Phycobiliprotein active peptide with function of reducing blood sugar as well as preparation method and application of phycobiliprotein active peptide

Abstract

The embodiment of the invention relates to a phycobiliprotein active peptide with a hypoglycemic function and a preparation method and application thereof, and belongs to the technical field of biological active peptides. According to the phycobiliprotein active peptide with the blood sugar reducing function, the amino acid sequence of the phycobiliprotein active peptide is Ser-Tyr-Phe-Asp-Arg-Ala, Ile-Ala-Ser-Tyr-Phe-Asp-Arg, Gly-Tyr-Tyr-Leu-Arg or Val-Ala-Phe-Gly-Arg-Phe, and the amino acid sequence of the phycobiliprotein active peptide with the blood sugar reducing function is shown in the description. The phycobiliprotein active peptide in the embodiment of the invention is separated from spirulina phycobiliprotein, and the spirulina phycobiliprotein is an important protein resource in spirulina and a high-quality source of bioactive peptide, and has wider application; the phycobiliprotein active peptide is low in molecular weight, and compared with a chemically synthesized hypoglycemic preparation, food-borne peptides are safer and more efficient, free of toxic and side effects and easy to digest and absorb; the phycobiliprotein active peptide has high binding affinity with the alpha-glucosidase and the DPP-IV, can form a very stable ligand receptor compound, and has a good inhibition effect on the alpha-glucosidase and the DPP-IV; the compound has excellent inhibition performance on alpha-glucosidase, alpha-amylase and DPP-IV (dipeptidyl peptidase-IV).

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of bioactive peptides, and in particular to a phycobiliprotein active peptide with blood sugar lowering function and a preparation method and application thereof. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar caused by abnormal insulin metabolism or insufficient secretion. The absolute or relative lack of insulin secretion causes metabolic disorders of carbohydrates, proteins, fats, and the destruction of oxidative balance. Complications faced by diabetic patients include cardiovascular disease, kidney disease, retinopathy, and nerve damage. Types of diabetes include type 1 diabetes, type 2 diabetes, and gestational diabetes. Type 2 diabetes is the most common type of diabetes, accounting for more than 90% of diabetic patients.

[0003] Under the increasing pressure of patients, drugs for the treatment of type 2 diabetes are emerging in an endless stream, mainly sulfadiazine, dimethoate, benzoic acid derivatives, glycosidase inhibitors and thiazolidinediones. Although these drugs can temporarily relieve diabetes, long-term use will produce toxic side effects.

[0004] In recent years, natural products have attracted widespread attention due to their potential therapeutic effects. Studies have shown that some components in Spirulina may regulate blood sugar levels through different mechanisms, such as improving insulin resistance, promoting insulin secretion, or inhibiting the absorption of carbohydrates in the intestine. As a blue-green algae, Spirulina is not only rich in protein, vitamins, minerals and essential fatty acids, but also contains a variety of bioactive substances, such as phycocyanin, polysaccharides and carotenoids. Among them, phycobiliprotein is an important pigment protein in Spirulina, which has multiple physiological activities such as regulating immune function, anti-oxidation and anti-tumor. However, there are relatively few studies on the active peptides of phycobiliprotein in Spirulina in lowering blood sugar.

[0005] With the development of bioinformatics, database-assisted computer simulation methods have been successfully applied to predict and design bioactive peptides with different activities, such as DPP-IV inhibitory peptides, ACE inhibitory peptides, antioxidant peptides, etc. Molecular docking technology is a commonly used bioinformatics technology. Its principle is to simulate the geometric model of molecules through chemometric methods and calculate the energy of the model, and then find the optimal binding mode between small molecules (ligands) and biological macromolecules (receptors). Therefore, molecular docking is widely used to simulate peptide-receptor protein interactions and helps to further understand the targeting mechanism of peptides. Summary of the invention

[0006] In view of this, the embodiments of the present application provide a phycobiliprotein active peptide with blood sugar lowering function and its preparation method application. The phycobiliprotein active peptide is expected to provide a new treatment option for diabetic patients, while opening up a new way for the high-value utilization of Spirulina resources, and can effectively overcome the defects of the above-mentioned prior art.

[0007] According to a first aspect of an embodiment of the present application, a phycobiliprotein active peptide having a blood sugar lowering function is provided, wherein the amino acid sequence of the phycobiliprotein active peptide is Ser-Tyr-Phe-Asp-Arg-Ala, Ile-Ala-Ser-Tyr-Phe-Asp-Arg, Gly-Tyr-Tyr-Leu-Arg or Val-Ala-Phe-Gly-Arg-Phe.

[0008] Ser means the English name is Serine, and the Chinese name is serine;

[0009] Tyr means the English name is Tyrosine, and the Chinese name is tyrosine;

[0010] Phe means Phenylalanine in English and phenylalanine in Chinese;

[0011] Asp means Asparagine in English and Asparagine in Chinese;

[0012] Arg means the English name is Arginine and the Chinese name is Arginine;

[0013] Ala means Alanine in English and Alanine in Chinese;

[0014] Ile means the English name is Isoleucine and the Chinese name is Isoleucine;

[0015] Gly means Glycine in English and glycine in Chinese;

[0016] Leu means the English name is Leucine, and the Chinese name is leucine;

[0017] Val means the English name is Valine and the Chinese name is valine.

[0018] The active peptides in the present application are obtained from the above amino acids by solid phase synthesis or directed bio-enzymatic hydrolysis and purification.

[0019] In some embodiments that may include the above embodiments, the amino acid sequence of the phycobiliprotein active peptide is Ser-Tyr-Phe-Asp-Arg-Ala, abbreviated as SYFDRA, and its molecular formula is C 34 H47 N9O 11 , molecular weight is 757.78Da.

