A sea cucumber sand spit active peptide with xanthine oxidase inhibitory activity and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and in particular relates to a sea cucumber sand spit active peptide with xanthine oxidase inhibitory activity, and its preparation method and application. The sea cucumber sand spit active peptide provided by the present invention contains at least one of FPF and WFP, has high xanthine oxidase inhibitory activity, can achieve uric acid lowering function by inhibiting xanthine oxidase activity, and can be used to prepare uric acid lowering drugs. The sea cucumber sand spit active peptide is prepared from the sand spit, a by-product of sea cucumber processing, as a raw material through bio-enzymatic hydrolysis technology under a specific process, which not only solves the problem of low utilization rate of sea cucumber processing by-products, but also reduces resource waste. The present invention provides a green and sustainable auxiliary intervention program for patients with hyperuricemia and gout, and at the same time provides an innovative way to high-value utilization of marine processing waste.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a sea cucumber sand spit active peptide having xanthine oxidase inhibitory activity, and a preparation method and application thereof. Background Art

[0002] With changes in dietary patterns and the prevalence of metabolic syndrome, hyperuricemia has become the third most common metabolic disease after hypertension and diabetes. Abnormally elevated serum uric acid levels not only induce gouty arthritis but are also directly associated with serious complications such as renal impairment and cardiovascular disease. Xanthine oxidase (XOD), the rate-limiting enzyme in uric acid biosynthesis, is a key target for controlling uric acid production. Currently, both allopurinol and febuxostat, the leading clinical drugs, are XOD inhibitors. However, while both effectively inhibit enzyme activity, long-term use can lead to severe allergic reactions and liver and kidney damage, and treatment costs can reach as high as 2,000-5,000 yuan per person per year. Therefore, the development of safe and economical XOD inhibitors is urgent.

[0003] Bioactive peptides are short functional peptides composed of 2-20 amino acids linked by specific sequences. Their small molecular weight, strong targeting, high safety, and high metabolic stability make them ideal candidates for the development of new enzyme inhibitors. Marine biological resources, due to their unique metabolic pathways, often produce active peptide structures rarely seen in terrestrial organisms. Sea cucumbers are rich in protein and are an excellent marine biological raw material for obtaining active peptides. However, existing research has mostly focused on the antioxidant and immunomodulatory functions of sea cucumber body wall peptides, and the exploration of their uric acid-lowering activity is still in its early stages. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a sea cucumber spit active peptide with xanthine oxidase inhibitory activity, as well as its preparation method and application. The sea cucumber spit active peptide provided by the present invention has a significant xanthine oxidase inhibitory effect and can be used to prepare anti-gout drugs or drugs for treating other hyperuricemia-related diseases. The preparation method provided by the present invention is efficient and environmentally friendly, making it suitable for industrial implementation.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a sea cucumber sand spit active peptide having xanthine oxidase inhibitory activity. The sea cucumber sand spit active peptide contains oligopeptide WFP, and the amino acid sequence of the oligopeptide WFP is Trp-Phe-Pro (i.e., tryptophan-phenylalanine-proline).

[0007] Molecular docking results showed that oligopeptide WFP formed two hydrogen bonds with Gln768 and Gly1261 in the active center of xanthine oxidase, and there were 12 hydrophobic interactions. The results of in vitro xanthine oxidase inhibitory activity assay showed that the half-inhibitory concentration IC50 of oligopeptide WFP for xanthine oxidase inhibition was 100%. 50 The results showed that the oligopeptide WFP had significant xanthine oxidase inhibitory activity.

[0008] In addition, the molecular weight of oligopeptide WFP is 448.5 Da. The smaller molecular weight allows both to be directly absorbed through the intestines without relying on enzymatic digestion and decomposition, making it particularly suitable for patients with hyperuricemia and impaired gastrointestinal function.

[0009] This invention provides a safe and reliable new active ingredient for the treatment of gout and other hyperuricemia-related diseases, and expands new applications for sea cucumber active peptides. Furthermore, the oligopeptide WFP is derived from sea cucumber processing waste, spit. Therefore, the discovery of this sea cucumber spit active peptide increases the application value of sea cucumber spit as a raw material source for active pharmaceutical ingredients, providing a new approach for its resource utilization.

[0010] By combining the oligopeptide WFP with a carrier protein or encapsulation technology, it can be developed into a highly effective and low-toxic xanthine oxidase inhibitor.

[0011] Preferably, the sea cucumber sand spit active peptide further contains oligopeptide FPF, and the amino acid sequence of the oligopeptide FPF is: Phe-Pro-Phe (i.e., alanine-proline-phenylalanine).

[0012] Molecular docking results showed that oligopeptide FPF formed five hydrogen bonds with Glu1262, Gln768, Phe912, and Ser1081 in the active center of xanthine oxidase, and there were eight hydrophobic interactions. The results of in vitro xanthine oxidase inhibitory activity assay showed that the half-inhibitory concentration IC50 of oligopeptide FPF for xanthine oxidase inhibition was 2.37. 50 The results show that the oligopeptide FPF also has significant xanthine oxidase inhibitory activity. So far, FPF has not been found to have the effect of inhibiting xanthine oxidase activity. In addition, the experiment found that when the oligopeptide FPF was used in combination with the oligopeptide WFP, the half-inhibitory concentration IC 50 It is 0.49±0.04mg / mL, showing a synergistic effect.

