Polypeptide with xanthine oxidase inhibitory activity and preparation method and application thereof

Abstract

The invention discloses a polypeptide with xanthine oxidase inhibitory activity and a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The polypeptide is derived from jellyfish, the amino acid sequence is LGALWPPM, the molecular weight is 884.10 Da, and the polypeptide is obtained by hydrolyzing the jellyfish through simulated gastric juice and simulated intestinal juice and has xanthine oxidase inhibitory activity. The polypeptide LGALWPPM has the beneficial effects that the maximum inhibition rate of the polypeptide LGALWPPM on xanthine oxidase reaches 96.84%, the IC50 value is 4.654 mM, and compared with most of existing xanthine oxidase inhibitory peptides, the activity is better, and the inhibition effect is better. Meanwhile, due to the unique dose response characteristic and the higher maximum inhibition ratio of the peptide, a new research idea is further developed for research and development of a novel xanthine oxidase inhibitor, certain development potential is shown, and the peptide can be developed into uric acid reducing drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to a small molecule bioactive peptide and its preparation method and application, in particular to a polypeptide with xanthine oxidase inhibitory activity and its preparation method and application, and belongs to the technical field of bioactive peptides. BACKGROUND

[0002] Gout is a metabolic disease caused by purine metabolism disorder or uric acid excretion disorder, and its core pathological mechanism is the abnormal increase of serum uric acid level, which leads to the deposition of urate crystals in joints and surrounding tissues, causing severe pain, joint deformity and even kidney damage. In recent years, the prevalence of gout has been on the rise, and it is one of the chronic diseases affecting people's health.

[0003] Xanthine oxidase (XOD) is a key rate-limiting enzyme in the purine metabolic pathway. It can catalyze the conversion of hypoxanthine to xanthine and further to uric acid. Therefore, inhibiting the activity of xanthine oxidase to treat gout is a common method in clinical treatment. Currently, the commonly used xanthine oxidase inhibitors include allopurinol, febuxostat, etc. These xanthine oxidase inhibitors are all chemical synthetic inhibitors, which can effectively reduce uric acid levels, but long-term use can easily cause liver and kidney toxicity, allergic reactions and other side effects, and has limitations in clinical application. Therefore, developing natural, low-toxicity and high-efficiency xanthine oxidase inhibitory peptides has become a research hotspot in the field of functional foods and biological medicines, and has important clinical value and social significance.

[0004] Chinese patent CN112920255A discloses a novel blue scad xanthine oxidase inhibitory peptide and its preparation method. The amino acid sequence of the xanthine oxidase inhibitory peptide is FPSV, and its inhibitory activity on xanthine oxidase can reach 94.17%.

[0005] Chinese patent CN114044802A discloses a preparation method and application of a xanthine oxidase inhibitory peptide. The amino acid sequence of the xanthine oxidase inhibitory peptide is FWF, which can continuously and stably inhibit the activity of xanthine oxidase, and the inhibition rate on xanthine oxidase is 20.69%.

[0006] Chinese patent CN118307658A discloses three whey protein polypeptides with xanthine oxidase inhibitory activity, and their preparation methods and applications. The amino acid sequences of the three whey protein polypeptides with xanthine oxidase inhibitory activity are KIDAL, KFDKAL and KGYGGV, respectively, and their inhibitory activities on xanthine oxidase are 83%, 75% and 72%, respectively.

[0007] At present, there is no xanthine oxidase inhibiting peptide with the same or similar amino acid sequence as the present case, and the existing xanthine oxidase inhibiting peptide has the highest inhibitory activity on xanthine oxidase, which only reaches 94.17%, and there is still room for improvement.

[0008] Jellyfish is a kind of marine organism rich in collagen, polysaccharide and bioactive peptide and other active ingredients, which is widely distributed, and its nutritional and medicinal value has been widely recognized. However, at present, the systematic mining and activity verification research of xanthine oxidase inhibiting peptide in jellyfish protein simulated digestion products is still relatively scarce, and a mature development system has not yet been formed. SUMMARY

[0009] The purpose of the present application is to provide a small molecule bioactive peptide derived from jellyfish with high inhibitory activity on xanthine oxidase and its preparation method and application.

