoes ultrasonic-assisted alkali dissolution and acid precipitation to extract protein;

[0021] 2) The grape seed protein obtained in step 1) is enzymatically degraded to release protein peptides;

[0022] 3) The hydrolysate obtained from enzymatic hydrolysis in step 2) is purified by ultrafiltration and RP-HPLC, and then obtained by rotary evaporation and freeze-drying.

[0023] To obtain 100% grape seed protein peptides, the grape seed extraction process described above preferably includes the following steps:

[0024] Using defatted grape seeds from wine fermentation byproducts as raw material, whole grape seeds were ground into powder under liquid nitrogen protection. The powder was mixed with 50% anhydrous ethanol and allowed to stand in the dark for 1 hour. The top phase was removed by filtration to obtain defatted and dephenolized grape seed powder. The defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-to-liquid ratio of 1:16. The pH was adjusted to 11 with 6 M sodium hydroxide, and ultrasonic extraction was performed while stirring. The lower solid phase was removed by filtration. PVPP (cross-linked polyvinylpyrrolidone) was then added to the resulting filter and reacted for 30 minutes. The lower solid phase was removed to obtain a grape seed protein alkaline solution. The pH of the alkaline solution was then adjusted to the isoelectric point 3.5 with hydrochloric acid, refrigerated for 2 hours, centrifuged to obtain an acidic precipitate, and freeze-dried to obtain grape seed protein powder.

[0025] In a preferred embodiment of the present invention, the grape seed powder and 50% anhydrous ethanol are mixed in a solid-liquid ratio of 1:5.

[0026] In a preferred embodiment of the present invention, the ultrasonic wave used for extraction is 40 Hz with a power of 200 W and a reaction time of 40 min. In this embodiment of the present invention, extraction using ultrasonic technology at a specific frequency can significantly improve extraction efficiency and reduce time costs.

[0027] In a preferred embodiment of the present invention, the amount of cross-linked polyvinylpyrrolidone used to adsorb polyphenols is 4% (w / w), which can significantly reduce the polyphenol content in grape seed protein. Due to the significant reduction in anthocyanins, the resulting protein powder is also purer in color.

[0028] In a preferred embodiment of the present invention, step 2) specifically involves the following enzymatic hydrolysis steps:

[0029] The grape seed protein powder obtained in step 1) was dissolved in a 0.01M phosphate buffer (PBS) system and adjusted to the optimal reaction conditions of 45℃ and pH=10.0. Alkaline protease was added at an enzyme / protein concentration ratio of 4000U / g for the next enzymatic hydrolysis. The pH and temperature of the entire reaction system were kept stable throughout the process. The reaction was terminated by boiling water bath. A 15% trichloroacetic acid solution was added to precipitate the protein, and the excess protein was removed by centrifugation. The concentrate of grape seed protein hydrolysate was obtained by freeze-drying.

[0030] In a preferred embodiment of the present invention, it has been verified that alkaline protease is the enzyme with the greatest ACE inhibition potential, and the optimal enzymatic hydrolysis time is 4 h. Under this enzymatic hydrolysis method, the resulting grape seed protein hydrolysate has the highest degree of hydrolysis and the strongest ACE inhibition potential.

[0031] To ensure the best inhibitory effect of the obtained grape seed protein solution, the ultrafiltration operation in step 3) is as follows:

[0032] The concentrated grape seed protein hydrolysate obtained in step 2) was purified using a Labscale TFF ultrafiltration system with a molecular weight cutoff of 5 kda to obtain grape seed protein hydrolysate with a molecular weight cutoff of <5 kda. It was then purified again using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kda to obtain grape seed protein hydrolysate with a molecular weight cutoff of 3-5 kda and <3 kda.

[0033] In a preferred embodiment of the present invention, the reversed-phase HPLC in step 3) can be:

[0034] Grape seed protein hydrolysate with a concentration of <3 kDa was separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was HPLC-grade acetonitrile. The detection wavelength was 214 nm. Elution peaks were collected for 9-12 min, and the solvent was removed by rotary evaporation to obtain crude grape seed protein peptides.

