Bullfrog skin source peptide as well as purification method and application thereof

Abstract

The invention discloses bullfrog skin-derived peptide and a purification method and application thereof.The molecular weight of the bullfrog skin-derived peptide is 1035.52 Da, and the amino acid sequence of the bullfrog skin-derived peptide is shown as SEQ ID NO.1. The bullfrog skin-derived peptide is extracted through an enzymolysis means to construct a polypeptide library, then the polypeptide library is screened through computer-aided screening, then screened peptide fragments are evaluated so as to be optimized, and the bullfrog skin-derived peptide is obtained. And finally obtaining the purified bullfrog skin source peptide. According to the bullfrog skin source peptide and the purification method and application thereof, the bullfrog skin source peptide has good blood sugar reducing and oxidation resisting functions, has the application potential of type II diabetes mellitus drugs, and is safe and free of toxic and side effects. The amino acid sequence is short, the operability is high, and the social popularization and application value is good.

Description

Technical Field

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

[0002] Currently, food-derived active substances are widely used to alleviate T2DM due to their low side effects and high activity. Among them, active peptides have attracted much attention due to their advantages such as small molecular weight, easy absorption and good stability. Natural plants and animals rich in protein are important potential sources of bioactive peptides, and the penultimate position of proline or alanine at the N-terminus of active peptides is extremely important for inhibiting DPP-IV enzyme activity. DPP-IV is a glycoprotein with serine exopeptidase activity, which mainly acts on incretin (GIP and GLP-1), destroying its chain segments and making 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. In addition, inhibiting DPP-IV enzyme activity has also been shown to be an effective and safe method for the treatment of T2DM. DPP-IV inhibitors can reduce blood glucose concentration by promoting insulin secretion. Common DPP-IV inhibitors such as sitagliptin, linagliptin, saxagliptin and alogliptin are used in the treatment of T2DM. However, high-frequency use of these drugs may have adverse effects on human tissues and organs and increase the risk of serious complications such as nasopharyngitis, cystitis, and inflammatory bowel disease. Therefore, it is imperative to explore natural alternatives to DPP-IV inhibitory drugs.

[0003] Bullfrog is a large edible frog with tender meat, delicious taste and rich nutrition. It is a high-protein, low-fat and low-cholesterol nutritious food and is very popular among the public. Therefore, the bullfrog industry has developed rapidly and has become one of the popular aquaculture species. Studies have shown that collagen hydrolysate extracted from salmon skin has high DPP-IV inhibitory activity, and the active tetrapeptide GPAE with hypoglycemic effect was found from it, showing significant anti-diabetic potential. The hydrolysate 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 hydrolysate with DPP-IV enzyme have been elucidated. In addition to aquatic animals, collagen hydrolysate of terrestrial animal skin also shows DPP-IV inhibitory effect. Collagen 9 peptide GPAGPIGPV and collagen 10 peptide GPAGPOGFPG were isolated from sheepskin collagen hydrolysate, which have high DPP-IV enzyme inhibitory activity. In addition, 5.9% to 20.9% of the peptides in deerskin protein hydrolysate contain proline at the penultimate position, indicating that deerskin collagen is most likely a potential source of DPP-IV inhibitory peptides. Therefore, animal skins rich in collagen are an excellent source of hypoglycemic peptides. Studies have found that bullfrog skin collagen is rich in glycine, proline, and hydroxyproline, indicating that bullfrog skin-derived protein hydrolysates may have DPP-IV inhibitory activity and have the potential for application as hypoglycemic peptides. However, current research on bullfrog skin mainly focuses on collagen extraction technology, gel properties, and the antioxidant efficacy of protein hydrolysates, and lacks research on the exploration of 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-derived peptide having the ability to significantly inhibit DPP-IV enzyme activity and having good ABTS free radical scavenging activity and FRAP antioxidant activity, as well as a purification method and application thereof.

[0005] The technical solution of the present invention to solve the above technical problems is: A bullfrog skin-derived peptide, with a molecular weight of 1035.52 Da and an amino acid sequence as shown in SEQ ID NO: 1. Specifically, the amino acid sequence is Arg-Gly-Phe-Asn-Gly-Leu-Asp-Gly-Ala-Lys (RGFNGLDGAK).

