Hog short peptide palppprr and application thereof

Abstract

The present application relates to a kind of highland barley short peptide PALPPPRR and its application.The preparation method includes: by computer virtual screening from highland barley seed protein hydrolysate obtain the candidate polypeptide sequence PALPPPRR with high binding capacity of alpha-glucosidase, then chemical synthesis is obtained the polypeptide.The polypeptide screened by the present application reaches-8.02 kcal·mol ‑1 The in vitro enzyme activity inhibition experiment proves that it has excellent anti-hyperglycemic activity, and is concentration-dependent.The highland barley protein peptide is natural, safe, and has clear structure, and can be used to prepare low glycemic index functional food, alpha-glucosidase inhibitor or auxiliary hypoglycemic drug, and provides a new effective component for dietary intervention of metabolic diseases such as diabetes and obesity.

Description

Technical Field

[0001] This invention belongs to the field of functional foods, and in particular relates to a short barley peptide, PALPPPRR, and its applications. Background Technology

[0002] With the increasing incidence of diabetes, obesity, and other metabolic diseases, people are paying more and more attention to healthy eating, especially the growing need to control blood sugar levels. Therefore, low-GI (low glycemic index) foods, as a healthy dietary approach that can effectively regulate blood sugar levels, have attracted the attention of consumers and researchers alike.

[0003] In the development of low-GI foods, utilizing natural protein hydrolysates to achieve stable postprandial blood glucose control is an important technical approach. Compared to synthetic drugs, food-derived peptides generally have higher safety profiles and are less likely to produce significant adverse reactions. Highland barley is an important food crop in plateau regions, possessing high nutritional value, particularly rich in protein and other nutrients. It plays a vital role in regulating blood glucose and improving lipid metabolism, making it a valuable source of bioactive substances.

[0004] Studies have shown that barley protein possesses α-glucosidase inhibitory activity, indicating significant potential for blood glucose regulation. However, natural protein hydrolysates are mixtures, typically containing multiple polypeptide sequences. Therefore, screening for characteristic peptide sequences with anti-glycemic potential from barley protein hydrolysates is a crucial issue for further developing and utilizing barley as a low-GI functional raw material. Summary of the Invention

[0005] The technical problem addressed by this invention is that the composition of natural barley protein hydrolysate is complex, and the specific characteristic peptide sequences that inhibit α-glucosidase are unclear, making it difficult to accurately apply it to the development of low-GI functional foods. Existing technologies lack rapid and low-cost methods for screening highly active α-glucosidase inhibitory peptides from barley protein; traditional experimental screening methods are time-consuming and inefficient. There is a need to provide a barley-derived polypeptide with a well-defined structure, high safety, and excellent inhibitory activity for the prevention or adjuvant treatment of metabolic diseases such as diabetes and obesity.

[0006] In view of the technical problems existing in the prior art, this invention designs a short barley peptide, PALPPPRR, and its applications. Based on the good low-GI activity of barley protein hydrolysates, this invention uses virtual screening and molecular docking techniques to screen for polypeptide sequences with low-GI potential, and verifies the low-GI activity of the related barley peptides, thereby achieving the effect of preventing and treating diabetes and related diseases.

[0007] It should be noted that, in this invention, unless otherwise specified, the specific meaning of "comprising" in relation to composition definition and description includes both open-ended meanings such as "comprising," "including," etc., and closed-ended meanings such as "composed of," etc., and similar meanings.

[0008] To solve the aforementioned technical problems, the present invention adopts the following solution:

[0009] A short peptide derived from highland barley, wherein the amino acid sequence of the short peptide is PALPPPRR.

[0010] Furthermore, the barley short peptides are derived from barley hydrolysis products or synthesized through chemical synthesis.

[0011] The present invention also discloses the application of the above-mentioned barley short peptide in the preparation of low glycemic index functional foods.

[0012] The present invention also discloses the application of the above-mentioned barley short peptide in the preparation of α-glucosidase inhibitors.

[0013] The present invention also discloses the application of the above-mentioned barley short peptide in the preparation of adjuvant hypoglycemic drugs.

[0014] The principle behind the inhibition of α-glucosidase by the barley short peptide PALPPPRR obtained in this invention is as follows:

[0015] The obtained polypeptide PALPPPRR binds stably to the enzyme by forming hydrogen bonds, hydrophobic interactions, and electrostatic interactions with the amino acid residues at the active site of α-glucosidase, occupying the substrate binding site, thereby inhibiting the enzyme's hydrolytic activity on glycoside substrates, reducing the glucose production rate, and achieving stable postprandial blood glucose levels.

[0016] This invention provides a short barley peptide, PALPPPRR, and its application, which has the following beneficial effects:

[0017] 1. This invention utilizes computer-aided virtual screening and molecular docking techniques to search for the polypeptide with the best affinity for α-glucosidase in barley protein hydrolysate, the polypeptide sequence of which is PALPPPRR. The binding energy between this polypeptide and α-glucosidase is -8.02 kcal / mol. -1 This indicates a strong interaction and high structural stability between the peptide and α-glucosidase. The inhibition rate assay of α-glucosidase demonstrates that the artificially synthesized peptide with the PALPPPRR sequence possesses excellent anti-hyperglycemic activity.

[0018] 2. This invention is characterized by its simplicity, speed, and low cost. By using computer-aided molecular docking, it accurately screens peptides with high potential to inhibit hyperglycemia, providing theoretical guidance for the inhibition of α-glucosidase and its mechanism of action.

[0019] 3. This invention overcomes the disadvantage of the complex composition of barley protein hydrolysate and has a well-defined polypeptide sequence.

