A soybean protein small molecule peptide with the effect of lowering blood pressure, blood sugar, and cholesterol, and its preparation and application.

Abstract

This invention discloses a soybean protein small molecule peptide with the effect of lowering blood pressure, blood sugar, and cholesterol, as well as its preparation and application, belonging to the field of small molecule peptide technology. The soybean protein small molecule peptide is obtained by targeted enzymatic hydrolysis of soybean protein isolate, containing the bioactive peptide PLEGFPF with the effect of lowering blood pressure, blood sugar, and cholesterol. Functional verification shows that the bioactive peptide PLEGFPF and the soybean protein small molecule peptide containing this peptide have the function of lowering blood lipids; it has the function of lowering blood sugar, with a lower effective concentration and better effect compared with the positive drug metformin; it has the function of lowering blood pressure, with a lower effective concentration and better effect compared with the positive drug captopril. Food-derived bioactive peptides have high biosafety; therefore, they can be applied to the development of related products for lowering or assisting in lowering blood pressure, blood sugar, and cholesterol, with good market prospects and application potential.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a soybean protein small molecule peptide with the effect of lowering blood pressure, blood sugar, and cholesterol, as well as its preparation and application. Background Technology

[0002] The "three highs" refers to hypertension, hyperglycemia, and hyperlipidemia, corresponding to chronic diseases characterized by abnormally elevated blood pressure, blood sugar, and blood lipids. These three conditions often coexist and influence each other. The diagnostic criteria for hypertension are ≥140 / 90 mmHg; hyperglycemia refers to elevated blood glucose levels; and hyperlipidemia manifests as abnormal cholesterol or triglycerides. There is a close pathological link between the three: hyperlipidemia leads to lipid deposition in the vascular endothelium, forming atherosclerotic plaques, causing arteriosclerosis and elevated blood pressure; diabetic patients often have lipid metabolism disorders, increasing the risk of hypertension; and obese hypertensive patients are prone to developing diabetes. Currently, drug treatment is an important measure for controlling the "three highs." In addition to drug treatment, consuming foods that regulate blood lipids, blood sugar, and blood pressure daily is also an economical and effective means of prevention.

[0003] Bioactive peptides (BAPs) are a class of peptide compounds derived from proteins that are beneficial or physiologically active in the life processes of organisms. They are a collective term for various peptides, ranging from dipeptides to complex linear and cyclic structures, formed by different compositions and arrangements of the 20 natural amino acids in proteins. Enzymatic hydrolysis is a common technique in the food industry for producing food-derived peptides. Bioactive peptides produced by enzymatic hydrolysis of proteins have the advantages of high safety and no side effects.

[0004] For example, soybean peptides are small molecule peptides obtained from soybean protein through enzymatic hydrolysis, microbial fermentation, or physical processing. Compared with intact protein, they have higher digestibility and functional activity. Soybean peptide powder contains a variety of bioactive peptides with various physiological activities. Numerous studies have revealed that soybean peptides have antioxidant, blood pressure lowering, cholesterol lowering, blood lipid lowering, anti-fatigue, and immune-enhancing bioactivities (Shen Jianhua. Preparation methods and bioactivity research progress of soybean peptides. Industrial Microbiology, 2025, 55(4):52-57).

[0005] Innovations in preparation technology have driven the development of soybean peptide research. Different preparation methods result in soybean peptides with different functional characteristics, purity, and physicochemical properties. For example, the traditional Bacillus subtilis fermentation process can degrade soybean protein into peptides with ACE inhibitory activity. Patent document CN118325998A discloses the preparation of soybean peptides by fermenting soybeans with a compound fermentation method using a combination of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium longum subsp. infantis, and Bacillus subtilis. The resulting soybean peptide products have excellent effects in enhancing immunity and lowering blood pressure, blood sugar, and cholesterol.

[0006] Therefore, using compound enzymatic hydrolysis technology to regulate the degree of hydrolysis to endow soybean peptides with more biological activity, and analyzing the peptide spectrum to discover highly efficient peptides related to the regulation of the three highs (hypertension, hyperlipidemia, and hyperglycemia) for application in the development of products that lower the three highs, is of great significance for the prevention and treatment of the three highs. Summary of the Invention

[0007] The purpose of this invention is to provide a natural small molecule bioactive peptide that regulates blood lipids, blood sugar, and blood pressure, and to apply it to the development of related products for lowering the three highs or helping to maintain healthy levels of blood lipids, blood sugar, and blood pressure.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention employs a complex enzyme consisting of alkaline protease and trypsin, and a complex enzyme consisting of neutral protease and bromelain, to sequentially enzymatically hydrolyze soybean protein isolate. The hydrolysate is then purified by activated carbon adsorption, filtration through a 600-mesh filter cloth, and separation using polyethylene macroporous adsorption resin to obtain soybean protein hydrolysate. The peptide sequences in the soybean protein hydrolysate are analyzed using LC-MS / MS peptide mapping. Molecular docking technology is then used to investigate peptides that can simultaneously bind to lipid-lowering targets SRC, blood glucose-lowering targets DPP-IV, and blood pressure-lowering targets ACE. A candidate peptide was screened, and its amino acid sequence was identified by mass spectrometry as Pro-Leu-Glu-Gly-Phe-Pro-Phe (PLEGFPF, abbreviated as PF-7), with a molecular weight of 805.4 Da. Further functional verification using the artificially synthesized peptide PLEGFPF revealed that this peptide possesses biological activity in regulating blood lipids, blood glucose, and blood pressure.

