Egg white-derived dipeptidyl peptidase-4 inhibitory peptide and application thereof

Abstract

The application discloses an egg white-derived dipeptidyl peptidase-4 (DPP-IV) inhibiting peptide and application thereof. The amino acid sequence of the inhibiting peptide is shown as SEQ ID NO. 5. The peptide is obtained by expanding the egg white peptide spectrum through probiotic intervention on egg ducks, combining enzymolysis, mass spectrometry identification, toxicity prediction, structure characteristic screening and molecular docking. In vitro activity verification shows that the inhibiting rate of the peptide on DPP-IV reaches 71.34+ / -1.62% at a concentration of 2 mg / mL, which is significantly better than that of other homologous peptides. The DPP-IV inhibiting peptide is safe in source and outstanding in activity, and can be used for preparing a medicine for assisting in reducing blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dipeptidyl peptidase-4 (DPP-IV) inhibitory peptide derived from egg white and its application. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized primarily by hyperglycemia, with type 2 diabetes accounting for a large proportion. Long-term abnormal blood sugar levels can lead to various complications, such as cardiovascular disease, kidney disease, and retinopathy, severely impacting patients' quality of life and even their lives. While existing hypoglycemic drugs can control blood sugar to some extent, long-term use may still be accompanied by gastrointestinal discomfort, hypoglycemia risk, liver and kidney burden, or other adverse reactions. Therefore, developing safe, well-defined, and suitable-for-long-term intake natural hypoglycemic active substances has become an important direction in the development of adjunctive hypoglycemic drugs.

[0003] Dipeptidyl peptidase-4 (DPP-IV) is an important target for regulating postprandial blood glucose. DPP-IV degrades glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), two incretin hormones that play crucial roles in glucose homeostasis: they promote glucose-dependent insulin secretion, inhibit glucagon release, delay gastric emptying, and promote pancreatic β-cell proliferation and survival. Inhibiting DPP-IV activity prolongs the half-life of GLP-1 and GIP, enhancing their physiological functions and thus effectively controlling postprandial blood glucose. Based on this mechanism, DPP-IV inhibitors have become an important class of drugs for the treatment of type 2 diabetes. For example, early studies have demonstrated that small-molecule DPP-IV inhibitors such as isoleucyl thiazolidine can effectively reduce postprandial blood glucose levels in mammals through oral administration.

[0004] In recent years, DPP-IV inhibitory peptides have become a hot topic in drug research due to their advantages such as natural origin, low side effects, and safe metabolites. Compared with chemically synthesized drugs, bioactive peptides are particularly suitable for daily intervention in people with prediabetes or mild diabetes. Currently, researchers have screened functional peptides with DPP-IV inhibitory activity from various food raw materials. For example, DPP-IV inhibitory peptides can be obtained from pine pollen through enzymatic hydrolysis, ultrafiltration, liquid chromatography separation, and mass spectrometry identification; peptides with DPP-IV inhibitory activity can also be obtained from eggshell membrane hydrolysate through a combination of enzymatic hydrolysis, ultrafiltration, HPLC-MS / MS identification, and molecular docking technology; and specific bioactive peptide sequences of egg yolk-derived hypoglycemic peptides have been obtained through enzymatic hydrolysis, liquid chromatography separation, and molecular docking screening.

[0005] Regarding aquatic product sources, DPP-IV inhibitory peptides from oysters have been shown to have good digestive stability and hypoglycemic effects. Oyster peptides rich in branched-chain amino acids, prepared via an alcohol precipitation-desugaring coupled with activated carbon adsorption process, exhibit DPP-IV inhibitory IC50. 50 The concentration reached 0.38 mg / mL, and oral administration for 4 weeks in a type 2 diabetic mouse model resulted in a 41.7% decrease in fasting blood glucose. Furthermore, the DPP-IV inhibitory peptide derived from fish skin (molecular weight <1500 Da) also exhibited good inhibitory activity, with an IC50 value of [missing value]. 50 The concentration was less than 0.55 mg / mL. Deer-derived DPP-IV inhibitory peptides (such as GPAGPXGVXGL) have also been reported to have hypoglycemic activity and possess the potential to be developed as drug lead compounds. These findings indicate that various animal-derived raw materials can serve as good sources of DPP-IV inhibitory peptides.

