XOD inhibitory peptide and its application

Abstract

The present invention belongs to the field of biotechnology and discloses a XOD inhibitory peptide and application. The XOD inhibitory peptide provided by the present invention is selected from one of the peptides whose amino acid sequences are shown in SEQ ID NO.1-2. The XOD inhibitory peptide provided by the present invention has a high XOD inhibition rate and can be used to prepare a product for reducing uric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides and relates to a XOD inhibitory peptide and application thereof. Background Art

[0002] Hyperuricemia is a metabolic syndrome caused by purine metabolism disorder. When the uric acid level in the blood or tissue fluid reaches above its saturation concentration, sodium urate crystals are easily produced, inducing arthritis and tissue destruction, namely gout. Experts widely believe that gout is a chronic, systemic disease with a clear causal relationship with chronic kidney disease and kidney stones, and increases the risk of hypertension, new-onset diabetes, and coronary heart disease. Among them, xanthine oxidase (XOD), as a key enzyme in the production of uric acid, can catalyze hypoxanthine to produce xanthine, and further convert it into uric acid. The food-borne XOD inhibitory peptide currently obtained from aquatic products has no side effects, is easily absorbed, and has a wide range of sources. Among them, fish meat is rich in protein and is an excellent source for the preparation of XOD inhibitory peptides.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a XOD inhibitory peptide and application thereof. The XOD inhibitory peptide provided by the present invention has XOD inhibitory activity and can be used in the preparation of uric acid regulating products.

[0005] To achieve the above object, the first technical solution provided by the present invention is:

[0006] The XOD inhibitory peptide is selected from one of the peptides whose amino acid sequences are shown in SEQ ID NO.1-2.

[0007] Preferably, the white mullet XOD inhibitory peptide is extracted from white mullet.

[0008] Preferably, the extraction method is: homogenizing the white mullet, simulating human in vitro digestion and enzymatic hydrolysis to obtain an enzymatic solution; using ultrafiltration to separate the enzymatic solution to retain ultrafiltration components with a molecular weight cutoff of less than 3000Da; using gel chromatography to chromatograph the ultrafiltration components; and separating and purifying the components obtained after chromatography.

[0009] The second technical solution provided by the present invention is: use of any of the above-mentioned XOD inhibitory peptides in food.

[0010] The third technical solution provided by the present invention is: use of any of the above-mentioned XOD inhibitory peptides in the preparation of uric acid regulating products.

[0011] The above-mentioned XOD inhibitory peptide can also be synthesized using amino acids as raw materials by solid phase synthesis method, which is well known in the art.

[0012] The XOD inhibitory peptide provided by the present invention has strong XOD inhibitory activity and is a polypeptide with a certain function of reducing uric acid.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The XOD inhibitory peptide provided by the present invention has high XOD inhibitory activity, has a certain function of reducing uric acid, and has good application prospects in the field of drugs with uric acid reducing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the chromatogram of the ultrafiltration component with a molecular weight below 3000Da after being separated by Sephadex G-15 gel chromatography;

[0016] Figure 2 is the XOD activity inhibition rate of chromatographic components G1, G2, and G3;

[0017] Figure 3 is the high performance liquid chromatogram of the chromatography component G2;

[0018] Figure 4 is the high performance liquid chromatogram of the chromatography component G3;

[0019] Figure 5 is the secondary mass spectrum of the sequence ALKKKMQM;

[0020] Figure 6 is the secondary mass spectrum of the sequence YGTWRKPSSL;

[0021] Figure 7 Ultra-high performance liquid chromatogram for purity verification of the synthetic compound of sequence SEQ ID NO.1;

[0022] Figure 8 Ultra-high performance liquid chromatogram for purity verification of the synthetic compound of sequence SEQ ID NO.2;

[0023] Fig. 9 The results of XOD inhibition activity of the compound of sequence SEQ ID NO.1;

[0024] Fig.10 The XOD inhibitory activity results of the compound with sequence SEQ ID NO.2 are shown. DETAILED DESCRIPTION

[0025] The present invention is further explained below in conjunction with the accompanying drawings and examples. The purpose of the present invention is to use white mullet meat as raw material, undergo proteolysis, separate and screen out proteins with clear sequences. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.

[0026] The first embodiment of the present invention provides a XOD inhibitory peptide selected from one of the peptides having amino acid sequences as shown in SEQ ID NO. 1-2.

[0027] Among them, SEQ ID NO.1 is ALKKKMQM;

[0028] SEQ ID NO.2 is YGTWRKPSSL.

[0029] In the embodiment of the present invention, the bioactive peptide is extracted from white mullet. In fact, it is also possible to use amino acids as raw materials and adopt chemical methods to synthesize peptides with amino acid sequences of SEQ ID NO.1-2, which is well known in the art.

