A tartary buckwheat protein source oligopeptide, and an extraction and screening method and application thereof

Abstract

The application discloses a tartary buckwheat protein source oligopeptide and an extraction and screening method and application thereof, and belongs to the technical field of DPP-IV inhibiting peptides. Eleven oligopeptides capable of being combined with a DPP-IV enzyme activity pocket are screened from tartary buckwheat through an integrated model of peptidomics + virtual screening. Activity and safety verification is carried out through DPP-IV inhibiting kinetics experiments and a Caco-2 cell model. Results show that the tartary buckwheat protein source oligopeptide of the application exhibits significant DPP-IV inhibiting activity in vitro and in a cell level, and cell toxicity experiments prove that the oligopeptide has no significant cell toxicity in an effective dose range and has good biological safety. The tartary buckwheat protein source oligopeptide provided by the application has high activity and good safety, can be applied to the development of blood sugar reducing drugs, special medical foods or health foods as a functional ingredient, and provides technical support for high value utilization of coarse grain resources.

Description

Technical Field

[0001] This invention relates to the field of DPP-IV inhibitory peptide technology, specifically to a buckwheat protein-derived oligopeptide, its extraction and screening method, and its application. Background Technology

[0002] Dipeptidyl peptidase-IV (DPP-IV) is a key target for the treatment of type 2 diabetes. It weakens the insulin-secreting biological activity of glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP) by specifically degrading them. Therefore, developing highly effective DPP-IV inhibitors to prolong the duration of action of endogenous GLP-1 / GIP is an effective strategy for blood glucose regulation. While current first-line clinical drugs (such as sitagliptin and vildagliptin, small-molecule chemically synthesized drugs) are effective, long-term use may cause nasopharyngitis, headaches, and gastrointestinal adverse reactions, and their synthesis costs are relatively high.

[0003] Bioactive peptides produced from food-derived proteins through enzymatic hydrolysis or fermentation have become potential natural alternatives due to their high safety and few side effects. Tartary buckwheat, as a crop with both medicinal and edible properties, has high protein nutritional value and has been proven to be an excellent source for preparing DPP-IV inhibitory peptides. However, the development of existing tartary buckwheat-derived DPP-IV inhibitory peptides still faces the following bottlenecks: (1) Insufficient screening efficiency and abundance of active peptides. Due to the randomness of traditional enzymatic hydrolysis processes, the sequences with clear DPP-IV inhibitory activity identified from tartary buckwheat protein are extremely limited (such as LHIVGPDK and LAGQS reported in the literature). (2) The inhibitory activity needs to be improved. The IC50 of the reported tartary buckwheat active peptides' DPP-IV inhibitory activity is limited. 50 The values ​​are mostly in the hundreds of micromoles or even millimoles. For example, the small peptides LHIVGPDK (J Food Sci, 2024), LAGQS, and LREIDDADK (Food Chem, 2025) have IC50 values ​​in the hundreds of micromoles or even millimoles. 50 The values ​​were 1.61 mM, 783.1 μM, and 425.9 μM, respectively. These values ​​are significantly lower than the nanomolar to micromolar levels required for clinical applications, making it difficult to directly convert them into highly efficient functional factors.

[0004] Therefore, there is an urgent need to develop highly active DPP-IV inhibitory peptides derived from tartary buckwheat to address the problem that existing active peptides are scarce in number and have weak inhibitory activity, making it difficult to meet the development needs of functional products. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a buckwheat protein-derived oligopeptide, its extraction and screening method, and its application, thereby solving the problems of scarce active peptides, weak inhibitory activity, and difficulty in meeting the development needs of functional products.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A buckwheat protein-derived oligopeptide is provided, wherein the buckwheat protein-derived oligopeptide is any one of the following: A1. Having an amino acid sequence as shown in SEQ ID NO.1-10 and an amino acid sequence as shown in any of the LPIs; A2. A derivative obtained by chemical or enzymatic modification of the amino acid sequence described in A1, which has DPP-IV inhibitory activity; A3. A mutant that has at least 80% sequence identity with the amino acid sequence described in A1 and retains DPP-IV inhibitory activity.