[0009] Therefore, the present invention provides a novel bioactive peptide PLEGFPF, wherein the amino acid sequence of the bioactive peptide PLEGFPF is Pro-Leu-Glu-Gly-Phe-Pro-Phe.

[0010] This invention also provides a method for preparing the bioactive peptide PLEGFPF, which can be prepared by solid-phase synthesis. The specific method includes: using an Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as a solid-phase carrier, and sequentially introducing phenylalanine, proline, glycine, glutamic acid, leucine, and proline residues to extend the peptide chain from the C-terminus to the N-terminus, thereby synthesizing the heptapeptide PLEGFPF in a solid phase.

[0011] The bioactive peptide PLEGFPF can also be obtained by enzymatic hydrolysis of soy protein isolate. The specific method includes: mixing soy protein isolate powder with water at a mass ratio of 1:10-15, adding 0.2% (by weight of the soy protein isolate powder) of a complex protein A composed of alkaline protease and trypsin at a mass ratio of 2:1, homogenizing to obtain a pretreatment solution; heating the pretreatment solution to 50-55℃, adding 1% (by weight of the soy protein isolate powder) of a complex protein B composed of alkaline protease and trypsin at a mass ratio of 8:7, hydrolyzing for 3.5 hours, then adding 0.3% (by weight of the soy protein isolate powder) of a complex protein C composed of neutral protease and bromelain at a mass ratio of 1:1.2, continuing hydrolysis for 3 hours to obtain an enzymatic hydrolysate; and separating the heptapeptide PLEGFPF from the hydrolysate.

[0012] The present invention also provides the application of the bioactive peptide PLEGFPF in the preparation of lipid-lowering and / or blood glucose-lowering and / or blood pressure-lowering products.

[0013] This invention demonstrates that the heptapeptide PLEGFPF has regulatory effects on blood lipids, blood glucose, and blood pressure. In a hyperlipidemia model, intervention with heptapeptide PLEGFPF significantly reduced triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels. In a hyperglycemia model, intervention with heptapeptide PLEGFPF significantly reduced blood glucose levels, with an effective concentration that was 1 / 20th that of the positive control drug metformin. In a hypertension model, intervention with heptapeptide PLEGFPF significantly reduced blood flow activity and angiotensin II levels, with an effective concentration that was 1 / 20th that of the positive control drug captopril. Therefore, it can be applied to the development of products that lower or assist in lowering the "three highs" (hyperlipidemia, hyperglycemia, and hypertension), or help maintain healthy levels of blood lipids, blood glucose, and blood pressure.

[0014] Furthermore, the product may be, but is not limited to, pharmaceuticals and health foods. Specifically, the pharmaceuticals are drugs for treating hyperlipidemia and / or diabetes and / or hypertension, used to lower at least one of the following: blood lipids, blood sugar, and blood pressure. The health foods are used to help maintain at least one of the following: blood lipid levels, blood sugar levels, and blood pressure levels.

[0015] Another object of the present invention is to provide a soybean protein small molecule peptide with the effect of lowering blood pressure, blood sugar, and cholesterol, wherein the preparation method of the soybean protein small molecule peptide includes: (1) Mix soy protein isolate powder with water at a mass ratio of 1:10-15, add 0.1-0.3% of the weight of soy protein isolate powder of complex protein A composed of alkaline protease and trypsin at a mass ratio of 2:1, homogenize to obtain a pretreatment solution; (2) Heat the pretreatment solution to 50-55℃, add 0.5-1.5% of the weight of soy protein isolate powder, a complex protein B composed of alkaline protease and trypsin in a mass ratio of 8:7, and enzymatically hydrolyze for 3-4 hours. Then add 0.2-0.4% of the weight of soy protein isolate powder, a complex protein C composed of neutral protease and bromelain in a mass ratio of 1:1.2, and continue enzymatic hydrolysis for 2.5-3.5 hours to obtain the enzymatic hydrolysate. (3) Add activated carbon to the enzymatic hydrolysate for adsorption, then filter it through a 600-mesh filter cloth. The filtered liquid is adsorbed by polyethylene macroporous adsorption resin and eluted with sodium hydroxide solution of 0.01% by mass and volume. The eluent is then desalted, concentrated, sterilized and dried to obtain the soybean protein small molecule peptide; which contains the bioactive peptide PLEGFPF.