[0006] Egg white resources, due to their rich protein composition, high biocompatibility, and wide availability, have a solid foundation for the development of bioactive peptides. Duck egg white, as an important animal-derived protein resource, has a high protein content and a balanced amino acid composition, possessing good potential for developing hypoglycemic bioactive peptides. However, existing research has largely focused on the utilization of chicken egg white, with relatively little research on the development and utilization of functional peptides derived from duck egg white, especially lacking in-depth exploration of the systematic screening and functional verification of DPP-IV inhibitory peptides derived from duck egg white.

[0007] Based on this, the present invention expands the range of identifiable peptides in egg white through front-end regulation methods (such as probiotic intervention), and then integrates all egg white peptide sequences in the sample, combined with toxicity prediction, peptide length and molecular weight limitation, structural feature screening, molecular docking and in vitro DPP-IV inhibitory activity verification, to screen out duck egg white-derived DPP-IV inhibitory peptides with well-defined structures and outstanding activities. This will help to make up for the lack of existing egg white-derived hypoglycemic peptide development and provide a new source of active ingredients for adjuvant hypoglycemic drugs. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a polypeptide derived from duck egg white with significant DPP-IV inhibitory activity, and to provide the application of the polypeptide in assisting in lowering blood sugar.

[0009] In a first aspect, the present invention provides a duck egg white-derived DPP-IV inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.5, namely Leu-Pro-Trp-Ile-Ser-Leu-Arg (LPWISLR).

[0010] The inhibitory peptide binds to the active site residues of DPP-IV via hydrogen bonds, hydrophobic interactions, and / or salt bridges, with a binding energy of -8.10 kcal / mol.

[0011] In a second aspect, the present invention provides a composition comprising the duck egg white-derived DPP-IV inhibitory peptide and acceptable excipients, carriers or excipients.

[0012] Thirdly, the present invention provides the application of the duck egg white-derived DPP-IV inhibitory peptide or a combination thereof in the preparation of adjuvant hypoglycemic drugs.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a duck egg white-derived DPP-IV inhibitory peptide. This peptide exhibits an inhibition rate of 71.34 ± 1.62% against DPP-IV at a concentration of 2 mg / mL, with an IC50 of 0.36 mg / mL. Molecular docking analysis shows that LPWISLR stably binds to the active site of DPP-IV through multiple interactions, including hydrogen bonds, hydrophobic interactions, and salt bridges, with a binding energy of -8.10 kcal / mol. Duck egg white is an edible protein resource, and the inhibitory peptide provided by this invention is safe and suitable for long-term consumption. This invention also expands the peptide spectrum through probiotic intervention and, combined with multidimensional screening criteria (toxicity, length, molecular weight, structural features, and molecular docking), improves the success rate of active peptide discovery. Attached Figure Description

[0014] Figure 1 The inhibition rate curve of the egg white-derived DPP-IV inhibitory peptide LPWISLR on DPP-IV enzyme activity measured in Example 2 was used to calculate the half-maximal inhibitory concentration (IC50). 50 The results showed IC 50 The value was 0.36 mg / mL.

[0015] Figure 2 The mass spectrum of the synthesized and purified egg white-derived DPP-IV repressor peptide LPWISLR in Example 2 shows the parent ion peak ([M+H)). + The measured molecular weight and characteristic fragment ions were highly consistent with the theoretical molecular weight (884.06 Da), verifying that the peptide sequence was correct and the purity met the standard.

