AU2021103416A4

AU2021103416A4 - Polypeptide, composition, compound preparation for lowering uric acid, preparation method and use thereof

Info

Publication number: AU2021103416A4
Application number: AU2021103416A
Authority: AU
Inventors: Zhongli Pei; Mingzhan Tian; Wei Wei; Jiuxun Zhang; Xiping Zhang; Xuejun Zhang
Original assignee: Zhongshiduqing Shandong Biotech Co Ltd
Current assignee: Zhongshiduqing Shandong Biotech Co Ltd
Priority date: 2020-06-29
Filing date: 2021-06-17
Publication date: 2021-08-05
Anticipated expiration: 2029-06-17
Also published as: CN111704650A; CN111704650B

Abstract

OF THE DISCLOSURE The present disclosure provides a polypeptide, a composition, a compound preparation for lowering uric acid, a preparation method and use thereof, and belongs to the technical field of biomedicine. The polypeptide includes a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1; the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2. The present disclosure provides a skipjack collagen peptide; the skipjack collagen peptide includes anserine, carnosine, the first polypeptide, and the second polypeptide. The present disclosure further provides a compound preparation; the compound 0 preparation includes the skipjack collagen peptide, kudzuvine root powder, chrysanthemum powder, coix seed powder, and galangal powder. The polypeptide, composition and compound preparation provided by the present disclosure are derived from natural raw materials of animals and plants, and can lower uric acid effectively. ABSTRACT DRAWING U Uric acid content 16 14.86 14.56 14.26 14 12.43 12.91 12.63 12.39 12.3 12 11.09 9 10 8 D 4 2 0 0& Skipjack collagen peptide Skipjack collagen peptide (pure product) (compound) Concentration (pg/mL) FIG. 8

Description

POLYPEPTIDE, COMPOSITION, COMPOUND PREPARATION FOR LOWERING URIC ACID, PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

[01] The present disclosure relates to the technical field of biomedicine, and in particular to a polypeptide, a composition, a compound preparation for lowering uric acid, a preparation method and use thereof.

BACKGROUND ART

[02] Uric acid, the main metabolite of birds and reptiles, is slightly soluble in water and easy to form crystals. The main product in normal human urine is urea with a small amount of uric acid. Normally, there is approximately 1,200 mg of uric acid in the body, approximately 600 mg of which is newly produced every day, and 600 mg of which is excreted, being in a balanced state. However, if the body produces too much to be excreted or the uric acid excretion mechanism is degraded, too much uric acid will retain in the body. When the blood uric acid level is greater than 7 mg/DL, the body fluid will become acidic and normal functions of human cells will be influenced. If left unattended for a long time, gout will be developed.

[03] At present, uric acid lowering mainly depends on pharmaceutical chemicals, such as allopurinol and probenecid. Although allopurinol and probenecid are effective in lowering uric acid, both drugs have certain damages to liver function. Therefore, in the process of lowering uric acid, in order to avoid liver dysfunction, most people are treated by dietary structure adjustment, weight loss, abstinence or administration of diuretics, so as to slowly lower uric acid levels. Currently, there is a lack of uric acid lowering active pharmaceutical ingredients obtained from natural animal and plant raw materials.

SUMMARY

[04] An objective of the present disclosure is to provide a polypeptide, a composition, a compound preparation for lowering uric acid, a preparation method and use thereof. The polypeptide, composition and compound preparation provided by the present disclosure are derived from natural raw materials of animals and plants, and can lower uric acid effectively.

[05] To achieve the above objective, the present disclosure provides the following technical solutions:

[06] The present disclosure provides a polypeptide for lowering uric acid, where the polypeptide includes a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1; the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2.

[07] The present disclosure provides a skipjack collagen peptide including the polypeptide according to the above solution, where the skipjack collagen peptide includes anserine, carnosine, a first polypeptide, and a second polypeptide; the anserine, the carnosine, the first polypeptide, and the second polypeptide have a mass ratio of (1.2-1.6): (7.0-7.4): (2.7-3.1): (3.8-4.2);

[08] the anserine has a chemical structural formula shown in Formula I:

0 H

0 ~~

NN

1091 FormulaI1; 1101 the caruosine has achemical structural formula shown in FormulaI11: H N

[11] NH 2 Formula II.

[12] Preferably, the anserine, the carnosine, the first polypeptide, and the second polypeptide may have a mass ratio of 1.4:7.2:2.9:4.0.

[13] The present disclosure further provides a method for preparing the skipjack collagen peptide according to the above solution, including the following steps:

[14] 1) mixing minced skipjack meat with water and holding at 50-55°C for 0.5-1.5 h to obtain a mixture;

[15] 2) after adjusting the mixture to pH 6.5-7.5, mixing the mixture with papain, and enzymatically hydrolyzing at 58-62°C for 2.5-3.5 h to obtain an enzymatic hydrolysate; where based on 100 parts by weight of minced skipjack meat, there are 2.5-3.5 parts by weight of papain;

[16] 3) adjusting the enzymatic hydrolysate to pH 4.8-5.2, and inactivating the enzyme at 105-115°C for 3-8 min to obtain an inactivated enzymatic hydrolysate; and

[17] 4) mixing the inactivated enzymatic hydrolysate with activated carbon, holding at 55-60°C for 0.5-1.5 h, and filtering a resulting mixture through a 30,000 Da ceramic membrane and a 1 Kd organic film successively, collecting a filtrate, and freeze-drying the filtrate to obtain the skipjack collagen peptide.

[18] Preferably, after collecting the filtrate in step 4), the filtrate may be further freeze-dried.

