A pentapeptide IK5 with uric acid-lowering activity and a preparation method and application thereof

Abstract

The application discloses a pentapeptide IK5 with uric acid reducing activity as well as a preparation method and application thereof, and belongs to the technical field of small molecular peptides. The amino acid sequence of the pentapeptide IK5 is ITGYK, and the pentapeptide IK5 can be prepared by a solid-phase synthesis method and an enzymolysis method. The pentapeptide IK5 is identified from a protease enzymolysis liquid of a fine weak red winged seaweed, has potential interaction with xanthine oxidase, and can reduce the uric acid content of zebrafish by 44.4% (to 5.93+ / -0.47 mu mol / g protein) at a concentration of 100 mu g / mL. Compared with ananeine (which can reduce the uric acid content of zebrafish by 25.7%), the pentapeptide IK5 has better uric acid reducing activity. Compared with allopurinol (which can reduce the uric acid content of zebrafish to 4.61+ / -1.11 mu mol / g protein), the uric acid reducing ability of the pentapeptide IK5 and the allopurinol has no significant difference. The pentapeptide IK5 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 pentapeptide IK5 with uric acid reducing activity as well as a preparation method and application thereof. BACKGROUND

[0002] The prevention and treatment of hyperuricemia (HUA) is facing a transition from symptom control to source intervention. In this process, xanthine oxidase (XOD) as the core target of the uric acid synthesis pathway, the development of its inhibitors has not been limited to the simple drug efficacy, but increasingly focuses on the safety of long-term use, the sustainability of the source and the mildness of the effect. Although existing chemical drugs can effectively control uric acid, their potential side effects limit their application scenarios in daily prevention and long-term management, which leaves a broad space for the development of food-derived uric acid reducing peptides.

[0003] Currently, the development of food-derived uric acid reducing peptides has obvious path dependence in protein sources, and a large number of studies are focused on conventional terrestrial proteins. For example, Gong Weixiang et al. studied the preparation of walnut uric acid reducing peptides in the article "Response surface optimization of ultrasonic-assisted enzyme method for preparing walnut uric acid reducing peptides". Lin Xia reported the alleviating effect of silkworm chrysalis protein peptides on hyperuricemia in mice in the paper "Efficacy of silkworm chrysalis protein peptides in reducing uric acid and anti-photoaging". Algal proteins contain active fragments with novel structures and unique functions due to their different evolutionary paths and unique survival pressures from terrestrial organisms. Gao Lifang et al. reported PAR2 inhibitory peptides PAGR and PAR obtained from algal protein hydrolysate in the article "Computer simulation screening of edible algal protein source PAR2 inhibitory peptides". Wu Jingna et al. reported that LVLLFLFGE had good ACE inhibitory activity in the article "Screening and stability evaluation of angiotensin-converting enzyme inhibitory peptides from red hair algae". However, this huge resource treasure has not been systematically explored and developed.

[0004] Solieria tenuis is an important economic seaweed rich in high-quality protein. At present, the utilization level of these high-quality proteins is low, and most of them have not been used for high-value utilization, resulting in great waste of resources. Converting Solieria tenuis protein into high-activity uric acid reducing peptides through enzymatic hydrolysis technology is an innovative practice to realize the full utilization of marine resources. SUMMARY

[0005] The purpose of the present application is to provide a small molecule peptide identified from Solieria tenuis protein enzymatic hydrolysate, which has novel sequence structure and strong uric acid reducing activity, as well as a preparation method and application thereof.

[0006] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows:

[0007] A pentapeptide IK5 with uric acid reducing activity, the amino acid sequence of the pentapeptide IK5 is ITGYK.

[0008] The preparation method of the aforementioned pentapeptide IK5 with uric acid reducing activity adopts a solid-phase synthesis method, uses Fmoc-protected amino acids as raw materials, and selects polystyrene resin as a solid-phase carrier to synthesize the pentapeptide IK5.

[0009] The preparation method of the aforementioned pentapeptide IK5 with uric acid reducing activity adopts an enzymatic hydrolysis method, and the method is specifically as follows:

[0010] (1) The fine weak red lingzhi is put into water, and after being heated to 40 DEG C, yeast is added, and enzymatic hydrolysis is carried out at the temperature for 2h;

[0011] (2) Continue to heat, and after being heated to 48 DEG C, alkaline protease and neutral protease are added, and enzymatic hydrolysis is carried out at the temperature for 2h;

[0012] (3) Continue to heat, and after being heated to 58 DEG C, papain is added, and enzymatic hydrolysis is carried out at the temperature for 3h;

[0013] (4) Continue to heat, and after being heated to 85 DEG C, keep for 30 min;

[0014] (5) The enzyme solution is left to precipitate, the supernatant is taken and centrifuged, and the supernatant after centrifugation is spray dried to obtain fine weak red lingzhi peptide, which contains pentapeptide IK5;

[0015] Wherein, the amount ratio of fine weak red lingzhi, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2 by mass.