[0007] This invention provides a method for extracting and screening the above-mentioned buckwheat protein-derived oligopeptides, comprising the following steps: (1) Extraction and sequence identification of buckwheat peptides; (2) Screening of buckwheat protein-derived oligopeptides: ① Select peptides from the peptides extracted and identified in step (1) that have 3-7 amino acids, with the first amino acid being hydrophobic and the second amino acid being proline; ② Based on the BIOPEP-UWM database, predict the peptides selected in step ①, and select peptides with a score ≥ 0.5; ③ The peptides selected in step ② are subjected to DPP-IV inhibitory activity prediction, and peptides with a score ≥0.5 are preferred; ④ Perform hydrophobicity prediction on the peptides selected in step ③, and preferentially select peptides with hydrophobicity ≥ -0.5; ⑤ Perform toxicity prediction on the peptides selected in step ④, and select non-toxic peptides. ⑥ Perform sensitization prediction on the peptides selected in step ⑤, and select peptides that are not sensitizing; ⑦ Perform molecular docking simulations on the peptides selected in step ⑥, and select peptides with the optimal binding energy ≤ -5 kcal / mol.

[0008] Furthermore, step (1), the extraction of buckwheat peptides, specifically includes the following steps: S1. Mix the hulled buckwheat seed powder with petroleum ether and stir overnight. After defatting, filter and air dry to obtain defatted buckwheat powder. S2. Buckwheat gluten powder was extracted from the defatted buckwheat powder in step S1 using the Osborne method. S3. Prepare a protein solution from the buckwheat gluten powder obtained in step S2, then add alkaline protease, adjust the pH value to 7 with alkaline solution, and then perform enzymatic hydrolysis to obtain an enzymatic hydrolysate. S4. The enzymatic hydrolysate from step S3 is subjected to ultrafiltration. Protein fractions with a molecular weight <3kDa are selected for sequencing. The obtained raw data is then analyzed. After spectral analysis, peptide sequences with a length range of 2-14 amino acids are obtained for subsequent screening.

[0009] Furthermore, step ⑦, the molecular docking simulation, specifically includes the following steps: B1. Download the crystal structure of the DPP-IV complex from the PDB database, and then remove its water molecules, chloride ions, sodium ions and proligands, retaining only the DPP-IV protein backbone; B2. Convert the peptide sequence selected in step ⑥ into a 3D structure; B3. Prepare the 3D structure obtained in step B2, including dehydration and hydrogenation and charge distribution, and then perform molecular docking to construct a docking grid box for the protein structure. B4. Perform three-dimensional visualization and force analysis on the molecular docking results of step B3.

[0010] This invention provides an application of the above-mentioned buckwheat protein-derived oligopeptide in the preparation of DPP-IV inhibitors.

[0011] The present invention provides a DPP-IV inhibitor, comprising at least one of the above-mentioned buckwheat protein-derived oligopeptides.

[0012] Furthermore, DPP-IV inhibitors also include pharmaceutically acceptable carriers, excipients, matrices, or additives.

[0013] Furthermore, DPP-IV inhibitors are available in dosage forms such as granules, powders, capsules, tablets, mixtures, or oral liquids.

[0014] This invention provides the application of the above-mentioned DPP-IV inhibitor in the preparation of drugs, dietary supplements or foods with DPP-IV inhibitory function.

[0015] The present invention also provides a drug, dietary supplement or food having DPP-IV inhibitory function, including the above-mentioned DPP-IV inhibitor.

[0016] The present invention has the following beneficial effects: (1) This invention obtains multiple novel peptides with micromolar-level inhibitory activity in one step, enriching the library of highly active peptides derived from tartary buckwheat. Prior art has reported tartary buckwheat peptide IC50... 50 The activity values ​​are mostly in the hundreds of micromoles to millimoles range (e.g., 1.61 mM for LHIVGPDK), indicating low and limited activity. This invention, through targeted screening and validation, obtained 11 novel highly active peptides. Among them, the representative peptide IPVT showed a high IC50 value in the Caco-2 cell model. 50The value reached 69.56 μM, and its inhibitory activity was improved by an order of magnitude compared with the existing technology.

