CN116731103B - Small molecule peptide and application thereof in inhibiting xanthine oxidase - Google Patents
Small molecule peptide and application thereof in inhibiting xanthine oxidase Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a small molecular peptide and application thereof in inhibiting xanthine oxidase, and belongs to the technical field of enzymolysis processing products. The amino acid sequence of the small molecule peptide is shown as SEQ ID NO. 2. The application of the small molecular peptide in preparing medicines for inhibiting xanthine oxidase and medicines for inhibiting uric acid level is provided. The invention obtains the small molecular peptide with high inhibition activity to xanthine oxidase through experimental study and screening. The invention has important significance for treating hyperuricemia.
Description
Technical Field
The invention relates to a small molecular peptide and application thereof in inhibiting xanthine oxidase, belonging to the technical field of enzymolysis processing products.
Background
Oyster is one of the largest cultivated shellfish in the world, is a high-quality protein library, and has rich nutritive value. At present, most of oyster processing modes are direct eating, the industrial value of the oyster is difficult to improve, and oyster polypeptide processing is an important direction of deep processing.
During purine metabolism, xanthine Oxidase (XO) catalyzes the conversion of the intermediate hypoxanthine to xanthine, which then continues to catalyze the conversion of xanthine to uric acid. Hyperuricemia and its complications, including kidney stones and cardiovascular disease, can be caused by excessive uric acid production and reduced excretion due to high purine diets and alcohol intake. The traditional medicine for treating hyperuricemia often has serious toxic and side effects, so the development cost is low, the toxic and side effects are small, and the food-source uric acid reducing peptide easy to absorb has important significance for treating hyperuricemia.
Disclosure of Invention
Aiming at the prior art, the invention provides a small molecular peptide and application thereof in inhibiting xanthine oxidase. The invention has important significance for treating hyperuricemia and improves the additional yield of oyster.
The invention is realized by the following technical scheme:
A small molecule peptide having the amino acid sequence: IASGFF as shown in SEQ ID NO. 2.
The application of the small molecular peptide in preparing medicines for inhibiting xanthine oxidase and medicines for inhibiting uric acid level is provided.
Experimental research shows that oyster polypeptide has xanthine oxidase inhibiting (uric acid reducing) activity, so that oyster polypeptide has potential as functional product for inhibiting uric acid level. Further, by LC-MS/MS identification, the small molecular peptide is obtained by molecular butt screening, and has higher inhibitory activity on xanthine oxidase, so that the small molecular peptide has the potential of being a functional product for inhibiting uric acid level.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: schematic of the effect of protease on xanthine oxidase inhibition.
Fig. 2: the influence of enzymolysis time on xanthine oxidase inhibition rate is shown schematically.
Fig. 3: schematic of the effect of enzyme addition on xanthine oxidase inhibition.
Fig. 4: the effect of feed liquid ratio on xanthine oxidase inhibition is shown schematically.
Fig. 5: schematic of the effect of initial pH on xanthine oxidase inhibition.
Fig. 6: schematic diagram of the effect of enzymolysis temperature on xanthine oxidase inhibition rate.
Fig. 7: the separation result of the enzymolysis liquid through G-15 sephadex column chromatography is shown in the schematic diagram.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods and detection methods known in the prior art unless otherwise specified.
EXAMPLE 1 optimization of oyster enzymolysis Process
1. Sieve enzyme
(1) Selecting compound flavourzyme, papain, bromelain, neutral protease, alkaline protease and acid protease to carry out enzymolysis on the raw materials: opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; water was added to 8g of oyster meat homogenate at a feed to liquid ratio of 1:3 (g: ml). Dividing into 6 parts, respectively adding compound flavor protease, papain, bromelain, neutral protease, alkaline protease and acid protease, wherein the addition amount is 2000U/g (enzyme activity/oyster meat homogenate), regulating the initial pH value to the optimal pH value of each protease (the optimal pH values of the compound flavor protease, the papain, the bromelain, the neutral protease, the alkaline protease and the acid protease are 6.5,6.5,6.5,6.5 and 10.5,3.0 respectively) by using sodium hydroxide solution or hydrochloric acid, and reacting for 4 hours under the conditions of the optimal temperature of each protease (the optimal temperature of the compound flavor protease, the papain, the bromelain, the neutral protease, the alkaline protease and the acid protease is 50 ℃, the temperature is 55 ℃, the temperature is 50 ℃, the temperature is 45 ℃) and the speed is 200rpm to obtain an enzymolysis product. Sampling, boiling for 10min, inactivating enzyme, cooling to room temperature, centrifuging at 8000rpm for 10min, and measuring xanthine oxidase inhibition rate. Three parallel groups are provided.
