Bullfrog skin-derived peptide LGPQR as well as purification method and application thereof

Abstract

The invention discloses bullfrog skin-derived peptide LGPQR and a purification method and application thereof.The molecular weight of the bullfrog skin-derived peptide LGPQR is 570.34 Da, and the amino acid sequence of the bullfrog skin-derived peptide LGPQR is shown as SEQ ID NO.1. The bullfrog skin-derived peptide LGPQR is obtained by extracting bullfrog skin enzymatic hydrolysate through an enzymolysis means to construct a polypeptide library, then screening the polypeptide library through computer-aided screening, and then evaluating the screened peptide fragments so as to be optimal. Finally, the purified bullfrog skin source peptide LGPQR is obtained. According to the bullfrog skin-derived peptide LGPQR as well as the purification method and the application thereof, the bullfrog skin-derived peptide LGPQR has relatively good functions of reducing blood sugar and resisting oxidation, has application potential of type II diabetes medicines, 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 specifically relates to a bullfrog skin-derived peptide LGPQR 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] Studies have shown that collagen hydrolysates extracted from salmon skin have high DPP-IV inhibitory activity, and the active tetrapeptide GPAE with hypoglycemic effect was found, 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 hydrolysates from terrestrial animal skins also show DPP-IV inhibitory effects. 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%~20.9% of the peptides in deerskin protein hydrolysate contain proline at the penultimate position, indicating that deerskin collagen is very likely to be a potential source of DPP-IV inhibitory peptides. In summary, animal skins rich in collagen are an excellent source of hypoglycemic peptides, and the hypoglycemic effect and action pathway of active peptides are closely related to their source species and peptide amino acid sequence.

[0004] Bullfrog is a large edible frog with tender meat, delicious taste and rich nutrition. It is a high-protein, low-fat, 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 found that bullfrog skin collagen is rich in glycine, proline and hydroxyproline, which indicates that the protein hydrolysate from bullfrog skin may have DPP-IV inhibitory activity and has the potential for application as a hypoglycemic peptide. However, the current research on bullfrog skin mainly focuses on collagen extraction technology, gel properties and the antioxidant effect of protein hydrolysates, and lacks research on the exploration of bullfrog skin-derived DPP-IV inhibitory peptides. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a bullfrog skin-derived peptide LGPQR which has the ability to significantly inhibit DPP-IV enzyme activity and has good ABTS free radical scavenging activity and FRAP antioxidant activity, as well as a purification method and application thereof.

[0006] The technical solution of the present invention to solve the above technical problems is: A bullfrog skin-derived peptide LGPQR has a molecular weight of 570.34 Da and an amino acid sequence as shown in SEQ ID NO: 1. Specifically, the amino acid sequence is Leu-Gly-Pro-Gln-Arg (LGPQR).

[0007] The present invention also discloses a method for purifying the bullfrog skin-derived peptide LGPQR, 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 7 parts and deionized water was added to adjust the pH to 5.5-11 to prepare 7 parts of the prepared solution. Bromelain, flavor protease, composite protease, alkaline protease, papain, trypsin and neutral protease were added to the 7 parts of the prepared solution at an enzyme / substrate ratio of 1.8-6.3%, and then the 7 parts of the prepared solution were placed at 20-60 ℃ for enzymatic hydrolysis for 2-6 h; Preparation of crude enzymatic extracts: 7 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 7 portions of crude enzymatic extracts; Activity determination: the hydrolysis degree, nitrogen recovery rate and DPP-IV enzyme inhibition activity of 7 enzymatic crude extracts were determined. The enzyme inhibition activity of the enzymatic crude extract corresponding to papain was significantly higher, and it was selected as the target enzymatic crude extract; Peptide screening: Use a mass spectrometer to analyze and measure the peptides contained in the target enzymatic hydrolysate to construct a peptide library. Use computer-aided 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 best peptides were selected from the screened peptides, and the two peptides with the highest affinity were selected for site analysis using pymol software to predict the sequence information and physicochemical properties of the peptides. The two peptides were then solid-phase synthesized to obtain corresponding synthetic peptides, which were respectively formulated into peptide solutions and then verified for DPP-IV enzyme inhibition activity, ABTS free radical scavenging activity and FRAP antioxidant activity. The peptide with the best final verification result was the bullfrog skin-derived peptide LGPQR purified by the present invention.

[0008] 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.