[0017] (2) The buckwheat protein oligopeptides provided by this invention are a class of natural DPP-IV inhibitory peptides, which are safer to consume, have a smaller molecular weight, are hydrophobic, are more easily absorbed by the human body, have a fast metabolic rate, are non-toxic, have good affinity with target proteins, and can form stable complexes, thus showing good prospects for hypoglycemic applications. This invention expands the high-value utilization pathway of buckwheat protein, transforming low-value-added or underutilized buckwheat protein resources into high-purity peptides with clear DPP-IV inhibitory activity.

[0018] (3) Significantly improved screening efficiency and reduced R&D costs. Addressing the problems of high blindness and long identification cycles in existing technologies for buckwheat peptide screening, this invention constructs an integrated model of "peptidomics + virtual screening." By pre-excluding sequences with no potential activity through computer simulation, the range of candidate peptides to be verified is reduced from tens of thousands to a dozen or so, significantly reducing the workload and reagent consumption of wet experiments (such as peptide synthesis and activity testing), thus achieving efficient and low-cost extraction of active peptides from buckwheat protein.

[0019] (4) The structural characteristics and mechanism of action of the active peptides were elucidated, guiding rational design. Existing technologies mostly only focus on activity detection and lack in-depth understanding of structure-activity relationships. This invention, through molecular docking and ADMET analysis, clarified the key binding mode between the screened peptides and the S1 pocket of the DPP-IV enzyme, revealing the molecular basis for their highly efficient inhibitory effect, and providing a theoretical basis for subsequent structure-based peptide modification (such as structural optimization and stability improvement). Attached Figure Description

[0020] Figure 1 The mass spectrum of IPETEI, an oligopeptide derived from tartary buckwheat protein; Figure 2 The mass spectrum of IPGGLL, an oligopeptide derived from tartary buckwheat protein; Figure 3 The mass spectrum of IPTFVE, an oligopeptide derived from tartary buckwheat protein; Figure 4 The mass spectrum of IPVAL, an oligopeptide derived from tartary buckwheat protein; Figure 5 The mass spectrum of IPVT, an oligopeptide derived from tartary buckwheat protein; Figure 6 The mass spectrum of LPI, an oligopeptide derived from tartary buckwheat protein; Figure 7 The mass spectrum of LPILE, an oligopeptide derived from tartary buckwheat protein; Figure 8 The mass spectrum of VPIA, an oligopeptide derived from tartary buckwheat protein; Figure 9The mass spectrum of VPIT, an oligopeptide derived from tartary buckwheat protein; Figure 10 The mass spectrum of VPTFF, an oligopeptide derived from tartary buckwheat protein; Figure 11 The mass spectrum of VPVEK, an oligopeptide derived from tartary buckwheat protein; Figure 12 Lineweaver Burk double inverse plots of buckwheat protein-derived oligopeptides VPVEK, IPETEI, IPGGLL, and LPI; Figure 13 Lineweaver Burk double inverse plots of buckwheat protein-derived oligopeptides VPIA, IPVT, IPTFVE, and IPVAL; Figure 14 Lineweaver Burk double inverse plots of buckwheat protein-derived oligopeptides VPIT, VPTFF, and LPILE; Figure 15 Figure showing the effect of buckwheat protein-derived oligopeptides on the viability of Caco-2 cells; Figure 16 Molecular docking diagram of IPETEI and IPGGLL; Figure 17 Molecular docking diagram of IPTFVE and IPVAL; Figure 18 Molecular docking diagram of IPVT and LPILE; Figure 19 This is a molecular docking diagram of VPIA and LPI; Figure 20 Molecular docking diagram of VPIT and VPTFF; Figure 21 This is a molecular docking diagram of VPVEK. Detailed Implementation

[0021] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] This invention uses an integrated model of "peptidomics + virtual screening" to screen 11 buckwheat protein-derived oligopeptides that can bind to the S1 pocket of DPP-IV enzyme. The amino acid sequences of these 11 oligopeptides are as follows: IPTFVE (SEQ ID NO.1), IPETEI (SEQ ID NO.2), IPVAL (SEQ ID NO.3), VPTFF (SEQ ID NO.4), IPGGLL (SEQ ID NO.5), LPILE (SEQ ID NO.6), VPIA (SEQ ID NO.7), VPVEK (SEQ ID NO.8), IPVT (SEQ ID NO.9), VPIT (SEQ ID NO.10), and LPI.