[0016] This invention demonstrates that the soybean protein small molecule peptides have the effect of regulating blood lipids, blood glucose, and blood pressure. In a hyperlipidemia model, intervention with the soybean protein small molecule peptides significantly reduced triglyceride levels. In a hyperglycemia model, intervention with the soybean protein small molecule peptides significantly reduced blood glucose levels, exhibiting a lower effective concentration and superior efficacy compared to the positive control drug metformin. In a hypertension model, intervention with the soybean protein small molecule peptides significantly reduced blood flow activity and angiotensin II levels, exhibiting a lower effective concentration and superior efficacy compared to the positive control drug captopril.

[0017] Therefore, the present invention provides the application of the aforementioned soybean protein small molecule peptides in the preparation of products that lower blood lipids and / or lower blood sugar and / or lower blood pressure.

[0018] Furthermore, the product may be, but is not limited to, pharmaceuticals or health foods.

[0019] Specifically, the present invention provides a pharmaceutical composition for lowering the levels of the three highs: high blood lipids, high blood sugar, and high blood pressure. The pharmaceutical composition comprises an effective dose of a bioactive peptide PLEGFPF with the amino acid sequence Pro-Leu-Glu-Gly-Phe-Pro-Phe or a small molecule peptide of soybean protein containing the bioactive peptide PLEGFPF, and a pharmaceutically acceptable carrier.

[0020] The bioactive peptide PLEGFPF or the soybean protein small molecule peptide in the pharmaceutical composition provided by the present invention can be used as the sole active ingredient for lowering blood pressure, blood sugar, and cholesterol, or it can be combined with other active ingredients that have the same effect.

[0021] In this invention, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of this invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject.

[0022] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, and lubricants.

[0023] The present invention uses the bioactive peptide PLEGFPF or the soybean protein small molecule peptide as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares the formulation according to the formulation preparation method described in pharmaceutical science.

[0024] Furthermore, the pharmaceutical composition may be in the form of, but is not limited to, an oral formulation. Specifically, the formulation may be, but is not limited to, an oral liquid, capsule, tablet, granule, or powder.

[0025] This invention provides a health food that helps maintain healthy levels of blood lipids, blood sugar, and blood pressure. The health food includes a bioactive peptide PLEGFPF with the amino acid sequence Pro-Leu-Glu-Gly-Phe-Pro-Phe or a small molecule peptide of soybean protein containing the bioactive peptide PLEGFPF, as well as food science-acceptable excipients.

[0026] In this invention, the food-grade excipients that are acceptable in food science are those capable of delivering an effective dose of the active substance of this invention without interfering with the bioactivity of the active substance.

[0027] Furthermore, the dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

[0028] The beneficial effects of this invention are as follows: This invention provides a bioactive peptide PLEGFPF with effects of lowering blood pressure, blood sugar, and cholesterol, as well as a soybean protein small molecule peptide containing this peptide. The peptide can be obtained through artificial synthesis or targeted enzymatic hydrolysis of soybean protein isolate. Functional validation in a hyperlipidemia model shows that the bioactive peptide PLEGFPF and the soybean protein small molecule peptide containing this peptide have lipid-lowering functions; functional validation in a hyperglycemia model shows that the bioactive peptide PLEGFPF and the soybean protein small molecule peptide containing this peptide have blood sugar-lowering functions, with a lower effective concentration and better effect compared to the positive control drug metformin; functional validation in a hypertension model shows that the bioactive peptide PLEGFPF and the soybean protein small molecule peptide containing this peptide have blood pressure-lowering functions, with a lower effective concentration and better effect compared to the positive control drug captopril. Furthermore, food-derived bioactive peptides have high biosafety; therefore, they can be applied to the development of products for lowering or assisting in lowering blood pressure, blood sugar, and cholesterol, showing good market prospects and application potential. Attached Figure Description

[0029] Figure 1The figure shows the effect of soybean peptides prepared in Example 1 on zebrafish triglycerides. The # symbol indicates a significant difference compared with the blank group, where # means p < 0.05 and ### means p < 0.001; the * symbol indicates a significant difference compared with the model group, where * means p < 0.05 and ** means p < 0.01.

[0030] Figure 2 The figure shows the effect of soybean peptides prepared in Example 1 on blood glucose levels in high-sugar zebrafish. The # symbol indicates a significant difference compared with the blank group, and #### indicates p < 0.0001; the * symbol indicates a significant difference compared with the model group, and *** indicates p < 0.001.

[0031] Figure 3 The figure shows the effect of soybean peptides prepared in Example 1 on blood flow velocity in zebrafish. The # symbol indicates a significant difference compared with the blank group, and ### indicates p < 0.001; the * symbol indicates a significant difference compared with the model group, and * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0032] Figure 4 The figure shows the effect of soybean peptides prepared in Example 1 on angiotensin II in zebrafish. The # symbol indicates a significant difference compared with the blank group, and ### indicates p < 0.001; the * symbol indicates a significant difference compared with the model group, and ** indicates p < 0.01, *** indicates p < 0.001.

[0033] Figure 5 This is a total ion diagram of soybean peptides.

