An oat polypeptide and its application in synergistic hypoglycemic effect with oat beta-glucan

Abstract

This invention belongs to the field of biotechnology, specifically relating to an α-glucosidase inhibitory peptide and its applications. The invention uses oat bran as raw material to extract protein, prepares oat bioactive peptides through enzymatic hydrolysis, and uses bioinformatics analysis to screen for the α-glucosidase inhibitory peptide RELGGFF, which exhibits an IC50 value of 531.90±5.23 μg / mL against α-glucosidase. Furthermore, the inhibitory effect of the combined use of the α-glucosidase inhibitory peptide RELGGFF and oat β-glucan on α-glucosidase activity is investigated, and its application can be explored in the preparation of products that inhibit α-glucosidase activity, in the preparation of hypoglycemic products, or in the preparation of products for treating diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an α-glucosidase inhibitory peptide and its applications. Background Technology

[0002] Diabetes is a long-term chronic disease characterized by persistent hyperglycemia, a metabolic disorder that severely impacts people's lives and well-being. There are two main forms of diabetes: type 1 and type 2. Insulin-dependent diabetes mellitus (ADQ) occurs primarily because the pancreatic beta cells cannot produce enough insulin, and its causes can be genetic or autoimmune. Uncontrolled hyperglycemia is a central problem for patients with type 2 diabetes, damaging the cardiovascular system and increasing the risk of cardiovascular disease, kidney damage, and retinal disease.

[0003] Type 2 diabetes is characterized by glucose homeostasis disturbance, leading to hyperglycemia. Type 2 diabetes can regulate postprandial carbohydrate absorption by inhibiting starch digestion and reducing glucose uptake in the small intestine. Alpha-amylase and alpha-glucosidase play crucial roles in glucose metabolism and maintaining normal physiological functions in the body's circulation, thus becoming effective targets.

[0004] Currently, some medications treat diabetes by regulating postprandial carbohydrate absorption, such as acarbose, vogglitazone, or miglitol. However, these medications can have adverse effects on the body, causing digestive problems such as bloating, cramps, and kidney damage, limiting their use in specific populations. These side effects have spurred research into natural enzyme inhibitors to find effective treatments for diabetes.

[0005] Oat bran is a byproduct of oat processing, rich in bioactive components such as protein and oat beta-glucan. However, most of it is used as animal feed, resulting in a significant waste of this valuable resource. This application aims to extract bioactive peptides from oat bran to obtain natural hypoglycemic active ingredients. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention extracts proteins from oat bran, prepares oat bioactive peptides via enzymatic hydrolysis, screens α-glucosidase inhibitory peptides using bioinformatics analysis, and further investigates the inhibitory effect of the combined use of α-glucosidase inhibitory peptides and oat β-glucan on α-glucosidase activity.

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

[0008] The second technical solution provided by the present invention is the application of the α-glucosidase inhibitory peptide RELGGFF described in the first technical solution, particularly its application in inhibiting α-glucosidase activity, and more particularly in its application in the preparation of products that inhibit α-glucosidase activity, or in the preparation of hypoglycemic products, or in the preparation of products for treating diabetes.

[0009] The products mentioned include, but are not limited to, health supplements or pharmaceuticals.

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

[0011] Furthermore, the composition uses the inhibitory peptide RELGGFF as the active ingredient;

[0012] Furthermore, the composition contains the 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, the synergistic inhibitory effect of the inhibitory peptide RELGGFF in the composition is best when the concentration of the inhibitory peptide RELGGFF is 800 μg / mL and the concentration of oat β-glucan is 50 mg / mL, with a CI value of 0.7107.

[0015] The fourth technical solution provided by the present invention is the application of the composition described in the third technical solution, particularly in the inhibition of α-glucosidase activity, and more particularly in the preparation of products that inhibit α-glucosidase activity, or in the preparation of hypoglycemic products, or in the preparation of products for treating diabetes.

