Abdominal distention hippocampal peptide and application thereof in preparation of uric acid reducing and gout resisting products

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a swelling hippocampal peptide and application thereof in preparation of uric acid reducing and gout resisting products. The amino acid sequence is shown as SEQ ID NO.1-5. The uric acid reducing effect is verified by adopting an in-vitro XOD inhibition experiment and a cell experiment; furthermore, the uric acid reducing and gout resisting activity of the compound is verified through a hyperuricemia animal model, and a theoretical basis is provided for high-valued utilization of the belly-swelling hippocampus and development of uric acid reducing and gout resisting products.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of bioactive peptides, and particularly to a swelling-belly seahorse peptide and its application in the preparation of products for reducing uric acid and treating gout. Background Art

[0002] Gout is a chronic metabolic disease caused by abnormal uric acid metabolism. Its pathological basis is excessive production or insufficient excretion of uric acid in the body, resulting in an increase in serum uric acid level (hyperuricemia), and then the formation of urate crystals deposited in joints, soft tissues and kidneys, triggering inflammatory reactions, joint damage and renal dysfunction. Although current anti-gout drugs have strong uric acid-lowering activity, they can also cause severe allergic reactions and cardiovascular diseases. Therefore, developing natural anti-gout substances with high efficiency, low toxicity and novel action mechanisms to relieve uric acid has potential application value in health foods.

[0003] In recent years, bioactive peptides have become an important direction in the research and development of anti-gout compounds due to their multi-target effects and low side effects. The swelling-belly seahorse ( Hippocampus abdominalis ), as a high-value marine organism, has developed rapidly in China's mariculture industry in recent years. It not only has traditional medicinal values (such as tonifying the kidney and strengthening yang, anti-inflammatory and detumescence), but also has become an important raw material for preparing bioactive peptides because of its rich high-quality protein and active ingredients. Chinese Patent CN117899119A discloses a preparation method and application of a high-dissolution seahorse powder. Through the instantaneous low-temperature spray drying technology, the active ingredients, nutritional components, appearance color and natural smell of the raw seahorse are retained to the greatest extent. Then, through the enzymatic hydrolysis and extraction of the micropowder, the macromolecular proteins that are difficult to absorb in the seahorse are degraded into small molecules that are easy to absorb, so that the high-dissolution seahorse powder prepared by the present invention can better exert its medicinal effect. However, the seahorse powder of this invention has a complex composition, containing steroid compounds, carbohydrate compounds, amino acids and polypeptides, and the specific species of seahorse is not clear. Moreover, with the changes of time and environment, the types and contents of seahorse proteins also change continuously. For example, there are great differences in the protein types and contents between large seahorses and small seahorses, pregnant seahorses and non-pregnant seahorses. Therefore, further research is needed.

[0004] In summary, there is no report on the application research of swelling-belly seahorse peptides in metabolic diseases (such as hyperuricemia, gout), and the specific functional protein components have not been clarified. Therefore, it remains to be studied whether swelling-belly seahorse short peptides can be obtained by enzymatic hydrolysis, proteins with determined sequences can be obtained, and then a method for obtaining swelling-belly seahorse products with uric acid-lowering and anti-gout effects in a more controllable manner. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the application research of seahorse peptides in metabolic diseases (such as hyperuricemia and gout) has not been reported, and the specific functional protein components have not been clarified, which are the technical and market gaps that need to be filled.

[0006] To solve the above problems, the present invention provides a seahorse peptide and its application in the preparation of products for reducing uric acid and treating gout. By using the combined technology of composite enzyme-directed enzymatic hydrolysis and membrane separation and purification process, seahorse polypeptides are efficiently prepared. Five potential seahorse active peptide sequences with xanthine oxidase inhibitory activity and analgesic effects are disclosed by using LC-MS / MS mass spectrometry technology and activity prediction software. The uric acid-lowering effect is verified by in vitro XOD inhibition experiments and cell experiments; further, the uric acid-lowering and anti-gout activities are verified by a hyperuricemic animal model, providing a theoretical basis for the high-value utilization of seahorses and the development of products for reducing uric acid and treating gout.

