A hexapeptide TE6 having xanthine oxidase inhibitory activity, and a preparation method and application thereof

Abstract

The application discloses a hexapeptide TE6 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 hexapeptide TE6 is TIATVE, and the hexapeptide TE6 can be prepared by a solid-phase synthesis method and an enzymolysis method. The hexapeptide TE6 is identified from a protease enzymolysis liquid of a fine red alga, has potential interaction with xanthine oxidase, and has an xanthine oxidase inhibition rate of 44.74% at a concentration of 0.1 mg / mL. Compared with anane peptide (the xanthine oxidase inhibition rate of which is 36.03%), the xanthine oxidase inhibition rate of the hexapeptide TE6 is significantly increased (p<0.05), and the xanthine oxidase inhibiting activity of the hexapeptide TE6 is stronger, so that the hexapeptide TE6 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 hexapeptide TE6 with xanthine oxidase inhibitory activity, its preparation method, and its application. Background Technology

[0002] Hyperuricemia (HUA) is a growing global health problem. Prolonged high levels of uric acid in the body lead to the deposition of urate crystals in the kidneys, causing complications of hyperuricemia. Xanthine oxidase (XOD) is a core catalyst in the uric acid synthesis pathway in the body, and its activity directly determines the rate of uric acid production. Therefore, inhibiting XOD activity is a key step in blocking the production of endogenous uric acid.

[0003] As a oxidoreductase containing molybdenum pterin cofactor, xanthocyanin-derived oxygen (XOD) is a key target in clinical uric acid-lowering therapy. Although clinical drugs such as allopurinol and febuxostat have significant XOD inhibitory effects, potential severe hypersensitivity reactions and cardiovascular risks limit long-term use in some patients. Against this backdrop, exploring safe and effective XOD-inhibiting peptides from natural food resources has become an important direction in drug development. Proteins from different sources can release structurally diverse bioactive peptides after enzymatic hydrolysis. It is noteworthy that different enzymatic hydrolysis strategies lead to significant differences in the bioactive peptide profiles.

[0004] The functions of bioactive peptides from different natural sources in regulating uric acid have been reported. Yang et al., in "Identification of novel xanthine oxidase inhibitory peptides from Takifuguobscurus: Peptidomic analysis, molecular docking, and dynamics simulation," reported the isolation of peptides with XOD inhibitory activity from the enzymatic hydrolysate of *Takifuguobscurus*, among which WAAFPPDVAGN exhibited the strongest XOD inhibitory activity. Qi et al., in "Novel xanthine oxidase inhibitory peptides derived from whey protein: identification, in vitro inhibition mechanism and in vivo activity validation," reported the identification of ALPM and LWM from whey protein hydrolysate, finding that both possess in vitro XOD inhibitory activity and in vivo uric acid-lowering effects. In her paper, "Preparation, Properties, and Uric Acid-Lowering Activity of Xanthine Oxidase Inhibiting Peptides from Skipjack Tuna Dark Flesh," Dai Qingfei reported the isolation of four bioactive peptides (KFLR, ERFR, KFLK, and FEKAF) from the enzymatic hydrolysates of skipjack tuna dark flesh, all exhibiting significant XOD inhibitory activity. Notably, a review article published in *Food Science and Human Wellness*, titled "Food-derived bio-functional peptides for the management of hyperuricemia and associated mechanism," systematically reviewed relevant research on food-derived uric acid-lowering peptides, providing a comprehensive overview of reported uric acid-lowering peptide sequences. No peptides with the same sequence as those described in this invention were found. These naturally derived uric acid-lowering peptides show broad application prospects, but further research is needed to identify bioactive peptides with novel sequences and stronger uric acid-lowering activities.

[0005] Solieria tenuis, a species of red algae, is a warm-temperate economic seaweed. It is rich in protein and has a balanced ratio of essential amino acids. Its protein can be used as a high-quality raw material for the preparation of various bioactive peptides. Summary of the Invention

[0006] 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 *Pteris vittata*, as well as a method for preparing the small molecule peptide and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A hexapeptide TE6 with xanthine oxidase inhibitory activity, wherein the amino acid sequence of the hexapeptide TE6 is TIATVE.

[0009] The aforementioned method for preparing hexapeptide TE6 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 hexapeptide TE6 in a solid phase.

[0010] The aforementioned application of the hexapeptide TE6, which has xanthine oxidase inhibitory activity, in the preparation of formulations that inhibit xanthine oxidase activity.