[0016] The aforementioned pentapeptide IK5 with uric acid reducing activity is applied to the preparation of uric acid reducing drugs.

[0017] The application has the advantages that the application first discloses and verifies that the active peptide (pentapeptide IK5) with the sequence ITGYK has uric acid reducing activity, the structure sequence is different from common sequences, provides a new core efficacy component and material basis for developing uric acid reducing drugs from fine weak red lingzhi, and finds that the pentapeptide IK5 (concentration 100 μg / mL) can reduce the uric acid content of zebrafish by 44.4% by testing the uric acid reducing activity of the pentapeptide IK5 by using a zebrafish hyperuricemia model, compared with the classic uric acid reducing peptide - gmetidine (under the same concentration, can reduce the uric acid content of zebrafish by 25.7%), the uric acid reducing activity of the pentapeptide IK5 is stronger, and the pentapeptide IK5 can be used for preparing uric acid reducing drugs to relieve hyperuricemia. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a schematic diagram of the binding mode of the pentapeptide IK5 and xanthine oxidase;

[0019] Figure 2 Figure 4 is a graph of the results of uric acid content calculation of each group, wherein * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and ns represents p > 0.05. DETAILED DESCRIPTION

[0020] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples.

[0021] I. Preparation of fine weak red fur algae protein peptide

[0022] The method for preparing the fine weak red fur algae protein peptide specifically comprises the following steps:

[0023] (1) 100 g of fine weak red fur algae is placed in 1000 mL of water, and 1 g of yeast is added after being warmed to 40°C, and enzymatic hydrolysis is carried out at this temperature for 2 h;

[0024] (2) Continue to warm up, and after being warmed to 48°C, 3 g of alkaline protease and 2 g of neutral protease are added, and enzymatic hydrolysis is carried out at this temperature for 2 h;

[0025] (3) Continue to warm up, and after being warmed to 58°C, 2 g of papain is added, and enzymatic hydrolysis is carried out at this temperature for 3 h;

[0026] (4) Continue to warm up, and after being warmed to 85°C, keep for 30 min;

[0027] (5) The enzyme hydrolysate 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 powdery product, i.e., fine weak red fur algae protein peptide.

[0028] II. Obtain polypeptide sequences in fine weak red fur algae protein peptide

[0029] The fine weak red fur algae protein peptide obtained above is subjected to mass spectrometry determination by LC-MS / MS, and the results of mass spectrometry determination are analyzed by mass spectrometry analysis software to obtain a plurality of polypeptide sequences.

[0030] The LC-MS / MS determination conditions are as follows:

[0031] (1) Liquid phase method: the chromatographic column is C18, 3 μm, 250 mm x 75 μm (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 300 nL / min, the injection volume is 1 μL, the chromatographic gradient is 70 min, and the specific elution gradient is: 0-55 min, A phase from 95% uniformly reduced to 65%; 55-63 min, A phase from 65% uniformly reduced to 50%, 63-64 min, A phase from 50% uniformly reduced to 0; 64-70 min, keep 0% A phase.

[0032] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, first-order resolution 120000, AGC setting 310, scan range 110-2000 m / z. MIPS mode is peptide, select valence 1-6, secondary resolution 17500, separation window 1.6 m / z.

[0033] III. Screening active peptides with peak area > 4.00 x 10 8 and amino acid number ≤ 6

[0034] From the above, 32 active peptides with peak area > 4.00 x 10 8 and amino acid number ≤ 6 were finally screened from the obtained polypeptide sequences, and the screening results are shown in Table 1-1 and Table 1-2.

[0035] Table 1-1 Active peptides with high abundance in fine weak red frond algae protein peptides (I)

[0036]

[0037] Table 1-2 Active peptides with high abundance in fine weak red frond algae protein peptides (II)

[0038]

[0039] IV. Screening active peptides with strong binding ability to xanthine oxidase

[0040] 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. Active peptide sequences with strong binding ability to xanthine oxidase were screened.

[0041] The 3D structure of xanthine oxidase can be downloaded from RCSB protein database (PDB ID: 1FIQ). The docking score represents the docking result, 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.

[0042] The molecular docking results of the above 32 active peptides with xanthine oxidase are shown in Table 2-1 and Table 2-2.

[0043] Table 2-1 Prediction results of interaction between active peptides and xanthine oxidase (I)

[0044]

[0045] Table 2-2 Prediction results of interaction between active peptides and xanthine oxidase (II)

[0046]

[0047] V. Molecular docking analysis

[0048] Among the 32 active peptides listed in Table 2-1 and Table 2-2, the docking scores of IDWR (referred to as tetrapeptide IR4, SEQ ID NO: 30) and ITGYK (referred to as pentapeptide IK5, SEQ ID NO: 15) are the largest, which are 103.0304 kcal / mol and 95.0491 kcal / mol, respectively. In the present application, ITGYK (pentapeptide IK5) is selected for further molecular docking analysis.