[0023] Example 1: Extraction of tartary buckwheat peptides Using tartary buckwheat as raw material, peptides were extracted from tartary buckwheat using enzymatic hydrolysis. The specific experimental steps are as follows: (1) Preparation of defatted buckwheat flour: The dehulled buckwheat grains are crushed, passed through a 100-mesh sieve, and defatted with petroleum ether at a material-to-liquid ratio of 1:5 (w / v) in a fume hood at room temperature for 24 hours. After the solvent is evaporated, defatted buckwheat flour is obtained.

[0024] (2) Extraction of buckwheat gluten powder (Osborne method): Defatted buckwheat powder was extracted in three stages, with a material-to-water ratio of 1:10 (m / v) for each stage. The specific extraction conditions and separation steps are as follows: ① Mix defatted buckwheat powder with pure water, stir at 40 ℃ for 2 hours, centrifuge at 8000 r / min for 15 minutes, and collect the precipitate; ② Mix the precipitate from step ① with 2% NaCl solution, stir at 40 ℃ for 2 hours, centrifuge at 8000 r / min for 15 minutes, and collect the precipitate; ③ Mix the precipitate from step ② with 0.05 M NaOH solution, stir at 40 °C for 2 hours, centrifuge at 8000 r / min for 15 minutes, and take the supernatant.

[0025] ④ Adjust the pH of the supernatant obtained above to 4.0 with 1M HCl solution, let it stand, centrifuge at 8000 r / min for 15 minutes, collect the precipitate, reconstitute it and adjust the pH to 7.0, freeze dry to obtain buckwheat gluten protein powder, and store it at -20 ℃ for later use.

[0026] (3) Prepare a protein solution with a substrate concentration of 5% by preparing buckwheat gluten powder, then add 5% alkaline protease, adjust the pH value to 7 with 1 M NaOH solution, and react at 55 °C for 1 hour to obtain the enzymatic hydrolysate.

[0027] (4) The enzyme digest was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain ultrafiltration fractions with a molecular weight ≤3 kDa. Fractions <3 kDa were selected for sequencing (using NanoLC MS / MS). After desalting with a C18 stage-tip and vacuum drying at 45 °C, the samples were processed under the following conditions: column: 150 μm.d. × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600 nL / min; analysis time for each fraction: 66 min. The raw data were analyzed using PepOS 3.4. A total of 1238 reliable oligopeptides and small peptides were obtained from the final analysis, with a length range of 2-14 amino acids.

[0028] Example 2: Screening and Identification of Buckwheat Protein-Derived Oligopeptides The following steps were taken to screen for buckwheat protein-derived oligopeptides with DPP-IV inhibitory activity from the peptides obtained in Example 1 using computer methods: (1) Small peptides with 3-7 amino acids, where the first amino acid is hydrophobic and the second amino acid is proline, were screened from the peptides screened in Example 1. (2) Based on the BIOPEP-UWM database, predict the peptides selected in step (1) and select peptides with a score ≥0.5.

[0029] (3) Based on the StackDPPIV model, the DPP-IV inhibitory activity of the peptides selected in step (2) was predicted, and peptides with a score ≥0.5 were selected.

[0030] (4) Perform hydrophobicity prediction on the peptides selected in step (3) (https: / / www.gravy-calculator.de / index.php), and preferably select peptides with hydrophobicity ≥ -0.5; (5) Perform toxicity prediction on the peptides selected in step (4) (https: / / webs.iiitd.edu.in / raghava / toxinpred3 / ), and select non-toxic peptides; (6) Perform sensitization prediction on the peptides selected in step (5) (https: / / allercatpro.bii.a-star.edu.sg), and select peptides that are not sensitizing.

