A hexapeptide il6 with uric acid-lowering activity and a preparation method and application thereof
By extracting and solid-phase synthesizing hexapeptide IL6 from red algae agar residue, the problems of large side effects and waste of red algae resources of existing XOD inhibitors are solved, and stronger uric acid-lowering activity is achieved, which is suitable for the preparation of uric acid-lowering drugs.
Patent Information
- Application Number
- CN202610115691.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-15
- Estimated Expiration
- 2046-01-28
AI Technical Summary
Existing XOD inhibitors such as allopurinol and febuxostat may cause adverse reactions during the process of lowering uric acid, and the protein resources of agar waste residue, a by-product of red algae processing, have not been effectively utilized.
The hexapeptide IL6 with the amino acid sequence IGGSIL was extracted from the protein in the waste residue of red algae agar and prepared by solid-phase synthesis. Its potential interaction with xanthine oxidase was utilized to prepare uric acid-lowering drugs.
Hexapeptide IL6 showed stronger uric acid-lowering activity in a zebrafish hyperuricemia model, exhibiting better uric acid-lowering effects compared to the classic peptide anserine, and significantly reducing uric acid levels in zebrafish.
Smart Images
Figure CN121591836B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a hexapeptide IL6 with uric acid-lowering activity, its preparation method, and its application. Background Technology
[0002] Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder, closely related to gout and various cardiovascular complications, and its prevalence has shown a significant upward trend in recent years. In the uric acid production pathway, xanthine oxidase (XOD) is a key rate-limiting enzyme, catalyzing the conversion of hypoxanthine to xanthine and further to uric acid, thus becoming an important target for controlling uric acid levels. Currently used XOD inhibitors (such as allopurinol and febuxostat) can effectively lower uric acid, but may cause adverse reactions such as skin rashes and abnormal liver function. Therefore, the development of food-derived XOD inhibitory peptides with safe sources and low side effects has become a research hotspot.
[0003] XOD-inhibiting peptides obtained from proteins of different sources through enzymatic hydrolysis exhibit significant differences in structure and activity. YuqunWu et al., in their article "Purification, identification, and computational analysis of xanthine oxidase inhibitory peptides from kidney bean," reported the isolation of DWYDIK from kidney bean hydrolysate, which possessed good XOD inhibitory activity (inhibition rate 68.63%). Longjun Shu et al., in their article "Short Hexapeptide Optimized from Rice-Derived Peptide 1 Shows Promising Anti-hyperuricemia Activities," revealed the uric acid-lowering activity of AAAAGA in mice. Chen M et al., in their article "Anserine beneficial effects in hyperuricemic rats by inhibiting XOD, regulating uric acid transporter and repairing hepatorenal injury," reported the uric acid-lowering function of the classic uric acid-lowering peptide, anserine. Meanwhile, Yanxin Wang et al. summarized the common uric acid-lowering peptide sequences reported in recent literature in their article "Food-derived bio-functional peptides for the management of hyperuricemia and associated mechanism".
[0004] Marine organisms, due to their unique living environment, often possess protein amino acid sequences with special structures not found in terrestrial organisms, making them an excellent source for discovering novel bioactive peptides. Currently, the protein resources of agar residue, a byproduct of red algae processing, are not being effectively utilized. During agar extraction, approximately 60%-70% of the algal protein remains in the residue and is directly discarded. These proteins provide ideal raw materials for the preparation of uric acid-lowering peptides, offering greater economic benefits than other protein sources. Systematic exploration of red algae agar residue and the discovery of peptides with specific uric acid-lowering activities can achieve high-value transformation of red algae processing waste. Summary of the Invention
[0005] The purpose of this invention is to provide a small molecule peptide with a novel sequence structure and strong uric acid-lowering activity, which was identified from the protein hydrolysate of red algae agar residue, as well as the preparation method and application of the small molecule peptide.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A hexapeptide IL6 with uric acid-lowering activity, wherein the amino acid sequence of the hexapeptide IL6 is IGGSIL.
[0008] The aforementioned method for preparing hexapeptide IL6 with uric acid-lowering activity employs a solid-phase synthesis method, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize hexapeptide IL6 in a solid phase.
[0009] The aforementioned application of hexapeptide IL6 with uric acid-lowering activity in the preparation of uric acid-lowering drugs.
