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. The uric acid reducing effect of the compound is proved through in-vitro xanthine oxidase (XOD) and adenosine deaminase (ADA) inhibition experiments and cell experiments; on the basis, the uric acid reducing and gout resisting activity of the peptide sequence is further verified by means of a hyperuricemia animal model. The achievement provides a solid theoretical support for high-value development and utilization of the hippocampus japonicus and research and development of uric acid reducing and gout resisting related products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of preparation and application of biological peptides, and particularly relates to a Hippocampus abdominalis peptide and application thereof in preparation of products for reducing uric acid and resisting gout. BACKGROUND

[0002] In modern society, gout as a common chronic metabolic disease brings many troubles to the life of patients. The pathological basis is abnormal uric acid metabolism. When the body generates too much uric acid or excretes insufficiently, the serum uric acid level will be elevated, forming hyperuricemia. The deposition of urate crystals in joints, soft tissues and kidneys can cause a series of adverse consequences, such as inflammatory reaction, joint damage and even renal dysfunction.

[0003] As a high-value marine organism, Hippocampus abdominalis is undergoing a key breakthrough in the research and development of bioactive peptides in the field of resisting gout. The inventors have previously developed a Hippocampus abdominalis peptide powder, which has been verified to have uric acid-lowering effect by in vitro XOD inhibition experiment and cell experiment. Further, the uric acid-lowering and anti-gout activity has been verified by a high uric acid animal model, providing a theoretical basis for the high-value utilization of Hippocampus abdominalis and the development of products for reducing uric acid and resisting gout. However, this is still insufficient to solve the problem of complex composition and unclear target of traditional Hippocampus powder, and more proteins with determined sequences need to be identified and clarified to provide a molecular basis for obtaining Hippocampus abdominalis products with uric acid-lowering and anti-gout in a more controllable way. SUMMARY

[0004] The technical problem to be solved by the present application is that the prior art has insufficient research on Hippocampus abdominalis peptides in reducing uric acid and resisting gout, and more proteins with determined sequences need to be identified and clarified to provide a molecular basis for obtaining Hippocampus abdominalis products with uric acid-lowering and anti-gout in a more controllable way.

[0005] To solve the above problems, the present application provides a Hippocampus abdominalis peptide and application thereof in preparation of products for reducing uric acid and resisting gout. The Hippocampus abdominalis polypeptide is efficiently prepared by using a composite enzyme directional enzymolysis technology combined with a membrane separation and purification process. The LC-MS / MS mass spectrometry technology and activity prediction software are used to disclose one Hippocampus abdominalis active peptide sequence with xanthine oxidase inhibition activity and anti-pain effect. The uric acid-lowering effect is verified by in vitro XOD and ADA preparation experiment and cell experiment. Further, the uric acid-lowering and anti-gout activity is verified by a high uric acid animal model, providing a theoretical basis for the high-value utilization of Hippocampus abdominalis and the development of products for reducing uric acid and resisting gout.

[0006] To achieve the above purpose, the present application realizes the following technical means: a Hippocampus abdominalis peptide, the amino acid sequence of which is shown in SEQ ID NO. 1: SEQ ID NO. 1: GWNIPIGTL.

[0007] The application of the above-mentioned pachybrachia hippocampus peptide in preparing products for reducing uric acid and resisting gout. The essence of high uric acid is abnormal purine metabolism in the body, which leads to excessive generation or insufficient excretion of purine substances. XOD is a key rate-limiting enzyme for uric acid synthesis, which can catalyze the generation of uric acid through the combination of the active site molybdenum atom and xanthine, and ADA is an important aminotransferase in the purine metabolism process. Through the transformation of adenosine, it participates in the synthesis of uric acid, so the activity of XOD and ADA can be used as an important indicator for monitoring whether the uric acid metabolism is abnormal, so by inhibiting the activity of XOD and ADA, the generation of uric acid can be effectively reduced. In the present application, it is found through molecular docking that the peptide segment represented by SEQ ID NO. 1 can be combined with XOD and ADA proteins with energy of-7.6 kcal / mol and-5.2 kcal / mol, respectively. The structure of the peptide segment can be embedded into the active pocket of XOD and ADA, and by interacting with the key amino acid residues, van der Waals force, conventional hydrogen bond, and hydrophobic interaction force are formed to inhibit the activity of XOD and ADA, and finally the purpose of relieving hyperuricemia is achieved.

[0008] Further, the amino acid sequence of the pachybrachia hippocampus peptide represented by SEQ ID NO. 1 is artificially synthesized.