[19] The present disclosure further provides a compound preparation for lowering uric acid, including the following raw materials: 80-88 parts by weight of the skipjack collagen peptide according to the above solution or prepared by the preparation method, 3-8 parts by weight of kudzuvine root powder, 3-8 parts by weight of chrysanthemum powder, 2-5 parts by weight of coix seed powder, and 2-5 parts by weight of galangal powder.

[20] Preferably, dosage form of the compound preparation may include granules, powder, tablet, capsule or decoction.

[21] The present disclosure further provides use of the polypeptide according to above solution, the skipjack collagen peptide, the skipjack collagen peptide prepared by the preparation method, or the compound preparation in the preparation of a health food for lowering uric acid.

[22] The present disclosure further provides use of the polypeptide, the skipjack collagen peptide, the skipjack collagen peptide prepared by the preparation method, or the compound preparation in the preparation of a medicament for treating gout.

[23] The present disclosure has the following beneficial effects: The present disclosure provides a polypeptide for lowering uric acid, where the polypeptide includes a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1; the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2. The first polypeptide and the second polypeptide of the present disclosure are isolated from the skipjack collagen peptide. The first polypeptide and the second polypeptide of the present disclosure reduce the production rate of uric acid by inhibiting hypoxanthine invertase. Both the first polypeptide and the second polypeptide of the present disclosure have an excellent uric acid lowering effect.

BRIEF DESCRIPTION OF THE DRAWINGS

[24] FIG. 1 is a chromatogram of carnosine standard;

[25] FIG. 2 is a chromatogram of anserine standard;

[26] FIG. 3 illustrates the detection results of the content of anserine and carnosine in skipjack collagen peptide;

[27] FIG. 4 is a flowchart of preparing a compound preparation from skipjack collagen peptide in Example 2;

[28] FIG. 5 is a chromatogram of separation and purification of skipjack collagen peptide in Example 3;

[29] FIG. 6 is a mass spectrum of identification of an amino acid sequence of GHAFTYL;

[30] FIG. 7 is a mass spectrum of identification of an amino acid sequence of TDEVVVFY;

[31] FIG. 8 illustrates the experimental results of the uric acid-reducing effect of the skipjack collagen peptide (pure product) and the compound preparation.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[32] The present disclosure provides a polypeptide for lowering uric acid, where the polypeptide 'I includes a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1, specifically: GHAFTYL (Gly-His-Ala-Phe-Thr-Tyr-Lue); the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2, specifically: TDEVVVFY (Thr-Asp-Glu-Val-Val-Val- Phe-Tyr).

[33] In the present disclosure, the first polypeptide and the second polypeptide may be isolated from skipjack collagen peptide. In the specific implementation of the present disclosure, the first polypeptide and the second polypeptide, as well as carnosine and anserine standards, are synthesized by Shanghai Qiangyao Biological Technology Co., Ltd. The first polypeptide and the second polypeptide of the present disclosure may have an advantage of freedom from being destroyed by hydrolysis when taken orally.

[34] The present disclosure provides a skipjack collagen peptide including the polypeptide according to the above solution; the skipjack collagen peptide includes anserine (p-alanyl-1-methyl L-histidine), carnosine (P-alanyl-L-histidine), a first polypeptide, and a second polypeptide; the anserine, the camosine, the first polypeptide, and the second polypeptide have a mass ratio of (1.2-1.6): (7.0-7.4): (2.7-3.1): (3.8-4.2), and preferably 1.4:7.2:2.9:4.0; the anserine, carnosine, first polypeptide and second polypeptide in the present disclosure may all inhibit hypoxanthine oxidase.

[35] The anserine has a chemical structural formula shown in Formula I:

0 H H2NOH

0 N

/N--J

[36] Formula I;

[37] the carnosine has a chemical structural formula shown in Formula II: H N

0

HN

[38] NH 2 Formula II.

[39] In the present disclosure, functions of the anserine are to inhibit hypoxanthine oxidase activity and reduce the production of uric acid.

[40] In the present disclosure, functions of the carnosine are to inhibit hypoxanthine oxidase activity and reduce the production of uric acid.

[41] The present disclosure further provides a method for preparing the skipjack collagen

A peptide according to the above solution, including the following steps:

[42] 1) mixing minced skipjack meat with water and holding at 50-55°C for 0.5-1.5 h to obtain a mixture;

[43] 2) after adjusting the mixture to pH 6.5-7.5, mixing the mixture with papain, and enzymatically hydrolyzing at 58-62°C for 2.5-3.5 h to obtain an enzymatic hydrolysate; where based on 100 parts by weight of minced skipjack meat, there are 2.5-3.5 parts by weight of papain;

[44] 3) adjusting the enzymatic hydrolysate to pH 4.8-5.2, and inactivating the enzyme at 105-115°C for 3-8 min to obtain an inactivated enzymatic hydrolysate; and

[45] 4) mixing the inactivated enzymatic hydrolysate with activated carbon, holding at 55-60°C for 0.5-1.5 h, and filtering a resulting mixture through a 30,000 Da ceramic membrane and a 1 Kd organic film successively, collecting a filtrate, and freeze-drying the filtrate to obtain the skipjack collagen peptide.

[46] In the present disclosure, minced skipjack meat is mixed with water, and held at 50-55°C for 0.5-1.5 h to obtain a mixture; the minced skipjack meat and the water may preferably have a mass ratio of 1:(1.5-2.5), and more preferably 1:2; the water may preferably include deionized water; the function of holding may be intended to better remove the fishy odor of the minced skipjack meat. In the present disclosure, the holding may preferably be conducted for 1 h.

