Tetrapeptide IR4 with uric acid reducing activity as well as preparation method and application thereof

Abstract

The invention discloses tetrapeptide IR4 with uric acid reducing activity and a preparation method and application thereof, and belongs to the technical field of small molecule peptide. The amino acid sequence of the tetrapeptide IR4 is IDWR, and the tetrapeptide IR4 can be prepared through a solid-phase synthesis method and an enzymolysis method. The tetrapeptide IR4 is identified from porphyra haitanensis proteolysis liquid, has potential interaction with xanthine oxidase, can reduce the uric acid content of zebra fish by 33.6% (reduced to 5.93 + / -0.47 [mu] mol / g protein) when the concentration is 100 [mu] g / mL, and has better uric acid reducing activity compared with goose carnosine (which can reduce the uric acid content of zebra fish by 25.7%). Compared with allopurinol (the uric acid content of zebra fish can be reduced to 4.61 + / -1.11 mu mol / g protein), the tetrapeptide IR4 and allopurinol have no significant difference in uric acid reducing capacity, and the tetrapeptide IR4 can be used for preparing uric acid reducing drugs and relieving hyperuricemia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of small molecule peptides, and particularly relates to a tetrapeptide IR4 with uric acid reducing activity as well as a preparation method and application thereof. BACKGROUND

[0002] The incidence of hyperuricemia (HUA) is increasing year by year worldwide, and is showing a trend of younger. In the uric acid generation pathway, xanthine oxidase (XOD) as a key catalytic enzyme is an important target for controlling endogenous uric acid levels. However, the XOD inhibitors widely used in clinical practice (such as allopurinol and febuxostat) have potential adverse reactions such as skin reactions and abnormal liver function, which cannot meet the needs of patients for long-term medication. Therefore, the development of food-derived uric acid reducing peptides with safe sources and low side effects has become an important direction of current research.

[0003] In the development of uric acid reducing peptides, the active peptides obtained from different protein sources differ significantly in structure and efficiency. Xiao-Tong Li et al. reported the uric acid reducing activity of sheep bone bioactive peptides in the article "In vitro and in vivo uric acid reducing activity and mechanism of action of sheep bone bioactive peptides". Xiao-Dong Pei et al. reported a uric acid reducing peptide GNQQVHLQSQDM derived from feather keratin protease hydrolysate in the article "Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities", with an XOD inhibition IC 50 value of 12.15 mg / mL. Chen Q reported a quantitative structure-activity relationship prediction model of 192 uric acid reducing peptides in the article "Virtual screening, activity mechanism and prediction model of pig internal organ uric acid reducing peptides". Chen Y et al. systematically summarized the obtaining, activity and mechanism of action of uric acid reducing peptides in the article "Anti-hyperuricemia bioactive peptides: a review on obtaining, activity, and mechanism of action". Existing researches are mostly focused on terrestrial animal milk proteins or part of plant proteins, and the reported active peptides have certain limitations in sequence novelty and activity intensity. In contrast, algal proteins often have unique amino acid composition and functional potential due to their special living environment. Feng Y et al. reported the functional activity and application research progress of algal active peptides in the article "Functional activity and application research progress of algal active peptides".

[0004] Porphyra haitanensis is a large economic alga with high yield and wide food, and high protein content. At present, the deep processing and utilization of Porphyra haitanensis mainly focuses on instant food and polysaccharide extraction, and the rich protein resources have not been fully developed in the aspect of active peptides. SUMMARY

[0005] The purpose of the present application is to provide a small molecule peptide with novel sequence structure and strong uric acid lowering activity identified from Porphyra haitanensis protease solution, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned target, the technical scheme adopted by the present application is as follows: A tetrapeptide IR4 with uric acid lowering activity, the amino acid sequence of the tetrapeptide IR4 is IDWR.

[0007] The preparation method of the aforementioned tetrapeptide IR4 with uric acid lowering activity adopts solid phase synthesis method, uses Fmoc protected amino acid as raw material, and selects polystyrene resin as solid phase carrier to synthesize the tetrapeptide IR4.

