Tea-derived dpp-iv inhibiting peptide, screening method and application thereof

Abstract

The application discloses tea source DPP-IV inhibiting peptides, a screening method and application thereof, and belongs to the technical field of bioactive peptides. Specifically, novel DPP-IV inhibiting peptides APWLEPLR, LAFDLIR and EAAWGLAR are screened and identified from tea proteins by virtual enzymolysis and in combination with computer-aided tools. Research shows that APWLEPLR exhibits significant DPP-IV inhibiting activity in vitro and does not exhibit obvious cytotoxicity in Caco-2 cells, showing good safety. The application successfully screens and verifies bioactive peptides APWLEPLR, LAFDLIR and EAAWGLAR with good DPP-IV inhibiting activity, and the research not only provides a scientific basis for developing novel natural DPP-IV inhibiting peptides, but also provides a new idea for developing functional tea products.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a tea-derived DPP-Ⅳ inhibitory peptide, its screening method, and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a complex metabolic disease prevalent worldwide, characterized by chronic hyperglycemia and insulin resistance, and has become one of the most serious threats to public health. It is predicted that the number of people with diabetes globally will reach 783 million by 2045. Therefore, the intervention and treatment of diabetes has become a global research hotspot. Dipeptidyl peptidase-IV (DPP-IV) is an important therapeutic target for type 2 diabetes. DPP-IV inhibitors, as a novel oral hypoglycemic agent, work by attenuating the activity of DPP-IV to inhibit the degradation of endogenous glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), thereby promoting insulin secretion and lowering blood glucose levels. However, long-term use of synthetic DPP-IV inhibitors, such as sitagliptin, vildagliptin, and saxagliptin, can cause various adverse reactions, including gastrointestinal reactions, allergies, pancreatitis, and lipid metabolism disorders. Therefore, developing food-derived, safe, and effective DPP-IV inhibitors has become an important research direction for the treatment of type 2 diabetes mellitus (T2DM).

[0003] In recent years, dietary DPP-IV inhibitory peptides have attracted widespread attention due to their high activity, easy absorption, and good safety profile. Many researchers have successfully prepared and identified naturally occurring, highly active DPP-IV inhibitory peptides using plant and animal-derived proteins. For example, two novel DPP-IV inhibitory tripeptides (WPR and MPR) derived from sesame protein, and three DPP-IV inhibitory peptides (LAHMGPY, VPSIHDLF, and VAHPAPYF) derived from shrimp head hydrolysate are reversible competitive DPP-IV inhibitory peptides. Peptides (LKPTPEGDL and LKPTPEGDLEIL) isolated and identified from bovine whey protein inhibit DPP-IV activity in a non-competitive manner, thereby achieving a blood glucose-lowering effect. These studies indicate that dietary DPP-IV inhibitory peptides can effectively control blood glucose and are an important option for diabetes intervention.

[0004] tea( Camellia sinensis (L.) KuntzeTea, with its unique aroma and flavor, has become the world's most consumed beverage. It is not only rich in bioactive substances such as protein, polysaccharides, and polyphenols, but also possesses various physiological functions including lowering blood sugar, anti-oxidation, and lowering blood pressure. Although tea is rich in protein (20%-30%), its proportion of non-water-soluble proteins (glutenin, globulin, prolamins) is relatively high, leading to low utilization of tea protein and resource waste. Tea protein contains high levels of hydrophobic amino acids such as phenylalanine, leucine, and valine. Studies have shown that hydrophobic amino acids are key structural features for the hypoglycemic activity of plant-derived hypoglycemic peptides. Currently, studies have identified various bioactive peptides from tea. For example, Cao et al. successfully identified novel ACE inhibitory peptides (FPFPRPP, PPPRGP, PFPRPPH, LGHPW, LKFPDF) from Wuyi rock tea; prepared uric acid-lowering peptides from Yinghong No. 9 tea using enzymatic hydrolysis; and identified the DPP-IV inhibitory dipeptide Val-Leu from green tea. In summary, while some progress has been made in the research of bioactive peptides derived from tea, research on DPP-IV inhibitory peptides from tea remains relatively limited. Existing studies are mostly confined to traditional enzymatic hydrolysis methods, failing to select proteases specifically for hydrolysis based on the structural characteristics of DPP-IV inhibitory peptides. This results in the discovery of highly active peptides being both somewhat indiscriminate and inefficient. Regarding the exploration of inhibition mechanisms, most studies rely solely on theoretical predictions using molecular docking, followed by analysis of the DPP-IV inhibitory capacity (IC50) of the peptides. 50 While analyses of the interaction between DPP-IV and enzyme inhibition kinetics have been conducted, in-depth investigations have not been carried out. For example, there are few reports on spectroscopic (UV, fluorescence) analyses of the interaction between peptides and DPP-IV, or on observations of the microstructure of peptides after interaction with free DPP-IV using atomic force microscopy (AFM). At the cellular level, although existing studies have investigated the cellular inhibitory capacity (in situ IC50) of DPP-IV inhibitory peptides... 50 The levels of GLP-1 secretion were measured, but the exploration of GLP-1 secretion-related signaling pathways is still mainly based on network pharmacology predictions, lacking cell experimental verification. The specific molecular mechanisms need to be studied in depth.

[0005] The discovery of conventional bioactive peptides relies on enzymatic digestion, chromatographic separation, and mass spectrometry identification, which suffers from bottlenecks such as long cycles, high costs, and unpredictable activity predictions. Thanks to advancements in proteomics and peptidomics, and innovations in bioinformatics tools, virtual screening and activity probability prediction based on whole protein sequences have become possible. By integrating multi-omics data with computer-aided simulations, targeted discovery of potential bioactive peptides from food sources can be achieved, significantly shortening the research and development cycle and laying a theoretical foundation for the green and efficient development of food-derived peptides. Researchers have already successfully used these technologies to develop and identify various bioactive peptides. For example, Chen et al. developed a novel DPP-IV inhibitory peptide from cannabis seeds using a combination of multi-omics analysis and molecular docking; flavor peptides were successfully identified from shiitake mushroom extract by integrating peptidomics analysis and computer simulation methods; similarly, Liao et al. discovered an ACE inhibitory peptide in soft-shell turtle meat using computer-simulated digestion and virtual screening techniques. The comprehensive application of these computer technologies not only overcomes the shortcomings of traditional experimental methods but also enables the efficient and accurate development and identification of bioactive peptides.

[0006] Therefore, this invention aims to identify novel DPP-Ⅳ inhibitory peptides by virtually enzymatically hydrolyzing tea proteins and using computer-aided tools, while exploring their inhibition mechanism against DPP-Ⅳ using enzyme inhibition kinetics, ultraviolet spectroscopy, fluorescence spectroscopy, and molecular docking systems. Summary of the Invention

[0007] The purpose of this invention is to provide a tea-derived DPP-IV inhibitory peptide, its screening method, and its application, thereby addressing the problems existing in the prior art. This research not only provides a scientific basis for the development of novel natural DPP-IV inhibitory peptides but also offers new ideas for the development of functional tea products.