[47] In the present disclosure, after the mixture is obtained, the mixture is adjusted to pH 6.5-7.5, mixed with papain, and enzymatically hydrolyzed at 58-62°C for 2.5-3.5 h to obtain an enzymatic hydrolysate; based on 100 parts by weight of minced skipjack meat, there may be 2.5-3.5 parts by weight and more preferably 2 parts by weight of papain. The present disclosure uses papain for enzymatic hydrolysis to obtain the highest target product content. Through enzymatic hydrolysis, the protein in the minced skipjack meat changes from a large molecular weight protein to a low molecular weight peptide.

[48] In the present disclosure, the mixture may preferably be adjusted to pH 7; the enzymatic hydrolysis may preferably be conducted at 60°C for 3 h, and NaOH may preferably be a reagent used for adjusting the pH.

[49] In the present disclosure, after the enzymatic hydrolysate is obtained, the enzymatic hydrolysate was adjusted to 4.8-5.2, and inactivated at 105-115°C for 3-8 min to obtain an inactivated enzymatic hydrolysate; in the present disclosure, the enzymatic hydrolysate may preferably be adjusted to pH 5; the enzyme inactivation may preferably be conducted at 110°C for 5 min; HCl may preferably be a reagent used for adjusting the pH.

[50] In the present disclosure, after the inactivated enzymatic hydrolysate is obtained, the inactivated enzymatic hydrolysate is mixed with activated carbon, held at 55-60°C for 0.5-1.5 h, and filtered through a 30,000 Da ceramic membrane and a 1 Kd organic film successively; a filtrate is collected and freeze-dried to obtain the skipjack collagen peptide; the material of the organic film may preferably include polyvinyl chloride (PVC); the inactivated enzymatic hydrolysate and the activated carbon may preferably have a mass ratio of 100:4; the pH value may preferably be 5.0 in the holding; the holding may preferably be conducted for 1 h; the holding may preferably be conducted at 58°C; the function of the holding may be intended to enable the activated carbon to better deodorize and decolorize; the freeze-drying procedure may preferably be: treating at -49°C for 6 h, warming up to -45°C for 4 h, warming up to -40°C for 4 h, warming up to -35°C for 4 h, warming up to -30°C for 4 h, warming to -25°C for 4 h, warming up to -20°C for 4 h, warming up to -15°C for 3 h, warming up to -10°C for 3 h, warming up to -5°C for 2 h, warming up to 0°C for 2 h, warming up to 5°C for 2 h, warming up to 10°C for 2 h, and warming up to 15°C for 2 h.

[51] In the present disclosure, the ceramic membrane filters out large molecular weight proteins and fats, and the 1,000 Da organic film removes part of heavy metals and salts, and the quality of the polypeptide may be improved after two filtrations.

[52] The present disclosure further provides a compound preparation for lowering uric acid, including the following raw materials: 80-88 parts by weight of the skipjack collagen peptide according to the above solution or prepared by the preparation method, 3-8 parts by weight of kudzuvine root powder, 3-8 parts by weight of chrysanthemum powder, 2-5 parts by weight of coix seed powder, and 2-5 parts by weight of galangal powder; preferably, the compound preparation may include 84 parts by weight of skipjack collagen peptide, 5 parts by weight of kudzuvine root powder, 5 parts by weight of chrysanthemum powder, 3 parts by weight of coix seed powder, and 3 parts by weight of galangal powder; the compound preparation may preferably be 0.5 mm in particle size; in the present disclosure, the kudzuvine root powder, chrysanthemum powder, coix seed powder, and galangal powder may be conventionally commercially available.

[53] The present disclosure has no special restriction on the method for preparing the compound preparation, and the compound preparation of the present disclosure may be obtained after mixing the raw materials uniformly; after the mixing, it may be preferable to further include pulverization.

[54] In the present disclosure, the dosage form of the compound preparation may preferably include powder. In the present disclosure, the dosage form of the compound preparation may preferably include powder. The compound preparation of the present disclosure may have excellent water solubility, and active pharmaceutical ingredients may not be decomposed after administration.

[55] The present disclosure further provides use of the polypeptide according to above solution, the skipjack collagen peptide, the skipjack collagen peptide prepared by the preparation method, or the compound preparation in the preparation of a health food for lowering uric acid; the dosage form of the health food may preferably include powder.

[56] The present disclosure further provides use of the polypeptide, the skipjack collagen peptide, the skipjack collagen peptide prepared by the preparation method, or the compound preparation in the preparation of a medicament for treating gout; the dosage form of the medicament may preferably include powder.

[57] An administration method of the medicine or health food of the present disclosure may preferably include the following steps: the medicine or health food is brewed with water at 40°C before oral administration; the frequency of the oral administration is 3 times/day, and the dose of the medicament or health food is preferably 6-10 g, and more preferably 8 g each time. The present disclosure has no special restriction on brewing time and method, as long as the medicine or health food may be uniformly mixed. The present disclosure has no special restriction on the amount of water, as long as the compound preparation may be fully dissolved.

[58] The technical solutions of the present disclosure will be described below clearly and completely in conjunction with the examples of the present disclosure. It is clear that the described examples are only a part of, not all of, the examples of the present disclosure. Based on the examples of the present disclosure, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present disclosure.

[59] Example 1 Method for preparing skipjack protein peptide

[60] 1. Frozen skipjacks were taken out of a cold storage, washed 3 times with deionized water, and soaked to thaw. The skipjacks were minced by a meat grinder to obtain minced skipjack meat.

