Natural DPP-IV inhibitory peptide derived from Spirulina, preparation method and application thereof

Abstract

The present invention discloses a natural DPP-IV inhibitory peptide derived from spirulina, a preparation method and an application thereof, and belongs to the technical field of bioactive peptides. The natural DPP-IV inhibitory peptide is derived from spirulina, has an amino acid sequence of IASY or IAGIDE, is obtained by enzymolysis of spirulina with neutral protease, and has DPP-IV inhibitory activity. The benefits of the present invention are: (1) it is derived from edible spirulina and is a type of natural DPP-IV inhibitory peptide, which is safer for the human body; (2) it is composed of 4 amino acids and 6 amino acids, respectively, has a small molecular weight, and is more easily absorbed by the human body; (3) IC 50 The values ​​are 0.22 mg / mL and 0.38 mg / mL respectively, which are more active than most existing DPP‑IV inhibitory peptides and have outstanding hypoglycemic activity. They can be used in the field of hypoglycemic drugs or compositions and auxiliary hypoglycemic functional foods.

Description

Technical Field

[0001] The present invention relates to a small molecule bioactive peptide and a preparation method and application thereof, and in particular to a natural DPP-IV inhibitory peptide derived from Spirulina and having DPP-IV inhibitory activity and a preparation method and application thereof, belonging to the technical field of bioactive peptides. Background Art

[0002] Diabetes is a disease that seriously threatens human health. It is mainly divided into two types: insulin-dependent diabetes (type I diabetes) and non-insulin-dependent diabetes (type II diabetes). Among them, type II diabetes patients account for more than 95% of the total number of patients. Therefore, the prevention and treatment of type II diabetes is the focus of diabetes research.

[0003] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin polypeptide (GIP) are the main members of the incretin family. They stimulate and protect pancreatic β cells, promote the synthesis and secretion of insulin, and reduce postprandial blood sugar. Dipeptidyl peptidase-IV (DPP-IV) can cleave a variety of peptide hormones including GLP-1 and GIP and rapidly inactivate them. DPP-IV inhibitors increase the levels of incretin GLP-1 and GIP in the body by inhibiting the activity of DPP-IV, thereby indirectly promoting insulin secretion and alleviating the symptoms of hyperglycemia in patients with type 2 diabetes.

[0004] Currently, clinically used DPP-IV inhibitors include sitagliptin, saxagliptin, vildagliptin, alogliptin and linagliptin, etc. Although these drugs have good DPP-IV inhibitory effects, frequent intake can cause serious adverse reactions, such as liver damage, excessive hypotension, nausea and vomiting, allergic reactions, etc. Therefore, it is particularly important to find DPP-IV inhibitors with high efficiency and low toxicity and side effects. Summary of the invention

[0005] The purpose of the present invention is to provide two natural DPP-IV inhibitory peptides derived from spirulina, which are safe to eat and have high DPP-IV inhibitory activity, as well as preparation methods and applications thereof.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] The natural DPP-IV inhibitory peptide derived from Spirulina is derived from Spirulina, and the amino acid sequence is IASY or IAGIDE.

[0008] The method for preparing the aforementioned Spirulina-derived natural DPP-IV inhibitory peptide comprises the following steps:

[0009] (1) washing, drying, crushing and sieving the spirulina in sequence to obtain spirulina powder;

[0010] (2) adding spirulina powder to water, and sequentially performing soaking, crushing, solid-liquid separation, ultra-high-speed centrifugation of the filtrate, and spray drying of the supernatant to obtain phycocyanin powder;

[0011] (3) Add phycocyanin powder to water at a ratio of 1 kg: 30 L, adjust the pH value to 7, add a certain amount of neutral protease to make the mass concentration of neutral protease 3%, react at 50 ° C for 3 h, and obtain an enzymatic solution;

[0012] (4) heating the enzymatic hydrolysate to inactivate the enzyme, and centrifuging to obtain the supernatant to obtain the inactivated enzymatic hydrolysate;

[0013] (5) ultrafiltration treatment of the enzyme-killed hydrolyzate using an ultrafiltration membrane with a molecular weight cutoff of 3 kD to obtain an ultrafiltration fraction with a molecular weight ≤ 3 kD;

[0014] (6) The ultrafiltration fractions with a molecular weight of ≤3 kD are sequentially subjected to low-temperature concentration and freeze-drying treatment to obtain phycocyanin peptide powder, which contains polypeptides with the amino acid sequences of IASY and IAGIDE.

