Dipeptidyl peptidase-iv inhibiting peptides and uses thereof

Abstract

The application discloses dipeptidyl peptidase-IV inhibiting peptides and application thereof, two dipeptidyl peptidase-IV inhibiting peptides are as follows: one heptapeptide FPGPIRF, the amino acid sequence of the heptapeptide FPGPIRF is Phe-Pro-Gly-Pro-Ile-Arg-Phe; one octapeptide VPFPRLHF, the amino acid sequence of the octapeptide VPFPRLHF is Val-Pro-Phe-Pro-Arg-Leu-His-Phe. The polypeptide of the application is synthesized by using a polypeptide synthesizer and adopting a solid-phase synthesis method. In-vitro dipeptidyl peptidase-IV inhibiting activity detection shows that the two synthetic polypeptides provided by the application have good inhibiting effect on the activity of dipeptidyl peptidase-IV, and can be applied to the field of biological pharmacy.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to dipeptidyl peptidase-IV inhibitory peptides and their applications. Background Technology

[0002] In recent years, with the rapid development of the global economy and the improvement of people's living standards, unhealthy lifestyles and dietary habits have gradually become common, and diabetes has become a highly prevalent disease. Statistics show that in 2020, the prevalence of diabetes among adults in 31 provinces of China reached 11.2%, ranking first in the world (according to the new diagnostic criteria of the World Health Organization). It is estimated that between 2017 and 2045, the increase in diabetes will exceed 50%, with approximately 693 million people suffering from diabetes. Type 2 diabetes is the most common type of diabetes, which not only affects people's quality of life but also causes a series of complications, constantly threatening people's health. There are many mechanisms of diabetes pathogenesis, one of which involves inhibiting enzymes related to the blood glucose response, such as dipeptidyl peptidase-IV. Studies have shown that dipeptidyl peptidase-IV participates in the regulation of postprandial blood glucose concentration, inhibiting the blood glucose response by regulating its activity, making it a new target for the treatment of type 2 diabetes. Therefore, inhibiting the activity of dipeptidyl peptidase-IV can effectively lower blood glucose and achieve the goal of preventing and treating diabetes.

[0003] Bioactive peptides, also known as functional peptides, are widely available, easily absorbed, and highly safe, while also possessing a wealth of active functions, including antiviral, anti-photoaging, hypoglycemic, and immune-enhancing effects. Wang Junbo et al. extracted and validated marine collagen peptides from deep-sea salmon, achieving a reduction in fasting insulin levels and improving the biological activity of insulin. Hypoglycemic bioactive peptides have a certain hypoglycemic effect and, compared to hypoglycemic drugs, have the advantage of fewer side effects; however, the sources of these bioactive peptides are currently limited in the market, and the preparation process is cumbersome. Summary of the Invention

[0004] This invention selects dipeptidyl peptidase-IV as the research object to determine the in vitro hypoglycemic activity of the synthetic peptide. The purpose of this invention is to provide dipeptidyl peptidase-IV inhibitory peptides and their applications. The two dipeptidyl peptidase-IV inhibitory peptides in this invention can be applied in the field of biopharmaceuticals.

[0005] The two synthetic peptides provided by this invention have good hypoglycemic activity and are simple to prepare with high purity.

[0006] A dipeptidyl peptidase-IV inhibitory peptide, wherein the dipeptidyl peptidase-IV inhibitory peptide comprises one or more of the following two peptides: a heptapeptide FPGPIRF, whose amino acid sequence is Phe-Pro-Gly-Pro-Ile-Arg-Phe; and an octapeptide VPFPRLHF, whose amino acid sequence is Val-Pro-Phe-Pro-Arg-Leu-His-Phe.

[0007] Further, the dipeptidyl peptidase-IV inhibitory peptide can inhibit the activity of dipeptidyl peptidase-IV.

[0008] Further, the dipeptidyl peptidase-IV inhibitory peptide is solid-phase synthesized by a standard Fmoc protocol.

[0009] The present invention also provides use of the dipeptidyl peptidase-IV inhibitory peptide, and the use of the dipeptidyl peptidase-IV inhibitory peptide in biopharmaceuticals, specifically: use of any one of the heptapeptide FPGPIRF and the octapeptide VPFPRLHF in preparation of hypoglycemic drugs.