[0034] Figure 6 This is the primary mass spectrum of the heptapeptide PLEGFPF.

[0035] Figure 7 This is a secondary mass spectrum of the heptapeptide PLEGFPF. In the figure, b2 represents the second b-type fragment ion generated by the N-terminal cleavage of the peptide, y2 represents the second y-type fragment ion generated by the C-terminal cleavage of the peptide, b3 represents the third b-type fragment ion generated by the N-terminal cleavage of the peptide, b4 represents the fourth b-type fragment ion generated by the N-terminal cleavage of the peptide, and b5 represents the fifth b-type fragment ion generated by the N-terminal cleavage of the peptide.

[0036] Figure 8 The figure shows the effect of heptapeptide PLEGFPF on zebrafish triglycerides. The # symbol indicates a significant difference compared with the blank group, and ### indicates p < 0.001; the * symbol indicates a significant difference compared with the model group, and * indicates p < 0.05, ** indicates p < 0.01.

[0037] Figure 9The effect of heptapeptide PLEGFPF on total cholesterol in zebrafish is shown in the figure. The # symbol indicates a significant difference compared with the blank group, and ## indicates p < 0.01; the * symbol indicates a significant difference compared with the model group, and *** indicates p < 0.001.

[0038] Figure 10 The figure shows the effect of heptapeptide PLEGFPF on low-density lipoprotein in zebrafish. The # symbol indicates a significant difference compared with the control group, where # means p < 0.05; the * symbol indicates a significant difference compared with the model group, where ** means p < 0.01 and *** means p < 0.001.

[0039] Figure 11 The effect of heptapeptide PLEGFPF on blood glucose levels in high-sugar zebrafish is shown in the figure. The # symbol indicates a significant difference compared with the control group, and #### indicates p < 0.0001; the * symbol indicates a significant difference compared with the model group, and **** indicates p < 0.0001.

[0040] Figure 12 The effect of heptapeptide PLEGFPF on blood flow velocity in zebrafish is shown in the figure. The # symbol indicates a significant difference compared with the control group, and ### indicates p < 0.001; the * symbol indicates a significant difference compared with the model group, and * indicates p < 0.05, ** indicates p < 0.01.

[0041] Figure 13 The figure shows the effect of heptapeptide PLEGFPF on angiotensin II in zebrafish. The # symbol indicates a significant difference compared with the blank group, and ## indicates p < 0.01; the * symbol indicates a significant difference compared with the model group, and * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0044] The soy protein isolate used in the following examples was purchased from Linyi Shansong Biological Products Co., Ltd.; alkaline protease, trypsin, neutral protease, and bromelain were purchased from Nanning Pangbo Bioengineering Co., Ltd.; and the polyethylene macroporous adsorption resin was HPD-800.

[0045] Example 1: Preparation of soybean peptides This embodiment provides a method for preparing soybean peptides by enzymatic hydrolysis of soybean protein isolate using a complex protease. The specific steps are as follows: 1. Feeding pretreatment: Add 1850 kg of water to the reactor, then slowly add 150 kg of soy protein isolate, stir evenly, then add 300 g of complex proteinase A (200 g of alkaline proteinase and 100 g of trypsin), and homogenize at 10000 r / min for 20 min. 2. Enzymatic hydrolysis: Heat the liquid to 53℃, add 1.5 kg of compound proteinase B (800 g of alkaline proteinase and 700 g of trypsin) and hydrolyze for 3.5 hours. Then add 450 g of compound proteinase C (200 g of neutral proteinase and 250 g of bromelain) and continue hydrolysis for 3 hours. 3. Adsorption filtration: Heat the liquid to 80℃, add 20 kg of activated carbon for adsorption for 30 min, and then filter through a 600-mesh filter cloth. 4. Adsorption separation: The filtered liquid is adsorbed by pre-activated polyethylene macroporous adsorption resin for 30 min, and then eluted with 0.01% sodium hydroxide solution by mass / volume. 5. Nanofiltration: The eluent is desalted by nanofiltration using a 200 Da nanofiltration membrane; 6. Concentration: After desalting, the liquid is concentrated to a sugar content of 25° and then sterilized and dried to produce soybean peptide powder.

[0046] Example 2: The effect of soybean peptides on lowering blood pressure, blood sugar, and cholesterol in zebrafish I. Evaluation of lipid-lowering efficacy Wild-caught AB zebrafish at 5 dpf were placed in 6-well plates containing culture medium, 30 fish per well, with 3 wells per group, divided into a blank group, a model group, and different dose treatment groups. The intervention lasted for 72 h. After the intervention, the zebrafish were washed once with PBS, collected in 1.5 mL sterile centrifuge tubes, and ground with a certain amount of PBS. After grinding, the supernatant was collected by centrifugation and the triglyceride (TG) was measured (kit purchased from Nanjing Jiancheng Bioengineering Institute).