[0020] In some embodiments that may include the above embodiments, the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 3.504 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.8603 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 8.344 mg / mL;

[0021] The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of -20.3 to 11.7% on α-glucosidase in a concentration range of 0 to 2 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 27.7 to 62.5% on α-amylase in a concentration range of 0 to 2 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 31.1 to 55.8% on DPP-IV in a concentration range of 0 to 10 mg / mL.

[0022] In some embodiments that may include the above embodiments, the amino acid sequence of the phycobiliprotein active peptide is Ile-Ala-Ser-Tyr-Phe-Asp-Arg, abbreviated as IASYFDR, and its molecular formula is C 40 H 58 N 10 O 12 , molecular weight is 870.94Da.

[0023] In some embodiments that may include the above embodiments, the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 1.046 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.1292 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 0.9089 mg / mL;

[0024] The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 3.46-65.1% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of 48.7-66.8% on α-amylase in a concentration range of 0-2 mg / mL; the inhibition rate of 18.6-97.3% on DPP-IV in a concentration range of 0-10 mg / mL.

[0025] In some embodiments that may include the above embodiments, the amino acid sequence of the phycobiliprotein active peptide is Gly-Tyr-Tyr-Leu-Arg, abbreviated as GYYLR, and its molecular formula is C 32 H 46 N8O8, molecular weight is 670.75Da.

[0026] In some embodiments that may include the above embodiments, the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 0.1148 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.3080 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 4.6770 mg / mL;

[0027] The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 49.2-87.9% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of 39.6-68.1% on α-amylase in a concentration range of 0-2 mg / mL; the inhibition rate of 33.2-55.7% on DPP-IV in a concentration range of 0-10 mg / mL.

[0028] In some embodiments that may include the above embodiments, the amino acid sequence of the phycobiliprotein active peptide is Val-Ala-Phe-Gly-Arg-Phe, abbreviated as VAFGRF, and its molecular formula is C 34 H 49 N9O7, with a molecular weight of 695.81 Da; and

[0029] The phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 0.7824 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.8297 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 0.9468 mg / mL;

[0030] The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 22.4-63.7% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of phycobiliprotein active peptide with hypoglycemic function on α-amylase is 22.8-69.5% in a concentration range of 0-2 mg / mL; the inhibition rate of phycobiliprotein active peptide with hypoglycemic function on DPP-IV is 31.4-94.0% in a concentration range of 0-10 mg / mL.

[0031] The second aspect of the present application also provides a method for preparing the above-mentioned phycobiliprotein active peptide with hypoglycemic function, comprising the following steps:

[0032] The phycobiliprotein active peptides are extracted from the phycobiliprotein of Spirulina by solid phase synthesis or directed bio-enzymatic hydrolysis and purification.

[0033] The third aspect of the embodiments of the present application also provides the use of the above-mentioned phycobiliprotein active peptide with hypoglycemic function in the preparation of medicines or foods for preventing or treating type 2 diabetes.

[0034] Preferably, α-glucosidase and DPP-IV crystal structure are selected and the α-glucosidase and DPP-IV crystal structure are pretreated.

[0035] Preferably, a 3D structure of the phycobiliprotein active peptide is constructed, and the constructed 3D structure is pretreated.

[0036] Preferably, the 3D structure of the pretreated phycobiliprotein active peptide is molecularly docked with the pretreated α-glucosidase and DPP-IV crystals, respectively.

[0037] Preferably, the α-glucosidase inhibition rate of the phycobiliprotein active peptide obtained by solid phase synthesis at a concentration of 0 to 2 mg / mL is determined, and the half-maximal inhibitory concentration IC50 value of the phycobiliprotein active peptide on α-glucosidase is obtained.

[0038] Specifically, the determination of the α-glucosidase inhibition rate comprises the following steps:

[0039] (A1) preparing the synthetic peptide into gradient concentration sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL;

[0040] (A2) Add 50 μL of sample solutions of different concentrations and 50 μL of 1 U / mL α-glucosidase solution into a 96-well plate and mix well;

[0041] (A3) shake at 37°C for 15 min;

[0042] (A4) Add 100 μL of substrate solution (1 mM p-NPG solution);

[0043] (A5) Incubate at 37°C on a shaker for 30 min;

[0044] (A6) Add 100 μL of Na2CO3 solution to terminate the reaction;

[0045] (A7) measuring absorbance at 405 nm;

[0046] (A8) Negative control group used 50 μL of PBS buffer instead of 50 μL of sample solutions of different concentrations;

[0047] (A9) In the positive control group, 50 μL of acarbose solution with the same concentration as the synthetic peptide was used to replace 50 μL of sample solutions with different concentrations;

[0048] (A10) background group, 50 μL of PBS buffer was used instead of 50 μL of 1 U / mL α-glucosidase solution;

[0049] (A11) The remaining operations are the same as above;

[0050] (A12) The inhibition rate of α-glucosidase is obtained by formula (1).

[0051]

[0052] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0053] Preferably, the α-amylase inhibition rate of the phycobiliprotein active peptide obtained by solid phase synthesis at a concentration of 0-2 mg / mL is determined, and the half inhibition concentration IC50 value of the phycobiliprotein active peptide on α-amylase is obtained.