[0010] In order to achieve the above-mentioned target, the technical scheme adopted by the present application is as follows: A polypeptide with xanthine oxidase inhibitory activity, the amino acid sequence of the polypeptide is LGALWPPM.

[0011] The preparation method of the aforementioned polypeptide with xanthine oxidase inhibitory activity adopts enzymatic hydrolysis method, which comprises the following steps: (1) Clean fresh jellyfish is soaked in deionized water at 4℃ for 48h, and then washed with phosphate buffer; (2) The jellyfish is cut into pieces and added to the phosphate buffer, and then homogenized in an ice bath using a high-speed homogenizer to obtain a jellyfish homogenate, which is diluted and used as needed; (3) The pH value of the jellyfish homogenate is adjusted to 3.0, then the simulated gastric juice is added, and after mixing evenly, it is digested in a constant temperature oscillator at 37℃, 180rpm for 6h; (4) The pH value of the gastric digestion product is adjusted to 7.0, then the simulated intestinal juice is added, and after mixing evenly, it is digested in a constant temperature oscillator at 37℃, 180rpm for 6h; (5) The intestinal digestion product is filtered with a 0.22µm sterile filter membrane, and then boiled in a 100℃ water bath to obtain a hydrolysate; (6) The cooled hydrolysate is transferred to a centrifuge tube, and the hydrolysate is centrifuged at 4℃, and the supernatant is collected, and the supernatant is further centrifuged at 4℃ using a ultrafiltration tube with a molecular weight cut-off of 5kDa, and the filtrate is collected to obtain a jellyfish peptide solution, which contains the aforementioned polypeptide with xanthine oxidase inhibitory activity.

[0012] Preferably, in step (2), the ratio of the amount of jellyfish pieces to phosphate buffer is 1g:9mL; the jellyfish homogenate is mixed with the simulated gastric juice at a volume ratio of 1:1.

[0013] Preferably, in step (3), the pH value of the mixed solution is adjusted to 3.0±0.1 every 1h during the experiment.

[0014] Preferably, in step (4), the gastric digestion product is mixed with the simulated intestinal fluid at a volume ratio of 3:1; and the pH value of the mixed solution is adjusted to 7.0±0.1 every 1h during the experiment.

[0015] The method for preparing the aforementioned polypeptide with xanthine oxidase inhibitory activity adopts a solid-phase synthesis method, and comprises the following steps: The polypeptide powder is obtained by using Fmoc-protected amino acids as raw materials, polystyrene resin as a solid-phase carrier, and Fmoc solid-phase synthesis strategy for solid-phase synthesis.

[0016] The aforementioned polypeptide LGALWPPM with xanthine oxidase inhibitory activity is applied to preparation of a xanthine oxidase inhibitor.

[0017] The present application has the following advantages: (1) The xanthine oxidase inhibitory peptide provided by the present application is derived from edible jellyfish, and is a kind of natural xanthine oxidase inhibitory peptide, which is safer for human body; (2) The xanthine oxidase inhibitory peptide provided by the present application is composed of 8 amino acids (LGALWPPM), and has a smaller molecular weight (884.10 Da), which is more easily absorbed by human body; (3) The xanthine oxidase inhibitory peptide provided by the present application has higher stability in a simulated digestive environment, which breaks through the bottleneck of easy degradation and difficult activity maintenance of natural active peptides; (4) The maximum inhibition rate of the xanthine oxidase inhibitory peptide provided by the present application on xanthine oxidase reaches 96.84%, and the IC 50 value of the xanthine oxidase inhibitory peptide on xanthine oxidase is 4.654 mM, which is more excellent in activity performance compared with most of the existing xanthine oxidase inhibitory peptides. Meanwhile, the unique dose-response characteristics and the higher maximum inhibition rate of the xanthine oxidase inhibitory peptide further open up a new research idea for the research and development of new xanthine oxidase inhibitors, and show certain development potential, which can be developed into a drug for reducing uric acid. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a partial three-dimensional structure diagram of molecular docking of the polypeptide LGALWPPM and xanthine oxidase; Figure 2 is a calculation result diagram of the xanthine oxidase inhibition rate of the polypeptide LGALWPPM under different concentrations. DETAILED DESCRIPTION

[0019] The present application will be specifically described below in combination with the drawings and examples.