[0015] The use of the aforementioned natural DPP-IV inhibitory peptide derived from Spirulina in the preparation of a DPP-IV inhibitor.

[0016] The present invention is beneficial in that:

[0017] (1) The two DPP-IV inhibitory peptides provided by the present invention are derived from edible Spirulina and are a type of natural DPP-IV inhibitory peptides that are safer for the human body;

[0018] (2) The two DPP-IV inhibitory peptides provided by the present invention are respectively composed of 4 amino acids (Ile-Ala-Ser-Tyr) and 6 amino acids (Ile-Ala-Gly-Ile-Asp-Glu), have a smaller molecular weight, and are more easily absorbed by the human body;

[0019] (3) Two DPP-IV inhibitory peptide ICs provided by the present invention 50 The values ​​are 0.22 mg / mL and 0.38 mg / mL respectively, which are more active than most existing DPP-IV inhibitory peptides, with outstanding hypoglycemic activity, and can be used in the field of hypoglycemic drugs or compositions and auxiliary hypoglycemic functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the mass spectrum of IASY;

[0021] Figure 2 It is a comparison chart of the DPP-IV inhibition rate of each polypeptide at the same concentration in Table 3;

[0022] Figure 3 is the half-maximal inhibitory concentration (IC) of each peptide in Table 3 for DPP-IV 50 )Comparison chart;

[0023] Figure 4 It is the kinetic curve diagram of IASY. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0025] 1. Preparation of Phycocyanin Peptide Powder

[0026] Using spirulina as raw material, phycocyanin peptide powder is prepared by enzymatic hydrolysis. The specific preparation method is as follows:

[0027] 1. Wash, dry, crush and sieve the spirulina in sequence to obtain spirulina powder;

[0028] 2. Adding spirulina powder to water, sequentially performing soaking, crushing, solid-liquid separation, ultra-high-speed centrifugation of the filtrate, and spray drying of the supernatant to obtain phycocyanin powder;

[0029] 3. Add phycocyanin powder to water at a ratio of 1 kg: 30 L, adjust the pH value to 7 with a 1 mol / L NaOH solution, then add a certain amount of neutral protease to make the mass concentration of neutral protease 3%, react at 50 ° C for 3 hours, and obtain an enzymatic solution;

[0030] 4. Heat the enzymatic hydrolysate at 80°C for 10 min to inactivate the enzyme, then centrifuge at 7000 r / min for 15 min, take the supernatant, and obtain the inactivated enzymatic hydrolysate;

[0031] 5. Ultrafiltration was performed on a portion of the enzyme-killed hydrolysate using ultrafiltration membranes with molecular weight cutoffs of 3kD and 10kD, respectively, to obtain three ultrafiltration fractions: molecular weight ≤ 3kD, molecular weight in the range of 3kD (not included) to 10kD (inclusive), and molecular weight > 10kD;

[0032] 6. The other part of the enzyme-killed hydrolysate that was not subjected to ultrafiltration treatment and the three ultrafiltration fractions with molecular weight ≤3kD, molecular weight in the range of 3kD (exclusive) to 10kD (inclusive) and molecular weight >10kD were sequentially subjected to low-temperature concentration and freeze-drying treatment to obtain phycocyanin peptide powder 1 (obtained by freeze-drying the enzyme-killed hydrolysate), phycocyanin peptide powder 2 (obtained by freeze-drying the ultrafiltration fraction with a molecular weight ≤3kD), phycocyanin peptide powder 3 (obtained by freeze-drying the ultrafiltration fraction with a molecular weight in the range of 3kD (exclusive) to 10kD (inclusive)), and phycocyanin peptide powder 4 (obtained by freeze-drying the ultrafiltration fraction with a molecular weight >10kD).