[0035] In a preferred embodiment of the present invention, the grape seed protein peptide with specific ACE-inhibiting function prepared by the above method includes a polypeptide with the following amino acid sequence: Phe-Phe-Tyr-Pro-Leu;

[0036] In a preferred embodiment of the present invention, the grape seed protein peptide with ACE inhibition function prepared by the above method comprises a polypeptide with the following amino acid sequence: Ser-Gly-Met-Phe-Pro-Phe-Pro;

[0037] In a preferred embodiment of the present invention, the grape seed protein peptide with ACE inhibition function prepared by the above method comprises a polypeptide with the following amino acid sequence: Trp-Phe-Phe-Pro-Gly-Pro-Arg.

[0038] Grape seed protein peptides containing any one of the above three polypeptides have good ACE inhibitory activity, with an ACE inhibitory activity (Ic50 value) of less than 0.5 μg / mL.

[0039] The present invention also provides an expression vector or recombinant microorganism, wherein the expression vector or recombinant microorganism contains at least one of the above-mentioned grape seed protein peptides.

[0040] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0041] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0042] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprising any one or more of the above-mentioned grape seed protein peptides: Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR);

[0043] The aforementioned grape seed protein peptides can significantly inhibit ACE activity.

[0044] In one embodiment of the present invention, the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field.

[0045] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate, etc.; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0046] The present invention also provides a medicine comprising an effective dose of at least one of the above-mentioned grape seed protein peptides.

[0047] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0048] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0049] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0050] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0051] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0052] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0053] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0054] The present invention also provides the use of at least one or more of the above-mentioned grape seed protein peptides in the preparation of pharmaceuticals; which are used to inhibit ACE activity, lower blood pressure, prevent or treat hypertension or diseases that benefit from ACE inhibition, or prevent or treat chronic metabolic syndrome with obesity, low immunity or cardiovascular and cerebrovascular diseases.

[0055] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0056] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0057] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0058] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0059] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0060] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0061] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0062] The present invention also provides a method for preparing the above-mentioned pharmaceutical product, the method comprising mixing at least one of the above-mentioned grape seed protein peptides with at least one acceptable excipient.

[0063] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0064] Beneficial effects

[0065] (1) The present invention obtained grape seed protein peptides with ACE inhibitory activity, specifically Phe-Phe-Tyr-Pro-Leu or Ser-Gly-Met-Phe-Pro-Phe-Pro or Trp-Phe-Phe-Pro-Gly-Pro-Arg, which have novel structures and are easy to prepare. They can be obtained from grape seed protein or synthesized artificially.

[0066] (2) The three polypeptides obtained in this invention have strong ACE inhibitory activity; among them, the half-maximal inhibitory concentration (IC50) of Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR) is high. 50 The half-maximal inhibitory concentration (IC50) was 25.27 ± 0.7393 nmol / L; the half-maximal inhibitory concentration (IC50) of Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) was 25.27 ± 0.7393 nmol / L. 50 The half-maximal inhibitory concentration (IC50) of Phe-Phe-Tyr-Pro-Leu (FFYPL) was 66.39 ± 26.13 nmol / L; the half-maximal inhibitory concentration (IC50) of Phe-Phe-Tyr-Pro-Leu (FFYPL) was 66.39 ± 26.13 nmol / L. 50 The value was 81.71 ± 5.847 nmol / L;

[0067] (3) The grape seed protein peptide of the present invention can be used in pharmaceuticals; as a preventive or adjunctive treatment for patients with hypertension or chronic metabolic syndrome with obesity, cardiovascular and cerebrovascular diseases, it has broad application prospects in the pharmaceutical field. Attached Figure Description

[0068] Figure 1 The graph shows the peptide concentration results of grape seed protein under different hydrolytic enzymes and different hydrolysis times.

[0069] Figure 2 The figure shows the ACE inhibition rate of grape seed protein under different hydrolytic enzymes and different hydrolysis times. Detailed Implementation

[0070] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are commercially available.