[0020] I. Preparation of jellyfish homogenate Firstly, rinse fresh jellyfish with deionized water (remove surface impurities), and soak the rinsed jellyfish in 4℃ deionized water for 48h (desalt and remove soluble proteins).

[0021] Then, rinse the soaked jellyfish with phosphate buffer solution (10mM, pH 7.4) for 3 times.

[0022] After that, cut the rinsed jellyfish into pieces with a volume less than 1mm³.

[0023] Next, take 100mg of the cut pieces and transfer them to a 1mL centrifuge tube containing 900µL of phosphate buffer solution (10mM, pH 7.4), and homogenize them with a high-speed homogenizer (Tissue Master TM , Shanghai Biotech Co., Ltd.) under ice bath, 6000rpm conditions for 6 times, 30s each time, with a 15s interval between each homogenization (to prevent the instrument from overheating), to obtain a jellyfish homogenate.

[0024] Finally, adjust the concentration of the jellyfish homogenate to 10% (w / v) with phosphate buffer solution (10mM, pH 7.4).

[0025] II. Preparation of jellyfish peptide solution Firstly, take 15mL of the jellyfish homogenate (concentration of 10%, w / v), adjust the pH value to 3.0 with 0.1M hydrochloric acid solution, and add 15mL of simulated gastric juice to the jellyfish homogenate at a volume ratio of 1:1, mix well, and then digest in a constant temperature shaker at 37℃, 180rpm for 6h (simulated gastric digestion process) to obtain a gastric digestion product. During the experiment, adjust the pH value of the mixture to 3.0±0.1 every 1h with 0.1M hydrochloric acid solution.

[0026] Then, slowly add 1M sodium bicarbonate solution to the mixture to adjust the pH value to 7.0 (terminate the gastric digestion process).

[0027] After that, take 15mL of the gastric digestion product, add 5mL of simulated intestinal juice to the gastric digestion product at a volume ratio of 3:1, and digest in a constant temperature shaker at 37℃, 180rpm for 6h (simulated intestinal digestion process) to obtain an intestinal digestion product. During the experiment, adjust the pH value of the mixture to 7.0±0.1 every 1h with 0.1M sodium hydroxide solution.

[0028] Next, take 10mL of the intestinal digestion product, filter it with a 0.22µm sterile filter membrane, and boil it in a 100℃ water bath for 15min (inactivate residual enzymes) to obtain a hydrolysate.

[0029] Finally, the hydrolysis solution cooled to room temperature was transferred to a centrifuge tube, and the hydrolysis solution was centrifuged (6000 rpm, 15 min) at 4°C, and the supernatant was collected. The supernatant was further centrifuged (10000 g, 15 min) at 4°C using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the filtrate was collected to obtain a jellyfish peptide solution.

[0030] Preparation method of simulated gastric fluid: 3.6 mg of pepsin was dissolved in 100 mL of phosphate buffer (10 mM, pH 7.4), and the pH value was adjusted to 3.0 with 0.1 M hydrochloric acid solution.

[0031] Preparation method of simulated intestinal fluid: 1.75 g of trypsin, 2.0 g of cholate, and 3.25 mg of trypsin were dissolved in 100 mL of sodium bicarbonate solution (1 M), and the pH value was adjusted to 7.0 with 0.1 M sodium hydroxide solution.

[0032] III. Identification of polypeptide sequences LC-MS / MS analysis was performed using a Thermo Fisher EASY-nLC 1200-Q Exactive mass spectrometry system, and the peptide spectrum of the jellyfish peptide solution was analyzed.

[0033] Chromatographic separation was performed using a reversed-phase C18 column (0.15 mm x 150 mm, RP-C18, Column Technology Inc.). The mobile phase A was 0.1% formic acid aqueous solution (v / v), and the mobile phase B was 0.1% formic acid-acetonitrile aqueous solution (acetonitrile: water = 21:4, v / v). The column was initially equilibrated with 95% A solution, and then the sample was injected into a Zorbax 300SB-C18 peptide trap column (Agilent Technologies, Wilmington, DE) via an automatic sampler, and then transferred to the analytical column for separation. The gradient program was set as follows: within 0-50 min, B was linearly increased from 4% to 50%, within 50-54 min, B was increased from 50% to 100%, and within 54-60 min, B was maintained at 100%.