[0016] Figure 3 In Example 2, a three-dimensional interaction diagram of the egg white-derived DPP-IV inhibitory peptide LPWISLR obtained by molecular docking with the active site of DPP-IV is shown. The diagram illustrates the stable binding of LPWISLR (represented by a rod-shaped model) with key amino acid residues of DPP-IV (such as Asp-545, Val-546, Trp-629, Tyr-547, Tyr-631, etc.) through hydrogen bonds (represented by dashed lines), hydrophobic interactions, and salt bridges. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally implemented under conventional conditions or according to the methods recommended in the manufacturer's operation manual.

[0018] Example 1: Screening of DPP-IV inhibitory peptides 1. Test Methods 1.1 Sample Preparation Healthy laying ducks of similar weight and size, aged 32 weeks, were selected, and 2% Lactobacillus plantarum solution (live bacteria content of 5×10⁻⁶) was added to their basal diet. 9 –2×10 10 Ducks were fed a continuous diet (CFU / mL) for 8 weeks. After 8 weeks, duck eggs were collected and the egg whites were separated. 10 μL of egg white was taken and 20 μL of lysis buffer was added, along with protease inhibitor at a ratio of 1:50. The mixture was sonicated for 0.5 minutes, followed by centrifugation at 12,000 rpm for 20 minutes. The supernatant was collected to obtain the total protein solution from the duck egg white.

[0019] 1.2 Proteolytic digestion and mass spectrometry identification 15 μg of total duck egg white protein solution was subjected to TCEP reduction, IAA alkylation, and short-term denaturation at 95℃. Trypsin was then added and the mixture was hydrolyzed overnight at 37℃ with shaking. The reaction was terminated by acidification with formic acid. The peptide mixture was desalted using a C18 HPLC system, eluted, and vacuum dried. The peptides were then separated using a Vanquish Neo UHPLC system and detected by DIA using an Orbitrap Astral mass spectrometer. Mass spectrometry data were analyzed using DIA-NN 1.9.2 software in conjunction with a duck protein database to obtain peptide sequence information.

[0020] 1.3 Peptide Screening The identified peptides were then screened as follows: ① Use ToxinPred 3.0 to assess toxicity and screen for non-toxic peptides; ② Retain peptides with a length of 2–18 amino acids and a molecular weight of 0.2–2.0 kDa; ③ Screen peptides with at least three of the following structural features: C-terminus is Lys or Arg; contains hydrophobic amino acid residues; contains Pro residues; contains at least one aromatic amino acid from Trp, Phe or Tyr; N-terminus contains at least two hydrophobic or aromatic amino acid residues at positions 1–4. ④ Peptides with a binding energy of less than -7.0 kcal / mol to the active site of DPP-IV were screened through molecular docking.

[0021] 1.4 Molecular docking The DPP-IV crystal structure with PDB ID 2QT9 from the RCSB Protein Data Bank was used as the receptor model. The receptor protein was preprocessed using AutoDock Tools: water of crystallization and the original ligand were removed, polar hydrogen was added, and atomic charges were assigned, then saved in PDBQT format. The three-dimensional conformation of the target peptide was constructed using a cloud platform (https: / / cloud.yinfotek.com), optimized for energy minimization, and set as a flexible ligand before being converted to PDBQT format. Molecular docking was performed using AutoDockTools-1.5.7, with the docking box center coordinates set to x=42.01, y=48.931, z=42.778 to examine the binding of the peptide to the DPP-IV active region. The docking results were visualized and analyzed using PyMOL software.

[0022] 2. Test Results Through the above step-by-step screening, six candidate DPP-IV inhibitory peptides were finally obtained, namely: VAVFDTGLSEK, LAWVPVAGSTGYR, LAEGGPQR, FDFFNYAGIHR, LPWISLR, and GPHFLPFNVK.

[0023] The characteristics of the six peptides are shown in Table 1. The results show that the peptide length ranges from 7 to 13 amino acids; the molecular weight ranges from 0.8 to 1.4 kDa; all peptides are non-toxic; the binding energy ranges from -8.89 to -7.64 kcal / mol, all of which meet the requirement of being less than -7.0 kcal / mol; and all peptides meet the structural feature screening criteria (at least three criteria).