[0072] The defatted grape seed powder involved in the following examples can be prepared by the following method: grape seed powder is mixed with n-hexane at a solid-liquid ratio of 1:5, allowed to stand, and then filtered to obtain defatted grape seed powder.

[0073] Alternatively, defatted grape seeds, a byproduct of wine fermentation, can be used as raw materials.

[0074] The sources of the raw materials involved in the following embodiments are as follows:

[0075] PPVP (crosslinked polyvinylpyrrolidone) was purchased from Shanghai Mairui Biochemical Technology Co., Ltd., product number M86828-100G; Folin-Ciocalteu was purchased from Shanghai Titan Technology Co., Ltd., product number 4140369A.

[0076] The alkaline protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 200 U / mg and a product number of S10154-100g.

[0077] Papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 800 U / mg and product number S10011-100g.

[0078] The pepsin was purchased from Merck Life Science in China, with an enzyme activity of ≥500 U / mg, and the product number was 77160-25g.

[0079] Bromelain was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 300 U / mg and product number S10009-100g.

[0080] The neutral protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 100 U / mg and a product number of S10013-250g.

[0081] The BCA protein concentration assay kit (enhanced version) was purchased from Beyotime Biotechnology Co., Ltd., product number: P0010S;

[0082] The angiotensin-converting enzyme (ACE) inhibitor activity assay kit was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., catalog number: A502.

[0083] The measurement methods involved in the following examples are as follows:

[0084] Methods for measuring ACE (angiotensin-converting enzyme) inhibitory activity:

[0085] The method was based on the Angiotensin-Converting Enzyme Inhibitor Activity Assay Kit (ACE Kit-WST, catalog number: A502) from Dongren Chemical Technology (Shanghai) Co., Ltd. 3-hydroxybutyryl-glycyl-glycyl-glycine (3HB-GGG) was used as a substrate to screen for ACE inhibitors. Under the action of ACE and aminoacylase, 3HB-GGG was cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The ACE inhibition rate was calculated by detecting the absorbance of 3HB. The kit included ACE enzyme working solution, substrate dilution buffer, and chromogenic solution. Activity was measured using a 96-well plate.

[0086] Sample: Add 20 μl of sample, 20 μl of substrate, and 20 μl of enzyme working solution to the well in sequence;

[0087] Negative control: Add 20 μl of pure water, 20 μl of substrate, and 20 μl of enzyme working solution to the wells in sequence;

[0088] Background blank: Add 40 μl of pure water and 20 μl of substrate to the well in sequence;

[0089] Sample blank: Add 20 μl of sample and 240 μl of pure water to the well in sequence;

[0090] Place the above wells in a 37℃ constant temperature incubator and let them stand for 60 min. Add 200 μl of colorimetric solution to each well of the sample, negative control, and background blank, and incubate at room temperature for 10 min. Measure the absorbance at 450 nm using an ELISA reader.

[0091] Ic 50 The half-maximal inhibitory concentration (WMC), i.e., the sample concentration at which the ACE inhibition rate is 50%, was used to dilute the test sample into five concentration gradients spanning 50% based on the preliminary experimental inhibition rate data. The ACE inhibition rate at each gradient was measured, and the data were imported into a GraghPad nonlinear fitting curve to obtain Ic. 50 value.

[0092] Calculation formula:

[0093]

[0094] Methods for determining BCA protein concentration:

[0095] The method was based on the instructions for the Beyotime Biotechnology BCA Protein Concentration Assay Kit (Enhanced Version) (Catalog No.: P0010S). The kit contains BCA working solution, protein standard (20 mg BSA), and protein standard preparation solution. Activity was measured using a 96-well plate.

[0096] Dissolve the protein standard in protein standard preparation solution to prepare a 0.5 mg / ml protein standard solution. Then dilute it with distilled water to prepare concentrations of 0.4, 0.3, 0.2, 0.1, 0.05, and 0.025 mg / ml. Add 20 μl of protein standard and sample solution to each well of a 96-well plate, followed by 200 μl of BCA working solution. Incubate at 37°C for 25 min, and measure the absorbance using an A562 microplate reader. Construct a protein standard curve and calculate the protein concentration of the sample based on the standard curve.