[0034] Mass spectrometry analysis was performed using a Q Exactive HF-X mass spectrometer (Thermo Fisher) in positive ion mode. The full scan range was m / z 350-1800, and the resolution was 70000 (@ m / z 200). After each full scan (MS1), the top 10 high-abundance parent ions were collected for fragment spectrum (MS2), and the fragment scan resolution was 17500 (@ m / z 200). The ion injection time was 20 ms (MS1) and 60 ms (MS2), respectively, and the target ion mass was set to 3 x 10 6 (MS1) and 5 x 10 5(MS2). In data-dependent acquisition (DDA) mode, fragmentation was performed using high-energy collision dissociation (HCD) with a normalized collision energy of 28%. Mass spectrometry data were acquired and processed using Xcalibur software (Thermo Scientific).

[0035] Through peptide spectrum analysis, a total of 220 polypeptides were obtained from the jellyfish peptide solution.

[0036] Four, preliminary screening of polypeptides with potential xanthine oxidase inhibitory activity using bioinformatics tools The novelty of polypeptides was queried using BIOPEP and UniProt databases. If a polypeptide sequence has been reported, further screening work will not be carried out.

[0037] The biological activity of polypeptides was predicted using the Peptide Ranker tool. When the score of a polypeptide exceeds 0.5, it is considered that the polypeptide has potential biological activity.

[0038] The digestion-resistant properties of polypeptides were predicted using the Peptide Cutter tool. If there are no sites in the polypeptide sequence that can be cut by pepsin (Pepsin pH1.3 and pH>2.0, EC3.4.23.1), trypsin (Trypsin, EC3.4.21.4) and chymotrypsin (Chymotrypsin, EC3.4.21.1), it is considered that the polypeptide sequence has potential resistance to gastrointestinal digestion.

[0039] The cell membrane penetration of polypeptides was predicted using the CPP pred tool. When the score of a polypeptide sequence exceeds 0.5, it is considered to have the potential for complete transmembrane absorption.

[0040] The stability and physicochemical properties of polypeptides in blood were evaluated using the Plife Pred tool. If the half-life of a polypeptide sequence is higher than 800s, it is considered that the polypeptide has certain stability in blood.

[0041] The potential allergenicity of polypeptides was predicted using the Aller TOP v.2.1 tool, and the potential toxicity of polypeptides was predicted using the Toxin Pred tool. Only sequences that are not considered to have potential allergenicity and toxicity can be subjected to subsequent synthesis and verification.

[0042] Among the 220 polypeptides obtained by mass spectrometry identification, 17 were reported sequences, and the remaining 203 sequences were not reported. Based on Peptide Ranker score (> 0.5), digestion stability and transmembrane ability, 120 candidate polypeptides were screened from the 203 unreported polypeptides, and 99 high-risk polypeptides were eliminated by AllerTOP v.2.0 toxicity / allergy prediction, and finally 21 high-quality polypeptides were obtained.

[0043] V. Virtual screening of polypeptides with the strongest xanthine oxidase inhibitory activity by molecular docking The 21 high-quality polypeptides screened in the previous process were used as ligands, and xanthine oxidase was used as the receptor. Molecular docking technology was used to analyze the interaction sites and interaction forces between the 21 polypeptides and xanthine oxidase.

[0044] The three-dimensional structure of xanthine oxidase (PDB ID: 1FIQ) was obtained from the PDB database (http: / / www.rcsb.org / ). The three-dimensional structures of the 21 high-quality polypeptides were constructed using the Pymol program. The 21 high-quality polypeptides were subjected to semi-flexible docking with xanthine oxidase using Auto dock software. Center (x, y, z) = (28.634, 30.078, 101.35). The results of molecular docking were expressed as binding energy values, and the conformation with the smallest binding energy was selected as the best binding site.

