A pentapeptide VE5 having xanthine oxidase inhibitory activity, and a preparation method and application thereof

Abstract

The application discloses a pentapeptide VE5 with xanthine oxidase inhibitory activity and a preparation method and application thereof, and belongs to the technical field of small-molecule peptides. The amino acid sequence of the pentapeptide VE5 is VSIVE, and the pentapeptide VE5 can be prepared by a solid-phase synthesis method and an enzymolysis method. The pentapeptide VE5 is identified from a porphyra haitanensis protease hydrolysate, has potential interaction with xanthine oxidase, and has an xanthine oxidase inhibition rate of 67.88% at a concentration of 0.1 mg / mL. Compared with ananeuropeptide (the xanthine oxidase inhibition rate of which is 36.03%), the xanthine oxidase inhibition rate is extremely significantly increased (p<0.001), and the xanthine oxidase inhibitory activity is stronger, so that the pentapeptide VE5 can be used for preparing an xanthine oxidase activity inhibiting preparation and relieving hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a pentapeptide VE5 with xanthine oxidase inhibitory 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. Long-term elevated uric acid levels are a significant risk factor for gouty arthritis, uric acid nephropathy, and cardiovascular disease. Uric acid production primarily depends on the catalytic action of xanthine oxidase (XOD). Therefore, directly inhibiting XOD activity is one of the effective strategies for controlling endogenous uric acid production at its source. While clinically available XOD inhibitors (such as allopurinol and febuxostat) have proven efficacy, they also carry significant risks associated with their use. These limitations have prompted researchers to seek XOD inhibitors derived from natural foods with a better safety profile to meet the market demand for long-term prevention and adjunctive regulation.

[0003] Discovering peptides with XOD inhibitory activity from dietary proteins is currently a hot topic in functional peptide research. Compared with chemical drugs, dietary bioactive peptides generally have better biocompatibility and lower risk of side effects, making them suitable for drug development. However, existing research mostly focuses on terrestrial animal milk or plant proteins, and the activity and structural novelty of the peptides obtained are often limited.

[0004] Marine ecosystems harbor biodiversity far exceeding that of terrestrial ecosystems, and their unique environment has endowed marine proteins with special amino acid sequences and functional properties. Porphyra haitanensis, a widely cultivated and consumed large economic red algae, is an excellent source of protein. Currently, the deep processing and utilization of Porphyra haitanensis mainly focuses on ready-to-eat foods and polysaccharide extraction, while its abundant protein resources have not yet been fully and effectively developed at the bioactive peptide level. This makes Porphyra haitanensis a highly promising and yet-to-be-explored new XOD-inhibiting peptide resource. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule peptide with a novel sequence structure and strong xanthine oxidase inhibitory 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 pentapeptide VE5 with xanthine oxidase inhibitory activity, wherein the amino acid sequence of the pentapeptide VE5 is VSIVE.

[0008] The aforementioned method for preparing the pentapeptide VE5 with xanthine oxidase inhibitory 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 pentapeptide VE5 in a solid phase.

[0009] The aforementioned application of the pentapeptide VE5, which has xanthine oxidase inhibitory activity, in the preparation of preparations that inhibit xanthine oxidase activity.

[0010] The advantages of this invention are as follows: This invention discloses and verifies for the first time that an active peptide with a VSIVE sequence (pentapeptide VE5) has xanthine oxidase inhibitory activity. Its structural sequence is different from common sequences, providing a novel core active ingredient and material basis for developing xanthine oxidase inhibitory preparations derived from Porphyra yezoensis. In vitro xanthine oxidase inhibitory activity tests showed that the xanthine oxidase inhibition rate of pentapeptide VE5 (concentration 0.1 mg / mL) was 67.88%, which is significantly increased (p<0.001) compared with the classic uric acid-lowering peptide—gossypol (at the same concentration, the xanthine oxidase inhibition rate is 36.03%). The xanthine oxidase inhibitory activity is stronger and can be used to prepare xanthine oxidase inhibitory preparations to alleviate hyperuricemia. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the binding mode between the pentapeptide VE5 and xanthine oxidase;

[0012] Figure 2 The graph shows the results of the xanthine oxidase inhibition rate detection of pentapeptide VE5 and anserine, where *** indicates p<0.001. Detailed Implementation

[0013] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0014] I. Preparation of Porphyra yezoensis protein peptides

[0015] The method for preparing Porphyra yezoensis protein peptides specifically includes the following steps:

[0016] (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;

[0017] (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.

[0018] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.

[0019] (4) Continue to heat up to 85℃ and hold for 30 minutes;

[0020] (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.

[0021] II. Obtaining the polypeptide sequence from the Porphyra yezoensis protein peptide

[0022] 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.

[0023] The LC-MS / MS determination conditions are as follows:

[0024] (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%.