[0010] The advantages of this invention are as follows: The hexapeptide IL6 provided by this invention was identified from the protein hydrolysate of red algae agar waste. Molecular docking revealed that hexapeptide IL6 has a potential interaction with xanthine oxidase. Using a zebrafish hyperuricemia model, the uric acid-lowering activity of hexapeptide IL6 was tested, and it was found that hexapeptide IL6 (concentration 100 μg / mL) can reduce the uric acid content of zebrafish by 34.9%. Compared with the classic uric acid-lowering peptide, anserine (at the same concentration, it can reduce the uric acid content of zebrafish by 25.7%), hexapeptide IL6 has stronger uric acid-lowering activity and can be used to prepare uric acid-lowering drugs to alleviate hyperuricemia. Attached Figure Description
[0011] Figure 1 This is a schematic diagram of the binding mode between hexapeptide IL6 and xanthine oxidase;
[0012] Figure 2 The graph shows the uric acid content results for each group. In the graph, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and ns indicates p>0.05. Detailed Implementation
[0013] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.
[0014] 1. Collect red algae agar waste
[0015] Agar was extracted from Gracilaria verrucosa, a red algae, according to the method described in the local standard DB35T2159-2023, and the waste residue of the red algae agar was collected.
[0016] II. Preparation of protein peptides from red algae agar residue
[0017] The method for preparing protein peptides from red algae agar residue includes the following steps:
[0018] (1) Put 100g of red algae agar waste into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze at this temperature for 2h;
[0019] (2) Continue heating until the temperature reaches 48°C. Then add 3g of alkaline protease and 2g of neutral protease and hydrolyze at this temperature for 2 hours.
[0020] (3) Continue to heat up to 58°C, then add 2g of papain and hydrolyze at this temperature for 3 hours;
[0021] (4) Continue to heat up to 85℃ and hold for 30 minutes;
[0022] (5) Let the enzymatic hydrolysate stand to precipitate, take the supernatant and centrifuge at 8000 rpm for 30 min, and spray dry the supernatant after centrifugation to obtain a powdered product, which is the red algae agar waste protein peptide.
[0023] III. Obtaining the polypeptide sequence from the protein peptides in the waste residue of red algae agar
[0024] The red algae agar waste protein peptides obtained above were analyzed by LC-MS / MS mass spectrometry. The mass spectrometry results were analyzed using mass spectrometry analysis software to obtain several polypeptide sequences.
[0025] The LC-MS / MS determination conditions are as follows:
[0026] (1) Liquid chromatography method: The chromatographic column is C18, 3μm, 250mm×75μm (Eksigent). The mobile phase A is water containing 0.1% formic acid and the mobile phase B is acetonitrile containing 0.1% formic acid. The flow rate is 300nL / min and the injection volume is 1μL. The chromatographic gradient is 70min. The specific elution gradient is as follows: 0-55min, phase A decreases uniformly from 95% to 65%; 55-63min, phase A decreases uniformly from 65% to 50%; 63-64min, phase A decreases uniformly from 50% to 0%; 64-70min, phase A is maintained at 0%.
[0027] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120,000, AGC set to 310, scan range of 110-2000 m / z. MIPS mode is peptide, valence state 1-6 is selected, secondary resolution is 17,500, separation window is 1.6 m / z.
[0028] IV. Screening peak area ≥ 5.00 × 10 6 Active peptides with ≤6 amino acids
[0029] From the several polypeptide sequences obtained above, 62 peak areas ≥ 5.00 × 10⁻⁶ were finally selected. 6 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1, 1-2, and 1-3.
[0030] Table 1-1 High-abundance bioactive peptides in red algae agar waste residue (Part 1)
[0031]
[0032] Table 1-2 High-abundance bioactive peptides in red algae agar waste residue (II)
[0033]
[0034] Table 1-3 High-abundance bioactive peptides in red algae agar waste residue (Part III)
[0035]
[0036] V. Screening for bioactive peptides with strong binding affinity to xanthine oxidase
[0037] Using Discovery Studio software, the active peptide sequences in Tables 1-1, 1-2, and 1-3 were molecularly docked with xanthine oxidase. Before docking, the 2D structure of the active peptides was converted into a 3D structure by minimizing energy, and active peptide sequences with strong binding ability to xanthine oxidase were screened.
