Casein-derived hypoglycemic peptide and application thereof

Abstract

The invention provides a casein source blood sugar reducing peptide and application thereof. According to the invention, through research and analysis on protein hydrolysate, two polypeptides with DPP-IV inhibitory activity are found, and the amino acid sequences of the polypeptides are respectively APFPEV and FPEVF. After high-purity polypeptide is obtained through a chemical synthesis method, a liquid chromatograph-mass spectrometer is used for characterization, the effect of the polypeptide is verified through a DPP-IV inhibition experiment, experimental results find that the two polypeptides can effectively inhibit DPP-IV enzyme and are high in inhibition activity, the IC50 value of the polypeptide APFPEV is 224.67 mu M, the IC50 value of the polypeptide FPEVF is 328.86 mu M, and it is proved that the polypeptide has a better inhibition effect compared with similar polypeptides.

Description

Technical Field

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

[0002] The core goal of diabetes treatment is to control blood sugar and delay complications. At present, a system combining drug intervention, lifestyle management and technical assistance has been formed. Traditional drugs such as insulin and metformin still occupy a basic position, but new hypoglycemic drugs (such as SGLT-2 inhibitors and GLP-1 receptor agonists) have rapidly become popular because of their cardiorenal protective effects. Insulin delivery technology continues to be optimized, and smart insulin pumps and closed-loop artificial pancreas systems achieve precise regulation through dynamic blood sugar monitoring, significantly improving the quality of life of patients with type 1 diabetes. Stem cell therapy and gene editing technology (such as CRISPR) have entered the clinical trial stage in the field of islet regeneration, bringing hope for the cure of diabetes. Current diabetes medications include biguanides, α-glycoside inhibitors, DPP-IV inhibitors, etc. Among them, dipeptidyl peptidase (DPP-IV) inhibitors have obvious efficacy and no adverse reactions such as hypoglycemia and obesity, becoming a hot spot 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 the activity of dipeptidyl peptidase-IV (DPP-IV). At present, research mainly focuses on the mining of peptides from natural sources and artificial design optimization. Natural peptides are mostly isolated from the hydrolysates of food proteins (such as whey, fish, and beans), 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 means to optimize peptide activity and selectivity. In addition, advances in enzymatic hydrolysis, fermentation technology, and chemical synthesis methods have improved the yield and stability of peptides. However, existing DPP-IV inhibitory peptides generally have problems such as low oral bioavailability and short half-life in vivo, which limits their transformation into clinical drugs.

[0004] At present, domestic and foreign scholars have prepared food-derived DPP-IV inhibitory peptides from a variety of animal and plant proteins, which have safe and efficient hypoglycemic effects. Among them, there are many potential DPP-IV inhibitory peptides in casein sequences, but the number of reported DPP-IV inhibitory peptides is limited, and further research and development and application are urgently needed. 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 the use of the casein-derived hypoglycemic peptide.

[0007] The purpose of the present invention is achieved through the following technical solutions: A casein-derived hypoglycemic peptide, which is at least one of the following polypeptides: APFPEV, whose amino acid sequence is Ala-Pro-Phe-Pro-Glu-Val and molecular weight is 658.33; FPEVF, its amino acid sequence is Phe-Pro-Glu-Val-Phe, and its molecular weight is 637.31.

[0008] The casein-derived hypoglycemic peptide is extracted and separated from casein.

[0009] The casein-derived hypoglycemic peptide is prepared by chemical synthesis.

[0010] Application of the casein-derived hypoglycemic peptide in the preparation of a DPP-IV inhibitor.

[0011] Application of the casein-derived hypoglycemic peptide in the preparation of drugs for treating diabetes.

[0012] Application of the casein-derived hypoglycemic peptide in the preparation of food with hypoglycemic effect.

