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

Abstract

The application 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, has an amino acid sequence of LGALWPPM, a molecular weight of 884.10 Da, is obtained by hydrolysis of the jellyfish in simulated gastric juice and simulated intestinal juice, and has xanthine oxidase inhibitory activity. The polypeptide LGALWPPM has a maximum inhibition rate of 96.84 % on xanthine oxidase, and has an IC 50 value of 4.654 mM. Compared with most existing xanthine oxidase inhibitory peptides, the activity of the polypeptide is better. Meanwhile, the unique dose response characteristics and the high maximum inhibition rate of the polypeptide further open up a new research idea for the research and development of new xanthine oxidase inhibitors, and show certain development potential, and the polypeptide can be developed into a drug for reducing uric acid.

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 a maximum inhibitory activity of only 94.17% on xanthine oxidase, which still has room for improvement.

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

[0009] The present application aims to provide a small molecule bioactive peptide derived from jellyfish with high inhibitory activity on xanthine oxidase, as well as its preparation method and application.

[0010] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows:

[0011] A polypeptide with xanthine oxidase inhibitory activity, the amino acid sequence of the polypeptide is LGALWPPM.

[0012] The preparation method of the aforementioned polypeptide with xanthine oxidase inhibitory activity adopts enzymatic hydrolysis method, comprising the following steps:

[0013] (1) Clean fresh jellyfish is soaked in deionized water at 4℃ for 48h, and then washed with phosphate buffer;

[0014] (2) The jellyfish is cut into pieces and added to the phosphate buffer, and homogenized in an ice bath using a high-speed homogenizer to obtain a jellyfish homogenate, which is diluted and reserved;

[0015] (3) The pH value of the jellyfish homogenate is adjusted to 3.0, then simulated gastric juice is added, and after mixing evenly, it is digested in a constant temperature oscillator at 37℃, 180rpm for 6h;

[0016] (4) The pH value of the gastric digestion product is adjusted to 7.0, then simulated intestinal juice is added, and after mixing evenly, it is digested in a constant temperature oscillator at 37℃, 180rpm for 6h;

[0017] (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;

[0018] (6) The cooled hydrolysate is transferred to a centrifuge tube, and the hydrolysate is centrifuged at 4℃, and the supernatant is collected, which is further centrifuged at 4℃ using a 5kDa molecular weight cutoff ultrafiltration tube, and the filtrate is collected to obtain a jellyfish peptide solution, which contains the aforementioned polypeptide with xanthine oxidase inhibitory activity.

[0019] Preferably, in step (2), the ratio of the amount of jellyfish pieces to phosphate buffer is 1g:9mL; the ratio of the jellyfish homogenate to simulated gastric juice is 1:1 by volume.

[0020] Preferably, in step (3), the pH of the mixture is adjusted to 3.0±0.1 every 1h during the experiment.

[0021] Preferably, in step (4), the ratio of the gastric digestion product to simulated intestinal juice is 3:1 by volume; the pH of the mixture is adjusted to 7.0±0.1 every 1h during the experiment.

[0022] The method for preparing the aforementioned polypeptide with xanthine oxidase inhibitory activity adopts solid-phase synthesis, and comprises the following steps:

[0023] The polypeptide powder is obtained by adopting Fmoc solid-phase synthesis strategy using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier.

[0024] The aforementioned polypeptide LGALWPPM with xanthine oxidase inhibitory activity is applied to the preparation of a xanthine oxidase inhibitor.

[0025] The present application has the following advantages:

[0026] (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 the human body;

[0027] (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 the human body;

[0028] (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;

[0029] (4) The xanthine oxidase inhibitory peptide provided by the present application has a maximum inhibition rate of 96.84% on xanthine oxidase, and an IC 50 value of 4.654mM on xanthine oxidase, which is more excellent in activity than 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

[0030] Figure 1is a partial three-dimensional structure diagram of the polypeptide LGALWPPM docking with xanthine oxidase;

[0031] Figure 2 is a calculation result diagram of xanthine oxidase inhibition rate of the polypeptide LGALWPPM at different concentrations. DETAILED DESCRIPTION

[0032] The application will be described in detail below in combination with the drawings and examples.