[0008] To achieve the above objectives, the present invention provides the following solution: In a first aspect, the present invention provides a tea-derived DPP-Ⅳ inhibitory peptide, wherein the amino acid sequence of the tea-derived DPP-Ⅳ inhibitory peptide is any one of APWLEPLR, LAFDLIR, or EAAWGLAR.

[0009] Secondly, the present invention also provides a method for screening the aforementioned tea-derived DPP-Ⅳ inhibitory peptide, comprising the following steps: Search the UniProtKB database Camellia sinensis var. sinensis ( Chinese tea High-confidence protein sequences were obtained from the Swiss-Prot category, and relevant sequences were supplemented from the TrEMBL section. The Align multiple sequence alignment tool in UniProt was used for sequence alignment analysis, and finally tea protein sequences with similarity of less than 90% were screened out. The BIOPEP-UWM database was used to predict potential bioactive peptides in the tea protein sequences. The tea protein sequence was virtually enzymatically digested using the PeptideCutter online tool to obtain bioactive peptides. The bioactive peptides were screened using the PeptideRanker and BIOPEP databases to obtain the tea-derived DPP-Ⅳ inhibitory peptide.

[0010] Thirdly, the present invention also provides the application of the tea-derived DPP-Ⅳ inhibitory peptide in the preparation of DPP-Ⅳ inhibitors.

[0011] Preferably, the DPP-IV inhibitor further comprises one or more pharmaceutically acceptable carriers.

[0012] Fourthly, the present invention also provides the application of the tea-derived DPP-Ⅳ inhibitory peptide in the preparation of a diabetes treatment drug.

[0013] Preferably, the dosage form of the drug is selected from injections, solutions, emulsions, sprays, granules, powders, capsules, pills, tablets, patches, or sustained-release formulations.

[0014] Fifthly, the present invention also provides a DPP-Ⅳ inhibitor, wherein the DPP-Ⅳ inhibitor includes the tea-derived DPP-Ⅳ inhibitory peptide.

[0015] Preferably, the DPP-Ⅳ inhibitor also includes a pharmaceutically acceptable carrier.

[0016] In a sixth aspect, the present invention also provides a diabetes treatment drug, wherein the diabetes treatment drug includes the tea-derived DPP-Ⅳ inhibitory peptide.

[0017] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers.

[0018] This invention utilizes bioinformatics tools to predict and analyze the amino acid distribution and the frequency (A value) of DPP-Ⅳ active fragments in tea proteins. Based on this, virtual enzymatic hydrolysis of tea proteins is performed, and novel tea-derived DPP-Ⅳ inhibitory peptides are identified using computer-aided tools. Furthermore, the specific inhibitory mechanism against DPP-Ⅳ is explored using enzyme inhibition kinetics, spectroscopy (UV spectroscopy, fluorescence spectroscopy), atomic force microstructure analysis, and molecular docking and molecular dynamics simulation systems. In addition, its inhibitory ability at the Caco-2 cell level and its stability in in vitro simulated gastrointestinal digestion are further evaluated. This invention not only identifies a novel and highly efficient tea-derived DPP-Ⅳ inhibitory peptide but also provides a theoretical basis for the high-value utilization of tea proteins and a technical reference for the development of functional foods.

[0019] The present invention discloses the following technical effects: This invention identifies novel DPP-IV inhibitory peptides APWLEPLR, LAFDLIR, and EAAWGLAR through virtual enzymatic hydrolysis of tea proteins combined with computer-aided screening. Studies show that APWLEPLR exhibits significant DPP-IV inhibitory activity in vitro and shows no significant cytotoxicity in Caco-2 cells, demonstrating its good safety profile. This invention successfully screened and validated the bioactive peptides APWLEPLR, LAFDLIR, and EAAWGLAR with good DPP-IV inhibitory activity. This research not only provides a scientific basis for the development of novel natural DPP-IV inhibitory peptides but also offers new ideas for the development of functional tea products. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Analysis of potential bioactive peptides in tea proteins; where A is the phylogenetic tree of tea protein sequences from the UniProt database, B is the amino acid composition of tea proteins, and C is the frequency of known bioactive peptides in tea proteins. Figure 2 The structure-activity relationship of the tea-derived DPP-Ⅳ inhibitory peptide is shown; where A is the peptide length, B is the molecular weight distribution, C is the N-terminal amino acid distribution, and D is the virtual screening flowchart. Figure 3 The DPP-IV inhibitory activity of the synthetic peptides is represented by A, where A represents the DPP-IV inhibitory activity of three peptides (APWLEPLR, EAAWGLAR, LAFDLIR) at the same concentration, and B represents the DPP-IV inhibitory activity of APWLEPLR at different concentrations. Figure 4 The enzyme inhibition mechanism of APWLEPLR and DPP-Ⅳ was analyzed; where A is the result of the inhibition reversibility judgment, B is the Lineweaver-Burk plot of different concentrations of APWLEPLR and DPP-Ⅳ, C is the second-order curve of the slope of the Lineweaver-Burk plot against different concentrations of APWLEPLR, and D is the second-order curve of the intercept of the Lineweaver-Burk plot against different concentrations of APWLEPLR. Figure 5Spectral analysis of the interaction between APWLEPLR and DPP-Ⅳ at different concentrations; where A is the UV absorption spectrum of the interaction between APWLEPLR and DPP-Ⅳ at different concentrations, B is the fluorescence spectrum of the interaction between APWLEPLR and DPP-Ⅳ at different concentrations (298.15 K), C is the fluorescence spectrum of the interaction between APWLEPLR and DPP-Ⅳ at different concentrations (304.15 K), D is the fluorescence spectrum of the interaction between APWLEPLR and DPP-Ⅳ at different concentrations (310.15 K), E is the Stern-Volmer curve of APWLEPLR and DPP-Ⅳ, and F is the double logarithmic equation of APWLEPLR and DPP-Ⅳ. Figure 6 The images are atomic force microscopy (AFM) images of DPP-Ⅳ and APWLEPLR and DPP-Ⅳ; where A is a 2D AFM image of DPP-Ⅳ, B is a 3D AFM image of DPP-Ⅳ, C is a 2D AFM image of APWLEPLR and DPP-Ⅳ, and D is a 3D AFM image of APWLEPLR and DPP-Ⅳ. Figure 7 The diagram shows the molecular docking results of APWLEPLR and DPP-Ⅳ; where A is the three-dimensional binding conformation diagram and B is the two-dimensional interaction diagram (where green dashed lines represent hydrogen bond interactions, purple circles represent hydrophobic / van der Waals interactions, and red circles represent salt bridge / ionic bond interactions). Figure 8 The results are shown in the molecular dynamics simulations of APWLEPLR and DPP-Ⅳ. Among them, A is the RMSD plot of APWLEPLR and DPP-Ⅳ, B is the RMSF plot of APWLEPLR and DPP-Ⅳ, C is the Rg plot of APWLEPLR and DPP-Ⅳ, D is the SASA plot of APWLEPLR and DPP-Ⅳ, E is the Hbond plot of APWLEPLR and DPP-Ⅳ, F is the residue binding energy contribution of DPP-Ⅳ, G is the residue binding energy contribution of APWLEPLR, H is the 3D landscape plot of the free energy of the interaction between APWLEPLR and DPP-Ⅳ, and I is the 2D landscape plot of the free energy of the interaction between APWLEPLR and DPP-Ⅳ. Figure 9 Effects of sitagliptin and APWLEPLR on cell viability and their effect on the in situ IC50 of DPP-IV 50 Where A represents the effect of sitagliptin on cell viability, B represents the effect of APWLEPLR on cell viability, and C represents the in situ IC50 of sitagliptin on DPP-IV. 50 D is the in-situ IC of APWLEPLR for DPP-Ⅳ. 50 ; Figure 10The in vitro simulated gastrointestinal digestion stability of APWLEPLR is shown in Figure A, where A represents the changes in the inhibitory activity of APWLEPLR against DPP-IV after simulating the stomach, intestine, and gastrointestinal digestion, and B represents the dynamic changes in the inhibitory activity of APWLEPLR against DPP-IV during the simulated gastrointestinal digestion process. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1 All experiments were repeated three times under the same conditions, and results are expressed as mean ± standard deviation (SD). All data plots were created and significance analyzed using GraphPad Prism software. p <0.05 is considered statistically significant.