[0030] In a specific embodiment, the specific method for extracting the bioactive peptides is as follows: removing the by-products of the white mullet, adding ultrapure water 2-6 times the mass of the fish meat for homogenization to obtain a homogenous material; adjusting the pH of the homogenous material to 2.0 with HCl, adding pancreatic enzyme at 1%-4% of the mass of the homogenous material, simulating intestinal digestion for 2.5 hours under 37°C water bath conditions, placing it in boiling water for 10 minutes to terminate digestion, and cooling it to room temperature to obtain an enzymatic solution; centrifuging the above enzymatic solution to obtain the supernatant and vacuum freeze-drying to obtain a crude peptide powder of the white mullet; ultrafiltration separation of the above crude peptide powder, retaining an ultrafiltration component G with a molecular weight of less than 3000Da, freeze-drying and storing it at -18°C; preparing the above ultrafiltration component G into a solution with a concentration of 10-50 mg / mL, chromatographing it using Sephadex G-15 dextran gel chromatography, and freeze-drying the obtained components and storing them at -18°C; selecting the components with XOD inhibitory activity after chromatography through XOD inhibitory activity assay, and separating and purifying the components using RP-HPLC method.

[0031] In a further embodiment, the centrifugation conditions are 4°C, 8000xg, and time 20 min.

[0032] In a further embodiment, the Sephadex G-15 dextran gel chromatography method has a sample loading amount of (2-4) mL, an elution rate of (1-2) mL / min, and a detection wavelength of 220 nm.

[0033] Furthermore, the sample loading volume is 4 mL.

[0034] In the following specific implementation of the present invention, the ultrafiltration fractions were chromatographed using Sephadex G-15 dextran gel chromatography, and the fractions having XOD inhibitory activity after chromatography were selected, and further separated using RP-HPLC, and then identified by LC-MS / MS.

[0035] Among them, the determination of XOD inhibitory activity is through the determination of xanthine oxidase inhibitory activity, and the main determination method is spectrophotometry. The test basis is that uric acid has a characteristic absorption peak at 295nm.

[0036] The specific method for determining XOD inhibitory activity is as follows:

[0037] Solution preparation: The catalytic reaction is carried out in a 200 mM (pH=7.5) phosphate buffer solution. Xanthine solution: Accurately weigh xanthine and add a small amount of 1.0 M NaOH solution to dissolve it, add 100 mL of phosphate buffer solution, and adjust the pH to 7.5 with HCl solution to obtain a substrate solution (0.42 mM). XOD working solution: Dilute XOD with an initial activity of 100 U with phosphate buffer solution to a final concentration of 0.10 U / mL.

[0038] Chromatographic conditions: The chromatographic column was a Symmetry C18 column, the mobile phase was a potassium dihydrogen phosphate aqueous solution at pH=6.5, the flow rate was 1.0 mL / min, the detection wavelength was 290 nm, and the injection volume was 20 μL. At the same time, phosphate buffer was used as a blank control.

[0039] Preparation of uric acid standard solution: dilute the uric acid standard to 20, 40, 80, 180, and 360 mmol / L uric acid standard solutions. The injection volume is 20 μL, and liquid phase detection is performed at 290 nm to obtain a uric acid standard curve.

[0040] When measuring the XOD inhibitory activity of the sample, first add 50 µL of XOD solution (0.50 U / mL) and 50 µL of phosphate buffer solution (200 mM, pH=7.8) or a certain concentration of white mullet XOD inhibitory peptide solution to the microwells in the 96-well plate, incubate at 25°C for 5 min, then add 150 µL of substrate solution to start the catalytic reaction. After 30 min of reaction, add 80 mL of 1M hydrochloric acid to stop the enzymatic reaction. Then dilute 10 times with buffer, filter the diluted mixture with a 0.22μm nylon membrane and perform HPLC analysis. Phosphate buffer (200 mM, pH=7.5) was used as a negative control.

[0041] The calculation formula of XOD activity inhibition rate is as follows: XOD inhibition rate (%) = (1 − B / A) × 100%, where A – uric acid content of positive control (allopurinol) reaction; B – uric acid content of sample reaction;

[0042] In order to make the technical solution of the present invention clearer, the extraction and effect of XOD inhibitory peptides are described in detail below through multiple specific examples.

[0043] Example 1

[0044] The specific steps of the method for extracting, separating and purifying XOD inhibitory peptides are as follows:

[0045] The by-products of white mullet are removed, and ultrapure water with an amount 2-6 times the weight of the fish meat is added for homogenization to obtain a homogenous material; the pH of the homogenous material is adjusted to 2.0 with HCL, and pancreatic enzyme is added at a weight of 1%-4% of the weight of the homogenous material, and intestinal digestion is simulated for 2.5 hours in a water bath at 37° C., and the material is placed in boiling water for 10 minutes to terminate the digestion, and then cooled to room temperature to obtain an enzymolysis solution; the enzymolysis solution is centrifuged to obtain the supernatant, and vacuum freeze-dried to obtain a crude peptide powder of white mullet; the crude peptide powder is separated by ultrafiltration, and an ultrafiltration component G with a molecular weight of less than 3000 Da is retained, and the ultrafiltration component G is freeze-dried and stored at -18° C.