[0031] This study ultimately screened 11 oligopeptides with DPP-IV inhibitory activity, and their mass spectra are shown below. Figure 1-11 The prediction results of steps (1) to (6) are shown in Table 1.

[0032] Table 1. Computer prediction results of DPP-IV inhibitory oligopeptides derived from tartary buckwheat.

[0033] As shown in Table 1, the 11 oligopeptides selected by screening all had high BIOPEP-UWM and StackDPPIV scores for DPP-IV activity prediction, were generally hydrophobic, and were non-toxic and non-sensitizing.

[0034] (7) Molecular docking was performed on the 11 buckwheat protein-derived oligopeptides screened in step (6). The specific steps are as follows: ① Download the crystal structure of the DPP-IV complex from the PDB database, ID 4PNZ. Use PyMoL software to remove water molecules, chloride ions, sodium ions and proligands, retaining only the DPP-IV protein backbone.

[0035] ② Use ChemDraw and Chem3D to convert the peptide sequences of the 11 buckwheat protein oligopeptides screened in step (6) into 3D structures and save them in pdb format.

[0036] ③ Use AutoDock for structure preparation, including dehydration and hydrogenation, charge distribution, and conversion to PDBQT format. Use AutoDock Vina for docking and construct a docking grid box for the protein structure, with a size of 92 Å × 50 Å × 54 Å. The coordinates of the DPP-Ⅳ active site are: x = 30.936, y = 45.831, z = 27.828. Other parameters are left at their default settings.

[0037] ④ The docking results from step ③ were visualized in three dimensions and subjected to force analysis using Pymol and PLAP. The results are shown in Table 2-3. Figure 16-21 As shown.

[0038] Table 2 Binding energy of DPP-IV inhibitory oligopeptides derived from tartary buckwheat

[0039] Table 3. Bonding between buckwheat protein-derived oligopeptides and DPP-IV enzyme.

[0040] As shown in Table 2-3, the molecular docking binding energies of these 11 DPP-IV inhibitory peptides range from -6 kcal / mol to -7.9 kcal / mol, indicating that they can all bind to DPP-IV. Generally, a binding energy < -5 kcal / mol is considered a good docking, and the lower the value, the more stable the peptide. Hydrogen bonds between the peptide and DPP-IV contribute to the stability of the enzyme-peptide complex and are closely related to the inhibitory ability of DPP-IV activity. A maximum of 11 hydrogen bonds were formed. The binding mode of the peptide to DPP-IV is driven by multiple non-covalent interactions. In addition to forming a hydrogen bond network with amino acid residues, it also contacts nonpolar residues on the receptor surface through hydrophobic interactions and forms salt bridges with charged residues through electrostatic interactions, all contributing to stabilizing the complex conformation and achieving the inhibitory effect.

[0041] Example 3: Detection of in vitro enzyme-level inhibitory activity of buckwheat protein-derived oligopeptides Eleven buckwheat protein-derived oligopeptides (purity >95%) were synthesized via solid-phase synthesis. The in vitro DPP-IV inhibitory activity of these eleven buckwheat protein-derived oligopeptides was detected using the following method: Diprotin A (IPI) was used as a positive control in the DPP-IV in vitro inhibition experiment, serving as a reference for the inhibitory effect of the samples on DPP-IV. 25 μL of oligopeptide solutions of different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mg / mL) and 1.6 mM Gly-Pro-pNA solution were added sequentially, mixed, and incubated at 37 ℃ for 10 min. Then, 50 μL of 10 U / L DPP-IV solution was added, and the mixture was incubated at 37 ℃ with shaking for 60 min. The reaction was then terminated by adding 100 μL of pH 4.0, 1 M HAC-NaAC solution, and the absorbance was measured at 405 nm. The inhibition rate was calculated using the following formula:

[0042] In the formula: A1 is the sample group, A2 is the background group without DPP-IV, A3 is the negative control group without sample solution, and A4 is the blank group without sample and DPP-IV.