[0011] The advantages of this invention are as follows: This invention discloses and verifies for the first time that an active peptide with the TIATVE sequence (hexapeptide TE6) has xanthine oxidase inhibitory activity. Its structural sequence is different from common sequences, providing a novel core active ingredient and material basis for developing drugs derived from *Hymenopterus xanthipes* to alleviate hyperuricemia. In vitro xanthine oxidase inhibitory activity tests showed that hexapeptide TE6 (concentration 0.1 mg / mL) had a xanthine oxidase inhibition rate of 44.74%, which is significantly increased (p<0.05) compared with the classic uric acid-lowering peptide—gossyptide (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 preparations that inhibit xanthine oxidase activity to alleviate hyperuricemia. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the binding mode between hexapeptide TE6 and xanthine oxidase;

[0013] Figure 2 The graph shows the results of xanthine oxidase inhibition rate detection of hexapeptide TE6 and anserine, where * indicates 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 *Pteris vittata* protein peptides

[0016] The method for preparing *Pteris vittata* protein peptides specifically includes the following steps:

[0017] (1) Put 100g of weak red pheasant into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze 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 - fine and weak red winged vegetable protein peptide.

[0022] II. Obtaining the polypeptide sequence from the protein peptide of *Gnaphalium affine*.

[0023] The obtained *Gnaphalium affine* 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 > 2.00 × 10 8 Active peptides with ≤6 amino acids

[0028] From the several polypeptide sequences obtained above, 78 peak areas > 2.00 × 10⁻⁶ were finally screened. 8 The screening results for bioactive peptides with ≤6 amino acids are shown in Tables 1-1, 1-2, 1-3, and 1-4.

[0029] Table 1-1 High-abundance bioactive peptides in *Gnaphalium affine* protein peptides (Part 1)

[0030]

[0031] Table 1-2 High-abundance bioactive peptides in *Gnaphalium affine* protein peptides (Part II)

[0032]

[0033] Table 1-3 High-abundance bioactive peptides in *Gnaphalium affine* protein peptides (Part III)

[0034]

[0035] Table 1-4 High-abundance bioactive peptides in *Gnaphalium affine* protein peptides (Part 4)

[0036]

[0037] IV. Screening for bioactive peptides with strong binding affinity to xanthine oxidase

[0038] Using Discovery Studio software, the active peptide sequences in Tables 1-1, 1-2, 1-3, and 1-4 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.

[0039] 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.

[0040] The molecular docking results of the above 78 bioactive peptides with xanthine oxidase are shown in Tables 2-1, 2-2, 2-3 and 2-4.

[0041] Table 2-1 Predicted results of the interaction between bioactive peptides and xanthine oxidase (I)

[0042]

[0043] Table 2-2 Predicted results of the interaction between bioactive peptides and xanthine oxidase (II)

[0044]

[0045] Table 2-3 Predicted results of interactions between bioactive peptides and xanthine oxidase (Part III)

[0046]

[0047] Table 2-4 Predicted results of interactions between bioactive peptides and xanthine oxidase (IV)

[0048]

[0049] V. Molecular docking analysis

[0050] Among the 78 bioactive peptides listed in Tables 2-1, 2-2, 2-3, and 2-4, TIATVE (denoted as hexapeptide TE6, SEQ ID NO: 7) had the highest docking score of 107.5060 kcal / mol. Therefore, TIATVE (hexapeptide TE6) was selected for further molecular docking analysis.

[0051] Analysis revealed that the binding mode of hexapeptide TE6 to xanthine oxidase is as follows: Figure 1 As shown, the molecular docking is as follows:

[0052] The hexapeptide TE6 interacts with xanthine oxidase through 7 HH bonds, 4 CH bonds, 2 salt bridges, and 5 electrostatic interactions, with 14 amino acid residues involved in the interaction between hexapeptide TE6 and xanthine oxidase.

[0053] VI. Evaluation of the xanthine oxidase inhibitory activity of hexapeptide TE6

[0054] 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 TE6 (purity >90%).

[0055] The hexapeptide TE6 or anserine (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare TE6 solution and anserine solution with a concentration of 0.1 mg / mL, respectively.

[0056] Sample group: Take 20 μL of TE6 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.

[0057] 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.

[0058] 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. Isocratic elution conditions were A: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.

[0059] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0060]

[0061] Where A represents the uric acid concentration in the control group and B represents the uric acid concentration in the sample group.

[0062] Calculations showed that at a concentration of 0.1 mg / mL, the xanthine oxidase inhibition rates of hexapeptide TE6 and anserine were 44.74±3.6% and 36.03±1.9%, respectively. (See the comparison below.) Figure 2 .

[0063] 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 hexapeptide TE6 was significantly increased to 44.74±3.6% (p<0.05).

[0064] Therefore, it can be seen that hexapeptide TE6 has stronger xanthine oxidase inhibitory activity and can be used to prepare preparations that inhibit xanthine oxidase activity to alleviate hyperuricemia.

[0065] 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 hexapeptide TE6 with xanthine oxidase inhibitory activity, characterized in that, The amino acid sequence of the hexapeptide TE6 is TIATVE.

2. The method for preparing the hexapeptide TE6 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 hexapeptide TE6.

3. The use of the hexapeptide TE6 with xanthine oxidase inhibitory activity as described in claim 1 in the preparation of drugs to relieve hyperuricemia.

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