[0024] Table 1: Statistical Information of Each Peptide Segment

[0025] Example 2: Verification of the DPP-IV inhibitory effect of the peptide 1. Test Methods 1.1 Experimental Materials and Reagents The peptides to be tested: The six candidate peptides (VAVFDTGLSEK, LAWVPVAGSTGYR, LAEGGPQR, FDFFNYAGIHR, LPWISLR, and GPHFLPFNVK) obtained from the screening in Example 1 were synthesized by solid-phase synthesis and purified by high-performance liquid chromatography (HPLC) with a purity of ≥95%.

[0026] Positive control: Sitagliptin, a known DPP-IV inhibitor.

[0027] DPP-IV enzyme: recombinant human DPP-IV, activity ≥0.1 U / mL.

[0028] Substrate: Gly-Pro-AMC (7-amino-4-methylcoumarin-labeled glycyl-proline), a DPP-IV specific fluorescent substrate.

[0029] Buffer solution: 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl.

[0030] Sample solvent: deionized water or buffer solution.

[0031] 1.2 Experimental Grouping and Sample Addition The experiment was conducted in 96-well black microplates, with 3 replicates per group. The specific sample loading protocol is shown in Table 2.

[0032] Table 2: Experimental Grouping and Sample Addition Scheme

[0033] 1.3 Operating Procedures (1) Sample addition and pre-incubation: Add DPP-IV enzyme working solution (add an equal volume of buffer to blank wells), sample solvent, or test sample (peptide concentration of 2 mg / mL) or sitagliptin solution to each well of the 96-well plate as described above. Shake the plate for 3 seconds using a microplate reader, then incubate at 37°C for 10 minutes to allow the inhibitor (if present) to fully bind with the DPP-IV enzyme.

[0034] (2) Adding a substrate to initiate the reaction: Prepare a working reaction solution by mixing the substrate Gly-Pro-AMC with buffer at a volume ratio of 1:9, and add 170 μL to each well. Immediately shake the plate to mix for 3 seconds, and continue incubation at 37°C for 30 minutes to allow the DPP-IV enzyme to catalyze the release of the fluorescent AMC group from the substrate.

[0035] (3) Fluorescence detection: The fluorescence value of each well was detected using a fluorescence microplate reader. The excitation wavelength (Ex) was 360 nm and the emission wavelength (Em) was 460 nm. The fluorescence intensity (F) of each well was recorded.

[0036] 1.4 Inhibition rate Calculate the DPP-IV inhibition rate using the following formula:

[0037] in: F blank: Fluorescence value of blank wells (no DPP-IV enzyme, no inhibitor) F pair: Fluorescence value of control wells (containing DPP-IV enzyme, without inhibitor) F assay: Fluorescence value of the assay well or positive control well (containing DPP-IV enzyme, or an inhibitor). 1.5 IC 50 calculate The test peptides were prepared into solutions of different concentrations (2.00, 1.00, 0.50, 0.10, and 0.05 mg / mL), and their inhibition rates against DPP-IV enzyme activity were measured. The inhibition rate was determined using LPWISLR concentrations. The independent variable is the DPP-IV inhibition rate. Using DPP-IV as the dependent variable, a logistic dose-response model with bottom=0% and top=100% constraints was used for nonlinear regression fitting to calculate the half-maximal inhibitory concentration (IC50) of the analyte peptide against DPP-IV. 50 The fitted equation is as follows:

[0038] In the formula, The value represents the DPP-IV inhibition rate, expressed as % . The concentration of the polypeptide to be tested is expressed in mg / mL. The concentration of the target peptide when the DPP-IV inhibition rate reaches 50%; Let be the Hill slope.

[0039] 1.6 Statistical Analysis One-way ANOVA was performed using SPSS software (version 26.0), and Tukey's HSD test was used for multiple comparisons between groups. The significance level was set at P < 0.05. All data are expressed as mean ± standard deviation (Mean ± SD).