[61] 2. 100 kg of the minced skipjack meat obtained in step 1 was weighed, 200 kg of deionized water was added, and the solution was stirred evenly; the temperature was rose to 55°C and held for 1 h, and minced skipjack meat was obtained.

[62] 3. The minced skipjack meat in step 2 was adjusted to pH 7 with 1 M NaOH, warmed up to °C, mixed with 3% papain (3 kg), and enzymatically hydrolyzed for 3 h to obtain an enzymatic hydrolysate.

[63] 4. The enzymatic hydrolysate in step 3 was adjusted to pH 5 with 1 M HCl, warmed up to 110°C, held for 5 min, and inactivated to obtain an inactivated enzymatic hydrolysate.

[64] 5. The inactivated enzymatic hydrolysate in step 4 was cooled to 55°C, mixed with activated carbon, held for 1 h, filtered through a 30,000 Da ceramic membrane and a 1Kd organic film successively, freeze-dried for 24 h (a freeze dryer was used to freeze-dry the liquid into a powder), and packed into a box.

[65] 6. The anserine and carnosine in the skipjack collagen peptide freeze-dried in step 5 was detected, and components thereof were further separated and purified; the specific detection process was as follows:

[66] High-performance liquid chromatography (HPLC) was used for detection, and the detection method of the present disclosure could quickly detect the content of anserine and camosine in unknown samples. Using porous filler as stationary phase, sample components were separated according to the difference in relative molecular mass thereof, and detected at 210 nm (the ultraviolet absorption wavelength of the peptide bond); using a special data processing software for '7 determining the relative molecular mass distribution in gel chromatography (GPC software), chromatograms and data thereof were processed to calculate the content of anserine and carnosine.

[67] Column: XBridge@ C18 5 m 4.6 x 250 mm Column.

[68] HPLC mobile phase preparation and analysis methods: A: pH 9.5, aqueous potassium dihydrogen phosphate solution (10% by weight); B: water; C: water; D: aqueous methanol solution (90% by volume).

[69] The detection procedure is shown in Table 1.

[70] Table 1 The procedure for the detection of the content of anserine and carnosine Time A% B% C% D% 10 5 0 0 95 15 50 0 0 50 20 50 0 0 50 22 5 0 0 95 30 5 0 0 95

[71] Detecting wavelength: 210 nm. Flow rate: 0.7 mL/min. Detection time: 30 min. Injection volume: 10 pL.

[72] Treatment of standards and samples

[73] 1 mg of a standard was dissolved in 1 mL of water to prepare a peptide standard solution with a mass concentration of 1 mg/mL; the peptide standard solution was filtered through an organic film with a pore size of 0.45 [m, and the sample was injected to obtain a chromatogram of the standard. FIG. 1 is a chromatogram of carnosine standard, where the carnosine can be determined in the sample according to the fact that the peak time of the carnosine standard (3.480) is basically consistent with the that of the sample (3.479), and the content of carnosine in the sample is determined by the external standard method and in accordance with the peak area thereof. FIG. 2 is a chromatogram of anserine standard, where the anserine can be determined in the sample according to the fact that the peak time of the anserine standard (4.028) is basically consistent with the that of the sample (3.975), and the content of anserine in the sample is determined by the external standard method and in accordance with the peak area thereof; FIG. 3 illustrates the detection results of the content of anserine and camosine in skipjack collagen peptide.

[74] Accurately, 100 mg of the sample was weighed in a 10 mL volumetric flask, diluted to volume with water, sonicated for 10 min to fully dissolve and mix the sample. The sample was filtered through a 0.45 m organic film to obtain a filtrate, and the filtrate was analyzed to obtain a chromatogram of the sample, as shown in FIG. 3.

[75] The content of anserine and carnosine in the sample is calculated according to the following calculation formula:

A(%) = x 100%

[761 B2*C

[77] A = the content of analyte in the sample;

[78] B = the chromatographic peak area of analyte in the sample solution;

[79] B2 = the chromatographic peak area of analyte standard in the standard solution;

[80] C= sample concentration;

[81] C2 = standard concentration.

[82] After detection, the content of anserine in skipjack protein peptide was 1.4%, and the content of carnosine was 7.2%.

[83] Example 2 A compound preparation prepared based on the skipjack collagen peptide prepared in Example 1 (skipjack collagen peptide compound)

[84] Weights of all raw materials were as follows: 84 kg of skipjack collagen peptide, 5 kg of kudzuvine root powder, 5 kg of chrysanthemum powder, 3 kg of coix seed powder, 3 kg of galangal powder.

[85] The preparation method (see FIG. 4 for the flow chart) consisted of the following steps:

[86] 1) The skipjack collagen peptide, kudzuvine root powder, chrysanthemum powder, coix seed powder, and galangal powder were successively added into a mixer for stirring thoroughly; the mixture was dispensed into packs to obtain the product, 8 g per pack.

[87] Example 3 Sequence identification of polypeptide with uric acid lowering activity

[88] 1. Separation and purification of skipjack collagen peptide

[89] The skipjack collagen peptide prepared in Example 1 was dissolved in water to prepare a 120 mg/mL solution, which was separated and purified by Sephadex G-50 column (3 * 100 cm). The mobile phase was 10% methanol, and the flow rate was 1 mL/min; the absorbance was measured at 214 nm; fractions were collected according to the chromatographic peak (FIG. 5), and each fraction was concentrated and freeze-dried to powder with a vacuum drier.

[90] 2. The different fractions of the skipjack collagen peptide obtained in step 1 were subjected to activity identification (identification was carried out using a xanthine oxidase in vitro inhibitory activity detection system).

