Tetrapeptide with hypoglycemic effect and preparation and application thereof

Abstract

The invention discloses tetrapeptide with a hypoglycemic effect as well as preparation and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the tetrapeptide LLRP is Leu-Leu-Arg-Pro, and the tetrapeptide LLRP has DPP-IV inhibitory activity, can effectively reduce the blood sugar level and has no obvious side effect, so that the tetrapeptide LLRP can be applied to preparation of drugs or food for reducing blood sugar. The invention further provides polypeptide containing the peptide fragment LLRP and wheat peptide rich in the polypeptide, tetrapeptide LLRP with the effect of reducing blood sugar is generated after digestion of gastrointestinal tracts, and the tetrapeptide LLRP is applied to preparation of drugs or food for reducing blood sugar. The invention provides a new scheme and theoretical basis for treatment of diabetes, and has good market prospect and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a tetrapeptide with blood sugar lowering effect and the preparation and application thereof. Background Art

[0002] Diabetes mellitus (DM) is a syndrome of a series of metabolic disorders such as protein, fat, water and electrolytes caused by absolute or relative insulin deficiency and reduced insulin sensitivity of target tissue cells, with hyperglycemia as the main sign. Type 2 diabetes mellitus (T2DM) is the most common type of diabetes, and its main characteristics are insulin resistance and insufficient insulin secretion. With the increasing number of diabetes patients worldwide, finding effective blood sugar-lowering treatment options has become a hot topic in medical research.

[0003] In recent years, dipeptidyl peptidase-IV (DPP-IV) inhibitors have been widely used in clinical practice as a new type of hypoglycemic drug. One of the key mechanisms for regulating postprandial blood glucose levels is the inhibition of DPP-IV. DPP-IV is an important enzyme that can degrade peptide hormones such as glucagon-like peptide-1 (GLP-1), thereby affecting insulin secretion and blood glucose levels. By inhibiting the activity of DPP-IV, the half-life of GLP-1 can be prolonged and its biological activity can be enhanced, thereby increasing insulin secretion and lowering postprandial blood glucose levels. However, the DPP-IV inhibitors currently available on the market still have some shortcomings, such as side effects and drug resistance. Therefore, finding new DPP-IV inhibitors remains an important direction in diabetes research.

[0004] Among many bioactive molecules, peptides have gradually attracted attention due to their good biocompatibility and targeting. In recent years, domestic and foreign scholars have continuously isolated peptide substances with hypoglycemic function from natural animals, plants and human bodies, and studied their structure and mechanism of action, opening up a new path for the prevention and treatment of diabetes. For example, patent document CN118546209A discloses a new active oligopeptide KLGKFF obtained from the protein decomposition product obtained by the synergistic treatment of soybean meal enzymes and bacteria. The oligopeptide has a significant inhibitory effect on the DPP-IV enzyme and can increase the glucose consumption of insulin-resistant HePG2 cells.

[0005] Wheat peptides are made from wheat protein (gluten powder) through biological enzymatic hydrolysis, refining, spray drying and other processes. Patent document CN114990180A discloses that wheat peptides are obtained by enzymatic hydrolysis of gluten powder using alkaline protease, complex enzyme and flavor protease in sequence, and then filtered through 0.25 μm and 10 nm membranes and dried. The wheat peptides have the effect of assisting in lowering blood sugar. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out high-efficiency peptide segments related to the hypoglycemic effect, it will provide a basis for the development of new hypoglycemic preparations. Summary of the invention

[0006] The purpose of the present invention is to provide a natural small molecule bioactive peptide with hypoglycemic effect, and to apply the peptide to the development of related products for hypoglycemic effect.

[0007] To achieve the above object, the present invention adopts the following technical solutions: The present invention utilizes alkaline protease, neutral protease and flavor protease to perform biological enzymolysis on wheat gluten powder in sequence, and then utilizes ZGC108DQ gel strong acid cation exchange resin to adsorb the enzymolysis solution, and obtains clarified wheat peptides after desorption with alkali solution. The peptide sequences of clarified wheat peptides before and after gastrointestinal simulated digestion are analyzed by LC-MS / MS peptide spectrum analysis technology, and it is found that the clarified wheat peptides are rich in polypeptides containing LLRP peptide segments, and the digestion product is tetrapeptide LLRP, and the amino acid sequence of peptide segment LLRP is Leu-Leu-Arg-Pro (SEQ ID NO.1). Molecular docking technology is used to explore the interaction between peptide segments and dipeptidyl peptidase-IV (DPP-IV), and the results show that tetrapeptide LLRP and peptide segments containing LLRP have higher molecular docking energy with DPP-IV. Further artificial synthesis of tetrapeptide LLRP is carried out, and functional verification shows that the peptide segment has DPP-IV inhibitory activity and hypoglycemic function.