[0097] In the following examples, (w / v) refers to g / ml.

[0098] Example 1: Grape Seed Protein Extraction Process

[0099] 1. Preparation of grape seed protein

[0100] 1) Select defatted Cabernet Sauvignon grape seeds from wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection; thus, defatted grape seed powder is prepared.

[0101] The defatted grape seed powder was mixed with a 50% (w / v) ethanol aqueous solution at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was repeated twice to obtain defatted and phenol-free grape seed powder.

[0102] 2) Dissolve the defatted and dephenolized grape seed powder obtained in step 1) in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16 (w / v), adjust the pH to 11 with 6 M sodium hydroxide, and extract with ultrasound (frequency 40 kHz) for 40 min while stirring. Filter to remove the lower solid phase. Then add PVPP (cross-linked polyvinylpyrrolidone) to the filtrate at a solid-liquid ratio of 4% (w / v), mix and react for 30 min, remove the lower solid phase to obtain grape seed protein alkaline solution;

[0103] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point 3.5 with 6M hydrochloric acid, refrigerate at 4 ℃ for 2 h, and centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to reconstitute the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0104] 2. Optimization of grape seed protein extraction process

[0105] To verify the effects of ethanol extraction pretreatment and polyvinylpyrrolidone (PVP) impurity removal on the efficiency of grape seed protein preparation, and to select defatted Cabernet Sauvignon grape seeds from the same four groups of wine fermentation by-products, the whole grape seeds were ground into powder under liquid nitrogen protection to obtain defatted grape seed powder (preparation method is the same as step 1). This invention designed four sets of control experiments (Table 1):

[0106] Table 1: Four Controlled Trials

[0107]

[0108] The specific method is as follows:

[0109] 1) Preparation of Grape Seed Protein #1:

[0110] The defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16. The pH was adjusted to 11 with 6 M sodium hydroxide, and the mixture was extracted for 40 min by ultrasonication (40 kHz) while stirring. The lower solid phase was removed by filtration. Then, 4% pvpp (cross-linked polyvinylpyrrolidone) was added to the filtrate, and the mixture was reacted for 30 min. The lower solid phase was removed to obtain an alkaline solution of grape seed protein, which was then freeze-dried to obtain grape seed protein #1 (GSP #1).

[0111] 2) Preparation of Grape Seed Protein #2:

[0112] The defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16. The pH was adjusted to 11 with 6 M sodium hydroxide. The mixture was extracted for 40 min with stirring and ultrasonication (frequency 40 kHz). The lower solid phase was removed by filtration to obtain grape seed protein alkaline solution. The solution was freeze-dried to obtain grape seed protein #2 (GSP #2).

[0113] 3) Preparation of Grape Seed Protein #3:

[0114] The defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was repeated twice to obtain defatted and phenol-free grape seed powder. Next, it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6 M sodium hydroxide. Extraction was performed for 40 min with stirring and ultrasonication (frequency 40 kHz). The lower solid phase was removed by filtration. Then, 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) was added to the filtrate, and the mixture was reacted for 30 min. The lower solid phase was removed to obtain a grape seed protein alkaline solution, which was freeze-dried to obtain grape seed protein #3 (GSP #3).

[0115] 4) Preparation of Grape Seed Protein #4:

[0116] The defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was repeated twice to obtain defatted and phenol-free grape seed powder. Then, it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6 M sodium hydroxide. Extraction was performed for 40 min with stirring and ultrasonication (frequency 40 kHz). The lower solid phase was removed by filtration to obtain an alkaline solution of grape seed protein, which was then freeze-dried to obtain grape seed protein #4 (GSP #4).

[0117] The protein content was determined using the Kjeldahl method (GB5009.5-2016) of the standard standard for the determination of protein in food.