[0038] The 3D structure of xanthine oxidase can be downloaded from the RCSB protein database (PDB ID: 1FIQ). Docking results are expressed as docking scores; the higher the docking score, the stronger the binding affinity between the active peptide and xanthine oxidase, and the more likely it is to inhibit xanthine oxidase activity.
[0039] The molecular docking results of the above 62 active peptides with xanthine oxidase are shown in Tables 2-1, 2-2 and 2-3.
[0040] Table 2-1 Predicted results of the interaction between bioactive peptides and xanthine oxidase (I)
[0041]
[0042] Table 2-2 Predicted results of interactions between bioactive peptides and ACE (Part II)
[0043]
[0044] Table 2-3 Predicted results of interactions between bioactive peptides and ACE (Part III)
[0045]
[0046] VI. Molecular docking analysis
[0047] Among the 62 bioactive peptides mentioned above, YEEI (tetrapeptide YI4, SEQ ID NO: 44), YEEL (tetrapeptide YL4, SEQ ID NO: 45), and IGGSIL (hexapeptide IL6, SEQ ID NO: 4) had the highest docking scores, at 103.065 kcal / mol, 102.331 kcal / mol, and 98.7686 kcal / mol, respectively. Their peak areas were similar, at 8.44 × 10⁻⁶ kcal / mol. 6 8.44×10 6 7.62×10 6 .
[0048] According to the study "Anti-hyperuric acid peptides derived from fish hydrolysates based on an in vivo hyperuricemia model and in vitro xanthine oxidase inhibitory activity," the hydrophobic peptide WML is more likely to enter the active site of xanthine oxidase than the hydrophilic peptide PGACSN, possibly due to hydrophobic interactions. Therefore, the hydrophilicity / hydrophobicity of the tetrapeptides YI4 (YEEI), YL4 (YEEL), and IL6 (IGGSIL) were predicted.
[0049] The hydrophilicity and hydrophobicity prediction results of tetrapeptide YI4, tetrapeptide YL4, and hexapeptide IL6 are as follows: The average hydrophilicity coefficients (GRAVY) of tetrapeptide YI4, tetrapeptide YL4, and hexapeptide IL6 are -0.95, -1.13, and 1.87, respectively (negative values represent hydrophilicity, with smaller values indicating greater hydrophilicity; positive values represent hydrophobicity, with larger values indicating greater hydrophobicity). This means that only hexapeptide IL6 is a hydrophobic peptide, which is more likely to enter the active site of xanthine oxidase than tetrapeptide YI4 (a hydrophilic peptide) and tetrapeptide YL4 (a hydrophilic peptide).
[0050] Therefore, the hexapeptide IL6 (IGGSIL), which has the strongest hydrophobicity, was selected for further molecular docking analysis.
[0051] Analysis revealed that the binding mode of hexapeptide IL6 to xanthine oxidase is as follows: Figure 1 As shown, the molecular docking is as follows:
[0052] The hexapeptide IL6 interacts with xanthine oxidase through 9 HH bonds, 2 CH bonds, and 1 electrostatic interaction, with 15 amino acid residues involved in the interaction between hexapeptide IL6 and xanthine oxidase.
[0053] VII. Evaluation of the uric acid-lowering activity of hexapeptide IL6
[0054] The uric acid-lowering activity of hexapeptide IL6 was evaluated using a zebrafish hyperuricemia model, confirming that hexapeptide IL6 has a uric acid-lowering function.
[0055] A solid-phase synthesis method was adopted, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize hexapeptide IL6 (purity >90%).
[0056] Experimental fish: 300 healthy (normally hatched, able to swim normally) 5-day-old wild-type zebrafish (AB strain) were randomly divided into 5 groups, with 6 replicates in each group.
[0057] The experiment was conducted using six-well plates, with 10 zebrafish placed in each well and a culture medium volume of 4 mL per well. The specific experimental groups are as follows:
[0058] (1) Control group: no treatment was given to the zebrafish.
[0059] (2) Model group: Zebrafish were first treated with potassium oxonate for 24 hours, and then treated with sodium xanthine for 24 hours. The final concentration of potassium oxonate was 600 μM and the final concentration of sodium xanthine was 30 μM. A zebrafish hyperuricemia model was constructed.
[0060] (3) Positive control group: First, a zebrafish hyperuricemia model was constructed according to the method of the model group, and then zebrafish were treated with allopurinol for 24 hours. The final concentration of allopurinol was 2mM.
