Casein-derived hypoglycemic peptide and its application

Abstract

The present invention provides a casein-derived hypoglycemic peptide and its application. The present invention studied and analyzed protein hydrolysates and discovered two polypeptides with DPP-IV inhibitory activity, whose amino acid sequences were APFPEV and FPEVF, respectively. After obtaining high-purity polypeptides by chemical synthesis, they were characterized by liquid chromatography-mass spectrometry and their effects were verified by DPP-IV inhibition experiments. The experimental results showed that both polypeptides could effectively inhibit the DPP-IV enzyme and had strong inhibitory activity. The IC value of the polypeptide APFPEV was 0.04. 50 The value is 224.67 μM, and the IC 50 The value was 328.86 μM, which proved that it had a better inhibitory effect than similar peptides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a casein-derived blood sugar-lowering peptide and application thereof. Background Art

[0002] Diabetes treatment focuses on controlling blood sugar and delaying complications. Currently, a system combining pharmacological intervention, lifestyle management, and technological assistance has emerged. Traditional medications such as insulin and metformin remain fundamental, but newer glucose-lowering drugs (such as SGLT-2 inhibitors and GLP-1 receptor agonists) are rapidly gaining popularity due to their cardioprotective and renal protective properties. Insulin delivery technology continues to improve. Smart insulin pumps and closed-loop artificial pancreas systems, using continuous glucose monitoring, enable precise regulation, significantly improving the quality of life for patients with type 1 diabetes. Stem cell therapy and gene editing technologies (such as CRISPR) are entering clinical trials for pancreatic islet regeneration, offering hope for a cure. Current diabetes medications include biguanides, α-glucosidase inhibitors, and DPP-IV inhibitors. Dipeptidyl peptidase (DPP-IV) inhibitors, with their proven efficacy and lack of adverse effects such as hypoglycemia and obesity, have become a hot topic in diabetes research and development.

[0003] DPP-IV inhibitory peptides are a class of bioactive peptides that delay incretin degradation and improve metabolic disorders in type 2 diabetes by inhibiting dipeptidyl peptidase-IV (DPP-IV). Currently, research focuses on discovering peptides from natural sources and optimizing their design. Natural peptides are often isolated from hydrolysates of food proteins (such as whey, fish, and legumes), and their activity depends on specific amino acid sequences (such as short peptides containing Pro, Ala, or Leu at the N-terminus). Computer-assisted screening, molecular docking, and structure-activity relationship analysis have become important tools for optimizing peptide activity and selectivity. Furthermore, advances in enzymatic hydrolysis, fermentation technology, and chemical synthesis methods have improved peptide yield and stability. However, existing DPP-IV inhibitory peptides generally suffer from low oral bioavailability and short in vivo half-life, which hinder their translation into clinical drugs.

[0004] Currently, researchers at home and abroad have prepared food-derived DPP-IV inhibitory peptides from a variety of animal and plant proteins, demonstrating safe and effective hypoglycemic effects. While casein sequences contain numerous potential DPP-IV inhibitory peptides, the number reported is limited, necessitating further research and development. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a casein-derived hypoglycemic peptide.

[0006] Another object of the present invention is to provide applications of the casein-derived hypoglycemic peptide.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A casein-derived hypoglycemic peptide, which is at least one of the following polypeptides:

[0009] APFPEV, whose amino acid sequence is Ala-Pro-Phe-Pro-Glu-Val and molecular weight is 658.33;

[0010] FPEVF, its amino acid sequence is Phe-Pro-Glu-Val-Phe, and its molecular weight is 637.31.

[0011] The casein-derived hypoglycemic peptide is extracted and separated from casein.

[0012] The casein-derived hypoglycemic peptide is prepared by chemical synthesis.

[0013] Application of the above casein-derived hypoglycemic peptide in the preparation of DPP-IV inhibitors.

[0014] Application of the above-mentioned casein-derived hypoglycemic peptide in the preparation of diabetes therapeutic drugs.

[0015] Application of the casein-derived hypoglycemic peptide in the preparation of food with hypoglycemic effect.