[0033] 2. Detection of DPP-IV inhibitory activity of the original enzymatic hydrolysate and three ultrafiltration fractions

[0034] The specific method for determining the DPP-IV inhibition rate is as follows:

[0035] 1. Dissolve phycocyanin peptide powder 1, phycocyanin peptide powder 2, phycocyanin peptide powder 3 and phycocyanin peptide powder 4 in purified water to obtain 4 sample solutions with a concentration of 5 mg / mL;

[0036] 2. Sample group: 50 μL of 5 mg / mL sample solution and 40 μL of 625 μg / L DPP-IV solution were added to a 96-well ELISA plate and incubated at 37°C for 10 min.

[0037] 3. Sample blank group: replace the DPP-IV solution with Tris-HCl buffer with a concentration of 0.1 M and a pH value of 8.0. Specifically, 50 μL of the sample solution with a concentration of 5 mg / mL and 40 μL of Tris-HCl buffer were added to a 96-well ELISA plate at the same time and incubated at 37°C for 10 min.

[0038] 4. Control group: pure water was used instead of sample solution. Specifically, 50 μL of pure water and 40 μL of 625 μg / L DPP-IV solution were added to a 96-well ELISA plate at the same time and incubated at 37°C for 10 min.

[0039] 5. Control blank group: replace the sample solution with pure water, and replace the DPP-IV solution with Tris-HCl buffer with a concentration of 0.1M and a pH value of 8.0. Specifically, 50 μL of pure water and 40 μL of Tris-HCl buffer were added to a 96-well ELISA plate at the same time, and incubated at 37°C for 10 min.

[0040] 6. After the incubation of each group, add 40 μL of 312.5 mg / L glycyl-prolyl-p-nitroaniline (Gly-Pro-pNA) solution to the 96-well ELISA plate and react at 37°C for 30 min.

[0041] 7. After the reaction is completed, add 100 μL of 0.1 M NaHCO3 solution to the 96-well ELISA plate to terminate the reaction;

[0042] 8. Detect the absorbance of each group of reaction solutions at 405 nm, and calculate the DPP-IV inhibition rate of each sample according to the following formula:

[0043]

[0044] Among them, A1 is the absorbance value of the sample group, A2 is the absorbance value of the sample blank group, A3 is the absorbance value of the control group, and A4 is the absorbance value of the control blank group.

[0045] The calculation results of DPP-IV inhibition rate of samples such as phycocyanin peptide powder 1, phycocyanin peptide powder 2, phycocyanin peptide powder 3 and phycocyanin peptide powder 4 at a concentration of 5 mg / mL are shown in Table 1.

[0046] Table 1 Calculation results of DPP-IV inhibition rate of samples

[0047]

[0048] It can be seen from Table 1 that at the same concentration, phycocyanin peptide powder 2 (obtained by freeze-drying the ultrafiltration fraction with a molecular weight ≤ 3 kD) has the highest DPP-IV inhibitory activity, so it was selected for the next step of affinity ultrafiltration screening.

[0049] 3. Isolation of small molecule bioactive peptides with DPP-IV inhibitory activity from phycocyanin peptide powder 2

[0050] Affinity ultrafiltration was used to separate small molecule bioactive peptides with DPP-IV inhibitory activity from phycocyanin peptide powder 2. The separation method is as follows:

[0051] 1. Add phycocyanin peptide powder 2 and DPP-IV to water respectively, so that the concentration of phycocyanin peptide powder 2 is 10 mg / mL and the concentration of DPP-IV is 625 μg / L, to obtain phycocyanin peptide powder solution and DPP-IV solution respectively;