[0013] FPF has a molecular weight of 409.5 Da and can also be directly absorbed through the intestines, independent of enzymatic digestion. Furthermore, this oligopeptide FPF can also be obtained from sea cucumber spit, further expanding the application areas of sea cucumber active peptides and increasing their application value.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned sea cucumber sand mouth active peptide, which specifically comprises the following steps:

[0015] S1, demineralizing the sea cucumber spit, drying the demineralized material at 40-45° C. to a moisture content of ≤0.15% w / w, and crushing the material to a particle size distribution D90 ≤0.15 mm to obtain a demineralized sea cucumber spit powder;

[0016] S2, mixing the sea cucumber sand mouth demineralized powder obtained in S1 with water at a material-liquid ratio of 1: (15-30), g: mL, and enzymolyzing with papain and flavor protease for 1.5-3 h; after the enzymolysis is completed, inactivating the enzyme and cooling to obtain an enzymolysis solution;

[0017] S3, centrifuging the enzymatic hydrolyzate obtained in S2, taking the supernatant, concentrating and adding ethanol to make the ethanol volume concentration be 70% to 90%, taking the supernatant after precipitation for 60 to 72 hours, intercepting through a 3 kDa ultrafiltration membrane, collecting the permeate, concentrating and drying to obtain the crude sea cucumber sand mouth peptide;

[0018] S4. Dissolve the crude sea cucumber gizzard peptide obtained in S3 in ultrapure water, separate and purify it by gel chromatography, collect the effluent with a molecular weight less than 1 kDa, and dry it to obtain the sea cucumber gizzard active peptide.

[0019] This preparation method utilizes a combination of papain and flavor protease for hydrolysis, which has a synergistic effect in producing peptides with xanthine oxidase inhibitory activity. Compared to using only one enzyme, hydrolysis with this combination of papain and flavor protease produces a product with higher xanthine oxidase inhibitory activity.

[0020] Preferably, the demineralization method described in S1 is: immersing the sea cucumber sand spit in a citric acid aqueous solution with a mass concentration of 4% to 8%, applying an ultrasonic frequency of 20 to 40 kHz and synchronous mechanical stirring in a temperature range of 45 to 60°C, separating the solid and liquid after 30 to 45 minutes and recovering the solid material, the mass volume ratio of the sea cucumber sand spit to the citric acid aqueous solution is 1: (20 to 25), g: mL; repeating this process until the liquid phase of the solid-liquid separation is colorless and transparent and no precipitate is precipitated after standing, and the inorganic residue on the surface of the solid phase is confirmed to be completely removed by visual inspection.

[0021] Preferably, the temperature of the enzymatic hydrolysis in S2 is 50±2° C. and the pH is 7.5±0.3.

[0022] Preferably, the amount of papain added in S2 is 8000-12000 U / g protein, wherein "protein" refers to the mass of protein in the defatted sea cucumber sand spit powder.

[0023] More preferably, the amount of papain added in S2 is 9000-11000 U / g protein, and more preferably 10000 U / g protein.

[0024] Preferably, the amount of flavor protease added in S2 is 4000-8000 U / g protein, wherein "protein" refers to the mass of protein in the defatted sea cucumber sand spit powder.

[0025] More preferably, the amount of flavor protease added in S2 is 5000-7000 U / g protein, and more preferably 6000 U / g protein.

[0026] Preferably, the material-liquid ratio in S2 is 1:(20-25), g:mL. More preferably, the material-liquid ratio is 1:23, g:mL.

[0027] Preferably, the enzymatic hydrolysis time in S2 is 1.8 to 2.5 h, and more preferably 2 h.

[0028] Preferably, the enzyme inactivation method in S2 is 95-100°C for 10-12 min. A further preferred enzyme inactivation method is 100°C for 10 min.

[0029] Preferably, the centrifugation parameters in S3 are: rotation speed 8000-10000 rpm, centrifugation time 15-20 min, temperature 23-27° C. More preferably, the centrifugation parameters are: rotation speed 8000 rpm, centrifugation time 15 min, temperature 25° C.

[0030] Preferably, the volume concentration of ethanol after adding ethanol in S3 is 75% to 85%, and more preferably 80%.

[0031] Preferably, the drying method in S3 is freeze drying.

[0032] Preferably, the chromatographic column of the gel chromatography in S4 is TK-Col 16 / 60 GF30, the detection wavelength of the ultraviolet detector is 220 nm, and the equilibration buffer is phosphate buffer.

[0033] Preferably, the injection concentration of the gel chromatography in S4 is 5-20 mg / mL.

[0034] Preferably, the preparation method further comprises: S5, separating and purifying the obtained components by preparative reverse-phase high performance liquid chromatography, collecting the effluent according to the peak time of oligopeptide FPF and oligopeptide WFP, and obtaining the purified sea cucumber sand mouth active peptide after drying.

[0035] Further preferably, the chromatographic conditions of the preparative reversed-phase high performance liquid chromatography are as follows: the chromatographic column is Agilent Prep C18, with specifications of 100 Å, 2 µm, 75 µm inner diameter × 150 mm length, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, the elution gradient, and the gradient elution program is set as follows: 0-8 min mobile phase B linearly increases from 5% to 10%, 8-33 min mobile phase B linearly increases from 10% to 15%, 33-43 min mobile phase B linearly increases from 15% to 28%, 43-50 min mobile phase B linearly increases from 28% to 40%, 50-60 min mobile phase B linearly increases from 40% to 95% and maintains until 65 min, 66-70 min returns to the initial ratio of 5%, the flow rate is constant at 250 nL / min, the column temperature is 40°C, and the detection wavelength is 220 nm; the effluent from 8 to 12 min is collected.

