Bioactive peptides with uric acid-lowering effects, preparations and applications thereof

Abstract

The present invention discloses bioactive peptides, preparations and applications thereof with uric acid lowering efficacy, and belongs to the technical field of bioactive peptides. The amino acid sequence of the bioactive peptide with uric acid lowering efficacy of the present invention is at least one of CPGVC and FPTLVRPT. The present invention obtains two bioactive peptides with uric acid lowering efficacy by separation and purification from phycocyanin, and preliminarily identifies their components and sequences by liquid chromatography-mass spectrometry. Chemical synthesis is performed according to the identified peptide sequence, and the uric acid lowering activity of the synthesized single peptide and compound peptide is detected. These bioactive peptides have good xanthine oxidase inhibitory activity, and therefore have good application prospects in the preparation of products with uric acid lowering efficacy, laying a foundation for the high added value utilization of phycocyanin and promoting the research and development and application of functional active peptides.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and in particular relates to bioactive peptides with uric acid lowering effects, preparations and applications thereof. Background Art

[0002] Gout is a metabolic disorder caused by high uric acid levels in the blood that seriously affects the quality of life of modern people. The incidence rate is gradually increasing. The current clinical treatment mechanism of gout drugs includes inhibiting the production of uric acid, promoting the excretion of uric acid and decomposing uric acid. Among them, xanthine oxidase (XO) is a key enzyme in the human body that catalyzes hypoxanthine to generate xanthine, and then generates uric acid or directly catalyzes xanthine to uric acid. Therefore, the rate of uric acid generation depends on the activity of XO to a certain extent. At present, drugs such as allopurinol and febuxostat are commonly used to inhibit XO activity, and the treatment effect is relatively significant, but there is a risk of inducing gout in the early stage of treatment, and there are certain toxic and side effects. With the improvement of people's health awareness, finding drug substitutes with less toxic and side effects has become a current research and development hotspot. A variety of natural products have been shown to have uric acid-lowering activity, such as flavonoids, phenolic acids, polysaccharides, bioactive peptides, etc. Among them, bioactive peptides have attracted much attention due to their low preparation cost, low sample cost, easy absorption, and high safety.

[0003] Bioactive peptides are specific protein fragments that have a positive impact on the body's health. Compared with complete proteins, small molecule peptide fragments have multiple advantages: they are easier to be absorbed and utilized by the human body, have high activity, and can play a physiological role even at low concentrations; they have a small molecular weight, are easy to modify and transform, and can be obtained through artificial chemical synthesis and other means. Compared with single amino acids, small molecule peptides not only have special physiological activities, but also have unique advantages in absorption channels and speed.

[0004] Phycocyanin is a functional protein in Spirulina that has anti-tumor, anti-inflammatory and antioxidant effects. However, as a biological macromolecule, phycocyanin has poor stability, is easily inactivated, and is not easily digested and absorbed by the human body. In order to overcome these problems, appropriate proteases can be selected to hydrolyze phycocyanin to obtain small molecule peptides, which have higher digestibility and functional activity. This method can not only enhance the bioavailability of phycocyanin, but also further enhance its application potential in health and pharmaceutical products.

[0005] Therefore, developing bioactive peptides with uric acid-lowering effects from phycocyanin can not only provide natural and safe uric acid-lowering products, but also promote the intensive utilization and industrial development of phycocyanin resources, which is of great significance for promoting human health and economic development. Summary of the invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to provide a bioactive peptide, a preparation and an application thereof having the effect of lowering uric acid.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A bioactive peptide with uric acid lowering effect, wherein the amino acid sequence of the bioactive peptide is at least one of CPGVC and FPTLVRPT.

[0009] Application of the above-mentioned bioactive peptides with uric acid lowering effect in the preparation of products with uric acid lowering effect.

[0010] Based on the above scheme, the product is a drug.

[0011] Based on the above scheme, the product further comprises pharmaceutically acceptable excipients.