[0007] To achieve the above object, the present invention is realized by the following technical means: An application of a seahorse peptide in the preparation of products for reducing uric acid and treating gout, wherein the seahorse peptide comprises polypeptides with amino acid sequences shown in SEQ ID NO.1-5: SEQ ID NO.1: NSGFDF.

[0008] SEQ ID NO.2: FSGFLLKSM.

[0009] SEQ ID NO.3: NPVPYW.

[0010] SEQ ID NO.4: WNIPIG.

[0011] SEQ ID NO.5: FGDFLS.

[0012] The preparation method of the above seahorse peptide comprises the following steps: Take fresh seahorses, wash them, freeze-dry them, and crush them into powder; add distilled water, add 3% composite protease and stir evenly, adjust the pH = 7.5 with HCL solution and NaOH solution respectively, and enzymatically hydrolyze at 50 °C for 5 h; After centrifuging the enzymatic hydrolysate, take the supernatant, filter it through a 0.45 μm microporous filter membrane, and use an ultrafiltration tube with a cut-off molecular weight of 3 kDa to retain peptide segments with a molecular weight of 3 kDa; freeze-dry the peptide solution to obtain seahorse peptide powder.

[0013] Further, distilled water is added in a ratio of 1:9 of the material-liquid ratio.

[0014] Further, the concentrations of both the HCL solution and the NaOH solution are 1 mol / L.

[0015] Further, the enzyme activity of the complex protease is 100 U / mg.

[0016] Further, the end point control of enzymatic hydrolysis: the degree of hydrolysis DH ≥ 25%, and the proportion of the molecular weight distribution of 500 - 3000 Da ≥ 80%.

[0017] Further, the enzymatic hydrolysate is centrifuged at 4000 g / min for 15 min at 4°C, and then the supernatant is taken.

[0018] Further, the complex protease is 1.2 million U / mg, product number: S10155, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0019] The above amino acid sequence is the Hippocampus abdominalis peptide shown in SEQ ID NOs. 1 - 5.

[0020] Further, the Hippocampus abdominalis peptide shown in SEQ ID NOs. 1 - 5 with the above amino acid sequence is artificially synthesized.

[0021] The essence of hyperuricemia is the abnormal purine metabolism of the body, resulting in excessive production or insufficient excretion of purine substances. XOD and ADA are two key uric acid metabolism enzymes widely expressed in the liver and play important roles in the process of uric acid production. Based on this, inhibiting the activity of XOD can effectively reduce the production of uric acid. In the present invention, through cell experiments and animal experiments, it is found that the hippocampal peptide segment is more likely to bind to XOD through hydrophobic interaction and intermolecular bonding, thereby changing its spatial structure, competitively binding with the substrate, and then inhibiting the activity of XOD, ultimately achieving the purpose of alleviating hyperuricemia.

[0022] The beneficial effects of the present invention are as follows: The present invention has screened five hippocampal peptide sequences with xanthine oxidase inhibitory activity and analgesic effects, verified the uric acid-lowering effect through in vitro XOD inhibition experiments and cell experiments; further verified its uric acid-lowering and anti-gout activities through a hyperuricemic animal model, providing a theoretical basis for the high-value utilization of Hippocampus abdominalis and the development of uric acid-lowering and anti-gout products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Docking result diagram of the peptide segment of SEQ ID NO.1 and the XOD receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide segment of SEQ ID NO.1 and the XOD receptor protein; B is the 2D structure of the docking of the peptide segment of SEQ ID NO.1 and the XOD receptor protein.

[0024] Figure 2: Docking result diagram of the peptide of SEQ ID NO.2 with the XOD receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide of SEQ ID NO.2 with the XOD receptor protein; B is the 2D structure of the docking of the peptide of SEQ ID NO.2 with the XOD receptor protein.

