A tetrapeptide with hypoglycemic effect, its preparation and application

Abstract

The present invention discloses a tetrapeptide with hypoglycemic effect, its preparation and application, belonging to the technical field of biomedicine. The amino acid sequence of the tetrapeptide LLRP is Leu-Leu-Arg-Pro. The tetrapeptide LLRP has DPP-IV inhibitory activity, can effectively reduce blood sugar level, and has no obvious side effects. Therefore, it can be applied to the preparation of drugs or health foods for reducing blood sugar. The present invention also provides polypeptides containing the peptide segment LLRP and wheat peptides rich in such polypeptides, which generate the hypoglycemic tetrapeptide LLRP after gastrointestinal digestion, and are applied to the preparation of drugs or health foods for reducing blood sugar. The present invention provides a new scheme and theoretical basis for the treatment of diabetes, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a tetrapeptide having hypoglycemic effect, its preparation and application. Background Art

[0002] Diabetes mellitus (DM) is a syndrome of a series of metabolic disorders of proteins, fats, water and electrolytes caused by absolute or relative insulin deficiency and reduced insulin sensitivity of target tissue cells, among which hyperglycemia is the main hallmark. Type 2 diabetes (T2DM) is the most common type of diabetes, and its main characteristics are insulin resistance and insulin deficiency. With the continuous increase in the number of global diabetes patients, finding effective hypoglycemic treatment regimens has become a hot topic in medical research.

[0003] In recent years, dipeptidyl peptidase-IV (DPP-IV) inhibitors, as a new type of hypoglycemic drug, have been widely used in clinical practice. One of the key mechanisms for regulating postprandial blood glucose levels is the inhibitory effect 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, its biological activity can be enhanced, thereby increasing insulin secretion and reducing postprandial blood glucose levels. However, currently available DPP-IV inhibitors on the market still have some deficiencies, such as side effects, drug resistance, etc. 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 properties. In recent years, scholars at home and abroad have continuously isolated peptide substances with hypoglycemic functions from natural animals and plants as well as the human body, and studied their structures and mechanisms of action, opening up a new way 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 and enzyme, and this oligopeptide has a significant inhibitory effect on DPP-IV enzyme and can increase the glucose consumption of insulin-resistant HePG2 cells.

[0005] Wheat peptides are prepared from wheat protein (gluten) through processes such as biological enzymolysis, refining, and spray drying. Patent document CN114990180A discloses that gluten is enzymolyzed successively using alkaline protease, compound enzyme, and flavor protease, and then dried after membrane filtration through 0.25 μm and 10 nm membranes to obtain wheat peptides, which have the effect of assisting in reducing blood sugar. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out highly effective peptide segments related to the blood sugar-lowering effect, it will provide a basis for the development of new blood sugar-lowering preparations. Summary of the Invention

[0006] The object of the present invention is to provide a natural small-molecule bioactive peptide with a blood sugar-lowering effect and apply it to the development of related blood sugar-lowering products.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention successively uses alkaline protease, neutral protease, and flavor protease to perform biological enzymolysis on wheat gluten, then uses ZGC108DQ gel strong acid cation exchange resin to adsorb the enzymolysis solution, and obtains clarified wheat peptides after desorbing with an alkali solution. The LC-MS / MS peptide spectrum analysis technology is used to analyze the polypeptide sequences of the clarified wheat peptides before and after gastrointestinal simulated digestion, and it is found that the clarified wheat peptides are rich in polypeptides containing the LLRP peptide segment, and the digestion product is the tetrapeptide LLRP. The amino acid sequence of the peptide segment LLRP is Leu-Leu-Arg-Pro (SEQ ID NO.1). The molecular docking technology is used to explore the interaction between the peptide segment and dipeptidyl peptidase-IV (DPP-IV). The results show that the tetrapeptide LLRP and the peptide segments containing LLRP have a high molecular docking energy with DPP-IV. Further, the tetrapeptide LLRP is artificially synthesized, and functional verification finds that this peptide segment has DPP-IV inhibitory activity and the function of reducing blood sugar.

