A bulbus ophionorus peptide and its application in preparation of uric acid-lowering and anti-gout products

Abstract

The present application relates to the preparation and application field of biological peptide, and more particularly to a hippocampus abdominalis peptide and its application in preparation of products for reducing uric acid and resisting gout. The amino acid sequence is shown as SEQ ID NO. 1, and the present application confirms the uric acid reducing effect through in vitro xanthine oxidase (XOD) and adenosine deaminase (ADA) inhibition experiments and cell experiments; on this basis, the uric acid reducing and gout resisting activity of the peptide sequence is further verified by means of a high uric acid animal model. This achievement provides a solid theoretical support for high-value development and utilization of hippocampus abdominalis, and research and development of products for reducing uric acid and resisting gout.

Description

Technical Field

[0001] This invention relates to the field of preparation and application of bioactive peptides, and particularly to a bloated hippocampal peptide and its application in the preparation of products for lowering uric acid and treating gout. Background Technology

[0002] In modern society, gout, a common chronic metabolic disease, brings many problems to patients' lives. Its pathological basis lies in abnormal uric acid metabolism. When too much uric acid is produced or too little is excreted in the body, serum uric acid levels will rise, leading to hyperuricemia. Furthermore, the deposition of urate crystals in joints, soft tissues, and kidneys can trigger a series of adverse consequences, such as inflammatory reactions, joint damage, and even kidney dysfunction.

[0003] The research and development of bioactive peptides from the abdominal seahorse (Hippocampus abdominalis), a high-value marine organism, is making significant strides in the field of anti-gout research. The inventors have previously developed an abdominal seahorse peptide powder, verifying its uric acid-lowering effect using in vitro XOD inhibition and cell experiments. Further verification of its uric acid-lowering and anti-gout activities was conducted using a hyperuricemic animal model, providing a theoretical basis for the high-value utilization of abdominal seahorse and the development of uric acid-lowering and anti-gout products. However, this is still insufficient to address the issues of complex composition and unclear target sites in traditional seahorse powder. It is necessary to identify and clarify more proteins with defined sequences to provide a molecular basis for obtaining abdominal seahorse products with uric acid-lowering and anti-gout properties in a more controllable manner. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the existing technology has insufficient research on the effects of expanding hippocampal peptides on lowering uric acid and preventing gout. It is necessary to identify and clarify more proteins with defined sequences to provide a molecular basis for obtaining expanding hippocampal products with uric acid-lowering and gout-preventing effects in a more controllable manner.

[0005] To address the aforementioned issues, this invention provides a bloated hippocampal peptide and its application in the preparation of uric acid-lowering and anti-gout products. A composite enzyme-directed enzymatic hydrolysis technique combined with membrane separation purification is employed to efficiently prepare the hippocampal peptide. Using LC-MS / MS mass spectrometry and activity prediction software, a hippocampal active peptide sequence exhibiting xanthine oxidase inhibitory activity and analgesic effects is disclosed. The uric acid-lowering effect is verified using in vitro XOD and ADA assays and cell experiments. Furthermore, its uric acid-lowering and anti-gout activities are validated using a hyperuricemic animal model, providing a theoretical basis for the high-value utilization of bloated hippocampus and the development of uric acid-lowering and anti-gout products.

[0006] To achieve the above objectives, the present invention utilizes the following technical means: a bloating hippocampal peptide, the amino acid sequence of which is shown in SEQ ID NO.1:

[0007] SEQ ID NO.1:

[0008] GWNIPIGTL.

[0009] The above-mentioned expanded hippocampal peptide is used in the preparation of uric acid-lowering and anti-gout products. Hyperuricemia is essentially caused by abnormal purine metabolism, leading to excessive production or insufficient excretion of purine substances. XOD is a key rate-limiting enzyme in uric acid synthesis, catalyzing the production of uric acid through the binding of molybdenum atoms at its activation site to xanthine. ADA is a crucial aminotransferase in purine metabolism, participating in uric acid synthesis through the conversion of adenosine. Therefore, the activities of XOD and ADA can serve as important indicators for monitoring abnormal uric acid metabolism; thus, inhibiting the activities of XOD and ADA can effectively reduce uric acid production. In this invention, molecular docking results showed that the peptide segment shown in SEQ ID NO.1 binds to XOD and ADA proteins with energies of -7.6 kcal / mol and -5.2 kcal / mol, respectively. The structures of the peptide segments can be inserted into the active pockets of XOD and ADA, inhibiting their activities through interactions with key amino acid residues, forming van der Waals forces, conventional hydrogen bonds, and hydrophobic interactions, ultimately achieving the goal of alleviating hyperuricemia.

