A tetrapeptide IR4 with uric acid-lowering activity, and a preparation method and application thereof
Tetrapeptide IR4 was prepared by enzymatic hydrolysis and solid-phase synthesis of Porphyra yezoensis protein resources, which solved the problems of large side effects and limited active peptide sequences of existing uric acid-lowering drugs, and achieved a highly efficient and safe uric acid-lowering effect. Tetrapeptide IR4 showed significant uric acid-lowering activity in zebrafish model.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-15
AI Technical Summary
Existing uric acid-lowering drugs such as allopurinol and febuxostat have adverse reactions such as skin reactions and abnormal liver function, which cannot meet the needs of long-term medication. Furthermore, existing bioactive peptides have limitations in terms of sequence novelty and activity intensity, and algal protein resources have not been fully utilized in the development of bioactive peptides.
Tetrapeptide IR4 with an IDWR sequence was prepared using solid-phase synthesis and enzymatic hydrolysis. Utilizing the protein resources of *Porphyra yezoensis*, tetrapeptide IR4 with highly efficient uric acid-lowering activity was obtained through multi-step enzymatic hydrolysis by yeast, alkaline protease, neutral protease, and papain, combined with solid-phase synthesis technology.
Tetrapeptide IR4 exhibited 33.6% uric acid-lowering activity in a zebrafish hyperuricemia model, which was significantly better than that of traditional anserine peptide. It also had uric acid-lowering ability comparable to allopurinol, providing a safe and effective uric acid-lowering drug solution.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a tetrapeptide IR4 with uric acid-lowering activity, its preparation method, and its application. Background Technology
[0002] The incidence of hyperuricemia (HUA) is rising globally and showing a trend towards affecting younger people. Xanthine oxidase (XOD), a key catalytic enzyme in the uric acid production pathway, is an important target for controlling endogenous uric acid levels. However, widely used XOD inhibitors (such as allopurinol and febuxostat) have potential adverse reactions such as skin reactions and abnormal liver function, failing to meet the needs of patients requiring long-term medication. Therefore, the development of safe, low-side-effect food-derived uric acid-lowering peptides has become an important research direction.
[0003] In the development of urate-lowering peptides, bioactive peptides obtained from different protein sources show significant differences in structure and efficacy. Li Xiaotong et al. reported the urate-lowering activity of sheep bone bioactive peptides in their article "In Vivo and In Vitro Uric Acid-Lowering Activity and Mechanism of Action of Sheep Bone Bioactive Peptides." Xiao-Dong Pei et al. reported the urate-lowering peptide GNQQVHLQSQDM derived from feather keratin protein hydrolysate in their article "Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities," highlighting its XOD inhibition IC50. 50 The value was 12.15 mg / mL. Chen Qian reported quantitative structure-activity relationship prediction models for 192 urate-lowering peptides in her article "Virtual Screening, Activity Mechanism and Predictive Model Study of Uric Acid-Lowering Peptides from Porcine Viscera". Chen Y et al. systematically summarized the acquisition, activity, and mechanism of action of urate-lowering peptides in their article "Anti-hyperuricemia bioactive peptides: a review on obtaining, activity, and mechanism of action". Existing research mainly focuses on terrestrial animal milk proteins or some plant proteins, and the reported active peptides have certain limitations in terms of sequence novelty and activity intensity. In contrast, algal proteins, due to their special living environment, often possess unique amino acid composition and functional potential. Feng Yuyao et al. reported multiple functions of algal active peptides in their article "Research Progress on Functional Activity and Application of Algal Active Peptides".
[0004] Porphyra haitanensis, a large economic algae with abundant production and wide edibility, is high in protein. Currently, the deep processing and utilization of Porphyra haitanensis mainly focuses on ready-to-eat foods and polysaccharide extraction; its rich protein resources have not yet been fully and effectively developed at the bioactive peptide level. 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 is identified from the protein hydrolysate of *Porphyra yezoensis*, 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 tetrapeptide IR4 with uric acid-lowering activity, wherein the amino acid sequence of the tetrapeptide IR4 is IDWR.
[0008] The aforementioned method for preparing the tetrapeptide IR4 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 the tetrapeptide IR4 in a solid phase.