[0016] The products mentioned include, but are not limited to, health supplements or pharmaceuticals.

[0017] Beneficial effects:

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

[0019] (2) This invention investigated the interaction between the α-glucosidase inhibitory peptide RELGGFF and oat β-glucan on α-glucosidase. The IC50 values ​​of RELGGFF and oat β-glucan on α-glucosidase were 531.90 ± 5.23 μg / mL and 67.61 ± 1.15 mg / mL, respectively. The synergistic effect of the two was best when the concentration of RELGGFF was 800 μg / mL and the concentration of oat β-glucan was 50 mg / mL, with a CI value of 0.7107. Attached Figure Description

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

[0021] Figure 2 This is a baseline peak diagram of the active peptide from liquid chromatography-mass spectrometry analysis.

[0022] Figure 3 This is a mass spectrometry chromatogram of RELGGFF.

[0023] Figure 4 The curve shows the inhibition rate of RELGGFF against α-glucosidase.

[0024] Figure 5 The curve shows the inhibition rate of oat β-glucan against α-glucosidase. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0026] The method for determining α-glucosidase inhibitory activity involved in this invention is as follows:

[0027] The inhibitory effect of the test sample on α-glucosidase activity was evaluated based on the principle that p-nitrophenol (PNP), the product of α-PNPG hydrolysis by α-glucosidase, has a specific absorption peak at 405 nm. An appropriate amount of the reaction stop solution was taken and the OD value was measured at 405 nm to calculate the inhibition rate. Each sample was tested in at least three parallel trials. The specific experimental steps are as follows:

[0028] As shown in Table 1, 200 μL of sample solutions of different concentrations and 200 μL of α-glucosidase working solution (1.5 U / mL) were mixed and incubated in a water bath at 37℃ for 5 min. Then, 200 μL of PNPG (2.5 mmol / L) was added and mixed thoroughly, and the mixture was incubated in a water bath at 37℃ for another 15 min. Finally, 800 μL of Na2CO3 (0.2 mol / L) was added to terminate the reaction, stopping the α-glucosidase hydrolysis. Based on the principle that p-nitrophenol (PNP) has specific absorption at 405 nm, 160 μL of the reaction termination solution was added to a 96-well plate, and the OD value at 405 nm was measured using a microplate reader. The effect of the peptide on α-glucosidase activity was evaluated by detecting the amount of p-nitrophenol (PNP) generated by the hydrolysis of p-nitrophenyl-α-D-glucosidase after the addition of the peptide and calculating the inhibition rate. Each sample was measured in triplicate.

[0029] The formula for calculating the inhibition rate is as follows:

[0030] α-glucosidase inhibition rate (%) = 1

[0031] Table 1. Assay system for α-glucosidase inhibitory activity

[0032]

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

[0034] Example 1: Obtaining the peptide RELGGFF

[0035] (1) Citric acid pretreatment

[0036] Oat bran powder was degreased using a hexane solution. The oat bran powder and hexane were mixed at a ratio of 1:3 (w / v) and degreased by shaking at 50°C for 4 hours. After degreasing, the liquid was poured into a petri dish and air-dried in a fume hood. The air-dried powder was stored at 4°C for later use.

[0037] Weigh 5 g of defatted oat bran into a beaker, add 0.50% citric acid, and mix thoroughly. Soak at room temperature (25°C) for 60 min, then filter the oat bran through cheesecloth. After filtration, dry in a 50°C oven to obtain defatted oat flour.

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

[0039] Defatted oat flour was dissolved in distilled water at a ratio of 1:10 (w / v), stirred until homogeneous, and the pH was adjusted to 10 with 1.0 mol / L NaOH solution. Extraction was performed at 25 °C with shaking for 90 min, followed by water bath extraction at 50 °C for 30 min. The supernatant was centrifuged at 5000 rpm for 20 min to remove the lower precipitate. The pH of the collected supernatant was adjusted to 4 with 1.0 mol / L hydrochloric acid solution, and the mixture was allowed to stand for 30 min to precipitate the protein. The precipitate was collected after centrifugation at 5000 rpm for 15 min. All extracted proteins were vacuum-frozen for 48 h to obtain oat protein powder.