[0025] Figure 3 : Docking result diagram of the peptide of SEQ ID NO.3 with the XOD receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide of SEQ ID NO.3 with the XOD receptor protein; B is the 2D structure of the docking of the peptide of SEQ ID NO.3 with the XOD receptor protein.

[0026] Figure 4 : Docking result diagram of the peptide of SEQ ID NO.4 with the XOD receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide of SEQ ID NO.4 with the XOD receptor protein; B is the 2D structure of the docking of the peptide of SEQ ID NO.4 with the XOD receptor protein.

[0027] Figure 5 : Docking result diagram of the peptide of SEQ ID NO.5 with the XOD receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide of SEQ ID NO.5 with the XOD receptor protein; B is the 2D structure of the docking of the peptide of SEQ ID NO.5 with the XOD receptor protein.

[0028] Figure 6 : Effects of each peptide on the uric acid level in the supernatant of the HK-2 cell model; Note: ## p < 0.01 compared with the blank group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0029] Figure 7 : Kidney weight and kidney index of mice; Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group; A is the kidney weight; B is the kidney index.

[0030] Figure 8 : Serum uric acid level of mice; Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0031] Figure 9 : Serum urea nitrogen level of mice; Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0032] Figure 10: Serum creatinine level in mice; Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0033] Figure 11 : Levels of XOD and ADA in mouse liver; where A is the level of XOD and B is the level of ADA; Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group. Detailed implementation mode

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] In addition, all materials used in the embodiments of the present invention are obtained by purchasing from the market unless otherwise specified.

[0036] Example 1: This example provides a puffed belly seahorse polypeptide prepared by a composite enzyme-directed enzymatic hydrolysis technology combined with a membrane separation technology. The polypeptide segments are identified by LC-MS / MS, and a novel peptide segment with significant xanthine oxidase inhibitory activity and uric acid excretion-promoting effect is screened by molecular docking technology. The specific steps are as follows: Preparation of puffed belly seahorse peptide enzymatic hydrolysate: Take 100 g of fresh puffed belly seahorses, wash them, freeze-dry them, and crush them into powder. Add distilled water according to the solid-liquid ratio of 1:9 (m / v), add 3% composite protease and stir evenly. Adjust the pH = 7.5 with HCL solution (1 mol / L) and NaOH solution (1 mol / L) respectively, and enzymatically hydrolyze at 50 °C for 5 h. Control of the enzymatic hydrolysis end point: Degree of hydrolysis (DH) ≥ 25%, and the molecular weight distribution is concentrated in the range of 500 - 3000 Da (accounting for ≥ 80%). After the enzymatic hydrolysate is centrifuged (centrifuged at 4 °C, 4000 g / min for 15 min), the supernatant is taken and filtered through a 0.45 μm microporous membrane, and the peptide segments with a molecular weight cut-off of 3 kDa are retained by an ultrafiltration tube with a molecular weight cut-off of 3 kDa. The peptide solution is freeze-dried to obtain puffed belly seahorse peptide powder. The composite protease (1.2 million U / mg, S10155) is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0037] LC-MS / MS identification of enzymatically digested hippocampal active peptide segments: The sample was redissolved in 200 μL of 0.1% TFA solution and mixed well by shaking. In the solution containing the sample, the pipette tip rinsed with 0.1% TFA solution was used to pipette up and down 15 - 20 times to adsorb the sample onto the C18 column. The salts on the C18 column were washed away, and elution was performed 3 times in 50% acetonitrile + 0.1% TFA solution until desalting was complete. The hippocampal peptides were analyzed for the sequence of the eluted peptide segments using the UltiMate 3000 UHPLC system (Thermo Fisher Scientiiffc), with an ACQUITY UPLC Peptide CSH C18 column (2.1×100 mm, 1.7 μm) and a TripleTOF 5600+ system. The hippocampal protein database was searched through NCBI. The Peptide Ranker was used to predict the activity of the identified peptide segments, and peptide sequences with higher scores were screened out.