[0008] Therefore, the present invention provides a bioactive peptide with hypoglycemic effect, wherein the bioactive peptide is a tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP, and the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro.

[0009] The polypeptide containing the core peptide LLRP can produce a tetrapeptide LLRP after digestion in the gastrointestinal tract. Further, the amino acid sequence of the polypeptide containing the core peptide LLRP is at least one of DNPQQGVPLLRPL (SEQ ID NO.2), THEEQQGVPLLRPL (SEQ ID NO.3), RPLLRPL (SEQ ID NO.4), QEQQQGVPLLRPL (SEQ ID NO.5), KQLLRPL (SEQ ID NO.6), THEQGLQLLRPL (SEQ ID NO.7), GVPLLRPR (SEQ ID NO.8), and KQLLRPR (SEQ ID NO.9).

[0010] The present invention also provides a method for preparing the bioactive peptide, which can be prepared by solid phase synthesis. Specifically, the method for preparing the tetrapeptide LLRP includes: adopting the Fmoc solid phase synthesis strategy, using Fmoc protected amino acids as raw materials, selecting Wang resin as a solid phase carrier, sequentially introducing proline, arginine, leucine, and leucine residues, extending the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the tetrapeptide LLRP.

[0011] The present invention also provides the use of the bioactive peptide in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar. The present invention has shown that the tetrapeptide LLRP has a blood sugar lowering effect. In an in vitro experiment, the tetrapeptide LLRP exhibited significant DPP-IV inhibitory activity; in a hyperglycemic zebrafish model, the blood sugar lowering effect of the tetrapeptide LLRP after intervention was better than that of the positive control metformin. Therefore, it can be applied to the development of related products for lowering blood sugar.

[0012] Furthermore, the diabetes is type 2 diabetes.

[0013] Another object of the present invention is to provide a wheat peptide with hypoglycemic effect, wherein the wheat peptide is rich in a polypeptide having a core peptide segment LLRP, and the amino acid sequence of the peptide segment LLRP is Leu-Leu-Arg-Pro; The preparation method of the wheat peptide comprises: (1) Enzymatic hydrolysis: wheat gluten and water were mixed at a mass ratio of 1:15-30, the pH was adjusted to 8.0±0.2, and 0.5% of alkaline protease based on the total weight of the gluten was added for enzymatic hydrolysis for 15-30 min; then, shearing was performed at a speed of 10000-15000 r / min for 15-30 min; then, 0.5%-3.0% of alkaline protease based on the total weight of the gluten and 0.5%-2.0% of neutral protease based on the total weight of the gluten were added for further enzymatic hydrolysis for 4-6 h, and after the end, 0.1%-1.5% of flavor protease based on the total weight of the gluten was added for 15-30 min; (2) Separation: The enzymatic hydrolysate is centrifuged at a speed of 4000-6000 r / min, and the supernatant is taken. A strong acid cationic resin is added to the supernatant, and the pH is adjusted to 3.0. The mixture is stirred for adsorption and then filtered. The filter cake is redissolved in a sodium hydroxide solution with a mass concentration of 1%-5% for desorption. The supernatant is nanofiltered, concentrated, sterilized, and dried to obtain the wheat peptide.

[0014] The wheat peptide provided by the present invention contains a polypeptide having a core peptide segment LLRP, which generates a tetrapeptide LLRP having a blood sugar lowering effect after being digested by the gastrointestinal tract, thereby exerting a blood sugar lowering function.

[0015] Furthermore, the polypeptide having a core peptide segment LLRP is at least one of the peptide segments having amino acid sequences as shown in SEQ ID NO.2 to SEQ ID NO.9.

[0016] The present invention also provides the use of the wheat peptide in preparing medicines for preventing or treating diabetes or foods for assisting in lowering blood sugar. The wheat peptide exerts a blood sugar lowering effect after being digested by the gastrointestinal tract.

[0017] The present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an effective dose of a tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP or the wheat peptide, wherein the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro.

