DPP-IV (dipeptidyl peptidase-IV) inhibition tetrapeptide capable of being orally absorbed as well as derivative and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an orally absorbable DPP-IV (dipeptidyl peptidase-IV) inhibitory tetrapeptide as well as a derivative and application thereof. The amino acid sequence of the tetrapeptide is Ile-Pro-Gly-Pro, the molecular weight of the tetrapeptide is 382.46 Da, the isoelectric point of the tetrapeptide is 5.52, and the tetrapeptide is an acidic tetrapeptide. The structure of the derivative comprises KLIPGPA, KAIPGPA, RLIPGPA, KMIPGPA and RIPGPGF (Region-Induced Pharmacopoeia Growth Factor). The tetrapeptide has good DPP-IV inhibitory activity, can tolerate digestion of stomach and intestine digestive juice after oral administration, is absorbed by intestinal tracts, and has a good hypoglycemic effect. The tetrapeptide derivative can be hydrolyzed by intestinal digestive enzyme to generate IPGP after being orally taken, and has a good hypoglycemic effect after being absorbed by intestinal tracts. The DPP-IV inhibitory peptide IPGP and the derivative thereof can be used for preparing hypoglycemic functional products, and have good application potential in the aspect of maintaining the stability of blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an orally absorbable DPP-IV inhibitory tetrapeptide and its derivatives and their applications. Background Technology

[0002] Diabetes mellitus is a metabolic disease with a high incidence rate and significant harm. It has become the third leading chronic non-communicable disease threatening human health and life, after malignant tumors and cardiovascular diseases. Among diabetic patients, 95% have type 2 diabetes mellitus (T2DM). T2DM is caused by partial damage to pancreatic β-cells, leading to relative insulin insufficiency or insulin resistance. Incretins are a group of polypeptide hormones in the intestine, playing a crucial role in maintaining glucose homeostasis. Two typical incretins are glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). When glucose is ingested, GLP-1 and GIP can delay gastric emptying, enhance satiety, inhibit glucagon release, promote pancreatic β-cell proliferation and differentiation to secrete insulin, and lower hyperglycemia. However, GLP-1 and GIP are rapidly cleaved and inactivated by dipeptidyl peptidase-IV (DPP-IV) in the body after release, with a very short half-life. Therefore, reducing DPP-IV secretion or inhibiting its activity is considered an important strategy for treating T2DM.

[0003] Oral administration is considered the optimal and most convenient method of drug delivery due to higher patient compliance, lower immunogenicity risk, and lower production costs. However, resistance to gastrointestinal digestion and effective transintestinal transport are two key limiting factors for the function of traditionally delivered bioactive peptides. The rat everted intestinal sac model (ERGSM) is an effective model for studying the digestion and absorption of nutrients or drugs in the intestine, and for exploring the role of intestinal enzymes in transintestinal transport. It has the advantages of being simple to operate, low in cost, and allows for rapid screening to obtain bioactive peptides with good absorption properties.

[0004] Studies have shown that some bioactive peptides derived from food proteins, obtained through enzymatic hydrolysis or microbial fermentation, can exhibit significant DPP-IV inhibitory activity, such as quinoa protein-derived peptide VAYPL, clam peptide LTWR, camel milk peptide LPAAP, salmon peptide LDKVFR, bluefin tuna peptide GPSGGGYDV, and casein peptide VPYPQ. Analysis of the structure-activity relationship of these peptides shows that peptides with a proline (Pro) residue at the second N-terminal site generally exhibit stronger DPP-IV inhibitory activity. Chinese patent CN120424164A, published on August 5, 2025, discloses a polypeptide with DPP-IV inhibitory activity and its applications. This invention obtained pure peptides APFP and MPFP through solid-phase synthesis, and measured their DPP-IV inhibition IC50 values ​​to be 153.08 μM and 296.50 μM, respectively, and their DPP-IV inhibitory activity was stronger than that of certain fragments in their sequences. The peptides APFP and MPFP discovered in this invention have strong DPP-IV inhibitory activity and can be used in the preparation of hypoglycemic drugs or foods, showing broad application prospects in industrial applications.