[0036] The third aspect of the present invention provides the use of oligopeptide WFP, oligopeptide FPF, the above-mentioned sea cucumber sand spit active peptide, or the sea cucumber sand spit active peptide prepared by the above-mentioned preparation method in the preparation of uric acid-lowering drugs.

[0037] Preferably, the uric acid-lowering drug is a xanthine oxidase inhibitor.

[0038] Preferably, the uric acid-lowering drug is an oral preparation.

[0039] Preferably, the ingredients of the uric acid-lowering drug also include pharmaceutically acceptable excipients, such as microcrystalline cellulose, magnesium stearate, lactose, etc.

[0040] Further preferably, the uric acid-lowering drug is a sustained-release preparation or a nano-delivery system, and these two dosage forms can improve the bioavailability of the active peptide of sea cucumber sand mouth.

[0041] The beneficial effects of the present invention are:

[0042] (1) Small molecule absorption advantage: The molecular weight of FPF and WFP is less than 1000 Da, and they can be directly absorbed through the intestine without the need for digestive enzymes. They are particularly suitable for patients with high uric acid who have impaired gastrointestinal function, providing them with a high-quality nitrogen source and amino acid supplement with high bioavailability and low gastrointestinal irritation. Compared with some traditional drugs that may cause gastrointestinal discomfort (such as allopurinol and febuxostat), small molecule peptides generally show significantly better gastrointestinal tolerance, which helps to reduce the burden on the digestive system at the nutritional support level and improve the patient's overall nutritional status and compliance.

[0043] (2) Multi-scenario application potential: FPF half-inhibitory concentration IC for xanthine oxidase inhibition 50The half-inhibitory concentration (IC) of WFP for xanthine oxidase inhibition was 1.56±0.1 mg / mL. 50 The serum uric acid content of the two drugs was 0.37±0.025 mg / mL, and both drugs had high xanthine oxidase inhibitory activity. They can be used for the treatment of patients with severe hyperuricemia and for the daily management of uric acid in hyperuricemia, and have a wide range of clinical applications.

[0044] (3) Efficient preparation method: The large-scale preparation of high-purity peptides is achieved through the combined technology of "composite enzymatic hydrolysis-membrane separation-chromatographic purification", breaking through the limitations of low efficiency and high impurities of traditional preparation processes.

[0045] (4) Efficient resource utilization and environmental protection value: Using the discarded sand spits from sea cucumber processing as raw materials, this traditional discarded part is converted into high-value-added active ingredients through bio-enzymatic hydrolysis technology under a specific process, reducing resource waste and environmental pollution, and helping to promote the transformation of the sea cucumber industry to a circular economy model.

[0046] (5) Significant economic benefits: As a cheap raw material, sand spits can be converted into high-value-added uric acid-lowering peptide products through the technology of the present invention, significantly increasing the income of the sea cucumber processing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a high performance liquid chromatogram of the oligopeptide FPF synthesized by the solid phase method in Example 5 of the present invention;

[0048] Figure 2 is a mass spectrum of the oligopeptide FPF synthesized by solid phase method in Example 5 of the present invention;

[0049] Figure 3 is a high performance liquid chromatogram of the oligopeptide WFP synthesized by the solid phase method in Example 6 of the present invention;

[0050] Figure 4 is a mass spectrum of the oligopeptide WFP synthesized by solid phase method in Example 6 of the present invention;

[0051] Figure 5 This is the inhibitory activity curve of oligopeptide FPF on xanthine oxidase based on the logarithmic concentration gradient in Test Example 1 of the present invention;

[0052] Figure 6 This is the inhibitory activity curve of the oligopeptide WFP on xanthine oxidase based on the logarithmic concentration gradient in Test Example 1 of the present invention;

[0053] Figure 7 This is the inhibitory activity curve of the mixed peptide of FPF and WFP based on the logarithmic concentration gradient on xanthine oxidase in Test Example 1 of the present invention;

[0054] Figure 8This is a diagram showing the interaction between oligopeptide FPF and xanthine oxidase molecules in Test Example 2 of the present invention;

[0055] Figure 9 This is a diagram of the interaction between the oligopeptide WFP and xanthine oxidase molecules in Test Example 2 of the present invention. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0057] The sea cucumber processing industry produces approximately 80,000 tons of spit byproducts annually. This hard tissue, composed of sea cucumber mouthparts, is rich in cross-linked collagen, which can be used to produce bioactive peptides. However, traditional enzymatic hydrolysis techniques are inefficient and have low protein utilization rates. Consequently, there is a significant technical gap in exploring the active properties of spit byproducts, resulting in their disposal as waste, causing significant resource waste and environmental pollution.

[0058] Through experimental research, the present invention has obtained oligopeptides FPF and WFP from sea cucumber spit, which exhibit xanthine oxidase inhibitory activity. Their amino acid sequences are Phe-Pro-Phe and Trp-Phe-Pro, respectively. Based on this, an embodiment of the present invention provides a sea cucumber spit active peptide with xanthine oxidase inhibitory activity, comprising the oligopeptide WFP. In a preferred embodiment, the sea cucumber spit active peptide also comprises the oligopeptide FPF.

