Sunflower disc uric acid lowering peptide as well as preparation method and application thereof

Abstract

The invention provides a sunflower disc uric acid lowering peptide as well as a preparation method and application thereof, and belongs to the field of vegetable protein polypeptides. The preparation method of the uric acid-reducing peptide of the sunflower disc comprises the following steps: step (1), airing and drying the sunflower disc, shearing, crushing, and sieving with a 40-mesh sieve to obtain sunflower disc powder; (2) soaking the sunflower disc powder in the step (1) by using an organic solvent, dialyzing and freeze-drying to obtain sunflower disc protein; (3) preparing a protein solution by using the sunflower disc protein in the step (2), and freeze-drying to obtain crude sunflower disc zymolyte; and (4) separating and purifying the zymolyte in the step (3) by using column chromatography, collecting eluent, and freeze-drying to obtain the sunflower disc polypeptide. The sunflower disc uric acid reducing peptide processed on the extracted and purified protein is high in purity, and the sunflower disc peptide with the high xanthine oxidase inhibition rate can be obtained through simple purification.

Description

Technical Field

[0001] The present invention relates to the technical field of plant protein polypeptides, in particular to a sunflower disc uric acid-lowering peptide and a preparation method and application thereof. Background Art

[0002] Uric acid is the end product of purine metabolism in the body. When its concentration is higher than 7mg / dL (male) or 6mg / dL (female), it will cause damage to the body and can be identified as a person with high uric acid. Currently, the drugs used clinically are divided into three categories: inhibiting uric acid synthesis (allopurinol and febuxostat), promoting uric acid excretion (probenecid and benzbromarone) and uricase. These drugs are only allowed to be used by people with high uric acid who have gout and related symptoms, and will cause side effects such as liver and kidney damage. There is currently no clear intervention for asymptomatic people with high uric acid, but long-term high uric acid will increase the risk of cardiovascular and kidney diseases.

[0003] In recent years, researchers have been committed to developing safe and effective natural functional compounds to alleviate hyperuricemia, such as polyphenols, alkaloids and saponins. However, these compounds are low in content and difficult to purify, and their preparation is challenging. Bioactive peptides and protein hydrolysates extracted from food proteins have attracted much attention due to their advantages such as easy absorption, low cost, and diverse biological activities, and a large number of literatures have shown their potential to lower uric acid. Sunflower disks, as a processing byproduct with a large output, have great development value. Therefore, the functional development of sunflower disk protein peptides is of great significance. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a sunflower disc uric acid-lowering peptide and a preparation method and application thereof, which solves the problem of low purity of the existing sunflower disc uric acid-lowering peptide extraction.

[0005] A method for preparing a sunflower disc uric acid-lowering peptide of the present invention comprises the following steps:

[0006] Step (1): after drying the sunflower discs, shearing, crushing, and passing through a 40-mesh sieve to obtain sunflower disc powder;

[0007] Step (2): soaking the sunflower disc powder in step (1) with an organic solvent, placing it in a fume hood for 10 hours to evaporate the solvent, adding distilled water, stirring and extracting, centrifuging and filtering under reduced pressure, retaining the filtrate, dialyzing and freeze-drying to obtain sunflower disc protein;

[0008] Step (3): using the sunflower disk protein in step (2) to prepare a protein solution, boiling it in a water bath and cooling it, performing enzymolysis twice using a protease, centrifuging and retaining the supernatant, decolorizing the supernatant, centrifuging and filtering, and freeze-drying to obtain a crude enzymolyzate of the sunflower disk;

[0009] Step (4): using column chromatography to separate and purify the enzymatic hydrolysate in step (3), collecting the eluate, and freeze-drying to obtain the sunflower disc polypeptide.

[0010] Preferably, step (2) can also be: adding distilled water to the sunflower disk powder in step (1), stirring and extracting, centrifuging, filtering under reduced pressure to retain the supernatant, adding an appropriate amount of ammonium sulfate to the extract, stirring for 10-15 minutes to fully dissolve the ammonium sulfate, precipitating for 10-18 hours, centrifuging to collect the precipitate, re-dissolving and dialysis, and then freeze-drying to obtain sunflower disk protein.

[0011] Preferably, the organic solvent in step (2) is a mixed solution of n-hexane and anhydrous ethanol in equal proportions, and the immersion time in step (2) is 10-20 minutes.

[0012] Preferably, the proteases used in the two enzymolysis steps in step (3) are alkaline protease 2.4L and papain, respectively, and the enzymolysis conditions are: enzyme addition amount is 0.5-5.0% of the total mass volume of the protein solution, enzymolysis pH is 8-10, enzymolysis temperature is 40-60°C, and enzymolysis time is 1.0-5.0h.