[0009] Therefore, the present invention provides a bioactive peptide with a blood sugar-lowering effect, and the bioactive peptide is the tetrapeptide LLRP or a polypeptide containing the core peptide segment LLRP, and the amino acid sequence of LLRP is Leu-Leu-Arg-Pro.

[0010] The polypeptide containing the core peptide segment LLRP can produce the tetrapeptide LLRP after gastrointestinal digestion. Further, the amino acid sequence of the polypeptide containing the core peptide segment 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), KQLLRPR (SEQ ID NO.9).

[0011] The present invention also provides a preparation method of the bioactive peptide, and the bioactive peptide can be prepared by solid-phase synthesis. Specifically, the preparation method of the tetrapeptide LLRP includes: adopting the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the solid-phase carrier, and introducing proline, arginine, leucine, and leucine residues in sequence to extend the peptide chain from the C-terminus to the N-terminus, and solid-phase synthesizing the tetrapeptide LLRP.

[0012] The present invention also provides the application of the bioactive peptide in the preparation of drugs for preventing or treating diabetes or health foods for assisting in reducing blood sugar. The research of the present invention shows that the tetrapeptide LLRP has a blood sugar-lowering effect. In in vitro experiments, the tetrapeptide LLRP exhibits significant DPP-IV inhibitory activity; in the hyperglycemic zebrafish model, the blood sugar-lowering effect after the intervention of the tetrapeptide LLRP is better than that of the positive control metformin. Therefore, it can be applied to the development of related products for reducing blood sugar.

[0013] Further, the diabetes is type 2 diabetes.

[0014] Another object of the present invention is to provide a wheat peptide with a blood sugar-lowering effect. The wheat peptide is rich in a polypeptide containing the core peptide segment LLRP, and the amino acid sequence of the peptide segment LLRP is Leu-Leu-Arg-Pro;

[0015] The preparation method of the wheat peptide includes:

[0016] (1) Enzymatic hydrolysis: Mix wheat gluten and water at a mass ratio of 1:15 - 30, adjust the pH to 8.0 ± 0.2, add alkaline protease accounting for 0.5% of the total weight of gluten and hydrolyze for 15 - 30 min; then shear at a rotational speed of 10000 - 15000 r / min for 15 - 30 min; then add alkaline protease accounting for 0.5% - 3.0% of the total weight of gluten and neutral protease accounting for 0.5% - 2.0% of the total weight of gluten and continue to hydrolyze for 4 - 6 h. After completion, add flavor protease accounting for 0.1% - 1.5% of the total weight of gluten and act for 15 - 30 min;

[0017] (2) Separation: Centrifuge the enzymatic hydrolysate at a rotational speed of 4000 - 6000 r / min and take the supernatant. Add strongly acidic cation resin to the supernatant, adjust the pH to 3.0, stir and adsorb, then filter. Dissolve the filter cake in sodium hydroxide solution with a mass concentration of 1% - 5% for desorption. Take the supernatant and obtain the wheat peptide through nanofiltration, concentration, sterilization, and drying.

[0018] The wheat peptide provided by the present invention contains a polypeptide with a core peptide segment LLRP, which generates a tetrapeptide LLRP with hypoglycemic effect after gastrointestinal digestion, thereby exerting a hypoglycemic function.

[0019] Furthermore, the polypeptide with a core peptide segment LLRP is at least one of the peptide segments shown in the amino acid sequences of SEQ ID NO.2 - SEQ ID NO.9.

[0020] The present invention also provides the application of the wheat peptide in the preparation of a drug for preventing or treating diabetes or a health food for assisting in hypoglycemia. The wheat peptide exerts a hypoglycemic effect after gastrointestinal digestion.

[0021] The present invention provides a pharmaceutical composition for preventing or treating diabetes. The pharmaceutical composition includes an effective dose of tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP or the wheat peptide, and the amino acid sequence of LLRP is Leu - Leu - Arg - Pro.