[0033] I. Preparation of jellyfish homogenate

[0034] Firstly, rinse the fresh jellyfish with deionized water (remove surface impurities), and soak the rinsed jellyfish in 4℃ deionized water for 48h (desalt and remove soluble proteins).

[0035] Then, rinse the soaked jellyfish with phosphate buffer solution (10mM, pH 7.4) for 3 times.

[0036] After that, cut the rinsed jellyfish into pieces with a volume less than 1mm³.

[0037] 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 Biyun Tian Biotechnology Co., Ltd.) under ice bath, 6000rpm conditions for 6 times, 30s each time, with an interval of 15s between each homogenization (to prevent the instrument from overheating), to obtain jellyfish homogenate.

[0038] Finally, adjust the concentration of the jellyfish homogenate to 10% (w / v) with phosphate buffer solution (10mM, pH 7.4).

[0039] II. Preparation of jellyfish peptide solution

[0040] Firstly, take 15mL of 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 the 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.

[0041] Then, slowly add 1M sodium bicarbonate solution to the mixture to adjust the pH value to 7.0 (terminate the gastric digestion process).

[0042] Then, 15 mL of the gastric digestion product was taken and 5 mL of simulated intestinal fluid was added to the gastric digestion product at a volume ratio of 3:1, and the mixture was digested in a constant-temperature oscillator at 37°C and 180 rpm for 6 h (simulated intestinal digestion), to obtain an intestinal digestion product. During the experiment, the pH value of the mixture was adjusted to 7.0 ± 0.1 every 1 h with 0.1M sodium hydroxide solution.

[0043] Next, 10 mL of the intestinal digestion product was filtered with a 0.22 µm sterile filter membrane and boiled in a 100°C water bath for 15 min (to inactivate residual enzymes), to obtain a hydrolyzate.

[0044] Finally, the hydrolyzate cooled to room temperature was transferred to a centrifuge tube, and the hydrolyzate 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 cut-off of 5 kDa, and the filtrate was collected, to obtain a jellyfish peptide solution.

[0045] 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 0.1M hydrochloric acid solution.

[0046] Preparation method of simulated intestinal fluid: 1.75 g of pancreatin, 2.0 g of cholate, and 3.25 mg of trypsin were dissolved in 100 mL of sodium bicarbonate solution (1M), and the pH value was adjusted to 7.0 with 0.1M sodium hydroxide solution.

[0047] III. Identification of polypeptide sequences

[0048] 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.

[0049] Chromatographic separation was performed using a reversed-phase C18 column (0.15 mm x 150 mm, RP-C18, Column Technology Inc.), with mobile phase A being 0.1% formic acid aqueous solution (v / v) and B being 0.1% formic acid-acetonitrile aqueous solution (acetonitrile:water = 21:4, v / v). The column was initially equilibrated with 95% A, and then the sample was injected into a Zorbax 300SB-C18 peptide trap column (Agilent Technologies, Wilmington, DE) via an automatic injector, 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%.

[0050] Mass spectrometry analysis was performed on a Q Exactive HF-X mass spectrometer (Thermo Fisher) in positive ion mode, with a full scan range of m / z 350-1800 and a resolution of 70000 (@ m / z 200). After each full scan (MS1), the top 10 high-abundance parent ions were collected for fragment spectrum (MS2), with a fragment scan resolution of 17500 (@ m / z 200), ion injection times of 20 ms (MS1) and 60 ms (MS2), and target ion quantities of 3x10 6 (MS1) and 5x10 5 (MS2), respectively. In the data-dependent acquisition (DDA) mode, high-energy collisional dissociation (HCD) was used for fragmentation, with a normalized collision energy of 28%. Mass spectrometry data were collected and processed using Xcalibur software (Thermo Scientific).

[0051] A total of 220 polypeptides were obtained from the jellyfish peptide solution through peptide mapping.

