Oat polypeptide and application thereof in synergic hypoglycemic with oat beta-glucan

Abstract

The invention belongs to the technical field of biology, and particularly relates to an alpha-glucosidase inhibitory peptide and application thereof. According to the invention, oat bran is used as a raw material to extract protein, the oat active peptide is prepared by a biological enzymolysis method, the alpha-glucosidase inhibitory peptide RELGGFF is analyzed and screened by utilizing bioinformatics, and the IC50 value of the alpha-glucosidase inhibitory peptide RELGGFF to alpha-glucosidase reaches 531.90 + / -5.23 [mu] g / mL. The invention further explores the inhibition effect of the combination of the alpha-glucosidase inhibitory peptide RELGGFF and oat beta-glucan on the activity of alpha-glucosidase, and applies the alpha-glucosidase inhibitory peptide RELGGFF and oat beta-glucan to the preparation of products for inhibiting the activity of alpha-glucosidase, or to the preparation of products for reducing blood sugar, or to the preparation of products for treating diabetes.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an alpha-glucosidase inhibitory peptide and application thereof. Background Art

[0002] There are two main forms of diabetes, type 1 and type 2. Insulin-dependent diabetes is caused by the inability of pancreatic beta cells to produce enough insulin, which is caused by genetic diseases or autoimmune reactions. Uncontrolled high blood sugar is the core problem of patients with type 2 diabetes, which damages the cardiovascular system and increases the risk of cardiovascular disease, kidney damage and retinal disease.

[0003] Type 2 diabetes is characterized by dysregulated glucose homeostasis, leading to hyperglycemia. Type 2 diabetes can regulate postprandial carbohydrate absorption by inhibiting starch digestion and reducing glucose uptake in the small intestine. α-Amylase and α-glucosidase play a vital role in glucose metabolism in the human body and maintaining normal physiological functions, making them effective targets.

[0004] At present, some drugs treat diabetes by regulating the absorption of carbohydrates after meals, such as acarbose, voglitose or miglitol, but these therapeutic drugs have adverse effects on the human body, causing adverse reactions in the digestive system such as bloating, cramps and kidney damage, which limits their application in special populations. The occurrence of these side effects has prompted the research on natural enzyme inhibitors to find effective products for the treatment of diabetes.

[0005] Oat bran is a byproduct of oat processing, rich in active ingredients such as protein and oat β-glucan, but most of it is used as feed, resulting in a large waste of high-quality resources. This application will use oat bran as a raw material to extract bioactive peptides in order to obtain natural hypoglycemic active ingredients. Summary of the invention

[0006] In order to solve the above technical problems, the present invention uses oat bran as raw material to extract protein, prepares oat active peptides by biological enzymatic hydrolysis, screens out α-glucosidase inhibitory peptides by bioinformatics analysis, and further explores the inhibitory effect of α-glucosidase inhibitory peptides and oat β-glucan on α-glucosidase activity.

[0007] One of the technical solutions provided by the present invention is an α-glucosidase inhibitory peptide, the amino acid sequence of which is Arg-Glu-Leu-Gly-Gly-Phe-Phe, referred to as RELGGFF. The α-glucosidase inhibitory peptide RELGGFF is an oat active peptide prepared by extracting protein from oat bran as a raw material and by a biological enzymatic hydrolysis method, and the IC50 value for α-glucosidase reaches 531.90±5.23μg / mL.

[0008] The second technical solution provided by the present invention is the use of the α-glucosidase inhibitory peptide RELGGFF described in the first technical solution, especially the use in inhibiting the activity of α-glucosidase, more especially the use in preparing the product of inhibiting the activity of α-glucosidase, or the use in preparing the product of lowering blood sugar, or the use in preparing the product of treating diabetes;

[0009] The products include but are not limited to food, health products or medicines.

[0010] The third technical solution provided by the present invention is a composition comprising α-glucosidase inhibitory peptide RELGGFF;

[0011] Further, the composition has the inhibitory peptide RELGGFF as an active ingredient;

[0012] Furthermore, the composition comprises inhibitory peptide RELGGFF and oat β-glucan;

[0013] Furthermore, the mass ratio of the inhibitory peptide RELGGFF to oat β-glucan in the composition is 1:30-300;

[0014] Preferably, when the concentration of the inhibitory peptide RELGGFF in the composition is 800 μg / mL and the concentration of oat β-glucan is 50 mg / mL, the synergistic inhibition effect of the two on α-glucosidase is the best, with a CI value of 0.7107.