[0025] (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.

[0026] III. Screening peak area > 5.00 × 10 8 Active peptides with ≤6 amino acids

[0027] From the several polypeptide sequences obtained above, 24 peak areas > 5.00 × 10⁻⁶ were finally selected. 8 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1 and 1-2.

[0028] Table 1-1 High-abundance bioactive peptides in Porphyra yezoensis protein peptides (Part 1)

[0029]

[0030] Table 1-2 High Abundance Bioactive Peptides in Porphyra yezoensis Protein Peptides (Part 2)

[0031]

[0032] IV. Screening for bioactive peptides with strong binding affinity to xanthine oxidase

[0033] 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.

[0034] 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.

[0035] The molecular docking results of the above 24 active peptides with xanthine oxidase are shown in Tables 2-1 and 2-2.

[0036] Table 2-1 Predicted results of the interaction between bioactive peptides and xanthine oxidase (I)

[0037]

[0038] Table 2-2 Predicted results of interactions between bioactive peptides and xanthine oxidase (II)

[0039]

[0040] V. Molecular docking analysis

[0041] Among the 24 bioactive peptides listed in Tables 2-1 and 2-2, VSIVE (denoted as pentapeptide VE5, SEQ ID NO: 19) and IDWR (denoted as tetrapeptide IR4, SEQ ID NO: 22) showed the highest docking scores with xanthine oxidase, at 103.5200 kcal / mol and 107.8660 kcal / mol, respectively. In this invention, VSIVE (pentapeptide VE5) was selected for further molecular docking analysis.

[0042] Analysis revealed that the binding mode of the pentapeptide VE5 to xanthine oxidase is as follows: Figure 1 As shown, the molecular docking is as follows:

[0043] The pentapeptide VE5 interacts with xanthine oxidase through 6 HH bond interactions, 6 CH bond interactions, 1 salt bridge interaction, and 3 electrostatic interactions. Thirteen amino acid residues are involved in the interaction between the pentapeptide VE5 and xanthine oxidase.

[0044] VI. Evaluation of the xanthine oxidase inhibitory activity of the pentapeptide VE5

[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 pentapeptide VE5 (purity >90%).

[0046] The pentapeptide VE5 or anserine (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare VE5 solution and anserine solution with a concentration of 0.1 mg / mL, respectively.

[0047] Sample group: Take 20 μL of VE5 solution or anserine peptide solution and mix it with 20 μL of xanthine oxidase solution (0.05 U / mL) at room temperature for 3 min. Then add 60 μL of xanthine solution (0.4 mmol / L) and react at 37℃ for 25 min. Finally, add 32 μL of hydrochloric acid (1 mol / L) to terminate the reaction.

[0048] Control group: Take 20 μL of PBS buffer and mix it with 20 μL of xanthine oxidase solution (0.05 U / mL) at room temperature for 3 min, then add 60 μL of xanthine solution (0.4 mmol / L), react at 37℃ for 25 min, and finally add 32 μL of hydrochloric acid (1 mol / L) to terminate the reaction.

[0049] The concentration of uric acid in the reaction system was determined by high-performance liquid chromatography (HPLC). 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. Mobile phase B consisted of HPLC-grade acetonitrile. The isocratic elution conditions were A:B = 85:15 (V / V), a flow rate of 1.0 mL / min at 25 °C, an injection volume of 10 μL per sample, and a run time of 15 min. The column was equilibrated with the mobile phase for at least 30 min before injection.

[0050] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0051]

[0052] Where A represents the uric acid concentration in the control group and B represents the uric acid concentration in the sample group.

[0053] Calculations showed that at a concentration of 0.1 mg / mL, the xanthine oxidase inhibition rates of pentapeptide VE5 and anserine were 67.88±2.4% and 36.03±1.9%, respectively. (See the comparison below.) Figure 2 .

[0054] Depend on Figure 2It can be seen that, at the same concentration, compared with the positive control anserine peptide, the xanthine oxidase inhibition rate of pentapeptide VE5 was significantly increased to 67.88±2.4% (p<0.001).

[0055] Therefore, it can be seen that the pentapeptide VE5 has stronger xanthine oxidase inhibitory activity and can be used to prepare preparations that inhibit xanthine oxidase activity to alleviate hyperuricemia.

[0056] 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 pentapeptide VE5 with xanthine oxidase inhibitory activity, characterized in that, The amino acid sequence of the pentapeptide VE5 is VSIVE.

2. The method for preparing the pentapeptide VE5 with xanthine oxidase inhibitory activity as described in claim 1, characterized in that, 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 pentapeptide VE5.

3. The use of the pentapeptide VE5 with xanthine oxidase inhibitory activity as described in claim 1 in the preparation of a drug for relieving hyperuricemia.

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