[91] (1) Preparation of solutions

[92] 0.2 mol/L (pH 7.5) phosphate buffer solution (PBS): 30.0838 g of Na2HPO4•12H20 and 2.4962 g of NaH2PO4•2H20 were accurately weighed, dissolved in deionized water, and diluted to 500 mL.

[93] Xanthine: 6.4 mg of xanthine was weighed accurately, dissolved with 1 mL of 1 M NaOH, and mixed with 100 mL of PBS; the pH was adjusted to 7.5 with 1 M HCl.

[94] Xanthine oxidase: 120 L of enzymatic hydrolysate was measured and diluted to 8 mL with

PBS.

[95] Mobile phase: 0.015 mol/L sodium dihydrogen phosphate solution.

[96] (2) Sample pretreatment

[97] Each sample was diluted to 40 mg/mL; 50 L of sample or PBS and 150 L of xanthine were added successively in a 96-well ELISA plate, and 3 parallel samples were made for each sample, with PBS group as blank control group. After holding at 25°C for 5 min, 50 L of xanthine oxidase was added, and the absorbance was read every 30 s, with a total of 50 readings for 25 min. After reading, the reaction was terminated with 80 L of 1 M HCl, and the reaction mixture was diluted 10-fold with ultrapure water and filtered through a 0.25 m waterborne membrane for detection.

[98] (3) Detection of uric acid production by HPLC

[99] Column: Zorbax Eclipse XDB-C18 column (5 m, 4.6 x 250 mm, Agilent),

[100] Liquid phase conditions: the eluent was 15% methanol + 85% ammonium dihydrogen phosphate solution, the injection volume was 20 L, the flow rate was 1 mL/min, the detecting wavelength was 290 nm, and the running time was 15 min.

[101] (4) Result analysis

[102] XOD inhibition rate = 1 - (Peak area of uric acid in the blank control group/Peak area of uric acid in the sample group)

[103] The inhibition rate of the different fractions was verified by the xanthine oxidase in vitro inhibitory activity detection system used by different fractions obtained in step 1, and it was found that the chromatographic peak with a retention time of 12.5 min had an excellent effect on promoting lead excretion. First of all, this component was further separated and purified by HPLC, followed by structure identification.

[104] Table 2 The xanthine oxidase in vitro inhibitory activity detection system used by different fractions Retentiontime 0-5min 6-10min 10-15min 15-20min 20-25min

Inhibition rate 5.1% 13.2% 26.9% 3.2% 1.5%

[105] 3. Purity and structure identification: Nanoscale liquid chromatography-Q EXACTIVE tandem mass spectrometry system was used to identify the purity and structure of the different fractions obtained in step 1.

[106] Chromatographic conditions:

[107] (1) Mobile phase: Phase A: 100% purified water + 0.1% formic acid; Phase B: 100% acetonitrile + 0.1% formic acid;

[108] (2) Flow rate of the mobile phase: 300 nL/min;

[109] (3) Injection volume: 1 pL of supernatant;

1n

[110] (4) The mobile phase gradient program is shown in Table 3:

[111] Table 3 Mobile phase gradient program Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0 A (%) 97 97 63 10 10 97 97 B (%) 3 3 37 90 90 3 3

[112] Using the nanoscale liquid chromatography-Q EXACTIVE tandem mass spectrometry system, the structure of the sample was identified as single-strand polypeptides of 807.9 Da GHAFTYL (Gly-His-Ala-Phe-Thr-Tyr-Lue) and 971.05 Da TDEVVVFY (Thr-Asp-Glu-Val-Val-Val-Phe-Tyr). The proportions of the two single peptide chains in the skipjack collagen peptide were 2.9 and 4.0%, respectively, as determined by LC-MC technology. FIG. 6 and FIG. 7 show mass spectra of identification of amino acid sequences of 807.9 Da GHAFTYL (Gly-His-Ala-Phe-Thr-Tyr-Lue) and 971.05 Da TDEVVVFY (Thr-Asp-Glu-Val-Val-Val-Phe-Tyr),respectively.

[113] Example 4In vivo simulated digestion of first polypeptide and second polypeptide

[114] The first polypeptide and the second polypeptide ingested into the human body need to pass through the gastrointestinal tract to be absorbed and utilized; for this reason, the in vitro digestion stability analysis was conducted on the first polypeptide and the second polypeptide: pepsin and trypsin were used to simulate gastrointestinal digestion, uric acid lowering peptide (1 g) was injected into 3 mL of 0.1 mol/L PBS, mixed with 6 mL of pepsin-guar gum mixture, adjusted to pH 1.5 with 2 mol/L HCl, and stirred magnetically in a thermostatic water bath at 37°C for 30 min; the above solution was mixed with 10 mL of PBS and adjusted to pH 6.9 with NaOH solution; 125 L of MgCl2-CaCl2 and 125 L of pancreatin were added, respectively, while making up to 50 mL with water and incubating on a shaker at 37°C for 120 min. The enzymes were inactivated in a boiling water bath, and the sample was centrifuged at 8,000 rpm for 10 min to collect a supernatant; after lyophilization of the supernatant, the inhibition rate of hypoxanthine oxidase was determined and mass spectrometry was performed.

[115] After in vivo simulated digestion, the single-strand polypeptides of 807.9 Da GHAFTYL (Gly-His-Ala-Phe-Thr-Tyr-Lue) and 971.05 Da TDEVVVFY (Thr-Asp-Glu-Val-Val-Val-Phe-Tyr) appeared very stable, with stability of 86.2% and 91.2%, respectively, as shown in Table 4.