(2) Measurement of in vitro XO inhibition: xanthine solution (1.5 mmol/l) and xanthine oxidase solution (0.1U/ml) were prepared separately with PBS buffer at pH=7.4. To a 1.5ml centrifuge tube, 125. Mu.l of the sample, 150. Mu.l of PBS buffer and 100. Mu.l of xanthine oxidase solution were added, incubated at 37℃for 15min, 125. Mu.l of xanthine solution were added, incubation was continued for 20min, and finally 100. Mu.l of hydrochloric acid (1 mol/l) was added to terminate the reaction. Controls were set up without sample, without xanthine oxidase and with the sample, respectively. The absorbance at 290nm was measured.
The formula for calculating the in vitro XO inhibition rate is as follows:
In vitro XO inhibition (%) = [ (1- (Abs 1-Abs3)/(Abs2-Abs4 ] ×100%).
Wherein Abs 1: absorbance of a living assay system containing xanthine, xanthine oxidase and sample; abs 2: contains xanthine and xanthine oxidase, and does not contain the absorbance value of a living system of which the sample is a measurement system; abs 3: absorbance of a living assay containing xanthine substrate and sample, without xanthine oxidase; abs 4: absorbance of the living system containing xanthine, without xanthine oxidase and sample.
(3) The results are shown in FIG. 1. As shown in the figure, the in vitro XO inhibition rate of the enzymolysis product of the acid protease is highest and can reach 67.62 percent, which is superior to other five proteases, so that the acid protease is selected for the following single factor optimization experiment.
2. Optimization of enzymolysis time
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; adding water into 4g of oyster meat homogenate, wherein the ratio of feed to liquid is 1:3 (g: ml), adjusting the initial pH value to 3, adding acid protease according to the enzyme adding amount of 2000U/g, and reacting for 2-6 h at 40 ℃ and 200 rpm; sampling at 2h, 3h, 4h, 5h and 6h respectively, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (measurement method is same as above), wherein each group is provided with three parallel groups.
The effect of different enzymolysis times on the in vitro XO inhibition rate of the enzymolysis product is shown in figure 2. Along with the prolongation of the enzymolysis time, the XO inhibition rate of the enzymolysis product shows a trend of rising and then falling, and the highest XO inhibition rate reaches 58.11% when the XO inhibition rate reaches 4 hours; there was a significant improvement over the 3h group (P < 0.05) and no significant difference over the 5h group (P > 0.05). Therefore, the enzymolysis time is 4h for the next optimization.
3. Optimization of enzyme addition
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; adding water into 4g oyster meat homogenate, wherein the feed-liquid ratio is 1:3 (g: ml), regulating the initial pH value to 3, dividing into 5 parts, adding acid protease according to the enzyme adding amount of 1000U/g, 2000U/g, 3000U/g, 4000U/g and 5000U/g respectively, and reacting for 4 hours at 40 ℃ and 200 rpm; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (measurement method is same as above), wherein three groups are arranged in parallel.
The effect of different enzyme addition amounts on the in vitro XO inhibition rate of the enzymatic hydrolysate is shown in FIG. 3. Along with the increase of the enzyme adding amount, the XO inhibition rate of the product reaches 58.74% when the enzyme adding amount is 3000U/g, and the inhibition activity of the group is obviously higher than that of other groups (P is less than 0.05), and the XO inhibition rate is reduced when the enzyme adding amount is increased. Thus, the amount of acid protease added was 3000U/g to be optimized in the next step.
4. Optimization of feed-liquid ratio
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate 4g; dividing into 5 parts, adding water into oyster meat homogenate, wherein the feed liquid ratio is 1:2, 1:3, 1:4, 1:5 and 1:6 (g: ml), adjusting the initial pH value to be 3, adding acid protease according to the enzyme adding amount of 3000U/g, and reacting for 4 hours at 40 ℃ and 200 rpm; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (measurement method is same as above), wherein three groups are arranged in parallel.