[0010] The abbreviations of the synthetic peptides described in the present invention are FPGPIRF and VPFPRLHF, respectively, their molecular weights are 832.4595 Da and 1011.565 Da in sequence, their purities are 95.97% and 95.47% in sequence, and their sequences are: Phe-Pro-Gly-Pro-Ile-Arg-Phe and Val-Pro-Phe-Pro-Arg-Leu-His-Phe in sequence. Wherein,[]

[0011] Asp represents the corresponding residue of the amino acid whose English name is Aspartic acid and Chinese name is aspartic acid;

[0012] Trp represents the corresponding residue of the amino acid whose English name is Tryptophan and Chinese name is tryptophan;

[0013] Val represents the corresponding residue of the amino acid whose English name is Valine and Chinese name is valine;

[0014] Ser represents the corresponding residue of the amino acid whose English name is Serine and Chinese name is serine;

[0015] Leu represents the corresponding residue of the amino acid whose English name is Leucine and Chinese name is leucine;

[0016] Gly represents the corresponding residue of the amino acid whose English name is Glycine and Chinese name is glycine;

[0017] Pro represents the corresponding residue of the amino acid whose English name is Proline and Chinese name is proline;

[0018] Glu represents the corresponding residue of the amino acid whose English name is Glutamic acid and Chinese name is glutamic acid;

[0019] His represents the corresponding residue of the amino acid whose English name is Histidine and Chinese name is histidine;

[0020] Phe represents the corresponding residue of the amino acid whose English name is Phenylalanine and Chinese name is phenylalanine;

[0021] Thr represents the corresponding residue of the amino acid whose English name is Threonine and Chinese name is threonine;

[0022] Tyr represents the corresponding residue of the amino acid whose English name is Tyrosine and Chinese name is tyrosine;

[0023] Arg represents the corresponding residue of the amino acid whose English name is Arginine and Chinese name is arginine;

[0024] Gln represents the corresponding residue of the amino acid whose English name is Glutarnine and Chinese name is glutamine.

[0025] The amino acid sequence of the present invention is synthesized by a reasonable peptide synthesis method through resin screening according to the standard Fmoc scheme. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymer resin, and then the N-terminus of this amino acid is used as the starting point of synthesis to form a peptide bond with the carboxyl group of another amino acid molecule. By repeating this operation, the target polypeptide product can be obtained. After the completion of solid-phase synthesis, the protecting group is removed, and the peptide chain is separated from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the synthesis is carried out from the C-terminus to the N-terminus (amino terminus).

[0026] The present invention studied that the synthetic peptide FPGPIRF at concentrations of 1 mmol / L and 5 mmol / L was mixed with dipeptidyl peptidase-IV and GP-AMC solution and reacted at 37°C for 30 min, then the fluorescence intensity was detected by a microplate reader and the inhibition rate was calculated.

[0027] The inhibition rate of said heptapeptide FPGPIRF against dipeptidyl peptidase-IV can reach 55.58%±1.73% at a concentration of 1 mmol / L, and can reach 86.94%±2.57% at a concentration of 5 mmol / L, which can be applied in the preparation of biomedical field.

[0028] This invention investigated the reaction of synthetic peptides VPFPRLHF at concentrations of 1 mmol / L and 5 mmol / L with dipeptidyl peptidase-IV and GP-AMC solutions at 37°C for 30 min, followed by detection of fluorescence intensity and calculation of inhibition rate using an enzyme-linked immunosorbent assay (ELISA) reader.

[0029] The octapeptide VPFPRLHF exhibits an inhibition rate of 74.00% ± 2.52% against dipeptidyl peptidase-IV at a concentration of 1 mmol / L; and an inhibition rate of 93.73% ± 2.19% against dipeptidyl peptidase-IV at a concentration of 5 mmol / L. The inhibitory concentration (IC50) of the octapeptide VPFPRLHF against dipeptidyl peptidase-IV is 0.56865 mM, making it suitable for applications in the biopharmaceutical field.