[0028] 1. Materials and Reagents Dipeptidyl peptidase DPP-IV (1346 U / L) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; glycine-proline-p-nitroaniline hydrochloride (Gly-Pro-PNA), sitagliptin, and Tris-HCl buffer were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; sodium acetate, PBS buffer, and CCK-8 assay kit were purchased from Beijing Lanjieke Technology Co., Ltd.; DMEM high-glucose medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; double antibiotics and fetal bovine serum were purchased from Beyotime Biotechnology Co., Ltd.; all other reagents were of analytical grade.

[0029] 2. Homology analysis of tea proteins and prediction of potential bioactive peptides Tea( Camellia sinensis (L.) Kuntze The protein sequence data were obtained from the UniProt database (https: / / www.uniprot.org / ). The specific search and screening process is as follows: First, protein sequences were obtained from the reviewed (Swiss-Prot) category to obtain high-confidence protein sequences. Then, relevant sequences were supplemented from the unreviewed (TrEMBL) section using keywords. Sequence alignment analysis was performed using the Align multiple sequence alignment tool in UniProt. Finally, tea protein sequences with a similarity of less than 90% were selected for subsequent analysis.

[0030] Potential bioactive peptides in tea protein sequences were predicted using the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php). The protein sequences were submitted using the built-in "Bioactive Peptides" tool, and their bioactivity potential was assessed using the "Calculation" function. The formula for calculating the frequency (A) of bioactive fragments is as follows: A = a × N (1); Where 'a' represents the number of fragments with specific activity, and 'N' represents the total number of amino acid residues in the protein.

[0031] Results: Using keywords in UniProtKB Camellia sinensis var. sinensis (China tea)A search was conducted, yielding two high-confidence protein sequences from the approved (Swiss-Prot) category, and 20 related sequences were added from the unapproved (TrEMBL) section. After manual verification to remove redundant and incomplete sequences, a high-quality, non-redundant dataset of 16 protein sequences was finally obtained. The UniProt accession numbers, names, and amino acid lengths of the selected proteins are shown in Table 1. Since highly similar protein sequences often originate from the same parent protein and exhibit extensive sequence overlap, containing a large number of identical amino acid fragments, a large number of identical peptides will be generated during enzymatic digestion. Therefore, the built-in Align function of UniProt was used to perform multi-sequence homology comparison analysis on these 16 tea protein sequences. Figure 1 As shown in Figure A, apart from A0A4S4ESS1 and A0A4S4DBB3, which are highly similar homologous sequences (74.69%), the homology scores of the remaining tea protein sequences are all below 30. Therefore, 15 protein sequences with low similarity were finally selected for subsequent bioactive peptide prediction analysis.

[0032] The type, quantity, and sequence of amino acids determine the function of bioactive peptides. Previous studies have shown that peptides rich in hydrophobic amino acids such as alanine (Ala), leucine (Leu), proline (Pro), and glycine (Gly) exhibit good DPP-IV inhibitory activity, α-glucosidase inhibitory activity, antioxidant activity, and uric acid-lowering activity. Therefore, analyzing the amino acid distribution in tea proteins and identifying potential bioactive fragments is of great significance for the targeted discovery of functional peptides. Figure 1 As shown in Figure B, tea protein is rich in hydrophobic amino acids such as Ala, Gly, Leu, and Val. This indicates that tea protein can serve as a high-quality raw material for the preparation of bioactive peptides.

[0033] To further clarify which bioactive peptide is most suitable for the development of tea proteins, the frequency (A value) of various active fragments in the tea protein sequences was analyzed using the BIOPEP-UWM database. A higher A value indicates a richer content of this type of bioactive peptide fragment in the protein, suggesting that the protein is most suitable for developing this type of bioactive peptide. The results showed that the DPP-Ⅳ inhibitory active fragment had the highest frequency (A value) among all tea protein sequences, ranging from 0.5784 to 0.7104. Figure 1 The results showed that tea proteins were most suitable for developing DPP-IV inhibitory peptides, followed by α-glucosidase and α-amylase inhibitory peptides.

[0034] Table 1. Tea protein sequences from the UniProt database 3. Virtual enzymatic hydrolysis of tea protein, structure-activity analysis and screening of DPP-IV inhibitory peptides 3.1 Virtual Enzymatic Hydrolysis The tea protein was virtually hydrolyzed using the online tool PeptideCutter (https: / / web.expasy.org / peptide_cutter / ), with trypsin selected to simulate the gastrointestinal digestion process and repetitive peptides removed.

[0035] 3.2 Peptide Screening Bioactive peptides were screened using the PeptideRanker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) and the BIOPEP database. Non-toxic and non-allergenic peptides were selected for further research using ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html / ) and AllerTOP v.2.0 (https: / / www.ddg-pharmfac.net / AllerTOP / ).

[0036] Results: The specific cleavage sites of proteases determine the peptide length and sequence structure of their hydrolysates, thus affecting the bioactivity of the hydrolysates. Therefore, it is crucial to select appropriate proteases for enzymatic hydrolysis. Trypsin can specifically cleave the Arg and Lys nucleotides at the C-terminus of peptides, thereby releasing peptides with hydrophobic amino acids at the N-terminus. Therefore, to rapidly obtain the amino acid sequence of the tea-derived DPP-Ⅳ inhibitory peptide, this invention utilizes the online enzymatic hydrolysis tool PeptideCutter to virtually hydrolyze these 15 tea proteins using trypsin. After removing single and repetitive peptides, 717 peptides were ultimately obtained for subsequent screening.