[0012] A preparation with uric acid lowering effect, wherein the active ingredient is at least one of the bioactive peptides shown in CPGVC and FPTLVRPT.

[0013] Based on the above scheme, the concentration of the active ingredient is 1 mg / mL.

[0014] On the basis of the above scheme, the uric acid lowering agent has a xanthine oxidase inhibitory effect.

[0015] The method for preparing a bioactive peptide having the effect of lowering uric acid from phycocyanin comprises the following steps:

[0016] (1) dissolving phycocyanin powder in ultrapure water to prepare a protein solution, adding protease to perform enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, centrifuging to obtain a supernatant;

[0017] (2) ultrafiltration of the supernatant obtained by the above enzymatic hydrolysis to obtain protein hydrolyzate with a molecular weight range of <3 kDa, 3-10 kDa, and >10 kDa;

[0018] (3) taking the protease hydrolysate with a molecular weight of <3 kDa and further purifying it by gel permeation chromatography to obtain multiple polypeptide components;

[0019] (4) determining the uric acid-lowering activity of the polypeptide components obtained in step (3), selecting the component with the highest uric acid-lowering activity and further purifying it by reverse phase HPLC to obtain multiple further purified polypeptide components;

[0020] (5) Determine the uric acid-lowering activity of the multiple polypeptide components further purified in step (4), select the component with the highest uric acid-lowering activity, and determine the amino acid sequence of the resulting peptide fragment by liquid chromatography-mass spectrometry.

[0021] Based on the above scheme, the phycocyanin in step (1) is Spirulina phycocyanin.

[0022] On the basis of the above scheme, the protease in step (2) is alkaline protease, the addition amount of the alkaline protease is 2000U / g, the enzymatic hydrolysis conditions are 50°C, pH 10.5, and the enzymatic hydrolysis is 6h.

[0023] Advantages of the technical solution of the present invention

[0024] The present invention separates and purifies two bioactive peptides with uric acid-lowering efficacy from phycocyanin by enzymatic hydrolysis, ultrafiltration, gel chromatography, and liquid chromatography separation techniques, and preliminarily identifies its components and sequences by liquid chromatography-mass spectrometry. Chemical synthesis is performed based on the identified peptide sequences, and the uric acid-lowering activity of the synthesized single peptides and compound peptides is tested. These bioactive peptides have good xanthine oxidase inhibitory activity, and therefore have good application prospects in the preparation of products with uric acid-lowering efficacy, laying a foundation for the high added value utilization of phycocyanin and the promotion of the research and development and application of functional active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Degree of hydrolysis of different phycocyanin hydrolysates;

[0026] Figure 2 The results of xanthine oxidase inhibition rate determination of different phycocyanin hydrolysates;

[0027] Figure 3 Electrophoresis analysis of the product obtained by alkaline protease enzymatic hydrolysis of phycocyanin (where M is a protein marker with a molecular weight of 3.3-20.1 KDa; 1 is the product obtained by alkaline protease enzymatic hydrolysis of phycocyanin)

[0028] Figure 4 The results of xanthine oxidase inhibition rate determination of different molecular weight products obtained by alkaline protease hydrolysis of phycocyanin;

[0029] Figure 5 Superdex of components with molecular weight <3KDa TM Increase 10 / 300 GL gel chromatogram;

[0030] Figure 6 From Superdex TM Increase 10 / 300 GL The results of xanthine oxidase inhibition rate determination of each component collected during filtration;

[0031] Figure 7 Superdex loaded on C18 column TMRP-HPLC chromatogram of the active fraction of Increase 10 / 300 GL;

[0032] Figure 8 The results of xanthine oxidase inhibition rate determination of each component collected from RP-HPLC;

[0033] Fig. 9 Mass spectrometry analysis results of phycocyanin peptide CPGVC;

[0034] Fig.10 Mass spectrometry analysis results of phycocyanin peptide FPTLVRPT;