[0059] The present invention also provides a method for preparing the above-mentioned sea cucumber sand mouth active peptide, which specifically comprises the following steps:

[0060] S1, demineralizing the sea cucumber spit, drying the demineralized material at 40-45° C. to a moisture content of ≤0.15% w / w, and crushing the material to a particle size distribution D90 ≤0.15 mm to obtain a demineralized sea cucumber spit powder;

[0061] S2, mixing the sea cucumber sand mouth demineralized powder obtained in S1 with water at a material-liquid ratio of 1: (15-30), g: mL, and enzymolyzing with papain and flavor protease for 1.5-3 h; after the enzymolysis is completed, inactivating the enzyme and cooling to obtain an enzymolysis solution;

[0062] S3, centrifuging the enzymatic hydrolyzate obtained in S2, taking the supernatant, concentrating and adding ethanol to make the ethanol volume concentration be 70% to 90%, taking the supernatant after precipitation for 60 to 72 hours, intercepting through a 3 kDa ultrafiltration membrane, collecting the permeate, concentrating and drying to obtain the crude sea cucumber sand mouth peptide;

[0063] S4. Dissolve the crude sea cucumber gizzard peptide obtained in S3 in ultrapure water, separate and purify it by gel chromatography, collect the effluent with a molecular weight less than 1 kDa, and dry it to obtain a sea cucumber gizzard oligopeptide containing the sea cucumber gizzard active peptide.

[0064] The embodiments of the present invention also provide the use of oligopeptide WFP, oligopeptide FPF, the above-mentioned sea cucumber sand spit active peptide, or the sea cucumber sand spit active peptide prepared by the above-mentioned preparation method in the preparation of uric acid-lowering drugs.

[0065] The solutions of the present invention are described below through specific embodiments.

[0066] Xanthine oxidase used in the following examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Oligopeptides were synthesized by Hefei Sener Biotechnology Co., Ltd.

[0067] Unless otherwise specified in the following examples, all solutions mentioned use water as the solvent.

[0068] Unless otherwise specified, the raw materials, reagents, drugs, or instruments used in the following examples are all commercially available products. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0069] Example 1

[0070] This embodiment provides a sea cucumber sand mouth active peptide having xanthine oxidase inhibitory activity and a preparation method thereof, comprising the following steps:

[0071] S1. Raw material pretreatment: thaw the frozen sea cucumber spit to obtain the raw material; then perform gradient demineralization on the raw material - immerse the raw material in a 5% mass concentration of citric acid aqueous solution and control the material-liquid ratio to 1:20 (g:mL), apply 30kHz ultrasonic frequency and synchronous mechanical stirring in the temperature range of 45~60℃, perform solid-liquid separation after 30 minutes and recover the solid material; repeat the demineralization process until the three end points are met at the same time: the liquid phase of the solid-liquid separation is colorless and transparent, no precipitate is precipitated after the liquid phase is allowed to stand, and the inorganic residue on the surface of the solid phase is confirmed to be completely removed by visual inspection; then the demineralized wet-based material is dried by hot air at 40-45℃ to a moisture content of ≤0.15%w / w; finally, the dried material is treated with air flow milling equipment to control the particle size distribution D90≤0.15 mm to obtain sea cucumber spit demineralized powder.

[0072] S2. Composite enzymatic hydrolysis: Add the demineralized sea cucumber sand spit powder prepared in S1 to distilled water at a material-liquid ratio of 1:23 (g:mL). Adjust the pH and temperature. Add papain at a dosage of 10,000 U / g protein and flavor protease at a dosage of 6,000 U / g protein for enzymatic hydrolysis. After 2 h of enzymatic hydrolysis, inactivate the mixture by boiling in water for 10 min and cool to obtain the enzymatic hydrolyzate.

[0073] S3. Ultrafiltration separation: The enzymatic hydrolysate obtained in S2 was centrifuged (8000 rpm, 25°C, for 15 min), the supernatant was taken, concentrated, and ethanol was added to make the volume concentration of ethanol 80% after the enzymatic hydrolysate was added. After precipitation for 72 h, the supernatant was taken, retained by a 3 kDa ultrafiltration membrane, and the permeate was collected, concentrated, and dried to obtain the crude sea cucumber sand mouth peptide.

[0074] S4. Gel filtration chromatography separation: The crude sea cucumber gizzard peptide obtained in S3 was dissolved in ultrapure water, separated and purified by gel chromatography, and the effluent with a molecular weight of less than 1 kDa was collected and dried to obtain sea cucumber gizzard active peptide containing FPF and WFP.

[0075] S5. Preparative reverse-phase HPLC separation and purification: The chromatographic conditions included: an Agilent PrepC18 column with a specification of 100 Å, 2 µm, 75 μm inner diameter × 150 mm length, mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the elution gradient was set as follows: 0-8 min mobile phase B linearly increased from 5% to 10%, 8-33 min mobile phase B linearly increased from 10% to 15%, 33-43 min mobile phase B linearly increased from 15% to 28%, 43-50 min mobile phase B linearly increased from 28% to 40%, 50-60 min mobile phase B linearly increased from 40% to 95% and maintained until 65 min, and 66-70 min restored to the initial proportion of 5%, the flow rate was constant at 250 nL / min, the column temperature was 40°C, and the detection wavelength was 220 nm; the effluent from 8 to 12 min was collected.

[0076] The active peptides from sea cucumber sand spout obtained by S4 were separated by reverse phase high performance liquid chromatography and detected by mass spectrometry using a high resolution mass spectrometer. The primary mass spectrometry parameters included a resolution of 70,000, a scanning range of m / z 300-1400, and an automatic gain control (AGC) of 3×10 6 , maximum injection time 60 ms and ion source voltage 2.0 kV (positive ion mode); secondary mass spectrometry parameters were resolution 17,500, AGC 5×10 4The maximum injection time was 80 ms, and the collision energy (NCE) was 27. The top 20 most intense precursor ions were selected for fragmentation, and a dynamic exclusion time of 30 s was set. Data retrieval and analysis were performed using PEAKS Studio software, matching the UniProtKB database (version UP000230750_2024_12_30). Mass tolerances were ±10 ppm for precursor ions and ±0.02 Da for fragment ions. Dynamic modifications included methionine oxidation (M) and N-terminal acetylation (N-term). The false positive rate (FDR) threshold was ≤1%.