The effect of different feed liquid ratios on the in vitro XO inhibition rate of the enzymatic hydrolysate is shown in FIG. 4. Along with the reduction of the feed liquid ratio, the XO inhibition rate is continuously increased, the inhibition rate is highest when the feed liquid ratio is 1:5, and is 59.14 percent, compared with the prior selection (feed liquid ratio is 1:3), the XO inhibition rate can be obviously improved (P is less than 0.05), and compared with the feed liquid ratio of 1:6, the XO inhibition rate is not obviously different (P is more than 0.05). Therefore, the feed-liquid ratio is 1:5 for the next optimization.
5. Optimization of initial pH
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; adding water into 4g of oyster meat homogenate, dividing the oyster meat homogenate into 6 parts according to the feed-liquid ratio of 1:5 (g: ml), respectively adjusting the initial pH values to 1,2, 3, 4, 5 and 6, adding acid protease according to the enzyme adding amount of 3000U/g, and reacting for 4 hours at 40 ℃ and 200 rpm; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (measurement method is same as above), wherein three groups are arranged in parallel.
The effect of different initial pH on in vitro XO inhibition of the enzymatic hydrolysate is shown in FIG. 5. The optimum pH range of the acidic protease is 2-4, and pH value deviation from this range changes the protease activity. The product XO inhibition was highest at pH 2, 68.35% and significantly higher than in the other groups (P < 0.05); as the pH increases, the XO inhibitory activity of the product continues to show a decreasing trend. Therefore, the selection of pH 2 continues the next optimization.
6. Optimization of enzymolysis temperature
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; adding water into 4g oyster meat homogenate, wherein the ratio of feed to liquid is 1:5 (g: ml), regulating the initial pH value to 3, adding acid protease according to 3000U/g of enzyme adding amount, dividing into 5 parts, and reacting for 4 hours at 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ under 200rpm respectively; sampling, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging at 8000rpm for 10min, collecting supernatant, and measuring XO inhibition rate (measurement method is same as above), wherein three groups are arranged in parallel.
The effect of different enzymolysis temperatures on the in vitro XO inhibition rate of the enzymolysis products is shown in figure 6. The XO inhibition rate shows a trend of rising and then falling along with the rising of the temperature, the XO inhibition activity reaches the strongest value at the temperature of 40 ℃ and is 68.62%, and meanwhile, the XO inhibition activity of the group can be found to be obviously higher than that of other groups (P is less than 0.05). Therefore, the optimal enzymolysis temperature is selected to be 40 ℃.
7. Orthogonal test optimization
And determining the level of 5 factors including enzymolysis time, enzyme adding amount, feed-liquid ratio, initial pH value and enzymolysis temperature according to the single factor experimental result, setting a 5-factor 4 level orthogonal test, and measuring the in-vitro XO inhibition rate to obtain the optimal enzymolysis condition. The experimental factors and levels are detailed in Table 1.
TABLE 1 level of orthogonal experimental design factors
The results of oyster enzymolysis orthogonal optimization experiments are shown in table 2. From the analysis of the R value, the primary and secondary effects of the enzymolysis time, the enzyme adding amount, the feed liquid ratio, the enzymolysis temperature and the initial pH on the basic nitrogen content of the enzymolysis liquid ammonia are as follows: a is larger than C and larger than B and larger than E, namely the enzymolysis time has the greatest influence, and then the influence of the initial pH on the basic nitrogen content of the liquid ammonia for enzymolysis is the smallest after the enzyme addition amount and the feed liquid ratio. Finally, the optimized combination is A2B2C3D2E3, namely the enzymolysis time is 4 hours, the enzyme adding amount is 3000U/g, the feed-liquid ratio is 1:5, the initial pH is 2, and the enzymolysis temperature is 40 ℃.
Table 2 orthogonal optimization table
EXAMPLE 2 Ultrafiltration, gel filtration chromatography and component Activity analysis
Opening oyster shell, taking meat, pulping to obtain oyster meat homogenate; adding water into 4g oyster meat homogenate, adjusting the initial pH value to 3 with the feed-liquid ratio of 1:5 (g: ml), adding acid protease according to the enzyme adding amount of 3000U/g, and reacting for 4 hours at 40 ℃ and 200rpm to obtain enzymolysis liquid.