[0018] The tetrapeptide LLRP or the polypeptide containing the core peptide LLRP or the wheat peptide in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that exerts a hypoglycemic effect, or can be compounded with other active ingredients that have a hypoglycemic effect.

[0019] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject.

[0020] Furthermore, the pharmaceutically acceptable carrier includes: one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0021] The present invention uses the tetrapeptide LLRP or the polypeptide containing the core peptide LLRP as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to the preparation preparation method recorded in pharmaceutics. Further, the preparation form of the pharmaceutical composition can be but not limited to an oral preparation. Specifically, the preparation form can be but not limited to an oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0022] The present invention provides a health food for assisting in lowering blood sugar, comprising a tetrapeptide LLRP or a polypeptide containing a core peptide LLRP as an active ingredient, and a food-based acceptable auxiliary material, wherein the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro. The food-based acceptable auxiliary material is a food-grade auxiliary material that can deliver an effective dose of the active substance of the present invention and does not interfere with the biological activity of the active substance.

[0023] Furthermore, the dosage form of the health food is beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0024] The present invention has the following beneficial effects: (1) The present invention provides a novel bioactive peptide LLRP with DPP-IV inhibitory activity. Animal experiments have shown that the tetrapeptide LLRP can effectively lower blood sugar levels without obvious side effects. Therefore, it can be used to prepare drugs or foods for lowering blood sugar.

[0025] (2) The present invention provides a polypeptide having a peptide segment LLRP and a wheat peptide rich in such a polypeptide, which generates a tetrapeptide LLRP having a hypoglycemic effect after digestion in the gastrointestinal tract, and can be used to prepare a drug or food for hypoglycemic effect. The present invention provides a new solution and theoretical basis for the treatment of diabetes, and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the secondary mass spectrum of the tetrapeptide LLRP.

[0027] Figure 2 Schematic diagram of the binding interaction between the tetrapeptide LLRP and DPP-IV.

[0028] Figure 3 The hypoglycemic effect of the tetrapeptide LLRP in the hyperglycemic zebrafish model.

[0029] Figure 4 Comparison of the hypoglycemic effects of the tetrapeptide LLRP and PSLL in the hyperglycemic zebrafish model. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Wheat protein (gluten powder) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0033] Example 1: Preparation of clarified wheat peptides Preparation of clarified wheat peptides by enzymatic hydrolysis: Wheat protein (gluten) and water were put into the enzymatic hydrolysis reaction tank at a ratio of 1:20, the pH of the feed solution was adjusted to 8.0±0.2, and 0.5% alkaline protease (derived from Bacillus licheniformis) of the total weight of gluten was added for enzymatic hydrolysis for 30 min, and then the shearing machine was turned on with a shearing speed of 15000 r / min for 30 min.

[0034] After the shearing was completed, the pH of the feed solution was adjusted to 8.0±0.2, and 2.0% of the total weight of gluten by alkaline protease (derived from Bacillus licheniformis) and 1.0% of the total weight of gluten by neutral protease (derived from Bacillus subtilis) were added to continue enzymatic hydrolysis for 5 h. After the end, 0.8% of the total weight of gluten by flavor protease was used for 30 min.

[0035] After the enzymatic hydrolysis, the solution was centrifuged in a disc centrifuge (speed 6000 r / min) and the supernatant was retained.

[0036] ZGC108DQ gel strong acid cationic resin was added to the supernatant after butterfly separation, the pH was adjusted to 3.0, and the mixture was stirred and adsorbed for 2 h. After the adsorption, the filter cake was passed through a plate and frame filter, and the filter cake was redissolved in a 3.0% sodium hydroxide solution for desorption for 40 min. The supernatant was nanofiltered and concentrated by low-temperature vacuum to a liquid concentration of 32°Bx. The concentrated liquid was sterilized and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0037] Example 2: Screening of active peptides In this example, we simulated the gastrointestinal digestion of wheat peptides in vitro and identified the related peptides before and after digestion by LC-MS / MS, used molecular docking technology to explore their specific mechanism of action with dipeptidyl peptidase-IV (DPP-IV), and screened out highly effective DPP-IV inhibitory peptides.