[0037] The DPP-IV inhibitory activity of peptides is closely related to their amino acid sequence, composition, and physicochemical properties. Analysis of the 717 peptides obtained from the virtual enzymatic digestion revealed that these peptides are mainly composed of 2-10 amino acid residues. Figure 2 (A). In terms of molecular weight, most of the obtained bioactive peptides are <1000 Da ( Figure 2 (B). Studies have shown that low molecular weight peptides bind more strongly to the DPP-Ⅳ active site and generally exhibit higher inhibitory activity. Regarding primary structure, such as... Figure 2 As shown in Figure C, most peptides have hydrophobic amino acids at their N-terminus (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val), with the largest number of peptides having Leu at the N-terminus. This is consistent with the structural characteristics of peptides with high DPP-IV inhibitory activity, meaning that peptides with hydrophobic amino acids at the N-terminus can enhance their DPP-IV inhibitory activity by interacting with the catalytic site of the enzyme through hydrophobic interactions.

[0038] To screen for the peptide with the strongest DPP-IV inhibitory activity from 717 peptides, this invention utilizes a computer-aided method for stepwise screening. The specific process is detailed below. Figure 2 First, 348 peptides were initially screened based on chain length of 2-8 kDa and molecular weight <1000 Da. Second, based on previous research (the second amino acid at the N-terminus being Pro or Ala is a typical structural feature of peptides with high DPP-IV inhibitory activity), 54 potential active peptides were further screened. Since good water solubility is crucial for the full absorption of DPP-IV inhibitory peptides to exert their hypoglycemic effect, and the hydrophobicity of the peptide also has a certain impact on its DPP-IV inhibitory activity, excessive hydrophobicity will significantly reduce its water solubility. Therefore, this invention further used the proportion of hydrophobic residues (>65%) as a screening criterion, based on good water solubility, and obtained 16 peptides that met the requirements. Finally, using online tools such as PeptideRanker, ToxinPred, and AllergenCatPro, peptides with a Peptide Ranker activity score >0.6 and that are non-toxic and non-sensitizing were further screened from the 16 peptides, ultimately yielding three potential DPP-IV inhibitory peptides: APWLEPLR (SEQ ID NO.1), LAFDLIR (SEQ ID NO.2), and EAAWGLAR (SEQ ID NO.3).

[0039] 4. Solid-phase synthesis of peptides The selected peptide sequences were synthesized by Anhui Guoping Pharmaceutical Co., Ltd., and the purity of the synthesized peptides was >95%.

[0040] 5. DPP-Ⅳ inhibitory activity assay After thoroughly mixing the sample solution (200 μL) with the DPP-Ⅳ enzyme solution (100 μL), incubate at 37°C for 10 min. Then add Gly-Pro-PNA solution (50 μL) and incubate at 37°C for 60 min. Finally, add sodium acetate solution (200 μL) to terminate the reaction, and measure the absorbance at 405 nm. The DPP-Ⅳ inhibition rate is calculated using the following formula: (2); Where A1 represents the sample group, A2 represents the sample blank group, A3 represents the control group, and A4 represents the control blank group. The half-maximal inhibitory concentration (IC50) is... 50 The value can be obtained in GraphPad Prism software based on the curve fitting equation of the DPP-Ⅳ inhibition rate as a function of different sample concentrations.

[0041] Results: To verify the in vitro DPP-IV inhibitory activity of the three peptides obtained in section 3.2, the above peptides (purity > 95%) were synthesized using solid-phase synthesis technology, and this indicator was then detected. Figure 3 As shown in Figure A, at the same concentration of 1000 μM, all three peptides exhibited certain DPP-IV inhibitory activity, with inhibition rates all exceeding 20%. Among them, APWLEPLR showed the strongest inhibitory ability, with a DPP-IV inhibition rate of 73.97 ± 1.63%, significantly higher than the other peptides. p <0.05). This may be because the peptide conforms to the two major structural features of highly efficient DPP-IV repressive peptides, namely, the N-terminus is a hydrophobic amino acid and the second position of the N-terminus is Pro.

[0042] Further analysis was conducted on the effect of concentration on the inhibitory activity of APWLEPLR against DPP-Ⅳ. Within a certain concentration range, the inhibitory ability of APWLEPLR against DPP-Ⅳ gradually increased with increasing concentration. Figure 3 The concentration-dependent characteristic (IC50) was indicated by the result of nonlinear fitting using GraphPad software. 50 The concentration was 546.6 ± 4.92 μM. This indicates that the APWLEPLR screened in this invention has good DPP-IV inhibitory ability and can be further studied as a novel DPP-IV inhibitory peptide.

[0043] 6. Analysis of the inhibitory mechanism of APWLEPLR on DPP-Ⅳ 6.1 Reversibility determination With the substrate Gly-Pro-PNA concentration fixed at 1 mM, the enzymatic reaction rates of different peptide concentrations (0, 500, 750, 1000, and 1250 μM) at different DPP-Ⅳ concentrations were determined. The experimental methods were standard and will not be described in detail here.

[0044] Results: Enzyme inhibitors are classified into reversible and irreversible inhibitors. Reversible inhibitors bind to enzymes via non-covalent bonds, while irreversible inhibitors bind to enzymes via covalent bonds; both types cause enzyme inactivation. Figure 4 As shown in Figure A, the enzyme reaction rate increases linearly with increasing DPP-Ⅳ concentration. Regardless of the presence of APWLEPLR, all fitted curves intersect at the origin, and the slope of the curves is negatively correlated with the APWLEPLR concentration, indicating that the inhibitory effect of this peptide on DPP-Ⅳ is reversible. Therefore, it can be inferred that APWLEPLR reversibly binds to DPP-Ⅳ through non-covalent interactions (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions), thereby reducing the enzyme's catalytic activity and ultimately slowing down the reaction rate.

[0045] 6.2 Enzyme inhibition kinetics assay With the DPP-Ⅳ concentration fixed at 0.05 U / mL, the initial rate of enzyme reaction was determined for different concentrations of peptides (0, 400, 600, 800, 1000 μM) at different concentrations of Gly-Pro-PNA. The experimental methods were standard and will not be described in detail here.

[0046] The competitive inhibition formula is as follows: (3); The formula for mixed-type suppression is as follows: (4); in, V This represents the initial reaction rate; V max This represents the maximum initial reaction rate; i [S] represents the inhibitor concentration; [S] represents the substrate concentration. K m It is the Michaelis constant; K ic This is the competitive inhibition dissociation constant; K iu It is a non-competitive inhibition dissociation constant.

