Bullfrog skin peptide with DPP-IV (dipeptidyl peptidase-IV) inhibitory activity as well as purification method and application thereof

Abstract

The invention discloses bullfrog skin peptide with DPP-IV inhibitory activity and a purification method and application thereof.The molecular weight of the bullfrog skin peptide is 681.40 Da, and the amino acid sequence of the bullfrog skin peptide is shown as SEQ ID NO.1. The bullfrog skin peptide is extracted through an enzymolysis means, then components containing peptide fragments are extracted through a column chromatography purification mode, finally the components are evaluated and screened, and the bullfrog skin peptide with the DPP-IV inhibitory activity is obtained. The purified bullfrog skin peptide with the DPP-IV inhibitory activity is obtained. The bullfrog skin peptide with the DPP-IV inhibitory activity has the advantages of being strong in DPP-IV enzyme inhibitory effect, safe and free of toxic and side effects, has the application potential of hypoglycemic peptides, is short in amino acid sequence and strong in operability, and has good social popularization and application value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptide drugs, and in particular relates to a bullfrog skin peptide with DPP-IV inhibitory activity and a purification method and application thereof. Background Art

[0002] At present, the incidence of chronic metabolic disease type 2 diabetes mellitus (T2DM) has increased significantly, and long-term use of chemical drugs is often accompanied by adverse side effects. Food-derived active peptides have gradually become a new research direction for the development of healthy hypoglycemic functional factors due to their low side effects, wide sources, high activity and easy absorption. Among them, dipeptidyl peptidase IV (DPP-IV) inhibitory peptides can effectively improve diabetic symptoms and are commonly used drugs for the treatment of T2DM. DPP-IV is a glycoprotein with serine exopeptidase activity. It mainly acts on incretin, destroys its chain segments, and makes it lose the ability to stimulate insulin secretion and maintain postprandial blood sugar balance. Therefore, inhibiting DPP-IV enzyme is one of the important targets for the effective treatment of diabetes. Animal skin is rich in collagen, and its content can reach about 80% of the total protein. The hydrolysis product of proline-rich collagen is a potential inhibitor of DPP-IV. Fish skin is rich in collagen. Studies have shown that by enzymatic hydrolysis of salmon skin gelatin, active tetrapeptide GPAE with hypoglycemic effect was discovered. The hydrolyzate of Alaska pollock skin after simulated gastrointestinal digestion also showed strong DPP-IV inhibitory ability. In addition, the structure-activity relationship and inhibitory mechanism of tilapia skin hydrolyzate with DPP-IV enzyme have also been elucidated. Therefore, animal skin rich in collagen is an excellent source of DPP-IV inhibitory peptides.

[0003] In recent years, the bullfrog industry has developed rapidly and has become one of the popular aquaculture species. In 2023, the national bullfrog production exceeded one million tons, and breeding was spread across more than 20 provinces and cities across the country. There are more than 50,000 related catering companies, and the industry output value exceeds 100 billion yuan. It is an important aquaculture species in my country. Studies have shown that bullfrog skin collagen is rich in glycine, proline and hydroxyproline, that is, the protein hydrolysate from bullfrog skin may have DPP-IV inhibitory activity and has the application potential of hypoglycemic peptides. However, the current research on aquatic animal skin-derived DPP-IV inhibitory peptides is mainly focused on fish skin, and there is a lack of related research on bullfrog skin-derived DPP-IV inhibitory peptides. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a bullfrog skin peptide with DPP-IV inhibitory activity and a purification method and application thereof, fill the gap in the research of bullfrog skin-derived DPP-IV inhibitory peptides, and obtain new DPP-IV inhibitory peptides.

[0005] The technical solution of the present invention to solve the above technical problems is: A bullfrog skin peptide with DPP-IV inhibitory activity, with a molecular weight of 681.40 Da and an amino acid sequence as shown in SEQ ID NO: 1. Specifically, the amino acid sequence is Val-Gly-Pro-Val-Gly-Pro-Arg (VGPVGPR).