[0008] The preparation method of the aforementioned tetrapeptide IR4 with uric acid lowering activity adopts enzyme hydrolysis method, and the specific steps are as follows: (1) put Porphyra haitanensis into water, heat to 40℃, then add yeast, and hydrolyze at this temperature for 2h; (2) continue to heat, heat to 48℃, then add alkaline protease and neutral protease, and hydrolyze at this temperature for 2h; (3) continue to heat, heat to 58℃, then add papain, and hydrolyze at this temperature for 3h; (4) continue to heat, heat to 85℃, and keep for 30min; (5) precipitate the hydrolysis solution, centrifuge the supernatant, and spray dry the centrifuged supernatant to obtain Porphyra haitanensis protein peptide, wherein the tetrapeptide IR4 is contained; wherein the mass ratio of Porphyra haitanensis, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2.

[0009] The application of the aforementioned tetrapeptide IR4 with uric acid lowering activity in preparing uric acid lowering drugs.

[0010] The application has the advantages that the application first discloses and verifies that the active peptide (tetrapeptide IR4) with the IDWR sequence has uric acid reducing activity, the structure sequence is different from common sequences, and provides a new core efficacy component and material basis for developing the uric acid reducing drug from Porphyra haitanensis. The uric acid reducing activity of the tetrapeptide IR4 is tested by using the zebrafish hyperuricemia model, and it is found that the tetrapeptide IR4 (concentration 100 μg / mL) can reduce the uric acid content of the zebrafish by 33.6%, and compared with the classic uric acid reducing peptide (carnosine) (under the same concentration, the uric acid content of the zebrafish can be reduced by 25.7%), the uric acid reducing activity of the tetrapeptide IR4 is stronger, and the tetrapeptide IR4 can be used for preparing the uric acid reducing drug and relieving hyperuricemia. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a schematic diagram of the binding mode of the tetrapeptide IR4 and xanthine oxidase; Figure 2 is a graph of the uric acid content detection results of each group, wherein * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and ns represents p>0.05. DETAILED DESCRIPTION

[0012] The application will be specifically introduced below in combination with the drawings and specific embodiments.

[0013] I. Preparation of Porphyra haitanensis protein peptide The method for preparing the Porphyra haitanensis protein peptide specifically includes the following steps: (1) 100 g of Porphyra haitanensis is put into 1000 mL of water, 1 g of yeast is added after the temperature is raised to 40℃, and the enzyme hydrolysis is carried out at the temperature for 2 h; (2) continue to raise the temperature, 3 g of alkaline protease and 2 g of neutral protease are added after the temperature is raised to 48℃, and the enzyme hydrolysis is carried out at the temperature for 2 h; (3) continue to raise the temperature, 2 g of papain is added after the temperature is raised to 58℃, and the enzyme hydrolysis is carried out at the temperature for 3 h; (4) continue to raise the temperature, and keep the temperature at 85℃ for 30 min; (5) the enzyme hydrolysis liquid is left to precipitate, the supernatant is taken out and centrifuged at 8000 rpm for 30 min, and the supernatant after centrifugation is spray dried to obtain a powder product, i.e. the Porphyra haitanensis protein peptide.

[0014] II. Obtain polypeptide sequences in the Porphyra haitanensis protein peptide The Porphyra haitanensis protein peptide obtained above is subjected to mass spectrometry by using LC-MS / MS, and the mass spectrometry result is analyzed by using a mass spectrometry analysis software to obtain a plurality of polypeptide sequences.

[0015] The LC-MS / MS determination conditions are as follows: (1) Liquid phase method: the chromatographic column is C18, 3pm, 250mmx75pm (Eksigent), the mobile phase A is ultrapure water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, the flow rate is 300nL / min, the injection volume is 1pL, the 70min chromatographic gradient, the specific elution gradient is: 0-55min, A phase from 95% to 65% uniformly; 55-63min, A phase from 65% to 50% uniformly, 63-64min, A phase from 50% to 0% uniformly; 64-70min, keep 0% A phase.

[0016] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, first-order resolution is 120000, AGC setting is 310, scanning range is 110-2000m / z. The MIPS mode is peptide, the selection valence is 1-6, the second-order resolution is 17500, and the separation window is 1.6m / z.