[0013] Preferably, activated carbon is used for decolorization in step (3).

[0014] Preferably, the chromatography column filler in step (4) is Sephadex G-50.

[0015] An application of a sunflower disc uric acid-lowering peptide, and use of the sunflower disc uric acid-lowering peptide in the preparation of functional foods;

[0016] Use of sunflower disc uricosuric peptide in the preparation of xanthine oxidase inhibitors;

[0017] Use of sunflower disc uric acid-lowering peptide in the preparation of uric acid-lowering drugs.

[0018] The present invention provides a sunflower disc uric acid-lowering peptide and its preparation method and application.

[0019] Beneficial effects:

[0020] The sunflower disc uric acid lowering peptide processed by the present invention on the extracted and purified protein has high purity, and the sunflower disc peptide with high xanthine oxidase inhibition rate can be obtained after simple purification. At present, the polypeptide has a good xanthine oxidase inhibition rate and potential uric acid lowering effect after computer virtual screening, artificial synthesis and in vitro verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The high and low dose groups significantly reduced the levels of serum creatinine and urea nitrogen. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Embodiment 1:

[0024] The embodiment of the present invention provides a method for preparing a sunflower disc uric acid-lowering peptide, comprising the following steps:

[0025] Step (1): after drying the sunflower discs, shearing, crushing, and passing through a 40-mesh sieve to obtain sunflower disc powder;

[0026] Step (2): soaking the sunflower disc powder in step (1) with an organic solvent, placing it in a fume hood for 10 hours to evaporate the solvent, adding distilled water, stirring and extracting, centrifuging and filtering under reduced pressure, retaining the filtrate, dialyzing and freeze-drying to obtain sunflower disc protein;

[0027] Step (3): using the sunflower disk protein in step (2) to prepare a protein solution, boiling it in a water bath and cooling it, performing enzymolysis twice using a protease, centrifuging and retaining the supernatant, decolorizing the supernatant, centrifuging and filtering, and freeze-drying to obtain a crude enzymolyzate of the sunflower disk;

[0028] Step (4): using column chromatography to separate and purify the enzymatic hydrolysate in step (3), collecting the eluate, and freeze-drying to obtain the sunflower disc polypeptide.

[0029] The organic solvent in step (2) is a mixed solution of n-hexane and anhydrous ethanol in equal proportions, and the immersion time in step (2) is 10-20 minutes.

[0030] The proteases used in the two enzymolysis steps in step (3) are 2.4 L alkaline protease and papain, respectively. The enzymolysis conditions are: the enzyme addition amount is 0.5% of the total mass volume of the protein solution, the enzymolysis pH is 8, the enzymolysis temperature is 40° C., and the enzymolysis time is 1.0.

[0031] In step (3), activated carbon is used for decolorization.

[0032] In step (4), the chromatography column filler is Sephadex G-50.

[0033] Embodiment 2:

[0034] The embodiment of the present invention provides a method for preparing a sunflower disc uric acid-lowering peptide, comprising the following steps:

[0035] Step (1): after drying the sunflower discs, shearing, crushing, and passing through a 40-mesh sieve to obtain sunflower disc powder;

[0036] Step (2): adding distilled water to the sunflower disk powder in step (1), stirring and extracting, centrifuging, filtering under reduced pressure to retain the supernatant, adding an appropriate amount of ammonium sulfate to the extract, stirring for 15 minutes to fully dissolve the ammonium sulfate, precipitating for 18 hours, centrifuging to collect the precipitate, re-dissolving and dialysis, and freeze-drying to obtain sunflower disk protein, the organic solvent is a mixed solution of n-hexane and anhydrous ethanol in equal proportions, and the soaking time in step (2) is 20 minutes;

[0037] Step (3): using the sunflower disk protein in step (2) to prepare a protein solution, boiling water bath and cooling, using protease to perform enzymolysis twice, centrifuging and retaining the supernatant, using activated carbon to decolorize the supernatant, centrifugally filtering, and freeze-drying to obtain a crude enzymolysate of the sunflower disk, wherein the proteases used in the two enzymolysis are alkaline protease 2.4L and papain, respectively, and the enzymolysis conditions are as follows: the enzyme addition amount is 5.0% of the total mass volume of the protein solution, the enzymolysis pH is 10, the enzymolysis temperature is 60°C, and the enzymolysis time is 5.0h;

[0038] Step (4): using column chromatography to separate and purify the enzymatic hydrolysate in step (3), wherein the chromatographic column filler is Sephadex G-50, collecting the eluate, and freeze-drying to obtain the sunflower disc polypeptide.

