An octapeptide derived from soybean protein with the effect of lowering blood pressure, blood sugar, and cholesterol, and its preparation and application.

Abstract

This invention discloses an octapeptide derived from soybean protein with the function 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 octapeptide sequence is YEGNWGPL, which can be obtained artificially or by targeted enzymatic hydrolysis of soybean protein isolate. Functional verification in a hyperlipidemia model shows that the octapeptide YEGNWGPL has the function of lowering blood lipids; functional verification in a hyperglycemia model shows that the octapeptide YEGNWGPL has the function of lowering blood sugar, with a significantly better effect than the drug metformin; functional verification in a hypertension model shows that the octapeptide YEGNWGPL has the function of lowering blood pressure, with a significantly better effect than the antihypertensive drug captopril; moreover, food-derived bioactive peptides have high biosafety, therefore, they can be applied to the development of related products for lowering blood pressure, blood sugar, and cholesterol 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 small molecule octapeptide derived from soybean protein that has the effect of lowering blood pressure, blood sugar, and cholesterol, as well as its preparation method and application. Background Technology

[0002] The "three highs" refers to hypertension, hyperglycemia, and hyperlipidemia, all of which are chronic diseases. Hyperlipidemia can cause vascular embolism, hypertension can cause cerebral hemorrhage and cerebral hemorrhage, and hyperglycemia can cause diabetes. These three conditions often coexist due to metabolic abnormalities and influence each other. For example, hyperlipidemia accelerates arteriosclerosis and indirectly raises blood pressure, while diabetes is often accompanied by other metabolic disorders, increasing the risk of hypertension. Therefore, developing nutritional foods that improve and lower the "three highs" to achieve bodily balance is crucial for improving the health of middle-aged and elderly people.

[0003] Food-derived bioactive peptides are a class of small-molecule peptide compounds with beneficial physiological functions, prepared from food raw materials through enzymatic hydrolysis, fermentation, separation, and purification techniques. Their development and application in functional foods hold broad promise. For example, soybean peptides are small-molecule protein fragments or amino acid chains with a molecular weight below 6000 Da and a length of 2-20 amino acids, obtained from soybean protein through hydrolysis, separation, and purification. These small-molecule soybean peptides have low viscosity, good solubility, good water absorption, and low osmotic pressure, making them easier for the body to digest, absorb, and utilize than soybean protein, exhibiting superior and broader biological activities. Functional studies have shown that soybean peptides possess antioxidant, anticancer, antihypertensive, and cholesterol-lowering physiological activities (Wang Jinling et al. Progress in the preparation and functional research of soybean peptides. China Brewing, 2022, 41(1): 25-31.).

[0004] The physiological activity of bioactive peptides is closely related to their structure, including the molecular weight, amino acid composition, amino acid configuration, and hydrophobicity of the peptides. Different preparation methods will result in different functional characteristics, purity, and physicochemical properties of the soybean peptides. For example, patent document CN105614899A provides a method for preparing low-molecular-weight soybean peptides, which includes: using soybean protein isolate as raw material, first adding a complex protease 1 obtained by combining papain, bromelain, and 3942 neutral protease, and hydrolyzing for 1-2 hours; then adding a complex protease 2 obtained by combining fig protease and Aspergillus niger, and hydrolyzing for 1-3 hours; and finally adding 537 acidic protease and hydrolyzing for 1-2 hours. This method ensures the formation and retention of functional peptide fragments with antioxidant, blood pressure lowering, blood lipid lowering, and cholesterol lowering properties through targeted hydrolysis of soybean protein isolate.