[0030] Compared with the prior art, the present invention has the following advantages and technical effects:

[0031] This invention synthesizes two polypeptides, FPGPIRF and VPFPRLHF, for the first time, and detects the inhibitory activity of the two synthetic polypeptides on dipeptidyl peptidase-IV. The in vitro detection of dipeptidyl peptidase-IV inhibitory activity shows that the two synthetic polypeptides provided by this invention have a good inhibitory effect on dipeptidyl peptidase-IV, and the synthetic polypeptides provided by this invention have a certain hypoglycemic activity. Attached Figure Description

[0032] Figure 1 This is the HPLC chromatogram of the synthesized polypeptide Phe-Pro-Gly-Pro-Ile-Arg-Phe.

[0033] Figure 2 MS image of the synthesized polypeptide Phe-Pro-Gly-Pro-Ile-Arg-Phe.

[0034] Figure 3 This is the HPLC chromatogram of the synthesized polypeptide Val-Pro-Phe-Pro-Arg-Leu-His-Phe.

[0035] Figure 4 MS image of the synthesized polypeptide Val-Pro-Phe-Pro-Arg-Leu-His-Phe.

[0036] Figure 5 This is a curve showing the fitted activity of sitagliptin against dipeptidyl peptidase-IV.

[0037] Figure 6Bar chart showing the inhibitory activity of different concentrations of the synthetic peptides Phe-Pro-Gly-Pro-Ile-Arg-Phe and Val-Pro-Phe-Pro-Arg-Leu-His-Phe against dipeptidyl peptidase-IV.

[0038] Figure 7 The curve showing the fitted activity of the synthesized peptide Val-Pro-Phe-Pro-Arg-Leu-His-Phe against dipeptidyl peptidase-IV. Detailed Implementation

[0039] The present invention will be further illustrated below with specific examples, but the implementation and protection scope of the present invention are not limited thereto.

[0040] Solid-phase synthesis of FPGPIRF

[0041] Using a high-molecular-weight resin (Shanghai Taopu Biotechnology Co., Ltd.), following the characteristic amino acid sequence Phe-Pro-Gly-Pro-Ile-Arg-Phe, the carboxyl group of Phe was first covalently linked to a resin. Then, the amino group of Phe and the carboxyl group of Arg underwent a dehydration reaction. After this treatment, Ile was added, and the amino group of Ile reacted with the carboxyl group of Pro. Amino acids were added sequentially from right to left. After adding the last Phe amino acid, the resin was excised to obtain the target polypeptide FP GPIRF. Purification was performed using high-performance liquid chromatography (HPLC). Figure 1 As shown in the figure, the chromatographic column was a Kromasil C18-5, 4.6*150mm. Mobile phase A: acetonitrile solution containing 0.1% trifluoroacetic acid (TFA); mobile phase B: 100% aqueous solution containing 0.1% trifluoroacetic acid (TFA). Within 30 min, the concentration of phase A increased from 5.0% to 90.0% (v / v), and the concentration of phase B increased from 95.0% to 10.0% (v / v). The flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. The solution was rapidly frozen in liquid nitrogen and freeze-dried to obtain the final product, requiring a purity of over 95%, and its structure was identified by MS (e.g., ...). Figure 2 (As shown). By Figure 1 and Figure 2 It can be seen that the main peak FPGPIRF accounts for a very large percentage, with only a small number of impurity peaks present in the sample. Combined with the mass spectrometry data, the purity of the synthesized peptide FPGPIRF is 95.97%, and the molecular weight is matched.

[0042] Solid-phase synthesis of VPFPRLHF

[0043] Using a high-molecular-weight resin (Shanghai Taopu Biotechnology Co., Ltd.), following the characteristic amino acid sequence Val-Pro-Phe-Pro-Arg-Leu-His-Phe, the carboxyl group of Phe was first covalently linked to a resin. Then, the amino group of Phe and the carboxyl group of His underwent a dehydration reaction. After this treatment, Leu was added, and the amino group of Leu reacted with the carboxyl group of Arg. Amino acids were added sequentially from right to left. After adding the last Val amino acid, the resin was excised to obtain the target polypeptide VPFPRLHF. High-performance liquid chromatography (HPLC) was used for analysis. Figure 3 Purification was performed using a Kromasil C18-5 column (4.6 x 150 mm). Mobile phase A consisted of acetonitrile containing 0.1% trifluoroacetic acid (TFA); mobile phase B consisted of a 100% aqueous solution containing 0.1% TFA. Within 30 minutes, the concentration of phase A increased from 5.0% to 90.0% (v / v), and phase B increased from 95.0% to 10.0% (v / v). The flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. The product was then rapidly frozen in liquid nitrogen and freeze-dried to obtain a final product with a purity of 95% or higher. The structure was identified by MS (e.g., [structure not specified]). Figure 4 (As shown). By Figure 3 and Figure 4 It can be seen that the main peak VPFPRLHF accounts for a very large percentage, with only a small number of impurity peaks present in the sample. Combined with the mass spectrometry data, the purity of the synthesized peptide VPFPRLHF is 95.47%, and the molecular weight is matched.