[0009] The preparation method of the powder comprising the above-mentioned pachybrachia hippocampus peptide comprises the following steps: Fresh pachybrachia hippocampus is taken, washed, freeze-dried, and crushed into powder; distilled water is added, 3% complex protease is added and stirred uniformly, HCL solution and NaOH solution are used to adjust pH = 7.5, and enzyme hydrolysis is carried out at 50℃ for 5 h. After centrifugation of the enzyme hydrolysate, the supernatant is filtered through a 0.45 μm microporous filter, and a peptide segment with a molecular weight of 3 kDa is intercepted by using an ultrafiltration tube with a molecular weight cut-off of 3 kDa; the peptide solution is freeze-dried to obtain pachybrachia hippocampus peptide powder.

[0010] Further, distilled water is added in a ratio of 1:9.

[0011] Further, the concentration of the HCL solution and the NaOH solution is 1 mol / L.

[0012] Further, the enzyme activity of the complex protease is 120 million U / mg.

[0013] Further, the enzyme hydrolysis end point control is that the degree of hydrolysis DH is ≥25%, and the molecular weight distribution ratio of 500-3000 Da is ≥80%.

[0014] Further, the supernatant is taken after the enzyme hydrolysate is centrifuged at 4℃ at 4000 g / min for 15 min.

[0015] Further, the complex protease 120 million U / mg, item number: S10155, is purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0016] The amino acid sequence is the pachyptila hippocampus peptide shown in SEQ ID NO. 1.

[0017] Further, the amino acid sequence is the pachyptila hippocampus peptide shown in SEQ ID NO. 1.

[0018] The beneficial effects of the present application are: The present application successfully screens a hippocampus peptide sequence, which not only has xanthine oxidase inhibitory activity, but also has anti-pain effect. In the research, through the in vitro xanthine oxidase (XOD) inhibition experiment and cell experiment, the uric acid lowering effect is confirmed; on this basis, with the help of high uric acid animal model, the uric acid lowering and anti-gout activity of the peptide sequence are further verified. This achievement provides a solid theoretical support for the high-value development and utilization of pachyptila hippocampus, as well as the research and development of uric acid lowering and anti-gout related products. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : The 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.

[0020] Figure 2 : The docking result diagram of the peptide segment of SEQ ID NO. 1 and the ADA receptor protein molecule; wherein, A is the 3D structure of the docking of the peptide segment of SEQ ID NO. 1 and the ADA receptor protein; B is the 2D structure of the docking of the peptide segment of SEQ ID NO. 1 and the ADA receptor protein.

[0021] Figure 3 : The influence of the peptide segment on the uric acid level of the supernatant of the HK-2 cell model; note: ## compared with the blank group p < 0.01; ** compared with the model group, p < 0.01.

[0022] Figure 4 : The serum uric acid level of mice; note: ## compared with the control group p < 0.01; ** compared with the model group, p < 0.01.

[0023] Figure 5 : The serum urea nitrogen level of mice; note: ## compared with the control group p < 0.01; ** compared with the model group, p < 0.01.

[0024] Figure 6: Serum creatinine levels in mice; Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group.

[0025] Figure 7 : XOD and ADA levels in the livers of mice; wherein, A is the XOD level, and B is the ADA level; Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0027] In addition, each material used in the embodiments of the present application is commercially available, unless otherwise specified.

[0028] Example 1 The present example provides a pufferfish polypeptide powder prepared by a complex enzyme directional enzymolysis technology combined with a membrane separation technology, and identifies the peptide segments of the polypeptide by LC-MS / MS, and screens out a new peptide segment with significant xanthine oxidase inhibitory activity and uric acid excretion promoting effect by using molecular docking technology. Specifically, the steps include the following steps: Pufferfish peptide enzymolysis liquid preparation: 100 g of fresh pufferfish was taken, washed, freeze-dried, and pulverized into powder. Distilled water was added at a solid-liquid ratio of 1:9 (m / v), 3% complex protease was added and stirred uniformly, HCL solution (1 mol / L) and NaOH solution (1 mol / L) were used to adjust the pH to 7.5, and the enzymolysis was carried out at 50°C for 5 h. Enzymolysis end point control: degree of hydrolysis (DH) ≥ 25%, and molecular weight distribution was concentrated in 500-3000 Da (accounting for ≥ 80%). After centrifugation (4°C, 4000 g / min for 15 min), the supernatant was filtered through a 0.45 μm microporous filter, and the peptide segments with a molecular weight of 3 kDa were intercepted by an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The peptide solution was freeze-dried to obtain pufferfish peptide powder. The complex protease (1.2 million U / mg, S10155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] LC-MS / MS identification of active hippocampus peptide segments after enzymolysis: The sample was re-dissolved in 200 μL of 0.1% TFA solution and shaken to mix. The gun head was rinsed with 0.1% TFA solution and then repeatedly blown for 15-20 times in the sample solution to adsorb the sample onto the C18 column. The salt substances on the C18 column were washed away, and the desalination was completed by eluting three times in 50% acetonitrile + 0.1% TFA solution. The hippocampus peptide was analyzed for sequence of eluted peptide segments by UltiMate 3000 UHPLC system (Thermo Fisher Scientific), using ACQUITY UPLC peptide CSH C18 column (2.1x100 mm, 1.7 μm) and TripleTOF 5600+ system. The hippocampus protein database was searched by NCBI. The identified peptide segments were predicted for activity using Peptide Ranker, and the peptide sequences with higher scores were screened.