[0035] Fig.11 Molecular weight plot of the mass spectrum of peptide CPGVC;

[0036] Fig.12 Molecular weight plot of the mass spectrum of the peptide FPTLVRPT;

[0037] Fig.13 The results of determination of xanthine oxidase inhibition rate of chemically synthesized peptides CPGVC, FPTLVRPT and two composite peptides (CPGVC+FPTLVRPT). DETAILED DESCRIPTION

[0038] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0040] In the following embodiments,

[0041] 1. Determination of degree of hydrolysis (DH)

[0042] The degree of hydrolysis of phycocyanin was determined by the o-phthalaldehyde (OPA) method, and the specific method is as follows: 80 mg of OPA was dissolved in 2 mL of β-mercaptoethanol, 5 mL of 10% SDS (w / v) and 92.8 mL of 0.1 M sodium tetraborate to prepare a 100 mL OPA reagent solution. 4 mL of phycocyanin hydrolysate (alkaline protease, papain, trypsin, pepsin, neutral protease and acid protease hydrolysate) was mixed with 4 mL of OPA reagent, incubated at room temperature for 2 minutes, and the absorbance at a wavelength of 340 nm was measured.

[0043] Phycocyanin was placed in 6M HCl and reacted at 4°C for 24 hours. The amount of free amino acids was determined using a serine standard curve as the amount of free amino acids produced by the complete hydrolysis of phycocyanin.

[0044] The degree of hydrolysis of phycocyanin was calculated according to the following formula:

[0045] Degree of hydrolysis (%) = [(NH 2 ) t -(NH 2 ) t0 ] / (NH 2 ) T

[0046] Where: (NH 2 ) t represents the amount of free amino acids in the enzymatic hydrolysis product at time t; (NH 2 ) t0 Represents the amount of free amino acids that have not been hydrolyzed by enzymes; (NH 2 ) T Represents the amount of free amino acids after complete hydrolysis.

[0047] 2. Determination of xanthine oxidase (XO) inhibitory activity

[0048] Take 70.2061g 4-aminoantipyrine, 0.018g horseradish peroxidase, and 0.5594g phenol, dilute to 1L with Tris-HCl buffer to prepare a color developing solution; add 50μL of XOD (0.52U / mL) solution and 50μL of the sample to be tested to each well of a 96-well plate, shake for 30s and incubate at 37°C for 10min; after incubation, add 100μL of xanthine solution (2.0mmol / L) and 100μL of color developing solution.

[0049] The mixture was sample group A1, the buffer-replaced xanthine solution was recorded as A0, and the buffer-replaced sample was recorded as A2. The 96-well plate was then placed at 37°C for 15 min, 30 μL of NaOH (1.0 mol / L) was added to terminate the reaction, and the absorbance at 508 nm was measured.

[0050] The uric acid-lowering activity was calculated using the following formula:

[0051] Xanthine oxidase (XO) inhibitory activity (%) = [A2-(A1-A0)] / A2×100

[0052] Wherein, A1 is the absorbance of the sample group with the mixture as the sample, A0 is the absorbance of the sample group without xanthine, and A2 is the absorbance of the buffer group containing xanthine.

[0053] The phycocyanin in the following examples is Spirulina phycocyanin purchased from Zhejiang Binmei Biotechnology Co., Ltd.

[0054] Example 1

[0055] A bioactive peptide having the effect of lowering uric acid, wherein the bioactive peptide comprises a peptide having an amino acid sequence shown in at least one of SEQ ID NO: 1 and SEQ ID NO: 2:

[0056] SEQ ID NO: 1: CPGVC;

[0057] SEQ ID NO:2: FPTLVRPT.