[0005] 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

[0006] The purpose of this invention is to provide a natural small molecule biopeptide with the effect of lowering blood pressure, blood sugar, and cholesterol, which can be used to develop safe and effective new products for lowering blood pressure, blood sugar, and cholesterol.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention uses soy protein isolate as raw material, and sequentially employs a complex enzyme consisting of alkaline protease and trypsin, and a complex enzyme consisting of neutral protease and bromelain, for enzymatic hydrolysis. 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 soy protein hydrolysate. The peptide sequences in the soy 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 target SRC, blood glucose-lowering target DPP-IV, and blood pressure-lowering target ACE. A candidate peptide was screened, and its amino acid sequence was identified by mass spectrometry as Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu (YEGNWGPL, abbreviated as YL-8), with a molecular weight of 934.4185 Da. Further functional verification using the artificially synthesized peptide YEGNWGPL revealed that this peptide possesses biological activity in regulating blood lipids, blood glucose, and blood pressure.

[0008] Therefore, the present invention provides a bioactive peptide YEGNWGPL, wherein the amino acid sequence of the bioactive peptide YEGNWGPL is Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu.

[0009] This invention also provides a method for preparing the bioactive peptide YEGNWGPL, 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 leucine, proline, glycine, tryptophan, asparagine, glycine, glutamic acid, and tyrosine residues to extend the peptide chain from the C-terminus to the N-terminus, thereby synthesizing the octapeptide YEGNWGPL in a solid phase.

[0010] The bioactive peptide YEGNWGPL 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 53°C, 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 octapeptide YEGNWGPL from the hydrolysate.

[0011] The present invention also provides the application of the bioactive peptide YEGNWGPL in the preparation of lipid-lowering and / or blood sugar-lowering and / or blood pressure-lowering products.

[0012] This invention demonstrates that the octapeptide YEGNWGPL has regulatory effects on blood lipids, blood glucose, and blood pressure. In a hyperlipidemia model, administration of octapeptide YEGNWGPL significantly reduced triglyceride (TG) levels. In a hyperglycemia model, administration of octapeptide YEGNWGPL significantly reduced blood glucose levels, with an effective concentration that is 1 / 10 that of the hypoglycemic drug metformin. In a hypertension model, administration of octapeptide YEGNWGPL significantly reduced blood flow activity and angiotensin II levels, with an effective concentration that is 1 / 20 that of the antihypertensive drug captopril. Therefore, it can be applied to the development of products that lower or assist in lowering the levels of the "three highs" (hyperlipidemia, hyperglycemia, and hypertension), or to maintain healthy levels of blood lipids, blood glucose, and blood pressure.

[0013] Furthermore, the product may be, but is not limited to, pharmaceuticals or health foods.

[0014] Specifically, the product is a drug for treating hyperlipidemia and / or diabetes and / or hypertension, and its purpose is to lower blood lipids, lower blood sugar, and lower blood pressure by at least one of these.

[0015] Alternatively, the product may be a health food product that assists in lowering blood lipids and / or blood sugar and / or blood pressure. The purpose of the health food product is to assist in lowering blood lipids to maintain healthy blood lipid levels, assist in lowering blood sugar to maintain healthy blood sugar levels, and assist in lowering blood pressure to maintain healthy blood pressure levels, at least one of the following:

[0016] Another object of the present invention is to provide a pharmaceutical composition for lowering the levels of the "three highs" (hyperlipidemia, hyperglycemia, and hypertension), wherein the pharmaceutical composition comprises an effective dose of a bioactive peptide YEGNWGPL with the amino acid sequence Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu, and a pharmaceutically acceptable carrier.

[0017] The bioactive peptide YEGNWGPL in the pharmaceutical composition provided by this 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.

[0018] 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.

[0019] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, and lubricants.

[0020] This invention uses the bioactive peptide YEGNWGPL as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a formulation according to the formulation preparation method described in pharmaceutical science.

[0021] 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.

[0022] Another objective of this invention is to provide a health food for maintaining healthy levels of blood lipids, blood sugar, and blood pressure, wherein the health food comprises a bioactive peptide YEGNWGPL with the amino acid sequence Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu, and food science-acceptable excipients.

[0023] 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.