[0015] The fourth technical solution provided by the present invention is the use of the composition described in the third technical solution, especially the use in inhibiting the activity of α-glucosidase, more particularly the use in preparing a product for inhibiting the activity of α-glucosidase, or the use in preparing a product for lowering blood sugar, or the use in preparing a product for treating diabetes;

[0016] The products include but are not limited to food, health products or medicines.

[0017] Beneficial effects:

[0018] (1) The present invention uses oat bran as raw material, prepares and separates α-glucosidase inhibitory peptide RELGGFF through biological enzymatic hydrolysis, and the IC50 value of α-glucosidase reaches 531.90±5.23 μg / mL.

[0019] (2) The present invention investigates the interaction between the α-glucosidase inhibitory peptide RELGGFF and oat β-glucan on α-glucosidase. The IC50 values ​​of α-glucosidase of RELGGFF and oat β-glucan were 531.90±5.23μg / mL and 67.61±1.15mg / mL, respectively. When the concentration of RELGGFF was 800μg / mL and the concentration of oat β-glucan was 50mg / mL, the synergistic effect of the two was the best, with a CI value of 0.7107. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The inhibitory effect of different molecular weight components on α-glucosidase after ultrafiltration.

[0021] Figure 2 This is the base peak diagram of LC-MS analysis of active peptides.

[0022] Figure 3 This is the mass spectrum analysis diagram of RELGGFF.

[0023] Figure 4 This is the inhibition rate curve of RELGGFF on α-glucosidase.

[0024] Figure 5 This is the inhibition rate curve of oat β-glucan on α-glucosidase. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0026] The α-glucosidase inhibitory activity determination method of the present invention is as follows:

[0027] The inhibitory effect of the sample on the activity of α-glucosidase was evaluated by using the principle that the product of α-PNPG hydrolyzed by α-glucosidase, p-nitrophenol (PNP), has a specific absorption peak at 405nm. Take an appropriate amount of the reaction termination solution and measure the OD value at 405nm to calculate the sample inhibition rate. Each sample was tested at least 3 times in parallel. The specific experimental steps are as follows:

[0028] As shown in Table 1, 200 μL of sample solution of different concentrations and 200 μL of α-glucosidase working solution (1.5 U / mL) were taken, mixed and incubated in a water bath at 37°C for 5 min, then 200 μL of PNPG (2.5 mmol / L) was added and mixed evenly, and continued to be incubated in a water bath at 37°C for 15 min, and finally 800 μL of Na2CO3 (0.2 mol / L) was added to terminate the reaction, so that the α-glucosidase hydrolysis reaction was stopped, and 160 μL of the reaction termination solution was taken to a 96-well plate according to the principle that p-nitrophenol (PNP) was specifically absorbed at 405 nm, and the OD value of the wavelength of 405 nm was measured in an ELISA instrument, and the amount of p-nitrophenol (PNP) generated by the hydrolysis of p-nitrophenyl-α-D-glucoside (α-PNPG) by α-glucosidase after the addition of the polypeptide was detected, and the inhibition rate was calculated to evaluate the effect of the polypeptide on the activity of α-glucosidase. Each sample was measured in parallel 3 times.

[0029] The inhibition rate calculation formula is as follows:

[0030]

[0031] Table 1 α-glucosidase inhibitory activity assay system

[0032]

[0033] The present application will be further explained below through specific embodiments.

[0034] Example 1 Obtaining the polypeptide RELGGFF

[0035] (1) Citric acid pretreatment

[0036] Use n-hexane solution to defatted oat bran powder. Mix oat bran powder and n-hexane in a ratio of 1:3 (w / v), shake and defatted at 50°C for 4 hours. After defatting, pour the liquid into a watch glass and air-dry it in a fume hood. Store the air-dried powder at 4°C for later use.