[0009] The aforementioned method for preparing the tetrapeptide IR4 with uric acid-lowering activity is an enzymatic hydrolysis method, specifically as follows: (1) Porphyra yezoensis is placed in water, heated to 40°C, and yeast is added. The mixture is hydrolyzed at this temperature for 2 hours. (2) The temperature is further increased to 48°C, and alkaline protease and neutral protease are added. The mixture is hydrolyzed at this temperature for 2 hours. (3) The temperature is further increased to 58°C, and papain is added. The mixture is hydrolyzed at this temperature for 3 hours. (4) The temperature is further increased to 85°C and maintained for 30 minutes. (5) The hydrolysate is allowed to stand and precipitate. The supernatant is centrifuged, and the supernatant after centrifugation is spray-dried to obtain Porphyra yezoensis protein peptide, which contains tetrapeptide IR4. The mass ratio of Porphyra yezoensis, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2.
[0010] The aforementioned application of the tetrapeptide IR4 with uric acid-lowering activity in the preparation of uric acid-lowering drugs.
[0011] The advantages of this invention are as follows: This invention discloses and verifies for the first time that an active peptide (tetrapeptide IR4) with an IDWR sequence has uric acid-lowering activity. Its structural sequence is different from common sequences, providing a novel core active ingredient and material basis for developing uric acid-lowering drugs derived from Porphyra yezoensis. Using a zebrafish hyperuricemia model, the uric acid-lowering activity of tetrapeptide IR4 was tested and found that tetrapeptide IR4 (concentration 100 μg / mL) can reduce the uric acid content of zebrafish by 33.6%. 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%), tetrapeptide IR4 has stronger uric acid-lowering activity and can be used to prepare uric acid-lowering drugs to alleviate hyperuricemia. Attached Figure Description
[0012] Figure 1 This is a schematic diagram of the binding mode between the tetrapeptide IR4 and xanthine oxidase;
[0013] Figure 2 This is a graph showing the uric acid content test results for each group. In the graph, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and ns represents p>0.05. Detailed Implementation
[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.
[0015] I. Preparation of Porphyra yezoensis protein peptides
[0016] The method for preparing Porphyra yezoensis protein peptides specifically includes the following steps:
[0017] (1) Put 100g of laver into 1000mL of water, heat it to 40℃ and add 1g of yeast, and enzymatically hydrolyze it at this temperature for 2h;
[0018] (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.
[0019] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.
[0020] (4) Continue to heat up to 85℃ and hold for 30 minutes;
[0021] (5) Let the enzyme hydrolysate stand to precipitate, take the supernatant and centrifuge at 8000 rpm for 30 min, and spray dry the supernatant after centrifugation to obtain the powdered product - Lagerstroemia indica protein peptide.
[0022] II. Obtaining the polypeptide sequence from the Porphyra yezoensis protein peptide
[0023] The obtained Porphyra protein peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry software to obtain several polypeptide sequences.
[0024] The LC-MS / MS determination conditions are as follows:
[0025] (1) Liquid chromatography method: The chromatographic column is C18, 3μm, 250mm×75μm (Eksigent). The mobile phase A is ultrapure water containing 0.1% formic acid, and the mobile phase B is acetonitrile containing 0.1% formic acid. The flow rate is 300nL / min, the injection volume is 1μL, and 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%.
[0026] (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.
[0027] III. Screening peak area > 5.00 × 10 8 Active peptides with ≤6 amino acids
[0028] From the several polypeptide sequences obtained above, 24 peak areas > 5.00 × 10⁻⁶ were finally screened out. 8 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1 and 1-2.
[0029] Table 1-1 High-abundance bioactive peptides in Porphyra yezoensis protein peptides (Part 1)
[0030]
[0031] Table 1-2 High Abundance Bioactive Peptides in Porphyra yezoensis Protein Peptides (Part 2)
[0032]
[0033] IV. Screening for bioactive peptides with strong binding affinity to xanthine oxidase
[0034] Using Discovery Studio software, the active peptide sequences in Tables 1-1 and 1-2 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.
[0035] 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.
[0036] The molecular docking results of the above 24 bioactive peptides with xanthine oxidase are shown in Table 2.