[0022] In the pharmaceutical composition provided by the present invention, tetrapeptide LLRP or a polypeptide containing a core peptide segment LLRP or the wheat peptide can be used as the only active ingredient exerting a hypoglycemic effect, or can be compounded with other active ingredients having a hypoglycemic effect.

[0023] The pharmaceutical composition further includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver the active substance in an effective dose of the present invention, does not interfere with the biological activity of the active substance, and has no toxic or side effects on the host or subject.

[0024] Furthermore, the pharmaceutically acceptable carrier includes one or more of fillers, wetting agents, disintegrants, binders, or lubricants.

[0025] The present invention uses the tetrapeptide LLRP or a polypeptide containing the core peptide segment LLRP as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to the preparation method of the preparation recorded in pharmacy. Furthermore, the dosage form of the pharmaceutical composition can be, but is not limited to, an oral preparation. Specifically, the dosage form can be, but is not limited to, oral liquid, capsule, microcapsule powder, tablet, granule, or emulsion.

[0026] The present invention provides a health food for assisting in reducing blood sugar, including the tetrapeptide LLRP or a polypeptide containing the core peptide segment LLRP as the active ingredient, and a food-grade excipient acceptable in food science. The amino acid sequence of LLRP is Leu-Leu-Arg-Pro. The food-grade excipient acceptable in food science is a food-grade excipient that can deliver the active substance of the effective dose of the present invention without interfering with the biological activity of the active substance.

[0027] Furthermore, the dosage form of the health food is beverage, oral liquid, capsule, microcapsule powder, tablet, granule, or emulsion.

[0028] The beneficial effects of the present invention are as follows:

[0029] (1) The present invention provides a new bioactive peptide LLRP with DPP-IV inhibitory activity. Through animal experiments, it is verified that the tetrapeptide LLRP can effectively reduce the blood sugar level and has no obvious side effects. Therefore, it can be applied to the preparation of drugs or health foods for reducing blood sugar.

[0030] (2) The present invention provides a polypeptide with the peptide segment LLRP and a wheat peptide rich in such polypeptides, which generates the tetrapeptide LLRP with a blood sugar-lowering effect after gastrointestinal digestion, and can be applied to the preparation of drugs or health foods for reducing blood sugar. The present invention provides a new solution and theoretical basis for the treatment of diabetes and has good market prospects and application potential. Description of the Drawings

[0031] Figure 1 It is the secondary mass spectrum of the tetrapeptide LLRP.

[0032] Figure 2 It is the schematic diagram of the binding of the tetrapeptide LLRP to DPP-IV.

[0033] Figure 3 It is the blood sugar-lowering effect of the tetrapeptide LLRP in the hyperglycemic zebrafish model.

[0034] Figure 4Comparison of the hypoglycemic effects of tetrapeptide LLRP and PSLL in a hyperglycemic zebrafish model. Detailed implementation mode

[0035] 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 used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0036] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0037] Wheat protein (gluten) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.

[0038] Example 1: Preparation of clarified wheat peptide

[0039] Preparation of clarified wheat peptide by enzymatic hydrolysis: Put wheat protein (gluten) and water into the enzymatic hydrolysis reaction tank at a ratio of 1:20, adjust the pH of the feed liquid to 8.0±0.2, add 0.5% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten and carry out enzymatic hydrolysis for 30 min, then start the shearing machine with a shearing speed of 15000 r / min and shear for 30 min.

[0040] After the shearing is completed, adjust the pH of the feed liquid to 8.0±0.2, add 2.0% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten and 1.0% of neutral protease (derived from Bacillus subtilis) based on the total weight of gluten, and continue enzymatic hydrolysis for 5 h. After completion, use 0.8% of flavor protease based on the total weight of gluten and act for 30 min.

[0041] After the enzymatic hydrolysis is completed, centrifuge and separate through a disc centrifuge (rotation speed 6000 r / min), and intercept the supernatant.