[0038] Molecular docking to screen active peptide segments: Using xanthine oxidase (XOD) as the key target protein, the top 5 peptide segments ranked by activity prediction were selected for homology modeling and molecular docking with the main binding active sites of the above two proteins respectively, with the XOD self-ligand as the negative control. Peptide sequence homology modeling: The Chemdraw3D 18.1 software was used to input the peptide sequence for 3D modeling to generate the peptide segment structure. The energy minimization of the peptide structure was performed through the CHARMm program in the molecular simulation software Discover Studio 2019.

[0039] Pre-treatment of the receptor: The X-ray crystal structure of bovine XOD protein (PDBID: 3NS1) was downloaded from the PDB database. The existing ligand was removed using the Discover Studio 2019 software, and chemical bond correction and charge addition were performed. Further, the active pocket was searched for and the pocket site was covered. Molecular docking of the receptor and peptide segments was performed through the CDOCKER program. Based on the results of the highest CDOCKEREnergy score, indicators such as binding free energy, hydrogen bonds, hydrophobic groups, and charged groups were analyzed, and the docking score was calculated.

[0040] Compound protease was used to enzymatically hydrolyze Hippocampus abdominalis to obtain Hippocampus abdominalis bioactive peptides. Data were collected by LC-MS / MS and the original files were generated. According to the search in the NCBI database, Proteome Discoverer software was used to analyze the peptide fingerprint map. A total of 1232 different peptide sequences were matched. Further screening resulted in 5 peptides with a residual local confidence greater than 80% and a PeptideRanker score of the peptide sequence greater than 0.8, namely NSGFDF, FSGFLLKSM, NPVPYW, WNIPIG, and FGDFLS. By comparing with the bioactive peptides already discovered in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php), it was found that none of the above 5 peptides had been reported, and they were considered new bioactive peptide sequences. The results are shown in Table 1.

[0041] Table 1 Identification and activity prediction of Hippocampus abdominalis bioactive peptide sequences: ; a From PeptideRanker ( http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0042] Molecular docking can effectively calculate the binding sites between the above new peptides and receptor proteins. The above 5 peptides were selected for homology modeling and molecular docking with XOD protein, and the interaction between the peptides and the amino acid residues in the active center of XOD was analyzed. The docking results are shown from A-B in Figure 1 to A-B in Figure 5 . The target protein used in this experiment is the XOD protein from cow's milk, and its receptor itself has a quercetin ligand with a binding energy of -8.7. The following docking modes between the peptides and xanthine oxidase are the best poses after simulation. The docking scores of the five peptides with XOD protein are: NSGFDF (-223.09), FSGFLLKSM (-263.29), NPVPYW (-206.31), WNIPIG (-199.24), and FGDFLS (-248.74). The structures of the 5 peptides can be embedded in the active pocket of XOD, and by interacting with key amino acid residues, conventional hydrogen bonds and hydrophobic interactions are formed to block the binding of XOD to the substrate. The above results indicate that the above 5 hippocampal bioactive peptide segments are good XOD inhibitors at the molecular docking level and have the potential to be developed into multi-target anti-hyperuricemic active ingredients.

[0043] Example 2: In this example, peptide segments were artificially synthesized, and the in vitro XOD inhibitory activity of the above peptide segments and their effects on in vitro cell models were explored. The specific steps are as follows: The five bioactive peptides predicted in Example 1 were biosynthesized separately by Shanghai Sangon Biotech Co., Ltd. through the Fmoc-solid phase peptide synthesis method. After HPLC and MS sequence analysis, the purity of the five peptide segments, namely NSGFDF (NF6), FSGFLLKSM (FM9), NPVPYW (NW6), WNIPIG (WG6), and FGDFLS (FS6), was greater than 98.89%. Chemically synthesized polypeptides have the characteristics of high purity and good safety in clinical applications due to controllable raw materials and processes. However, during the synthesis process, due to the large number of steps involved and the use of various solvents, it is inevitable to have a certain impact on the biological activity of the polypeptide. Therefore, it is necessary to further verify the biological activity of the synthesized polypeptide.