[0010] Furthermore, the bloated hippocampal peptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized.

[0011] The preparation method of the above-mentioned expanded hippocampal peptide powder includes the following steps:

[0012] Fresh bloated seahorses were taken, washed, freeze-dried, and pulverized into powder. Distilled water was added, and 3% compound protease was added and stirred evenly. The pH was adjusted to 7.5 with HCl solution and NaOH solution, respectively, and enzymatic hydrolysis was carried out at 50℃ for 5 h.

[0013] After centrifugation, the supernatant of the enzymatic hydrolysate was filtered through a 0.45 μm microporous membrane and then a 3 kDa molecular weight cutoff ultrafiltration tube was used to retain peptides with a molecular weight cutoff of 3 kDa. The peptide solution was freeze-dried to obtain expanded hippocampal peptide powder.

[0014] Furthermore, distilled water is added at a material-to-liquid ratio of 1:9.

[0015] Furthermore, the concentrations of both the HCl solution and the NaOH solution are 1 mol / L.

[0016] Furthermore, the enzyme activity of the complex protease is 1.2 million U / mg.

[0017] Furthermore, the enzymatic hydrolysis endpoint is controlled as follows: degree of hydrolysis DH ≥ 25%, and molecular weight distribution of 500-3000 Da ≥ 80%.

[0018] Furthermore, the enzymatic hydrolysate was centrifuged at 4000 g / min for 15 min at 4°C, and the supernatant was collected.

[0019] Furthermore, the complex protease, 1.2 million U / mg, catalog number: S10155, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] The above amino acid sequence is the bloated hippocampal peptide shown in SEQ ID NO.1.

[0021] Furthermore, the bloated hippocampal peptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention successfully screened a hippocampal peptide sequence that not only possesses xanthine oxidase inhibitory activity but also anti-analgesic effects. In the study, its uric acid-lowering effect was confirmed through in vitro xanthine oxidase (XOD) inhibition experiments and cell experiments. Furthermore, the uric acid-lowering and anti-gout activities of this peptide sequence were further verified using a hyperuricemic animal model. This achievement provides solid theoretical support for the high-value development and utilization of the abdominal-expanding hippocampus, as well as the research and development of uric acid-lowering and anti-gout related products. Attached Figure Description

[0024] Figure 1 Figure 1 shows the docking results of the SEQ ID NO.1 peptide with the XOD receptor protein; where A is the 3D structure of the docking of the SEQ ID NO.1 peptide with the XOD receptor protein; and B is the 2D structure of the docking of the SEQ ID NO.1 peptide with the XOD receptor protein.

[0025] Figure 2 Figure 1 shows the docking results of the SEQ ID NO.1 peptide with the ADA receptor protein; where A is the 3D structure of the docking of the SEQ ID NO.1 peptide with the ADA receptor protein; and B is the 2D structure of the docking of the SEQ ID NO.1 peptide with the ADA receptor protein.

[0026] Figure 3 Effect of peptides on uric acid levels in the supernatant of HK-2 cell model; Note: ## p < 0.01 compared with the blank group; ** p < 0.01 compared with the model group.

[0027] Figure 4 Serum uric acid levels in mice; Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group.

[0028] Figure 5: Serum urea nitrogen level in mice; Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group.

[0029] Figure 6 : Serum creatinine level in mice; Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group.

[0030] Figure 7 : Mouse liver XOD and ADA levels; where A is XOD level and B is ADA level; Note: ## p < 0.01 compared with control group; ** p < 0.01 compared with model group. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0033] Example 1:

[0034] This example provides a method for preparing expanded hippocampal polypeptide powder using a combination of enzymatic targeted hydrolysis and membrane separation technology. The peptides were identified by LC-MS / MS, and novel peptides exhibiting significant xanthine oxidase inhibitory activity and uric acid excretion-promoting effects were screened using molecular docking technology. The specific steps include the following:

[0035] Preparation of enzymatic hydrolysate of expanded hippocampus peptides: 100 g of fresh expanded hippocampus was washed, freeze-dried, and pulverized into powder. Distilled water was added at a material-to-liquid ratio of 1:9 (m / v), and 3% complex protease was added and stirred evenly. The pH was adjusted to 7.5 with HCl solution (1 mol / L) and NaOH solution (1 mol / L), respectively, and enzymatic hydrolysis was carried out at 50°C for 5 h. Hydrolysis endpoint control: degree of hydrolysis (DH) ≥ 25%, molecular weight distribution concentrated in the 500-3000 Da range (≥ 80%). After centrifugation (4°C, 4000 g / min for 15 min), the supernatant was filtered through a 0.45 μm microporous membrane, and then ultrafiltration was performed using a 3 kDa molecular weight cutoff ultrafiltration tube to retain peptides with a molecular weight cutoff of 3 kDa. The peptide solution was freeze-dried to obtain expanded hippocampus peptide powder. The complex protease (1.2 million U / mg, S10155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] LC-MS / MS identification of active hippocampal peptides after enzymatic digestion: The sample was reconstituted in 200 μL of 0.1% TFA solution and vortexed to mix. The pipette tip, after being rinsed with 0.1% TFA solution, was repeatedly pipetted 15-20 times to adsorb the sample onto the C18 column. Salts on the C18 column were washed away by eluting three times in 50% acetonitrile + 0.1% TFA solution until desalting was complete. The hippocampal peptides were analyzed using an UltiMate 3000 UHPLC system (Thermo Fisher Scientific) with an ACQUITY UPLC peptide CSH C18 column (2.1 × 100 mm, 1.7 μm) and a TripleTOF 5600+ system to determine the sequence of the eluted peptides. A hippocampal protein database search was performed using NCBI. Peptide Ranker was used to predict the activity of the identified peptides, and peptide sequences with higher scores were selected.

[0037] Molecular docking screening of active peptides: Using xanthine oxidase (XOD) and adenosine deaminase (ADA) as key target proteins, peptides were homology-modeled and molecularly docked with the major binding active sites of XOD and ADA proteins, respectively. Their respective ligands served as negative controls. Peptide sequence homology modeling: Peptide sequences were input into Chemdraw3D 18.1 software for 3D modeling to generate peptide structures. Energy minimization of the peptide structure was performed using the CHARMm program in Discover Studio 2019.

[0038] Receptor pretreatment: X-ray crystal structures of the XOD protein (PDBID: 3NS1) and ADA protein (PDBID: 3IAR) were downloaded from the PDB database. Existing ligands were removed using Discover Studio 2019 software, and chemical bonds were corrected and charges were added. Active pockets were further identified and covered. Molecular docking of the receptor and peptide was performed using the CDOCKER program. The docking score was calculated based on the highest CDOCKER Energy score, combined with indicators such as free energy, hydrogen bonds, hydrophobic groups, and charged groups.

[0039] Bioactive peptides from inflated hippocampus were obtained by enzymatic hydrolysis using a complex protease. Data were acquired by LC-MS / MS and raw files were generated. Peptide fingerprints were analyzed using Proteome Discover software based on a search of the NCBI database. Further screening identified one peptide, GWNIPIGTL, with a residual local confidence score greater than 80% and a peptide sequence Peptide Ranker score greater than 0.8. Comparison with previously discovered bioactive peptides in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php) revealed that this single peptide sequence had not been previously reported and was therefore considered a novel bioactive peptide sequence. The results are shown in Table 1.

[0040] Table 1. Sequence identification and activity prediction of active peptides from the distended hippocampus.

[0041] sequence Mass-to-nucleus ratio m / z electric charge Source protein Source protein sequence <![CDATA[Peptide activity score a > GWNIPIGTL 970.54 1 PREDICTED: serotransferrin-like isoform X1 XP_019748407.1 0.8

[0042] a From PeptideRanker ( http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0043] Molecular docking can effectively calculate the binding sites between the aforementioned novel peptides and receptor proteins. Homology modeling was performed on the peptides, and molecular docking was conducted with XOD and ADA proteins, respectively, to analyze the interactions between the peptides and the amino acid residues at their active sites. The docking results between the peptides and XOD protein are shown below. Figure 1 As shown in Figure AB, the target protein XOD used in this experiment has a quercetin ligand in its receptor, with a binding energy of -8.7 kcal / mol. The docking mode between the peptide and xanthine oxidase in the figure represents the optimal posture after simulation. The GWNIPIGTL (GL9) peptide binds to XOD protein with an energy of -7.6 kcal / mol. The peptide structure can insert into the active pocket of XOD, forming conventional hydrogen bonds and hydrophobic interactions with key amino acid residues, thus blocking XOD from binding to the substrate. The docking results of the peptide with ADA protein are shown in the figure. Figure 2 As shown in Figure AB, the docking mode between the peptide and adenosine deaminase in the figure represents the optimal posture after simulation. The binding energy of the GL9 peptide to ADA protein is -5.2 kcal / mol. The peptide structure interacts with ADA protein through multiple valence bonds, including van der Waals forces, hydrogen bonds, alkyl bonds, and π-alkyl bonds, forming a stable conformation and thus inhibiting ADA activity. These results indicate that GL9 is a good inhibitor of XOD and ADA and has the potential to be developed into a multi-target uric acid-lowering active ingredient.