[0042] Add ZGC108DQ gel strong acid cation resin to the supernatant after disc separation, adjust the pH to 3.0, stir and adsorb for 2 h. After the adsorption is completed, pass through a plate and frame filter press, take the filter cake and redissolve it in a sodium hydroxide solution with a mass concentration of 3.0% for desorption for 40 min. Take the supernatant and after nanofiltration, concentrate it by low-temperature vacuum to a feed liquid concentration of 32°Bx. The concentrated feed liquid is sterilized and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0043] Example 2: Screening of bioactive peptide segments

[0044] In this example, in vitro simulated gastrointestinal digestion of wheat peptides and LC-MS / MS were used to identify peptide segments with correlation before and after digestion. Molecular docking technology was used to explore the specific mechanism of their interaction with dipeptidyl peptidase-IV (DPP-IV), and efficient DPP-IV inhibitory peptides were screened out.

[0045] 1. In vitro simulated digestion

[0046] Take 10 mL of wheat peptide sample solution (preheated in a 37°C water bath), add 9 mg / mL NaCl solution (physiological saline, preheated in a 37°C water bath), adjust the pH value to 2 with 1 mol / L HCl, add 4 mL of gastric juice (0.4 g of pepsin dissolved in 0.01 mol / L HCl solution) to the system, and simulate gastric digestion in a 37°C water bath with shaking at 120 r / min for 2 h. Pre-adjust the pH value of the digested sample to 6.5. Pipette 2 mL of the gastric digested sample into a centrifuge tube, and add 4 mL of intestinal juice (0.02 g of trypsin and 0.12 g of porcine bile salt are respectively dissolved in 10 mL of 0.1 mol / L NaHCO 3 solution to obtain 2 mg / mL trypsin solution and 12 mg / mL bile solution, and mix the pancreatic juice and bile evenly at a ratio of 1:1), adjust the pH to 7.0, and simulate intestinal digestion in a 37°C water bath with shaking at 120 r / min for 2 h. All experiments were carried out in the dark and anaerobic conditions. The samples after simulated digestion were freeze-dried and stored at -80°C until analysis.

[0047] 2. Sequence identification of wheat peptides

[0048] The wheat peptide samples before and after simulated digestion were analyzed for polypeptide sequencing using LC-MS / MS method. The analysis was completed by Beijing Bio-Tech Pack Technology Co., Ltd. First, the samples were subjected to reduction alkylation and desalting, and then the treated samples were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry results. After de novo analysis by PEAKS Studio 10.6, the peptide sequence analysis results were obtained.

[0049] Analysis of the peptide profiles of wheat peptides before and after digestion found that before digestion, wheat peptides were rich in multiple peptide segments containing the LLRP sequence, and after digestion, they contained the LLRP peptide segment. After sorting, it is shown in Table 1.

[0050] Table 1. Peptide segments containing LLRP before and after digestion

[0051]

[0052] 3. Screening of active peptides with potential hypoglycemic function

[0053] Dipeptidyl peptidase-IV (DPP-IV) can weaken the hypoglycemic effect by degrading glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Inhibiting DPP-IV activity can prolong the activity of these incretin hormones, thereby promoting insulin secretion, inhibiting glucagon release, and achieving blood glucose control. DPP-IV inhibitors have become a core class of drugs for treating type 2 diabetes by selectively blocking the activity of this enzyme and enhancing the function of the endogenous incretin system to lower blood glucose. Therefore, in this example, the peptide segments obtained by digesting wheat peptides were molecularly docked with DPP-IV to screen for small peptide segments that may have hypoglycemic effects.

[0054] Peptide segments meeting the criteria were screened according to the conditions of average local confidence (ALC) greater than 95%, peak area greater than 2×10 6 , and PeptideRanker score greater than 0.8. Subsequently, the peptide segments were molecularly docked with dipeptidyl peptidase-IV (DPP-IV).