[0047] Grouping: 1) Blank group: 5 mL of system water / well. System water formula: NaCl 35 g, NaHCO3 2 g, CaCl2 1 g, KCl 0.5 g, diluted to 1 L with water; 2) Model group: Egg yolk powder (final concentration of 0.2% by mass / volume in system water) + 5 mL system water / well. A high-fat diet model was established. 3) Intervention group: Egg yolk powder (final concentration of 0.2% by mass / volume percentage in system water) + different concentrations of soybean peptide (final concentrations of 100, 50, 20, and 10 μg / mL) + 5 mL of system water / well.

[0048] The results are as follows Figure 1 The results showed that 0.2% egg yolk powder significantly increased the TG content in the model group, indicating the successful establishment of the 0.2% high-fat zebrafish model. Intervention with soybean peptides significantly reduced the triglyceride content in zebrafish, demonstrating the significant lipid-lowering effect of soybean peptides. Specifically, intervention with 50 μg / mL soybean peptides reduced the TG content in high-fat zebrafish by 48.96%.

[0049] II. Evaluation of hypoglycemic efficacy Wild-caught AB zebrafish at 4 dpf were placed in 6-well plates containing culture medium, with 10 zebrafish per group (3 wells per group), divided into a blank group, a model group, and groups receiving different doses of the drug. The intervention lasted for 24 hours. After the intervention, the zebrafish were washed three times with PBS, collected in 1.5 mL sterile centrifuge tubes, and ground with a certain amount of PBS. After grinding, the supernatant was collected by centrifugation, and the blood glucose content was measured (the kit was purchased from Nanjing Jiancheng Biotechnology Institute).

[0050] Grouping: (1) Blank group: 5 mL of system water / well; (2) Model group: alloxan (final concentration in system water 333 μM) + glucose (final mass-volume percentage concentration in system water 2.67%) + 5 mL system water / well; (3) Positive control group: alloxan (final concentration in system water 333 μM) + glucose (final concentration in system water 2.67% by mass / volume percentage) + metformin (final concentration in system water 5 μg / mL) + 5 mL system water / well; (4) Intervention group: alloxan (final concentration in system water 333 μM) + glucose (final concentration in system water 2.67% by mass / volume percentage) + different concentrations of soybean peptide (final concentration in system water 1, 5, 10, 20 μg / mL) + 5 mL system water / well.

[0051] The results are as follows Figure 2 As shown, compared to the control group, the glucose content in the zebrafish in the model group was significantly increased, indicating successful model establishment. Metformin, as a positive control, significantly reduced glucose content. Soybean peptides at concentrations ranging from 1 to 20 μg / ml significantly reduced glucose content in zebrafish, indicating that soybean peptides have a significant hypoglycemic effect. Specifically, compared to the hyperglycemic model group, 20 μg / mL soybean peptides reduced blood glucose levels by 52.36%.

[0052] III. Evaluation of the blood pressure lowering effect: 3.1 Measurement of blood flow activity Wild-caught AB strain zebrafish embryos at 3 dpf were placed in six-well plates containing 6 mL of culture water, 15 zebrafish per well, and cultured in an incubator at 28°C for three consecutive days. Ten zebrafish were randomly selected from each experimental group and placed under a heart rate and blood flow analysis system to record the blood flow video of the zebrafish. The blood flow activity (%) of the zebrafish was analyzed.

[0053] Specific dosing groups: (1) Blank group: Propylthiouracil (PTU, with 120 μL of 1.5 g / L PTU solution added) + system water; (2) Model group: N-nitro-L-arginine methyl ester (L-NAME) (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + system water; (3) Positive control group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + captopril (final concentration 40 μg / mL) + system water; (4) Dosage group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + different concentrations of soybean peptide (final concentration 10, 20, 30, 40, 50 μg / mL) + system water.

[0054] 3.2 Measurement of Angiotensin II (Ang II) Levels Wild-caught AB strain zebrafish embryos at 3 dpf were placed in six-well plates containing 6 mL of culture water, 30 embryos per well, in triplicate, and administered the drug for 3 consecutive days. After drug administration, each group of fish was washed twice with PBS and collected in 1.5 mL Eppendorf tubes. 200 μL of PBS buffer was added, and the mixture was homogenized, centrifuged, and the supernatant was collected. The supernatant was then analyzed using a zebrafish angiotensin II (Ang II) ELISA kit.

[0055] Specific dosing groups: (1) Blank group: PTU (120 μL of 1.5 g / L PTU solution added) + system water; (2) Model group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of 1.5 g / L PTU solution added) + system water; (3) Positive control group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + captopril (final concentration 40 μg / mL) + system water; (4) Drug administration group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + soybean peptides of different concentrations (final concentrations of 10, 20, 30, 40, 50 μg / mL) + system water.

[0056] The results are as follows Figure 3 , Figure 4 As shown, compared to the control group, the blood flow activity and angiotensin II levels in the model group zebrafish were significantly increased, indicating successful model establishment. The positive control group, captopril (an antihypertensive drug), significantly reduced blood flow activity and angiotensin II levels in zebrafish. Soybean peptide concentrations within the range of 10-50 μg / mL significantly reduced blood flow activity and angiotensin II levels in zebrafish, indicating that soybean peptides have a significant antihypertensive effect. Specifically, compared to the hypertension model group, 10 μg / mL of soybean peptides significantly reduced blood flow activity by 45.04% and angiotensin II levels by 19.12% in zebrafish.