[0030] Molecular docking screening of active peptide segments: Xanthine oxidase (XOD) and adenosine deaminase (ADA) were used as key target proteins, and the peptide segments were homologous modeling and molecular docking with the main binding active sites of the above XOD protein and ADA protein, and their respective ligands were used as negative controls. Peptide sequence homology modeling: the peptide sequence was input into Chemdraw3D 18.1 software for 3D modeling to generate peptide segment structure. The structure of the peptide was energy minimized by CHARMm program in molecular simulation software Discover Studio 2019.

[0031] Receptor pretreatment: The X-ray crystal structure of XOD protein (PDB ID: 3NS1) and the X-ray crystal structure of ADA protein (PDB ID: 3IAR) were downloaded from PDB database, and Discover Studio 2019 software was used to remove existing ligands and correct chemical bonds and add charges. Further search for active pockets and cover pocket sites, and perform molecular docking of receptors and peptide segments by CDOCKER program, and analyze the results according to the highest value of CDOCKER Energy score, calculate docking score according to the indexes of binding free energy, hydrogen bond, hydrophobic group and charged group, etc.

[0032] The active peptide of Hipposcarus longipes was obtained by protease enzyme hydrolysis. The data were collected by LC-MS / MS and the original file was generated. According to the NCBI database search, the peptide fingerprint was analyzed by Proteome Discover software. Further screening found that there was only one peptide segment with a residual local confidence greater than 80% and a peptide segment sequence Peptide Ranker score greater than 0.8, i.e. GWNIPIGTL. By comparing the active peptides found in the Bioactive peptides database (https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php), it was found that the above-mentioned one peptide segment had not been reported, and it was considered to be a new active peptide sequence. The results are shown in Table 1.

[0033] Table 1 Identification of active peptide sequence of Hipposcarus longipes and activity prediction Sequence Mass m / z Charge Source protein Source protein sequence Peptide activity score a ]] GWNIPIGTL 970.54 1 PREDICTED: serotransferrin-like isoform X1 XP_019748407.1 0.8

[0034] a from PeptideRanker ( http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0035] Molecular docking can effectively realize the calculation of the binding site between the above-mentioned new peptide and the receptor protein. The above-mentioned peptide segment was homologous modeling and molecular docking with XOD and ADA proteins respectively, and the interaction between the peptide segment and the active center amino acid residues of the two was analyzed. The docking results of the peptide segment and XOD protein are shown in Figure 1 A-B of the figure, the target protein XOD used in the experiment has its own quercetin ligand, and the binding energy is-8.7 kcal / mol. The docking mode between the peptide segment and xanthine oxidase in the figure is the best pose after simulation. The binding energy of GWNIPIGTL (GL9) peptide segment and XOD protein is-7.6 kcal / mol. The structure of the peptide segment can be embedded into the active pocket of XOD, and by interacting with the key amino acid residues, it forms conventional hydrogen bonds and hydrophobic interaction forces, thereby blocking the binding of XOD and the substrate. The docking results of the peptide segment and ADA protein are shown in Figure 2 A-B of the figure, the docking mode between the peptide segment and xanthine oxidase in the figure is the best pose after simulation. The binding energy of GWNIPIGTL (GL9) peptide segment and XOD protein is-7.6 kcal / mol. The structure of the peptide segment can be embedded into the active pocket of XOD, and by interacting with the key amino acid residues, it forms conventional hydrogen bonds and hydrophobic interaction forces, thereby blocking the binding of XOD and the substrate. The docking results of the peptide segment and ADA protein are shown in Figure 2 A-B of the figure, the docking mode between the peptide segment and xanthine oxidase in the figure is the best pose after simulation. The binding energy of GWNIPIGTL (GL9) peptide segment and XOD protein is-7.6 kcal / mol. The structure of the peptide segment can be embedded into the active pocket of XOD, and by interacting with the key amino acid residues, it forms conventional hydrogen bonds and hydrophobic interaction forces, thereby blocking the binding of XOD and the substrate. The docking results of the peptide segment and ADA protein are shown in