[0017] III. Screening of 24 active peptides with peak area >5.00x10 8 and amino acid number ≤6 From the above obtained several polypeptide sequences, 24 active peptides with peak area >5.00x10 8 and amino acid number ≤6 were finally screened out, and the screening results are shown in Table 1-1 and Table 1-2.

[0018] Table 1-1 High-abundance active peptides in Porphyra haitanensis protein peptides (I)

[0019] Table 1-2 High-abundance active peptides in Porphyra haitanensis protein peptides (II)

[0020] IV. Screening of active peptides with strong xanthine oxidase binding ability The active peptide sequences in Table 1-1 and Table 1-2 were docked with xanthine oxidase using Discovery Studio software, and the 2D structure of the active peptide was converted to 3D structure by energy minimization before docking. The active peptide sequences with strong xanthine oxidase binding ability were screened.

[0021] The 3D structure of xanthine oxidase can be downloaded from RCSB protein database (PDB ID: 1FIQ). The docking result is expressed by docking score, the larger the docking score, the stronger the binding ability of the active peptide to xanthine oxidase, and the more likely to inhibit the activity of xanthine oxidase.

[0022] The molecular docking results of the above 24 active peptides with xanthine oxidase are shown in Table 2.

[0023] Table 2 Predicted results of interaction of active peptides with xanthine oxidase

[0024] V. Molecular docking analysis Among the 24 active peptides listed in Table 2, the docking scores of VSIVE (referred to as the pentapeptide VE5, SEQ ID NO: 19) and IDWR (referred to as the tetrapeptide IR4, SEQ ID NO: 22) were the largest, being 103.5200 kcal / mol and 107.866 kcal / mol, respectively. In the present application, IDWR (tetrapeptide IR4) was selected for further molecular docking analysis.

[0025] Analysis showed that the binding mode of tetrapeptide IR4 with xanthine oxidase was as shown in Figure 1 The molecular docking is as follows: Eleven H-H bond interactions, three C-H bond interactions, one salt bridge interaction, and one electrostatic interaction were formed between tetrapeptide IR4 and xanthine oxidase, and 15 amino acid residues were involved in the interaction between tetrapeptide IR4 and xanthine oxidase.

[0026] VI. Evaluation of the uric acid-lowering activity of tetrapeptide IR4 The uric acid-lowering activity of tetrapeptide IR4 was evaluated using a zebrafish hyperuricemia model, and it was confirmed that tetrapeptide IR4 has the function of lowering uric acid.

[0027] Fmoc-protected amino acids were used as raw materials, polystyrene resin was selected as the solid phase carrier, and solid phase synthesis was used to synthesize tetrapeptide IR4 (purity > 90%).

[0028] Experimental fish: 300 healthy (normally hatched and able to swim normally) 5-day-old wild-type zebrafish (AB) strains were randomly and evenly divided into 5 groups, with 6 replicates in each group.

[0029] A six-well culture plate was used for the experiment, with 10 zebrafish placed in each well, and the volume of the culture solution in the well was 4 mL. The specific experimental grouping is as follows: (1) Control group: No treatment was given to the zebrafish.

[0030] (2) Model group: The zebrafish were first treated with potassium oxonate for 24 h, and then treated with sodium xanthate for 24 h. The final concentration of potassium oxonate was 600 μM, and the final concentration of sodium xanthate was 30 μM, thereby constructing a zebrafish hyperuricemia model.

[0031] (3) Positive control group: A zebrafish hyperuricemia model was first constructed according to the method of the model group, and then the zebrafish were treated with allopurinol for 24 h. The final concentration of allopurinol was 2 mM.

[0032] (4) The group of carnosine: First, the high uric acid model of zebrafish was constructed according to the method of the model group, and then the zebrafish was treated with carnosine for 24 hours, and the final concentration of carnosine was 100 μg / mL.

[0033] (5) The IR4 group: First, the high uric acid model of zebrafish was constructed according to the method of the model group, and then the zebrafish was treated with the tetrapeptide IR4 synthesized by solid phase for 24 hours, and the final concentration of the tetrapeptide IR4 was 100 μg / mL.