[0057] Example 3: Discovery of the active ingredients in soybean peptides that lower blood pressure, blood sugar, and cholesterol The peptide spectra of soybean peptides were commissioned to Zhejiang University for peptide spectrum analysis. The total ion mass spectrum of soybean peptides is shown below. Figure 5 As shown, the full peptide profile data of soybean peptides were obtained by checking the protein database.

[0058] Key targets for lowering blood lipids identified through network pharmacology screening include SRC (non-receptor tyrosine kinase), AKT1 (serine / threonine kinase), MMP9 (matrix metalloproteinase), CASP3 (cysteine ​​protease), CASP8 (cysteine ​​protease), and IL1β (pro-inflammatory cytokine).

[0059] Peptides obtained from soybean peptide peptiography using SRC targets were classified according to their relative abundance >10. 7 Peptides with a score >0.5 were initially screened. The peptides after initial screening were then subjected to molecular docking screening for peptides with lipid-lowering effects. Potential peptides obtained from docking were then subjected to docking screening for hypoglycemic targets (DPP-IV) and hypotensive targets (ACE). Finally, common peptides were selected as potential active peptides for lowering blood pressure, blood sugar, and cholesterol. The specific screening results are shown in Table 1.

[0060] Table 1. Potential bioactive peptides that can bind to three targets simultaneously: lowering blood lipids, lowering blood sugar, and lowering blood pressure. The heptapeptide PLEGFPF underwent primary and secondary structure analysis by mass spectrometry, and the results are as follows: Figure 6 and Figure 7 As shown, the [M+H]+ ion signal of the heptapeptide PLEGFPF in the primary structure is 806.407 m / z, which is basically consistent with the molecular weight of heptapeptide PLEGFPF, which is 805.4 Da. The amino acid sequence of heptapeptide PLEGFPF in the secondary structure is Pro-Leu-Glu-Gly-Phe-Pro-Phe.

[0061] Example 4: Synthesis of the active peptide PLEGFPF In this embodiment, the peptide PLEGFPF was synthesized artificially. Specifically, it was synthesized by Shenzhen Borunsida Biotechnology Co., Ltd., with a purity of ≥98%.

[0062] The specific synthesis method includes the following steps: S1. Weigh Fmoc-Phe-Wang Resin and place it in a glass reaction column. Add DCM to swell the solution for 30 min, then remove the DCM under reduced pressure. S2. Wash the resin three times with DMF, add 20% piperidine / DMF solution and react for 20 min to remove the protecting group fmoc, remove the solution under reduced pressure, and wash with DMF six times. S3. Weigh out the second amino acid Fmoc-Pro-OH and TBTU respectively and add them to the resin. Dissolve them in DMF and add DIEA. React for 30 min. Take the resin to perform a color reaction and observe the color of the solution and the color of the resin. If the solution is bright yellow and the resin is yellow, it means that the reaction is complete. Remove the solvent under reduced pressure. S4. Repeat steps S2 and S3 to couple the corresponding amino acids in sequence until the last amino acid Fmoc-Pro(otbu)-oh is attached. Then wash three times each with DMF, DCM and methanol, and dry the resin. S5. Add lysis buffer to remove resin and amino acid side chain protecting groups, filter with sand core, add diethyl ether to the filtrate to precipitate, centrifuge and wash the solid 3 times, dry and detect by MS to obtain heptapeptide PLEGFPF.

[0063] Example 5: The efficacy of heptapeptide PLEGFPF in lowering blood pressure, blood sugar, and cholesterol in a zebrafish model. I. Evaluation of lipid-lowering efficacy Wild-caught AB zebrafish (5 dpf) were placed in 6-well plates containing culture medium, 30 fish per well, with 3 wells per group. The groups were divided into a control group, a model group, and different dose treatment groups. The intervention lasted 72 h. After the intervention, the zebrafish were washed once with PBS, collected in 1.5 mL sterile centrifuge tubes, and ground with PBS. After grinding, the supernatant was collected and used to determine triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) (kits purchased from Nanjing Jiancheng Bioengineering Institute).

[0064] Grouping: (1) Blank group: 5 mL of system water / well; (2) Model group: Egg yolk powder (final concentration 0.2% by mass / volume) + 5 mL system water / well; (3) Intervention group: Egg yolk powder (final concentration of 0.2% by mass / volume percentage) + different concentrations of peptide PLEGFPF (final concentrations of 20, 10, 5, 1, 0.5, 0.25 μg / mL) + 5 mL of system water / well.