[0036] Example 2: This example synthesizes a peptide segment and explores the XOD inhibitory activity of the peptide segment in vitro and its effect on a cell model in vitro, including the following steps: One bioactive peptide of the swollen hippocampus predicted in Example 1 was biosynthesized by Fmoc-poly peptide solid-phase synthesis by Shanghai Shenguo Co., Ltd. The purity of GL9 peptide segment was greater than 98.89% after HPLC and MS sequence analysis. The chemically synthesized polypeptide has the characteristics of high purity and good safety in clinical application due to controllable raw materials and process. The biological activity of the synthesized polypeptide was further verified.

[0037] Xanthine oxidase inhibitory activity determination: (1) The xanthine oxidase inhibitory activity of the active peptide segment GL9 was determined by high performance liquid chromatography. In the 96-well plate, the blank group was added with 50 μL of PBS buffer per well, the positive control group was added with 50 μL of allopurinol (0.1 mg / mL) per well, the experimental group was added with 50 μL of sample peptide segment GL9 solution (25 mg / mL) synthesized in Example 1, and xanthine oxidase solution (0.025 U / mL) was added to each well, mixed well, and then incubated in a 37°C constant temperature water bath for 10 min. Then 50 μL (5 mmol / L) of xanthine stock solution was added and mixed well, and incubated at 37°C for 25 min. Finally, HCL (1 mol / L) was added to terminate the reaction, and the reaction solution was taken, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The above experiment was repeated three times in parallel.

[0038] HPLC chromatographic conditions: C18 chromatographic column (4.6 mm × 250 mm, 5 μm), sample size 20 μL, gradient elution program 0-3 min 95% A, 3-6 min 95%-0% A, 6-8 min 0%-95% A, 8-10 min 95% A, detection wavelength 254 nm.

[0039] Table 2 HPLC chromatographic conditions Chromatographic conditions Parameters Column temperature 30 ℃ Flow rate 1 mL / min Mobile phase A 0.1% (v / v) formic acid in water Mobile phase B Methanol

[0040] The inhibition rate of XOD was calculated using the following formula: ; (2) Calculation of IC50 value of XOD inhibitory activity peptide: Set GL9 peptide segment gradient concentration (2, 4, 8, 16, 32 mg / mL), respectively, in the 96-well plate to add the above 50 μL gradient concentration of peptide solution, the blank group each hole adds 50 μL PBS buffer. Each well was added 50 μL of xanthine oxidase solution (0.025 U / mL), oscillation mixed, in 37℃ constant temperature water bath oven preheating 10 min, then add 50 μL (5 mmol / L) xanthine stock solution, mix well, 37℃ incubation for 25 min, finally add 80 μL HCL (1 mol / L) to terminate the reaction, dilute with ultrapure water, through 0.22 μm filter membrane, use HPLC analysis of uric acid content. HPLC chromatographic conditions and XOD inhibition rate calculation method same as above step (1), parallel repeat 3 times, with the logarithm of peptide concentration and XOD inhibition rate as the horizontal and vertical coordinates to make scatter plot, fitting trend line to calculate XOD inhibition IC 50 Value.

[0041] HK-2 cell culture: normal human renal tubular epithelial cell line HK-2 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, and placed in a 37℃, 5% CO2 incubator for culture. The cells were subcultured when they reached the logarithmic growth phase.

[0042] In vitro uric acid reducing activity of active peptide segment: from the logarithmic growth of HK-2 cells, 2x10 5 Cells per well were seeded in a 6-well plate with a seeding volume of 2 mL. The seeded plate was placed in a 37℃, 5% CO2 incubator for incubation, with a duration of 24 h. Blank, model, GL9 peptide segment (1 mmol / L) and allopurinol (100 μmol / L) positive control groups were set up for pre-culture for 24 h. The blank and model groups were not treated, and each group had 3 replicate wells. The culture solution in the plate was aspirated, washed with PBS, and 4 mmol / L adenosine serum-free medium was added to each well of the model group, positive control group and peptide segment group, and serum-free medium was added to the control group for incubation for 24 h. 0.01 U / mL of xanthine oxidase (dissolved in PBS) was added to each well, treated for 6 h, and the supernatant was collected. The uric acid content was determined using a kit.