[0037] Weigh 5 g of defatted oat bran in a beaker, add 0.50% citric acid, mix and stir thoroughly, soak at room temperature 25°C for 60 minutes, filter the oat bran with gauze, and dry in a 50°C oven to obtain defatted oatmeal powder.

[0038] (2) Extraction of oat protein by alkali dissolution and acid precipitation

[0039] Dissolve defatted oatmeal in distilled water at a ratio of 1:10 (w / v), stir evenly, adjust the pH to 10 with 1.0 mol / L NaOH solution, extract under oscillation at room temperature 25°C for 90 min, then extract in a water bath at 50°C for 30 min, and centrifuge at 5000 rpm for 20 min to remove the lower precipitate. Use 1.0 mol / L hydrochloric acid solution to adjust the pH value of the collected supernatant to 4, let it stand for 30 min to precipitate protein. Collect the precipitate after centrifugation at 5000 rpm for 15 min. All extracted proteins were vacuum frozen for 48 h to obtain oat protein dry powder.

[0040] (3) Protease hydrolysis to obtain oat peptides

[0041] The freeze-dried oat protein was fully mixed with distilled water at a ratio of 2% (w / v), and alkaline protease was added. The substrate concentration was 1.14%, the enzyme addition amount was 12700U / g, the solution pH was adjusted to 10.50, and the enzymatic hydrolysis was carried out at 50°C for 4.7h, and the enzyme was inactivated by heating at 85°C for 10min to obtain oat polypeptide enzymatic hydrolysate.

[0042] (4) Ultrafiltration

[0043] The oat polypeptide hydrolysate was centrifuged at 6000r / min for 15min to obtain the supernatant, filtered through a 0.45μm microfiltration membrane, and then separated with a 5KDa ultrafiltration membrane, the residual liquid and the filtrate were retained, and the filtrate was separated by a 3KDa ultrafiltration membrane to obtain the residual liquid and the filtrate, and finally oat polypeptide solutions with different molecular weights (>5KDa, 3-5KDa and <3KDa) were obtained, and the three polypeptide solutions with different molecular weights were vacuum freeze-dried and stored. The effects of three polypeptide components with different molecular weights (150μg / mL) on the inhibitory activity of α-glucosidase were determined.

[0044] The results are as follows Figure 1 As shown in the figure, it can be seen from the results that the effect of the polypeptide component on the activity of α-glucosidase is related to the molecular weight. The polypeptide components of different molecular weights have an inhibitory effect on the activity of α-glucosidase, but the shorter the molecular weight, the stronger the inhibitory effect on the activity of α-glucosidase. Among them, the α-glucosidase inhibition rate of the polypeptide component with a molecular weight of <3KDa is 24.96±0.56%, which is significantly higher than that of the polypeptide components with a molecular weight of 3-5KDa and >5KDa.

[0045] Therefore, the oat polypeptide components with molecular weight less than 3KDa were further separated and purified to screen out peptides with hypoglycemic activity for subsequent experiments.

[0046] (5) LC-MS / MS identification

[0047] The oat polypeptide components with molecular weight less than 3KDa were subjected to chromatographic separation and mass spectrometry identification according to relevant operations, and the results were analyzed and identified using the MaxQuant database to obtain the corresponding polypeptide sequences. A total of 655 polypeptide sequences were obtained through LC-MS analysis, and their base peak diagrams are shown in the figure below. Figure 2 shown.

[0048] (6) Bioinformatics analysis

[0049] ① Prediction of non-toxic and non-allergenic active peptides

[0050] 655 peptide sequences were obtained by LC-MS / MS sequencing. The oat protein sequences were downloaded from the NCBI website to screen the target peptides, and peptides derived from oats were obtained; the target peptides with scores greater than 0.5 were evaluated on the Peptide Ranker online software; the ToxinPred and AllerTOP v.2.0 online websites were used to analyze whether the peptides were non-toxic and non-allergenic. According to the results of peptide amino acid composition analysis, library comparison and computer analysis, 29 peptides with non-toxicity, non-allergenicity and activity scores above 0.5 were finally screened.