[0118] Weigh a thoroughly mixed sample of solid grape seed protein powder into a digestion tube, then add 0.4 g of copper sulfate, 6 g of potassium sulfate, and 20 ml of sulfuric acid into a digestion furnace for digestion. Once the furnace temperature reaches 420℃, continue digestion for 1 hour. At this point, the liquid in the digestion tube will be green and transparent. After cooling, add 50 ml of water and use an automatic Kjeldahl nitrogen analyzer for automatic liquid addition, distillation, titration, and recording of titration data. Multiply the acid consumption by a conversion factor of 6.25 to obtain the protein content.

[0119] The polyphenol content was determined using the Folin-phenol method:

[0120] First, a standard curve was established using the standard phenolic substance catechin. 0.1 mL of a 1 mg / mL sample was added to a 10 mL volumetric flask, followed by 7 mL of distilled water and shaking. Then, 0.5 mL of 1M Folin-Ciocalteu was added. After 1 min, 1.5 mL of 20% (v / v) sodium carbonate solution was added, mixed, and the volume was adjusted to 10 mL with distilled water. The reaction was carried out in the dark for 120 min, and the absorbance was measured at 765 nm. The results were expressed as equivalent to the catechin standard curve.

[0121] 5) Detection of experimental results

[0122] The protein content and polyphenol content of defatted grape seed powder and the above-obtained grape seed protein #1, #2, #3, and #4 were determined, and the results are shown in Table 2.

[0123] Table 2: Grape seed protein content under different extraction methods

[0124]

[0125] As can be seen from Table 2, Grape Seed Protein #3 (GSP #3) has the highest protein content and the lowest total phenol content, indicating that the impurity removal effect is good, the operation is simple, and the cost is low, making it an effective method for extracting grape seed protein.

[0126] Example 2: Preparation of concentrated grape seed protein hydrolysate

[0127] The preparation method of grape seed protein peptides with ACE inhibition is as follows:

[0128] 1. Preparation of grape seed protein

[0129] 1) Select defatted Cabernet Sauvignon grape seeds from wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0130] 2) The defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 hour. The top phase was removed by filtration, and the residue was repeated twice to obtain defatted and phenol-free grape seed powder. Then, it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6 M sodium hydroxide. Extraction was performed for 40 minutes with stirring and ultrasonication (frequency 40 kHz). The lower solid phase was removed by filtration. Next, 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) was added to the filtrate, and the mixture was reacted for 30 minutes. The lower solid phase was then removed to obtain a grape seed protein alkaline solution.

[0131] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point 3.5 with 6M hydrochloric acid, refrigerate at 4 ℃ for 2 h, and centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to reconstitute the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0132] 2. Preparation of concentrated grape seed protein hydrolysate with ACE inhibition

[0133] The grape seed protein powder obtained in step 1 was dissolved in a 0.01 M phosphate buffer (PBS) system. The magnetic stirrer connected to the pH electrode was adjusted to the optimal reaction conditions of 45 ℃, pH=10.0, and stirring speed of 40 rpm. Alkaline protease was added at an enzyme / protein concentration ratio of 4000 U / g for the next enzymatic hydrolysis. The pH and temperature of the entire reaction system were kept stable during the process. The reaction was terminated by boiling water bath after 4 h. 15% (v / v) 1M trichloroacetic acid solution was added to precipitate the protein, and the excess protein was removed by centrifugation at 12000 rpm. The concentrate of grape seed protein hydrolysate was obtained by freeze drying.

[0134] Example 3: Optimization of the preparation method of grape seed protein hydrolysate concentrate

[0135] The specific implementation method is the same as in Example 2, except that the reaction time of 4 h in step 2, (1) is replaced with 0.5, 1, 2, 3, 4, 5 or 6 h, and the alkaline protease in step 2, (2) is replaced with papain, pepsin or neutral protease, bromelain (Table 3). The remaining steps are the same as in Example 2, and grape seed protein hydrolysate concentrate is obtained. The ACE inhibition rate and peptide concentration of the grape seed protein hydrolysate concentrate prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease or bromelain for different reaction times are determined using the above-mentioned ACE inhibition activity determination method and protein concentration determination method.

[0136] The results are as follows Figures 1-2 As shown in Table 3.