[0040] (3) Oat polypeptides were obtained by enzymatic hydrolysis with protease.

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

[0042] (4) Ultrafiltration

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

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

[0045] Therefore, oat polypeptide components with a molecular weight <3 kDa were further separated, purified, and screened to identify peptides with hypoglycemic activity for subsequent experiments.

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

[0047] Oat polypeptide components with a molecular weight <3 kDa were separated by chromatography and identified by mass spectrometry according to relevant procedures. The results were then analyzed using the MaxQuant database to obtain the corresponding polypeptide sequences. A total of 655 polypeptide sequences were obtained through liquid chromatography-mass spectrometry analysis, and their peak chromatograms are shown below. Figure 2 As shown.

[0048] (6) Bioinformatics analysis

[0049] ①Prediction of non-toxic, non-allergenic bioactive peptides

[0050] 655 peptide sequences were obtained by LC-MS / MS sequencing. Oat protein sequences downloaded from the NCBI website were used to screen for target peptides, resulting in oat-derived peptides. Target peptides with a score greater than 0.5 were evaluated using the Peptide Ranker online software. The non-toxicity and non-allergenicity of the peptides were confirmed using ToxinPred and AllerTOP v.2.0 online analysis. Based on peptide amino acid composition analysis, library alignment, and computer analysis, 29 peptides were ultimately selected as non-toxic, non-allergenic, and with an activity score of 0.5 or higher.

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

[0052] The length of an active peptide chain affects its stability; therefore, peptides with fewer than 8 amino acids were selected for further experimental procedures. Active peptides can only exert their physiological effects and improve human health after being absorbed by the human digestive system. Therefore, the water solubility and ADMET properties of bioactive peptides are crucial for their functional activity in vivo. The admetSAR online model can be used to simulate and predict the ADMET properties of peptides, where HIA is the human gastrointestinal absorption index and BBB is the blood-brain barrier cross-linking index. Based on the results of water solubility, HIA, and BBB, peptides with good water solubility and ADMET properties were selected for further research.

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

[0054] ①Peptide ligand preparation

[0055] Based on Peptide Ranker evaluation, peptide hydrophilicity / hydrophobicity, and whether they are allergens, suitable peptides were selected for molecular docking. The peptide molecules were correctly protonated at pH 7.4 (referencing human pH: 7.35–7.45), and PDBOT files were generated using AutoDock software.

[0056] ② Preparation of α-glucosidase receptor

[0057] The protein structure of α-glucosidase (PDB ID: 3A4A) was located in the RCSB PDB database (http: / / www.rcsb.org / pdb) and downloaded. It was then preprocessed: water of crystallization was removed, non-standard peptide chains were removed, nonpolar hydrogens were combined, lone pairs of electrons were merged, solvent molecules were removed, all hydrogen atoms were added, Gasteiger charges were recalculated, and the atom type was changed to Autodock4. Finally, it was converted to pdbqt format for later use.

[0058] ③ Molecular docking methods and evaluation

[0059] AutoDock was used to dock the ligand peptide with the receptor α-glucosidase protein. The PDBOT files of both the α-glucosidase protein and the ligand peptide were opened, and the corresponding parameters, such as the GridBox binding site, docking algorithm, and docking parameters, were set according to the molecular docking procedure. AutoDock was then run to obtain the final docking results. Based on the binding energy of the docking results, the optimal binding conformation of the peptide and α-glucosidase was selected. The 3D image of the optimal conformation was viewed using PyMol software, and further analysis was performed on the optimal conformation.