[0043] IC 50 The half-maximal inhibitory concentration (HMCS) is the inhibitor concentration required to achieve a 50% inhibition rate, and it is a core indicator for measuring the inhibitory activity of DPP-IV. It is calculated by dividing the DPP-IV inhibitory peptide concentration by the logarithm (log n) of the HMCS concentration. 10 C) is plotted on the x-axis, and the corresponding DPP-IV inhibition rate is plotted on the y-axis. A dose-response curve is then plotted, and the equation is obtained through nonlinear regression fitting. In the formula: a is the slope of the regression equation, b is the intercept of the regression equation, and C is the inhibitor concentration. Substituting y=50% into the formula, we can determine the IC.50 value.

[0044] Based on the inhibition rate, the IC50 of 11 buckwheat protein-derived oligopeptides was calculated. 50 The values ​​are shown in Table 4. The IC50 values ​​of DPP-IV inhibition of the 11 screened oligopeptides are also shown. 50 The values ​​were all lower than the reported IC50 values ​​of the DPP-IV inhibitory peptide derived from tartary buckwheat. 50 Values, especially IPTFVE, and their IC 50 The values ​​were all below 100 μM, achieving a high level of in vitro inhibition of DPP-IV enzyme activity.

[0045] Table 4. In vitro enzymatic inhibitory activity (IC50) of buckwheat protein-derived oligopeptides 50 value)

[0046] Example 4: Lineweaver-Burk double reciprocal curves of buckwheat protein-derived oligopeptides In vitro enzymology IC based on oligopeptides 50 To determine the different concentrations of oligopeptides, Lineweaver-Burk double reciprocal curves were constructed using different concentrations of Gly-Pro-pNA (0.25–4 mM). A buffer solution was used instead of a blank control. 25 μL of oligopeptide solutions of different concentrations were added sequentially (see [link to relevant documentation]). Figure 12-14 The mixture was incubated at 37 °C for 10 minutes with 1.6 mM Gly-Pro-pNA solution. Then, 50 μL of 10 U / L DPP-IV solution was added, and the mixture was incubated at 37 °C with shaking for 60 minutes. The reaction was terminated by adding 100 μL of pH 4.0, 1 M HAC-NaAC solution, and the absorbance was measured at 405 nm.

[0047] Plot a double reciprocal curve with the reciprocal of substrate concentration (1 / [S]) on the x-axis and the reciprocal of initial reaction velocity (1 / V) on the y-axis. Perform linear fitting and determine the inhibition type of the inhibitor based on the intersection of the curves.

[0049] The results are as follows Figure 12-14 As shown, DPP Oligopeptides with strong IV-inhibiting activity exhibit multiple inhibition modes, including competitive, non-competitive, and mixed modes.

[0050] For IPVEK and IPVT, the slope of the double reciprocal curves gradually increases with increasing concentration, and all straight lines intersect at a point on the Y-axis, exhibiting typical competitive inhibition characteristics. Essentially, they compete with the substrate for binding to the active site of DPP-IV. For oligopeptides such as IPGGLL, LPI, VPIA, IPTFVE, IPVAL, VPIT, VPTFF, and LPILE, the slope and Y-intercept gradually increase with increasing concentration, intersecting at a point on the X-axis, exhibiting non-competitive inhibition. In this mode, the peptide does not compete with the substrate for the active site; it binds to the inactive site region of the enzyme molecule, inducing a change in the overall spatial conformation of DPP-IV, thereby inhibiting DPP-IV enzyme activity. For IPETEI, the slope and Y-intercept gradually increase with increasing concentration, intersecting at a point in the second quadrant, representing a mixed type of competitive and anti-competitive inhibition; it can bind to both DPP-IV enzyme and enzyme-substrate complexes. DPP-IV inhibitory peptides regulate enzyme activity through competitive, non-competitive, and mixed inhibition modes. Different peptide segments can exert inhibitory effects through competitive active pocket sites, enzyme conformational allosteric regulation, or dual binding mechanisms.