[0005] Sea cucumber is a high-protein, low-fat, cholesterol-free food with significant medicinal and health benefits. The protein content of the dried sea cucumber body wall can reach approximately 90%, and it is rich in a variety of amino acids. In particular, its body wall protein contains a relatively high amount of proline, which gives sea cucumber peptides prepared from it the potential for good DPP-IV inhibitory activity. Summary of the Invention

[0006] The purpose of this invention is to provide: an orally absorbable DPP-IV inhibitory tetrapeptide and its derivatives, which can help in the development of hypoglycemic products, and solve the technical problems of low oral hypoglycemic activity of traditional DPP-IV inhibitory peptides or combinations thereof.

[0007] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0008] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0009] The definition of the standard chemical term can be found in the reference "Research, Development and Application of Active Peptides", Science Press, May 2021, First Edition.

[0010] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0011] The term "IPGP" refers to a peptide with the amino acid sequence Ile-Pro-Gly-Pro.

[0012] The term "KLIPGPA" refers to the amino acid sequence of the peptide as Lys-Leu-Ile-Pro-Gly-Pro-Ala.

[0013] The term "KAIPGPA" refers to the peptide with the amino acid sequence Lys-Ala-Ile-Pro-Gly-Pro-Ala.

[0014] The term "RLIPGPA" refers to the amino acid sequence of the peptide, which is Arg-Leu-Ile-Pro-Gly-Pro-Ala.

[0015] The term "RIPGPGF" refers to the amino acid sequence of the peptide, which is Arg-Ile-Pro-Gly-Pro-Gly-Phe.

[0016] The term "KMIPGPA" refers to the amino acid sequence of the peptide as Lys-Met-Ile-Pro-Gly-Pro-Ala.

[0017] In a first aspect, the present invention provides a tetrapeptide having DPP-IV inhibitory activity, wherein the amino acid sequence of the tetrapeptide is Ile. Pro Gly Pro, with a molecular weight of 382.46 Da and an isoelectric point of 5.52, is an acidic tetrapeptide.

[0018] Preferably, the preparation method of the tetrapeptide includes both chemical synthesis and biosynthesis, specifically: (1) Chemical synthesis: The Fmoc solid-phase synthesis method was adopted, and the synthesis was carried out in the order of C-terminus → N-terminus. First, Fmoc-Pro-OH was loaded onto 2-CTC (2-chlorotriphenylmethyl) resin, and after deprotection with 20% piperidine / DMF (N,N-dimethylformamide), Gly, Pro and Ile were condensed sequentially. Each step was activated with HBTU / HOBt / DIPEA (benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate / 1-hydroxybenzotriazole / N,N-diisopropylethylamine). The completeness of the reaction was detected by ninhydrin. Finally, the peptide chain was cleaved with TFA (trifluoroacetic acid) mixture, and the crude product was obtained by ether precipitation. The pure product was obtained by HPLC purification and lyophilization. The product was verified by mass spectrometry and Edman degradation.

[0019] (2) Biosynthesis: The DNA sequence encoding Ile-Pro-Gly-Pro is artificially synthesized, inserted into an expression vector containing a promoter and tag, and transformed into host cells such as Escherichia coli, Bacillus, or yeast; transcription and translation are initiated by an inducer (such as IPTG (isopropyl-β-D-thiogalactoside)) to express a fusion protein containing the target peptide. After separation by affinity chromatography, the tag is cleaved by a specific protease, and then purified to obtain IPGP. Finally, the product is verified by mass spectrometry.

[0020] Secondly, the present invention also provides derivatives of the above-mentioned tetrapeptide, comprising the above-mentioned tetrapeptide core structure IPGP and amino acid residues at the N-terminus or C-terminus that are easily recognized by intestinal digestive enzymes.

[0021] Preferably, the amino acid sequence of the tetrapeptide derivative is Lys-Leu-Ile-Pro-Gly-Pro-Ala, Lys-Ala-Ile-Pro-Gly-Pro-Ala, Arg-Leu-Ile-Pro-Gly-Pro-Ala, Lys-Met-Ile-Pro-Gly-Pro-Ala and Arg-Ile-Pro-Gly-Pro-Gly-Phe.