[0054] Specifically, the determination of α-amylase inhibition rate comprises the following steps:

[0055] (B1) preparing the synthetic peptide into gradient concentration sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL;

[0056] (B2) Add 100 μL of sample solution of different concentrations and 100 μL of 1 U / mL α-amylase solution into a 1.5 mL centrifuge tube and mix well;

[0057] (B3) shake at 37°C for 10 min;

[0058] (B4) Add 200 μL of 2% pretreated starch solution (heat in a 95°C water bath for 8 min to denature it, wait until the temperature drops to room temperature, mix well and set aside);

[0059] (B5) shake at 37°C for 20 min;

[0060] (B6) Add 200 μL of reaction termination solution (0.4 M Na2CO3 solution containing 1% DNS and 12% sodium potassium tartrate);

[0061] (B7) heating in a water bath at 100°C for 15 min;

[0062] (B8) After the reaction is completed, cool to room temperature, pipette 200 μL of the reaction solution into a 96-well plate, and measure the absorbance at 540 nm;

[0063] (B9) Negative control group used 100 μL of PBS buffer instead of 100 μL of sample solutions of different concentrations;

[0064] (B10) The positive control group used 100 μL of acarbose solution with the same concentration as the synthetic peptide instead of 100 μL of sample solutions with different concentrations;

[0065] (B11) background group, 100 μL of PBS buffer was used instead of 100 μL of 1U / mL α-amylase solution;

[0066] (B12) The remaining operations are the same as above;

[0067] (B13) The inhibition rate of α-amylase is obtained by formula (2).

[0068]

[0069] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0070] Preferably, the DPP-IV inhibition rate of the phycobiliprotein active peptide obtained by solid phase synthesis at a concentration of 0 to 10 mg / mL is determined, and the half maximal inhibition concentration IC50 value of the phycobiliprotein active peptide on DPP-IV is obtained.

[0071] Specifically, the DPP-IV inhibition rate was determined using the Elabscience dipeptidyl peptidase 4 (DPP4) inhibitor screening test kit, all reagents were prepared according to the instructions, and the determination included the following steps:

[0072] (C1) preparing the synthetic peptide into gradient concentration sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL;

[0073] (C2) Add 20 μL of enzyme reagent and 30 μL of sample solution of different concentrations into a 96-well plate;

[0074] (C3) shake the plate for 3 seconds and incubate at 37°C for 10 minutes;

[0075] (C4) Add 170 μL of reaction working solution to each well;

[0076] (C5) Incubate at 37°C for 30 min, and measure the fluorescence value of each well with a fluorescence microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm;

[0077] (C6) Negative control group used 30 μL of PBS buffer instead of 30 μL of sample solutions with different concentrations;

[0078] (C7) The positive control group used 30 μL of sitagliptin solution of different concentrations instead of 30 μL of sample solution of different concentrations;

[0079] (C8) Background group, 20 μL of buffer was used instead of 20 μL of DPP-IV enzyme solution;

[0080] (C9) The remaining operations are the same as above;

[0081] (C10) The inhibition rate of DPP-IV was obtained by formula (3).

[0082]

[0083] Among them, F a is the fluorescence value of the negative control group; F0 is the fluorescence value of the background group; F b is the fluorescence value of the experimental group.

[0084] Preferably, the phycobiliprotein active peptide is added to food or medicine to prepare functional food or blood sugar-lowering medicine.

[0085] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0086] 1. In the examples of the present application, the phycobiliprotein active peptides are separated from Spirulina phycobiliproteins. Spirulina phycobiliproteins are important protein resources and high-quality sources of bioactive peptides in Spirulina and will have wider applications;

[0087] 2. The molecular weight of the phycobiliprotein active peptides in the examples of this application is relatively low. Compared with chemically synthesized hypoglycemic preparations, food-derived peptides are safer, more efficient, non-toxic and non-side effects, and easy to digest and absorb;

[0088] 3. The molecular docking results in the examples of this application show that the phycobiliprotein active peptide has a high binding affinity with α-glucosidase and DPP-IV, can form a very stable ligand receptor complex, and has a good inhibitory effect on α-glucosidase and DPP-IV;

[0089] 4. The phycobiliprotein active peptides in the examples of the present application have excellent inhibitory properties against α-glucosidase, α-amylase and DPP-IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0091] Figure 1 3D structure diagram of SYFDRA in Example 1 of the present application;

[0092] Figure 2 3D and 2D images of SYFDRA binding to the active center of α-glucosidase in Example 1 of the present application;

[0093] Figure 3 3D and 2D diagrams of the binding of SYFDRA to the active center of DPP-IV in Example 1 of the present application;

[0094] Figure 4 This is a graph showing the inhibition of α-glucosidase activity by SYFDRA in Example 1 of the present application;

[0095] Figure 5 This is a graph showing the inhibition of SYFDRA on α-amylase activity in Example 1 of the present application;

[0096] Figure 6 This is a graph showing the inhibition of DPP-IV activity by SYFDRA in Example 1 of the present application;

[0097] Figure 7 3D structure diagram of IASYFDR in Example 2 of the present application;

[0098] Figure 8 3D and 2D images of the binding of IASYFDR to the active center of α-glucosidase in Example 2 of the present application;

[0099] Fig. 9 3D and 2D images of the binding of IASYFDR to the active center of DPP-Ⅳ in Example 2 of the present application;

[0100] Fig.10 This is a graph showing the inhibition of α-glucosidase activity by IASYFDR in Example 2 of the present application;

[0101] Fig.11 This is a graph showing the inhibition of α-amylase activity by IASYFDR in Example 2 of the present application;

[0102] Fig.12 This is a graph showing the inhibition of DPP-Ⅳ activity by IASYFDR in Example 2 of the present application;

[0103] Fig.13 It is a 3D structure diagram of GYYLR in Example 3 of the present application;

[0104] Fig.14 3D and 2D diagrams of the binding of GYYLR to the active center of α-glucosidase in Example 3 of the present application;