[0038] 1. In vitro simulated digestion Take 10 mL of wheat peptide sample solution (preheated in a water bath at 37°C), add 9 mg / mL NaCl solution (normal saline, preheated in a water bath at 37°C), adjust the pH to 2 with 1 mol / L HCl, add 4 mL of gastric juice (0.4 g pepsin dissolved in 0.01 mol / L HCl solution) to the system, and simulate gastric digestion after shaking at 37°C water bath and 120 r / min for 2 h. The pH value of the digested sample was pre-adjusted to 6.5. Pipette 2 mL of gastric digested sample into a centrifuge tube, add 4 mL of intestinal juice (0.02 g trypsin and 0.12 g pig bile salt dissolved in 10 mL 0.1 mol / L NaHCO3 solution, respectively, to obtain 2 mg / mL trypsin solution and 12 mg / mL bile solution, pancreatic juice and bile mixed 1:1), adjust the pH to 7.0, and simulate intestinal digestion after shaking at 37°C water bath and 120 r / min for 2 h. All experiments were performed in the absence of light and oxygen, and the simulated digested samples were lyophilized and stored at −80°C until analysis.

[0039] 2. Sequence identification of wheat peptides The wheat peptide samples before and after simulated digestion were subjected to peptide sequencing analysis using the LC-MS / MS method, and the analysis was completed by Beijing Biotech Biotechnology Co., Ltd. First, the samples were reductively alkylated and desalted, and then the processed samples were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry results, which were analyzed by PEAKS Studio10.6Denovo to obtain the peptide sequence analysis results.

[0040] Analysis of the peptide spectra of wheat peptides before and after digestion revealed that wheat peptides were rich in multiple peptides containing LLRP sequences before digestion and contained LLRP peptides after digestion, which are shown in Table 1 after sorting.

[0041] Table 1. Peptides containing LLRP before and after digestion 3. Screening of active peptides with potential hypoglycemic function Dipeptidyl peptidase-IV (DPP-IV) can weaken the hypoglycemic effect of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) by degrading them. Inhibiting DPP-IV activity can prolong the activity of these incretin hormones, thereby promoting insulin secretion, inhibiting glucagon release, and achieving blood sugar control. DPP-IV inhibitors selectively block the activity of this enzyme and become a core drug for lowering blood sugar in the treatment of type 2 diabetes by enhancing the function of the endogenous incretin system. Therefore, in this example, the peptide fragments after wheat peptide digestion were molecularly docked with DPP-IV to screen small molecule peptide fragments that may have hypoglycemic effects.

[0042] According to the average local confidence (ALC) greater than 95%, the peak area greater than 2×10 6 , the peptides that met the conditions were screened with a PeptideRanker score greater than 0.8, and then the peptides were molecularly docked with dipeptidyl peptidase-IV (DPP-IV).

[0043] First, the crystal structure of DPP-IV (5CXV) was downloaded from the PDB protein database. The active center was defined by removing water molecules and adding hydrogen atoms from the receptor target using DiscoveryStudio software. The structure of the peptide segment of the selected wheat peptide was constructed by Discovery Studio, and its energy was minimized by the DPP-IV force field, and these peptides were defined as ligands. The constructed peptide was docked with DPP-IV by CDOCKER to simulate the binding mode, site and amino acid residues with the lowest binding energy and the highest binding degree, and screened according to the binding energy and the number of hydrogen bonds. Finally, two tetrapeptides (LLRP and PSLL) were determined, as shown in Table 2.

[0044] Table 2. Peptides with potential immunomodulatory activity in digested wheat peptides Peptide sequence ALC score (%) length Mass-to-charge ratio (m / z) Relative abundance Molecular weight (Da) PeptideRanker Score (-) Docking energy (kcal / mol) LLRP 96.8 4 498.34 4.67E+06 497.33 0.97 161.15 PSLL 99.3 4 429.27 2.88 E+06 428.26 0.50 113.65 The secondary mass spectrum of the tetrapeptide LLRP is as follows Figure 1 As shown in the figure, the fragmentation ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z-type ions break to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide, and the molecular weight is 497.33 Da. The tetrapeptide was further analyzed by secondary mass spectrometry using the in-source collision induced dissociation technique, and the primary structure of the tetrapeptide was determined to be Leu-Leu-Arg-Pro.

[0045] The 2D and 3D images of the molecular docking of tetrapeptide LLRP and DPP-IV are shown in Figure 2 As shown. Analysis of chemical bonds revealed that the tetrapeptide LLRP was mainly bound to DPP-IV through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), hydrophobic interactions (alkyl groups), and electrostatic interactions (including salt bridges and attractive charges), with a docking energy of -161.15 kcal / mol. DPP-IV formed eight van der Waals forces with amino acid residues ARG429, TYR457, ILE405, GLY406, SER458, LEU60, SER460, and LEU57, four hydrogen bonds with VAL459, ARG471, and SER59, one hydrophobic interaction with ILE418, and two electrostatic interactions with GLU408 and ARG471.