[0058] Example 2

[0059] The method for preparing a bioactive peptide having the effect of lowering uric acid from phycocyanin comprises the following steps:

[0060] (1) Take 5g of phycocyanin powder, prepare a protein solution with a concentration of 5% (w / v) with ultrapure water, use ultrasonic cell disruption treatment, set at a power of 450W for 30min, then add 2000U / g (2000U added per gram of phycocyanin powder) of alkaline protease, adjust the pH to 10.5, and perform enzymolysis at 50℃ for 6h. After the enzymolysis reaction is completed, the enzymolysis solution is boiled at 100℃ for 10min to terminate the reaction. The enzymolysis solution is centrifuged at 6000r / min and 4℃ for 15min, and the supernatant is retained.

[0061] Effects of ultrasound-assisted treatment and protease selection on the degree of hydrolysis of phycocyanin and the inhibition of xanthine oxidase activity related to uric acid-lowering activity

[0062] Phycocyanin was divided into a group using ultrasound-assisted treatment (set at a power of 450W for 30min) and a group without ultrasound-assisted treatment. Each group was then enzymatically hydrolyzed with alkaline protease, papain, trypsin, pepsin, neutral protease and acidic protease. The temperature and pH of the enzymatic hydrolysis were carried out at the optimal temperature and pH of each protease (Table 1), and the other conditions were the same as those of alkaline protease. After the enzymatic hydrolysis was completed, the degree of hydrolysis (DH, %) of the phycocyanin hydrolyzate by different proteases in the ultrasound-assisted treatment group and the group without ultrasound-assisted treatment was determined. The results are shown in Figure 1. Figure 1 The results showed that the hydrolysis ability of alkaline protease was the best in both the ultrasound-assisted treatment group and the non-ultrasound-assisted treatment group, and the ultrasound-assisted treatment group was better than the non-ultrasound-assisted treatment group ( Figure 1 ).

[0063] The effects of different proteases on the inhibition of xanthine oxidase activity related to anti-uric acid activity in the ultrasound-assisted group were then measured. The results showed that the anti-uric acid effect of alkaline protease hydrolysate was significant ( Figure 2 ).

[0064] Table 1 Optimal temperature and pH of different proteases

[0065]

[0066] (2) The supernatant obtained by the above enzymatic hydrolysis is fractionated into different molecular weights using an ultrafiltration centrifuge tube, with a molecular weight cutoff of 10 and 3 kDa. The fractions are collected as follows: >10 kDa, the peptide is retained but does not pass through the 10 kDa membrane; 3-10 kDa, the peptide penetrates the 10 kDa membrane but does not penetrate the 3 kDa membrane; <3 kDa, the peptide penetrates the 3 kDa membrane. Thus, the protease hydrolysate with a molecular weight range of <3 kDa, 3-10 kDa, and >10 kDa is obtained ( Figure 3 ).

[0067] The inhibitory effect of xanthine oxidase activity related to uric acid-lowering activity was determined for components of different molecular weights. The results showed that protein peptides with a smaller molecular weight <3KDa showed better inhibitory effects on xanthine oxidase ( Figure 4 ).

[0068] (3) Take the <3kDa enzymatic solution and run it on Superdex TM The product was further purified by filtration on an Increase 10 / 300 GL gel permeation column (10×300 mm). The column was eluted with deionized water (pH 10.5) and fractions were collected at a flow rate of 1 mL / min. The fractions were detected at 280 nm. Four peptide fractions were collected in the order of the peak elution time and named F1, F2, F3 and F4 ( Figure 5 ).

[0069] The four polypeptide components obtained above were tested for their inhibitory effects on xanthine oxidase activity, which is related to uric acid-lowering activity. The results showed that the four components all had the ability to inhibit xanthine oxidase ( Figure 6 ), among which component F3 has the most significant effect.