[0009] 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.

[0010] 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%.

[0011] 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.

[0012] 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.

[0013] In addition, the present invention also discloses an application of bullfrog skin-derived peptide LGPQR for preparing a drug for type II diabetes.

[0014] The present invention has the following beneficial effects: (1) The bullfrog skin-derived peptide LGPQR 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.

[0015] (2) The bullfrog skin-derived peptide LGPQR 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

[0016] Figure 1 The figure shows the hydrolysis degree, nitrogen recovery rate and DPP-IV enzyme inhibition activity corresponding to 7 enzymatic crude extracts prepared in the examples of the present invention.

[0017] Figure 2 It is a diagram showing the conformation of the LGPQR polypeptide in the DPP-IV enzyme pocket and the binding mode in the active site of the DPP-IV enzyme in the embodiments of the present invention.

[0018] Figure 3 It is a diagram showing the conformation of the INTPVK 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

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 2. ABTS·+ removal 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.

[0023] 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.

[0024] A method for purifying a bullfrog skin-derived peptide LGPQR 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, and then soaked in 0.05 M NaOH solution for 1.5 h to remove non-collagen and pigment in the bullfrog skin. After soaking, it was rinsed with running water until the pH was neutral, and then drained and cut into small pieces of 2*2 cm. 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.

[0025] Enzymolysis, frog skin gelatin was divided into 7 parts and deionized water was added to adjust the pH to 5.5-11 respectively to prepare 7 preparation solutions. Specifically, the pH of the first part was adjusted to 7.5, and the composite protease was added at an enzyme / substrate ratio of 4.8% (w / w), and the mixture was incubated at 50°C for 4 h. The pH of the second part was adjusted to 7.5, and the flavor protease was added at an enzyme / substrate ratio of 4.8% (w / w), and the mixture was incubated at 50°C for 4 h. The pH of the third part was adjusted to 6.5, and papain was added at an enzyme / substrate ratio of 4.8% (w / w), and the mixture was incubated at 55°C for 4 h. The pH of the fourth part was adjusted to 6.8, and bromelain was added at an enzyme / substrate ratio of 4.8% (w / w), and the mixture was incubated at 45°C for 4 h. The pH of the fifth part was adjusted to 10.5, and alkaline protease was added at an enzyme / substrate ratio of 4.8% (w / w), and the mixture was incubated at 45°C for 4 h. h, the sixth portion was adjusted to pH 7.0, neutral protease was added at an enzyme / substrate ratio of 4.8% (w / w), and incubated at 50 ℃ for 4 h, and the seventh portion was adjusted to pH 7.5, trypsin was added at an enzyme / substrate ratio of 4.8% (w / w), and incubated at 37 ℃ for 4 h to ensure that all 7 prepared solutions were fully enzymatically hydrolyzed.

[0026] For the preparation of crude enzymatic extracts, 7 portions of 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 7 crude enzymatic extracts.

[0027] Activity determination, the hydrolysis degree, nitrogen recovery rate and DPP-IV enzyme inhibition activity of 7 enzymatic crude extracts were determined. The hydrolysis degree can reflect the hydrolysis of bullfrog skin gelatin, while the nitrogen recovery rate can reflect the yield of bullfrog skin gelatin during the enzymatic hydrolysis process. These two parameters have important economic significance in the actual processing of bullfrog skin. Figure 1As shown in a, the hydrolysis degree of bullfrog skin gelatin by 7 different proteases varied, ranging from 9.98 to 16.36%. Among them, papain had the highest hydrolysis degree of bullfrog skin gelatin, which was 16.36±0.37%. Compared with the neutral protease with the lowest hydrolysis degree, the hydrolysis degree increased by 63.93%. The results of nitrogen recovery rate are shown in Figure 1 As shown in Figure b, the nitrogen recovery rates of the seven different proteases on bullfrog skin gelatin were not significantly different and the nitrogen recovery rates were relatively high, all exceeding 85%. There was no significant difference in the nitrogen recovery rates of flavor protease, composite protease, alkaline protease, papain and neutral protease ( P >0.05), all exceeding 96%. This indicates that the five enzymes can fully recover gelatin when hydrolyzing bullfrog skin gelatin. Figure 1 As shown in c, there are significant differences in the DPP-IV inhibition rates of bullfrog skin peptides obtained after hydrolyzing bullfrog skin gelatin with 7 different proteases ( P <0.05), the inhibition rate was 19.86-54.98%. Among them, the peptide hydrolyzed by papain had the highest enzyme inhibition activity, reaching 54.98±1.04%. Compared with the trypsin hydrolyzed peptide with the lowest enzyme inhibition activity, the enzyme inhibition rate was significantly increased by 176.84%. Therefore, the enzymatic crude extract obtained after papain hydrolysis was selected as the target enzymatic crude extract.