[0046] The molecular docking results show that the tetrapeptide LLRP can bind to DPP-IV and inhibit DPP-IV, thereby exerting a hypoglycemic effect.

[0047] We commissioned Jier Biochemical (Shanghai) Co., Ltd. to synthesize the peptides LLRP and PSLL with a purity of ≥98% for subsequent functional verification.

[0048] Example 3: In vitro DPP-IV inhibition rate of tetrapeptide LLRP The DPP-IV inhibition rate of the peptide was determined using the Cayman (manufacturer) DPP-IV inhibitor kit. The assay principle is as follows: The test uses the fluorescent substrate gly-pro-aminomethylcoumarin (AMC) to measure the activity of DPP-IV. DPP cleaves the peptide bond to release the free AMC group, generating fluorescence with an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm.

[0049] The DPP-IV inhibition rate of the tetrapeptides LLRP and PSLL at 1000 µM and 500 µM, respectively, was determined as follows: (1) 100% Initial Activity Wells - Add 30 µL of dilution buffer, 10 µL of DPP-IV, and 10 µL of pure water to three wells.

[0050] (2) Background wells - Add 40 µL of diluted buffer and 10 µL of pure water to three wells.

[0051] (3) Inhibitor Wells - Add 30 µL of diluted assay buffer, 10 µL of diluted DPP-IV, and 10 µL of inhibitor (intervention sample, 1000 µM and 500 µM concentrations of LLRP and PSLL) to three wells.

[0052] The specific operation is as follows: first, add 30 µL buffer, 10 µL DPP-IV enzyme and 10 µL sample to a 96-well plate and mix well, add 50 µL substrate solution to start the reaction, and then incubate at 37°C for 30 min, and read the fluorescence with an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm. Detect the fluorescence intensity and record the result as sample A. The result measured by using buffer instead of DPP-IV is recorded as blank A, and the result measured by using buffer instead of sample is recorded as control A. The calculation formula of DPP-IV inhibition rate is as follows: inhibition rate (%) = (control A-sample A) / (control A-blank A) × 100%.

[0053] The results are shown in Table 3. The DPP-IV inhibition rate of the tetrapeptide LLRP was as high as 84.29%±1.23% at 1000 μM, and its inhibition rate was also over 50% at 500 μM, indicating that LLRP has the potential to inhibit DPP-IV. However, the tetrapeptide PSLL, which also has a high molecular docking energy with DPP-IV, performed poorly in the in vitro DPP-IV inhibition experiment, and the inhibition rates at the measured concentrations of 1000 μM and 500 μM were both less than 10%.

[0054] Table 3. DPP-IV inhibition rate (%) of tetrapeptide LLRP and PSLL Example 4: Hypoglycemic effect of tetrapeptide LLRP in a hyperglycemic zebrafish model The hyperglycemic zebrafish model was used to determine the hypoglycemic effect of the tetrapeptide LLRP. Wild AB zebrafish of 4 days old were placed in a six-well plate, with 3 parallel wells per group and 10 fish per well. The group interventions were as follows: (1) Blank control group (NC): system water; (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water; (3) Metformin positive control group (Met): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water; (4) Tetrapeptide LLRP intervention group (LLRP-5, LLRP-1, LLRP-0.1): 333 μM alloxan + 2.67% glucose + 5 / 1 / 0.1 μg / mL tetrapeptide LLRP + system water.

[0055] After 24 h of intervention with the above intervention scheme, 4-day-old zebrafish were washed three times with PBS solution to clean the sugar solution on the surface of zebrafish. Use a disposable dropper to transfer the zebrafish in the six-well plate to a 1.5 mL centrifuge tube, remove the excess liquid in the centrifuge tube, add 0.1 mL of anhydrous ethanol, place in a cool place for 15 min, move into an oven for 120 min (60 ℃) until dried (crushed), and finally add 5 µL of ultrapure water to the centrifuge tube, shake it ultrasonically for 10 min, and draw 2 µL to measure the glucose value using a blood glucose meter.