[0077] After testing, the content of oligopeptide FPF in the sea cucumber sand mouth active peptides obtained by S4 was 8.93%, and the content of oligopeptide WFP was 2.31%.

[0078] Example 2

[0079] This embodiment provides a sea cucumber sand mouth active peptide having xanthine oxidase inhibitory activity and a preparation method thereof, comprising the following steps:

[0080] S1. Raw material pretreatment: same as in Example 1.

[0081] S2. Composite enzymatic hydrolysis: Add the defatted sea cucumber sand mouth powder prepared in S1 to distilled water with a solid-liquid ratio of 1:15 (g:mL). Adjust the pH and temperature. Add papain at a dosage of 8000 U / g protein and flavor protease at a dosage of 4000 U / g protein for enzymatic hydrolysis. After 1.5 h of enzymatic hydrolysis, inactivate the mixture by boiling in water for 10 min and cool to obtain the enzymatic hydrolyzate.

[0082] S3. Ultrafiltration separation: The enzymatic hydrolysate obtained in S2 was centrifuged (8000 rpm, 25°C, for 15 min), the supernatant was taken, concentrated, and ethanol was added to make the volume concentration of ethanol 70% after the enzymatic hydrolysate was added. After precipitation for 72 h, the supernatant was taken and retained by a 3 kDa ultrafiltration membrane. The permeate was collected, concentrated, and dried to obtain the crude sea cucumber sand mouth peptide.

[0083] S4. Gel filtration chromatography separation: The same as Example 1 was used to obtain sea cucumber spit active peptides containing oligopeptide FPF and oligopeptide WFP. After testing, the content of oligopeptide FPF in the obtained sea cucumber spit active peptides was 4.85%, and the content of oligopeptide WFP was 0.99%.

[0084] Example 3

[0085] This embodiment provides a sea cucumber sand mouth active peptide having xanthine oxidase inhibitory activity and a preparation method thereof, comprising the following steps:

[0086] S1. Raw material pretreatment: same as in Example 1.

[0087] S2. Composite enzymatic hydrolysis: Add the defatted sea cucumber sand mouth powder prepared in S1 to distilled water at a solid-liquid ratio of 1:30 (g:mL). Adjust the pH and temperature. Add papain at a dosage of 12,000 U / g protein and flavor protease at a dosage of 8,000 U / g protein for enzymatic hydrolysis. After 3 h of enzymatic hydrolysis, inactivate the mixture by boiling in water for 10 min and cool to obtain the enzymatic hydrolyzate.

[0088] S3. Ultrafiltration separation: The enzymatic hydrolysate obtained in S2 was centrifuged (8000 rpm, 25°C, for 15 min), the supernatant was taken, concentrated, and ethanol was added to make the volume concentration of ethanol 90% after the enzymatic hydrolysate was added. After precipitation for 72 h, the supernatant was taken, retained by a 3 kDa ultrafiltration membrane, and the permeate was collected, concentrated, and dried to obtain the crude peptide of sea cucumber sand mouth.

[0089] S4. Gel filtration chromatography separation: The same as Example 1 was used to obtain sea cucumber spit active peptides containing oligopeptide FPF and oligopeptide WFP. After testing, the content of oligopeptide FPF in the obtained sea cucumber spit active peptides was 4.86%, and the content of oligopeptide WFP was 1.21%.

[0090] Example 4

[0091] This embodiment provides a sea cucumber sand mouth active peptide having xanthine oxidase inhibitory activity and a preparation method thereof, comprising the following steps:

[0092] S1. Raw material pretreatment: same as in Example 1.

[0093] S2. Composite enzymatic hydrolysis: Add the defatted sea cucumber sand mouth powder prepared in S1 to distilled water at a solid-liquid ratio of 1:25 (g:mL). Adjust the pH and temperature. Add papain at a dosage of 9000 U / g protein and flavor protease at a dosage of 7000 U / g protein for enzymatic hydrolysis. After 2.5 h of enzymatic hydrolysis, inactivate the mixture by boiling in water for 10 min and cool to obtain the enzymatic hydrolyzate.

[0094] S3. Ultrafiltration separation: The enzymatic hydrolysate obtained in S2 was centrifuged (8000 rpm, 25°C, for 15 min), the supernatant was taken, concentrated, and ethanol was added to make the volume concentration of ethanol 80% after the enzymatic hydrolysate was added. After precipitation for 72 h, the supernatant was taken, retained by a 3 kDa ultrafiltration membrane, and the permeate was collected, concentrated, and dried to obtain the crude sea cucumber sand mouth peptide.

[0095] S4. Gel filtration chromatography separation: The same as Example 1 was used to obtain sea cucumber spit active peptides containing oligopeptide FPF and oligopeptide WFP. After testing, the content of oligopeptide FPF in the obtained sea cucumber spit active peptides was 5.72%, and the content of oligopeptide WFP was 1.48%.

[0096] Example 5

[0097] This example provides an oligopeptide FPF having xanthine oxidase inhibitory activity.

[0098] The conventional solid phase synthesis method was used to synthesize the peptide in the amino acid sequence of alanine-proline-phenylalanine to obtain the oligopeptide FPF. It was detected by LC-MS / MS, and the obtained HPLC chromatogram was as shown below. Figure 1 As shown in the mass spectrum Figure 2 shown.