[0028] For peptide screening, the nano-liquid chromatography EASY-nLC 1200 nano LC system was connected to a quadrupole-electrostatic field orbital trap high-resolution mass spectrometer Orbitrap mass spectrometry system (nLC 1200-ESI-qTOF-MS / MS) to analyze and identify the peptides. Specifically, the target enzymatic crude extract sample was prepared with Tri-HCL solution at a concentration of 25 μg / mL and filtered with a 0.22 μm filter membrane. The sample 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). A 28% step normalized collision energy (NCE) was used in the HCD collision cell to generate product ions. The raw mass spectrometry data files were obtained and 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 to construct a bullfrog skin peptide library.Autodock Vina 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 high-affinity, non-toxic, and cell-penetrating peptides were screened, and their sequences were LGPQR, INTPVK (SEQ ID NO: 2), RLDDVT (SEQ ID NO: 3), RGFDQ (SEQ ID NO: 4), LGPDGR (SEQ ID NO: 5), and RGPVGP (SEQ ID NO: 6), 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, six peptides with high affinity, non-toxic / allergenic and cell-penetrating properties were screened.

[0029]

[0030] 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, LGPQR and INTPVK, 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 file was saved in pdbqt format for later use. The results of the LGPQR peptide docking site analysis are shown in Figure 2As shown, amino acid residues 125Arg, 205Glu, 206Glu, 357Phe, 547Tyr, 554Lys, 630Ser, 631Tyr, 662Tyr, and 666Tyr on the DPP-IV enzyme protein chain bind to the LGPQR peptide segment through hydrogen bonds, Pi-Sigma, Pi-Alkyl, Alkyl, charge, and non-complementary interactions, respectively. Specifically, 125Arg and 662Tyr form hydrogen bonds with the C1-position O of the Gln amino acid at the 4th position of the N-terminus of the LGPQR peptide, respectively; 205Glu forms a hydrogen bond with the branched H of the Arg amino acid at the 1st position of the C-terminus of the LGPQR peptide, 554Lys forms a hydrogen bond with the C1-position O of the Leu amino acid at the 1st position of the N-terminus of the LGPQR peptide, 630Ser and 631Tyr form hydrogen bonds with the branched O of the Gln amino acid at the 4th position of the N-terminus of the LGPQR peptide, and 630Ser forms a hydrogen bond with the C1-position O of the Pro amino acid at the 3rd position of the N-terminus of the LGPQR peptide; 357Phe and 666Tyr form hydrogen bonds with the branched C-position O of the Arg amino acid at the 1st position of the C-terminus of the LGPQR peptide, respectively. The 547Tyr forms a Pi-Alkyl bond with the branched H on the Leu amino acid at the 1st position of the N-terminal of the LGPQR peptide, and the 554Lys forms an Alkyl bond with the branched C on the Leu amino acid at the 1st position of the N-terminal of the LGPQR peptide; 125Arg binds to the N1 H on the Gln amino acid at the 4th position of the N-terminal of the LGPQR peptide and the C1 O on the Arg amino acid at the 1st position of the C-terminal by charge attraction; 125Arg binds to the N1 H on the Gln amino acid at the 4th position of the N-terminal of the LGPQR peptide by non-complementary interaction, and 554Lys binds to the C1 H on the Leu amino acid at the 1st position of the N-terminal of the LGPQR peptide by non-complementary interaction. The results of the docking site analysis of the INTPVK peptide are shown in the figure. Figure 3As shown, amino acid residues 125Arg, 357Phe, 545Asp, 547Tyr, 554Lys, 629Trp, 630Ser, 662Tyr, and 666Tyr on the DPP-IV enzyme protein chain bind to the INTPVK peptide segment through hydrogen bonds, Pi-Alkyl, Alkyl, charge, salt bridge, and non-complementary interactions, respectively. Specifically, 125Arg forms a hydrogen bond with the C1-position O of the Thr amino acid at the 3rd position of the N-terminus of the INTPVK peptide, 545Asp forms a hydrogen bond and a salt bridge with the branch chain H of the Lys amino acid at the 1st position of the C-terminus of the INTPVK peptide, 547Tyr forms a hydrogen bond with the N1-position H and the branch chain O of the Asn amino acid at the 2nd position of the N-terminus of the INTPVK peptide, 554Lys forms a hydrogen bond with the branch chain O of the Lys amino acid at the 1st position of the C-terminus of the INTPVK peptide and the C1-position O of the Val amino acid at the 2nd position of the C-terminus, 629Trp forms a hydrogen bond with the C1-position O of the Lys amino acid at the 1st position of the C-terminus of the INTPVK peptide, 630Ser forms a hydrogen bond with the C1-position O and N2-position O of the Asn amino acid at the 2nd position of the N-terminus of the INTPVK peptide, The side chain O of Thr amino acid at position 3 of the end forms a hydrogen bond, 662Tyr and 666Tyr form hydrogen bonds with the side chain H of Asn amino acid at position 2 of the N-terminal of INTPVK peptide, respectively; 357Phe forms a Pi-Alkyl bond with the side chain C of Ile amino acid at position 1 of the N-terminal of INTPVK peptide, 547Tyr and 554Lys form Pi-Alkyl and Alkyl bonds with the side chain C of Val amino acid at position 5 of the N-terminal of INTPVK peptide, respectively; 554Lys binds to the C1 position of Lys amino acid at position 1 of the C-terminal of INTPVK peptide by charge attraction; 554Lys binds to the side chain H of Asn amino acid at position 2 of the N-terminal of INTPVK peptide by non-complementary interaction. Therefore, LGPQR and INTPVK polypeptides bind tightly to the receptor protein DPP-IV enzyme, achieving the activity inhibition effect of DPP-IV enzyme. The sequence information and physicochemical property predictions of LGPQR and INTPVK peptides are shown in Table 2.