[0052] Four, preliminary screening of polypeptides with potential xanthine oxidase inhibitory activity using bioinformatics tools

[0053] The novelty of the polypeptides was queried using the BIOPEP and UniProt databases. If a polypeptide sequence has been reported, further screening work will not be carried out.

[0054] The biological activity of the 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.

[0055] The digestion-resistant properties of the 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.

[0056] The cell membrane penetration of the polypeptides was predicted using the CPP pred tool. When the score of a polypeptide sequence exceeds 0.5, it is considered that it has the potential for complete transmembrane absorption.

[0057] The stability and physicochemical properties of the polypeptides in blood were evaluated using the Plife Pred tool. If the half-life of a polypeptide sequence is higher than 800 s, it is considered that the polypeptide has certain stability in blood.

[0058] The potential allergenicity of the polypeptides was predicted using the Aller TOP v.2.1 tool, and the potential toxicity of the polypeptides was predicted by the Toxin Pred tool. Only the sequences determined to be free of potential allergenicity and toxicity can be subjected to subsequent synthesis and verification.

[0059] Among the 220 polypeptides obtained through mass spectrometry identification, 17 were reported sequences, and the remaining 203 sequences were not reported. Based on Peptide Ranker scores (>0.5), digestion stability, and transmembrane ability, 120 candidate polypeptides were selected from the 203 unreported polypeptides. Further, 99 high-risk polypeptides were eliminated through AllerTOP v.2.0 toxicity / allergenicity prediction, and finally 21 high-quality polypeptides were obtained.

[0060] Five, virtual screening of polypeptides with the strongest xanthine oxidase inhibitory activity by molecular docking

[0061] 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.

[0062] 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.

[0063] Taking the binding energy of -9.0 kcal / mol as the screening threshold, one polypeptide with potential xanthine oxidase inhibitory activity, LGALWPPM (SEQ ID NO: 1), was finally screened out through molecular docking screening and based on the evaluation criteria of potential activity, safety, and biological accessibility.

[0064] The sequence and properties of the screened polypeptide are shown in Table 1.

[0065] Table 1 Sequence and properties of the screened polypeptide

[0066]

[0067] Through visual analysis, the interaction (local three-dimensional structure) of the polypeptide LGALWPPM with xanthine oxidase is shown in Figure 1 Figure 1 ​It can be seen that eight hydrogen bonds are formed between the polypeptide LGALWPPM and E45, E263, D360, R426 and K1228.

[0068] Six, verify the actual xanthine oxidase inhibitory activity of polypeptide LGALWPPM

[0069] 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 used for solid phase synthesis to obtain polypeptide powder.

[0070] To explore the inhibitory effect of polypeptide LGALWPPM on xanthine oxidase, gradient concentrations are set for experiments, and the specific operation is as follows:

[0071] 1. Preparation of 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;

[0072] 2. Sample group: take 100 μL of polypeptide solution, mix with 50 μL of 2.0 mM concentration xanthine substrate solution, and pre-incubate at 37°C in the dark for 15 min;

[0073] 3. Sample control group: take 100 μL of polypeptide solution, mix with 50 μL of 2.0 mM concentration xanthine substrate solution, and pre-incubate at 37°C in the dark for 15 min;

[0074] 4. Blank group: take 100 μL of phosphate buffer (10 mM, pH 7.4), mix with 50 μL of 2.0 mM concentration xanthine substrate solution, and pre-incubate at 37°C in the dark for 15 min;

[0075] 5. Blank control group: take 100 μL of phosphate buffer (10 mM, pH 7.4), mix with 50 μL of 2.0 mM concentration xanthine substrate solution, and pre-incubate at 37°C in the dark for 15 min;

[0076] 6. After the pre-incubation reaction is completed, 50 μL of 0.05 U / mL xanthine oxidase solution pre-incubated at 37°C for 30 min is added to the reaction system of the sample group and the blank group, and 50 μL of phosphate buffer (10 mM, pH 7.4) is added to the reaction system of the sample control group and the blank control group, and the incubation reaction is continued at 37°C in the dark for 15 min to ensure the integrity of the enzyme reaction process.