[0044] In vitro inhibitory activity of synthetic peptides against dipeptidyl peptidase-IV

[0045] 1. Preparation of reagents

[0046] 1) 0.1 mol / L pH=8.0 Tris-HCl buffer solution: Accurately weigh 12.114 g of tris(hydroxymethyl)aminomethane (Tris) and dissolve it in 800 mL of ultrapure water. Stir to dissolve, adjust the pH to 8.0 with concentrated hydrochloric acid, and make up to 1000 mL with distilled water.

[0047] 2) 0.05 μg / mL dipeptidyl peptidase-IV solution: Take 10 μg of dipeptidyl peptidase-IV and prepare it to 10 μg / mL with the above Tris-HCl buffer solution. After dissolving it completely, take 10 μL and add it to 1990 μL of distilled water.

[0048] 3) 100 μM glycyl-proline-7-amino-4-methylcoumarin hydrobromide (GP-AMC) solution: Prepare the substrate to 0.1 mM using Tris solution, dissolve it completely, and then add 10 μL to 9990 μL of distilled water.

[0049] 2. Experimental Procedure

[0050] Add 25 μL of peptide FPGPIRF or VPFPRLHF solution to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution, denoted as the peptide group. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, denoted as FLU. 多肽 Tris-HCl buffer was used instead of dipeptidyl peptidase-IV in the peptide group as the peptide background, denoted as FLU. 多肽背景 Tris-HCl buffer solution was used instead of the peptide solution in the peptide group as a negative control, denoted as FUL. 阴性对照 Tris-HCl buffer solution was used to replace dipeptidyl peptidase-IV in the negative control group as a negative background, denoted as FUL. 阴性背景 Sitagliptin was used as a positive control. The dipeptidyl peptidase-IV inhibition rate was calculated using the following formula.

[0051]

[0052] Add 25 μL of sitagliptin solution of different concentrations to each of the 96-well blackboards, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution, designated as the positive control group. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and record the value as FLU. 阳性药物 Tris-HCl buffer was used to replace dipeptidyl peptidase-IV in the positive control group as the drug background, denoted as FLU. 药物背景 Tris-HCl buffer solution was used as a negative control instead of sitagliptin in the positive control group, denoted as FUL. 阴性对照 Tris-HCl buffer solution was used to replace dipeptidyl peptidase-IV in the negative control group as a negative background, denoted as FUL. 阴性背景 The dipeptidyl peptidase-IV inhibition rate was calculated according to the following formula.

[0053]

[0054] Example 1

[0055] 25 μL of sitagliptin solution (0.0078125 μg / mL) was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the background in place of dipeptidyl peptidase-IV in the positive control group, and Tris-HCl buffer was used as the negative control in place of sitagliptin in the positive control group. The mixture was incubated precisely at 37°C for 30 min, and the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The inhibition rate of dipeptidyl peptidase-IV was calculated. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.0078125 ug / mL by 19.86% ± 3.42%.

[0056] Example 2

[0057] Add 25 μL of sitagliptin solution (0.015625 μg / mL) to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control, and Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.015625 ug / mL by 31.59% ± 3.53%.

[0058] Example 3

[0059] Add 25 μL of sitagliptin solution (0.03125 μg / mL) to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control, and Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.03125 ug / mL by 50.93% ± 0.63%.

[0060] Example 4

[0061] Add 25 μL of 0.0625 μg / mL sitagliptin solution to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AM C solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control, and Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.0625 ug / mL by 66.62% ± 1.69%.