[0105] Fig.15 3D and 2D diagrams of the binding of GYYLR to the active center of DPP-Ⅳ in Example 3 of the present application;

[0106] Fig.16 This is a graph showing the inhibition of α-glucosidase activity by GYYLR in Example 3 of the present application;

[0107] Fig.17 This is a graph showing the inhibition of α-amylase activity by GYYLR in Example 3 of the present application;

[0108] Fig.18 This is a graph showing the activity inhibition of GYYLR on DPP-Ⅳ in Example 3 of the present application;

[0109] Fig.19 3D structure diagram of VAFGRF in Example 4 of the present application;

[0110] Fig. 20 3D and 2D images of the binding of VAFGRF to the active center of α-glucosidase in Example 4 of the present application;

[0111] Fig.21 3D and 2D diagrams of the binding of VAFGRF to the active center of DPP-IV in Example 4 of the present application;

[0112] Fig. 22 This is a graph showing the inhibition of α-glucosidase activity by VAFGRF in Example 4 of the present application;

[0113] Fig.23 This is a graph showing the inhibition of α-amylase activity by VAFGRF in Example 4 of the present application;

[0114] Fig.24 This is a graph showing the inhibitory activity of VAFGRF on DPP-Ⅳ in Example 4 of the present application. DETAILED DESCRIPTION

[0115] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0116] Unless otherwise specified, the experimental methods used in the examples of this application are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0117] Example 1

[0118] (1) Molecular docking of the active peptide SYFDRA with α-glucosidase and DPP-4

[0119] α-glucosidase (PDB ID: 3wy1) and DPP-4 (PDB ID: 4a5s) were selected as receptor proteins, and SYFDRA was used as ligand molecules. The structure of the ligand molecule was drawn using ChemDraw software, and then the 2D structure was modified by MM2 energy minimization using Chem3D software. The 3D crystal structure of the receptor protein was downloaded from the PDB database to remove the solvent and the original ligand small molecule contained in the macromolecular receptor; the receptor protein was hydrogenated, the charge was calculated, and the atom type was set using Autodock software, and the charge of the ligand molecule was adjusted, the torsion center and torsion bond were detected, and the docking box coordinates and size were determined ((3wy1:(2.473,-18.215,10.254) 4a5s:(13.984,29.803,56.514) ), perform semi-flexible docking, predict the binding energy between the ligand small molecule and the protein, select the lowest binding energy as the optimal conformation, and use Pymol software to visualize the docking results.

[0120] (2) Synthesis of the active peptide SYFDRA and verification of its hypoglycemic activity in vitro

[0121] 1. Synthesis of peptides

[0122] The peptide synthesis experiment was commissioned to Shanghai Kepeptide Biotechnology Co., Ltd., and the method used was solid phase synthesis. The crude peptide samples synthesized need to be purified to obtain purer samples. The purity of the purified synthetic peptides was identified by HPLC, and the molecular weight was identified by MS.

[0123] 2. Verification of α-glucosidase inhibition effect

[0124] The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 50 μL of sample solutions of different concentrations and 50 μL of 1U / mL α-glucosidase solution were added to a 96-well plate, mixed evenly, and reacted in a 37°C constant temperature incubator for 15 minutes. Then 100 μL of substrate solution (1 mM p-NPG solution) was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 30 minutes, and then 100 μL of Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm. 50 μL of buffer solution and 50 μL of enzyme solution were added to the negative control group, 50 μL of sample solution and 50 μL of buffer solution were added to the background group, and 50 μL of acarbose solution and 50 μL of enzyme solution of the same concentration as the synthetic peptide were added to the positive control group, and the rest of the operations were the same as above. The calculation formula of α-glucosidase inhibition rate is as follows:

[0125]

[0126] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0127] 3. Verification of α-amylase inhibition effect

[0128] The synthetic peptide was prepared into gradient concentration sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 100 μL of sample solutions of different concentrations and 100 μL of 1U / mL α-amylase solution were added to a 1.5 mL centrifuge tube, mixed evenly, and reacted in a 37°C constant temperature incubator for 10 min. Then 200 μL of 2% pretreated (heated in a 95°C water bath for 8 min to denature it, and then mixed evenly for use when the temperature drops to room temperature) starch solution was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 20 min. 200 μL of reaction termination solution (0.4 M Na2CO3 solution containing 1% DNS and 12% sodium potassium tartrate) was added, and heated in a 100°C water bath for 15 min. After the reaction was completed, the temperature dropped to room temperature, 200 μL of the reaction solution was aspirated into a 96-well plate, and the absorbance was measured at 540 nm. The negative control group used 100 μL of PBS buffer instead of 100 μL of sample solutions of different concentrations, the positive control group used 100 μL of acarbose solution with the same concentration as the synthetic peptide instead of 100 μL of sample solutions of different concentrations, and the background group used 100 μL of PBS buffer instead of 100 μL of 1U / mL α-amylase solution, and the rest of the operations were the same as above.

[0129] The inhibition rate of α-amylase was obtained by the following formula.

[0130]

[0131] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0132] 4. Verification of DPP-Ⅳ inhibitory effect

[0133] The DPP-IV inhibition rate was determined by using the Elabscience dipeptidyl peptidase 4 (DPP4) inhibitor screening test kit. The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL. 20 μL of enzyme reagent and 30 μL of sample solutions of different concentrations were added to a 96-well plate, the plate was shaken for 3s, and incubated at 37°C for 10min. 170 μL of reaction working solution was added to each well, incubated at 37°C for 30min, and the fluorescence value of each well was detected by a fluorescence microplate reader at an excitation wavelength of 360nm and an emission wavelength of 460nm. The negative control group used 30 μL of PBS buffer instead of 30 μL of sample solution of different concentrations, the positive control group used 30 μL of sitagliptin solution of different concentrations instead of 30 μL of sample solution of different concentrations, and the background group used 20 μL of buffer instead of 20 μL of DPP-IV enzyme solution. The rest of the operations were the same as above.