[0077] 7. After the enzymatic reaction is completed, the absorbance of each reaction system is measured at a wavelength of 295 nm using an ELISA reader (uric acid has a characteristic absorption peak at this wavelength, while xanthine has lower absorption at this wavelength), and the xanthine oxidase inhibition rate is calculated based on the absorbance.

[0078] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0079]

[0080] 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.

[0081] The inhibition rates of different concentrations of the peptide LGALWPPM on xanthine oxidase were calculated as follows:

[0082] Table 2. Inhibition rate of xanthine oxidase by different concentrations of peptide LGALWPPM

[0083]

[0084] As shown in Table 2, the polypeptide LGALWPPM has a significant dose-dependent inhibitory effect on xanthine oxidase, exhibiting a linear dose-response relationship (R² > 0.99) in the concentration range of 1 mM to 11 mM, with a maximum inhibition rate of 96.84%, indicating that it has strong inhibitory potential for xanthine oxidase.

[0085] A graph showing the xanthine oxidase inhibitory activity of peptide LGALWPPM was obtained by plotting the concentration of the peptide LGALWPPM on the x-axis and the xanthine oxidase inhibition rate on the y-axis. Figure 2 ).

[0086] Depend on Figure 2 It can be seen that the half-maximal inhibitory concentration (IC50) of the polypeptide LGALWPPM on xanthine oxidase is... 50 The value is 4.654 mM.

[0087] In summary, the peptide LGALWPPM exhibits a high xanthine oxidase inhibitory activity of 96.84%, with an IC50 value of [missing information]. 50 The value was 4.654 mM. This peptide exhibits unique dose-response characteristics and excellent maximum inhibition rate, providing a new direction for the development of novel xanthine oxidase inhibitors and showing good development potential.

[0088] 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 the polypeptide LGALWPPM can be used as a new type of 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.

[0089] 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. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the protection scope of the present application.

Claims

1. A polypeptide having xanthine oxidase inhibitory activity, characterized by, The amino acid sequence of the polypeptide is LGALWPPM.

2. A method for producing the polypeptide having xanthine oxidase inhibitory activity according to claim 1, characterized by, The enzymatic method comprises the following steps: (1) Clean fresh jellyfish is soaked in deionized water at 4°C for 48 h, and then washed with phosphate buffer; (2) The jellyfish is cut into pieces and added to the phosphate buffer, and homogenized in an ice bath using a high-speed homogenizer to obtain a jellyfish homogenate, which is diluted and stored for later use; (3) The pH value of the jellyfish homogenate is adjusted to 3.0, and then simulated gastric juice is added, and the mixture is mixed uniformly and digested in a constant temperature oscillator at 37°C and 180 rpm for 6 h; (4) The pH value of the gastric digestion product is adjusted to 7.0, and then simulated intestinal juice is added, and the mixture is mixed uniformly and digested in a constant temperature oscillator at 37°C and 180 rpm for 6 h; (5) The intestinal digestion product is filtered with a 0.22 µm sterile filter membrane, and then boiled in a 100°C water bath to obtain a hydrolysate; (6) The cooled hydrolysate is transferred to a centrifuge tube, and the hydrolysate is centrifuged at 4°C, and the supernatant is collected, and the supernatant is further centrifuged at 4°C using a 5 kDa molecular weight cutoff ultrafiltration tube, and the filtrate is collected to obtain a jellyfish peptide solution, which contains the polypeptide with xanthine oxidase inhibitory activity according to claim 1.

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

4. The production method according to claim 2, characterized by, 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 value of the mixture is adjusted to 3.0±0.1 every 1 h during the experiment.

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

1.

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

8. A method for producing the polypeptide having xanthine oxidase inhibitory activity according to claim 1, characterized by, The solid-phase synthesis method comprises the following steps: 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 used for solid-phase synthesis to obtain a polypeptide powder.

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