[0031]

[0032] Subsequently, the two peptide segments 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. Both peptide segments LGPQR and INTPVK have certain DPP-IV enzyme inhibition activity, but compared with the INTPVK peptide segment, the DPP-IV enzyme inhibition activity of the LGPQR peptide segment is stronger, with a difference of about 11.5 times (the smaller the IC50 value, the stronger the enzyme inhibition activity). ABTS +The scavenging activity and FRAP values ​​represent the antioxidant capacity of the peptide. As shown in Table 3, the peptides LGPQR and INTPVK both have good antioxidant capacity, and the antioxidant capacity of the LGPQR peptide is stronger than that of the INTPVK peptide. The LGPQR peptide with the best final verification result is the bullfrog skin-derived peptide LGPQR purified by the present invention.

[0033]

[0034] 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 LGPQR, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for purifying the bullfrog skin-derived peptide LGPQR 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 7 parts and deionized water was added to adjust the pH to 5.5-11 to prepare 7 parts of the prepared solution. Bromelain, flavor protease, composite protease, alkaline protease, papain, trypsin and neutral protease were added to the 7 parts of the prepared solution at an enzyme / substrate ratio of 1.8-6.3%, and then the 7 parts of the prepared solution were placed at 20-60 ℃ for enzymatic hydrolysis for 2-6 h; Preparation of crude enzymatic extracts: 7 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 7 portions of crude enzymatic extracts; Activity determination: the hydrolysis degree, nitrogen recovery rate and DPP-IV enzyme inhibition activity of 7 enzymatic crude extracts were determined. The enzyme inhibition activity of the enzymatic crude extract corresponding to papain was significantly higher, and it was selected as the target enzymatic crude extract; Peptide screening: Use mass spectrometer to analyze and measure the peptides contained in the target enzymatic crude extract to construct a peptide library. Use computer-aided 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 LGPQR.

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 any one of claims 2 to 6, characterized in that: The DPP-IV enzyme inhibition activity is verified by measuring the DPP-IV enzyme activity inhibition experiment, the ABTS free radical scavenging activity is verified by measuring the ABTS·+ scavenging experiment, and the FRAP antioxidant activity is verified by measuring the FRAP assay experiment.

8. The purification method according to any one of claims 2 to 6, 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. An application of the bullfrog skin-derived peptide LGPQR as claimed in claim 1, characterized in that: Used for preparing drugs for type II diabetes.

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