[116] Table 4 Stability results of GHAFTYL and TDEVVVFY digested by pepsin and trypsin in vitro Peak area before Peak area after Stability rate Sample name enzymatic enzymatic hydrolysis hydrolysis (0)

Gly-His-Ala-Phe-Thr-Tyr-Lue 3,651.9 3,279.3 89.80 Thr-Asp-Glu-Val-Val-Val-Phe-Tyr 2,931.3 2,812.1 95.9% 1 1

[117] After Gly-His-Ala-Phe-Thr-Tyr-Lue was digested by pepsin-trypsin in vitro, the stability was 89.8%; after Thr-Asp-Glu-Val-Val-Val-Phe-Tyr was digested by pepsin-trypsin in vitro, the stability was 95.9%; it was indicated that Gly-His-Ala-Phe-Thr-Tyr-Lue and Thr-Asp-Glu-Val-Val-Val-Phe-Tyr were not easily destroyed by enzymatic hydrolysis in the presence of pepsin-trypsin. To this end, the inhibition rate of Gly-His-Ala-Phe-Thr-Tyr-Lue and Thr-Asp-Glu-Val-Val-Val-Phe-Tyr against hypoxanthine oxidase was determined. The results are shown in Table 5.

[118] Table 5 The determination results of the inhibition rate of Gly-His-Ala-Phe-Thr-Tyr-Lue and Thr-Asp-Glu-Val-Val-Val-Phe-Tyr against hypoxanthine oxidase Sample name Inhibition rate before Inhibition rate after enzymatic hydrolysis enzymatic hydrolysis Gly-His-Ala-Phe-Thr-Tyr-Lue 36.9% 39.2% Thr-Asp-Glu-Val-Val-Val-Phe-Tyr 42.9% 41.2%

[119] Example 5 Evaluation test of uric acid lowering effects of skipjack collagen peptide (Example 1) and skipjack collagen peptide compound (compound preparation) prepared in Example 2

[120] 1. The 5 dpf wild-type AB zebrafish was treated with potassium cyanate combined with xanthine to induce hypeluricemia, and a zebrafish model of hypeluricemia was established.

[121] In water, 5 dpf wild-type AB zebrafishes from Institute of Hydrobiology, Chinese Academy of Sciences were treated with 1.5 mL of 20 mM potassium cyanate (final concentration: 10mM) and 150 L of 10 mM (final concentration: 0.5 mM) xanthine for 24 h to hypeluricemia, and zebrafish models of hypeluricemia were established.

[122] 2. MTCs of the skipjack collagen peptide (pure product) prepared in Example 1 and the skipjack collagen peptide compound (compound preparation) prepared in Example 2 on zebrafishes with hypeluricemia were 125 and 250 g/mL (obtained by the maximal tolerance dose testing), respectively. The concentrations of the skipjack collagen peptide (pure product) for the evaluation of uric acid lowering effect were set to: 13.9, 41.7, and 125 [g/mL; the concentrations of the first polypeptide for the evaluation of uric acid lowering effect were set to: 4.0, 12.1, and 36.3 [g/mL; the concentrations of the second polypeptide for the evaluation of the uric acid lowering effect were set to: 5.6, 16.7, and 50.0 g/niL; the concentrations of the compound preparation for the evaluation of the uric acid lowering effect were set to: 16.5, 49.6, and 149 [g/mL.

[123] 3. Two hundred and seventy 5 dpf wild-type AB zebrafishes were randomly selected in a six-well plate, and 30 zebrafishes were treated in each well (experimental group); the zebrafishes were induced with potassium cyanate combined with xanthine to establish a model of hypeluricemia.

[124] The zebrafishes were administered in water with 13.9, 41.7, and 125 [g/niL skipjack 1) collagen peptide (pure product), 4.0, 12.1, and 36.3 pig/mL first polypeptide, 5.6, 16.7, and 50.0 pg/mL second polypeptide, and 16.5, 49.6, and 149 ptg/mL compound preparation, respectively;

[1251 the positive control drug allopurinol had a concentration of 136 ptg/mL, and the volume of each well (experimental group) was 3 mL;

[126] meanwhile, a normal control group (zebrafishes treated with water for fish farming) and a model control group were set up.

[127] After each experimental group was treated for one day, the zebrafishes were homogenized to collect supernatants, and the content of uric acid in the zebrafishes was determined by HPLC.

[128] Chromatographic conditions included: detector wavelength: y =288 nm, chromatographic column: Shimadzu C18 (5 pm, 4.6 x 250 mm); mobile phase: water (0.2% acetic acid): methanol (V:V)= 94:6; flow rate: 1.0 mL/min; column temperature: 30°C; injection volume: 10 pL.

[129] The statistical analysis results of the uric acid content were used to evaluate the uric acid lowering effects of the skipjack collagen peptide (pure product) and the compound preparation. The statistical processing results were expressed as mean SE. The calculation formula for uric acid lowering effect is as follows:

[130] Uric acid lowering effect (%)= _"__ "_°_ ~_ °°__ "__"_ x

100%

[131] 4. Statistical analysis was performed using analysis of variance and Dunnett's t-test, and p<0.05 indicated a significant difference. The results are shown in Table 6 and FIG. 8.

[132] Comparison of the uric acid content of zebrafishes in the model control group (14.86 ptg/mgprot) versus the normal control group (11.09 ptg/mgprot) (p<0.01) indicated that the model was successfully established. There was significant difference in uric acid content between zebrafishes in the positive control drug allopurinol 136 tg/mL group (12.43 ptg/mgprot) and the model control group (14.86 pg/mgprot) (p<0.05), and the allopurinol showed a 16% decrease in uric acid content in zebrafishes, indicating allopurinol had an uric acid lowering effect on zebrafishes.