[0051] ② Water solubility and ADEMT properties of active peptides

[0052] The length of the peptide chain of active peptides will affect the stability of the peptides, so peptides with less than 8 amino acids are selected for the next experimental operation. Active peptides can only play a physiological role in the body and improve human health after being absorbed by the human digestive system. Therefore, the water solubility and ADMET properties of bioactive peptides are the key to whether they can play functional activities in organisms. The admetSAR online model can be used to simulate and predict the ADMET properties of peptides, where HIA is the human gastrointestinal absorption index of the peptide, and BBB is the index of whether the active peptide can pass through the human blood-brain barrier. In view of this, according to the water solubility, HIA and BBB results of bioactive peptides, peptides with good water solubility and ADEMT properties are selected for subsequent research.

[0053] (7) Molecular docking simulation screening of inhibitory peptides

[0054] ① Preparation of peptide ligands

[0055] According to the Peptide Ranker evaluation, the hydrophilicity of the peptides and whether they are allergens, appropriate peptides were selected for molecular docking. The peptide molecules were correctly protonated at pH = 7.4 (reference human pH: 7.35-7.45), and the PDBOT files were output using AutoDock software.

[0056] ② Preparation of α-glucosidase receptor

[0057] The protein structure of α-glucosidase (PDB ID: 3A4A) was searched and downloaded from the RCSB PDB database (http: / / www.rcsb.org / pdb). It was preprocessed by removing crystal water, non-standard peptide chains, merging non-polar hydrogen, merging lone pairs of electrons, removing solvent molecules, adding all hydrogen atoms, recalculating Gasteiger charges and modifying to Autodock4 atom types, and converting it to pdbqt format for later use.

[0058] ③Molecular docking method and evaluation

[0059] AutoDock was used to dock the ligand peptide and the receptor α-glucosidase protein. The PDBOT files of the α-glucosidase protein and the ligand peptide were opened respectively. The corresponding specific parameters such as the binding site of GridBox, the docking algorithm, and the docking parameters were set according to the molecular docking steps. The final docking results were obtained by running AutoDock. The best binding conformation of the peptide and α-glucosidase was screened out according to the binding energy of the docking results. The 3D image of the best conformation was viewed using PyMol software and the best conformation was further analyzed.

[0060] The results are as follows:

[0061] Computer molecular simulation docking showed that the peptide to be studied can be docked with α-glucosidase. The negative binding energy indicates that the target bioactive peptide can form a stable complex with α-glucosidase. Among them, RELGGFF (7 peptides) has the lowest binding energy. The amino acid sequence composition of the peptide RELGGFF with the lowest binding energy and its binding energy with α-glucosidase are shown in Table 2.

[0062] Table 2 Amino acid sequence of polypeptide fragments and their binding energy with α-glucosidase

[0063]

[0064] The peptide RELGGFF was compared with the amino acid sequences of the existing functional peptides in the BIOPEP and AHTPDB data to ensure the novelty of the peptide (the mass spectrometry analysis of RELGGFF is shown in Figure 2). Figure 3 ).

[0065] At the same time, the instability index of RELGGFF was predicted based on the online tool for biological activity (https: / / web.expasyorg / protparam / ), and the result was 26.20. When the number of amino acids in a polypeptide is 5 or more, the stability index is less than 40, which is considered stable, and RELGGFF is relatively stable.

[0066] Other relevant properties of RELGGFF are shown in Tables 3 and 4.

[0067] Table 3 Prediction of non-toxic and non-allergenic active peptides

[0068]

[0069] Table 4 Water solubility and ADEMT properties of active peptides

[0070]

[0071] Since computer molecular simulation docking showed that RELGGFF had the lowest docking energy and the highest binding ability with α-glucosidase, RELGGFF was selected for artificial synthesis and further research.

[0072] Example 2 Determination of α-glucosidase inhibitory activity of polypeptide RELGGFF

[0073] (1) A synthesis company (Nanjing Yuanpeptide Biotechnology Co., Ltd.) was commissioned to synthesize the peptide RELGGFF based on the amino acid sequence of RELGGFF.

[0074] (2) According to the aforementioned α-glucosidase inhibitory activity assay method, the effect of the α-glucosidase inhibitory peptide RELGGFF on α-glucosidase activity at concentrations of 50, 100, 200, 400, and 800 μg / mL was determined.