[0047] Results: In reversible inhibition, based on the interactions between the enzyme, substrate, and inhibitor, it can be classified into competitive, non-competitive, and mixed inhibition. Figure 4 As shown in Figure B, the straight lines in the Lineweaver-Burk double reciprocal plot of the peptide APWLEPLR inhibiting DPP-Ⅳ intersect at a single point in the third quadrant. Furthermore, with increasing peptide concentration, the ordinate of the double reciprocal plot gradually increases, while the abscissa gradually decreases. Additionally, as shown in Table 2, V... max K continuously decreases m It continuously decreases, and the R-squared value of all fitted lines is... 2 All values ​​were greater than 0.95, indicating a good fit to the data and consistent with the characteristics of mixed inhibition (competitive + non-competitive). These results suggest that the peptide APWLEPLR is a mixed DPP-IV inhibitor, meaning that APWLEPLR can both competitively bind to DPP-IV and bind to the DPP-IV-substrate complex, ultimately weakening the catalytic activity of DPP-IV.

[0048] Based on the above results, the inhibition kinetic parameters of APWLEPLR on DPP-Ⅳ were further analyzed. For example... Figure 4 As shown in Figures C and D, the quadratic curves of APWLEPLR concentration versus slope (C) and intercept (D) exhibit a good linear relationship, indicating that APWLEPLR interacts with DPP-IV enzyme through a class of binding sites. Furthermore, Table 2 shows the competitive dissociation constant K for the interaction between APWLEPLR and DPP-IV. ic (294 μM) is higher than the non-competitive dissociation constant K. iu(133 μM) indicates that APWLEPLR has a higher affinity for the enzyme-substrate complex than for the free enzyme. Therefore, the novel peptide APWLEPLR screened in this invention is a DPP-Ⅳ mixed-type inhibitory peptide, which is more likely to bind to the enzyme-substrate complex.

[0049] Table 2 Enzyme inhibition kinetic parameters and inhibition types of APWLEPLR and DPP-Ⅳ 6.3 Ultraviolet Spectroscopy Scan After mixing DPP-Ⅳ solution (0.01 U / mL) with peptide solutions of different concentrations (0, 50, 75, 100, 125, 150 μM), the mixture was incubated at 37℃ for 10 min before measurement. The scanning wavelength was set to 190-300 nm with an interval of 1 nm.

[0050] Results: Conformational changes during protein-ligand interactions can be observed using ultraviolet spectroscopy. For example... Figure 5 As shown in Figure A, the UV absorption peak of DPP-Ⅳ is located at 210-220 nm (π-π* transition of the peptide bond). When interacting with different concentrations of APWLEPLR, the absorption peak intensity significantly increases, and is directly proportional to the APWLEPLR concentration, while the peak position exhibits a red shift. Furthermore, the absorption peak appearing at 280 nm after adding the peptide is a characteristic peak of aromatic amino acids in APWLEPLR, and the intensity of the absorption peak gradually increases with increasing peptide concentration. Overall, no new absorption peak appears after the interaction of APWLEPLR and DPP-Ⅳ, indicating that APWLEPLR and DPP-Ⅳ mainly interact in a non-covalent manner, causing a change in enzyme conformation. These results further support the conclusion that APWLEPLR is a reversible DPP-Ⅳ inhibitory peptide.

[0051] 6.4 Fluorescence quenching analysis, thermodynamic parameters and interaction force type analysis Different concentrations of peptide (0, 0.001, 0.005, 0.01, 0.05, 0.1 μM) were mixed with DPP-Ⅳ, and the fluorescence intensity was measured at 298.15 K, 304.15 K, and 310.15 K, respectively. The excitation wavelength was set to 280 nm, and the emission spectral range was 350–400 nm. The excitation and emission slit widths were both set to 5 nm.

[0052] Fluorescence quenching type was analyzed using the Stern-Volmer equation: (5); (6); in, F0 and F The values ​​represent the maximum fluorescence intensity without and with the addition of an inhibitor, respectively. K q The rate constant during bimolecular quenching; τ 0 The average fluorescence lifetime of free macromolecules (10 for DPP-IV) -8 s); [Q] The concentration of the inhibitor; K sv It is the fluorescence quenching constant; K a The binding constant; n This represents the number of binding sites.

[0053] The thermodynamic parameters are determined using the Vant Hoff equation, as shown in the following formula: (7); (8); in, T For temperature; R The gas constant is 8.314 J / (mol·K); ΔH For enthalpy change; ΔS It is an entropy change; ΔG For Gibbs free energy.

[0054] Results: Due to the presence of aromatic amino acid residues (such as tryptophan and tyrosine) in the DPP-Ⅳ molecule, the tryptophan (Trp) residue is the most sensitive to changes in the surrounding microenvironment, producing endogenous fluorescence upon excitation at 280 nm. When exogenous substances such as inhibitors interact with DPP-Ⅳ, fluorescence quenching occurs. This phenomenon not only reflects the conformational change of DPP-Ⅳ after interaction but also helps determine the mechanism of action between the inhibitor and the enzyme. Therefore, fluorescence spectroscopy was used to analyze the interaction between APWLEPLR and DPP-Ⅳ.

[0055] like Figure 5 As shown in Figure BD, when the excitation wavelength is 280 nm, the maximum emission characteristic absorption peaks of DPP-Ⅳ at 298.15 K, 304.15 K, and 310.15 K are all located at 325 nm. After adding different concentrations of APWLEPLR, the fluorescence intensity of DPP-Ⅳ gradually decreased, and this decrease was proportional to the concentration. However, no blue shift or red shift was observed, indicating that the peptide APWLEPLR can directly interact with DPP-Ⅳ to form a peptidase complex, causing quenching of its intrinsic fluorescence. Fluorescence quenching types include static quenching, dynamic quenching, and a combination of dynamic and static quenching, which can be determined based on the quenching rate constant K at different temperatures. q and quenching constant Ksv Changes were used to determine the type of quenching. To further explore the specific mechanism of fluorescence quenching, the relevant fluorescence quenching parameters at different temperatures (298.15 K, 304.15 K, 310.15 K) were analyzed using the Stern-Volmer equation. Figure 5 As shown in Figure E, the Stern-Volmer plot exhibits a good linear relationship at different temperatures, indicating that the quenching process may be a single static or dynamic quenching mechanism. Table 3 shows the quenching constant K... sv The overall trend is that it increases with increasing temperature, and the quenching rate constant K q The value is much smaller than the maximum scattering collisional quenching rate constant of biological macromolecules (2.0 × 10⁻⁶). 10 (M / s), which indicates that the quenching mechanism of APWLEPLR for DPP-Ⅳ is a dynamic quenching mechanism.

[0056] Thermodynamic parameters of the interaction between APWLEPLR and DPP-Ⅳ were further calculated using the van't Hoff and Srern-Volmer equations to clarify the molar ratio of their interaction and the driving force behind it. Table 3 shows that as temperature increases, the combination constant K... a Although it decreased slightly at 304.15 K, it increased significantly at 310.15 K, indicating that the higher the temperature, the stronger the binding tendency of APWLEPLR and DPP-Ⅳ, that is, the higher the stability of the formed complex.