[0099] Example 6

[0100] This example provides an oligopeptide WFP having xanthine oxidase inhibitory activity.

[0101] The conventional solid phase synthesis method was used to synthesize the peptide in the amino acid sequence of tryptophan-phenylalanine-proline to obtain the oligopeptide WFP. It was detected by LC-MS / MS, and the obtained HPLC chromatogram was as shown in FIG. Figure 3 As shown in the mass spectrum Figure 4 shown.

[0102] Example 7

[0103] This embodiment provides a sea cucumber sand spit active peptide, which is a mixed peptide prepared by compounding the oligopeptide FPF and oligopeptide WFP of Examples 4 and 5 at a molar ratio of 1:1.

[0104] Comparative Example 1

[0105] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0106] The preparation method is basically the same as that of Example 1, except that papain (enzyme dosage is 16000 U / g protein) is used alone for enzymatic hydrolysis.

[0107] Comparative Example 2

[0108] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0109] The preparation method is basically the same as that of Example 1, except that flavor protease (enzyme addition amount is 16000 U / g protein) is used alone for enzymatic hydrolysis.

[0110] Comparative Example 3

[0111] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0112] The preparation method is basically the same as that of Example 2, except that papain (enzyme dosage is 12000 U / g protein) is used alone for enzymatic hydrolysis.

[0113] Comparative Example 4

[0114] This embodiment provides a sea cucumber sand mouth active peptide and a preparation method thereof.

[0115] The preparation method is basically the same as that of Example 2, except that flavor protease (enzyme addition amount is 12000 U / g protein) is used alone for enzymatic hydrolysis.

[0116] Comparative Example 5

[0117] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0118] The preparation method is basically the same as that of Example 3, except that papain (enzyme dosage is 20,000 U / g protein) is used alone for enzymatic hydrolysis.

[0119] Comparative Example 6

[0120] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0121] The preparation method is basically the same as that of Example 3, except that flavor protease (enzyme addition amount is 20000 U / g protein) is used alone for enzymatic hydrolysis.

[0122] Comparative Example 7

[0123] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0124] This preparation method is basically the same as Example 4, except that no subsequent operation is performed after sugar removal, that is, no subsequent operation is performed after S3 reaches "taking the supernatant after 72 hours of precipitation".

[0125] Comparative Example 8

[0126] This comparative example provides a sea cucumber sand spit active peptide and a preparation method thereof.

[0127] The preparation method is basically the same as that in Example 1, except that no subsequent operation is performed after ultrafiltration, that is, no subsequent operation is performed after S3 is completed.

[0128] Test Example 1

[0129] The sea cucumber sand mouth active peptides prepared in Examples 1 to 4 and Comparative Examples 1 to 8, the oligopeptide FPF and oligopeptide WFP prepared in Examples 5 to 7, and the mixed peptides thereof were subjected to in vitro xanthine oxidase inhibitory activity assay.

[0130] Xanthine and xanthine oxidase were dissolved in phosphate buffer at pH 7.4, respectively, to prepare xanthine substrate solution with a final concentration of 0.48 mmol / L and xanthine oxidase solution with a final concentration of 0.02 U / mL. At the same time, the active peptides from sea cucumber sand mouth, oligopeptide FPF, oligopeptide WFP, and mixed peptides of FPF and WFP to be tested were dissolved in phosphate buffer at pH 7.4 and a concentration of 0.05 mol / L to obtain the sample solutions to be tested. 50 μL of sea cucumber gizzard active peptide solution and 50 μL of xanthine oxidase solution were added to a 96-well plate, shaken and mixed, and pre-incubated at 37°C for 30 min to achieve sufficient binding of sea cucumber gizzard active peptide, oligopeptide FPF, oligopeptide WFP and mixed peptides with the enzyme; then 150 μL of xanthine substrate solution was added to initiate the enzymatic reaction, and the incubation was continued at 37°C for 5 min. Finally, 80 μL of 1 mol / L hydrochloric acid solution was added to terminate the reaction system. Phosphate buffer was used as the blank control, and the absorbance value was measured at a wavelength of 290 nm using a UV-visible spectrophotometer. The xanthine oxidase inhibition rate was calculated by comparing the absorbance changes between the experimental group and the blank control group.

[0131] The results of in vitro xanthine oxidase inhibitory activity assay of oligopeptide FPF are as follows Figure 5 As shown in Figure 2, oligopeptide FPF has good xanthine oxidase inhibitory activity, and the activity increases in a dose-dependent manner. The IC 50 It is 1.56±0.1 mg / mL.

[0132] The results of in vitro xanthine oxidase inhibitory activity assay of oligopeptide WFP are as follows: Figure 6 As shown in Figure 2, oligopeptide WFP also has good xanthine oxidase inhibitory activity, and the activity increases in a dose-dependent manner. The IC 50 It is 0.37±0.025 mg / mL.

[0133] The results of in vitro xanthine oxidase inhibitory activity assay of the mixed peptides of FPF and WFP are as follows: Figure 7 As shown in Figure 2, the mixed peptide also has good xanthine oxidase inhibitory activity, and the activity increases in a dose-dependent manner. The IC 50 It is 0.49±0.04mg / mL.

[0134] The results of in vitro xanthine oxidase inhibition activity assays for the sea cucumber spit active peptides prepared in Examples 1 to 4 are shown in Table 1, and the results of in vitro xanthine oxidase inhibition activity assays for the sea cucumber spit active peptides prepared in Comparative Examples 1 to 8 are shown in Table 2. A comparison of the preparation methods and assay results between the different Examples and Comparative Examples is shown in Tables 3 to 6.