[133] The uric acid content of zebrafishes in the skipjack collagen peptide (pure product) 13.9, 41.7, and 125 tg/mL groups was 16.56, 14.91, and 14.63 tg/mgprot, respectively; the model control group was insignificantly different from the 13.9 pg/mL group (p>0.05)and significantly different from the 41.7 and 125 tg/mL groups (both p<O.05), and the uric acid lowering effect were 1.8%, 11.6%, and 13.2%, respectively, suggesting that the skipjack collagen peptide (pure product) had an uric acid lowering effect on zebrafishes at a concentration of 41.7 tg/mL.

[134] The uric acid content of zebrafishes in the first polypeptide (GHAFTYL) 4.0, 12.1, and 36.3 ptg/mL groups was 13.82, 12.91, and 12.76 tg/mgprot, respectively; the model control group was significantly different from the 4.0, 12.1, and 36.3 pg/mL groups (all p<0.05), and the uric acid lowering effects were 18.0%, 23.4%, and 24.3%, respectively, suggesting that the first polypeptide

1'2

(GHAFTYL) had an uric acid lowering effect on zebrafishes at a concentration of 4.0 [g/mL.

[135] The uric acid content of zebrafishes in the second polypeptide (TPEVVVFY) 5.6, 16.7, and 50.0 pg/mL groups was 13.56, 12.12, and 11.83 g/mgprot, respectively; the model control group was significantly different from the 5.6, 16.7, and 50.0 g/mL groups (all p<0.05), and the uric acid lowering effects were 19.6%, 28.1%, and 29.8%, respectively, suggesting that the second polypeptide (TPEVVVFY) had an uric acid lowering effect on zebrafishes at a concentration of 5.6 pg/mL.

[136] The uric acid content of zebrafishes in the compound preparation 16.5, 49.6, and 149 pg/mL groups was 15.76, 14.39, and 14.30 g/mgprot, respectively; the model control group was insignificantly different from the 14.26 g/mL group (p>0.05) and significantly different from the 49.6 and 149 g/mL groups (both p<0.05), and the uric acid lowering effect were 6.5%, 14.7%, and 15.2%, respectively, suggesting that the compound preparation had an uric acid lowering effect on zebrafishes at a concentration of 49.6 [g/mL.

[137] Table 6 Experimental results of uric acid lowering effects after treatment with the skipjack collagen peptide (pure product) and the compound preparation

Concentration Uric acid content ( g/mgprot) Uric acid lowering effect ([g/mL) (mean SE) °)

Normal control - 11.09 0.83 Model control - 16.86 0.39 Allopurinol 136 pg/mL 13.43 0.23* 20.0* 13.9 16.56 0.32 1.8 Skipjackcollagen 41.7 14.91 0.32 11.6 peptide (pure product) 125 14.63 0.27 13.2

First polypeptide 4.0 13.82 18.0 GHAFTYL 12.1 12.91 23.4 36.3 12.76 24.3 5.6 13.56 19.6 Second polypeptide 16.7 12.12 28.1 TDEVVVFY 50.0 11.83 29.8 16.5 15.76 0.37 6.5 Compound preparation 49.6 14.39 0.29 14.7 149 14.30 0.32 15.2

[138] The comparison of the uric acid content of zebrafishes treated with the skipjack collagen peptide (pure product) and the compound preparation versus the model control group is shown in

1A

FIG. 8, *p<0.05.

[139] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, and such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

[140] Exemplary embodiments of the present disclosure are described in the following statements:

Statement 1. A polypeptide for lowering uric acid, wherein the polypeptide comprises a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1; the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2.

Statement 2. A skipjack collagen peptide comprising the polypeptide according to Statement 1, wherein the skipjack collagen peptide comprises anserine, carnosine, a first polypeptide, and a second polypeptide; the anserine, the camosine, the first polypeptide, and the second polypeptide have a mass ratio of (1.2-1.6): (7.0-7.4): (2.7-3.1): (3.8-4.2); the anserine has a chemical structural formula shown in Formula I:

H2N OH 0 N~ N

Formula I; the carnosine has a chemical structural formula shown in Formula II: H N

N 0

H H2 Formula II.

Statement 3. The skipjack collagen peptide according to Statement 2, wherein the anserine, the carnosine, the first polypeptide, and the second polypeptide have a mass ratio of 1.4:7.2:2.9:4.0.

1 1z

Statement 4. A method for preparing a skipjack collagen peptide according to Statement 2 or 3, comprising the following steps: 1) mixing minced skipjack meat with water and holding at 50-55°C for 0.5-1.5 h to obtain a mixture; 2) after adjusting the mixture to pH 6.5-7.5, mixing the mixture with papain, and enzymatically hydrolyzing at 58-62°C for 2.5-3.5 h to obtain an enzymatic hydrolysate; wherein based on 100 parts by weight of minced skipjack meat, there are 2.5-3.5 parts by weight of papain; 3) adjusting the enzymatic hydrolysate to pH 4.8-5.2, and inactivating the enzyme at 105-115°C for 3-8 min to obtain an inactivated enzymatic hydrolysate; and 4) mixing the inactivated enzymatic hydrolysate with activated carbon, holding at 55-60°C for 0.5-1.5 h, and filtering a resulting mixture through a 30,000 Da ceramic membrane and a 1 Kd organic film successively, collecting a filtrate, and freeze-drying the filtrate to obtain the skipjack collagen peptide.

Statement 5. The preparation method according to Statement 4, wherein after collecting the filtrate in step 4), the filtrate is further freeze-dried.

