A pentapeptide LE5 having xanthine oxidase inhibitory activity and a preparation method and application thereof

Abstract

The application discloses a pentapeptide LE5 with xanthine oxidase inhibiting 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 LE5 is LGGVE, and the pentapeptide LE5 can be prepared by a solid-phase synthesis method and an enzymolysis method. The pentapeptide LE5 is identified from a Gracilaria lemaneiformis protein enzymolysis liquid, has potential interaction with xanthine oxidase, and has an xanthine oxidase inhibition rate of 60.58% at a concentration of 0.1 mg / mL. Compared with an anserine (xanthine oxidase inhibition rate of 36.03%), the xanthine oxidase inhibition rate is extremely significantly increased (p<0.001), and the xanthine oxidase inhibiting activity is stronger, so that the pentapeptide LE5 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 LE5 with xanthine oxidase inhibitory activity, its preparation method, and its application. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by disordered purine metabolism and / or reduced uric acid excretion, leading to abnormally high uric acid levels in the blood. Long-term hyperuricemia can cause urate crystals to deposit in joints, soft tissues, kidneys, and other sites, triggering various complications and seriously endangering human health. First-line uric acid-lowering drugs in clinical practice mainly include allopurinol and febuxostat, which inhibit uric acid production, and benzbromarone, which promotes uric acid excretion. These chemical drugs have significant side effects with long-term use. Therefore, developing safe, efficient, and naturally derived novel uric acid-lowering functional factors has become an important research direction in the fields of food science, pharmacy, and medicine.

[0003] Xanthine oxidase (XOD) is a key rate-limiting enzyme in the production of uric acid in the body, and inhibiting XOD activity is one of the effective strategies to reduce uric acid production at its source. Researchers have been searching for low-toxicity and highly effective XOD inhibitors from natural products, and various food-derived bioactive peptides from animal and plant proteins have been found to have XOD inhibitory activity. Among them, the most classic XOD inhibitory peptide is anserine isolated from bonito hydrolysate. These naturally derived XOD inhibitory peptides show broad application prospects, but further research is needed to identify bioactive peptides with novel sequences and stronger XOD inhibitory activity.

[0004] Eucheuma denticulatum, a type of red algae rich in protein, is widely used in the food industry. Except for a small portion that is directly processed into cold dishes, most of it is processed into carrageenan. If highly active functional peptides can be discovered from it, the added value of Eucheuma denticulatum will be greatly enhanced. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule peptide identified from the protein hydrolysate of *Euphorbia milii*, which has a novel sequence structure and strong xanthine oxidase inhibitory activity, as well as a method for preparing the small molecule peptide and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A pentapeptide LE5 with xanthine oxidase inhibitory activity, wherein the amino acid sequence of the pentapeptide LE5 is LGGVE.

[0008] The aforementioned method for preparing the pentapeptide LE5 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 LE5 in a solid phase.

[0009] The aforementioned application of the pentapeptide LE5, 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: The pentapeptide LE5 provided by this invention was identified from the protein hydrolysate of *Euphorbia milii*. Molecular docking revealed that pentapeptide LE5 has a potential interaction with xanthine oxidase. In vitro xanthine oxidase inhibitory activity tests showed that pentapeptide LE5 (concentration 0.1 mg / mL) inhibited xanthine oxidase by 60.58%, which is significantly higher than that of the classic uric acid-lowering peptide—gossypol (at the same concentration, the xanthine oxidase inhibition rate is 36.03%) (p<0.001). Pentapeptide LE5 has stronger xanthine oxidase inhibitory activity and can be used to prepare preparations that inhibit xanthine oxidase activity to alleviate hyperuricemia. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the binding mode between the pentapeptide LE5 and xanthine oxidase;

[0012] Figure 2 The graph shows the results of xanthine oxidase inhibition rate detection of pentapeptide LE5 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 Euphorbia lactea protein peptides

[0015] The method for preparing Euphorbia lactea protein peptides specifically includes the following steps:

[0016] (1) Put 100g of Euphorbia milii 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 - Kirin's protein peptide.

[0021] II. Obtaining the polypeptide sequence from the protein peptide of *Euphorbia milii*.

[0022] The obtained *Euphorbia lactea* 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 > 1.00 × 10 8 Active peptides with ≤6 amino acids

[0027] From the several polypeptide sequences obtained above, 20 peak areas > 1.00 × 10⁻⁶ were finally screened. 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 Peak areas of protein peptides from *Euphorbia milii* with an area >1.00×10⁻⁶ 8 Active peptides with ≤6 amino acids (I)

[0029]

[0030] Table 1-2 Peak areas of *Euphorbia milii* protein peptides >1.00×10⁻⁶ 8 Active peptides with ≤6 amino acids (II)

[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 20 bioactive peptides with xanthine oxidase are shown in Table 2.

[0036] Table 2. Predicted interaction results between bioactive peptides and xanthine oxidase

[0037]

[0038] V. Molecular docking analysis

[0039] Among the 20 bioactive peptides listed in Table 2, LGGVE (denoted as pentapeptide LE5, SEQ ID NO: 3) had the highest docking score, at 95.6996 kcal / mol. Therefore, LGGVE (pentapeptide LE5) was selected for further molecular docking analysis.

[0040] Analysis revealed that the binding mode of the pentapeptide LE5 to xanthine oxidase is as follows: Figure 1 As shown, the molecular docking is as follows:

[0041] The pentapeptide LE5 interacts with xanthine oxidase through 6 HH bonds, 3 CH bonds, and 3 electrostatic interactions, with 12 amino acid residues involved in the interaction between the pentapeptide LE5 and xanthine oxidase.

[0042] VI. Evaluation of the xanthine oxidase inhibitory activity of the pentapeptide LE5

[0043] 1. Solid-phase synthesis of pentapeptide LE5

[0044] 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 LE5 (purity >90%).

[0045] The pentapeptide LE5 or anserine (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare LE5 solution and anserine solution with a concentration of 0.1 mg / mL, respectively.

[0046] Sample group: Take 20 μL of LE5 solution or anserine 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.

[0047] 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.

[0048] 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.

[0049] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0050]

[0051] Where A represents the uric acid concentration in the control group and B represents the uric acid concentration in the sample group.

[0052] Calculations showed that at a concentration of 0.1 mg / mL, the xanthine oxidase inhibition rates of pentapeptide LE5 and anserine were 60.83±2.3% and 36.03±1.9%, respectively. (See the comparison below.) Figure 2 .

[0053] Depend on Figure 2 It can be seen that, at the same concentration, compared with the positive control anserine peptide, the xanthine oxidase inhibition rate of pentapeptide LE5 was significantly increased to 60.83±2.3% (p<0.001).

[0054] Therefore, it can be seen that the pentapeptide LE5 has stronger xanthine oxidase inhibitory activity and can be used to prepare preparations that inhibit xanthine oxidase activity to alleviate hyperuricemia.

[0055] 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 LE5, a pentapeptide with xanthine oxidase inhibitory activity, in the preparation of drugs to relieve hyperuricemia, wherein the amino acid sequence of LE5 is LGGVE.

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