[0046] The ultrafiltration component G was prepared into a solution with a concentration of 25 mg / mL and filtered through a 0.22 um aqueous microporous filter membrane. The solution was chromatographed using Sephadex G-15 dextran gel chromatography, with double distilled water as the eluent. The chromatography method was as follows: the elution flow rate was 1 mL / min, the detection wavelength was 220 nm, and the sample volume was 4 mL. The chromatographic results are shown in Figure 1 As shown, the horizontal axis represents the elution time (min), and the vertical axis represents the absorbance A. The three separated components were successively obtained in the chromatography collection and elution process, and were labeled G1, G2, and G3 in turn, and freeze-dried and stored at -18°C. The G1, G2, and G3 components obtained after chromatography were selected for XOD inhibition activity determination, and the results were shown in Figure 2 As shown in the figure, the XOD inhibition activity of components G2 and G3 was the best, and components G2 and G3 were selected for the next step of RP-HPLC separation and purification.

[0047] RP-HPLC was used to separate and purify the G2 and G3 components. The separation conditions of RP-HPLC were as follows: mobile phase A was water containing 0.1% TFA; mobile phase B was acetonitrile containing 0.1% TFA; gradient elution, i.e. 0min 2% B, 0-18min 2%-10% B, 18-36min 10%-20B, 36-55min 20%-30B, 55-70min 30%-40% B, analytical column was C18 (3μm, 100A), injection volume was 10μL; flow rate was 1mL / min.

[0048] The RP-HPLC separation patterns of the chromatographic components G2 and G3 are as follows: Figure 3-4 In the figure, the horizontal axis represents the elution time (min), and the vertical axis represents the response value (AU).

[0049] The purified components were identified by liquid chromatography-mass spectrometry. The liquid phase conditions were as follows: mobile phase A was water containing 0.1% formic acid; mobile phase B was water containing 0.1% formic acid and 80% CAN; gradient elution, i.e. 0min 4% B, 0-2min 4%-8% B, 2-45min 8%-28% B, 45-55min 28%-40% B, 55-56min 40%-95% B, 56-66min 95% B, analytical column was packed with Acclaim PepMap RPLC C18 (3μm, 150μm id×150mm); injection volume was 10μL; flow rate was 600nL / min. Mass spectrometry conditions: primary mass spectrometry parameters were Resolution 70000, AGCtarget 3e6, MaximumIT100 ms, Scanrange100to1500m / z; secondary mass spectrometry parameters were Resolution 17500, AGCtarget 1e5MaximumIT 50 ms, TopN20, nce / steppedNCE28.

[0050] The amino acid sequences of the identified anti-inflammatory peptides from white mullet are:

[0051] G2 component: Tyr-Gly-Thr-Trp-Arg-Lys-Pro-Ser-Ser-Leu (YGTWRKPSSL);

[0052] G3 component: Ala-Leu-Lys-Lys-Lys-Met-Gln-Me (ALKKKMQM).

[0053] like Figure 5-6 Table 1 shows the information of the above XOD inhibitory peptides.

[0054] Table 1 is the information of XOD inhibitory peptides

[0055] .

[0056] Example 2

[0057] In order to further verify the XOD inhibitory activity of the above XOD inhibitory peptide, the sequences of SEQ ID NO.1~2 were synthesized by solid phase synthesis method to obtain the synthetic compounds of SEQ ID NO.1~2.

[0058] The purity of the synthetic product was tested by the separation and purification method in Example 1. The test results are as follows: Figure 7-8 The purity of the synthetic products of sequence SEQID NO.1~2 was ≥98%.

[0059] Using allopurinol as a positive control, the XOD inhibitory activity of the above-mentioned SEQ ID NO.1~2 compounds and dexamethasone at different concentrations (0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL) was measured. The results are shown in Figure 9-10 As shown, the positive control group is the allopurinol group, ALKKKMQM is the synthetic sample of sequence SEQ ID NO.1, and YGTWRKPSSL is the synthetic sample of sequence SEQ ID NO.2.

[0060] according to Figure 9-10 The results show that the greater the concentration of the two samples, the higher the XOD inhibition rate. Fig. 9 In the experiment, when the concentration was 4 mg / mL, the XOD inhibitory activity of allopurinol in the control group was 89.23%, and the XOD inhibitory activity of the peptide ALKKKMQM was 71.37%, indicating that the sequence ALKKKMQM has good XOD inhibitory activity. Fig.10 In the experiment, the XOD inhibitory activity of allopurinol in the control group was 91.23%, and that of the peptide YGTWRKPSSL was 69.21%, indicating that the sequence YGTWRKPSSL has good XOD inhibitory activity.

[0061] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content of the present invention by a person skilled in the art should be considered as the protection scope of the claims of the present invention.

Claims

1. A XOD inhibitory peptide, characterized in that The peptide is selected from the group consisting of the peptides with amino acid sequences as shown in SEQ ID NO.1~2.

2. Use of the XOD inhibitory peptide according to claim 1 in the preparation of uric acid regulating drugs.

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