[0134] The inhibition rate of DPP-IV was obtained by the following formula.

[0135]

[0136] Among them, F a is the fluorescence value of the negative control group; F0 is the fluorescence value of the background group; F b is the fluorescence value of the experimental group.

[0137] After completing the above experiments, a series of data on the novel hypoglycemic active peptide SYFDRA discovered in this application and its inhibitory activity against α-glucosidase, α-amylase and DPP4 were obtained. Figure 1 Shown is the 3D structure diagram of SYFDRA;

[0138] Figure 2 and Figure 3 3D and 2D diagrams of SYFDRA binding to the active centers of α-glucosidase and DPP-Ⅳ, respectively; in order to more intuitively demonstrate the inhibitory activity of the present application on α-glucosidase, α-amylase and DPP4, a dose-response curve diagram was drawn, such as Figure 4 , Figure 5, Figure 6 shown.

[0139] Example 2

[0140] (1) Molecular docking of the active peptide IASYFDR with α-glucosidase and DPP-4

[0141] α-glucosidase (PDB ID: 3wy1) and DPP-4 (PDB ID: 4a5s) were selected as receptor proteins, and IASYFDR was used as ligand molecules. The structure of the ligand molecule was drawn using ChemDraw software, and then the 2D structure was modified by MM2 energy minimization using Chem3D software. The 3D crystal structure of the receptor protein was downloaded from the PDB database to remove the solvent and the original ligand small molecule contained in the macromolecular receptor; the receptor protein was hydrogenated, the charge was calculated, and the atom type was set using Autodock software, and the charge of the ligand molecule was adjusted, the torsion center and torsion bond were detected, and the docking box coordinates and size were determined (3wy1: (2.473, -18.215, 10.254) 4a5s:(13.984,29.803,56.514) ), perform semi-flexible docking, predict the binding energy between the ligand small molecule and the protein, select the lowest binding energy as the optimal conformation, and use Pymol software to visualize the docking results.

[0142] (2) Synthesis of the active peptide IASYFDR and verification of its hypoglycemic activity in vitro

[0143] 1. Synthesis of peptides

[0144] The peptide synthesis experiment was commissioned to Shanghai Kepeptide Biotechnology Co., Ltd., and the method used was solid phase synthesis. The crude peptide samples synthesized need to be purified to obtain purer samples. The purity of the purified synthetic peptides was identified by HPLC, and the molecular weight was identified by MS.

[0145] 2. Verification of α-glucosidase inhibition effect

[0146] The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 50 μL of sample solutions of different concentrations and 50 μL of 1U / mL α-glucosidase solution were added to a 96-well plate, mixed evenly, and reacted in a 37°C constant temperature incubator for 15 minutes. Then 100 μL of substrate solution (1 mM p-NPG solution) was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 30 minutes, and then 100 μL of Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm. 50 μL of buffer solution and 50 μL of enzyme solution were added to the negative control group, 50 μL of sample solution and 50 μL of buffer solution were added to the background group, and 50 μL of acarbose solution and 50 μL of enzyme solution of the same concentration as the synthetic peptide were added to the positive control group, and the rest of the operations were the same as above. The calculation formula of α-glucosidase inhibition rate is as follows:

[0147]

[0148] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0149] 3. Verification of α-amylase inhibition effect

[0150] The synthetic peptide was prepared into gradient concentration sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 100 μL of sample solutions of different concentrations and 100 μL of 1U / mL α-amylase solution were added to a 1.5 mL centrifuge tube, mixed evenly, and reacted in a 37°C constant temperature incubator for 10 min. Then 200 μL of 2% pretreated (heated in a 95°C water bath for 8 min to denature it, and then mixed evenly for use when the temperature drops to room temperature) starch solution was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 20 min. 200 μL of reaction termination solution (0.4 M Na2CO3 solution containing 1% DNS and 12% sodium potassium tartrate) was added, and heated in a 100°C water bath for 15 min. After the reaction was completed, the temperature dropped to room temperature, 200 μL of the reaction solution was aspirated into a 96-well plate, and the absorbance was measured at 540 nm. The negative control group used 100 μL of PBS buffer instead of 100 μL of sample solutions of different concentrations, the positive control group used 100 μL of acarbose solution with the same concentration as the synthetic peptide instead of 100 μL of sample solutions of different concentrations, and the background group used 100 μL of PBS buffer instead of 100 μL of 1U / mL α-amylase solution, and the rest of the operations were the same as above.

[0151] The inhibition rate of α-amylase was obtained by the following formula.

[0152]

[0153] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0154] 4. Verification of DPP-Ⅳ inhibitory effect

[0155] The DPP-IV inhibition rate was determined by using the Elabscience dipeptidyl peptidase 4 (DPP4) inhibitor screening test kit. The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL. 20 μL of enzyme reagent and 30 μL of sample solutions of different concentrations were added to a 96-well plate, the plate was shaken for 3s, and incubated at 37°C for 10min. 170 μL of reaction working solution was added to each well, incubated at 37°C for 30min, and the fluorescence value of each well was detected by a fluorescence microplate reader at an excitation wavelength of 360nm and an emission wavelength of 460nm. The negative control group used 30 μL of PBS buffer instead of 30 μL of sample solution of different concentrations, the positive control group used 30 μL of sitagliptin solution of different concentrations instead of 30 μL of sample solution of different concentrations, and the background group used 20 μL of buffer instead of 20 μL of DPP-IV enzyme solution. The rest of the operations were the same as above.