[0024] Furthermore, the dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

[0025] The beneficial effects of this invention are as follows: This invention provides a bioactive peptide, YEGNWGPL, with the function of lowering blood pressure, blood sugar, and cholesterol. This peptide can be obtained through artificial synthesis or targeted enzymatic hydrolysis of soy protein isolate. Functional validation in a hyperlipidemia model shows that the bioactive peptide YEGNWGPL has the function of lowering blood lipids; functional validation in a hyperglycemia model shows that the bioactive peptide YEGNWGPL has the function of lowering blood sugar, with a significantly better effect than the drug metformin; functional validation in a hypertension model shows that the bioactive peptide YEGNWGPL has the function of lowering blood pressure, with a significantly better effect than the antihypertensive drug captopril; moreover, 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, and have good market prospects and application potential. Attached Figure Description

[0026] Figure 1 This is the primary mass spectrum of the octapeptide YEGNWGPL.

[0027] Figure 2 This is a secondary mass spectrum of the octapeptide YEGNWGPL. In the figure, y2 represents the second y-type fragment ion generated by the C-terminus of the peptide, b2 represents the second b-type fragment ion generated by the N-terminus of the peptide, b5 represents the fifth b-type fragment ion generated by the N-terminus of the peptide, b6-NH3 represents the sixth deaminated fragment ion generated by the N-terminus of the peptide, and b6 represents the sixth b-type fragment ion generated by the N-terminus of the peptide.

[0028] Figure 3 The figure shows the effect of the octapeptide YEGNWGPL on triglyceride levels in a hyperlipidemic zebrafish model. The # symbol indicates a significant difference compared to the control group, and ## indicates... p <0.01, ### indicates p <0.001; * indicates a significant difference compared to the model group, * indicates p <0.05, ** indicates p <0.01.

[0029] Figure 4 The figure shows the effect of the octapeptide YEGNWGPL on blood glucose levels in a hyperglycemic zebrafish model. The # symbol indicates a significant difference compared to the control group, and #### indicates... p <0.0001; * indicates a significant difference compared to the model group, * indicates p <0.05, *** indicates p <0.001.

[0030] Figure 5 The figure shows the effect of the octapeptide YEGNWGPL on blood flow activity in a hypertensive zebrafish model. The # symbol indicates a significant difference compared to the control group, and ### indicates... p<0.001; * indicates a significant difference compared to the model group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0031] Figure 6 The figure shows the effect of the octapeptide YEGNWGPL on angiotensin II levels in a hypertensive zebrafish model. The # symbol indicates a significant difference compared to the control group, and ### indicates... p <0.001; * indicates a significant difference compared to the model group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, ns indicates no significant difference. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Example 1: Screening of active peptides In this embodiment, a complex protease was used to enzymatically hydrolyze soybean protein isolate. Peptides in the hydrolysate were identified by LC-MS / MS, and molecular docking technology was used to screen for candidate peptides with activities that lower blood pressure, blood sugar, and cholesterol.

[0036] 1. Enzymatic hydrolysis S1. 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. S2. Enzymatic hydrolysis: Heat the solution to 53℃, add 1.5 kg of complex proteinase B (800 g of alkaline proteinase and 700 g of trypsin) and hydrolyze for 3.5 hours. Then add 450 g of complex proteinase C (200 g of neutral proteinase and 250 g of bromelain) and continue hydrolysis for 3 hours. S3. Adsorption filtration: Heat the feed liquid to 80℃, add 20 kg of activated carbon for adsorption for 30 min, and then filter it through a 600-mesh filter cloth; S4. Adsorption separation: The filtered liquid is adsorbed by pre-activated polyethylene macroporous adsorption resin for 30 min, and then eluted with sodium hydroxide solution of 0.01% by mass / volume. S5. Nanofiltration: The eluent is desalted by nanofiltration using a 200 Da nanofiltration membrane; S6. Concentration: After desalination, the liquid is concentrated to a sugar content of 25° and then sterilized and dried to prepare soybean peptide powder.