[0065] The results are as follows Figures 8-10 The results showed that a 0.2% concentration of egg yolk powder significantly increased the levels of TG, TC, and LDL in the model group, indicating the successful establishment of a high-fat zebrafish model. Intervention with the heptapeptide PLEGFPF significantly reduced the levels of TG, TC, and LDL in high-fat zebrafish, demonstrating its significant lipid-lowering effect. Specifically, intervention with 1 μg / mL of heptapeptide PLEGFPF significantly reduced TG levels by 39.07%, TC levels by 47.37%, and LDL levels by 23.42% in high-fat zebrafish.

[0066] II. Evaluation of hypoglycemic efficacy Wild-caught AB zebrafish with a 4 dpf growth rate were placed in 6-well plates containing culture medium, with 10 zebrafish per group (3 wells per group), divided into a control group, a model group, and different dose treatment groups. The intervention lasted for 24 hours. After the intervention, the zebrafish were washed three times with PBS, collected in 1.5 mL sterile centrifuge tubes, and ground with a certain amount of PBS. After grinding, the supernatant was collected by centrifugation, and the blood glucose content was measured.

[0067] Grouping: (1) Blank group: 5 mL of system water / well; (2) Model group: alloxan (final concentration 333 μM) + glucose (final concentration 2.67% by mass / volume percentage) + 5 mL system water / well; (3) Positive control group: alloxan (final concentration 333 μM) + glucose (final concentration 2.67% by mass / volume) + metformin (final concentration 5 μg / mL) + 5 mL system water / well; (4) Intervention group: alloxan (final concentration 333 μM) + glucose (final concentration 2.67% by mass / volume percentage) + different concentrations of peptide PLEGFPF (final concentrations of 0.25, 0.5, 1, 5, 10, 20 μg / mL) + 5 mL of system water / well.

[0068] The results are as follows Figure 11 As shown, compared to the control group, the glucose content in the zebrafish in the model group was significantly increased, indicating successful modeling. Metformin, as a positive control drug, significantly reduced glucose levels. The heptapeptide PLEGFPF, at concentrations ranging from 0.25 to 20 μg / mL, significantly reduced glucose levels in zebrafish, indicating that PLEGFPF has a significant hypoglycemic effect. Furthermore, its effective concentration is 1 / 20th that of metformin, meaning its hypoglycemic effect is 20 times that of metformin. Specifically, compared to the hyperglycemic model group, 0.25 μg / mL of PLEGFPF reduced blood glucose levels in zebrafish by 41.84%.

[0069] III. Evaluation of the blood pressure lowering effect: 3.1 Measurement of blood flow activity Wild-caught AB strain zebrafish embryos at 3 dpf were placed in six-well plates containing 6 mL of culture water, 15 zebrafish per well, and cultured in an incubator at 28°C for three consecutive days. Ten zebrafish were randomly selected from each experimental group and placed in a heart rate and blood flow analysis system to record the blood flow video and analyze the blood flow activity of the zebrafish.

[0070] Specific dosing groups: (1) Blank group: PTU (120 μL of 1.5 g / L PTU solution added) + system water; (2) Model group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of 1.5 g / L PTU solution added) + system water; (3) Positive control group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + captopril (final concentration 40 μg / mL) + system water; (4) Drug administration group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + different concentrations of peptide PLEGFPF (final concentrations of 2, 4, 6, 8, 10 μg / mL) + system water.

[0071] 3.2 Measurement of Angiotensin II (Ang II) Levels Wild-caught AB strain zebrafish embryos at 3 dpf were placed in six-well plates containing 6 mL of culture water, 30 embryos per well, in triplicate, and administered the drug for 3 consecutive days. After drug administration, each group of fish was washed twice with PBS, collected in 1.5 mL Eppendorf tubes, and 200 μL of PBS buffer was added. The mixture was homogenized, centrifuged, and the supernatant was collected. The supernatant was then analyzed using a zebrafish angiotensin II (Ang II) ELISA kit.

[0072] Specific dosing groups: (1) Blank group: PTU (120 μL of 1.5 g / L PTU solution added) + system water; (2) Model group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of 1.5 g / L PTU solution added) + system water; (3) Positive control group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + captopril (final concentration 40 μg / mL) + system water; (4) Drug administration group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + different concentrations of peptide PLEGFPF (final concentrations of 2, 4, 6, 8, 10 μg / mL) + system water.

[0073] The results are as follows Figure 12 , Figure 13 As shown, compared to the control group, the blood flow activity and angiotensin II levels in the model group zebrafish were significantly increased, indicating successful model establishment. The positive control drug, captopril (an antihypertensive drug), significantly reduced blood flow activity and angiotensin II levels in zebrafish. The heptapeptide PLEGFPF, at concentrations ranging from 2 to 10 μg / mL, significantly reduced blood flow activity and angiotensin II levels in zebrafish, indicating that PLEGFPF has a significant antihypertensive effect. Furthermore, its effective concentration is 1 / 20th that of captopril, meaning its antihypertensive effect is 20 times that of captopril. Specifically, compared to the hypertension model group, 2 μg / mL of PLEGFPF significantly reduced blood flow activity by 39.85% and angiotensin II levels by 8.98% in zebrafish.