[0006] The present invention also discloses a method for purifying the bullfrog skin-derived peptide, 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 divided into three equal parts and deionized water was added to adjust the pH to 6.5-7.5 to prepare three preparation solutions. Bromelain, flavor protease and papain were added to the three preparation solutions at an enzyme / substrate ratio of 2.8-5.8%, and then the three preparation solutions were placed in a water bath at 40-60 °C for enzymatic hydrolysis for 2-6 h. Preparation of crude enzymatic extracts: three portions of enzymatic hydrolysates were placed in a boiling water bath to inactivate the enzyme, then cooled to room temperature, centrifuged at 4°C for 10-20 min, and the supernatant was obtained. The supernatant was freeze-dried to obtain three portions of crude enzymatic extracts; Peptide screening: Use mass spectrometry to analyze and measure the peptides contained in the three enzymatic crude extracts to construct a peptide library. Use computer-assisted software to perform affinity analysis, potential toxicity / allergen prediction, and potential cell penetration prediction on the peptides in the peptide library to screen out at least two peptides with high affinity, no toxicity, and cell penetration; The two peptides with the highest affinity were selected from the screened peptides, and the site analysis was performed using pymol software to predict the sequence information and physicochemical properties of the peptides. The two peptides were then subjected to solid phase synthesis to obtain the corresponding synthetic peptides, which were then prepared into peptide solutions and their DPP-IV enzyme inhibition activity, ABTS free radical scavenging activity and FRAP antioxidant activity were verified. The peptide with the best verification result was the purified bullfrog skin-derived peptide.

[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 step of establishing a polypeptide library, the computer-aided software includes Autodock Vina software, Toxin Pred software, Aller TOP software and CPPpred software.

[0009] Specifically, in the optimization step, the solid phase synthesis adopts solid phase chemical synthesis, and the synthetic peptide is a polypeptide solid powder with a purity of >95%.

[0010] Specifically, the DPP-IV enzyme inhibition activity is verified by a DPP-IV enzyme activity inhibition experiment, the ABTS free radical scavenging activity is verified by an ABTS·+ scavenging experiment, and the FRAP antioxidant activity is verified by a FRAP assay 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-derived peptide for preparing a medicine for type II diabetes.

[0013] The present invention has the following beneficial effects: (1) The bullfrog skin-derived peptide prepared by the present invention can significantly inhibit DPP-IV enzyme activity, and at the same time has good ABTS free radical scavenging activity and FRAP antioxidant activity, has application potential as a type II diabetes drug, and is safe and has no toxic side effects.

[0014] (2) The bullfrog skin-derived peptide prepared by the present invention has a short amino acid sequence, strong operability, is easy for industrial application, and has good social promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The base peak chromatograms corresponding to the three enzymatic hydrolysates prepared in the examples of the present invention are shown in FIG.

[0016] Figure 2 It is a diagram showing the conformation of the RGFNGLDGAK polypeptide in the DPP-IV enzyme pocket and the binding mode in the active site of the DPP-IV enzyme in the examples of the present invention.

[0017] Figure 3 It is a diagram showing the conformation of the RGEGLPA polypeptide in the DPP-IV enzyme pocket and the binding mode in the active site of the DPP-IV enzyme in the examples of the present invention. DETAILED DESCRIPTION

[0018] 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.

[0019] In the present invention, the DPP-IV enzyme inhibition activity is verified by a DPP-IV enzyme activity inhibition experiment, the ABTS free radical scavenging activity is verified by an ABTS·+ scavenging experiment, and the FRAP antioxidant activity is verified by a FRAP assay. The detailed process of the above experiments is as follows. The test materials used in the present invention are all common commercial products and can be purchased on the market.

[0020] 1.DPP-IV enzyme activity inhibition experiment: 80 μL of 0.5 mM p-nitroaniline substrate solution was mixed with 80 μL of peptide solution (final concentration 0.8 mg / mL), and the mixture was preheated at 37 °C for 10 min before adding 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) Where Slope represents the slope of the absorbance values ​​A1 and A2 at two time points T1 and T2 within the linear range.