[0156] The inhibition rate of DPP-IV was obtained by the following formula.

[0157]

[0158] Among them, F a is the fluorescence value of the negative control group; F0 is the fluorescence value of the background group; F b is the fluorescence value of the experimental group.

[0159] After completing the above experiments, a series of data on the novel hypoglycemic active peptide IASYFDR discovered in this application and its inhibitory activity against α-glucosidase, α-amylase and DPP4 were obtained. Figure 7 Shown is the 3D structure diagram of IASYFDR; Figure 8 and Fig. 9 3D and 2D diagrams of IASYFDR binding to the active center of α-glucosidase and DPP-Ⅳ, respectively; in order to more intuitively demonstrate the inhibitory activity of the present application on α-glucosidase, α-amylase and DPP4, a dose-response curve diagram was drawn, such as Fig.10 , Fig.11 , Fig.12 shown.

[0160] Example 3

[0161] (1) Molecular docking of the active peptide GYYLR with α-glucosidase and DPP-4

[0162] α-glucosidase (PDB ID: 3wy1) and DPP-4 (PDB ID: 4a5s) were selected as receptor proteins, and GYYLR was selected as ligand molecule. The structure of ligand molecule was drawn by ChemDraw software, and then the 2D structure was modified by MM2 energy minimization using Chem3D software. The 3D crystal structure of receptor protein was downloaded from PDB database, and the solvent and original ligand small molecule contained in the macromolecular receptor were removed; Autodock software was used to hydrogenate, calculate charge, and set atom type for receptor protein, adjust charge, detect torsion center and torsion bond for ligand molecule, and determine the coordinates and size of docking box (3wy1: (2.473, -18.215, 10.254) 4a5s:(13.984,29.803,56.514) ), perform semi-flexible docking, predict the binding energy between the ligand small molecule and the protein, select the lowest binding energy as the optimal conformation, and use Pymol software to visualize the docking results.

[0163] (2) Synthesis of the active peptide GYYLR and verification of its hypoglycemic activity in vitro

[0164] 1. Synthesis of peptides

[0165] The peptide synthesis experiment was commissioned to Shanghai Kepeptide Biotechnology Co., Ltd., and the method used was solid phase synthesis. The crude peptide samples synthesized need to be purified to obtain purer samples. The purity of the purified synthetic peptides was identified by HPLC, and the molecular weight was identified by MS.

[0166] 2. Verification of α-glucosidase inhibition effect

[0167] The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 50 μL of sample solutions of different concentrations and 50 μL of 1U / mL α-glucosidase solution were added to a 96-well plate, mixed evenly, and reacted in a 37°C constant temperature incubator for 15 minutes. Then 100 μL of substrate solution (1 mM p-NPG solution) was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 30 minutes, and then 100 μL of Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm. 50 μL of buffer solution and 50 μL of enzyme solution were added to the negative control group, 50 μL of sample solution and 50 μL of buffer solution were added to the background group, and 50 μL of acarbose solution and 50 μL of enzyme solution of the same concentration as the synthetic peptide were added to the positive control group, and the rest of the operations were the same as above. The calculation formula of α-glucosidase inhibition rate is as follows:

[0168]

[0169] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0170] 3. Verification of α-amylase inhibition effect

[0171] Prepare the synthetic peptide into gradient concentration sample solutions of 0.125mg / mL, 0.25mg / mL, 0.5mg / mL, 1mg / mL, 1.5mg / mL, and 2mg / mL. Add 100μL of sample solutions of different concentrations and 100μL of 1U / mL α-amylase solution to a 1.5mL centrifuge tube, mix well, and react in a 37℃ constant temperature incubator for 10min. Add 200μL of 2% pretreated (heat in a 95℃ water bath for 8min to denature it, wait until the temperature drops to room temperature, mix well for use) starch solution, mix well, and react in a 37℃ constant temperature incubator for 20min. Add 200μL of reaction termination solution (0.4M Na2CO3 solution containing 1% DNS and 12% sodium potassium tartrate), and heat in a 100℃ water bath for 15min. After the reaction is completed, cool to room temperature, aspirate 200μL of reaction solution into a 96-well plate, and measure the absorbance at 540nm. The negative control group used 100 μL of PBS buffer instead of 100 μL of sample solutions of different concentrations, the positive control group used 100 μL of acarbose solution with the same concentration as the synthetic peptide instead of 100 μL of sample solutions of different concentrations, and the background group used 100 μL of PBS buffer instead of 100 μL of 1U / mL α-amylase solution, and the rest of the operations were the same as above.

[0172] The inhibition rate of α-amylase was obtained by the following formula.

[0173]

[0174] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0175] 4. Verification of DPP-Ⅳ inhibitory effect

[0176] The DPP-IV inhibition rate was determined by using the Elabscience dipeptidyl peptidase 4 (DPP4) inhibitor screening test kit. The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL. 20 μL of enzyme reagent and 30 μL of sample solutions of different concentrations were added to a 96-well plate, the plate was shaken for 3s, and incubated at 37°C for 10min. 170 μL of reaction working solution was added to each well, incubated at 37°C for 30min, and the fluorescence value of each well was detected by a fluorescence microplate reader at an excitation wavelength of 360nm and an emission wavelength of 460nm. The negative control group used 30 μL of PBS buffer instead of 30 μL of sample solution of different concentrations, the positive control group used 30 μL of sitagliptin solution of different concentrations instead of 30 μL of sample solution of different concentrations, and the background group used 20 μL of buffer instead of 20 μL of DPP-IV enzyme solution. The rest of the operations were the same as above.