[0056] The results are as follows Figure 3 As shown in the figure, compared with the blank control group (NC group), the blood glucose level of the model control group (MC group) after 24 hours of modeling with 333 μM alloxan + 2.67% glucose was significantly increased ( p <0.0001), indicating that the hyperglycemia model was successfully established. After intervention with the positive drug 5 μg / mL metformin (Met group), there was a significant downregulation effect compared with the MC group ( p <0.01). Under the intervention of 5 μg / mL and 1 μg / mL concentrations of tetrapeptide LLRP, blood glucose levels were significantly reduced compared with the MC group ( p <0.01), and was downregulated to no significant difference with the NC group ( p >0.05), indicating that the tetrapeptide LLRP has a significant hypoglycemic effect, and its effect is equivalent to that of metformin (Met) at the same dosage.

[0057] Example 5: Comparison of the hypoglycemic effects of tetrapeptide LLRP and PSLL in a hyperglycemic zebrafish model The hyperglycemic zebrafish model was used to compare the hypoglycemic effects of the tetrapeptide LLRP and PSLL. Wild AB zebrafish of 4 days old were placed in a six-well plate, with 3 parallel wells per group, 10 fish per well, and grouped as follows: (1) Blank control group (NC): system water; (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water; (3) Tetrapeptide LLRP intervention group (LLRP): 333 μM alloxan + 2.67% glucose + 1 μg / mL tetrapeptide LLRP + system water; (4) Tetrapeptide PSLL intervention group (PSLL): 333 μM alloxan + 2.67% glucose + 1 μg / mL tetrapeptide PSLL + system water.

[0058] The intervention method and blood glucose measurement method were the same as the “Glycemic-lowering effect of tetrapeptide LLRP in a hyperglycemic zebrafish model”. Figure 4As shown in Figure 2, under the intervention of 1 μg / mL tetrapeptide LLRP and PSLL, compared with the model control MC group, the tetrapeptide LLRP significantly downregulated blood glucose levels ( p <0.01), while the tetrapeptide PSLL had no significant down-regulation effect ( p >0.05).

Claims

1. A bioactive peptide having a blood sugar lowering effect, characterized in that: The bioactive peptide is a tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP, and the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro.

2. The method for preparing the bioactive peptide according to claim 1, characterized in that: The bioactive peptide is prepared by solid phase synthesis.

3. Use of the bioactive peptide according to claim 1 in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar.

4. The use according to claim 3, characterized in that The diabetes is type 2 diabetes.

5. A wheat peptide having a blood sugar lowering effect, characterized in that: The wheat peptide is rich in polypeptides having a core peptide segment LLRP, and the amino acid sequence of the peptide segment LLRP is Leu-Leu-Arg-Pro; The preparation method of the wheat peptide comprises: (1) Enzymatic hydrolysis: wheat gluten and water are mixed at a mass ratio of 1:15-30, the pH is adjusted to 8.0±0.2, and 0.5% of alkaline protease based on the total weight of the gluten is added for enzymatic hydrolysis for 15-30 min; then, the mixture is sheared at a speed of 10000-15000 r / min for 15-30 min; then, 0.5%-3.0% of alkaline protease based on the total weight of the gluten and 0.5%-2.0% of neutral protease based on the total weight of the gluten are added for further enzymatic hydrolysis for 4-6 h, and after the end, 0.1%-1.5% of flavor protease based on the total weight of the gluten is added for 15-30 min; (2) Separation: The enzymatic hydrolysate is centrifuged at a speed of 4000-6000 r / min, and the supernatant is taken. A strong acid cationic resin is added to the supernatant, and the pH is adjusted to 3.

0. The mixture is stirred for adsorption and then filtered. The filter cake is redissolved in a sodium hydroxide solution with a mass concentration of 1%-5% for desorption. The supernatant is nanofiltered, concentrated, sterilized, and dried to obtain the wheat peptide.

6. The wheat peptide according to claim 5, characterized in that The polypeptide having a core peptide segment LLRP is at least one of the peptide segments with amino acid sequences as shown in SEQ ID NO.2 to SEQ ID NO.

9.

7. Use of the wheat peptide according to claim 5 or 6 in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar.

8. A pharmaceutical composition for preventing or treating diabetes, characterized in that: The pharmaceutical composition comprises an effective dose of a tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP, and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro.

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

10. A health food for assisting in lowering blood sugar, characterized in that: The invention comprises a tetrapeptide LLRP as an active ingredient or a polypeptide containing a core peptide segment LLRP, and excipients acceptable in food science, wherein the amino acid sequence of the LLRP is Leu-Leu-Arg-Pro.

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