Deep-sea longevity fish precise active peptide with uric acid reducing and anti-inflammatory effects as well as preparation method and application of deep-sea longevity fish precise active peptide

Abstract

The invention relates to the technical field of preparation of deep-sea longevity fish precise active peptides, in particular to a deep-sea longevity fish precise active peptide with uric acid reducing and anti-inflammatory effects as well as a preparation method and application of the deep-sea longevity fish precise active peptide. The preparation method comprises the following steps: carrying out medium-temperature pretreatment, carrying out step-by-step enzymolysis with alkaline protease and acid protease, centrifuging, filtering, carrying out ultrafiltration interception and the like, and screening out 10 non-reported non-toxic active peptide fragments. The active peptide has a multi-target synergistic effect, inhibits the activity of xanthine oxidase, regulates and controls the expression of uric acid resorption protein, regulates and controls the expression of uric acid transporter protein, realizes bidirectional regulation of uric acid metabolism, efficiently removes hydroxyl radicals, activates superoxide dismutase and reduces the content of inflammation-related factors. The technology is simple and easy to control, low in equipment requirement and suitable for large-scale production, the active ingredients are natural, safety is high, the active ingredients can serve as functional factors to be applied to functional food, health care products and the like, and safe and diversified intervention schemes are provided for hyperuricemia and related inflammation problems.

Description

Technical Field

[0001] This invention relates to the field of precision active peptide preparation technology of deep-sea long-lived fish, and particularly to a precision active peptide of deep-sea long-lived fish with uric acid-lowering and anti-inflammatory effects, its preparation method and application. Background Technology

[0002] With rapid socio-economic development, residents' dietary structure has shown a significant upgrading trend, with a substantial increase in the intake of high-purine foods, such as seafood, directly driving a continuous rise in the prevalence of hyperuricemia and related metabolic diseases. Hyperuricemia, as a core pathogenic factor in gouty arthritis, not only causes symptoms such as joint deformities and pain, but also has a close pathological link with many major diseases such as hypertension, coronary heart disease, and chronic kidney disease. It has now become the second leading metabolic disease globally after diabetes, posing a serious challenge to public health and safety. Therefore, the development of efficient and safe uric acid-lowering intervention products is urgently needed.

[0003] Deep-sea long-lived fish (scientific name: Atlantic spiny seabream) Hoplostethus atlanticus As a precious deep-sea fish resource, it is rich in high-quality protein, unsaturated fatty acids, nucleotide derivatives, vitamins, and various minerals. Of particular note is the small-molecule active peptide extracted from the long-lived fish using bio-enzymatic hydrolysis technology, which has been proven to have significant dual functions of lowering uric acid and anti-inflammation. Its mechanism of action is specifically manifested in a bidirectional regulatory effect: on the one hand, this precise active peptide can competitively inhibit the activity of XO, blocking the biosynthesis pathway of uric acid at its source; on the other hand, it can target and regulate the expression level of key uric acid transport proteins such as URAT1 / GLUT9 in kidney tissue, inhibiting the reabsorption of uric acid by the renal tubules and promoting the active secretion of uric acid, ultimately achieving purine metabolism balance in the body through urinary excretion, effectively reducing the concentration of uric acid in the body, and having a clear preventive and alleviating effect on hyperuricemia and its complications such as gout.

[0004] However, the current market for uric acid-lowering and anti-inflammatory products generally suffers from many technical defects, including: (1) limited sources of active ingredients, which mostly rely on chemical synthesis or scarce animal and plant resources, making it difficult to meet the needs of large-scale production; (2) high preparation costs, with existing extraction or synthesis processes being complex, resulting in high product prices and restricting popular application; (3) prominent safety risks, with some chemically synthesized products having obvious side effects, and long-term use may cause potential damage to liver and kidney function; (4) limited single function, with most products only targeting a single link of uric acid generation or excretion, failing to achieve systemic regulation of purine metabolism, and having limited overall intervention effect.