[0060] The results are as follows:

[0061] Computer-aided molecular simulations showed that the peptide under study can dock with α-glucosidase. Negative binding energies indicate that the target bioactive peptide can form a stable complex with α-glucosidase. The peptide with the lowest binding energy is RELGGFF (7-peptide). The amino acid sequence composition of RELGGFF and its binding energy with α-glucosidase are shown in Table 2.

[0062] Table 2. Amino acid sequences of polypeptide fragments and their binding energies to α-glucosidase

[0063]

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

[0065] Meanwhile, the instability index of RELGGFF was predicted using the online bioactivity tool (https: / / web.expasyorg / protparam / ), and the result was 26.20. Peptides with 5 or more amino acids are considered stable with a stability index less than 40; RELGGFF is relatively stable.

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

[0067] Table 3 Prediction of Non-Toxic, Non-Allergen Bioactive Peptides

[0068]

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

[0070]

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

[0072] Example 2: Assay of α-glucosidase inhibitory activity of peptide RELGGFF

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

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

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

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

[0077] Following the aforementioned method for determining α-glucosidase inhibitory activity, the effect of oat β-glucan at concentrations of 20, 30, 40, 50, and 60 mg / mL on α-glucosidase activity was determined. Before the determination, oat β-glucan needed to be dissolved in a 70°C water bath.

[0078] The results are as follows Figure 5As shown, the inhibitory effect of oat β-glucan on α-glucosidase increases with increasing concentration. The logarithm of the oat β-glucan concentration (X) was used to establish a linear regression equation between the concentration and the inhibition rate (Y), and the IC50 value was calculated. The IC50 value of oat β-glucan on α-glucosidase was 67.61 ± 1.15 mg / 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 α-glucosidase inhibitory activity assay system). Nine groups of RELGGFF and oat β-glucan with different concentration combinations were set up (see Table 5 for details). The effects of the combined action of different concentration combinations of RELGGFF and oat β-glucan on α-glucosidase activity were determined according to the aforementioned method. The results are shown in Table 5.

[0081] (2) The combination index (CI) method was used to evaluate the interaction between the repressive peptide and β-glucan. The CI values ​​of the inhibitory effects of the repressive peptide and β-glucan on α-glucosidase were calculated using CompuSyn 2.0 software. CI < 1 indicates a synergistic effect between the repressive peptide and β-glucan, CI = 1 indicates an additive effect between the repressive peptide and β-glucan, and CI > 1 indicates an antagonistic effect between the repressive peptide and β-glucan.

[0082] The results are shown in Table 5 below. The results show that the CI values ​​of different concentration combinations of RELGGFF and oat β-glucan are all less than 1, indicating a synergistic inhibitory effect between the inhibitory peptide RELGGFF and β-glucan on α-glucosidase. The combination with a RELGGFF concentration of 800 μg / mL and an oat β-glucan concentration of 50 mg / mL has the lowest CI value (0.7107), indicating the best synergistic effect at this concentration.

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

[0084]

[0085] Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The scope of this invention is not limited to the contents of the specification but must be determined according to the claims.

Claims

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

2. The application of the inhibitory peptide RELGGFF according to claim 1, characterized in that, It is used in the treatment of non-diseases by inhibiting α-glucosidase activity.

3. The application of the inhibitory peptide RELGGFF according to claim 1, characterized in that, It is used in the preparation of health products or medicines that help lower blood sugar.

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

5. The composition according to claim 4, characterized in that, The composition uses the inhibitory peptide RELGGFF as its active ingredient.

6. The composition according to claim 4, characterized in that, The composition contains the inhibitory peptide RELGGFF and oat β-glucan.

7. The composition according to claim 6, characterized in that, The mass ratio of the inhibitory peptide RELGGFF to oat β-glucan in the composition is 1:30-300.

8. The use of the composition of any one of claims 5-6 in the preparation of health products or pharmaceuticals that assist in lowering blood sugar.

9. Use of the composition of any one of claims 5-6 in the preparation of a medicament for treating diabetes.

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