[0022] Preferably, the preparation method of the tetrapeptide derivative includes both chemical synthesis and biosynthesis, specifically: (1) Chemical synthesis: The Fmoc solid-phase synthesis method was adopted, and the synthesis was carried out in the order of C-terminus → N-terminus. The C-terminal starting amino acid was first coupled with Fmoc and then loaded onto 2-CTC resin. After deprotection with 20% piperidine / DMF, the subsequent amino acids were condensed sequentially. Each step was activated with HBTU / HOBt / DIPEA, and the reaction was detected by ninhydrin. Finally, the peptide chain was cut with TFA mixture, and the crude product was obtained by ether precipitation. The pure product was obtained by HPLC purification and lyophilization. The product was verified by mass spectrometry and Edman degradation.

[0023] (2) Biosynthesis: The DNA sequence encoding each derived peptide is artificially synthesized, inserted into an expression vector containing a promoter and a tag, and transformed into Escherichia coli, Bacillus, or yeast host cells; transcription and translation are initiated by an inducer (such as IPTG) to express the fusion protein containing the target peptide; after separation by affinity chromatography, the tag is cleaved by a specific protease, and then purified to obtain each target peptide; finally, the product is verified by mass spectrometry.

[0024] Thirdly, the present invention also provides the application of the above-mentioned tetrapeptide with DPP-IV inhibitory activity and its derivatives in the preparation of products for maintaining stable blood glucose levels.

[0025] Preferably, the product is a pharmaceutical, health product, or food.

[0026] Preferably, the drug is administered orally or by injection.

[0027] Preferably, the dosage form of the medicine is tablets, powders, granules, decoctions, injections, capsules, or pills.

[0028] Preferably, the medicine further includes pharmaceutically acceptable excipients.

[0029] Fourthly, the present invention also provides a blood sugar lowering product, comprising the above-mentioned tetrapeptide with DPP-IV inhibitory activity.

[0030] Preferably, the functional product is a pharmaceutical, health product, or food.

[0031] Fifthly, the present invention also provides a DPP-IV inhibitory drug, comprising the above-mentioned tetrapeptide having DPP-IV inhibitory activity.

[0032] The present invention has the following beneficial effects: The tetrapeptide provided by this invention exhibits good DPP-IV inhibitory activity, is resistant to digestion by gastric and intestinal digestive juices after oral administration, and is absorbed by the intestines, thus demonstrating a good hypoglycemic effect. The tetrapeptide derivative, after oral administration, can be hydrolyzed by intestinal digestive enzymes to generate IPGP, which, after intestinal absorption, also exhibits a good hypoglycemic effect. The DPP-IV inhibitory peptide IPGP and its derivatives of this invention can be used to prepare hypoglycemic functional products and pharmaceuticals, showing good application potential in maintaining stable blood glucose levels. Attached Figure Description

[0033] Figure 1 The total ion chromatogram and the MS / MS spectrum of IPGP are shown. Figure 2 Lineweaver for the inhibition kinetics of IPGP against DPP-IV Burk curve; Figure 3 This is a diagram showing the docking mode analysis between the tetrapeptide IPGP and DPP-IV (amino acids are represented by standard three-letter abbreviations, and the numbers following them are the position numbers of the amino acids in the primary sequence of the DPP-IV protein, such as TRP A:629 indicating tryptophan at position 629 on the A chain of DPP-IV). Figure 4 Figure 1 shows the oral glucose tolerance test results of healthy mice after oral administration of IPGP and its derivatives. Compared with the blank control group p <0.01), A represents the changes in blood glucose levels in mice in different drug administration groups; B represents the area under the glucose tolerance test curve in mice in different drug administration groups.

[0034] Figure 5 The effect of IPGP intervention for 5 weeks on fasting blood glucose and serum insulin levels in T2DM mice (Figure 1). ## Compared with the blank control group p <0.01; Compared with the T2DM model group p <0.01), A represents fasting blood glucose (FBG) level; B represents serum insulin level. Detailed Implementation

[0035] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of protection of the present invention.