[0137] Table 3: ACE inhibition Ic of different hydrolytic enzymes of grape seed protein at different hydrolysis times 50 value

[0138]

[0139] The results showed that the alkaline protease hydrolysate exhibited the strongest biological activity and the highest peptide content at 3 h.

[0140] The ACE inhibition Ic of grape seed protein hydrolysate concentrates prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease, or bromelain for 3 hours was measured. 50 The values ​​and results are shown in Table 4:

[0141] Table 4: ACE inhibition Ic of grape seed protein hydrolysates after 3 hours by different hydrolytic enzymes 50 value

[0142]

[0143] Preliminary verification showed that alkaline protease hydrolysate had the most significant inhibitory effect on ACE enzyme. 50 The concentration was 19.82 ± 1.256 μg soluble protein hydrolysate / mL (pepsin Ic). 50 =21.98±0.018 μg soluble protein hydrolysate / mL, neutral protease Ic 50 =35.18±1.461 μg soluble protein hydrolysate / mL, bromelain Ic 50 =24.58±4.163 μg soluble protein hydrolysate / mL, papain Ic 50 =104.5±12.47 μg soluble protein hydrolysate / mL).

[0144] Example 4: Preparation of grape seed protein peptides with ACE inhibition

[0145] The preparation method of grape seed protein peptides with ACE inhibition is as follows:

[0146] 1. Preparation of grape seed protein

[0147] 1) Select defatted Cabernet Sauvignon grape seeds from wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0148] 2) The defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 hour. The top phase was removed by filtration, and the residue was repeated twice to obtain defatted and phenol-free grape seed powder. Then, it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6 M sodium hydroxide. Extraction was performed for 40 minutes with stirring and ultrasonication (frequency 40 kHz). The lower solid phase was removed by filtration. Next, 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) was added to the filtrate, and the mixture was reacted for 30 minutes. The lower solid phase was then removed to obtain a grape seed protein alkaline solution.

[0149] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point 3.5 with 6M hydrochloric acid, refrigerate at 4 ℃ for 2 h, and centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to reconstitute the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0150] 2. Preparation of grape seed protein peptides with ACE inhibition

[0151] 1) Dissolve the grape seed protein powder obtained in step 1 above in a 0.01 M phosphate buffer (PBS) system. Adjust the magnetic stirrer connected to the pH electrode to the optimal reaction conditions of 45 ℃, pH=10.0, and stirring speed of 40 rpm. Add alkaline protease at an enzyme / protein concentration ratio of 4000 U / g for the next enzymatic hydrolysis. Maintain the pH and temperature of the entire reaction system stable during the process. After 3 h of reaction, stop the reaction in a boiling water bath. Add 1M trichloroacetic acid solution with a final concentration of 15% (v / v) to precipitate the protein and centrifuge at 12000 rpm to remove excess protein. Freeze-dry to obtain grape seed protein hydrolysate concentrate.

[0152] 2) The concentrated grape seed protein hydrolysate obtained in step 1) was separated by ultrafiltration and purified using a Labscale TFF ultrafiltration system with a molecular weight cutoff of 5 kda to obtain grape seed protein hydrolysate with a molecular weight cutoff of <5 kda. The hydrolysate was then purified again using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kda to obtain grape seed protein hydrolysate with a molecular weight cutoff of 3-5 kda and <3 kda.

[0153] 3) The <3 kDa grape seed protein hydrolysate obtained in step 2) was separated by RP-HPLC reversed-phase high-performance liquid chromatography. Mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B was HPLC-grade acetonitrile. The detection wavelength was 214 nm. The elution peaks were: 0–5 min, 2% B; 5–9 min, 2%–7% B; 9–28 min, 7%–30% B; 28–40 min, 30%–50% B; 40–41 min, 50%–100% B; 41–45 min, 100% B; 45–46 min, 100%–2% B; 46–60 min, 2% B. The flow rate was set to 2 mL / min. The elution peaks at 9–12 min were collected, and the solvent was removed by rotary evaporation to obtain crude grape seed protein peptides.