The enzymolysis solution is prepared into a solution with the concentration of 80mg/mL by using a Vivaflow50R (Sartorius, germany) ultrafiltration membrane package to obtain a component smaller than 10kDa and a component smaller than 5kDa, and then freeze-drying, and adding water to obtain a sample as described below. After the ultrafiltration membrane bag is used, the ultrafiltration membrane bag is washed by NaOH solution with the concentration of 0.5mol/l, and then is sealed by 10% ethanol solution and placed in the environment of 4 ℃.
G-15
Adding proper amount of ultrapure water into the sephadex G-15, boiling for 2 hours, and removing froth and impurities; adding ultrapure water, stirring, and standing. The impurities of the upper layer are removed, and the above operation is repeated until the upper layer is free of impurities. And sucking off excessive water on the upper layer of the gel, draining the filler to a chromatographic column with the thickness of 26mm multiplied by 80cm by using a glass rod, avoiding layering and bubble occurrence, and flushing the column after filling by using pure water until the height of the chromatographic column is not changed any more.
Loading: the loading volume is 3ml; the mobile phase is ultrapure water, and the elution speed is 2ml/min; the peak fractions were collected and the activity of the different fractions was determined by lyophilization.
As shown in FIG. 7, the separation results of the enzymatic hydrolysate by the gel column show that the total of 5 elution peaks are designated as X1, X2, X3, X4 and X5, respectively. The eluents corresponding to elution peaks X1 to X5 were collected in order, lyophilized, and then mixed with water to prepare a solution having a concentration of 20mg/ml, and the XO inhibition rate of each component was measured, and the results are shown in Table 3. The inhibition rate of the eluent corresponding to the elution peak X3 is the highest,
The eluent corresponding to the elution peak X3 was selected for the next experiment.
TABLE 3 XO inhibition of different Components
EXAMPLE 3 LC-MS/MS Mass Spectrometry identification of oyster polypeptide X3 peptide fragment composition
1. Polypeptide extraction: the eluate corresponding to the elution peak X3 in example 2 was lyophilized, redissolved with 0.15% acetic acid solution, passed through a 10K ultrafiltration tube, washed 2 times with 5% acetic acid solution, and the obtained filtrate was desalted with C18StageTip and dried in vacuo to obtain a peptide fragment. After drying, the peptide was reconstituted with 0.1% TFA solution, absorbance at 260nm was measured, and peptide concentration was determined for LC-MS/MS analysis.
LC-MS/MS analysis
An appropriate amount of peptide fragment was taken from each sample and chromatographed using a nanoliter flow EasynLC1200 chromatography system (ThermoScientific). Buffer solution: the solution A is 0.1% formic acid aqueous solution, and the solution B is a mixed solution of formic acid, acetonitrile and water (the volume concentration of acetonitrile is 80%, and the concentration of formic acid is 0.1%). The column was equilibrated with 100% solution a. Samples were introduced into TrapColumn (100 μm. Times.20 mm,5 μm, C18, dr. MaischGmbH) and subjected to a gradient separation by chromatography column (75 μm. Times.150 mm,3 μm, C18, dr. MaischGmbH) at a flow rate of 300nl/min.
The liquid phase separation gradient is as follows: 0 to 2 minutes, the linear gradient of the liquid B is from 3 to 5 percent; 2-42 minutes, the linear gradient of the liquid B is from 5% to 25%;42 minutes to 52 minutes, the linear gradient of the liquid B is 25 percent to 45 percent; 52-55 minutes, the linear gradient of the liquid B is from 45% to 90%; 55-70 minutes, and the solution B is maintained at 90%.
After separation of the peptide fragments, DDA (data dependent acquisition) mass spectrometry was performed using a Q-ExactivePlus mass spectrometer (thermo scientific). Analysis duration was 70min, detection mode: positive ion, parent ion scan range: 300-1800 m/z, primary mass spectrum resolution: 70,000@m/z200, AGCTARGET:3e6, one stage MaximumIT:30ms.
Peptide fragment secondary mass spectrometry was collected as follows: secondary mass spectrum (MS 2 scan) of 20 highest intensity parent ions was triggered after each full scan (fullscan), secondary mass spectrum resolution: 17,500@m/z200, AGCTARGET:2e5, second stage MaximumIT:60ms, MS2 Activate type: HCD, isolationwindow:1.6m/z, normalizedcollisionenergy:30.