[0006] The present invention also discloses a method for purifying the bullfrog skin peptide having DPP-IV inhibitory activity, comprising the following steps: Extraction of frog skin gelatin: wash the bullfrog skin with clean water and drain it, then soak it in NaOH solution to remove non-collagen and pigment in the bullfrog skin, rinse it with running water until the pH is neutral, drain it and cut it into pieces, place the cut bullfrog skin in 80-100 ℃ water for water bath treatment for 2-5 hours, filter it after treatment to obtain frog skin gelatin; Enzymatic hydrolysis: frog skin gelatin was added with deionized water to adjust the pH to 6.5-7.5, papain was added at an enzyme / substrate ratio of 2.8-5.8%, and enzymatic hydrolysis was performed at 40-60 °C for 2-6 h; Preparation of crude enzymatic extract: the enzymatic hydrolysate was placed in a boiling water bath to inactivate the enzyme, then cooled to room temperature, centrifuged at 4°C for 10-20 min, the supernatant was obtained, and the supernatant was freeze-dried to obtain a crude enzymatic extract; Column chromatography purification: using Sephadex G25 gel chromatography column to purify the crude extract of enzymatic hydrolysis, obtaining the eluate and grouping and labeling the eluate to obtain 1 to 33 components; Screening, the DPP-IV enzyme inhibition rate of 1 to 33 components was determined respectively, and the component with the highest DPP-IV enzyme inhibition rate was selected. This component was the bullfrog skin peptide with DPP-IV inhibitory activity.

[0007] Specifically, in the step of preparing the enzymatic crude extract, the boiling water bath time is 10 to 20 minutes and the centrifugal force is 8000g.

[0008] Specifically, in the column chromatography purification step, the specification of the Sephadex G25 gel chromatography column used is Ф2.5cm×60 cm, the loading concentration is 150-250 mg / mL, the loading volume is 2-6 mL, the flow rate is 5 mL / 17 min, one tube is collected every 17 minutes, and every 5 tubes are combined into one component.

[0009] Specifically, in the screening step, before determining the DPP-IV enzyme inhibition rate, the absorbance values ​​of 1 to 33 components are first determined, the absorbance values ​​of each component at 214, 254 and 280 nm are tested, and the component with the highest absorbance value is selected for the DPP-IV enzyme inhibition rate determination.

[0010] Specifically, the DPP-IV enzyme inhibition rate is determined by a DPP-IV enzyme activity inhibition experiment.

[0011] Specifically, in the frog skin gelatin extraction step, the concentration of the NaOH solution is 0.01-0.08 M, and the soaking time is 0.5-2 h.

[0012] In addition, the invention also discloses an application of bullfrog skin peptide with DPP-IV inhibitory activity for preparing type II diabetes medicine.

[0013] The present invention has the following beneficial effects: (1) The purified bullfrog skin peptide of the present invention has the advantages of strong DPP-IV enzyme inhibition and safety without toxic side effects, has the application potential of hypoglycemic peptides, and fills the research gap in the preparation of bullfrog skin DPP-IV inhibitory peptides; (2) The bullfrog skin peptide purified by the present invention has a short amino acid sequence, is easy to apply industrially, has strong operability, and has good social promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the absorbance value detection and DPP-IV enzyme inhibition activity distribution diagram of each component F1~F33 in the embodiment of the present invention.

[0015] Figure 2 This is the mass spectrum of the bullfrog skin peptide with DPP-IV inhibitory activity prepared by the present invention.

[0016] Figure 3 The diagram shows the conformation of the bullfrog skin peptide with DPP-IV inhibitory activity prepared by the present invention in the DPP-IV enzyme pocket and the binding mode in the active site of the DPP-IV enzyme. DETAILED DESCRIPTION

[0017] The present invention is described in detail below with reference to the embodiments. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0018] The method used in the present invention to determine the DPP-IV enzyme inhibition rate is a DPP-IV enzyme activity inhibition experiment, and its specific operation process is as follows. The test materials used in the present invention are all common commercial products and can be purchased on the market.

[0019] 80 μL of 0.5 mM p-nitroaniline substrate solution was mixed with 80 μL of peptide solution at a final concentration of (0.8 mg / mL), and the mixture was preheated at 37 °C for 10 min, followed by the addition of 40 μL of DPP-IV enzyme solution (0.8 U / μL). The absorbance at 405 nm was measured every 2 minutes for 90 minutes. All reaction solutions were prepared using Tris-HCl buffer (100 mM, pH 8.0). Tris-HCl buffer was used as a control instead of the test sample. The DPP-IV inhibition rate was calculated using the following formula: DPP-IV inhibition rate (%) = (Slope control group - Slope sample group) * 100 / Slope control group Slope = (A2-A1) / (T2-T1) Wherein Slope represents the slope of the absorbance values ​​A1 and A2 at two time points T1 and T2 in the linear range. In the present invention, the peptide solution used in each experiment is prepared from any one of the 1 to 33 components prepared in the present invention.