[0055] First, the crystal structure of DPP-IV (5CXV) was downloaded from the PDB protein database. After removing water molecules and adding hydrogen atoms to the receptor target through DiscoveryStudio software, its active center was defined. The structures of the selected peptide segments of wheat peptides were constructed by Discovery Studio, and their energies were minimized by the DPP-IV force field. These peptides were defined as ligands. The constructed peptides were docked with DPP-IV using CDOCKER to simulate the binding mode, site, and amino acid residues involved with the lowest binding energy and highest binding degree, and screening was performed according to the binding energy and the number of hydrogen bonds. Finally, two tetrapeptides (LLRP and PSLL) were determined, as shown in Table 2.

[0056] Table 2. Peptide segments with potential immunomodulatory activity in digested wheat peptides

[0057] 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

[0058] Among them, the secondary mass spectrum of the tetrapeptide LLRP is as Figure 1 shown. The cleavage fragment ions of peptides include: N-terminal fragment ions (types a, b, c) and C-terminal fragment ions (types x, y, z). 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 cleavage at both ends, etc. The b and y series ions are the most common. The primary structure of the peptide can be deduced by analyzing the b or y series fragment ions of the peptide, with a molecular weight of 497.33 Da. Further, the tetrapeptide was analyzed by in-source collision-induced dissociation technology for secondary mass spectrometry to determine the primary structure of the tetrapeptide as Leu-Leu-Arg-Pro.

[0059] The 2D and 3D molecular docking diagrams of tetrapeptide LLRP and DPP-IV are as Figure 2 shown. Analysis of chemical bonds reveals that tetrapeptide LLRP binds to DPP-IV mainly 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 forms 8 van der Waals forces with amino acid residues ARG429, TYR457, ILE405, GLY406, SER458, LEU60, SER460, and LEU57, 4 hydrogen bonds with VAL459, ARG471, and SER59, 1 hydrophobic interaction with ILE418, and 2 electrostatic interactions with GLU408 and ARG471.

[0060] From the molecular docking results, it can be seen that tetrapeptide LLRP can bind to DPP-IV, thereby inhibiting DPP-IV and then playing a hypoglycemic role.

[0061] Peptide segments LLRP and PSLL were synthesized by GL Biochem (Shanghai) Ltd., with a purity ≥ 98%, for subsequent functional verification.

[0062] Example 3: In vitro DPP-IV inhibition rate of tetrapeptide LLRP

[0063] The DPP-IV inhibition rate of the peptide segments was measured using a Cayman DPP-IV inhibitor kit. The measurement principle is as follows: This 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.

[0064] The DPP-IV inhibition rates of tetrapeptide LLRP and PSLL were measured at 1000 µM and 500 µM respectively. The measurement method is as follows:

[0065] (1) 100% initial activity wells - Add 30 µL of diluted buffer, 10 µL of DPP-IV, and 10 µL of pure water to three wells.

[0066] (2) Background wells - Add 40 µL of diluted buffer and 10 µL of pure water to three wells.

[0067] (3) Inhibitor wells - Add 30 µL of diluted experimental buffer, 10 µL of diluted DPP-IV, and 10 µL of inhibitor (intervention samples, LLRP and PSLL at concentrations of 1000 µM and 500 µM) to three wells.

[0068] The specific operation is as follows: First, add 30 µL of buffer, 10 µL of DPP-IV enzyme, and 10 µL of sample to a 96-well plate and mix well. Then add 50 µL of substrate solution to start the reaction. After incubating at 37 °C for 30 min, read the fluorescence at an excitation wavelength of 350 - 360 nm and an emission wavelength of 450 - 465 nm. Record the fluorescence intensity as the result of sample A. Record the result measured using buffer instead of DPP-IV as A blank, and record the result measured using buffer instead of sample as A control. The calculation formula for the DPP-IV inhibition rate is as follows: Inhibition rate (%) = (A control - A sample) / (A control - A blank) × 100%.

[0069] 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 also exceeded 50% at 500 µM, indicating that LLRP has DPP-IV inhibition potential. 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, with inhibition rates less than 10% at the measured concentrations of 1000 µM and 500 µM.