3. Database retrieval
Adopting mass spectrum database searching software pfind; the following protein databases were used: derived from UniprotProtein Databas. Wherein MaxQuant the library search software analysis parameter set is shown in table 4.
Table 4pFind analysis parameter set
After mass spectrum data retrieval, the X3 component adopts PSMFDR-0.05 and ProteinFDR-0.05 as screening standards for peptide fragment and protein identification respectively to obtain 19 peptide fragment sequences and 14 protein sequences.
Example 4 peptide fragment screening, synthesis and validation
1. Treatment of ligands
The molecular structural formulas of amino acids and polypeptides are drawn by using chemdraw19.0 and chem3DPro14.0 software, and are optimized by an MM2 force field and stored as mo12.
2. Treatment of receptors
Downloading the crystal structure 1N5X of the XO from a PDB database, deleting the B chain in the 1N5X by using PyMOL software, and extracting the ligand febuxostat (TEI) and storing the ligand febuxostat (TEI) in pdbqt format for later use. The acceptor 1N5X was dehydrated, hydrogenated, the gasteiger charge calculated, and all atoms assigned to the AD4 type were saved as pdbqt format for use.
3. Molecular docking
Molecular docking was performed using vina functions of PyRx software to mimic interactions of amino acids, small polypeptide molecules, and macromolecular proteins. According to the position of the original ligand febuxostat in 1N5X, the docking center coordinates are set to be (96, 54, 39) (X, y, z), the box size is 25 multiplied by 25, and other parameters adopt default values. Then, the amino acid and polypeptide ligand are subjected to docking treatment with the XO crystal structure one by one.
Results: the 19 peptide fragments in example 3 were each subjected to Vina simulated docking with XO, and according to Vina score, six peptide fragments were selected from Gly-Gly-Tyr-Gly-Ile-Phe (GGYGIF) (shown as SEQ ID NO. 1), ile-Ala-Ser-Gly-Phe-Phe (IASGFF) (shown as SEQ ID NO. 2), ala-Leu-Ser-Gly-Ser-Trp (ALSGSW) (shown as SEQ ID NO. 3), val-Ala-Gln-Asp-Ser-Ser-Ser-Tyr (VAQDSSY) (shown as SEQ ID NO. 4), leu-His-Cys-Ser-Thr-Leu (LHCSTL) (shown as SEQ ID NO. 5) and Met-Ala-Ile-Gly-Leu-Trp (MAIGLW) (shown as SEQ ID NO. 6) were synthesized.
Example 5 peptide fragment Synthesis and Activity verification
1. Peptide fragment synthesis
Peptide fragments GGYGIF, IASGFF, ALSGSW, VAQDSSY, LHCSTL and MAIGLW were both synthesized by Fmoc solid phase synthesis from Biotechnology (Shanghai) Inc.
2. Activity verification
Measurement of XO inhibition: the polypeptide was formulated as a 3mg/ml solution and the in vitro XO inhibition of the synthesized polypeptide was determined according to the method described in example 1. IC 50 values were calculated from regression lines of XO activity inhibition percentage versus species concentration.
The results of verification of the relevant properties and activity of the peptide fragments are shown in Table 5.
TABLE 5 results of verification of the relative Properties and Activity of peptide fragments
Note that: (1) Since the molecular docking results were random, vina scores shown in the table were the average of the highest values of 10 docking results (negative values); (2) "-" means no XO inhibitory activity.
(2) The hydrophobicity and molecular weight of the peptide fragment are obtained by http:// www.tulane.edu/-biochem/WW/PepDraw/respectively.
As can be seen from table 5, each of the six peptide fragments has higher XO inhibitory activity, with IASGFF, VAQDSSY and LHCSTL exhibiting higher XO inhibitory activity.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (3)
1. A small molecule peptide, characterized in that: the amino acid sequence of the polypeptide is IASGFF as shown in SEQ ID NO. 2.
2. The use of the small molecule peptide of claim 1 for the preparation of a medicament for inhibiting xanthine oxidase.
3. The use of the small molecule peptide of claim 1 for the preparation of a medicament having uric acid level inhibiting effect.
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