[0154] 4) The crude grape seed protein peptide obtained in step 3) was identified by mass spectrometry, and the components of the grape seed protein peptide were determined by LC-MS / MS.

[0155] The conditions for LC-MS / MS are:

[0156] The high-performance liquid chromatograph (HPLC) was a Dionex U3000; the column was C18, 3µm, 100Å, 75µm x 15 cm; mobile phase A: 0.1% Formic acid in water; mobile phase B: 0.1% Formic acid in Acetonitrile; the flow rate was 600 nL / min, with the following flow rates: 0–8 min 8% B, 8–16 min 13% B, 16–39 min 28% B, 39–50 min 40% B, 50–51 min 95% B, 51–55 min 95% B, 55–56 min 6% B, and 56–60 min 6% B.

[0157] The mass spectrometer was a Thermo Scientific Q Exactive; resolution settings: Level 1 70,000@m / z 200, Level 2 17,500@m / z 200; precursor ion scan range: m / z 300-1400; daughter ion scan range: m / z 100; MS1 AGC: 3e6, ion implantation time: 60 ms; MS2 AGC: 5e4, ion implantation time: 80 ms; ion selection window: 3.0 m / z; fragmentation mode: HCD, energy NCE 27; data-dependent MS / MS: Top 20; dynamic exclusion time: 15 s.

[0158] 5) Screening of grape seed protein peptides with ACE inhibition

[0159] Through computer simulation and in vitro assays, a total of 5 grape seed protein peptides with ACE inhibitory effects were screened (Table 5).

[0160] The results of the analysis of grape seed protein peptide components are as follows: the amino acid sequences of the three most bioactive peptides are Phe-Phe-Tyr-Pro-Leu (FFYPL), Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP), or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0161] Example 5: In vitro assay of the ACE inhibitory capacity of grape seed protein peptides

[0162] The sample is the ACE inhibitory peptide obtained in Example 4 through hydrolysis, ultrafiltration, reversed-phase HPLC and mass spectrometry identification. Its amino acid sequence is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0163] Phe-Phe-Tyr-Pro-Leu (FFYPL), Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP), Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR), and two other screened peptides (Table 5) were synthesized by Shanghai Jier Biochemical Co., Ltd., with a purity ≥98%.

[0164] ACE inhibition of the three peptides FFYPL and SGMFPFP or WFFPGPR 50 Value detection.

[0165] Other grape seed protein peptides with ACE inhibitory effects obtained through screening were synthesized using the above method (Table 5).

[0166] Ic 50 The specific method for determining the value is as follows:

[0167] Sample concentration preparation: Dissolve 2 mg of peptide sample (FFYPL) in 100 μl of distilled water to obtain 20 mg / ml peptide (FFYPL), and then dilute it to obtain 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml and 0.01 mg / ml peptide (FFYPL).

[0168] 1 mg of peptide sample (SGMFPFP) was dissolved in 100 μl of distilled water to obtain 10 mg / ml peptide (SGMFPFP), which was then diluted to obtain 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml and 0.01 mg / ml peptide (SGMFPFP).

[0169] Dissolve 1 mg of peptide sample (WFFPGPR) in 1 ml of distilled water to obtain 1 mg / ml peptide (WFFPGPR), then dilute it to obtain 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml and 0.01 mg / ml peptide (WFFPGPR).

[0170] The method was based on the Angiotensin-Converting Enzyme Inhibitor Activity Assay Kit (ACE Kit-WST, catalog number: A502) from Dongren Chemical Technology (Shanghai) Co., Ltd. 3-hydroxybutyryl-glycyl-glycyl-glycine (3HB-GGG) was used as a substrate to screen for ACE inhibitors. Under the action of ACE and aminoacylase, 3HB-GGG was cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The ACE inhibition rate was calculated by detecting the absorbance of 3HB. The kit included ACE enzyme working solution, substrate dilution buffer, and chromogenic solution. Activity was measured using a 96-well plate.