[0037] 2. Molecular docking screening of active peptides The peptide profile of soybean peptides was commissioned to Zhejiang University for peptide profile analysis, and the full peptide profile data of soybean peptides was obtained by checking against the protein database.

[0038] 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).

[0039] 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. After initial screening, the peptides were 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 possible active peptides for lowering blood pressure, blood sugar, and cholesterol. The specific screening results are shown in Table 1.

[0040] Table 1. Potential bioactive peptides that can bind to three targets simultaneously: lowering blood lipids, lowering blood sugar, and lowering blood pressure.

[0041] The primary and secondary structures of the octapeptide YEGNWGPL were analyzed by mass spectrometry, and the results are as follows: Figure 1 and Figure 2As shown, the [M+H]+ ion signal of the octapeptide YEGNWGPL in the primary structure is 935.42157 m / z, which is basically consistent with the molecular weight of 934.4185 Da of the octapeptide YEGNWGPL. The amino acid sequence of the octapeptide YEGNWGPL in the secondary structure is Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu.

[0042] Example 2: Synthesis of the active peptide YEGNWGPL In this embodiment, the peptide YEGNWGPL was artificially synthesized. Specifically, it was synthesized by Shenzhen Borunsida Biotechnology Co., Ltd., with a purity of ≥98%.

[0043] The specific synthesis method includes the following steps: S1. Weigh Fmoc-Leu-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 sequentially until the last amino acid Fmoc-Tyr(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 send to MS to obtain octapeptide YEGNWGPL.

[0044] Example 3: Evaluation of the lipid-lowering efficacy of octapeptide YEGNWGPL 1. Experimental Methods Wild-caught AB zebrafish at 5 dpf were placed in 6-well plates containing culture medium, with 30 fish per well and 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.

[0045] 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, water to a final volume of 1 L; (2) Model group: Egg yolk powder (final concentration of 0.2% by mass / volume percentage in system water) + 5 mL system water / well. A high-fat model was established using a high-fat diet; (3) Intervention group: Egg yolk powder (final concentration of 0.2% by mass volume in system water) + different concentrations of peptide fragments (final concentration of 20, 10, 5, 1 μg / mL) + 5 mL system water / well.

[0046] 2. Experimental Results The results are as follows Figure 3 As shown, 0.2% egg yolk powder significantly increased the TG content in the model group, indicating that the 0.2% zebrafish high-fat model was successfully established. Intervention with octapeptide YEGNWGPL significantly reduced the triglyceride content in zebrafish, indicating that octapeptide YEGNWGPL has a significant lipid-lowering effect. In particular, intervention with 1 μg / mL octapeptide YEGNWGPL significantly reduced the TG content in high-fat zebrafish by 27.22%.

[0047] Example 4: Evaluation of the hypoglycemic efficacy of octapeptide YEGNWGPL 1. Experimental Methods 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.

[0048] 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) + peptides of different concentrations (0.25, 0.5, 1, 5, 10, 20 μg / mL) + 5 mL system water / well.

[0049] 2. Experimental Results The results are as follows Figure 4 As shown, compared with the control group, the glucose content in zebrafish in the model group was significantly increased, indicating successful modeling. Metformin, as a positive control drug, could significantly reduce glucose content. Octapeptide YEGNWGPL concentrations within the range of 0.25-20 μg / mL significantly reduced glucose content in zebrafish, indicating that octapeptide YEGNWGPL has a significant hypoglycemic effect. Specifically, compared with the hyperglycemic model group, 0.5 μg / mL of octapeptide YEGNWGPL reduced blood glucose content in zebrafish by 44.17%, and the effective concentration was 1 / 10 of that of the hypoglycemic drug metformin, indicating that the hypoglycemic effect of small molecule octapeptides derived from soybean protein is significantly better than that of metformin.

[0050] Example 5: Evaluation of the blood pressure-lowering efficacy of octapeptide YEGNWGPL I. Measurement of blood flow activity 1. Experimental Methods 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.