[0049] After analysis, the binding mode of pentapeptide IK5 and xanthine oxidase is shown in Figure 1 , and the molecular docking is as follows:

[0050] Pentapeptide IK5 and xanthine oxidase form 6 H-H bond interactions, 1 C-H bond interaction, 1 salt bridge interaction and 3 electrostatic interactions, and 14 amino acid residues are involved in the interaction between pentapeptide IK5 and xanthine oxidase.

[0051] VI. Evaluation of the uric acid-lowering activity of pentapeptide IK5

[0052] The uric acid-lowering activity of pentapeptide IK5 was evaluated using a zebrafish hyperuricemia model, and it was confirmed that pentapeptide IK5 has the function of lowering uric acid.

[0053] Solid phase synthesis method was used to synthesize pentapeptide IK5 (purity > 90%) using Fmoc-protected amino acids as raw materials and polystyrene resin as solid phase carrier.

[0054] Experimental fish: 300 healthy (normally hatched and able to swim normally) 5-day-old wild type zebrafish (AB) strain, randomly divided into 5 groups, with 6 replicates in each group.

[0055] 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:

[0056] (1) Control group: No treatment was given to the zebrafish.

[0057] (2) Model group: First, the zebrafish were treated with potassium oxonate for 24 h, and then treated with xanthine sodium salt for 24 h. The final concentration of potassium oxonate was 600 μM, and the final concentration of xanthine sodium salt was 30 μM, and a zebrafish hyperuricemia model was constructed.

[0058] (3) Positive control group: First, the zebrafish hyperuricemia model was constructed according to the method of the model group, and then the zebrafish was treated with allopurinol for 24 hours. The final concentration of allopurinol was 2 mM.

[0059] (4) Carnosine group: First, the zebrafish hyperuricemia model was constructed according to the method of the model group, and then the zebrafish was treated with carnosine for 24 hours. The final concentration of carnosine was 100 μg / mL.

[0060] (5) IK5 group: First, the zebrafish hyperuricemia model was constructed according to the method of the model group, and then the zebrafish was treated with the solid-phase synthesized pentapeptide IK5 for 24 hours. The final concentration of the pentapeptide IK5 was 100 μg / mL.

[0061] 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 samples were 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 later use.

[0062] 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 salt 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.

[0063] After calculation, the uric acid content (μmol / g protein) of each group was as follows:

[0064] Table 3 Uric acid content detection results of each group

[0065]

[0066] 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<0.01.

[0067] Plotting Table 3, it can be seen that: Figure 2

[0068] (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).

[0069] (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 pentapeptide IK5 (100 μg / mL) treatment can significantly reduce the uric acid content to 4.97 ± 0.66 μmol / g protein (p < 0.01).

[0070] (3) There is no significant difference in uric acid content between pentapeptide IK5 treatment and positive control allopurinol treatment (p > 0.05), but the uric acid content after pentapeptide IK5 treatment is significantly lower than that after carnosine treatment (p < 0.01).

[0071] At the same dose (100 μg / mL), pentapeptide IK5 reduces the uric acid content of zebrafish by 44.4%, and the classic uric acid-lowering peptide carnosine (β-alanyl-1-methyl L-histidine) reduces the uric acid content of zebrafish by 25.7%, and pentapeptide IK5 has better uric acid-lowering activity than carnosine.

[0072] At the same dose (100 μg / mL), pentapeptide IK5 reduces the uric acid content of zebrafish to 4.97 ± 0.66 μmol / g protein, and allopurinol reduces the uric acid content of zebrafish to 4.61 ± 1.11 μmol / g protein, and there is no significant difference (p > 0.05), and pentapeptide IK5 has the same uric acid-lowering ability as allopurinol.

[0073] In summary, pentapeptide IK5 can be used to prepare uric acid-lowering drugs to relieve hyperuricemia.

[0074] 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, those skilled in the art can make other different forms of changes or modifications. 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. Process for the preparation of the pentapeptide IK5 having uric acid-lowering activity, the amino acid sequence of which is ITGYK, characterized in that, The enzymatic hydrolysis is as follows: (1) Put the fine and weak red ling into water, heat to 40℃, then add yeast, and hydrolyze at the temperature for 2 hours; (2) Continue to heat, heat to 48℃, then add alkaline protease and neutral protease, and hydrolyze at the temperature for 2 hours; (3) Continue to heat, heat to 58℃, then add papain, and hydrolyze at the temperature for 3 hours; (4) Continue to heat, heat to 85℃, and keep for 30 minutes; (5) Let the hydrolysis solution stand and precipitate, centrifuge the supernatant, and spray dry the centrifuged supernatant to obtain fine and weak red ling protein peptide, which contains pentapeptide IK5; wherein the amount ratio of the fine and weak red ling, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2 by mass.

2. Application of pentapeptide IK5 having uric acid-lowering activity in the preparation of uric acid-lowering drugs, wherein the amino acid sequence of the pentapeptide IK5 is ITGYK.

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