[0044] Determination of xanthine oxidase inhibitory activity: The xanthine oxidase inhibitory activities of the bioactive peptide segments NF6, FM9, NW6, WG9, and FS6 were verified by high performance liquid chromatography. Take 50 μL of xanthine oxidase solution (0.025 U / mL), then add 50 μL of peptide segment solution (25, 50, 100, 200 μg / mL), mix well, preheat in a 37°C constant temperature water bath for 10 min, then add 50 μL of (5 mmol / L) xanthine stock solution and mix thoroughly. After incubating at 37°C for 25 min, finally add HCl (1 mol / L) to terminate the reaction. Take the reaction solution, filter it through a 0.22 μm filter membrane, and analyze it by HPLC. The above experiment was repeated in parallel 3 times. A scatter plot was made with the logarithm of the peptide concentration and the XOD inhibition rate as the abscissa and ordinate respectively. After fitting the trend line, the XOD inhibition IC 50 value was calculated.

[0045] HPLC chromatographic conditions: C18 chromatographic column (4.6 mm × 250 mm, μm), injection volume is 20 μL, gradient elution program: 95% A from 0 - 3 min, 95% - 0% A from 3 - 6 min, 0% - 95% A from 6 - 8 min, 95% A from 8 - 10 min, detection wavelength 254 nm.

[0046] Table 2 HPLC chromatographic conditions: 。

[0047] The inhibition rate of different peptide segments was calculated using the following formula: 。

[0048] HK-2 cell culture: The normal human renal tubular epithelial cell line HK-2 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, placed in an incubator at 37°C and 5% CO2. The cells were passaged when they grew to the logarithmic growth phase.

[0049] In vitro determination of the uric acid-lowering activity of active peptide segments: Samples were taken from HK-2 cells in logarithmic growth phase and inoculated into 96-well plates at a density of 3x10 4 cells per well, with an inoculation volume of 100 μL per well. The inoculated plates were placed in an incubator at 37°C and 5% CO2 for incubation for 24 h. A blank group (Control), a model group (Model), each active peptide segment group (100 μg / mL), and a positive control group (Positive) of allopurinol (100 μmol / L) were set up for pre-incubation for 24 h. The blank group and the model group were not treated, and each group had 3 replicate wells. The culture medium in the plates was aspirated, and after washing with PBS, 250 μL of 4 mmol / L adenosine serum-free medium was added to each well in the model group, the positive control group, and each peptide segment group, and the control group was incubated with serum-free medium for 24 h. 50 μL of 0.01 U / mL xanthine oxidase (dissolved in PBS) was added to each well, and the supernatant was collected after treatment for 6 h. A kit was used to measure the uric acid content.

[0050] The experimental results are shown in Table 3. The above 5 peptide segments all had in vitro XOD inhibitory activity, and all showed a concentration-dependent increase. At a concentration of 100 μg / mL, the inhibitory activities of each peptide segment were as follows: the IC 50 value of NSGFDF (NF6) was 22.66 ± 1.37 mmol / L, the IC 50 value of FSGFLLKSM (FM9) was 14.32 ± 1.02 mmol / L, the IC 50 value of NPVPYW (NW6) was 24.47 ± 1.21 mmol / L, the IC 50 value of WNIPIG (WG6) was 32.01 ± 2.63 mmol / L, and the IC 50 value of FGDFLS (FS6) was 12.68 ± 1.41 mmol / L. This also verified the results of molecular docking. The XOD inhibitory activity of hippocampal active peptide segments may be related to their ability to form stable binding conformations at the active sites. The above peptide segments have the ability to inhibit XOD activity and thus reduce the production of uric acid in vivo.