[0045] Taking the binding energy -9.0 kcal / mol as the screening threshold, and based on the potential activity, safety and biological accessibility evaluation criteria, one polypeptide with potential xanthine oxidase inhibitory activity, LGALWPPM (SEQ ID NO: 1), was finally screened out.

[0046] The sequence and properties of the screened polypeptide are shown in Table 1.

[0047] Table 1 Sequence and properties of the screened polypeptide

[0048] Visual analysis showed that the interaction (local three-dimensional structure) of the polypeptide LGALWPPM with xanthine oxidase was as shown in Figure 1 It can be seen from Figure 1 that the polypeptide LGALWPPM forms 8 hydrogen bond interactions with E45, E263, D360, R426 and K1228.

[0049] VI. Verification of the actual xanthine oxidase inhibitory activity of the polypeptide LGALWPPM The polypeptide LGALWPPM is synthesized by solid phase synthesis. Specifically, Fmoc-protected amino acids are used as raw materials, polystyrene resin is used as a solid phase carrier, and Fmoc solid phase synthesis strategy is adopted to synthesize the polypeptide powder.

[0050] To explore the inhibitory effect of the polypeptide LGALWPPM on xanthine oxidase, gradient concentrations are set for experiments, and the specific operation is as follows: 1. Prepare a polypeptide solution: dissolve the polypeptide powder obtained by solid phase synthesis in ultrapure water to prepare polypeptide solutions with concentrations of 1 mM, 3 mM, 5 mM, 7 mM, 9 mM, and 11 mM, respectively; 2. Sample group: take 100 μL of the polypeptide solution and mix it with 50 μL of a 2.0 mM xanthine substrate solution, and pre-incubate the reaction at 37°C in the dark for 15 min; 3. Sample control group: take 100 μL of the polypeptide solution and mix it with 50 μL of a 2.0 mM xanthine substrate solution, and pre-incubate the reaction at 37°C in the dark for 15 min; 4. Blank group: take 100 μL of phosphate buffer (10 mM, pH 7.4) and mix it with 50 μL of a 2.0 mM xanthine substrate solution, and pre-incubate the reaction at 37°C in the dark for 15 min; 5. Blank control group: take 100 μL of phosphate buffer (10 mM, pH 7.4) and mix it with 50 μL of a 2.0 mM xanthine substrate solution, and pre-incubate the reaction at 37°C in the dark for 15 min; 6. After the pre-incubation reaction is completed, add 50 μL of a 0.05 U / mL xanthine oxidase solution pre-incubated at 37°C for 30 min to the reaction system of the sample group and the blank group, and add 50 μL of phosphate buffer (10 mM, pH 7.4) to the reaction system of the sample control group and the blank control group, and continue to incubate the reaction at 37°C in the dark for 15 min to ensure the integrity of the enzyme reaction process; 7. After the enzyme reaction is completed, use an enzyme marker to measure the absorbance of each reaction system at a wavelength of 295 nm (uric acid has a characteristic absorption peak at this wavelength, while xanthine has lower absorption at this wavelength), and calculate the xanthine oxidase inhibition rate according to the absorbance.

[0051] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0052] Where A1 represents the absorbance of the sample group, A2 represents the absorbance of the blank group, A3 represents the absorbance of the sample control group, and A4 represents the absorbance of the blank control group.

[0053] The inhibition rate of xanthine oxidase by polypeptide LGALWPPM at different concentrations is calculated as follows: Table 2 Inhibition rate of xanthine oxidase by polypeptide LGALWPPM at different concentrations

[0054] As shown in Table 2, polypeptide LGALWPPM has a significant dose-dependent inhibitory effect on xanthine oxidase, and a linear dose-effect relationship (R2>0.99) is shown in the concentration range of 1 mM-11 mM, and the maximum inhibition rate can reach 96.84%, indicating that it has strong xanthine oxidase inhibition potential.

[0055] The xanthine oxidase inhibition activity of polypeptide LGALWPPM is plotted with the concentration of polypeptide LGALWPPM as the abscissa and the xanthine oxidase inhibition rate as the ordinate, and the xanthine oxidase inhibition activity result graph of polypeptide LGALWPPM is obtained. Figure 2 ).