[0044] Example 2:

[0045] This example demonstrates the artificial synthesis of peptides and investigates their in vitro XOD inhibitory activity and effects on in vitro cell models. The specific steps include:

[0046] A bioactive peptide from the bloating hippocampus predicted in Example 1 was biosynthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc-peptide solid-phase synthesis method. HPLC and MS sequence analysis showed that the purity of the GL9 peptide was greater than 98.89%. Chemically synthesized peptides, due to the controllability of raw materials and processes, exhibit high purity and good safety for clinical application. Further verification of the bioactivity of the synthesized peptide was conducted.

[0047] Assay for xanthine oxidase inhibitory activity:

[0048] (1) The xanthine oxidase inhibitory activity of the active peptide GL9 was determined by high performance liquid chromatography. In a 96-well plate, 50 μL of PBS buffer was added to each well of the blank group, 50 μL of allopurinol (0.1 mg / mL) was added to each well of the positive control group, and 50 μL of the sample peptide GL9 solution (25 mg / mL) synthesized in Example 1 was added to each well of the experimental group. 50 μL of xanthine oxidase solution (0.025 U / mL) was added to each well of each group and mixed well. The mixture was preheated in a 37℃ water bath for 10 min, and then 50 μL (5 mmol / L) of xanthine stock solution was added and mixed thoroughly. The mixture was kept at 37℃ for 25 min, and finally HCl (1 mol / L) was added to terminate the reaction. The reaction solution was filtered through a 0.22 μm filter membrane and analyzed by HPLC. The above experiment was repeated 3 times in parallel.

[0049] HPLC chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm), injection volume 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.

[0050] Table 2 HPLC Chromatographic Conditions

[0051] Chromatographic conditions parameter Column temperature 30 ℃ Flow rate 1 mL / min Mobile phase A 0.1% (v / v) formic acid aqueous solution Mobile phase B methanol

[0052] The inhibition rate of XOD is calculated using the following formula:

[0053] ;

[0054] (2) Calculation of the IC50 value of the XOD inhibitory active peptide:

[0055] Set up gradient concentrations of GL9 peptides (2, 4, 8, 16, 32 mg / mL), and add 50 μL of each concentration to a 96-well plate. For the blank control group, add 50 μL of PBS buffer to each well. Add 50 μL of xanthine oxidase solution (0.025 U / mL) to each well, vortex to mix, preheat in a 37℃ water bath for 10 min, then add 50 μL (5 mmol / L) xanthine stock solution, mix thoroughly, incubate at 37℃ for 25 min, and finally add 80 μL of HCl (1 mol / L) to terminate the reaction. Dilute with ultrapure water, filter through a 0.22 μm filter membrane, and analyze uric acid content by HPLC. The HPLC chromatographic conditions and XOD inhibition rate calculation method are the same as in step (1) above. The experiment was repeated three times in parallel. A scatter plot was plotted with the logarithm of peptide concentration and XOD inhibition rate as the x and y axes, respectively. After fitting a trend line, the IC50 of XOD inhibition was calculated. 50 value.

[0056] 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 and placed in a 37°C, 5% CO2 incubator. The cells were passaged after reaching the logarithmic growth phase.

[0057] In vitro uric acid-lowering activity assay of active peptides: Samples were taken from logarithmically grown HK-2 cells, at 2 x 10⁻⁶ wells per well. 5 Cells were seeded at a density of 1,000 cells / well in 6-well plates, with a seeding volume of 2 mL per well. The seeded plates were incubated at 37°C with 5% CO2 for 24 h. A blank control group, a model group, a GL9 peptide group (1 mmol / L), and a positive control group (100 μmol / L) were pre-cultured for 24 h. The blank control group and model group received no treatment. Each group had three replicates. The culture medium was aspirated from the plates, and after washing with PBS, 4 mmol / L adenosine serum-free medium was added to each well of the model group, positive control group, and peptide group. The control group was incubated with serum-free medium for 24 h. 0.01 U / mL xanthine oxidase (dissolved in PBS) was added to each well, and after 6 h of treatment, the supernatant was collected, and the uric acid content was determined using a kit.