[0057] The interaction forces between ligands and protein molecules are generally intermolecular forces, including van der Waals forces, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. According to the theory of Ross and Subramanian, when ΔH>0 and ΔS>0, the interaction is mainly driven by hydrophobic interactions; when ΔH<0 and ΔS<0, the interaction is mainly dominated by hydrogen bonds and van der Waals forces; when ΔH≈0 and ΔS>0, the interaction is essentially an electrostatic interaction; when ΔH<0 and ΔS>0, the interaction is dominated by hydrophobic interactions, accompanied by hydrogen bonds or van der Waals forces. Table 3 shows that the enthalpy change ΔH<0 and entropy change ΔS>0 of the binding of the peptide APWLEPLR to DPP-Ⅳ indicate that the main forces involved in their binding are hydrogen bonds and hydrophobic interactions, and that the binding is accompanied by an exothermic and entropy-increasing process. This result further confirms the determination of the inhibition type of APWLEPLR on DPP-Ⅳ and the UV scanning results of their interaction in this invention. Figure 5 (F). In addition, ΔG is negative at all temperatures, and its absolute value increases continuously with increasing temperature, indicating that the interaction between APWLEPLR and DPP-Ⅳ is spontaneous, and the higher the temperature, the stronger the tendency for them to spontaneously combine.

[0058] Table 3. Fluorescence quenching parameters and thermodynamic parameters of APWLEPLR-DPP-Ⅳ interaction at different temperatures. 6.5 Atomic Force Microscopy Analysis The peptide was mixed with DPP-Ⅳ solution and then uniformly added to mica sheets. The mixture was dried completely at room temperature for 24 h and observed using an atomic force microscope.

[0059] Results: Atomic force microscopy (AFM), as a high-resolution nanoscale imaging technique, can detect the surface morphology and mechanical properties of samples at the atomic scale and has been widely used in the visualization and functionalization analysis of biomacromolecules such as proteins. To further verify the binding mode of APWLEPLR and DPP-Ⅳ derived earlier, the morphological changes of DPP-Ⅳ before and after binding with APWLEPLR were observed using AFM.

[0060] Figure 6 These are 2D and 3D topographic images before and after the integration of DPP-Ⅳ and APWLEPLR. Among them, such as... Figure 6 As shown in Figure A, before binding to APWLEPLR, DPP-Ⅳ exhibits small, scattered, and relatively uniformly distributed bright spots, indicating that the enzyme conformation is stable at this stage. Its three-dimensional structure diagram (…) Figure 6 (B) exhibits relatively uniform sharp protrusions and a high surface roughness. When APWLEPLR interacts with DPP-Ⅳ, the morphology of the complex changes significantly. Figure 6 (CD). It can be observed that, compared with DPP-Ⅳ alone, the overall distribution uniformity of bright spots is reduced, and obvious aggregation and fusion are observed. Figure 6 (C) 3D structure display ( Figure 6 In the mica substrate, the enzyme molecules (DPP-IV) exhibit a distinctly swollen state, displaying rounded protrusions. These results indicate that the binding of APWLEPLR to DPP-IV affects the microenvironment surrounding the enzyme, disrupting its original stable state. This is consistent with previous analyses of the inhibitory kinetics and thermodynamic parameters of the two, further confirming the interaction between APWLEPLR and DPP-IV, forming a stable complex.

[0061] 7. Analysis of the inhibitory mechanism of APWLEPLR on DPP-Ⅳ at the molecular level 7.1 Molecular docking analysis As macromolecular ligands, peptides were used to predict their structures using AlphaFold. The APWLEPLR 3D structures in PDB format were then imported into a peptide database for energy minimization. Energy minimization is a computational chemistry method used to find the lowest-energy configuration of a molecule by adjusting the positions of atoms. By iteratively optimizing the distances and angles between these atoms, the energy of the entire molecular system is brought to its lowest possible value. This aims to simulate the shape and configuration that molecules naturally adopt without external forces. The AlphaFold-predicted 3D structure file of the protein DPP4_HUMAN (PDB ID: 5J3J) was imported into MOE software for pre-docking processing, including structural correction, hydrogen atom addition, and optimization of the protein side chains. Molecular docking was performed using the Dock module in MOEv2022.02. The process of finding the optimal matching mode based on the mutual recognition of geometric and energy matching was used. Binding conformations were scored and ranked, and suitable peptide-protein binding conformations were selected for interaction analysis.

[0062] Protein structures were visualized using a Cartoon model, while peptides and interacting amino acid residues were represented using a stick model. Red dashed lines indicate hydrogen bonds. The 2D interaction diagrams of the protein-peptide complexes were analyzed using the Ligand Interactions module in MOE v2022.02, and a Ligand Interactions Report was generated.

[0063] Results: Molecular docking was used to further analyze the binding energy, binding site, and interaction between the peptide APWLEPLR and DPP-Ⅳ. The free binding energy of APWLEPLR and DPP-Ⅳ was -10.71 kcal / mol. The lower the binding energy between the peptide and the active site of DPP-Ⅳ, the higher the binding affinity. These results indicate that APWLEPLR has a stronger binding affinity to the active site of DPP-Ⅳ.

[0064] Studies have shown that the active pocket of DPP-Ⅳ consists of three key sites: S1, S2, and S3. Site S1 is composed of hydrophobic residues such as Tyr547, Ser630, and Val711. The S2 pocket is a hollow region containing Glu205, Glu206, and Tyr662; S3 is composed of Ser209, Arg358, and Phe357. Figure 7As shown in Figure A, the peptide APWLEPLR binds to the active pocket of DPP-Ⅳ and forms a stable complex. This peptide not only forms hydrogen bonds and ionic interactions with key residues in the S2 pocket (Glu205, Glu206), but also interacts with Arg358 and Tyr547 in the S3 pocket. Furthermore, the nitrogen atom at the N-terminus of the peptide forms a strong ionic bond with Glu361 at the bottom of the S2 pocket, with a bond length of 2.91 Å, providing crucial electrostatic anchoring for the complex. APWLEPLR also forms a hydrophobic interaction with Arg125. Figure 7 The presence of B indicates that the peptide effectively occupies the S1′ subsite, thereby blocking substrate entry. This further explains why APWLEPLR is a reversible hybrid DPP-IV inhibitory peptide (capable of binding to both the active site of DPP-IV and the enzyme-substrate complex). These results not only support the conclusion from UV spectroscopy that APWLEPLR interacts with DPP-IV in a non-covalent manner, but also align with thermodynamic analysis showing that the interaction forces are primarily hydrogen bonds and hydrophobic interactions. In conclusion, the predicted interaction mode is a key reason for the strong DPP-IV inhibitory activity of APWLEPLR.

[0065] 7.2 Molecular Dynamics Simulation Analysis To investigate the binding stability, dynamic conformational changes, and interaction characteristics of APWLEPLR within the active site of DPP-Ⅳ, this invention employs molecular dynamics simulations for analysis. The binding affinity and stability between AR-8 and DPP-Ⅳ were comprehensively evaluated by analyzing seven key parameters: root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), number of hydrogen bonds (Hbonds) in the protein-ligand complex, residue interaction energy between APWLEPLR and DPP-Ⅳ, and Gibbs free energy landscape. The experimental methods are conventional and will not be detailed here.