[0051] 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) + 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) + octapeptide YEGNWGPL (final concentrations of 2, 4, 6, 8, 10 μg / mL) + system water.

[0052] 2. Experimental Results The results are as follows Figure 5 As shown, compared to the control group, the blood flow activity in the model group of zebrafish was significantly increased, indicating successful model establishment. The positive control group, captopril (an antihypertensive drug), significantly reduced blood flow activity in zebrafish. Octapeptide YEGNWGPL concentrations within the range of 2-10 μg / mL significantly reduced blood flow activity in zebrafish, indicating that octapeptide YEGNWGPL has a significant antihypertensive effect. Specifically, compared to the hypertension model group, 2 μg / mL of octapeptide YEGNWGPL significantly reduced blood flow activity in zebrafish by 38.34%, and the effective concentration was 1 / 20th that of the antihypertensive drug captopril, indicating that the antihypertensive effect of small molecule octapeptides derived from soybean protein is significantly better than that of the antihypertensive drug captopril.

[0053] II. Measurement of Angiotensin II (Ang II) Levels 1. Experimental Methods 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.

[0054] 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) Dosage group: L-NAME (final concentration 125 μg / mL) + PTU (120 μL of PTU solution with a concentration of 1.5 g / L added) + octapeptide YEGNWGPL (final concentrations of 2, 4, 6, 8, 10 μg / mL) + system water.

[0055] 2. Experimental Results The results are as follows Figure 6As shown, compared to the control group, the angiotensin II level in the model group zebrafish was significantly increased, indicating successful model establishment. The positive control group, captopril (an antihypertensive drug), significantly reduced the angiotensin II content in zebrafish. Octapeptide YEGNWGPL concentrations within the range of 2-10 μg / mL significantly reduced the angiotensin II content in zebrafish, indicating that octapeptide YEGNWGPL has a significant antihypertensive effect. Specifically, compared to the hypertension model group, 2 μg / mL of octapeptide YEGNWGPL significantly reduced the angiotensin II content in zebrafish by 21.06%, and the effective concentration was 1 / 20th that of the antihypertensive drug captopril, indicating that the antihypertensive effect of small molecule octapeptides derived from soybean protein is significantly better than that of the antihypertensive drug captopril.

[0056] 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. A bioactive peptide YEGNWGPL, characterized in that, The amino acid sequence of the bioactive peptide YEGNWGPL is Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu.

2. The method for preparing the bioactive peptide YEGNWGPL as described in claim 1, characterized in that, The bioactive peptide YEGNWGPL was prepared by solid-phase synthesis. Alternatively, it can be obtained by enzymatically hydrolyzing soy protein isolate.

3. The use of the bioactive peptide YEGNWGPL as described in claim 1 in the preparation of products that lower blood lipids and / or blood sugar and / or blood pressure.

4. The application as described in claim 3, characterized in that, The product is a medication for treating hyperlipidemia and / or diabetes and / or hypertension.

5. The application as described in claim 3, characterized in that, The product is a health food that helps lower blood lipids and / or blood sugar and / or blood pressure.

6. A pharmaceutical composition for lowering blood pressure, blood sugar, and cholesterol, characterized in that, The term "lowering the three highs" refers to reducing the levels of high blood lipids, high blood sugar, and high blood pressure. The pharmaceutical composition includes an effective dose of the bioactive peptide YEGNWGPL with the amino acid sequence Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: fillers, wetting agents, disintegrants, binders, and lubricants.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is in the form of an oral formulation.

9. A health food product for maintaining healthy levels of blood lipids, blood sugar, and blood pressure, characterized in that, The health food product includes the bioactive peptide YEGNWGPL with the amino acid sequence Tyr-Glu-Gly-Asn-Trp-Gly-Pro-Leu, as well as food-grade excipients.

10. The health food product as described in claim 9, characterized in that, The dosage form of the health food is tablets, hard capsules, soft capsules, oral solutions, granules, or powders.

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