[0034] After the experiment, the zebrafish was collected in a 1.5 mL EP tube, and the liquid in the EP tube was sucked out. Then, 40 μL of phosphate buffer (PBS) was added to each EP tube, and the collected zebrafish sample was mechanically broken on ice using a handheld tissue homogenizer until no obvious tissue clumps were observed. Subsequently, centrifugation was performed at 4°C and 15000 rpm for 15 min, and the supernatant was transferred to a new EP tube. Finally, the protein concentration and uric acid concentration of the supernatant were determined using a BCA kit and a high-performance liquid chromatograph. The remaining supernatant was stored in a -80°C refrigerator for standby.

[0035] When determining the uric acid concentration of the supernatant, a ZORBAX Original Phenyl chromatographic column (5 μm, 4.6 mm x 250 mm) was used, the mobile phase A was ultrapure water containing 0.52 mmol / L 1-pentane sulfonic acid sodium and 0.20 mol / L potassium phosphate dibasic, the pH was adjusted to 4.0 with phosphoric acid solution, and the B phase was high-performance liquid chromatography grade acetonitrile. The isocratic elution condition was A phase: B phase = 85:15 (V / V), the flow rate was 1.0 mL / min at 25°C, the injection volume of each sample was 10 μL, and the running time was 15 min. At least 30 min of column equilibration with the mobile phase was required before sample injection.

[0036] After calculation, the uric acid content (μmol / g protein) of each group was as follows: Table 3 Detection results of uric acid content of each group

[0037] Note: * is compared with the model group, # is compared with the positive control group, & is compared with the carnosine group, wherein * represents p<0.05, ** represents p<0.01, *** represents p<0.001, # represents p<0.05, and & represents p<0.05.

[0038] The graph of Table 3 is as follows: Figure 2 It can be seen that: (1) The modeling process increased the uric acid content from 3.43 ± 0.53 μmol / g protein to 8.94 ± 1.49 μmol / g protein (p < 0.001).

[0039] (2) Compared with the model group, allopurinol (2 mM) treatment can significantly reduce the uric acid content to 4.61 ± 1.11 μmol / g protein (p < 0.01), carnosine (100 μg / mL) treatment can significantly reduce the uric acid content to 6.64 ± 0.24 μmol / g protein (p < 0.05), and tetrapeptide IR4 (100 μg / mL) treatment can significantly reduce the uric acid content to 5.93 ± 0.47 μmol / g protein (p < 0.01).

[0040] (3) There was no significant difference in uric acid content between tetrapeptide IR4 treatment and positive control allopurinol treatment (p > 0.05), but the uric acid content after tetrapeptide IR4 treatment was significantly lower than that after carnosine treatment (p < 0.05).

[0041] At the same dose (100 μg / mL), tetrapeptide IR4 reduced the uric acid content of zebrafish by 33.6%, and the classic uric acid-lowering peptide, carnosine (β-alanyl-1-methyl L-histidine), reduced the uric acid content of zebrafish by 25.7%, and tetrapeptide IR4 had better uric acid-lowering activity than carnosine.

[0042] At the same dose (100 μg / mL), tetrapeptide IR4 reduced the uric acid content of zebrafish to 5.93 ± 0.47 μmol / g protein, and allopurinol reduced the uric acid content of zebrafish to 4.61 ± 1.11 μmol / g protein, with no significant difference (p > 0.05), and tetrapeptide IR4 had comparable uric acid-lowering ability to allopurinol.

[0043] In summary, tetrapeptide IR4 can be used to prepare uric acid-lowering drugs to relieve hyperuricemia.

[0044] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or modifications can be made by those skilled in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or modifications derived from the technical solutions of the present application still fall within the protection scope of the present application.

Claims

1. A tetrapeptide IR4 with uric acid-lowering activity, characterized in that, The amino acid sequence of the tetrapeptide IR4 is IDWR.

2. The method for preparing the tetrapeptide IR4 with uric acid-lowering activity as described in claim 1, characterized in that, The tetrapeptide IR4 was synthesized in a solid-phase manner using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier.

3. The method for preparing the tetrapeptide IR4 with uric acid-lowering activity as described in claim 1, 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.

4. The use of the tetrapeptide IR4 with uric acid-lowering activity as described in claim 1 in the preparation of uric acid-lowering drugs.

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