[0052] 2. Mix the phycocyanin peptide powder solution and DPP-IV solution in a volume ratio of 1:2 and incubate at 37°C for 1 hour;

[0053] 3. After the incubation, centrifuge at 4000r / min for 30min in a 10kD ultrafiltration centrifuge tube, centrifuge three times to remove the peptides that are not bound to DPP-IV and collect the precipitate (the complex formed by the binding of DPP-IV and peptides);

[0054] 4. Mix the collected precipitate with 70% acetonitrile solution in a volume ratio of 1:2, incubate at room temperature for 10 min, and then centrifuge at 4000 r / min for 30 min to release the peptides in the complex. Repeat (mixing precipitate with acetonitrile solution - incubation - centrifugation) 3 times, collect the filtrate and spray dry it to obtain Spirulina hydrolysate, which contains small molecule bioactive peptides with potential DPP-IV binding ability.

[0055] 4. Purification of Spirulina Hydrolysate

[0056] The above obtained spirulina hydrolysate was added with water to prepare a sample solution with a concentration of 7.6 mg / mL, and the sample was purified by high performance liquid chromatography using a gradient elution method. The specific conditions of high performance liquid chromatography were as follows:

[0057] 1. Liquid chromatography column: Agilent ZORBAX SB-C18 (5 μm, 0.15 mm × 150 mm);

[0058] 2. Detection wavelength: 228nm;

[0059] 3. Sample volume: 3 μL;

[0060] 4. Mobile phase flow rate: 0.25 mL / min;

[0061] 5. Mobile phase: Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution (freshly prepared before use);

[0062] 6. Gradient elution method: 0min~50min, the volume content of mobile phase B in the mobile phase is 4%~50%, and the volume content of mobile phase A in the mobile phase is 96%~50%; 50min~54min, the volume content of mobile phase B in the mobile phase is 50%~100%, and the volume content of mobile phase A in the mobile phase is 50%~0%; 54min~60min, the volume content of mobile phase B in the mobile phase is 100%.

[0063] 5. Mass spectrometry analysis of the purified product and determination of the amino acid sequence

[0064] The purified product was analyzed by mass spectrometry using a Q Exactive mass spectrometer (Thermo Fisher). The mass spectrometry analysis conditions were as follows:

[0065] 1. Analysis time: 60 minutes;

[0066] 2. Detection method: positive ion;

[0067] 3. Collection method of peptide and peptide fragment mass-to-charge ratio: collect 10 fragment spectra (MS 2 scan).

[0068] The amino acid sequences of 11 purified products with larger peak areas were detected by liquid chromatography-mass spectrometry (LC-MS / MS). The detection results are shown in Table 2.

[0069] Table 2 Peptide sequences with potential DDP-IV inhibitory activity from Spirulina

[0070]

[0071] Among them, the mass spectrum of IASY is shown in Figure 1 .Depend on Figure 1 It can be seen that the molecular weight of IASY is 452.73, which is close to its theoretical molecular weight of 452.51.

[0072] 6. Detection of DPP-IV inhibitory activity of each purified product

[0073] The 11 polypeptides listed in Table 2 were subjected to solid phase synthesis to obtain 11 polypeptide powders.

[0074] The 11 kinds of polypeptide powders were prepared into sample solutions with a concentration of 1 mg / mL with water, and the DPP-IV inhibition rates at a concentration of 1 mg / mL were detected using the same method as above.

[0075] The calculation results of DPP-IV inhibition rate of 11 peptide powders at a concentration of 1 mg / mL are shown in Table 3.

[0076] Table 3 DPP-IV inhibition rate of 11 peptide powders

[0077]

[0078] Table 3 is plotted to obtain a comparison of the DPP-IV inhibition rates of the samples at the same concentration, as shown in FIG. Figure 2 As shown. Figure 2 It can be seen that the two polypeptides Ile-Ala-Ser-Tyr (IASY) and Ile-Ala-Gly-Ile-Asp-Glu (IAGIDE) have higher DPP-IV inhibition rates.