[0016] The present invention has the following advantages and effects compared to the prior art:

[0017] The present invention discovered two casein-derived hypoglycemic peptides through research and analysis of protein hydrolysates, whose amino acid sequences are APFPEV and FPEVF. After obtaining high-purity peptides through chemical synthesis, they were characterized using liquid chromatography-mass spectrometry and their effects were verified by DPP-IV inhibition experiments. The experimental results showed that both peptides can effectively inhibit the DPP-IV enzyme with strong inhibitory activity. The IC value of peptide APFPEV is 0. 50 The value is 224.67 μM, and the IC 50 The value was 328.86 μM, which proved that it had better inhibitory effect than similar peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the secondary mass spectrum of the synthetic peptide APFPEV;

[0019] Figure 2 is the secondary mass spectrum of the synthetic peptide FPEVF;

[0020] Figure 3 This is a graph showing the results of the DPP-IV inhibitory activity assay of the synthetic peptide APFPEV;

[0021] Figure 4 This is a graph showing the results of the DPP-IV inhibitory activity assay of the synthetic peptide FPEVF. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0023] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0024] Example 1: Chemical synthesis of polypeptides

[0025] In previous experiments, our research group discovered several peptides with potential activity in protein hydrolysates. To further study their properties, we used conventional solid-phase synthesis to synthesize the corresponding pure peptides for subsequent experiments. The specific steps are as follows:

[0026] (1) Chemical synthesis of peptides:

[0027] The dichlororesin is swollen and washed, and the 9-fluorenylmethoxycarbonyl protecting group (Fmoc) is removed. Afterwards, amino acids are added for condensation. The deprotection-condensation process is repeated until all amino acids are attached. The resin is cleaved to obtain a crude active peptide, which is then purified by reverse-phase high-performance liquid chromatography to obtain pure APFPEV.

[0028] In addition, referring to the above steps, by adjusting the order of adding amino acids, a pure polypeptide with the amino acid sequence of FPEVF was obtained for subsequent experiments.

[0029] (2) Characterization of peptides

[0030] In order to verify the effect of peptide synthesis in step (1), the purity was analyzed by reversed-phase high performance liquid chromatography. The column model was SHIMADZU Inertsil ODS-SP, size 4.6×250 mm, 5 μm, mobile phase A: water containing 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile containing 0.1% TFA; flow rate 1.0 mL / min, detection wavelength 220 nm.

[0031] In addition, the obtained pure peptide was characterized by LC-MS, and the results were as follows Figure 1 、 Figure 2 As shown, Figure 1 Secondary mass spectrum of synthetic peptide APFPEV, Figure 2 This is the secondary mass spectrum of the synthetic peptide FPEVF.

[0032] The experimental results showed that Example 1 successfully synthesized the peptides APFPEV and FPEVF, wherein the purity of the peptide APFPEV was 97.23% and the purity of FPEVF was 95.29%.

[0033] Table 1 Area of ​​the elution peak of peptide APFPEV

[0034]

[0035] Table 2 Area of ​​the elution peak of polypeptide FPEVF

[0036]

[0037] Example 2 Determination of DPP-IV Inhibitory Activity of Polypeptides

[0038] In order to verify the DPP-IV inhibitory activity of the polypeptide synthesized in Example 1, an experiment was set up in this example to detect its inhibition rate on the DPP-IV enzyme. The specific implementation steps are as follows:

[0039] In a 96-well plate, add 80 μL of the gradient concentration solution prepared from the pure peptide sample prepared in Example 1 and 80 μL of 0.5 mM substrate Gly-Pro-pNA, mix well, place in a microplate reader and incubate at 37 ° C for 10 min, then add 40 μL of 12.5 mU / mL DPP-IV enzyme solution (preheated at 37 ° C for 3 min), shake for 30 s, and read at 405 nm, once every 2 minutes, for a total of 120 minutes to monitor the release of pNA. An equal volume of 100 mM pH 8.0 Tris-HCl buffer was used instead of the sample as a control group. All reagents or samples were prepared or diluted using this Tris-HCl buffer. The absorbance value was plotted against time, and within the linear range (R 2 >0.995) to obtain the slope of the curve, and then calculate the DPP-IV inhibitory activity of the sample according to the following formula.

[0040] DPP-IV inhibition rate (%) = (1 - Slope sample / Slope control ) × 100% ;

[0041] Slope sample Represents the slope of the sample group, Slope control represents the slope of the control group.

[0042] The IC was obtained by calculating the DPP-IV inhibition curve to obtain the concentration required for the sample to inhibit 50% of the DPP-IV enzyme activity. 50 value.

[0043] DPP-IV inhibitory activity of hexapeptide APFPEV at different concentrations Figure 3 As shown, after calculation IC 50 The value was 224.67 μM, while the DPP-IV inhibitory activity of pentapeptide FPEVF at different concentrations was as follows Figure 4 As shown, after calculation IC 50 The value is 328.86 μM.

[0044] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a casein-derived hypoglycemic peptide in the preparation of a DPP-IV inhibitor or a drug for treating diabetes, characterized in that: The casein-derived hypoglycemic peptide is one of the following polypeptides: APFPEV, whose amino acid sequence is Ala-Pro-Phe-Pro-Glu-Val and molecular weight is 658.33; FPEVF, whose amino acid sequence is Phe-Pro-Glu-Val-Phe and molecular weight is 637.31.

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