[0171] Sample: Add 20 μl of sample, 20 μl of substrate, and 20 μl of enzyme working solution to the well in sequence;

[0172] Negative control: Add 20 μl of pure water, 20 μl of substrate, and 20 μl of enzyme working solution to the wells in sequence;

[0173] Background blank: Add 40 μl of pure water and 20 μl of substrate to the well in sequence;

[0174] Sample blank: Add 20 μl of sample and 240 μl of pure water to the well in sequence;

[0175] Place the above wells in a 37℃ constant temperature incubator and let them stand for 60 min. Add 200 μl of colorimetric solution to each well of the sample, negative control, and background blank, respectively, and incubate at room temperature for 10 min. Measure the absorbance at 450 nm using an ELISA reader.

[0176] Ic 50The half-maximal inhibitory concentration (WMC), i.e., the sample concentration at which the ACE inhibition rate is 50%, was used to dilute the test sample into five concentration gradients spanning 50% based on the preliminary experimental inhibition rate data. The ACE inhibition rate at each gradient was measured, and the data were imported into a GraghPad nonlinear fitting curve to obtain Ic. 50 value.

[0177] Calculation formula:

[0178]

[0179] The other two ACE inhibitory peptides inhibit ACE Ic 50 The value detection method is the same as above.

[0180] The results are as follows:

[0181] Table 5: ACE inhibitory capacity of grape seed protein peptides

[0182]

[0183] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A grape seed protein peptide, characterized in that, The grape seed protein peptide is FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide, and its amino acid sequence is shown in SEQ ID NO.1~3 respectively.

2. An expression carrier, characterized in that, The expression vector encodes the grape seed protein peptide of claim 1.

3. The expression vector according to claim 2, characterized in that, The vector is selected from DNA vectors, RNA vectors, or viral vectors.

4. The expression vector according to claim 2, characterized in that, The vector is selected from plasmids, transposon vectors, or CRISPR / Cas9 vectors.

5. A recombinant microorganism, characterized in that, The recombinant microorganism expressed the grape seed protein peptide of claim 1.

6. The recombinant microorganism according to claim 5, characterized in that, The recombined microorganisms are bacteria or fungi.

7. A medicine, characterized in that, The medicine contains an effective dose of the grape seed protein peptide of claim 1.

8. The pharmaceutical product according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

9. The pharmaceutical product according to claim 8, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate.

10. The pharmaceutical product according to claim 9, characterized in that, Other excipients, such as flavoring agents and sweeteners, can also be added to medicines.

11. The pharmaceutical product according to claim 8, characterized in that, The dosage forms of the medicine are oral, injectable, and inhaled.

12. The pharmaceutical product according to claim 11, characterized in that, The oral dosage forms are tablets, capsules, granules, oral liquids, and oral suspensions.

13. The pharmaceutical product according to claim 11, characterized in that, The injectable dosage forms are injection solutions and injection powders for injection.

14. The pharmaceutical product according to claim 11, characterized in that, The inhalation dosage form is an aerosol or powder inhaler.

15. The use of the FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide according to claim 1 in the preparation of antihypertensive drugs.

16. The application according to claim 15, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

17. The application according to claim 16, characterized in that, The excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, mannitol; Fillers: starch, sucrose; wetting agents: glycerin; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red. Lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; Coating materials: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate.

18. The application according to claim 17, characterized in that, Other excipients, such as flavoring agents and sweeteners, can also be added to medicines.

19. The application according to claim 15, characterized in that, The dosage forms of the medicine are oral, injectable, and inhaled.

20. The application according to claim 19, characterized in that, The oral dosage forms are tablets, capsules, granules, oral liquids, and oral suspensions.

21. The application according to claim 19, characterized in that, The injectable dosage forms are injection solutions and injection powders for injection.

22. The application according to claim 19, characterized in that, The inhalation dosage form is an aerosol or powder inhaler.

23. An ACE inhibitor or antihypertensive drug, characterized in that, It uses the grape seed protein peptides FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide as described in claim 1 as its active ingredients.

24. A method for preparing the pharmaceutical product according to any one of claims 7 to 14, characterized in that, The method includes mixing the grape seed protein peptide of claim 1 with at least one acceptable excipient.

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