[0020] A method for purifying a bullfrog skin peptide having DPP-IV inhibitory activity according to an embodiment of the present invention comprises the following steps: For the extraction of frog skin gelatin, the bullfrog skin was washed with clean water and drained, then soaked in 0.05 M NaOH solution for 1.5 h to remove the non-collagen and pigment in the bullfrog skin. After soaking, it was rinsed with running water until the pH was neutral, and then cut into 2*2 cm small pieces for later use. The cut bullfrog skin can be better for subsequent operations and improve the extraction rate. The cut bullfrog skin was placed in 95 ℃ water for water bath treatment for 4 h, and filtered after treatment to obtain frog skin gelatin; filtering with two layers of cotton cloth can effectively remove the oil layer of the extract.

[0021] For enzymatic hydrolysis, frog skin gelatin was added with deionized water to adjust the pH to 6.5, papain was added at an enzyme / substrate (E / S) ratio (w / w) of 4.8%, and enzymatic hydrolysis was performed in a water bath at 55 °C for 5 h to fully hydrolyze the frog skin gelatin.

[0022] For the preparation of the crude enzymatic extract, the enzymatic hydrolysate was placed in a boiling water bath for 10 min to inactivate the enzyme, then cooled to room temperature, centrifuged at 4°C and a centrifugal force of 8000 g for 10 min, and the supernatant was obtained. The supernatant was freeze-dried at -80°C and a pressure of 5 Pa to obtain the crude enzymatic extract.

[0023] Column chromatography purification: A Sephadex G25 gel chromatography column with a specification of Ф2.5 cm×60 cm was used to purify the crude extract of the enzymatic hydrolysis. The loading concentration was set to 200 mg / mL, the loading volume was 5 mL, the flow rate was 5 mL / 17 min, and a test tube of eluate was collected every 17 min. Every 5 consecutive tubes of eluate were grouped and labeled to obtain 1 to 33 components, which were recorded as F1 to F33. Screening, the absorbance values ​​at 214, 254 and 280 nm were measured for 1 to 33 components, among which the absorbance at 214 nm can detect the peptide bond content in the component, the absorbance at 254 nm can detect the phenylalanine content in the component, and the absorbance at 280 nm can detect the tryptophan and tyrosine content in the component. This can effectively screen out components with low polypeptide content, thereby reducing the number of operations in the DPP-IV enzyme activity inhibition experiment. The absorbance values ​​of each component after the test are as follows Figure 1 As shown, from Figure 1 As can be seen from the line graph, the absorbance of components F4 to F17 at 214 nm, 254 nm and 280 nm is relatively high. Therefore, components F4 to F17 were subsequently selected for DPP-IV enzyme inhibition rate determination. It was found that only 10 components, F4-F13, had inhibitory activity. The results are as follows: Figure 1As shown in the middle bar graph, the activity range is 13.64-84.67%, among which the enzyme inhibition rate of the F8 component is the highest (reaching 84.67 ± 0.12%). Therefore, the F8 component prepared in this example is the bullfrog skin peptide with DPP-IV inhibitory activity obtained by purification of the present invention.

[0024] The prepared bullfrog skin peptide was analyzed and identified by nano-liquid chromatography EASY-nLC 1200 nano LC system coupled with quadrupole-electrostatic field orbital trap high-resolution mass spectrometer Orbitrap mass spectrometry system (nLC 1200-ESI-qTOF-MS / MS). The bullfrog skin peptide solution was prepared with Tri-HCL solution at a concentration of 25 μg / mL and filtered with a 0.22 μm filter membrane. The bullfrog skin peptide solution was first enriched by an Acclaim Pep MapTM 100 trap column (75 μm × 2 cm, nano Viper 2Pk C18, 3 μm, pore size 100 μm), and then separated by an Acclaim Pep MapTM RSLC column (75μm × 25 cm, nano ViperC18, 2 μm, pore size 100 μm). The mobile phases included mobile phase A (0.1% formic acid water) and mobile phase B (20% 0.1% formic acid water plus 80% acetonitrile). The elution gradient was: 0-2 min: 100% solvent B; 2-10 min: 100%-90% solvent B; 10-20 min, 90%-75% solvent B; 20-25 min, 75%-60% solvent B, 25-35 min, 60%-10% solvent B, 35-40 min, 10%-100% solvent B, 40-45 min, 100% solvent B, with a flow rate of 300 nL / min and an injection volume of 2 μL. The mass spectrometer system was in positive data acquisition mode with a full MS1 scan from 100 to 1500 m / z at a resolution of 70,000 FWHM, followed by an MS2 scan of the 20 most abundant ions in high energy collision dissociation (HCD) mode. The fragmentation mass spectra of the precursor ions were recorded at a resolution of 17,500 FWHM and a quadrupole separation window of 1.6 (m / z). The product ions were generated using a 28% step normalized collision energy (NCE) in the HCD collision cell. The raw mass spectrometry data files were analyzed using Proteome Discoverer v.2.3 software. The mass tolerance for MS1 was 0.005 Da, the mass tolerance for MS2 was 0.05 Da, and the peptide score threshold was set to 36. Figure 2In the mass spectrum shown, the ion of the target peptide is selected as the parent ion to collide with the inert gas, so that the peptide bond in the peptide chain is broken to form a series of ions. The N-terminal fragment ion series is marked as the b series, and the C-terminal fragment ion series is marked as the y series. After comprehensive analysis of these fragment ion series, the amino acid sequence of the peptide segment can be obtained as Val-Gly-Pro-Val-Gly-Pro-Arg. The sequence information and physicochemical properties of the peptide segment are shown in Table 1. The VGPVGPR peptide segment is non-toxic and non-allergenic. The peptide segment has a chain length of 7, belongs to a heptapeptide, and has a steric hindrance of 0.59, indicating that the VGPVGPR peptide segment has good stability.