[0070] (4) The fraction F3 obtained from the gel permeation chromatography was further purified using reverse phase HPLC on a C18 column. A linear gradient of acetonitrile (0-40%) containing 0.1% trifluoroacetic acid (TFA) was used at a flow rate of 1 mL / min. The elution peak was detected at 215 nm, and five polypeptide fractions were collected in the order of the peak elution time and named F3-I, F3-II, F3-III, F3-IV and F3-V. Figure 7The active peak was concentrated by rotary evaporator. The xanthine oxidase inhibition rate was determined. The results showed that F3-Ⅱ had a good effect on xanthine oxidase inhibition ( Figure 8 ).

[0071] (5) Determination of the amino acid sequence of F3-Ⅱ. Liquid chromatography-mass spectrometry was used, and the liquid phase was Easy nLC 1200 nanoliter liquid phase system. The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography (Acclaim PepMap RSLC, 75μm×25cm C18-2μm 100A). The gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 minutes. The mass spectrometer used a ThermoFisher Q Exactive system combined with a nanoliter spray Nano Flex ion source (ThermoFisher, USA), the spray voltage was 1.9kV, and the ion transfer tube heating temperature was 275℃.

[0072] The mass spectrometer scanning mode is in the information-dependent acquisition working mode (DDA, Data Dependent Analysis), the primary mass spectrometer scanning resolution is 70000, the scanning range is 350-2000m / z, and the maximum injection time is 100ms. In each DDA cycle, a maximum of 20 secondary spectra with charges of 2+ to 5+ are collected, and the maximum injection time of the secondary mass spectrometer ions is 50ms. The collision cell energy (high energy collision induced dissociation, HCD) is set to 28eV, which is applicable to all precursor ions, and the dynamic exclusion is set to 25s.

[0073] As a result, two peptides were obtained (Table 2). The secondary mass spectrometry analysis showed that the peptide had a molecular weight of 477.1716 and a sequence of CPGVC ( Fig. 9 ); a peptide with a molecular weight of 929.5334 and a sequence of FPTLVRPT( Fig.10 ).

[0074] Table 2 List of amino acid sequences of component F3-Ⅱ determined by LC-MS

[0075]

[0076] (6) The sequences CPGVC and FPTLVRPT in Table 2 above were chemically synthesized ( Fig.11 and Fig.12 ) to obtain bioactive peptides with the effect of lowering uric acid.

[0077] Example 3

[0078] A bioactive peptide preparation with uric acid lowering effect, wherein the active ingredient is at least one of the bioactive peptides with amino acid sequences of CPGVC and FPTLVRPT.

[0079] Using pure water as solvent, single peptide CPGVC, FPTLVRPT and composite peptide CPGVC+FPTLVRPT were prepared into a solution with a concentration of 1 mg / mL. In the composite peptide, the mass ratio of the two peptides was 1:1, and the concentration of each peptide was 1 mg / mL. The xanthine oxidase inhibition ability of single peptide CPGVC, FPTLVRPT and composite peptide CPGVC+FPTLVRPT solutions was determined. The results showed that the xanthine oxidase inhibition effect was: CPGVC>CPGVC+FPTLVRPT>FPTLVRPT( Fig.13 ).

[0080] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A bioactive peptide having uric acid lowering effect, characterized in that: The amino acid sequence of the bioactive peptide is at least one of CPGVC and FPTLVRPT.

2. Use of the bioactive peptide with uric acid lowering effect according to claim 1 in the preparation of a product with uric acid lowering effect, characterized in that: The product described is a drug.

3. The use of the bioactive peptide having uric acid lowering effect according to claim 2 in the preparation of a product having uric acid lowering effect, characterized in that: The product also contains pharmaceutically acceptable excipients.

4. A preparation having the effect of lowering uric acid, characterized in that: The active ingredient is at least one of the bioactive peptides shown by CPGVC and FPTLVRPT.

5. The preparation having uric acid lowering effect according to claim 4, characterized in that: The concentration of active ingredient is 1 mg / mL.

6. The preparation having uric acid lowering effect according to claim 4 or 5, characterized in that: The uric acid lowering effect has the effect of inhibiting xanthine oxidase.

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