[0051] Example 5: Detection of in situ cellular inhibitory activity of buckwheat protein-derived oligopeptides The screened DPP-IV inhibitory peptides were subjected to in situ inhibitory activity assays in Caco-2 cells. Caco-2 cells were cultured at 2 × 10⁻⁶ cells / cells. 4 100 μL of the culture medium was seeded into each well of a 96-well plate and cultured for 24 hours until cell adhesion. The old culture medium was then discarded, and the cells were washed twice with PBS. 25 μL of different concentrations of the sample (0, 0.125, 0.25, 0.5, 1, and 2 mg / mL) were added to 125 μL of PBS, mixed well, and incubated at 37 °C for 10 minutes. Finally, 50 μL of Gly-Pro-pNA (1 mM) solution was added, and incubation continued for 60 minutes. The absorbance at 405 nm was measured. DMEM medium was used as a blank. The inhibition rate was calculated using the following formula:

[0052] In the formula: A1 is the sample group, A2 is the background group without DPP-IV, A3 is the negative control group without sample solution, and A4 is the blank group without sample and DPP-IV.

[0053] IC 50 The half-maximal inhibitory concentration (HMCS) is the inhibitor concentration required to achieve a 50% inhibition rate, and it is a core indicator for measuring the inhibitory activity of DPP-IV. It is calculated by dividing the DPP-IV inhibitory peptide concentration by the logarithm (log n) of the HMCS concentration. 10C) is plotted on the x-axis, and the corresponding DPP-IV inhibition rate is plotted on the y-axis. A dose-response curve is then plotted, and the equation is obtained through nonlinear regression fitting. In the formula: a is the slope of the regression equation, b is the intercept of the regression equation, and C is the inhibitor concentration. Substituting y=50% into the formula, we can determine the IC. 50 The values ​​are shown in Table 2. The IC50 values ​​of the 11 selected oligopeptides in Caco-2 cells are also presented. 50 The values ​​were all lower than the reported IC50 values ​​of the DPP-IV inhibitory peptide derived from tartary buckwheat. 50 Values, especially LPI, VPIA, IPVT, VPTFF, and LPILE, their ICs 50 The values ​​were all below 100 μM, achieving a high in situ inhibition effect on DPP-IV activity.

[0054] Table 5. In situ inhibitory activity of buckwheat protein-derived oligopeptides on Caco-2 cells (IC50). 50 value)

[0055] Example 6: Effects of buckwheat protein-derived oligopeptides on Caco-2 cell activity The effect of buckwheat protein-derived oligopeptides on Caco-2 cell viability was assessed using a colorimetric assay based on Cell Counting Kit-8 (CCK-8). Caco-2 cells were cultured at 2 × 10⁶ cells / cells. 4 / 100 μL of the medium was seeded into each well of a 96-well plate and incubated overnight. Subsequently, the original medium was replaced with 100 μL of complete medium containing different concentrations (0, 0.125, 0.25, 0.5, 1, and 2 mg / mL) of the sample, and incubated overnight. After incubation, 10 μL of CCK-8 reagent was added to each well, and the plate was incubated for another 2 hours. The absorbance at 450 nm was then measured. DMEM medium was used as a blank. The inhibition rate was calculated using the following formula:

[0056] In the formula: A1 is the experimental group, A2 is the cell-free blank group, and A3 is the negative control group without sample solution.

[0057] The results are as follows Figure 15 As shown, in Caco-2 cells, 11 buckwheat protein-derived oligopeptides inhibited the IC50 in situ. 50 At concentrations up to 10 times the effective value, no significant cytotoxicity was observed, demonstrating good safety.