[0062] Example 5

[0063] Add 25 μL of 0.125 μg / mL sitagliptin solution to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control. Use Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.125 ug / mL by 73.22% ± 5.29%.

[0064] Example 6

[0065] Add 25 μL of sitagliptin solution (0.25 μg / mL) to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control, and Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.25 ug / mL by 77.32% ± 0.01%.

[0066] Example 7

[0067] Add 25 μL of 0.5 μg / mL sitagliptin solution to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Use Tris-HCl buffer to replace sitagliptin in the positive control group as the drug background, and Tris-HCl buffer to replace sitagliptin in the positive control group as the negative control, and Tris-HCl buffer to replace dipeptidyl peptidase-IV in the negative control group as the negative background. After precise incubation at 37°C for 30 min, immediately measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and calculate the dipeptidyl peptidase-IV inhibition rate. Figure 5 It was found that sitagliptin inhibited dipeptidyl peptidase-IV at a concentration of 0.5 μg / mL by 82.85% ± 0.84%. The IC50 of sitagliptin against dipeptidyl peptidase-IV was 0.02964 μg / mL.

[0068] Example 8

[0069] 25 μL of 1 mmol / L peptide FPGPIRF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the dipeptidyl peptidase-IV inhibition rate was calculated. Figure 6 It can be seen that FPGPIRF inhibited dipeptidyl peptidase-IV at a concentration of 1 mmol / L by 55.58% ± 1.73%.

[0070] Example 9

[0071] 25 μL of a 5 mmol / L peptide FPGPIRF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the dipeptidyl peptidase-IV inhibition rate was calculated. Figure 6 It can be seen that FPGPIRF inhibited dipeptidyl peptidase-IV by 86.94% ± 2.57% at a concentration of 5 mmol / L.

[0072] Example 10

[0073] 25 μL of a 1 mmol / L peptide VPFPRLHF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the inhibition rate of dipeptidyl peptidase-IV was calculated. Figure 6 It can be seen that VPFPRLHF inhibited dipeptidyl peptidase-IV by 74.00% ± 2.52% at a concentration of 1 mmol / L.

[0074] Example 11

[0075] 25 μL of a 5 mmol / L peptide VPFPRLHF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the inhibition rate of dipeptidyl peptidase-IV was calculated. Figure 6 It can be seen that VPFPRLHF inhibited dipeptidyl peptidase-IV by 93.73% ± 2.19% at a concentration of 5 mmol / L.

[0076] Example 12

[0077] 25 μL of a 2.5 mmol / L peptide VPFPRLHF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the dipeptidyl peptidase-IV inhibition rate was calculated. Figure 7 It can be seen that VPFPRLHF inhibited dipeptidyl peptidase-IV by 84.63% ± 1.72% at a concentration of 2.5 mmol / L.

[0078] Example 13

[0079] 25 μL of a 1.25 mmol / L peptide VPFPRLHF solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the dipeptidyl peptidase-IV inhibition rate was calculated. Figure 7 It can be seen that VPFPRLHF inhibited dipeptidyl peptidase-IV by 68.62% ± 3.90% at a concentration of 1.25 mmol / L.

[0080] Example 14

[0081] 25 μL of a 0.625 mmol / L peptide VPFPRLH F solution was added to a 96-well black plate, followed by 25 μL of dipeptidyl peptidase-IV solution and 50 μL of GP-AMC solution. Tris-HCl buffer was used as the peptide background in place of dipeptidyl peptidase-IV in the peptide group, and Tris-HCl buffer was used as the negative control in place of the peptide solution in the peptide group. Sitagliptin was used as the positive control. After precise incubation at 37°C for 30 min, the fluorescence intensity was immediately measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, and the dipeptidyl peptidase-IV inhibition rate was calculated. Figure 7 It can be seen that VPFPRLHF inhibited dipeptidyl peptidase-IV at a concentration of 0.625 mmol / L by 51.40% ± 3.29%.

Claims

1. A dipeptidyl peptidase-IV inhibitory peptide, characterized in that, The dipeptidyl peptidase-IV inhibitory peptide is a heptapeptide FPGPIRF, and the amino acid sequence of the heptapeptide FPGPIRF is Phe-Pro-Gly-Pro-Ile-Arg-Phe.

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