[0021] 2. ABTS·+ Clearance Experiment Add 10 uL of synthetic peptide solution to a 96-well plate, add 20 uL of enzyme solution and 170 uL of ABTS working solution, mix well, react at room temperature for 6 min, measure the absorbance at 405 nm, and record it as OD sample. Use 10 uL of distilled water as the control group, and its corresponding absorbance is recorded as OD control. Prepare 0.1mM, 0.2mM, 0.14mM, 0.8mM and 1mM Trolox to make a standard curve, and find the corresponding Trolox concentration on the standard curve according to the absorbance of the synthetic peptide solution, that is, the ABTS·+ scavenging activity of the synthetic peptide solution.

[0022] 3.FRAP assay experiment: Prepare FRAP working solution according to the ratio of FRAP Assay Buffer: TPTZ solution: ferric chloride solution = 10:1:1. Add 30 μL of synthetic peptide solution to a 96-well microplate, and then quickly add 264 μL of FRAP working solution with a pipette. Incubate the reaction mixture at 30 °C for 30 min, and measure its absorbance at 593 nm, which is recorded as OD sample. Use 30 μL of distilled water as the control group, and its corresponding absorbance value is recorded as OD control. Take different concentrations (0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.2 and 1.5 mM) of Fe 2+ Standard solution, draw a standard curve. This method is based on Fe 2+ Indicates the total antioxidant capacity. Find the corresponding Fe on the standard curve according to the sample absorbance 2+ concentration, that is, the antioxidant capacity of the sample.

[0023] A method for purifying a bullfrog skin-derived peptide according to an embodiment of the present invention comprises the following steps: For frog skin gelatin extraction, take the bullfrog skin, wash it with clean water and drain it, then soak it in 0.05 M NaOH solution for 1.5 hours to remove the non-collagen and pigment in the bullfrog skin. After soaking, rinse it with running water until the pH is neutral, drain it and cut it into 2*2cm small pieces. The cut bullfrog skin is placed in 95℃ water for 4 hours, 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.

[0024] Enzymatic hydrolysis: frog skin gelatin was divided into 3 equal parts and deionized water was added to adjust the pH to 6.5-7.5 respectively to prepare 3 prepared solutions. Specifically, the pH of the first part was adjusted to 6.8, bromelain was added at an enzyme / substrate ratio (w / w) of 4.8%, and the mixture was incubated at 45°C for 4 h. The pH of the second part was adjusted to 6.5, papain was added at an enzyme / substrate ratio (w / w) of 4.8%, and the mixture was incubated at 55°C for 4 h. The pH of the third part was adjusted to 7.5, flavor protease was added at an enzyme / substrate ratio (w / w) of 4.8%, and the mixture was incubated at 50°C for 4 h, so that all 3 prepared solutions were fully enzymatically hydrolyzed.

[0025] For the preparation of enzymatic crude extracts, three enzymatic hydrolysates were 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 degrees and a pressure of 5 Pa to obtain three enzymatic crude extracts, namely, bromelain enzymatic crude extract, flavor protease enzymatic crude extract and papain enzymatic crude extract, which were recorded as S1 to S3, respectively.