[0051] Table 3 In vitro XOD inhibitory activity of active peptide segments: .

[0052] The HK-2 cell experiment was as Figure 6As shown in the figure, a peptide segment with a concentration of 1 mmol / L was used as the intervention dose. The results of the uric acid content in the cell supernatant showed that the uric acid level in the model group was significantly higher than that in the blank group (P < 0.01), indicating that 5 mmol / L adenosine and 0.01 U / mL exogenous xanthine oxidase can be used to establish a hyperuricemic cell model. The uric acid content in the allopurinol group was significantly lower than that in the model group (P<0.01), indicating that 100 μmol / L allopurinol can be used as a positive drug control. At a concentration of 100 μg / mL, compared with the model group, all 5 peptide segments had the effect of reducing uric acid, among which FM9 peptide and FS6 had the best uric acid-lowering effect (P<0.01).

[0053] Example 3: In this example, a hyperuricemic mouse model was constructed to verify the two peptide segments with the best xanthine oxidase inhibitory activity in Example 2 in vivo, in order to clarify the uric acid-lowering and anti-gout effects of hippocampal active peptides in vivo. The specific steps are as follows: The hippocampal nonapeptide FSGFLLKSM (FM9) and hippocampal hexapeptide FGDFLS (FS6) in Example 2 were used in the animal model of this example.

[0054] Animal experiment: 40 healthy male C57bl / 6J mice (7 weeks old, body weight 20±1.0 g) were purchased from Jinan Pengyue Experimental Animal Technology Co., Ltd. The animal breeding environment was: the temperature was maintained at 21°C - 23°C, the humidity was maintained at 45% - 55%, and the light / dark cycle was 12 h light / 12 h dark. During the experiment, the mice could freely eat (AIN-93G type feed) and drink water. After 7 days of adaptive feeding of the mice, the above mice were randomly divided into a control group (Normal), a model group (Model), a positive drug group (Positive, febuxostat 5.2 mg / kg / d), a hippocampal nonapeptide FM9 group (100 mg / kg BW), and a hippocampal hexapeptide FS6 group (100 mg / kg BW). Except for the control group, each group was intragastrically administered a suspension of hypoxanthine + potassium oxonate diluted with 5% CMC-Na (hypoxanthine 1 g / kg + potassium oxonate 0.25 g / kg) every day to establish a hyperuricemia mouse model for 21 consecutive days. During the experiment, the body weight was recorded every 3 days, and the intragastric administration volume was adjusted.

[0055] At the same time of modeling, the corresponding test samples were intragastrically administered according to the body weight every afternoon. The model group and the control group were intragastrically administered an equal dose of carboxymethylcellulose sodium. After the experiment, the mice were fasted for 12 h, anesthetized with isoflurane, and then blood was collected by eye socket puncture, and the mice were sacrificed by cervical dislocation. After blood collection, the kidney tissue was quickly separated on ice, weighed, and the kidney index was calculated (organ index = organ mass / body weight x 100%). Determination of serum function indexes: The levels of serum uric acid (UA), serum creatinine (Cr), and blood urea nitrogen (BUN) in mice were detected by an automatic biochemical analyzer.

[0056] Determination of the contents of XOD and ADA in the liver: 0.1 g of liver tissue was homogenized at 4°C (adding 1% PMSF and phosphatase inhibitor), and the supernatant was taken after centrifugation. The determination was carried out strictly according to the instructions of the Elisa kit.

[0057] As Figure 7 shown in A - B below, compared with the control group, the kidney mass of the model group decreased significantly (P < 0.01). The double - kidney masses of the FS6 peptide group and the positive drug group were higher than those of the model group, but there was no significant difference in the FM9 peptide group. There was no significant difference in the kidney indices of mice in each group.