[0066] Results: Root Mean Square Deviation (RMSD) is a key indicator for measuring protein conformational changes and structural stability. A higher RMSD indicates greater stability of the complex during kinetic simulations. Figure 8As shown in Figure A, in the initial stage of the simulation (0-10 ns), the RMSD value rises rapidly, indicating that the APWLEPLR-DPP-Ⅳ complex underwent significant conformational rearrangement to adjust to a more stable binding mode. In the middle stage (10-40 ns), the rate of increase in RMSD slows down, indicating that the complex structure is gradually stabilizing. In the later stage (40-100 ns), the RMSD value tends to stabilize, with overall fluctuations remaining within 0.05 nm, proving that the complex has reached dynamic equilibrium. Therefore, the APWLEPLR-DPP-Ⅳ complex maintains a relatively stable conformation after 40 ns, indicating that the complex has good structural stability during the simulation.

[0067] Root mean square fluctuation (RMSF) can assess the flexibility of amino acid residues in a complex during molecular dynamics simulations. A higher RMSF value indicates greater fluctuation of the residue relative to its average position during the simulation, meaning the region is more flexible; conversely, a lower RMSF value indicates greater stability. The vast majority of residues in the complex exhibited low RMSF values ​​(approximately 0.1 nm–0.2 nm), indicating overall structural stability of the complex during simulations. However, Figure 8 The B-value shows obvious spikes near residues such as Arg200, Asn250, and Arg350, indicating that the residues in these regions have high local flexibility, further illustrating that these key residues are involved in the binding process of APWLEPLR and DPP-Ⅳ.

[0068] The radius of gyration (Rg) describes the compactness of a protein structure. A high Rg value indicates a more open protein conformation, while a low Rg value indicates a more compact protein. Figure 8 As shown in Figure C, the Rg values ​​of APWLEPLR and DPP-Ⅳ are low throughout the simulation, and the Rg curve fluctuates little overall, indicating that DPP-Ⅳ maintains a stable conformation after binding with APWLEPLR, and that the APWLEPLR-DPP-Ⅳ complex exhibits high compactness and stability. This result is consistent with the aforementioned RMSD and RMSF analyses, jointly demonstrating that a stable complex is formed between APWLEPLR and DPP-Ⅳ.

[0069] Solvent-accessible surface area (SASA) is used to assess the degree to which a protein surface is exposed to solvent molecules, and is a key indicator for evaluating the compactness of molecular complexes. A larger SASA value indicates a high degree of protein surface exposure and a looser structure, while a smaller SASA value indicates a more compact structure. Figure 8As can be seen from Figure D, although the SASA curve exhibits continuous fluctuations throughout the molecular dynamics simulation, the overall average value remains relatively stable without any obvious upward or downward trend. This indicates that the surface exposure state of the complex is in dynamic equilibrium, and the overall conformation remains highly stable and relatively compact.

[0070] Hydrogen bonds (Hbonds) are the main intermolecular forces maintaining the structural stability of protein-ligand complexes. Their number and duration dynamically reflect the interaction strength and stability at the binding interface. Throughout the kinetic simulation, the number of hydrogen bonds between APWLEPLR and DPP-Ⅳ was mainly concentrated around 10. Figure 8 The absence of prolonged and sustained hydrogen bond breakage indicates a stable and continuous hydrogen bond interaction between the two molecules. This result corroborates the conclusions of the present invention regarding ultraviolet scanning, inhibition kinetics, thermodynamics, and molecular docking, collectively demonstrating that hydrogen bonding is the primary driving force for the formation of a stable complex between APWLEPLR and DPP-Ⅳ.

[0071] Interaction energy decomposition analysis can clarify the energy contribution of key amino acids in the protein-peptide interaction process, where negative values ​​indicate favorable binding interactions, and lower values ​​indicate stronger binding contributions. For example... Figure 8 As shown in Figure F, Arg358 contributes the highest binding energy (-7.36 kcal / mol) in DPP-Ⅳ, making it the most critical active site. Residues such as Ser630, Phe357, and Glu206 are also important active sites. Figure 8 As shown in G, Arg8 contributes the strongest binding energy (-9.08 kcal / mol) when APWLEPLR binds to DPP-Ⅳ, indicating its crucial role in stabilizing the interaction between APWLEPLR and DPP-Ⅳ. Furthermore, residues such as Pro2 and Leu4 also provide strong binding energies (-3.5 to -5.0 kcal / mol), synergistically ensuring the high affinity and structural stability of the complex as a whole. Although the N-terminal Ala1 generates a positive energy loss of +5.36 kcal / mol, possibly due to unfavorable conformational changes or repulsive interactions, the strong binding energy contributions from the aforementioned residues compensate for this loss, collectively ensuring a stable and tight binding between APWLEPLR and DPP-Ⅳ.

[0072] Gibbs free energy landscapes are multidimensional energy surfaces constructed based on molecular dynamics simulations. They can be used to characterize the stability and dynamic behavior of protein-ligand pairs in different conformational states, with the dark blue area representing lower-energy conformations. For example... Figure 8As shown in Figure HI, a significant dark blue global energy minimum point exists in the figure, at which point the complex reaches its most stable conformation. This indicates that the APWLEPLR / DPP-Ⅳ complex has reached a stable state within 100 ns of the kinetic simulation. This result is consistent with the RMSD and Rg analyses mentioned above, further confirming that the APWLEPLR / DPP-Ⅳ complex has tended to a stable conformation and reached dynamic equilibrium within the simulation time.

[0073] In summary, based on the comprehensive analysis of RMSD, RMSF, Rg, SASA, hydrogen bonding, interaction energy decomposition, and free energy landscape, it is jointly demonstrated that APWLEPLR and DPP-Ⅳ form a compact complex with high binding affinity within a dynamic simulation time of 100 ns, further confirming the high stability of the binding between the two.

[0074] 8. In situ DPP-IV inhibition analysis of APWLEPLR 8.1 Cell Culture Caco-2 cells were cultured in T25 flasks in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies). Cells were incubated at 37°C and 5% CO2 until the logarithmic growth phase, and then used for subsequent experiments.

[0075] 8.2 Cell viability assay Cell suspension (1×10) 4 Cells were seeded at 100 μL per cell in 96-well plates and cultured for 24 h. The culture medium was then replaced with 100 μL of fresh medium containing different peptide concentrations (100, 200, 400, 800, 1600 μM). After another 24 h of culture, 10 μL of CCK-8 solution was added to each well, and the plates were cultured for another 2 h. The absorbance at 450 nm was then measured. Blank wells contained only medium, while control wells contained both peptide and medium. Cell viability was calculated using the following formula: (9).