[0135] Table 1 Results of in vitro xanthine oxidase inhibition activity assay of sea cucumber sand spit active peptides obtained in Examples 1 to 4

[0136]

[0137] Table 2 Results of in vitro xanthine oxidase inhibition activity determination of sea cucumber sand mouth active peptides obtained in comparative examples 1 to 8

[0138]

[0139] Table 3 Preparation methods and test results of Example 1 and Comparative Examples 1 and 2

[0140]

[0141] Table 4 Preparation methods and test results of Example 2 and Comparative Examples 3 and 4

[0142]

[0143] Table 5 Preparation methods and test results of Example 3 and Comparative Examples 5 and 6

[0144]

[0145] It can be seen from Tables 3 to 5 that under the same enzymatic hydrolysis parameter conditions as in the example, when a single enzyme is used to enzymatically hydrolyze the sea cucumber gizzard, the xanthine oxidase inhibition rate of the sea cucumber gizzard active peptide is low. When a composite enzyme of papain and flavor protease is used for hydrolysis, the inhibition rate of the obtained sea cucumber gizzard active peptide on xanthine oxidase is better than that when treated with a single protease, indicating that papain and flavor protease have a synergistic effect on the enzymatic hydrolysis effect of sea cucumber gizzard, and can enable the sea cucumber gizzard active peptide obtained by enzymatic hydrolysis to contain more polypeptides that have an inhibitory effect on xanthine oxidase.

[0146] Table 6 Preparation methods and test results of Example 4 and Comparative Examples 7 and 8

[0147]

[0148] It can be seen from the results in Table 6 that, compared with no purification method (Comparative Example 7) and the use of ultrafiltration alone as a purification method (Comparative Example 8), when ultrafiltration and gel chromatography separation are used in conjunction, the inhibition rate of the obtained sea cucumber sand spit active peptide on xanthine oxidase is significantly improved, indicating that the sea cucumber sand spit active peptide obtained after ultrafiltration and gel chromatography separation according to the present invention contains more polypeptides that have an inhibitory effect on xanthine oxidase.

[0149] Test Example 2

[0150] In this test example, the structures and action sites of oligopeptides FPF and WFP prepared in Examples 5 and 6 were analyzed.

[0151] To analyze the molecular interaction mechanism between the active ingredient and the target protein, this experiment used the AutoDock Vina platform based on a semi-flexible docking strategy for molecular docking analysis. The specific process is as follows:

[0152] (1) Target protein pretreatment:

[0153] The target protein crystal structure (resolution: 2.0 Å) was obtained from the RCSB Protein Data Bank (PDB ID: 2E1Q), retaining the original ligand binding site water molecules and cofactors. The structure was optimized using AutoDock Tools 1.5.7: polar hydrogen atoms were filled, Kollman joint atomic charges were assigned, AD4 atom types were defined, and a docking grid box (size: 20 × 20 × 20 Å) was constructed centered on the active pocket. 3 , lattice spacing 0.375 Å).

[0154] (2) Ligand molecular modeling:

[0155] The three-dimensional conformation of the target peptide was optimized using Avogadro 1.2.0 (MMFF94 force field), and the local charge distribution was calculated using the AMBER GAFF force field. The AutoDock Tools were used to identify rotatable bonds in the ligand (based on topological analysis), and a PDBQT file containing flexible torsional degrees of freedom was generated.

[0156] (3) Docking parameters and execution:

[0157] The Vina search space was set to cover the entire active pocket, with a defined energy range (energy_range = 4) and a maximum number of output conformations (num_modes = 20). The receptor protein rigidity was fixed, allowing conformational sampling of the ligand's flexible side chains and key functional groups. The Lamarckian genetic algorithm was used to optimize the binding pose.

[0158] (4) Combining pattern analysis and visualization:

[0159] Based on binding free energy ΔG, kcal·mol -1 Optimal binding conformations were screened, with a scoring formula integrating van der Waals forces, hydrogen bonding, hydrophobic interactions, and desolvation effects. Three-dimensional interaction maps were constructed using PyMOL 2.5.2 to analyze the hydrogen bonding network, π-π stacking, and electrostatic complementarity between the ligand and receptor. Two-dimensional LigPlot+ plots were generated using Discovery Studio 2021 to quantitatively analyze the contributions of bonding residues.

[0160] The results showed that the docking binding energy of oligopeptide FPF was -10.9 kcal / mol, and the docking binding energy of oligopeptide WFP was -10.8 kcal / mol.

[0161] like Figure 8 and Figure 9 As shown, oligopeptide FPF forms five hydrogen bonds with Glu1262, Gln768, Phe912, and Ser1081 in the active center of xanthine oxidase, resulting in eight hydrophobic interactions. Oligopeptide WFP forms two hydrogen bonds with Gln768 and Gly1261 in the active center of xanthine oxidase, resulting in 12 hydrophobic interactions. The number and length of hydrogen bonds and the hydrophobic interactions indicate that both oligopeptides FPF and WFP interact significantly with xanthine oxidase and can effectively inhibit enzyme activity.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of a sea cucumber spit active peptide having xanthine oxidase inhibitory activity in the preparation of a uric acid-lowering drug, characterized in that: The sea cucumber sand mouth active peptide contains oligopeptide WFP, and the amino acid sequence of the oligopeptide WFP is Trp-Phe-Pro.

2. The use according to claim 1, characterized in that The sea cucumber sand mouth active peptide further contains oligopeptide FPF, and the amino acid sequence of the oligopeptide FPF is: Phe-Pro-Phe.