[0058] Serum UA, Cr, and BUN are important indicators for measuring whether renal function is impaired. The research results are as Figures 8 - 10 shown. Compared with the control group, the contents of UA, Cr, and BUN in the serum of the model group increased significantly (P < 0.01); compared with the model group, the UA content and BUN level in the FM9 peptide group and the FS6 peptide group decreased significantly (P < 0.01), and the serum Cr content decreased significantly (P < 0.05), but there was no significant difference between the two different peptide treatments. The above results indicate that hippocampal active peptides have the effect of reducing uric acid.

[0059] XOD is the key rate - limiting enzyme for uric acid synthesis, which can catalyze the production of uric acid by combining with xanthine through the molybdenum atom at the active site. ADA is a crucial aminotransferase in the process of purine metabolism, participating in the synthesis of uric acid through the conversion of adenosine. Therefore, the activities of XOD and ADA can be used as important indicators for monitoring whether uric acid metabolism is abnormal. The contents of XOD and ADA in the liver tissues of mice in each experimental group are as Figure 11 shown in A - B below. The contents of XOD and ADA in the livers of model control group mice were significantly higher than those of the control group (P < 0.05). Compared with the model control group, the positive control group and each dose group of hippocampal peptides (FM9 and FS6) had a significant inhibitory effect on XOD in the liver tissues of mice (P < 0.01). There was a certain inhibitory effect on ADA enzyme, but the difference did not reach the significant level (P > 0.05). The above results indicate that part of the way for FM9 peptide and FM6 peptide to inhibit the uric acid content in the serum of mice is to reduce the XOD content in the serum.

[0060] Finally, it should be noted that although the above embodiments describe the specific implementation manners of the present invention, they do not limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. And all modifications or equivalent replacements should be included within the protection scope of the present invention.

Claims

1. Use of a hippocampus abdominalis peptide in the preparation of a product for reducing uric acid and treating gout, characterized in that: The said Hippocampus abdominalis peptide comprises polypeptides with the amino acid sequences shown in SEQ ID NO.1-5.

2. The preparation method of the belly-swelling hippocampal peptide according to claim 1, characterized in that It includes the following steps: Take fresh Hippocampus abdominalis, wash it and then freeze-dry it, and crush it into powder; add distilled water, add 3% complex protease and stir evenly, adjust the pH = 7.5 with HCL solution and NaOH solution respectively, and carry out enzymatic hydrolysis at 50 °C for 5 h; After the enzymatic hydrolysate is centrifuged, take the supernatant, filter it through a 0.45 μm microporous membrane, and use an ultrafiltration tube with a molecular weight cut-off of 3 kDa to retain peptide segments with a molecular weight of 3 kDa; freeze-dry the peptide solution to obtain Hippocampus abdominalis peptide powder.

3. The preparation method according to claim 2, characterized in that: Add distilled water at a ratio of 1:9 of the material-liquid ratio.

4. The preparation method according to claim 2, characterized in that: The concentrations of the said HCL solution and NaOH solution are both 1 mol / L.

5. The preparation method according to claim 2, characterized in that: The enzyme activity of the said complex protease is 100 U / mg.

6. The preparation method according to claim 2, characterized in that: Control of the enzymatic hydrolysis end point: the degree of hydrolysis DH≥25%, and the proportion of the molecular weight distribution of 500-3000 Da is ≥80%.

7. The preparation method according to claim 2, characterized in that: The enzymatic hydrolysate is centrifuged at 4000 g / min at 4 °C for 15 min, and then the supernatant is taken.

8. Hippocampus abdominalis peptide powder prepared by any of the methods according to claims 2-7.

9. The pygmy seahorse peptide according to claim 1, wherein: The amino acid sequences are shown in SEQ ID NO.1-5.

10. The ventrally enlarged hippocampal peptide according to claim 9, wherein: The said Hippocampus abdominalis peptide is artificially synthesized.

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