[0061] (4) Goose muscle peptide group: First, a zebrafish hyperuricemia model was constructed according to the model group method, and then zebrafish were treated with goose muscle peptide for 24 hours. The final concentration of goose muscle peptide was 100 μg / mL.
[0062] (5) IL6 group: First, a zebrafish hyperuricemia model was constructed according to the method of the model group. Then, zebrafish were treated with hexapeptide IL6 synthesized in solid phase for 24 hours. The final concentration of hexapeptide IL6 was 100 μg / mL.
[0063] After the experiment, zebrafish were collected in 1.5 mL EP tubes, and the liquid in the EP tubes was aspirated. Then, 40 μL of phosphate-buffered saline (PBS) was added to each EP tube, and the collected zebrafish samples were mechanically homogenized on ice using a handheld tissue homogenizer until no obvious tissue clumps were observed. Subsequently, the samples were centrifuged at 15,000 rpm for 15 min at 4 °C, and the supernatant was transferred to a new EP tube. Finally, the protein and uric acid concentrations of the supernatant were determined using a BCA kit and high-performance liquid chromatography. The remaining supernatant was stored at -80 °C for later use.
[0064] For determining the uric acid concentration in the supernatant: a ZORBAX Original Phenyl column (5 μm, 4.6 mm × 250 mm) was used. Mobile phase A consisted of ultrapure water containing 0.52 mmol / L sodium 1-pentanesulfonate and 0.20 mol / L dipotassium hydrogen phosphate, with the pH adjusted to 4.0 using phosphoric acid solution. Phase B consisted of HPLC-grade acetonitrile. Isocratic elution conditions were: Phase A:Phase B = 85:15 (V / V), flow rate of 1.0 mL / min at 25 °C, injection volume of 10 μL per sample, and run time of 15 min. The column was equilibrated with the mobile phase for at least 30 min before injection.
[0065] The calculated uric acid content (μmol / g protein) for each group is as follows:
[0066] Table 3. Results of uric acid content detection in each group
[0067]
[0068] Note: * indicates comparison with the model, # indicates comparison with the positive control group, and & indicates comparison with the goose muscle peptide group. Among them, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, # indicates p<0.05, and && indicates p<0.01.
[0069] Plot a graph of Table 3, from Figure 2 It can be known that:
[0070] (1) The modeling process increased the uric acid content from 3.43±0.53μmol / g protein to 8.94±1.49μmol / g protein (p<0.001).
[0071] (2) Compared with the model group, allopurinol (2mM) treatment significantly reduced uric acid content to 4.61±1.11μmol / g protein (p<0.01), anserine (100μg / mL) treatment significantly reduced uric acid content to 6.64±0.24μmol / g protein (p<0.05), and hexapeptide IL6 (100μg / mL) treatment significantly reduced uric acid content to 5.82±0.27μmol / g protein (p<0.01).
[0072] (3) There was no significant difference in uric acid content after treatment with hexapeptide IL6 and that after treatment with the positive control allopurinol (p>0.05), but the uric acid content after treatment with hexapeptide IL6 was significantly lower than that after treatment with anserine peptide (p<0.01).
[0073] At the same dose (100 μg / mL), hexapeptide IL6 reduced the uric acid content of zebrafish by 34.9%, while the classic uric acid-lowering peptide, anserine (β-alanyl-1-methyl-L-histidine), reduced the uric acid content of zebrafish by 25.7%. Hexapeptide IL6 has better uric acid-lowering activity than anserine.
[0074] At the same dose (100 μg / mL), hexapeptide IL6 reduced the uric acid content in zebrafish to 5.82 ± 0.27 μmol / g protein, while allopurinol reduced the uric acid content in zebrafish to 4.61 ± 1.11 μmol / g protein. There was no significant difference between the two (p>0.05), indicating that hexapeptide IL6 has a uric acid-lowering ability comparable to allopurinol.
[0075] In summary, hexapeptide IL6 can be used to prepare uric acid-lowering drugs to alleviate hyperuricemia.
[0076] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the protection scope of this invention.
Claims
1. The application of hexapeptide IL6 with uric acid-lowering activity in the preparation of drugs to relieve hyperuricemia, wherein the amino acid sequence of hexapeptide IL6 is IGGSIL.