3. The use according to claim 1, characterized in that The uric acid-lowering drug is a xanthine oxidase inhibitor; and / or The uric acid-lowering drug is an oral preparation; and / or The ingredients of the uric acid-lowering drug also include pharmaceutically acceptable excipients.

4. The method for preparing the sea cucumber sand mouth active peptide according to claim 1 or 2, wherein: The specific steps include: S1. Demineralize the sea cucumber spit, dry the demineralized material at 40-45°C to a moisture content of ≤0.15% w / w, and grind it to a particle size distribution of D90 ≤0.15 mm to obtain a demineralized sea cucumber spit powder; the demineralization method is: immerse the sea cucumber spit in a citric acid aqueous solution with a mass concentration of 4%-8%, apply an ultrasonic frequency of 20-40 kHz at a temperature range of 45-60°C and synchronously mechanically stir, separate the solid and liquid after 30-45 minutes and recover the solid material, the mass volume ratio of the sea cucumber spit to the citric acid aqueous solution is 1: (20-25), g: mL; repeat the demineralization steps until the liquid phase of the solid-liquid separation is colorless and transparent and no precipitate is precipitated after standing, and the inorganic residue on the surface of the solid phase is confirmed to be completely removed by visual inspection; S2, mixing the sea cucumber sand spit demineralized powder obtained in S1 with water at a material-liquid ratio of 1: (15-30), g: mL, and enzymolyzing with papain and flavor protease for 1.5-3 h; after the enzymolysis is completed, inactivating the enzymes and cooling to obtain an enzymatic solution; the amount of papain added is 8000-12000 U / g protein, and the amount of flavor protease added is 4000-8000 U / g protein, where the protein refers to the mass of the protein in the sea cucumber sand spit defatted powder; the enzymolysis temperature is 50±2° C., and the pH is 7.5±0.3; S3, centrifuging the enzymatic hydrolyzate obtained in S2, taking the supernatant, concentrating and adding ethanol to make the ethanol volume concentration be 70% to 90%, taking the supernatant after precipitation for 60 to 72 hours, intercepting through a 3 kDa ultrafiltration membrane, collecting the permeate, concentrating and drying to obtain the crude sea cucumber sand mouth peptide; S4. Dissolve the crude sea cucumber gizzard peptide obtained in S3 in ultrapure water, separate and purify it by gel chromatography, collect the effluent with a molecular weight of less than 1 kDa, and dry it to obtain a sea cucumber gizzard oligopeptide containing the sea cucumber gizzard active peptide; the chromatographic column of the gel chromatography is TK-Col 16 / 60 GF30.

5. The preparation method according to claim 4, characterized in that The material-liquid ratio in S2 is 1:(20-25), g:mL; and / or The enzymatic hydrolysis time in S2 is 1.8 to 2.5 hours; and / or The centrifugation parameters in S3 are: rotation speed 8000-10000 rpm, centrifugation time 15-20 min, temperature 23-27°C; and / or After adding ethanol to S3, the volume concentration of ethanol is 75% to 85%.

6. The preparation method according to claim 5, characterized in that The amount of papain added in S2 is 9000-11000 U / g protein; and / or The amount of flavor protease added in S2 is 5000-7000 U / g protein; and / or The material-liquid ratio in S2 is 1:23, g:mL; and / or The enzymatic hydrolysis time in S2 is 2 h; and / or The centrifugation parameters in S3 are: rotation speed 8000 rpm, centrifugation time 15 min, temperature 25°C; and / or The volume concentration of ethanol after adding ethanol in S3 is 80%.

7. The preparation method according to any one of claims 4 to 6, characterized in that The UV detector detection wavelength of the gel chromatography in S4 is 220 nm, and the equilibration buffer is phosphate buffer.

8. The preparation method according to any one of claims 4 to 6, characterized in that The preparation method further comprises: S5, separating and purifying the obtained components by using preparative reverse-phase high performance liquid chromatography, collecting the effluent according to the peak time of oligopeptide FPF and oligopeptide WFP, and obtaining the purified sea cucumber sand mouth active peptide after drying.

9. The preparation method according to claim 8, characterized in that The chromatographic conditions of the preparative reversed-phase high-performance liquid chromatography are as follows: the chromatographic column is an Agilent Prep C18 with specifications of 100 Å, 2 µm, 75 µm inner diameter × 150 mm length, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, and the elution gradient is set as follows: the mobile phase B is linearly increased from 5% to 10% from 0 to 8 min, the mobile phase B is linearly increased from 10% to 15% from 8 to 33 min, the mobile phase B is linearly increased from 15% to 28% from 33 to 43 min, the mobile phase B is linearly increased from 28% to 40% from 43 to 50 min, the mobile phase B is linearly increased from 40% to 95% from 50 to 60 min and maintained until 65 min, and the mobile phase B is restored to the initial proportion of 5% from 66 to 70 min, the flow rate is constant at 250 nL / min, the column temperature is 40°C, and the detection wavelength is 220 nm; the effluent from 8 to 12 min is collected.

10. Use of the oligopeptide FPF, the sea cucumber sand spit active peptide according to claim 1 or 2, or the sea cucumber sand spit active peptide prepared by the preparation method according to any one of claims 4 to 9 in the preparation of uric acid-lowering drugs.

11. The use according to claim 10, characterized in that The uric acid-lowering drug is a xanthine oxidase inhibitor; and / or The uric acid-lowering drug is an oral preparation; and / or The ingredients of the uric acid-lowering drug also include pharmaceutically acceptable excipients.

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