[0076] 8.3 In-situ DPP-Ⅳ activity assay Caco-2 cells were seeded in 96-well plates (2 × 10⁻⁶ cells per well). 4Cells were cultured at 100 μL per cell for 24 h. Cells were washed twice with 200 μL PBS to remove the washing buffer. Then, 50 μL of sample solution (100, 200, 400, 600, 800, 1600 μM) was added to each well, and the plate was incubated at 37°C for 10 min. Next, 25 μL of Ly-Pro-pNA (1 mM) was added to each well, and incubation continued for 60 min. Finally, 100 μL of sodium acetate solution was added to terminate the reaction. The absorbance was measured at 405 nm using a microplate reader, and the calculation formula was the same as in equation (2).

[0077] Results: Caco-2 cells can mimic the microenvironment of human intestinal epithelial cells and naturally express human DPP-IV at high levels, which can be used to evaluate the activity of DPP-IV inhibitory peptides at the cellular level. Sitagliptin, as a highly effective DPP-IV inhibitor, regulates blood glucose levels by protecting endogenous glucagon and enhancing its activity, thus exerting a hypoglycemic effect. Therefore, this invention uses sitagliptin as a positive control to evaluate the ability of APWLEPLR to inhibit DPP-IV at the cellular level. To eliminate the interference of active substances on cell viability, the effects of different concentrations of the positive control drug and APWLEPLR on Caco-2 cell viability were analyzed. Figure 9 As shown in Figure A, compared with the control group, different concentrations of sitagliptin had no significant effect on the viability of Caco-2 cells. p >0.05). Figure 9 The results showed that APWLEPLR had no significant effect on cell viability at low concentrations (100-800 μM). p >0.05), while when the APWLEPLR concentration was 1600 μM, the viability of Caco-2 cells was significantly lower than that of the control group ( p Although the concentration was <0.05%, the overall activity was still above 90%, indicating that APWLEPLR had no significant cytotoxicity within the experimental concentration range.

[0078] Further evaluation was conducted on the inhibitory effect of APWLEPLR on DPP-Ⅳ activity in Caco-2 cells. Figure 7 According to the CD, APWLEPLR and the positive control sitagliptin showed a significant concentration-dependent inhibition of intracellular DPP-IV, and their IC50 values ​​were significantly different. 50 The in situ IC50 values ​​were 578.6 ± 2.78 μM and 0.02 ± 3.08 μM, respectively. Compared with DPP-IV inhibitory peptides from other sources, APWLEPLR exhibited stronger inhibitory activity. The in situ IC50 value of APWLEPLR was... 50 Value and its in vitro IC 50The values ​​are quite similar, which may be because it has stronger resistance to the hydrolysis of small intestinal brush border proteins, thus exhibiting better DPP-IV inhibitory activity at the cellular level. These results indicate that APWLEPLR has good stability in the intestinal environment and can be further developed as a functional DPP-IV inhibitory peptide.

[0079] 9. In vitro simulated gastrointestinal digestive stability analysis Bioactive peptides are easily degraded and inactivated by gastrointestinal digestive enzymes; therefore, in vitro simulated gastrointestinal digestion experiments are a core method for assessing their stability. To investigate the digestibility of APWLEPLR, this invention performed mass spectrometry (MS) analysis on its digestion products. The experimental methods were conventional and will not be described in detail here.

[0080] Results: As shown in Table 4, in addition to the detection of three minor degradation fragments (PWLEPLR, WLEPLR, and APWLEPL), a large number of complete APWLEPLR sequences were also detected in the digestion solution; its ion abundance (6.55 × 10⁻⁶) was also observed. 11 Each segment was 1.08 × 10⁻⁶ higher than the others. 2 8.66×10 3 and 5.60×10 5 The result shows that APWLEPLR has excellent resistance to degradation during gastrointestinal digestion.

[0081] Despite its structural stability, the digestion process still has some impact on the bioactivity of APWLEPLR. Figure 10 Compared with the undigested group, the DPP-IV inhibitory activity of APWLEPLR decreased after digestion in the stomach, intestine, and whole gastrointestinal tract. Specifically, the DPP-IV inhibition rate of APWLEPLR significantly decreased to 41.30±0.49% after gastric digestion. p The concentration of α (<0.05) is mainly attributed to conformational changes induced by the strong acid environment. Notably, APWLEPLR maintained high activity during subsequent enteric and whole gastrointestinal digestion. This phenomenon is attributed, on the one hand, to APWLEPLR's resistance to trypsin hydrolysis, and on the other hand, may be related to the partial recovery of its active conformation under the pH conditions during enteric digestion. In summary, APWLEPLR exhibits good digestive stability and has broad application prospects in the development of hypoglycemic functional foods.

[0082] Table 4. Mass spectrometry identification of APWLEPLR peptide fragments after in vitro simulated gastrointestinal digestion. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A tea-derived DPP-Ⅳ inhibitory peptide, characterized in that, The amino acid sequence of the tea-derived DPP-Ⅳ inhibitory peptide is any one of APWLEPLR, LAFDLIR, or EAAWGLAR.

2. A method for screening tea-derived DPP-IV inhibitory peptides as described in claim 1, characterized in that, Includes the following steps: Search the UniProtKB database Camellia sinensis var. sinensis ( Chinese tea High-confidence protein sequences were obtained from the Swiss-Prot category, and relevant sequences were supplemented from the TrEMBL section. The Align multiple sequence alignment tool in UniProt was used for sequence alignment analysis, and finally tea protein sequences with similarity of less than 90% were screened out. The BIOPEP-UWM database was used to predict potential bioactive peptides in the tea protein sequences. The tea protein sequence was virtually enzymatically digested using the PeptideCutter online tool to obtain bioactive peptides. The bioactive peptides were screened using the PeptideRanker and BIOPEP databases to obtain the tea-derived DPP-Ⅳ inhibitory peptide.

3. The use of the tea-derived DPP-Ⅳ inhibitory peptide according to claim 1 in the preparation of DPP-Ⅳ inhibitors.

4. The application according to claim 3, characterized in that, The DPP-IV inhibitor also contains one or more pharmaceutically acceptable carriers.

5. The use of the tea-derived DPP-Ⅳ inhibitory peptide according to claim 1 in the preparation of a diabetes treatment drug.

6. The application according to claim 5, characterized in that, The dosage form of the drug is selected from injections, solutions, emulsions, sprays, granules, powders, capsules, pills, tablets, patches, or sustained-release preparations.

7. A DPP-IV inhibitor, characterized in that, The DPP-Ⅳ inhibitor includes the tea-derived DPP-Ⅳ inhibitory peptide as described in claim 1.

8. The DPP-Ⅳ inhibitor according to claim 7, characterized in that, The DPP-Ⅳ inhibitors also include pharmaceutically acceptable carriers.

9. A diabetes treatment drug, characterized in that, The diabetes treatment drug includes the tea-derived DPP-Ⅳ inhibitory peptide as described in claim 1.

10. The diabetes treatment drug according to claim 9, characterized in that, The drug also contains one or more pharmaceutically acceptable carriers.

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