[0070] Table 3. DPP-IV inhibition rates (%) of tetrapeptides LLRP and PSLL

[0071]

[0072] Example 4: Hypoglycemic effect of tetrapeptide LLRP in a hyperglycemic zebrafish model

[0073] Use a hyperglycemic zebrafish model to determine the hypoglycemic effect of tetrapeptide LLRP. Four-day-old wild AB strain zebrafish were placed in six-well plates, with 3 wells in parallel for each group and 10 fish in each well. The grouping interventions are as follows:

[0074] (1) Blank control group (NC): System water;

[0075] (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water;

[0076] (3) Metformin positive control group (Met): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water;

[0077] (4)Four-peptide LLRP intervention group (LLRP-5, LLRP-1, LLRP-0.1): 333 μM alloxan + 2.67% glucose + 5 / 1 / 0.1 μg / mL four-peptide LLRP + system water.

[0078] After 4-day-old zebrafish were intervened according to the above intervention protocol for 24 h, they were repeatedly washed 3 times with PBS solution to wash away the sugar solution on the surface of the zebrafish. The zebrafish in the six-well plate were transferred to a 1.5 mL centrifuge tube using a disposable pipette, the excess liquid in the centrifuge tube was aspirated, 0.1 mL of absolute ethanol was added, and after standing in a cool place for 15 min, it was transferred to an oven for 120 min (60 °C) until dried (mashed). Finally, 5 μL of ultrapure water was added to the centrifuge tube, shaken ultrasonically for 10 min, and 2 μL was aspirated to measure the glucose value using a glucometer.

[0079] The results were as Figure 3 shown. Compared with the blank control group (NC group), the blood glucose value of the model control group (MC group) after modeling with 333 μM alloxan + 2.67% glucose for 24 h increased significantly ( p <0.0001), indicating that a hyperglycemic model was successfully established. After intervention with the positive drug 5 μg / mL metformin (Met group), there was a significant down-regulation compared with the MC group ( p <0.01). Under the intervention of 5 μg / mL and 1 μg / mL concentrations of four-peptide LLRP, compared with the MC group, the blood glucose value was significantly down-regulated ( p <0.01), and it was down-regulated to no significant difference from the NC group ( p >0.05), indicating that four-peptide LLRP has a significant hypoglycemic effect, and its effect is equivalent to that of metformin (Met) with the same dosage.

[0080] Example 5: Comparison of hypoglycemic effects of four-peptide LLRP and PSLL in a hyperglycemic zebrafish model

[0081] The hypoglycemic effects of four-peptide LLRP and PSLL were compared using a hyperglycemic zebrafish model. Wild AB strain 4-day-old zebrafish were placed in a six-well plate, with 3 parallel wells in each group and 10 in each well. The grouping was as follows:

[0082] (1)Blank control group (NC): System water;

[0083] (2)Model group (MC): 333 μM alloxan + 2.67% glucose + system water;

[0084] (3)Four-peptide LLRP intervention group (LLRP): 333 μM alloxan + 2.67% glucose + 1 μg / mL four-peptide LLRP + system water;

[0085] (4)Tetrapeptide PSLL intervention group (PSLL): 333 μM alloxan + 2.67% glucose + 1 μg / mL tetrapeptide PSLL + system water.

[0086] The intervention method and blood glucose measurement method were the same as those in "Hypoglycemic effect of tetrapeptide LLRP in hyperglycemic zebrafish model". The results were as Figure 4 shown. Under the intervention of tetrapeptide LLRP and PSLL at a concentration of 1 μg / mL, compared with the model control MC group, tetrapeptide LLRP significantly down-regulated the blood glucose value ( p < 0.01), while tetrapeptide PSLL had no significant down-regulation effect ( p > 0.05).

Claims

1. The use of the tetrapeptide LLRP in the preparation of a drug for preventing or treating diabetes or a health food for assisting in lowering blood sugar, characterized in that: The amino acid sequence of LLRP is Leu-Leu-Arg-Pro.

2. The use according to claim 1, characterized in that The diabetes is type 2 diabetes.

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