[0040] 2. Experimental Results 2.1 Comparison of DPP-IV inhibition rates of different peptides Table 3 shows the inhibition rates of the six candidate peptides and the positive control sitagliptin against DPP-IV at a concentration of 2 mg / mL.

[0041] Table 3: DPP-IV inhibition rate of each peptide segment

[0042] Note: Different letters represent significant differences (P<0.05).

[0043] The results showed that the inhibition rate of LPWISLR (71.34%) was significantly higher than that of the other 5 peptides (P<0.05), demonstrating strong DPP-IV inhibitory activity.

[0044] 2.2 LPWISLR IC 50 value IC 50 Value measurement results are as follows Figure 1 As shown, the results indicate that LPWISLR has a better IC performance than DPP-IV. 50 The concentration was 0.36 mg / mL, indicating that the polypeptide can effectively inhibit DPP-IV enzyme activity and has potential value for developing hypoglycemic activity.

[0045] 2.3 Mass spectrometry identification of LPWISLR Mass spectrometry analysis of the synthesized and purified LPWISLR revealed a clear parent ion peak ([M+H)) in the mass spectrum. + The measured molecular weight of the measured fragment ions (884.06 Da) is in high agreement with the theoretical molecular weight (884.06 Da). Figure 2 The purity of the synthesized peptide is ≥95%, which proves that the synthesized peptide is correct.

[0046] 2.4 Molecular interaction mechanism between LPWISLR and DPP-IV To further elucidate the molecular basis of LPWISLR's inhibition of DPP-IV, molecular docking methods (same as in Example 1) were used to analyze its interaction mode with the active site of DPP-IV. Docking analysis showed that LPWISLR mainly binds stably to the active pocket of DPP-IV through the following interactions: LPWISLR forms a core interaction network with the aspartic acid (Asp-545), valine (Val-546), and tryptophan (Trp-629) of DPP-IV via its C-terminal arginine (Arg-7). The arginine (Arg-7)–aspartic acid (Asp-545) interaction primarily functions as a salt bridge, while the main chain atoms of the arginine (Arg-7)–valine (Val-546) and arginine (Arg-7)–tryptophan (Trp-629) interactions mainly form hydrogen bonds. Simultaneously, hydrophobic residues in LPWISLR, such as leucine (Leu-1), isoleucine (Ile-4), and leucine (Leu-6), form hydrophobic interactions with residues in the DPP-IV pocket, such as tryptophan (Trp-629), tyrosine (Tyr-547), and tyrosine (Tyr-631). Figure 3 Multiple non-covalent interactions collectively stabilize the binding conformation of LPWISLR in the active pocket of DPP-IV.

[0047] These interactions together result in LPWISLR binding tightly to the active site of DPP-IV (binding energy of -8.10 kcal / mol), effectively blocking substrate entry into the enzyme's active site, thereby exerting significant DPP-IV inhibitory activity.

[0048] 3. Conclusion In vitro DPP-IV inhibitory activity assays of six candidate peptides revealed that LPWISLR, derived from duck egg white, exhibited an inhibition rate of 71.34 ± 1.62% against DPP-IV at a concentration of 2 mg / mL, significantly superior to other screened peptides. Molecular docking analysis further revealed that LPWISLR stably binds to the DPP-IV active site through multiple interactions, including hydrogen bonds, hydrophobic interactions, and salt bridges. Therefore, LPWISLR is a structurally well-defined and highly active duck egg white-derived DPP-IV inhibitory peptide with the potential to be developed as an adjunct hypoglycemic agent.

Claims

1. A protein-derived dipeptidyl peptidase-4 inhibitory peptide, characterized in that, The amino acid sequence of the inhibitory peptide is shown in SEQ ID NO.

5.

2. A composition, characterized in that, The composition comprises the inhibitory peptide of claim 1 and acceptable excipients.

3. The use of the inhibitory peptide of claim 1 or the composition of claim 2 in the preparation of a drug for assisting in lowering blood sugar.

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