[0074] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Use of the biologically active peptide PLEGFPF for the preparation of a medicament for lowering blood lipids and / or blood sugar and / or blood pressure, characterized in that, The amino acid sequence of the bioactive peptide PLEGFPF is Pro-Leu-Glu-Gly-Phe-Pro-Phe; the effective concentration of bioactive PLEGFPF for lowering blood lipids is 0.5-1 μg / mL; the effective concentration of bioactive PLEGFPF for lowering blood glucose is 0.25-20 μg / mL; and the effective concentration of bioactive PLEGFPF for lowering blood pressure is 2-4 μg / mL.

2. Use according to claim 1, wherein The bioactive peptide PLEGFPF is prepared by solid-phase synthesis or by enzymatic hydrolysis of soybean protein isolate.

3. The application of the bioactive peptide PLEGFPF in the preparation of health foods that help maintain healthy levels of blood lipids and / or blood sugar and / or blood pressure, characterized in that, The amino acid sequence of the bioactive peptide PLEGFPF is Pro-Leu-Glu-Gly-Phe-Pro-Phe; the effective concentration of bioactive PLEGFPF for lowering blood lipids is 0.5-1 μg / mL; the effective concentration of bioactive PLEGFPF for lowering blood glucose is 0.25-20 μg / mL; and the effective concentration of bioactive PLEGFPF for lowering blood pressure is 2-4 μg / mL.

4. The application of a small molecule soybean protein peptide in the preparation of lipid-lowering and / or blood sugar-lowering and / or blood pressure-lowering drugs, characterized in that, The preparation method of the soybean protein small molecule peptides includes: (1) Mix soy protein isolate powder with water at a mass ratio of 1:10-15, add 0.1-0.3% of the weight of soy protein isolate powder of complex protein A composed of alkaline protease and trypsin at a mass ratio of 2:1, homogenize to obtain a pretreatment solution; (2) Heat the pretreatment solution to 50-55℃, add 0.5-1.5% of the weight of soy protein isolate powder, a complex protein B composed of alkaline protease and trypsin in a mass ratio of 8:7, and enzymatically hydrolyze for 3-4 hours. Then add 0.2-0.4% of the weight of soy protein isolate powder, a complex protein C composed of neutral protease and bromelain in a mass ratio of 1:1.2, and continue enzymatic hydrolysis for 2.5-3.5 hours to obtain the enzymatic hydrolysate. (3) Activated carbon is added to the enzymatic hydrolysate for adsorption, and then filtered through a 600-mesh filter cloth. The filtered liquid is adsorbed by polyethylene macroporous adsorption resin and eluted with sodium hydroxide solution of 0.01% by mass and volume. The eluent is then desalted, concentrated, sterilized and dried to obtain the soybean protein small molecule peptide, which contains the bioactive peptide PLEGFPF with the amino acid sequence Pro-Leu-Glu-Gly-Phe-Pro-Phe.

5. The application as described in claim 4, characterized in that, The drug comprises an effective dose of the aforementioned small molecule peptide of soybean protein, and a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the compound is ###0002### The pharmaceutically acceptable carriers include one or more of the following: fillers, wetting agents, disintegrants, binders, and lubricants.

7. The use according to claim 5, wherein the compound is ###0002### The drug is in the form of an oral formulation.

8. The use of a soy protein small molecule peptide in the preparation of a health food for helping to maintain a healthy level of blood lipid and / or blood sugar and / or blood pressure, characterized in that, The preparation method of the soybean protein small molecule peptides includes: (1) Mix soy protein isolate powder with water at a mass ratio of 1:10-15, add 0.1-0.3% of the weight of soy protein isolate powder of complex protein A composed of alkaline protease and trypsin at a mass ratio of 2:1, homogenize to obtain a pretreatment solution; (2) Heat the pretreatment solution to 50-55℃, add 0.5-1.5% of the weight of soy protein isolate powder, a complex protein B composed of alkaline protease and trypsin in a mass ratio of 8:7, and enzymatically hydrolyze for 3-4 hours. Then add 0.2-0.4% of the weight of soy protein isolate powder, a complex protein C composed of neutral protease and bromelain in a mass ratio of 1:1.2, and continue enzymatic hydrolysis for 2.5-3.5 hours to obtain the enzymatic hydrolysate. (3) Activated carbon is added to the enzymatic hydrolysate for adsorption, and then filtered through a 600-mesh filter cloth. The filtered liquid is adsorbed by polyethylene macroporous adsorption resin and eluted with sodium hydroxide solution of 0.01% by mass and volume. The eluent is then desalted, concentrated, sterilized and dried to obtain the soybean protein small molecule peptide, which contains the bioactive peptide PLEGFPF with the amino acid sequence Pro-Leu-Glu-Gly-Phe-Pro-Phe.

9. Use according to claim 8, wherein the compound is ###0002### The health food product includes the aforementioned soybean protein small molecule peptides, as well as food science-acceptable excipients.

10. Use according to claim 9, wherein the compound is ###0002### The dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

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