[0036] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0037] Example 1: Design and synthesis of DPP-IV-inhibited tetrapeptide IPGP The specific steps are as follows: (1) Based on the screening of rat everted intestinal sac model, combined with the core structural features of DPP-IV inhibitory peptide, computer-aided screening was used and verified by in vitro and mouse in vivo experiments. Finally, an oligopeptide composed of 4 amino acids, namely DPP-IV inhibitory tetrapeptide IPGP, was obtained.

[0038] The amino acid sequence of IPGP is: Ile Pro Gly Pro, with a theoretical molecular weight of 382.46 Da and an isoelectric point of 5.52, is an acidic tetrapeptide.

[0039] The DPP-IV inhibitory tetrapeptide was prepared by the following method: Chemical synthesis: The Fmoc solid-phase synthesis method was used, synthesizing in the order of C-terminus → N-terminus. First, Fmoc-Pro-OH was loaded onto 2-CTC resin, deprotected with 20% piperidine / DMF, and then Gly, Pro, and Ile were sequentially condensed. Each step was activated with HBTU / HOBt / DIPEA, and the completeness of the reaction was detected with ninhydrin. Finally, the peptide chain was cleaved with a TFA mixture, and the crude product was precipitated with diethyl ether. The crude product was then purified by HPLC and lyophilized to obtain the pure product. The product was verified by mass spectrometry and Edman degradation.

[0040] In addition, the DPP-IV inhibitory tetrapeptide prepared by the following method can also achieve the technical effects of the present invention: Biosynthesis: The DNA sequence encoding Ile-Pro-Gly-Pro is artificially synthesized, inserted into an expression vector containing a promoter and tag, and transformed into host cells such as Escherichia coli, Bacillus, or yeast; transcription and translation are initiated by an inducer (such as IPTG) to express a fusion protein containing the target peptide. After separation by affinity chromatography, the tag is cleaved by a specific protease, and then purified to obtain IPGP. Finally, the product is verified by mass spectrometry.

[0041] This invention uses a chemical synthesis method to prepare IPGP for subsequent experiments.

[0042] Example 2: Absorption evaluation of tetrapeptide IPGP in an everted rat intestinal sac model Healthy male 7-8 week old SD rats, weighing 200-220 g, were provided by Shanghai Silex Laboratory Animal Co., Ltd. The housing temperature was 22°C. At 25℃, relative humidity 60% ± 5%, and 12 hours of light per day, three rats were used. Prior to the experiment, the rats were fasted for 12 hours. Sixteen hours later, after anesthesia with chloral hydrate, the abdomen was incised along the midline, and the duodenum was removed (6 portions were measured starting 1 cm from the pylorus of the stomach). (7 cm) Placed in oxygen-saturated ice-cold Tyrode buffer, the mesentery was removed, and oxygen-saturated ice-cold Tyrode buffer was injected into the intestinal sac using a syringe to fully expel the contents of the intestinal lumen. Surface moisture was blotted dry with filter paper. The obtained small intestine was inserted into one end of a glass rod, and the intestinal segment was carefully turned inside out using the glass rod. After washing in oxygen-saturated ice-cold Tyrode buffer, surface moisture was blotted dry, and one end of the everted intestinal segment was tied tightly with silk thread. 2 mL of 37°C Tyrode buffer was injected into the other end using a syringe, and then tied tightly with silk thread to form a sealed everted intestinal segment. It was placed in 30 mL of sample solution containing 5 mg / mL (dissolved in 37°C Tyrode buffer), and mixed oxygen (95% O2-5% CO2, 30 mL) was introduced. Samples were taken from the outer (mucosal side) and inner (serosa side) sides of the everted intestinal segment after 120 min (40 bubbles / min) and analyzed by LC-MS / MS using an Agilent 1290 Infinity II LC in series with a 6545XT AdvanceBioLC / Q-TOF.