[0025]

[0026] Based on molecular docking, the software Autodock Vina was used to screen the biological activity of the bullfrog skin peptide with DPP-IV inhibitory activity identified by mass spectrometry. The spatial structure of the bullfrog skin peptide was drawn by ChemDraw 20.0 (Cambridge SoftCorporation, Cambridge, Massachusetts, USA). The structure of the bullfrog skin peptide was saved in mol2 format using Open Babel GUI, and the peptide molecular structure was constructed using the Tripos force field energy minimization principle. The protonation state and hydrogen orientation were optimized in pymol and saved as pdbqt files as ligand files. The Lamarkian genetic algorithm (LGA) was used for docking calculations, and energy range = 4.0, exhaustiveness = 24.0, and num modes = 30.0 were set. The 3D structure of the DPP-IV enzyme (PDB ID: 1wcy) was obtained from the PDB database, the binding site was determined using vina, the docking parameter config.txt file was set, the water molecules and solvent molecules in the protein were removed, the protein was hydrogenated using pymol, and it was saved in pdbqt format for later use. The results are shown in Figure 2. Figure 3As shown, amino acid residues 48Tyr, 125Arg, 205Glu, 206Glu, 357Phe, 547Tyr, 554Lys, 627Trp, 628Gly, 629Trp, 630Ser, 662Tyr, 666Tyr, 740His, and 752Tyr on the DPP-IV enzyme protein chain bind to the VGPVGPR peptide segment through hydrogen bonds, Pi-donor hydrogen bonds, Pi-Sigma, Pi-Alkyl, charge, and non-complementary interactions, respectively. Specifically, 125Arg forms a hydrogen bond with the C1 O of the Gly amino acid at the 2nd N-terminal position of the VGPVGPR peptide and the C1 O of the Val amino acid at the 4th N-terminal position, respectively; 205Glu forms a hydrogen bond with the N1 H of the Gly amino acid at the 2nd N-terminal position of the VGPVGPR peptide; 206Glu forms a hydrogen bond with the N2 H of the Gly amino acid at the 2nd N-terminal position of the VGPVGPR peptide; 554Lys forms a hydrogen bond with the C1 O of the Pro amino acid at the 2nd C-terminal position of the VGPVGPR peptide; 628Gly and 629Trp form a hydrogen bond with the N2 H of the Gly amino acid at the 2nd C-terminal position of the VGPVGPR peptide, respectively. The 630Ser forms a hydrogen bond with the C1-position O of the Arg amino acid at the C-terminus 1 of the VGPVGPR peptide, the 662Tyr forms a hydrogen bond with the C1-position O of the Gly amino acid at the N-terminus 2 of the VGPVGPR peptide and the proline ring of the Pro amino acid at the N-terminus 3, the 740His forms a hydrogen bond with the C1-position O of the Val amino acid at the N-terminus 4 of the VGPVGPR peptide, and the 752Tyr forms a hydrogen bond with the Arg at the C-terminus 1 of the VGPVGPR peptide. The H of the amino acid branch chain forms a hydrogen bond; 357Phe forms a Pi-donor hydrogen bond with the N1-position H of the Val amino acid at the N-terminal 1 of the VGPVGPR peptide, and forms a Pi-Sigma bond with the H of the Val amino acid branch chain at the N-terminal 1; 547Tyr forms a Pi-Alkyl bond with the C1-position O and proline ring of the Pro amino acid at the C-terminal 2 of the VGPVGPR peptide; 627Trp and 629Trp form a Pi-Alkyl bond with the C of the Arg amino acid branch chain at the C-terminal 1 of the VGPVGPR peptide, respectively; 66Tyr forms a Pi-Alkyl bond with the proline ring of the Pro amino acid at the 3rd position of the N-terminus of the VGPVGPR peptide segment; 554Lys binds to the C1-position O of the Arg amino acid at the 1st position of the C-terminus of the VGPVGPR peptide segment by mutual charge attraction; 48Tyr binds to the H of the Arg amino acid branch chain at the 1st position of the C-terminus of the VGPVGPR peptide segment by non-complementary interaction, so the bullfrog skin peptide with DPP-IV inhibitory activity prepared by the present invention is tightly bound to the receptor protein DPP-IV enzyme, thereby achieving the activity inhibition effect of the DPP-IV enzyme.