[0039] Embodiment three:

[0040] The embodiment of the present invention provides an application of a sunflower disc uric acid-lowering peptide:

[0041] Use of sunflower disc uricosuric acid-lowering peptide in preparing functional food;

[0042] Use of sunflower disc uricosuric peptide in the preparation of xanthine oxidase inhibitors;

[0043] Use of sunflower disc uric acid-lowering peptide in the preparation of uric acid-lowering drugs;

[0044] The alleviating effect of sunflower disc uricosuric peptide on hyperuricemia mice: The experimental design is as follows:

[0045] After one week of adaptive feeding of mice, 50 mice were randomly divided into two groups. One group of 10 mice was fed with normal feed and used as blank control; one group was gavaged with potassium oxonate and adenine at fixed time points in the morning and evening for ten consecutive days to construct a hyperuric acid model, and the mice in the control group were gavaged with an equal amount of normal saline. On the ninth day of modeling, the mice were fasted overnight in advance. On the tenth day, orbital blood was collected to measure the serum uric acid of the mice, and the mice with successful modeling were randomly divided into 4 groups of 8 in each group. To ensure the stability of the model, all mice need to continue to be gavaged with potassium oxonate and adenine daily. The specific groups are as follows: (1) Model group; (2) High-dose group: daily oral gavage of 40 mg / kg; (3) Low-dose group: daily oral gavage of 20 mg / kg; (4) Positive control group: daily oral gavage of 20 mg / kg allopurinol. After three weeks of continuous gavage, the mice were killed and serum and organs were collected;

[0046] like Figure 1 The experimental results obtained showed that sunflower disc uricosuric peptide could significantly reduce the serum uric acid level and XOD activity of hyperuricemia mice, significantly reduce the levels of serum creatinine and urea nitrogen, and alleviate kidney inflammation caused by increased uric acid. The pathological sections of the kidneys also showed the same results.

[0047] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a sunflower disc uric acid-lowering peptide, characterized in that: The following steps are involved: Step (1): after drying the sunflower discs, shearing, crushing, and passing through a 40-mesh sieve to obtain sunflower disc powder; Step (2): soaking the sunflower disc powder in step (1) with an organic solvent, placing it in a fume hood for 10 hours to evaporate the solvent, adding distilled water, stirring and extracting, centrifuging and filtering under reduced pressure, retaining the filtrate, dialyzing and freeze-drying to obtain sunflower disc protein; Step (3): using the sunflower disk protein in step (2) to prepare a protein solution, boiling it in a water bath and cooling it, performing enzymolysis twice using a protease, centrifuging and retaining the supernatant, decolorizing the supernatant, centrifuging and filtering, and freeze-drying to obtain a crude enzymolyzate of the sunflower disk; Step (4): using column chromatography to separate and purify the enzymatic hydrolysate in step (3), collecting the eluate, and freeze-drying to obtain the sunflower disc polypeptide.

2. The method for preparing the sunflower disc uricosuric acid-lowering peptide according to claim 1, characterized in that: The step (2) may also be: adding distilled water to the sunflower disc powder in step (1), stirring and extracting, centrifuging, filtering under reduced pressure and retaining the supernatant, adding an appropriate amount of ammonium sulfate to the extract, stirring for 10-15 minutes to fully dissolve the ammonium sulfate, precipitating for 10-18 hours, collecting the precipitate by centrifugation, re-dissolving, dialysis, and freeze-drying to obtain the sunflower disc protein.

3. The method for preparing the sunflower disc uricosuric acid-lowering peptide according to claim 1, characterized in that: The organic solvent in step (2) is a mixed solution of n-hexane and anhydrous ethanol in equal proportions, and the immersion time in step (2) is 10-20 minutes.

4. The method for preparing the sunflower disc uricosuric acid-lowering peptide according to claim 1, characterized in that: The proteases used in the two enzymolysis steps in step (3) are 2.4 L alkaline protease and papain, respectively. The enzymolysis conditions are: the enzyme addition amount is 0.5-5.0% of the total mass volume of the protein solution, the enzymolysis pH is 8-10, the enzymolysis temperature is 40-60°C, and the enzymolysis time is 1.0-5.0h.

5. The method for preparing the sunflower disc uricosuric acid-lowering peptide according to claim 1, characterized in that: In step (3), activated carbon is used for decolorization.

6. The method for preparing the sunflower disc uricosuric acid-lowering peptide according to claim 1, characterized in that: In step (4), the chromatography column filler is Sephadex G-50.

7. An application of a sunflower disc uric acid-lowering peptide, characterized in that: Use of sunflower disc uricosuric acid-lowering peptide in preparing functional food; Use of sunflower disc uricosuric peptide in the preparation of xanthine oxidase inhibitors; Use of sunflower disc uric acid-lowering peptide in the preparation of uric acid-lowering drugs.

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