[0020] 4. The barley peptide involved in this invention is entirely derived from pure natural barley seeds, will not have adverse effects on the human gastrointestinal tract, has high safety, and has a good inhibitory effect on α-glucosidase activity. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the interaction between the barley short peptide PALPPPRR obtained by screening in this invention and the α-glucosidase molecule.

[0022] Figure 2 : This is a graph showing the inhibitory activity of the barley short peptide PALPPPRR provided by the present invention against α-glucosidase. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0024] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0025] Example 1

[0026] Step 1: Determination of barley seed sequence

[0027] Relevant protein sequences derived from barley seeds were selected from the NCBI database.

[0028] Step 2: Virtual screening of highly bioactive barley peptides

[0029] The protein digestion simulation analysis of barley seed protein was performed using the Protein Digestion Simulator program.

[0030] Different barley peptide sequences were obtained by sequentially hydrolyzing and digesting protein sequences related to barley seeds using pepsin and trypsin-chymotrypsin complex enzymes.

[0031] The obtained barley peptides were simulated and screened using the PeptideRanker tool to predict the bioactivity potential of barley seed peptides.

[0032] ToxinPred was used to screen for barley peptides with no potential toxicity and a molecular weight of less than 2 kDa.

[0033] The structure of barley peptides was predicted using ColabFold: AlphaFold2 using MMseqs2, and high-reliability barley peptides were screened.

[0034] Molecular docking of AutoDock Vina with α-glucosidase yielded a barley peptide sequence of PALPPPRR (proline-alanine-leucine-proline-proline-arginine-arginine) with high potential for low GI properties.

[0035] Step 3: Evaluation of Molecular Docking Results

[0036] The interaction between the PALPPPRR sequence barley peptide and α-glucosidase was calculated using molecular docking analysis.

[0037] The PALPPPRR sequence of barley peptide, as a potential ligand inhibitor of α-glucosidase receptor, shows its interaction site with the α-glucosidase receptor. Figure 1 .

[0038] Step 4: Assay for activity that inhibits blood glucose elevation

[0039] Assay for α-glucosidase inhibitory activity:

[0040] First, prepare 0.1 mol / L phosphate buffer (PBS) at pH 6.8, 1.5 mmol / L p-nitrobenzene-α-D-glucopyranoside (PNPG) solution, 0.1 U / mL α-glucosidase solution, and barley peptide solutions of different concentrations (0.1, 0.5, 1, 2, 4 mg / mL).

[0041] In each experimental group, α-glucosidase solution, PBS, and barley peptide solution were added according to the dosage. After incubation at 37°C for 20 min, 1.5 mmol / L PNPG solution was added. After incubation at 37°C for 25 min, 0.2 mL of 1 mol / L Na2CO3 was added to terminate the reaction, and the absorbance at 405 nm was measured.

[0042] Control group: 0.1 mL PBS, 0.1 mL PNPG

[0043] Experimental blank group: 0.05 mL PBS, 0.05 mL barley peptide solution, 0.1 mL PNPG

[0044] Experimental group: 0.05 mL α-glucosidase solution, 0.05 mL barley peptide solution, 0.1 mL PNPG

[0045] Control group: 0.05 mL α-glucosidase solution, 0.05 mL PBS, 0.1 mL PNPG

[0046] The results of the inhibitory activity are shown in Figure 2 .

[0047] After obtaining data from 5 different concentrations, the formula was used.

[0048]

[0049] Calculate the α-glucosidase inhibition rate and plot the inhibition rate as a function of concentration.

[0050] In the formula: i is the inhibition rate, A1 is the absorbance of the experimental group at 405nm, A2 is the absorbance of the control group at 405nm, A3 is the absorbance of the blank group at 405nm, and A4 is the absorbance of the experimental blank group at 405nm.

[0051] Further comparison can be made using the accompanying diagrams in the instruction manual:

[0052] Figure 1 This is a schematic diagram illustrating the interaction between the barley short peptide PALPPPRR obtained by screening in this invention and the α-glucosidase molecule.

[0053] from Figure 1 It can be seen that PALPPPRR interacts with the amino acid residues of α-glucosidase, forming a total of 3 hydrogen bonds. Simultaneously, a total of 15 amino acid residues are involved in the interaction between PALPPPRR and α-glucosidase: Leu320, Asp520, Phe540, Trp578, Gly542, Phe357, Thr355, Asp359, Asp360, Thr355, Leu436, Ala435, Lys432, Tyr404, Trp399, and Glu398.

[0054] Figure 2 The graph shows the inhibitory activity of the barley short peptide PALPPPRR provided by this invention against α-glucosidase.

[0055] from Figure 2As can be seen from the inhibition rate curves of different concentrations of PALPPPRR against α-glucosidase, the concentration of PALPPPRR is positively correlated with the inhibition rate of α-glucosidase. Overall, the inhibition rate of α-glucosidase varies relatively gradually across different concentrations. However, the inhibition rate of α-glucosidase increases continuously with increasing PALPPPRR concentration, exceeding 50% at a concentration of 4 mg / mL. Therefore, increasing the concentration of PALPPPRR significantly enhances the inhibition rate of α-glucosidase and provides substantial benefits in inhibiting α-glucosidase activity.

[0056] The present invention has been described above by way of example with reference to the embodiments and accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A short peptide derived from highland barley, characterized in that: The amino acid sequence of the barley short peptide is PALPPPRR.

2. The barley short peptide according to claim 1, characterized in that: The barley short peptides mentioned above are derived from barley hydrolysis products or synthesized through chemical synthesis.

3. The application of the barley short peptide according to any one of claims 1-2 in the preparation of low glycemic index functional foods.

4. The use of the barley short peptide according to any one of claims 1-2 in the preparation of α-glucosidase inhibitors.

5. The use of the barley short peptide according to any one of claims 1-2 in the preparation of an adjunct hypoglycemic drug.

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