[0043] The experimental results are shown in Table 3. The in vitro XOD inhibitory activity of hippocampal nonapeptide GL9 was IC 50 =10.67 ± 2.34 mmol / L, which also verified the results of molecular docking. The high XOD inhibitory activity of hippocampal active peptide segment may be related to its ability to form a stable binding conformation in the active site.

[0044] Table 3 In vitro XOD inhibitory activity of active peptide segment Peptide sequence XOD inhibiting IC 50 Values (mmol / L) GWNIPIGTL 10.67±2.34

[0045] The results of HK-2 cell experiments are shown in Figure 3. Compared with the blank group, the uric acid content in the supernatant of the model group cells was significantly increased (0.268 mmol / L, P < 0.01), indicating that 4 mmol / L adenosine and 0.01 U / mL exogenous xanthine oxidase can be used for modeling of a high uric acid cell model. Compared with the model group, the uric acid content in the supernatant of the allopurinol group and the GL9 peptide segment group (intervention dose: 1 mmol / L) was 0.135 mmol / L and 0.154 mmol / L, respectively, which was reduced by 50% and 43% (P < 0.01), respectively, indicating that the GL9 peptide segment has strong uric acid-lowering activity.

[0046] Example 3: This example uses the xanthine oxidase inhibiting peptide in Example 2 to verify the in vivo efficacy of the hippocampus active peptide in lowering uric acid and resisting gout, by constructing a high uric acid mouse model, and specifically includes the following steps: The hippocampus nonapeptide GL9 in Example 2 is used in the animal model of this example.

[0047] 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. During the experiment, the mice could freely eat (AIN-93G type feed) and drink water. After 7 days of adaptive feeding, the mice were randomly divided into a control group, a model group, a positive control group (febuxostat 5.2 mg / kg / d), and a hippocampus nonapeptide GL9 group (100 mg / kg BW). Except for the control group, a high uric acid mouse model was established by continuously gavaging (21 days) 5% carboxymethylcellulose sodium (CMC-Na) diluted hypoxanthine + potassium oxonate suspension (hypoxanthine 1 g / kg + potassium oxonate 0.25 g / kg). The body weight was recorded every 3 days during the experiment, and the gavage volume was adjusted.

[0048] At the same time as modeling, the corresponding test samples were gavaged according to the body weight of the mice in the afternoon every day, and the model group and the control group were gavaged with the same dose of CMC-Na. After the experiment, the mice were fasted for 12 h, and the blood was taken by enucleation after isoflurane anesthesia, and the mice were executed by cervical dislocation. Serum function index determination: the levels of mouse blood uric acid (UA), serum creatinine (Cr), and serum urea nitrogen (BUN) were detected by a full-automatic biochemical analyzer.

[0049] Liver XOD and ADA content determination: 0.1 g of liver tissue was homogenized (1% phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor were added) at 4°C, centrifuged to take the supernatant, and strictly according to the Elisa kit instruction manual.

[0050] Serum UA, Cr, BUN are important indicators to measure whether the kidney function appears disorder. The research results are shown in FIG. 4, Figure 5 and Figure 6 As shown in FIG. 4, compared with the control group, the content of serum UA, Cr, BUN of the model group mice were significantly increased (P < 0.01), which increased by 1.6 times, 1.1 times and 2.2 times, respectively; compared with the model group, the content of UA and the level of BUN of GL9 peptide group were significantly reduced (P < 0.01), which reduced by 23% and 39%, respectively; the content of serum Cr of GL9 peptide group was significantly reduced (12%, P < 0.05). The above results show that the hippocampus active peptide has the effect of reducing uric acid.

[0051] The content of XOD and ADA in the liver tissue of mice is shown in FIG. 7 A-B. Compared with the control group, the content of XOD and ADA in the liver of the model group mice was significantly increased (P < 0.01), which increased by 52% and 85%, respectively. Compared with the model group, the content of XOD and ADA in the liver tissue of the positive control group and GL9 group mice was significantly reduced (P < 0.01), which reduced by 22% and 38% in GL9 group mice, respectively. The above results show that GL9 peptide inhibits the content of uric acid in serum of mice by reducing the level of XOD and ADA in liver.

[0052] Finally, it should be noted that the above examples describe the specific embodiments of the present application, but are not limited to the present application; those skilled in the art should understand that these are only illustrative, the protection scope of the present application is defined by the appended claims. Any modification or equivalent replacement should be included in the protection scope of the present application.

Claims

1. A type of bloating hippocampal peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the expanded hippocampal peptide according to claim 1 in the preparation of products for lowering uric acid and treating gout.

3. The bloating hippocampal peptide as described in claim 1, characterized in that: The bloated hippocampal peptide is artificially synthesized.

Images