[0058] The experimental results are shown in Table 3. The in vitro XOD inhibitory activity of hippocampal nonapeptide GL9 is IC50. 50 =10.67 ± 2.34 mmol / L, which also verifies the molecular docking results. The high efficiency of the hippocampal active peptide in inhibiting XOD may be related to its ability to form a stable binding conformation at the active site.

[0059] Table 3. In vitro XOD inhibitory activity of active peptides

[0060] peptide sequence <![CDATA[XOD Inhibiting IC 50 Value (mmol / L)]]> GWNIPIGTL 10.67±2.34

[0061] The results of the HK-2 cell experiments are shown in Figure 3. Compared with the blank group, the uric acid content in the cell supernatant of the model group 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 to establish a hyperuricemic cell model. Compared with the model group, the uric acid content in the cell supernatant of the allopurinol group and the GL9 peptide group (intervention dose: 1 mmol / L) was 0.135 mmol / L and 0.154 mmol / L, respectively, which were reduced by 50% and 43% (P < 0.01), indicating that the GL9 peptide has a strong uric acid-lowering activity.

[0062] Example 3:

[0063] This example demonstrates the in vivo validation of the xanthine oxidase inhibitory peptide from Example 2 by constructing a hyperuricemic mouse model, aiming to clarify the uric acid-lowering and anti-gout effects of hippocampal active peptides in vivo. The specific steps include the following:

[0064] The hippocampal nonapeptide GL9 from Example 2 was used in the animal model of this example.

[0065] Animal Experiments: Forty healthy male C57bl / 6J mice (7 weeks old, weighing 20±1.0 g) were purchased from Jinan Pengyue Experimental Animal Technology Co., Ltd. During the experiment, the mice had free access to food (AIN-93G type diet) and water. After 7 days of acclimatization, the mice were randomly divided into a control group, a model group, a positive control group (febuxostat 5.2 mg / kg / d), and a hippocampal nonapeptide GL9 group (100 mg / kg BW). Except for the control group, a mouse model of hyperuricemia was established by continuous gavage (21 days) of a 5% carboxymethyl cellulose sodium (CMC-Na) diluted hypoxanthine + potassium oxonate suspension (hypoxanthine 1 g / kg + potassium oxonate 0.25 g / kg). Body weight was recorded every 3 days during the experiment, and the gavage volume was adjusted.

[0066] During modeling, mice were administered the corresponding test sample by gavage daily in the afternoon according to their body weight. The model group and control group were administered the same dose of CMC-Na by gavage. After the experiment, the mice were fasted for 12 hours, anesthetized with isoflurane, enucleated to collect blood, and euthanized by cervical dislocation. Serum functional indicators were measured: serum uric acid (UA), serum creatinine (Cr), and serum urea nitrogen (BUN) levels in mice were detected using a fully automated biochemical analyzer.

[0067] Determination of liver XOD and ADA content: 0.1 g of liver tissue was homogenized at 4°C (with 1% benzyl sulfonyl fluoride (PMSF) and phosphatase inhibitor added), the supernatant was collected by centrifugation, and the determination was strictly carried out according to the ELISA kit instructions.

[0068] Serum UA, Cr, and BUN are important indicators for assessing whether renal function is impaired. The study results are shown in Figure 4. Figure 5 and Figure 6 As shown, compared with the control group, the serum levels of UA, Cr, and BUN in the model group mice were significantly increased (P < 0.01), increasing by 1.6-fold, 1.1-fold, and 2.2-fold, respectively. Compared with the model group, the UA and BUN levels in the GL9 peptide group were significantly decreased (P < 0.01), decreasing by 23% and 39%, respectively, and the serum Cr level in the GL9 peptide group was significantly decreased (12%, P < 0.05). These results indicate that hippocampal active peptides have a uric acid-lowering effect.

[0069] The levels of XOD and ADA in mouse liver tissue are shown in Figure 7 (AB). Compared with the control group, the levels of XOD and ADA in the liver of the model group mice were significantly increased (P < 0.01), by 52% and 85%, respectively. Compared with the model group, the levels of XOD and ADA in the liver tissue of both the positive control group and the GL9 group mice were significantly decreased (P < 0.01), with the levels of XOD and ADA in the liver tissue of the GL9 group mice decreasing by 22% and 38%, respectively. These results indicate that GL9 peptide inhibits serum uric acid levels in mice by reducing the levels of XOD and ADA in the liver.

[0070] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

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 uric acid-lowering and anti-gout drugs.

3. The swelling-inducing hippocampal peptide according to claim 1, characterized in that: The bloated hippocampal peptide is artificially synthesized.

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