[0013] Compared with the prior art, the present invention has the following advantages and effects: The present invention has discovered two casein-derived hypoglycemic peptides by studying and analyzing protein hydrolysates, and their amino acid sequences are APFPEV and FPEVF. After obtaining high-purity peptides by chemical synthesis, they are characterized by liquid chromatography-mass spectrometry, and their effects are verified by DPP-IV inhibition experiments. The experimental results show that both peptides can effectively inhibit DPP-IV enzymes and have strong inhibitory activity. The IC value of peptide APFPEV is 2.34, which is 1.33. 50 The value is 224.67 μM, and the IC 50 The value is 328.86 μM, which proves that it has better inhibitory effect than similar peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the secondary mass spectrum of the synthetic peptide APFPEV; Figure 2 is the secondary mass spectrum of the synthetic peptide FPEVF; Figure 3 This is a graph showing the results of the DPP-IV inhibitory activity assay of the synthetic peptide APFPEV; Figure 4 This is a graph showing the results of assaying the DPP-IV inhibitory activity of the synthetic peptide FPEVF. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0016] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0017] Example 1 Chemical synthesis of polypeptides In the previous experiments, our research group found several peptides with potential activity in protein hydrolysates. In order 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: (1) Chemical synthesis of peptides: The dichlororesin was swollen and washed, and the 9-fluorenylmethoxycarbonyl protecting group (Fmoc protecting group) was removed before adding amino acids for condensation reaction, and the deprotection-condensation process was repeated until all amino acids were connected. The resin was cut to obtain a crude active peptide, which was purified by reverse phase high performance liquid chromatography to obtain a pure peptide APFPEV.

[0018] In addition, referring to the above steps, by adjusting the order of adding amino acids, a pure polypeptide with an amino acid sequence of FPEVF was obtained for subsequent experiments.

[0019] (2) Characterization of peptides In order to verify the effect of peptide synthesis in step (1), the purity was analyzed by reverse phase high performance liquid chromatography. The chromatographic 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.

[0020] 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.

[0021] The experimental results show that Example 1 successfully synthesized the polypeptides APFPEV and FPEVF, wherein the purity of the polypeptide APFPEV was 97.23%, and the purity of FPEVF was 95.29%.

[0022] Table 1 Area of ​​the peptide APFPEV elution peak

[0023] Table 2 Area of ​​peptide FPEVF elution peak

[0024] Example 2 Determination of DPP-IV Inhibitory Activity of Peptides In order to verify the DPP-IV inhibitory activity of the polypeptide synthesized in Example 1, this example sets up an experiment to detect its inhibition rate on the DPP-IV enzyme, and the specific implementation steps are as follows: 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), oscillate for 30 s, and read at 405 nm, once every 2 min for a total of 120 min, so as 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.

[0025] DPP-IV inhibition rate (%) = (1 - Slope sample / Slope control )×100%; Slope sample Represents the slope of the sample group, Slope control represents the slope of the control group.

[0026] 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.

[0027] DPP-IV inhibitory activity of hexapeptide APFPEV at different concentrations Figure 3 As shown, after calculating 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 calculating IC 50 The value is 328.86 μM.

[0028] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A casein-derived hypoglycemic peptide, characterized in that: The casein-derived hypoglycemic peptide is at least one of the following polypeptides: APFPEV, whose amino acid sequence is Ala-Pro-Phe-Pro-Glu-Val and molecular weight is 658.33; FPEVF, the amino acid sequence is Phe-Pro-Glu-Val-Phe, and the molecular weight is 637.

31.

2. The casein-derived hypoglycemic peptide according to claim 1, characterized in that: The casein-derived hypoglycemic peptide is prepared by chemical synthesis.

3. Use of the casein-derived hypoglycemic peptide according to claim 1 or 2 in the preparation of a DPP-IV inhibitor.

4. Use of the casein-derived hypoglycemic peptide according to claim 1 or 2 in the preparation of a drug for treating diabetes.

5. Use of the casein-derived hypoglycemic peptide according to claim 1 or 2 in the preparation of food with hypoglycemic effect.

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