[0043] Software analysis detected IPGP on the serosa of the intestinal sac of everted rats, indicating that IPGP can resist degradation by rat small intestinal epithelial membrane peptidases to a certain extent, and is thus completely absorbed by the intestinal sac of everted rats. The total ion chromatogram and MS / MS spectrum of IPGP are shown below. Figure 1 As shown.

[0044] Example 3: In vitro inhibitory activity assay of DPP-IV All reaction solutions were prepared using Tris-HCl buffer (0.1 M, pH 8.0). 25 μL of Gly-Pro-p-NA (1.8 mM) was mixed with an equal volume of the sample solution and preheated at 37 °C for 10 min. 50 μL of DPP-IV solution (10 U / L) was added, and incubation continued at 37 °C for 1 h. Finally, 100 μL of sodium acetate buffer (1 M, pH 4.0) was added to terminate the reaction, and the absorbance at 405 nm was immediately measured. The DPP-IV inhibition rate was calculated as follows:

[0045] Calculations show that IPGP has an IC value of 100 kJ / m² for DPP-IV suppression. 50 The concentration was 96.41±15.64 μM, indicating good DPP-IV inhibitory activity.

[0046] Example 4: Kinetic Study of DPP-IV Inhibition by Tetrapeptide IPGP The activity of DPP-IV at substrate concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mM was measured under conditions of DPP-IV inhibitory peptide concentrations of 0, 100, and 200 µM. Lineweaver-Burk double reciprocal curves were plotted with the reciprocal of substrate concentration (1 / [PNPG]) on the x-axis and the reciprocal of initial reaction rate (1 / ΔOD / min) on the y-axis. Kinetic parameters were calculated using the Lineweaver-Burk plots to determine the inhibitory mode of DPP-IV by the DPP-IV inhibitory peptide. Results are shown below. Figure 2 .

[0047] With increasing substrate concentration, Km significantly increased after the addition of IPGP (100, 200 μM), but Vmax showed no significant difference compared to the group without IPGP, indicating that substrate binding to DPP-IV was interrupted and the affinity between the enzyme and substrate was reduced. These results suggest that IPGP is a competitive inhibitor of DPP-IV, which is related to its properties as a substrate analog.

[0048] Example 5: Analysis of the docking mode between tetrapeptide IPGP and DPP-IV The crystal structure of DPP-IV (PDB ID: 5I7U) was obtained from the PDB database (https: / / www.rcsb.org / ). Pymol 2.6 was used to remove redundant ligands and water molecules, and Autodock Vina 1.5.6 was used to add hydrogen atoms. DS2019 was used to construct the predicted peptide structure and to perform hydrogen addition and charge supplementation on the small molecule. Docking was performed using Autodock Vina 1.5.6, with the catalytic pocket of DPP-IV as the binding region. The grid center coordinates were set to (x: 13.553, y: 26.431, z: 55.854), and the grid size was (x: 47.25, y: 47.25, z: 47.25). After docking, the conformation with the highest number of repetitions and the best binding effect was selected as the output result, and Pymol 2.6 and DS2019 were used for visualization analysis. The docking results are shown below. Figure 3 As shown, IPGP forms two hydrogen bonds with two DPP-IV amino acid residues (Arg125; Trp629). In addition, it also establishes salt bridges, electrostatic interactions, and alkyl interactions with the amino acid residues of DPP-IV.

[0049] Example 6: Determination of the in situ activity of tetrapeptide IPGP in Caco-2 cell monolayers The Caco-2 cell suspension was diluted with culture medium to a density of 1×10⁻⁶. 5100 μL of the sample was seeded into each well of a 96-well plate and cultured for 24 h. The cells were washed twice with 100 µL of PBS, and 150 µL of the sample to be tested was added. The plate was incubated in an incubator for 10 min. Then, 50 µL of 4 mM Gly-Pro-PNA·HCl was added, mixed well, and incubated at 37 °C for 60 min. Finally, the absorbance at 405 nm was measured using a microplate reader. The DPP-IV inhibition rate was calculated according to the following formula.