[0075] The results are as follows Figure 4 As shown, it can be seen that the inhibitory effect of RELGGFF on α-glucosidase increases with increasing concentration. The logarithm of the RELGGFF added concentration (X) is taken, and a linear regression equation is established between the inhibition rate (Y) to calculate the IC50 value. The IC50 value of α-glucosidase of RELGGFF is 531.90±5.23μg / mL.

[0076] Example 3 Determination of α-glucosidase inhibitory activity of oat β-glucan

[0077] According to the aforementioned α-glucosidase inhibitory activity assay method, the effects of oat β-glucan at concentrations of 20, 30, 40, 50, and 60 mg / mL on α-glucosidase activity were determined. Before the assay, the oat β-glucan was first dissolved in a 70°C water bath.

[0078] The results are as follows Figure 5 As shown, it can be seen that the inhibitory effect of oat β-glucan on α-glucosidase increases with the increase of concentration. The logarithm of the added concentration of oat β-glucan (X) is taken, and a linear regression equation is established between the inhibition rate (Y) to calculate the IC50 value. The IC50 value of α-glucosidase of oat β-glucan is 67.61±1.15mg / mL.

[0079] Example 4 Determination of the inhibitory activity of oat β-glucan and polypeptide RELGGFF on α-glucosidase

[0080] (1) When RELGGFF was used in combination with oat β-glucan, the peptide concentrations were 200, 400, and 800 μg / mL, and the oat β-glucan concentrations were 40, 50, and 60 mg / mL, respectively (the above concentrations refer to the concentrations of RELGGFF and oat β-glucan in 200 μL of the sample in the α-glucosidase inhibitory activity assay system). Different concentrations of RELGGFF and oat β-glucan were set up, a total of 9 groups (see Table 5 for details). The effects of different concentrations of RELGGFF and oat β-glucan on α-glucosidase activity were determined according to the above method. The results are shown in Table 5.

[0081] (2) The Chou-Talalay method was used to calculate the combination index (CI) to evaluate the interaction between the inhibitory peptide and β-glucan. The CI value of the inhibitory effect of the inhibitory peptide and β-glucan on α-glucosidase was calculated by CompuSyn2.0 software. CI < 1 indicates that there is a synergistic effect between the inhibitory peptide and β-glucan, CI = 1 indicates that there is an additive effect between the inhibitory peptide and β-glucan, and CI > 1 indicates that there is an antagonistic effect between the inhibitory peptide and β-glucan.

[0082] The results are shown in Table 5 below. It can be seen from the results that the CI values ​​of RELGGFF and oat β-glucan at different concentrations are all less than 1, indicating that the inhibitory peptide RELGGFF and β-glucan have a synergistic effect on the inhibition of α-glucosidase. When the concentration of RELGGFF is 800 μg / mL and the concentration of oat β-glucan is 50 mg / mL, the CI value of the combination is the smallest, which is 0.7107. This shows that at this combined concentration, the synergistic effect between the two is the best.

[0083] Table 5 Effect of RELGGFF combined with oat β-glucan on α-glucosidase activity

[0084]

[0085] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of this invention. The technical scope of this invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. An α-glucosidase inhibitory peptide, characterized in that The inhibitory peptide is RELGGFF.

2. Use of the inhibitory peptide RELGGFF according to claim 1.

3. The use according to claim 2, characterized in that It is used in inhibiting the activity of α-glucosidase.

4. The use according to claim 2, characterized in that The invention is used in the preparation of products for inhibiting the activity of α-glucosidase.

5. The use according to claim 2, characterized in that It is used in the preparation of blood sugar lowering products, or in the preparation of diabetes treatment products.

6. A composition comprising the α-glucosidase inhibitory peptide RELGGFF.

7. The composition according to claim 6, characterized in that The composition uses the inhibitory peptide RELGGFF as an active ingredient.

8. The composition according to claim 6, characterized in that The composition comprises the inhibitory peptide RELGGFF and oat β-glucan.

9. The composition according to claim 8, characterized in that The mass ratio of the inhibitory peptide RELGGFF to oat β-glucan in the composition is 1:30-300.

10. Use of the composition of any one of claims 7 to 8 in preparing a product for inhibiting α-glucosidase activity, or in preparing a product for lowering blood sugar, or in preparing a product for treating diabetes.

Images