[0026] For peptide screening, the peptides were analyzed and identified using the nano-liquid chromatography EASY-nLC 1200 nano LC system coupled with a quadrupole-electrostatic field orbital trap high-resolution mass spectrometer Orbitrap mass spectrometry system (nLC 1200-ESI-qTOF-MS / MS). Specifically, the S1, S2, and S3 samples were prepared with Tris-HCL solution at a concentration of 25 μg / mL and filtered with a 0.22 μm filter membrane. The samples were first enriched with an Acclaim Pep MapTM 100 trap column (75 μm × 2 cm, nano Viper 2Pk C18, 3 μm, pore size 100 μm), and then separated with an Acclaim Pep MapTM RSLC column (75μm × 25 cm, nano Viper C18, 2μm, pore size 100 μm). The mobile phase included mobile phase A (0.1% formic acid in water) and mobile phase B (20% 0.1% formic acid in 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, the flow rate was 300 nL / min, and the injection volume was 2 μL. The mass spectrometry system adopted the positive data acquisition mode and the full MS1 scan with a resolution of 70,000 FWHM. The base peak chromatograms of the three samples were scanned as shown in the figure. Figure 1As shown, the range was from 100 to 1500 m / z, and then the 20 most abundant ions were scanned by MS2 in high energy collision dissociation (HCD) mode. The fragmentation mass spectra of the precursor ions were recorded at a resolution of 17500 FWHM and a quadrupole separation window of 1.6 (m / z). A step normalized collision energy (NCE) of 28% was used in the HCD collision cell to generate product ions. After obtaining the raw mass spectrometry data file, the results were analyzed using Proteome Discoverer v. 2.3 software. The mass tolerance of MS1 was 0.005 Da, the mass tolerance of MS2 was 0.05 Da, and the peptide score threshold was set to 36 to construct the bullfrog skin peptide library. AutodockVina software, Toxin Pred software, Aller TOP software, and CPPpred software were used to perform affinity analysis, potential toxicity / allergen prediction, and potential cell penetration prediction on peptides in the peptide library. A total of 6 peptides with high affinity, non-toxicity, and cell penetration were screened, and the sequences were RGFNGLDGAK, RGEGLPA, KNLVLT, PVGPR, SVGPVGPR, and MGPVGPR, as shown in Table 1. Autodock Vina software can give affinity analysis based on the amino acid composition and length of the peptide sequence. The larger the absolute value, the greater the binding probability. CPPpred software can score based on the number and position of certain specific amino acid residues in the peptide sequence, giving a value of 0-1. The larger the value, the greater the probability of potential cell penetration. Toxin Pred software and Aller TOP software can give potential toxicity and allergen predictions based on specific amino acid characteristics based on machine learning technology. Based on this, 6 peptides with high affinity, non-toxicity / allergens, and cell penetration were screened.

[0027]