[0050]

[0051] The results showed that Caco-2 cells inhibited the IC50 of DPP-IV in situ. 50 The value was 187.0 ± 13.51 μM. IPGP in-situ suppression of DPP-IV IC 50 The value increased by only 0.94 times compared to the in vitro inhibition, indicating that it has a strong ability to resist brush edge enzyme degradation and has the ability to be completely absorbed into the body.

[0052] Example 7: Design of IPGP-based derived peptides Based on the specific recognition sites of major intestinal digestive enzymes such as trypsin and aminopeptidase, and using an endocerection triggering and exocerection pruning strategy, a series of derived peptides were designed with IPGP as the core structure. These peptides were derived by introducing amino acid residues easily recognized by intestinal proteases at the N-terminus or C-terminus. The sequences are: KLIPGPA, KAIPGPA, RLIPGPA, KMIPGPA, and RIPGPGF. The expected interaction mechanism between these derived peptides and intestinal digestive enzymes is as follows: trypsin preferentially recognizes the K or R residue at the N-terminus of the peptide and cleaves it thereafter, converting KLIPGPA, KAIPGPA, RLIPGPA, and KMIPGPA to LIPGPA, AIPGPA, LIPGPA, and MIPGPA, respectively, while RIPGPGF is converted to the IPGPGF intermediate. Subsequently, brush border-associated aminopeptidases can progressively remove excess residues (such as L, A, or M) from the N-terminus, further pruning the intermediates to IPGPA. Finally, pancreatic or intestinal carboxypeptidases and carboxyterminal exopeptidases can cleave the terminal A from the C-terminus, thereby releasing the target core peptide IPGP. For the intermediate IPGPGF derived from RIPGPGF, it is expected that the terminal F and G will be removed sequentially from the C-terminus under the action of carboxypeptidase, thereby achieving the release of IPGP.

[0053] Example 8: Verification of oral glucose tolerance test in healthy mice Healthy male mice (C57BL / 6, six weeks old) were used in an oral glucose tolerance test to verify the short-term hypoglycemic effect of IPGP. Mice were housed in an SPF-grade animal room and fed a normal diet for one week. Mice were fasted overnight before treatment.

[0054] Fifty-six mice were randomly divided into a control group (N) and treatment groups (IPGP, KLIPGPA, KAIPGPA, RLIPGPA, KMIPGPA, and RIPGPGF groups), with eight mice in each group. The treatment groups were administered 100 mg / kg (converted to IPGP content) of each peptide via gavage, while the control group was administered an equal volume of physiological saline via gavage. Thirty minutes later, all mice were orally administered 2 g / kg glucose. Blood glucose levels were measured using a Roche glucometer before (0 min) and at 15, 30, 60, 90, and 120 min after glucose administration. Changes in blood glucose levels and the area under the glucose-time curve (AUC) are shown below. Figure 4 As shown in the figure. The results showed that 30 min after administration, the blood glucose levels of mice in each group were significantly lower than those in the control group, and then tended to be consistent after 2 h. The area under the glucose curve (AUC) within 2 h after administration was calculated. The results showed that the AUC of the IPGP group, KLIPGPA group, KAIPGPA group, RLIPGPA group, KMIPGPA group, and RIPGPGF group decreased by 14.91%, 22.18%, 15.60%, 19.45%, 13.02%, and 9.46%, respectively. This indicates that IPGP and its derivative peptides can exert hypoglycemic effects in mice and can be further developed into oral hypoglycemic products.

[0055] Example 9: Validation of hypoglycemic activity in T2DM mice T2DM mice (C57BL / 6, FBG ≥ 11.1 mmol / L) induced by a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ, 60 mg / kg) were randomly divided into four groups: T2DM model group (MC), positive control sitagliptin group (PC), low-dose IPGP group (DL), and high-dose IPGP group (DH). The blank control group (NC) and MC group mice were administered distilled water by gavage daily; PC group mice were administered sitagliptin 12.5 mg / kg by gavage daily; DL group mice were administered IPGP 25 mg / kg by gavage daily; and DH group mice were administered IPGP 50 mg / kg by gavage daily. Throughout the treatment period, NC group mice were fed a standard diet, while the other groups were fed a high-fat diet.