[0177] The inhibition rate of DPP-IV was obtained by the following formula.

[0178]

[0179] Among them, F a is the fluorescence value of the negative control group; F0 is the fluorescence value of the background group; F b is the fluorescence value of the experimental group.

[0180] After completing the above experiments, a series of data on the novel hypoglycemic active peptide GYYLR discovered in this application and its inhibitory activity on α-glucosidase, α-amylase and DPP4 were obtained. Fig.13 Shown is the 3D structure diagram of GYYLR;

[0181] Fig.14 and Fig.15 3D and 2D diagrams of GYYLR binding to the active centers of α-glucosidase and DPP-Ⅳ, respectively; in order to more intuitively demonstrate the inhibitory activity of the present application on α-glucosidase, α-amylase and DPP4, a dose-response curve diagram was drawn, such as Fig.16 , Fig.17 , Fig.18shown.

[0182] Example 4

[0183] (1) Molecular docking of the active peptide VAFGRF with α-glucosidase and DPP-4

[0184] α-glucosidase (PDB ID: 3wy1) and DPP-4 (PDB ID: 4a5s) were selected as receptor proteins, and VAFGRF was used as ligand molecules. The structure of the ligand molecule was drawn using ChemDraw software, and then the 2D structure was modified by MM2 energy minimization using Chem3D software. The 3D crystal structure of the receptor protein was downloaded from the PDB database to remove the solvent and the original ligand small molecule contained in the macromolecular receptor; the receptor protein was hydrogenated, the charge was calculated, and the atom type was set using Autodock software, and the charge of the ligand molecule was adjusted, the torsion center and torsion bond were detected, and the docking box coordinates and size were determined (3wy1: (2.473, -18.215, 10.254) 4a5s:(13.984,29.803,56.514) ), perform semi-flexible docking, predict the binding energy between the ligand small molecule and the protein, select the lowest binding energy as the optimal conformation, and use Pymol software to visualize the docking results.

[0185] (2) Synthesis of the active peptide VAFGRF and verification of its hypoglycemic activity in vitro

[0186] 1. Synthesis of peptides

[0187] The peptide synthesis experiment was commissioned to Shanghai Kepeptide Biotechnology Co., Ltd., and the method used was solid phase synthesis. The crude peptide samples synthesized need to be purified to obtain purer samples. The purity of the purified synthetic peptides was identified by HPLC, and the molecular weight was identified by MS.

[0188] 2. Verification of α-glucosidase inhibition effect

[0189] The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL. 50 μL of sample solutions of different concentrations and 50 μL of 1U / mL α-glucosidase solution were added to a 96-well plate, mixed evenly, and reacted in a 37°C constant temperature incubator for 15 minutes. Then 100 μL of substrate solution (1 mM p-NPG solution) was added, mixed evenly, and reacted in a 37°C constant temperature incubator for 30 minutes, and then 100 μL of Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm. 50 μL of buffer solution and 50 μL of enzyme solution were added to the negative control group, 50 μL of sample solution and 50 μL of buffer solution were added to the background group, and 50 μL of acarbose solution and 50 μL of enzyme solution of the same concentration as the synthetic peptide were added to the positive control group, and the rest of the operations were the same as above. The calculation formula of α-glucosidase inhibition rate is as follows:

[0190]

[0191] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0192] 3. Verification of α-amylase inhibition effect

[0193] Prepare the synthetic peptide into gradient concentration sample solutions of 0.125mg / mL, 0.25mg / mL, 0.5mg / mL, 1mg / mL, 1.5mg / mL, and 2mg / mL. Add 100μL of sample solutions of different concentrations and 100μL of 1U / mL α-amylase solution to a 1.5mL centrifuge tube, mix well, and react in a 37℃ constant temperature incubator for 10min. Add 200μL of 2% pretreated (heat in a 95℃ water bath for 8min to denature it, wait until the temperature drops to room temperature, mix well for use) starch solution, mix well, and react in a 37℃ constant temperature incubator for 20min. Add 200μL of reaction termination solution (0.4M Na2CO3 solution containing 1% DNS and 12% sodium potassium tartrate), and heat in a 100℃ water bath for 15min. After the reaction is completed, cool to room temperature, aspirate 200μL of reaction solution into a 96-well plate, and measure the absorbance at 540nm. The negative control group used 100 μL of PBS buffer instead of 100 μL of sample solutions of different concentrations, the positive control group used 100 μL of acarbose solution with the same concentration as the synthetic peptide instead of 100 μL of sample solutions of different concentrations, and the background group used 100 μL of PBS buffer instead of 100 μL of 1U / mL α-amylase solution, and the rest of the operations were the same as above.

[0194] The inhibition rate of α-amylase was obtained by the following formula.

[0195]

[0196] Among them, A a is the absorbance value of the negative control group; A0 is the absorbance value of the background group; A b is the absorbance value of the experimental group.

[0197] 4. Verification of DPP-Ⅳ inhibitory effect

[0198] The DPP-IV inhibition rate was determined by using the Elabscience dipeptidyl peptidase 4 (DPP4) inhibitor screening test kit. The synthetic peptide was prepared into a gradient concentration sample solution of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL. 20 μL of enzyme reagent and 30 μL of sample solutions of different concentrations were added to a 96-well plate, the plate was shaken for 3s, and incubated at 37°C for 10min. 170 μL of reaction working solution was added to each well, incubated at 37°C for 30min, and the fluorescence value of each well was detected by a fluorescence microplate reader at an excitation wavelength of 360nm and an emission wavelength of 460nm. The negative control group used 30 μL of PBS buffer instead of 30 μL of sample solution of different concentrations, the positive control group used 30 μL of sitagliptin solution of different concentrations instead of 30 μL of sample solution of different concentrations, and the background group used 20 μL of buffer instead of 20 μL of DPP-IV enzyme solution. The rest of the operations were the same as above.