Statement 6. A compound preparation for lowering uric acid, comprising the following raw materials: 80-88 parts by weight of the skipjack collagen peptide according to Statement 2 or 3 or prepared by the preparation method according to Statement 4 or 5, 3-8 parts by weight of kudzuvine root powder, 3-8 parts by weight of chrysanthemum powder, 2-5 parts by weight of coix seed powder, and 2-5 parts by weight of galangal powder.

Statement 7. The compound preparation according to Statement 6, wherein dosage form of the compound preparation comprises granules, powder, tablet, capsule or decoction.

Statement 8. Use of the polypeptide according to Statement 1, the skipjack collagen peptide according to Statement 2 or 3, the skipjack collagen peptide prepared by the preparation method according to Statement 4 or 5, or the compound preparation according to Statement 6 in the preparation of a health food for lowering uric acid.

Statement 9. Use of the polypeptide according to Statement 1, the skipjack collagen peptide according to Statement 2 or 3, the skipjack collagen peptide prepared by the preparation method according to Statement 4 or 5, or the compound preparation according to Statement 6 in the preparation of a medicament for treating gout.

9825912_1.txt 17 Jun 2021

Sequence Listing

<110> Zhongshi Duqing(Shandong) Biotech Co., Ltd

<120> POLYPEPTIDE, COMPOSITION, COMPOUND PREPARATION FOR LOWERING URIC ACID, PREPARATION METHOD AND USE THEREOF

<160> 2

<170> SIPOSequenceListing 1.0 2021103416

<210> 1 <211> 7 <212> PRT <213> Katsuwonus pelamis

<400> 1 Gly His Ala Phe Thr Tyr Leu 1 5

<210> 2 <211> 8 <212> PRT <213> Katsuwonus pelamis

<400> 2 Thr Asp Glu Val Val Val Phe Tyr 1 5

Page 1

Claims

WHAT IS CLAIMED IS:

1. A skipjack collagen peptide, comprising a polypeptide for lowering uric acid, wherein the polypeptide comprises a first polypeptide and/or a second polypeptide; the first polypeptide has an amino acid sequence shown in SEQ ID NO. 1; the second polypeptide has an amino acid sequence shown in SEQ ID NO. 2, wherein the skipjack collagen peptide comprises anserine, carnosine, a first polypeptide, and a second polypeptide; the anserine, the camosine, the first polypeptide, and the second polypeptide have a mass ratio of (1.2-1.6): (7.0-7.4): (2.7-3.1): (3.8-4.2); the anserine has a chemical structural formula shown in Formula I:

H2N OH OH 0

N 0

HN 2 Formula II.

2. The skipjack collagen peptide according to claim 1, wherein the anserine, the carnosine, the first polypeptide, and the second polypeptide have a mass ratio of 1.4:7.2:2.9:4.0.

3. A method for preparing a skipjack collagen peptide according to claim 1 or 2, comprising the following steps: 1) mixing minced skipjack meat with water and holding at 50-55°C for 0.5-1.5 h to obtain a mixture; 2) after adjusting the mixture to pH 6.5-7.5, mixing the mixture with papain, and enzymatically hydrolyzing at 58-62°C for 2.5-3.5 h to obtain an enzymatic hydrolysate; wherein based on 100 parts by weight of minced skipjack meat, there are 2.5-3.5 parts by weight of papain; 3) adjusting the enzymatic hydrolysate to pH 4.8-5.2, and inactivating the enzyme at 105-115 0 C for 3-8 min to obtain an inactivated enzymatic hydrolysate; and

1'7

4) mixing the inactivated enzymatic hydrolysate with activated carbon, holding at 55-60°C for 0.5-1.5 h, and filtering a resulting mixture through a 30,000 Da ceramic membrane and a 1 Kd organic film successively, collecting a filtrate, and freeze-drying the filtrate to obtain the skipjack collagen peptide; wherein after collecting the filtrate in step 4), the filtrate is further freeze-dried.

4. A compound preparation for lowering uric acid, comprising the following raw materials: -88 parts by weight of the skipjack collagen peptide according to claim 1 or 2 or prepared by the preparation method according to claim 3, 3-8 parts by weight of kudzuvine root powder, 3-8 parts by weight of chrysanthemum powder, 2-5 parts by weight of coix seed powder, and 2-5 parts by weight of galangal powder.

5. The compound preparation according to claim 4, wherein dosage form of the compound preparation comprises granules, powder, tablet, capsule or decoction.

1 Q

－1/4－ 17 Jun 2021 2021103416

FIG. 1

FIG. 2

FIG. 3

－2/4－ 17 Jun 2021

Weighing and preparing raw materials 2021103416

Kudzuvine root powder Skipjack collagen

Chrysanthemu

Coix seeds 3% peptide 84%

Galangal 3% m 5% 5%

Mixing well

Dispensing, 8 g/ pack

Brewing: at 50-55°C Brewing with warm water, and stirring uniformly

Administration: 3 times/day

FIG. 4

－3/4－ 17 Jun 2021 2021103416

FIG. 5

FIG. 6

FIG. 7

－4/4－ 17 Jun 2021

尿酸含量 16 14.86 14.56 14.26 14 12.91 12.63 12.43 12.39 12.3 12 11.09 尿酸含量 (（ μg/mgprot） )

10 2021103416

8

6

4

2

0 组照组 L .9 .7 5 .5 .6 9 对照 g/m 量13 量41 量12 量16 量49 量14 正常模对 136μ 低剂中剂高剂低剂中剂高剂嘌呤醇别鲣鱼胶原肽（纯品）鲣鱼胶原肽（复方）浓度 (（ μg/mL))

FIG. 8