[0079] The 11 kinds of peptide powders were prepared into sample solutions of different concentrations with water. The concentration of the peptide was used as the horizontal axis and the DPP-IV inhibition rate of the peptide was used as the vertical axis. The curves were drawn using Origin2021 software. GraphPad Prism was used for data conversion and least squares fitting to calculate the half-inhibitory concentration (IC50) of DPP-IV. 50 ).

[0080] The half inhibitory concentration (IC) of 11 peptide powders on DPP-IV 50 ) The calculation results are shown in Table 4.

[0081] Table 4 Half inhibitory concentration (IC) of 11 peptide powders on DPP-IV 50 )

[0082]

[0083] Plot Table 4 to obtain the IC values ​​of each sample for DPP-IV. 50Comparison chart, such as Figure 3 As shown. Figure 3 It can be seen that the two peptides IASY (Ile-Ala-Ser-Tyr) and IAGIDE (Ile-Ala-Gly-Ile-Asp-Glu) have high DPP-IV inhibitory activity, and the IC 50 The values ​​are 0.22 mg / mL and 0.38 mg / mL respectively, which are more active than most existing DPP-IV inhibitory peptides and have outstanding hypoglycemic activity.

[0084] Isoleucine-alanine-serine-tyrosine (Ile-Ala-Ser-Tyr, IASY), with a theoretical molecular weight of 452.51 and a molecular formula of C 21 H 32 N4O7, the structural formula is as follows:

[0085] .

[0086] Isoleucine-alanine-glycine-isoleucine-aspartic acid-glutamic acid (Ile-Ala-Gly-Ile-Asp-Glu, IAGIDE), the theoretical molecular weight is 616.67, the molecular formula is C 26 H 44 N6O 11 , the structural formula is as follows:

[0087] .

[0088] VII. Preliminary evaluation of the structure-activity relationship of the purified product by molecular docking

[0089] Taking IASY (Ile-Ala-Ser-Tyr) as an example, the structure-activity relationship of the purified product was studied by molecular docking.

[0090] The three-dimensional structure of DPP-IV (PDB code: 1X70) was obtained from the RCSB protein database (www.pdb.org), and the protein and ligand were prepared using the AutoDockTools program. The protein structure in the protein database is stored in the PDB format. Water molecules were removed, polar hydrogens were added, Coleman charges were calculated, and non-polar hydrogens were merged before saving as pdbqt format files. The 3D structure of the ligand was prepared using ChamBio 3D, and saved in mol2 format after energy minimization. The 3D structure of the ligand was then processed using AutoDockTools and saved as a pdbqt format file. The grid center was located at X=-15.9, Y=56.7, and Z=27.2, the size of the grid box was 50Å×50Å×50Å, and the grid spacing was 0.375Å. Docking was performed by the AutoDock4.2.6 program, and a total of 100 docking operations were performed to calculate the binding energy (docking score, -CiE) of IASY and DPP-IV. The best binding mode with the lowest binding energy was screened.

[0091] The calculated binding energy between IASY and DPP-IV is -6.4 kcal / mol.

[0092] The docking results were analyzed using PyMOL Molecular Graphics System software. The results showed that IASY formed hydrogen bonds (Tyr58, Trp62, Lys463, Phe461, Val459) and Anion-π interactions (interactions between anions and "electron-deficient" aromatic π systems) with the residues of DPP-IV, and bound to the active center of DPP-IV through hydrophobic interactions.

[0093] This suggests that IASY can bind to DPP-IV through multiple forces to exert its hypoglycemic activity.

[0094] 8. Study the inhibition type of purified products by inhibition kinetics

[0095] The inhibition type of the purified product was studied by inhibition kinetics based on double reciprocal plots of 1 / V and 1 / Gly-Pro-pNA (glycyl-prolyl-p-nitroaniline hydrochloride), using IASY (Ile-Ala-Ser-Tyr) as a representative.