[0056] Five weeks after oral administration, mice were fasted for 12 hours (with free access to water) and blood was collected via the orbital sinus. The blood was allowed to coagulate at room temperature, and serum was separated. Blood glucose levels were measured using a Roche glucometer. Insulin levels were detected using an enzyme-linked immunosorbent assay (ELISA).

[0057] The results obtained are as follows Figure 5As shown, after 5 weeks of intervention, FBG levels in mice in both the DL and DH groups were significantly reduced. Further analysis of serum insulin levels revealed that insulin levels in the MC group were significantly lower than those in the NC group, consistent with the characteristic insulin deficiency or impaired secretion in T2DM. In contrast, serum insulin levels in mice in the DL and DH groups significantly increased, indicating that IPGP intervention helps alleviate β-cell dysfunction, restore glucose-stimulated insulin secretion, and fundamentally improve glucose homeostasis regulation.

[0058] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A tetrapeptide with orally absorbable DPP-IV inhibitory activity, characterized in that, The aforementioned tetrapeptide has the amino acid sequence Ile-Pro-Gly-Pro, a molecular weight of 382.46 Da, an isoelectric point of 5.52, and is an acidic tetrapeptide.

2. The method for preparing the tetrapeptide according to claim 1, characterized in that, This includes the following two methods: (1) Chemical synthesis: Fmoc solid-phase synthesis was adopted, and the synthesis was carried out in the order of C-terminus → N-terminus. In the first step, Fmoc-Pro-OH was loaded onto 2-CTC resin and deprotected by 20% piperidine / DMF. In the second step, Gly, Pro and Ile were condensed sequentially. Each step was activated by HBTU / HOBt / DIPEA, and the reaction was tested for completeness with ninhydrin. Finally, the peptide chain was cleaved with TFA mixture, and the crude product was precipitated with ether. The crude product was purified by HPLC and lyophilized to obtain the pure product. (2) Biosynthesis: The DNA sequence encoding Ile-Pro-Gly-Pro is artificially synthesized, inserted into an expression vector containing a promoter and a tag, and transformed into Escherichia coli, Bacillus, or yeast host cells; transcription and translation are initiated by an inducer to express a fusion protein containing the target peptide. After separation by affinity chromatography, the tag is cleaved by a specific protease and then purified to obtain the IPGP tetrapeptide.

3. A derivative of a tetrapeptide having DPP-IV inhibitory activity, characterized in that, Contains the tetrapeptide core structure Ile as described in claim 1 Pro Gly Pro and amino acid residues at the N-terminus or C-terminus that are easily recognized by intestinal digestive enzymes.

4. The tetrapeptide derivative according to claim 3, characterized in that, The amino acid sequences of the tetrapeptide derivatives are Lys-Leu-Ile-Pro-Gly-Pro-Ala, Lys-Ala-Ile-Pro-Gly-Pro-Ala, Arg-Leu-Ile-Pro-Gly-Pro-Ala, Lys-Met-Ile-Pro-Gly-Pro-Ala, and Arg-Ile-Pro-Gly-Pro-Gly-Phe.

5. The use of the tetrapeptide with DPP-IV inhibitory activity as described in claim 1 or a derivative of the tetrapeptide as described in any one of claims 3-4 in the preparation of products for maintaining stable blood glucose levels.

6. The application according to claim 5, characterized in that, The product is a pharmaceutical, health product, or food; the pharmaceutical is administered orally or by injection.

7. The application according to claim 5, characterized in that, The dosage form of the medicine is tablets, powders, granules, decoctions, capsules, pills, or injections.

8. The application according to claim 5, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

9. A blood sugar lowering product, characterized in that, Includes the tetrapeptide with DPP-IV inhibitory activity as described in claim 1 or a derivative of the tetrapeptide as described in any one of claims 3-4.

10. A DPP-IV inhibitory drug, characterized in that, Includes the tetrapeptide with DPP-IV inhibitory activity as described in claim 1 or a derivative of the tetrapeptide as described in any one of claims 3-4.

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