[0058] In summary, the buckwheat protein-derived oligopeptide provided by this invention is a natural DPP-IV inhibitory peptide, which is safer to consume, has a smaller molecular weight, and is hydrophobic, non-toxic, non-allergenic, and has good affinity with target proteins, forming a stable complex. It has good prospects for hypoglycemic applications.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A buckwheat protein-derived oligopeptide, characterized in that, The buckwheat protein-derived oligopeptide is any one of the following: A1. Having an amino acid sequence as shown in SEQ ID NO.1-10 and an amino acid sequence as shown in any of the LPIs; A2. A derivative obtained by chemical or enzymatic modification of the amino acid sequence described in A1, which has DPP-IV inhibitory activity; A3. A mutant that has at least 80% sequence identity with the amino acid sequence described in A1 and retains DPP-IV inhibitory activity.

2. The method for extracting and screening buckwheat protein-derived oligopeptides according to claim 1, characterized in that, Includes the following steps: (1) Extraction and sequence identification of buckwheat peptides; (2) Screening of buckwheat protein-derived oligopeptides: ① Select peptides from the peptides extracted and identified in step (1) that have 3-7 amino acids, with the first amino acid being hydrophobic and the second amino acid being proline; ② Based on the BIOPEP-UWM database, predict the peptides selected in step ①, and select peptides with a score ≥ 0.5; ③ The peptides selected in step ② are subjected to DPP-IV inhibitory activity prediction, and peptides with a score ≥0.5 are preferred; ④ Perform hydrophobicity prediction on the peptides selected in step ③, and preferentially select peptides with hydrophobicity ≥ -0.5; ⑤ Perform toxicity prediction on the peptides selected in step ④, and select non-toxic peptides. ⑥ Perform sensitization prediction on the peptides selected in step ⑤, and select peptides that are not sensitizing; ⑦ Perform molecular docking simulations on the peptides selected in step ⑥, and select peptides with the optimal binding energy ≤ -5 kcal / mol.

3. The extraction and screening method according to claim 2, characterized in that, Step (1), extraction and sequence identification of buckwheat peptides, specifically includes the following steps: S1. Mix the hulled buckwheat seed powder with petroleum ether and stir overnight. After defatting, filter and air dry to obtain defatted buckwheat powder. S2. Buckwheat gluten powder was extracted from the defatted buckwheat powder in step S1 using the Osborne method. S3. Prepare a protein solution from the buckwheat gluten powder obtained in step S2, then add alkaline protease, adjust the pH value to 7 with alkaline solution, and then perform enzymatic hydrolysis to obtain an enzymatic hydrolysate. S4. The enzymatic hydrolysate from step S3 is subjected to ultrafiltration. Protein fractions with a molecular weight <3kDa are selected for sequencing. The obtained raw data is then analyzed. After spectral analysis, peptide sequences with a length range of 2-14 amino acids are obtained for subsequent screening.

4. The extraction and screening method according to claim 2, characterized in that, The molecular docking simulation described in step ⑦ specifically includes the following steps: B1. Download the crystal structure of the DPP-IV complex from the PDB database, and then remove its water molecules, chloride ions, sodium ions and proligands, retaining only the DPP-IV protein backbone; B2. Convert the peptide sequence selected in step ⑥ into a 3D structure; B3. Prepare the 3D structure obtained in step B2, including dehydration and hydrogenation and charge distribution, and then perform molecular docking to construct a docking grid box for the protein structure. B4. Perform three-dimensional visualization and force analysis on the molecular docking results of step B3.

5. The use of the buckwheat protein-derived oligopeptide according to claim 1 in the preparation of DPP-IV inhibitors.

6. A DPP-IV inhibitor, characterized in that, It includes at least one of the buckwheat protein-derived oligopeptides as described in claim 1.

7. The DPP-IV inhibitor according to claim 6, characterized in that, The DPP-IV inhibitors also include pharmaceutically acceptable carriers, excipients, matrices, or additives.

8. The DPP-IV inhibitor according to claim 6, characterized in that, The dosage forms of the DPP-IV inhibitors are granules, powders, capsules, tablets, mixtures, or oral liquids.

9. The use of the DPP-IV inhibitor according to any one of claims 6-8 in the preparation of a medicament, dietary supplement or food having DPP-IV inhibitory function.

10. A drug, dietary supplement, or food having DPP-IV inhibitory function, characterized in that, Includes the DPP-IV inhibitor as described in any one of claims 6-8.

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