[0056] As shown in Table 2, polypeptide LGALWPPM has a significant dose-dependent inhibitory effect on xanthine oxidase, and a linear dose-effect relationship (R2>0.99) is shown in the concentration range of 1 mM-11 mM, and the maximum inhibition rate can reach 96.84%, indicating that it has strong xanthine oxidase inhibition potential. Figure 2 50 The half-inhibitory concentration (IC 50 ) of polypeptide LGALWPPM for xanthine oxidase is 4.654 mM.

[0057] In summary, the xanthine oxidase inhibition activity of polypeptide LGALWPPM is as high as 96.84%, and the IC 50 value is 4.654 mM. The polypeptide has unique dose response characteristics and excellent maximum inhibition rate, not only providing a new direction for the research and development of new xanthine oxidase inhibitors, but also showing good development potential.

[0058] Further combined with the molecular docking results (binding energy < -9.0 kcal / mol) and the non-allergic properties predicted by Aller TOP v2.1, it is confirmed that polypeptide LGALWPPM can be used as a new high-safety xanthine oxidase inhibitor, which is expected to provide a new type of natural candidate component for the prevention and improvement of gout and hyperuricemia, and to open up a new way for the high-value utilization of jellyfish resources.

[0059] It should be noted that the above embodiments of the present application are only 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 variations can be made by those of ordinary skill in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical solutions of the present application still fall within the protection scope of the present application.

Claims

1. A polypeptide with xanthine oxidase inhibitory activity, characterized in that, The amino acid sequence of the polypeptide is LGALWPPM.

2. The method for preparing the polypeptide with xanthine oxidase inhibitory activity according to claim 1, characterized in that, The enzymatic hydrolysis method includes the following steps: (1) Soak clean, fresh jellyfish in deionized water at 4°C for 48 hours, then rinse with phosphate buffer. (2) Cut the jellyfish into small pieces, add them to the phosphate buffer solution, homogenize them with a high-speed homogenizer under ice bath conditions to obtain a jellyfish homogenate, and dilute it for later use. (3) Adjust the pH of the jellyfish homogenate to 3.0, then add simulated gastric juice, mix well, and digest in a constant temperature shaker at 37℃ and 180rpm for 6 hours; (4) Adjust the pH of the gastric digestion products to 7.0, then add simulated intestinal fluid, mix well, and digest for 6 hours in a constant temperature shaker at 37°C and 180 rpm. (5) Filter the intestinal digestion products through a 0.22µm sterile filter membrane, and then boil them in a 100℃ water bath to obtain hydrolysate; (6) Transfer the hydrolysate cooled to room temperature to a centrifuge tube, centrifuge the hydrolysate at 4°C, collect the supernatant, further centrifuge the supernatant using a 5kDa ultrafiltration tube at 4°C, collect the filtrate, and obtain a jellyfish peptide solution containing the polypeptide with xanthine oxidase inhibitory activity as described in claim 1.

3. The preparation method according to claim 2, characterized in that, In step (2), the ratio of jellyfish fragments to phosphate buffer is 1g:9mL.

4. The preparation method according to claim 2, characterized in that, In step (3), the jellyfish homogenate and the simulated gastric juice are mixed at a volume ratio of 1:

1.

5. The preparation method according to claim 2, characterized in that, In step (3), the pH of the mixture was adjusted to 3.0 ± 0.1 every 1 hour during the experiment.

6. The preparation method according to claim 2, characterized in that, In step (4), the gastric digestion products are mixed with simulated intestinal fluid at a volume ratio of 3:

1.

7. The preparation method according to claim 2, characterized in that, In step (4), the pH of the mixture was adjusted to 7.0 ± 0.1 every 1 hour during the experiment.

8. The method for preparing the polypeptide with xanthine oxidase inhibitory activity according to claim 1, characterized in that, The solid-phase synthesis method includes the following steps: Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, a solid-phase synthesis strategy based on Fmoc was adopted to obtain polypeptide powder.

9. The use of the polypeptide LGALWPPM with xanthine oxidase inhibitory activity as described in claim 1 in the preparation of xanthine oxidase inhibitors.

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