[0096] Substrate: Glycyl-prolyl-p-nitroaniline hydrochloride (Gly-Pro-pNA), prepared with water into substrate solutions with concentrations of 156.25 mg / L, 312.5 mg / L, 625 mg / L, and 1250 mg / L, respectively.

[0097] Enzyme: DPP-IV, prepared with water to a concentration of 625 μg / L enzyme solution.

[0098] Sample: IASY, prepared with water into sample solutions with concentrations of 0 mg / mL, 1 mg / mL, and 2 mg / mL.

[0099] Take 100 μL of sample solution and 80 μL of substrate solution, add them to a 96-well ELISA plate at the same time, and incubate at 37°C for 10 min. After the incubation, add 80 μL of enzyme solution to the 96-well ELISA plate. Place the ELISA plate in an ELISA reader every 10 min to detect the absorbance of the reaction solution in each ELISA well at 405 nm.

[0100] The reaction rate V of the enzyme-catalyzed reaction is calculated based on the absorbance value. The reciprocal of the substrate solution concentration (1 / S) is plotted as the horizontal axis and the reciprocal of the enzyme-catalyzed reaction rate (1 / V) is plotted as the vertical axis to obtain the kinetic curve of IASY, as shown in the figure below: Figure 4 shown.

[0101] Depend on Figure 4 It can be seen that: as the concentration of sample IASY increases, Km (Km represents the substrate concentration when the enzymatic reaction reaches half of the maximum reaction rate) increases accordingly, indicating that IASY has a competitive inhibition mode. At the same time, IASY has no intersection on the X-axis and Y-axis, indicating that IASY is a mixed inhibition mode.

[0102] In summary, the polypeptides IASY and IAGIDE provided by the present invention are derived from edible spirulina, are a type of natural DPP-IV inhibitory peptides, and are safer to eat; the two are composed of 4 amino acids and 6 amino acids respectively, have a small molecular weight, and are more easily absorbed by the human body; and, after testing, the IC 50 The values ​​are 0.22 mg / mL and 0.38 mg / mL respectively, and the DPP-IV inhibitory effect is outstanding. Therefore, the polypeptides IASY and IAGIDE provided by the present invention can be applied to the field of hypoglycemic drugs or compositions and auxiliary hypoglycemic functional foods.

[0103] It should be noted that the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Spirulina-derived natural DPP-IV inhibitory peptide, characterized in that: The natural DPP-IV inhibitory peptide is derived from Spirulina, and has an amino acid sequence of IASY.

2. The method for preparing the natural DPP-IV inhibitory peptide derived from Spirulina according to claim 1, characterized in that: The following steps are involved: (1) washing, drying, crushing and sieving the spirulina in sequence to obtain spirulina powder; (2) adding spirulina powder to water, and sequentially performing soaking, crushing, solid-liquid separation, ultra-high-speed centrifugation of the filtrate, and spray drying of the supernatant to obtain phycocyanin powder; (3) Add phycocyanin powder to water at a ratio of 1 kg: 30 L, adjust the pH value to 7, add a certain amount of neutral protease to make the mass concentration of neutral protease 3%, react at 50 ° C for 3 h, and obtain an enzymatic solution; (4) heating the enzymatic hydrolysate to inactivate the enzyme, centrifuging and collecting the supernatant to obtain an enzyme-inactivated enzymatic hydrolysate; (5) ultrafiltration treatment of the enzyme-killed hydrolyzate using an ultrafiltration membrane with a molecular weight cutoff of 3 kD to obtain an ultrafiltration fraction with a molecular weight ≤ 3 kD; (6) The ultrafiltration fractions with a molecular weight of ≤3 kD are sequentially subjected to low-temperature concentration and freeze-drying treatment to obtain phycocyanin peptide powder, which contains polypeptides with the amino acid sequences of IASY and IAGIDE.

3. Use of the natural DPP-IV inhibitory peptide derived from Spirulina according to claim 1 in the preparation of a drug for treating type II diabetes.

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