[0027] The enzymatic crude extract prepared in this example also has a certain DPP-IV enzyme inhibitory activity, but its inhibitory effect is lower than that of the bullfrog skin purified peptide with DPP-IV inhibitory activity prepared in this example. The present invention performs in vitro DPP-IV enzyme inhibitory activity assay on the above enzymatic crude extract and purified peptide. The assay results are shown in Table 2. It can be seen that the DPP-IV enzyme activity inhibition rate of the enzymatic crude extract is 23.64%~68.78%, while the DPP-IV enzyme activity inhibition rate of the purified peptide is 61.43%~82.17%. The inhibition rate increases significantly with the increase of sample concentration (1~4 mg / mL). At the same concentration, the inhibition rate of the purified peptide is always higher than that of the enzymatic crude extract, and the increase is extremely large. From this, it can be shown that the DPP-IV inhibitory peptide derived from bullfrog skin peptide has a good inhibitory effect on the DPP-IV enzyme.

[0028]

[0029] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A bullfrog skin peptide having DPP-IV inhibitory activity, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for purifying bullfrog skin peptide having DPP-IV inhibitory activity as claimed in claim 1, characterized in that: The steps include: Extraction of frog skin gelatin: wash the bullfrog skin with clean water and drain it, then soak it in NaOH solution to remove non-collagen and pigment in the bullfrog skin, rinse it with running water until the pH is neutral, drain it and cut it into pieces for later use, place the cut bullfrog skin in 80-100 ℃ water for water bath treatment for 2-5 hours, filter it after treatment to obtain frog skin gelatin; Enzymatic hydrolysis: frog skin gelatin was added with deionized water to adjust the pH to 6.5-7.5, papain was added at an enzyme / substrate ratio of 2.8-5.8%, and enzymatic hydrolysis was performed at 40-60 °C for 2-6 h; Preparation of crude enzymatic extract: the enzymatic hydrolysate was placed in a boiling water bath to inactivate the enzyme, then cooled to room temperature, centrifuged at 4°C for 10-20 min, the supernatant was obtained, and the supernatant was freeze-dried to obtain a crude enzymatic extract; Column chromatography purification: using Sephadex G25 gel chromatography column to purify the crude extract of the enzymatic hydrolysis, obtaining the eluate and grouping and labeling the eluate to obtain 1 to 33 components; Screening, the DPP-IV enzyme inhibition rate was determined for 1 to 33 components respectively, and the component with the highest DPP-IV enzyme inhibition rate was selected. This component was the purified bullfrog skin peptide with DPP-IV inhibitory activity.

3. The purification method according to claim 2, characterized in that: In the step of preparing the enzymatic crude extract, the boiling water bath time is 10 to 20 minutes and the centrifugal force is 8000 g.

4. The purification method according to claim 2, characterized in that: In the column chromatography purification step, the specification of the Sephadex G25 gel chromatography column used is Φ2.5 cm×60 cm, the loading concentration is 150-250 mg / mL, the loading volume is 2-6 mL, the flow rate is 5 mL / 17 min, one tube is collected every 17 minutes, and every 5 tubes are combined into one component.

5. The purification method according to claim 2, characterized in that: In the screening step, before the DPP-IV enzyme inhibition rate is determined, 1 to 33 components are first optically determined to test the absorbance values ​​of each component at 214, 254 and 280 nm, and the component with the highest absorbance value is selected for the DPP-IV enzyme inhibition rate determination.

6. The purification method according to claim 5, characterized in that: The DPP-IV enzyme inhibition rate is determined by a DPP-IV enzyme activity inhibition experiment.

7. The purification method according to any one of claims 2 to 7, characterized in that: In the frog skin gelatin extraction step, the concentration of the NaOH solution is 0.01-0.08 M, and the soaking time is 0.5-2 h.

8. A use of the bullfrog skin peptide having DPP-IV inhibitory activity as claimed in claim 1, characterized in that: Used for preparing drugs for type II diabetes.

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