[0028] The optimal selection was based on the absolute value of affinity predicted by Autodock Vina software. The absolute value reflects the degree of binding of the peptide as a ligand to the DPP-IV receptor. The larger the absolute value, the greater the binding affinity and the higher the probability of inhibitory activity. Based on this, the two peptides with the highest affinity, RGFNGLDGAK and RGEGLPA, were selected from the six peptides screened for molecular docking analysis using Autodock Vina, and the spatial structure of the peptide was drawn by ChemDraw 20.0 (Cambridge SoftCorporation, Cambridge, Massachusetts, USA). The peptide structure 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 nummodes = 30.0 were set. The 3D structure of DPP-IV enzyme was obtained from the PDB database (PDB ID: 1wcy), the binding site was determined using vina, the docking parameters config.txt file was set, the water molecules and solvent molecules in the protein were removed, the protein was hydrogenated using pymol, and the result was saved in pdbqt format for later use. Figure 2As shown, amino acid residues 122Lys, 125Arg, 205Glu, 357Phe, 456Tyr, 547Tyr, 553Gln, 554Lys, 556Asp, 560Arg, 585Tyr, 630Ser, 709Asp, 739Asp, and 740His on the DPP-IV enzyme protein chain are bound to the RGFNGLDGAK peptide segment by hydrogen bonds, Pi-Alkyl, Alkyl, Pi-Pi stacking, charge, salt bridges, and non-complementary interactions, respectively. Specifically, 122Lys forms a hydrogen bond with the C1-position O of the Lys amino acid at the 1st position of the C-terminus of the RGFNGLDGAK peptide, 125Arg forms a hydrogen bond with the C1-position O of the Ala amino acid at the 2nd position of the C-terminus of the RGFNGLDGAK peptide, 456Tyr and 553Gln form a hydrogen bond with the branched H of the Arg amino acid at the 1st position of the N-terminus of the RGFNGLDGAK peptide, 547Tyr forms a hydrogen bond with the N1-position H of the Phe amino acid at the 3rd position of the N-terminus of the RGFNGLDGAK peptide, 554Lys forms a hydrogen bond with the As amino acid at the 7th position of the N-terminus of the RGFNGLDGAK peptide, and 556Tyr forms a hydrogen bond with the N1-position H of the Phe amino acid at the 7th position of the N-terminus of the RGFNGLDGAK peptide. 585Tyr forms a hydrogen bond with the N1-position H of the Arg amino acid at the 1st position of the N-terminus of the RGFNGLDGAK peptide, 630Ser forms a hydrogen bond with the C1-position O of the Asn amino acid at the 4th position of the N-terminus of the RGFNGLDGAK peptide, 709Asp and 739Asp form hydrogen bonds with the side chain H of the Lys amino acid at the 1st position of the C-terminus of the RGFNGLDGAK peptide, respectively; 125Arg and 740His form Alkyl and Pi-Al bonds with the side chain C of the Lys amino acid at the 1st position of the C-terminus of the RGFNGLDGAK peptide, respectively. kyl bond, 357Phe forms a Pi-Pi stack with the branched ring of the Phe amino acid at the 3rd position of the N-terminal of the RGFNGLDGAK peptide, 547Tyr forms a Pi-Alkyl bond with the branched C of the Leu amino acid at the 6th position of the N-terminal of the RGFNGLDGAK peptide, 585Tyr forms a Pi-Alkyl bond with the branched C of the Arg amino acid at the 1st position of the N-terminal of the RGFNGLDGAK peptide; 122Lys and 125Arg respectively bind to the C1 position O of the Lys amino acid at the 1st position of the C-terminal of the RGFNGLDGAK peptide by mutual charge attraction, 55 4Lys and 560Arg bind to the branch chain O of the Asp amino acid at the 7th position of the N-terminal of the RGFNGLDGAK peptide segment by mutual charge attraction, 556Asp binds to the branch chain N of the Arg amino acid at the 1st position of the N-terminal of the RGFNGLDGAK peptide segment by mutual charge attraction, 709Asp and 739Asp bind to the branch chain H of the Lys amino acid at the 1st position of the C-terminal of the RGFNGLDGAK peptide segment by salt bridge and mutual charge attraction; 205Glu binds to the branch chain O of the Asn amino acid at the 4th position of the N-terminal of the RGFNGLDGAK peptide segment by non-complementary interaction. The results of the RGEGLPA peptide docking site analysis are shown in Figure 2. Figure 3As shown, amino acid residues 61Arg, 62Trp, 105Tyr, 106Ser, 107Ile, 109Pro, 117Glu, 156Thr, 157Trp, 159Pro, and 463Lys on the DPP-IV enzyme protein chain bind to the RGEGLPA peptide segment through hydrogen bonds, Alkyl, and non-complementary interactions, respectively. Specifically, 62Trp forms a hydrogen bond with the branch chain H of the Arg amino acid at the 1st position of the N-terminus of the RGEGLPA peptide, 105Tyr forms a hydrogen bond with the N1-position H of the Gly amino acid at the 2nd position of the N-terminus of the RGEGLPA peptide, 106Ser forms a hydrogen bond with the C1-position O of the Arg amino acid at the 1st position of the N-terminus and the N1-position H of the Glu amino acid at the 3rd position of the N-terminus of the RGEGLPA peptide, 107Ile forms a hydrogen bond with the C1-position O and the branch chain H of the Arg amino acid at the 1st position of the N-terminus of the RGEGLPA peptide, 156Thr forms a hydrogen bond with the N1-position H and C1-position O of the Gly amino acid at the 4th position of the N-terminus of the RGEGLPA peptide, and the N1-position H and C1-position O of the Leu amino acid at the 5th position of the N-terminus, and 157Trp It forms a hydrogen bond with the C1 position O of the Glu amino acid at the 3rd position of the N-terminus of the RGEGLPA peptide; 61Arg forms an Alkyl bond with the branched C of the Arg amino acid at the 1st position of the N-terminus of the RGEGLPA peptide, 109Pro forms an Alkyl bond with the branched C of the Ala amino acid at the 1st position of the C-terminus of the RGEGLPA peptide, and 159Pro forms an Alkyl bond with the N1 ring of the Pro amino acid at the 2nd position of the C-terminus of the RGEGLPA peptide; 117Glu binds to the branched O of the Glu amino acid at the 3rd position of the N-terminus of the RGEGLPA peptide by non-complementary interaction, and 463Lys binds to the branched N of the Arg amino acid at the 1st position of the N-terminus of the RGEGLPA peptide by non-complementary interaction. Therefore, the RGFNGLDGAK and RGEGLPA polypeptides bind tightly to the receptor protein DPP-IV enzyme, achieving the activity inhibition effect of the DPP-IV enzyme. The sequence information and physical and chemical property predictions of the RGFNGLDGAK and RGEGLPA peptides are shown in Table 2.