[0199] The inhibition rate of DPP-IV was obtained by the following formula.

[0200]

[0201] Among them, F a is the fluorescence value of the negative control group; F0 is the fluorescence value of the background group; F b is the fluorescence value of the experimental group.

[0202] After completing the above experiments, a series of data on the novel hypoglycemic active peptide VAFGRF discovered in this application and its inhibitory activity on α-glucosidase, α-amylase and DPP4 were obtained. Fig.19 Shown is the 3D structure diagram of VAFGRF;

[0203] Fig. 20 and Fig.21 3D and 2D diagrams of VAFGRF binding to the active centers of α-glucosidase and DPP-Ⅳ, respectively; in order to more intuitively demonstrate the inhibitory activity of the present application on α-glucosidase, α-amylase and DPP4, a dose-response curve diagram was drawn, such as Fig. 22 , Fig.23 , Fig.24shown.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A phycobiliprotein active peptide with hypoglycemic function, characterized in that: The amino acid sequence of the phycobiliprotein active peptide is Ser-Tyr-Phe-Asp-Arg-Ala, Ile-Ala-Ser-Tyr-Phe-Asp-Arg, Gly-Tyr-Tyr-Leu-Arg or Val-Ala-Phe-Gly-Arg-Phe.

2. The phycobiliprotein active peptide with hypoglycemic function according to claim 1, characterized in that: The amino acid sequence of the phycobiliprotein active peptide is Ser-Tyr-Phe-Asp-Arg-Ala, abbreviated as SYFDRA, and its molecular formula is C 34 H 47 N9O 11 , molecular weight is 757.78Da.

3. The phycobiliprotein active peptide with hypoglycemic function according to claim 2, characterized in that: The phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, with an IC50 value of 3.504 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, with an IC50 value of 0.8603 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, with an IC50 value of 8.344 mg / mL; The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of -20.3 to 11.7% on α-glucosidase in a concentration range of 0 to 2 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 27.7 to 62.5% on α-amylase in a concentration range of 0 to 2 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 31.1 to 55.8% on DPP-IV in a concentration range of 0 to 10 mg / mL.

4. The phycobiliprotein active peptide with hypoglycemic function according to claim 1, characterized in that: The amino acid sequence of the phycobiliprotein active peptide is Ile-Ala-Ser-Tyr-Phe-Asp-Arg, abbreviated as IASYFDR, and its molecular formula is C 40 H 58 N 10 O 12 , molecular weight is 870.94Da.

5. The phycobiliprotein active peptide with hypoglycemic function according to claim 4, characterized in that: The phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 1.046 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.1292 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 0.9089 mg / mL; The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 3.46-65.1% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of 48.7-66.8% on α-amylase in a concentration range of 0-2 mg / mL; the inhibition rate of 18.6-97.3% on DPP-IV in a concentration range of 0-10 mg / mL.

6. The phycobiliprotein active peptide with hypoglycemic function according to claim 1, characterized in that: The amino acid sequence of the phycobiliprotein active peptide is Gly-Tyr-Tyr-Leu-Arg, abbreviated as GYYLR, and its molecular formula is C 32 H 46 N8O8, molecular weight is 670.75Da.

7. The phycobiliprotein active peptide with hypoglycemic function according to claim 6, characterized in that: The phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, with an IC50 value of 0.1148 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, with an IC50 value of 0.3080 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, with an IC50 value of 4.6770 mg / mL; The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 49.2-87.9% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of 39.6-68.1% on α-amylase in a concentration range of 0-2 mg / mL; the inhibition rate of 33.2-55.7% on DPP-IV in a concentration range of 0-10 mg / mL.

8. The phycobiliprotein active peptide with hypoglycemic function according to claim 1, characterized in that: The amino acid sequence of the phycobiliprotein active peptide is Val-Ala-Phe-Gly-Arg-Phe, abbreviated as VAFGRF, and its molecular formula is C 34 H 49 N9O7, molecular weight 695.81Da; The phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-glucosidase, and the IC50 value is 0.7824 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on α-amylase, and the IC50 value is 0.8297 mg / mL; the phycobiliprotein active peptide with hypoglycemic function has inhibitory activity on DPP-IV, and the IC50 value is 0.9468 mg / mL; The phycobiliprotein active peptide with hypoglycemic function has an inhibition rate of 22.4-63.7% on α-glucosidase in a concentration range of 0-2 mg / mL; the inhibition rate of phycobiliprotein active peptide with hypoglycemic function on α-amylase is 22.8-69.5% in a concentration range of 0-2 mg / mL; the inhibition rate of phycobiliprotein active peptide with hypoglycemic function on DPP-IV is 31.4-94.0% in a concentration range of 0-10 mg / mL.

9. A method for preparing the phycobiliprotein active peptide with hypoglycemic function according to any one of claims 1 to 8, characterized in that: The following steps are involved: The phycobiliprotein active peptides are extracted from the phycobiliprotein of Spirulina by solid phase synthesis or directed bio-enzymatic hydrolysis and purification.

10. Use of the phycobiliprotein active peptide with hypoglycemic function according to any one of claims 1 to 8 in the preparation of medicines or foods for preventing or treating type 2 diabetes.

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