[0037] Table 2. Predicted Interactions Between Active Peptides and Xanthine Oxidase
[0038]
[0039] V. Molecular docking analysis
[0040] Among the 24 bioactive peptides listed in Table 2, VSIVE (denoted as pentapeptide VE5, SEQ ID NO: 19) and IDWR (denoted as tetrapeptide IR4, SEQ ID NO: 22) had the highest docking scores, at 103.5200 kcal / mol and 107.866 kcal / mol, respectively. In this invention, IDWR (tetrapeptide IR4) was selected for further molecular docking analysis.
[0041] Analysis revealed that the binding mode of the tetrapeptide IR4 to xanthine oxidase is as follows: Figure 1 As shown, the molecular docking is as follows:
[0042] The tetrapeptide IR4 interacts with xanthine oxidase through 11 HH bond interactions, 3 CH bond interactions, 1 salt bridge interaction, and 1 electrostatic interaction. Fifteen amino acid residues are involved in the interaction between the tetrapeptide IR4 and xanthine oxidase.
[0043] VI. Evaluation of the uric acid-lowering activity of tetrapeptide IR4
[0044] The uric acid-lowering activity of tetrapeptide IR4 was evaluated using a zebrafish hyperuricemia model, confirming that tetrapeptide IR4 has the function of lowering uric acid.
[0045] 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 the tetrapeptide IR4 (purity >90%).
[0046] 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.
[0047] 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:
[0048] (1) Control group: no treatment was given to the zebrafish.
[0049] (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.
[0050] (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.
[0051] (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.
[0052] (5) IR4 group: First, a zebrafish hyperuricemia model was constructed according to the method of the model group. Then, zebrafish were treated with tetrapeptide IR4 obtained by solid-phase synthesis for 24 hours. The final concentration of tetrapeptide IR4 was 100 μg / mL.
[0053] 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.
[0054] 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.
[0055] The calculated uric acid content (μmol / g protein) for each group is as follows:
[0056] Table 3. Results of uric acid content detection in each group
[0057]
[0058] Note: * indicates comparison with the model group, # 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<0.05.
[0059] Plot a graph of Table 3, from Figure 2 It can be known that:
[0060] (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).
[0061] (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 tetrapeptide IR4 (100μg / mL) treatment significantly reduced uric acid content to 5.93±0.47μmol / g protein (p<0.01).
[0062] (3) There was no significant difference in uric acid content after tetrapeptide IR4 treatment and uric acid content after positive control allopurinol treatment (p>0.05), but the uric acid content after tetrapeptide IR4 treatment was significantly lower than that after anserine treatment (p<0.05).
[0063] At the same dose (100 μg / mL), tetrapeptide IR4 reduced the uric acid content of zebrafish by 33.6%, while the classic uric acid-lowering peptide, anserine (β-alanyl-1-methyl-L-histidine), reduced the uric acid content of zebrafish by 25.7%. Tetrapeptide IR4 has better uric acid-lowering activity than anserine.
[0064] At the same dose (100 μg / mL), tetrapeptide IR4 reduced the uric acid content in zebrafish to 5.93 ± 0.47 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), and tetrapeptide IR4 has a uric acid-lowering ability comparable to allopurinol.
[0065] In summary, the tetrapeptide IR4 can be used to prepare uric acid-lowering drugs to alleviate hyperuricemia.
[0066] 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. A method for preparing a tetrapeptide IR4 with uric acid-lowering activity, wherein the amino acid sequence of the tetrapeptide IR4 is IDWR, characterized in that, The enzymatic hydrolysis method is used, as detailed below: (1) Put the seaweed into water, heat it to 40°C, add yeast, and enzymatically hydrolyze it at this temperature for 2 hours; (2) Continue heating until the temperature reaches 48°C. Then add alkaline protease and neutral protease and hydrolyze at this temperature for 2 hours. (3) Continue to heat up to 58°C and add papain. At this temperature, enzymatically hydrolyze for 3 hours. (4) Continue to heat up to 85℃ and hold for 30 minutes; (5) Let the enzymatic hydrolysate stand to precipitate, take the supernatant and centrifuge, and spray dry the supernatant after centrifugation to obtain the Porphyra yezoensis protein peptide, which contains tetrapeptide IR4; The ratio of the amounts of laver, yeast, alkaline protease, neutral protease and papain by mass is 100:1:3:2:
2.
2. The application of tetrapeptide IR4 with uric acid-lowering activity in the preparation of uric acid-lowering drugs, wherein the amino acid sequence of the tetrapeptide IR4 is IDWR.