[0029]

[0030] Subsequently, the two peptides were subjected to solid phase synthesis and solid phase chemical synthesis to obtain the corresponding synthetic peptides, which were polypeptide solid powders with a purity of >95%. The synthetic peptides were prepared into peptide solutions and then tested for DPP-IV enzyme inhibition activity, ABTS free radical scavenging activity and FRAP antioxidant activity. The results are shown in Table 3. The peptides RGFNGLDGAK and RGEGLPA both have certain DPP-IV enzyme inhibition activity. The DPP-IV enzyme inhibition activity of the RGFNGLDGAK peptide is about 3 times stronger than that of RGEGLPA (the smaller the IC50 value, the stronger the enzyme inhibition activity). ABTS +The scavenging activity and FRAP value represent the antioxidant capacity of the peptide segment. At the same time, the antioxidant capacity of the RGFNGLDGAK peptide segment is also better than that of the RGEGLPA peptide segment. The final verification result shows that the RGFNGLDGAK peptide segment with excellent antioxidant capacity is the bullfrog skin-derived peptide purified by the present invention.

[0031]

[0032] 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 scope of protection of the present invention.

Claims

1. A bullfrog skin-derived peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for purifying the bullfrog skin-derived peptide according to 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, 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 divided into three equal parts and deionized water was added to adjust the pH to 6.5-7.5 to prepare three preparation solutions. Bromelain, flavor protease and papain were added to the three preparation solutions at an enzyme / substrate ratio of 2.8-5.8%, and then the three preparation solutions were placed in a water bath at 40-60 °C for enzymatic hydrolysis for 2-6 h. Preparation of crude enzymatic extracts: three portions of enzymatic hydrolysates were placed in a boiling water bath to inactivate the enzyme, then cooled to room temperature, centrifuged at 4°C for 10-20 min, and the supernatant was obtained. The supernatant was freeze-dried to obtain three portions of crude enzymatic extracts; Peptide screening: Use mass spectrometry to analyze and measure the peptides contained in the three enzymatic crude extracts to construct a peptide library. Use computer-assisted software to perform affinity analysis, potential toxicity / allergen prediction, and potential cell penetration prediction on the peptides in the peptide library to screen out at least two peptides with high affinity, no toxicity, and cell penetration; The two peptides with the highest affinity were selected from the screened peptides, and the site analysis was performed using pymol software to predict the sequence information and physicochemical properties of the peptides. The two peptides were then subjected to solid phase synthesis to obtain the corresponding synthetic peptides, which were then prepared into peptide solutions and their DPP-IV enzyme inhibition activity, ABTS free radical scavenging activity and FRAP antioxidant activity were verified. The peptide with the best verification result was the purified bullfrog skin-derived peptide.

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 step of establishing a polypeptide library, the computer-aided software includes Autodock Vina software, Toxin Pred software, Aller TOP software and CPPpred software.

5. The purification method according to claim 2, characterized in that: In the optimization step, the solid phase synthesis adopts solid phase chemical synthesis.

6. The purification method according to claim 2, characterized in that: In the optimization step, the synthetic peptide is a polypeptide solid powder with a purity of >95%.

7. The purification method according to claim 2, characterized in that: In the optimization step, the DPP-IV enzyme inhibition activity is verified by a DPP-IV enzyme activity inhibition experiment, the ABTS free radical scavenging activity is verified by an ABTS·+ scavenging experiment, and the FRAP antioxidant activity is verified by a FRAP assay experiment.

8. 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.

9. A use of the bullfrog skin-derived peptide as claimed in claim 1, characterized in that: Used for preparing drugs for type II diabetes.

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