[0005] It is evident that developing a preparation process for the uric acid-lowering and anti-inflammatory active ingredients of deep-sea long-lived fish, and overcoming the technical bottlenecks of existing products such as limited sources, high costs, numerous side effects, or single functions, is a technical problem that urgently needs to be solved, and has broad application prospects and technical benefits. Summary of the Invention

[0006] To overcome the technical bottlenecks of existing products, such as limited sources, high costs, numerous side effects, or limited functionality, this invention provides a method for preparing precise active peptides from deep-sea long-lived fish, comprising the following steps: S1. Pretreatment: Remove the gills and internal organs of fresh deep-sea long-lived fish, mince it into fish paste, add water and mix, and heat treat at 54℃~56℃ for 0.5h~1.5h to obtain pre-hydrolysate. S2. First hydrolysis: Adjust the pH of the pre-hydrolysate to 8.0~9.0, add alkaline protease, and hydrolyze at 50℃~60℃ for 1h~3h. After inactivating the enzyme, the first hydrolysate is obtained. S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 2.5~3.5, add acidic protease, and hydrolyze at 45℃~65℃ for 1.5h~2.5h. After enzyme inactivation, the second hydrolysate is obtained. S4. Post-processing: The second hydrolysate is centrifuged, decolorized and deodorized with activated carbon, filtered with diatomaceous earth, ultrafiltered, nanofiltered, desalinated and concentrated, and spray-dried to obtain deep-sea long-lived fish precise active peptide powder. Among them, the molecular weight cutoff of ultrafiltration is <5000Da; the mass content of the concentrated deep-sea long-lived fish precise active peptides is 10%~15%.

[0007] In some embodiments, in step S1, the minced fish paste is mixed with water at a mass ratio of 1:(2~4); in step S2, the amount of alkaline protease added is 0.3%~0.7% of the mass of the fish paste; in step S3, the amount of acidic protease added is 0.5%~1.5% of the mass of the fish paste.

[0008] In some embodiments, the alkaline protease activity is 0.73 million U / g to 2.55 million U / g, and the acidic protease activity is 0.68 million U / g to 2.05 million U / g.

[0009] In some embodiments, in steps S2 and S3, the enzyme inactivation specifically involves inactivating the enzyme at 84°C to 86°C for 15 to 20 minutes.

[0010] In some embodiments, in step S4, the activated carbon decolorization and deodorization specifically involves: centrifuging the second hydrolysate to obtain the supernatant, heating it to 50°C~60°C, adding activated carbon for treatment for 0.5h~1.5h, centrifuging again to obtain the supernatant, and passing it through a diatomaceous earth and 0.45nm aqueous membrane; wherein the amount of activated carbon added is 3.0%~5.0% of the mass of fish foam.

[0011] In some embodiments, the centrifugation specifically refers to centrifuging at 5000 rpm for 5 min to 10 min.

[0012] The present invention also provides a precise active peptide from deep-sea long-lived fish, which is prepared according to the preparation method of the precise active peptide from deep-sea long-lived fish described above.

[0013] The present invention also provides a functional product, characterized in that its components include the deep-sea long-lived fish precise active peptides as described above.

[0014] Furthermore, the functional product includes at least one of the following functions: (1) Antioxidant; (2) Inhibits xanthine oxidase activity; (3) Inhibit the secretion of inflammatory factors; (4) Inhibits uric acid synthesis and reabsorption; (5) Promotes uric acid excretion.

[0015] The present invention also provides the application of the deep-sea long-lived fish precise active peptide composition as described above in the preparation of products for significantly scavenging hydroxyl free radicals, activating SOD activity, inhibiting xanthine oxidase activity in vitro, reducing ROS content in HK-2 cells, reducing uric acid content in HK-2 cells, reducing IL-1β content in HK-2 cells, reducing NO content in HK-2 cells, inhibiting the mRNA expression of XO, URAT1, and TNF-α, and promoting the mRNA expression of ABCG2.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention uses deep-sea long-lived fish as raw material and prepares precise active peptides from them through a specific process. Ten previously unreported, non-toxic, and precisely active peptides were screened. These active peptides can competitively inhibit XO activity with an inhibition rate exceeding 89%, blocking the uric acid production pathway at its source. They can also precisely regulate the expression of renal uric acid transport proteins, inhibiting URAT1-mediated uric acid reabsorption and promoting ABCG2-mediated uric acid excretion, achieving bidirectional regulation of uric acid metabolism. Simultaneously, these active peptides can efficiently scavenge hydroxyl radicals with a clearance rate of no less than 95% and activate superoxide dismutase activity at levels exceeding 580 U / mL. By reducing the levels of reactive oxygen species, interleukin-1β, and nitric oxide in HK-2 cells and inhibiting tumor necrosis factor mRNA expression, they break the vicious cycle of "high uric acid-oxidative stress-inflammation," truly achieving a synergistic effect of lowering uric acid and reducing inflammation. This addresses the industry pain point of existing products that only target a single link in uric acid metabolism and have limited overall intervention effects.

[0017] Compared to existing technologies that suffer from complex active ingredient extraction processes, high equipment dependence, and high production costs, this invention optimizes an integrated preparation process of "medium-temperature pretreatment - stepwise enzymatic hydrolysis - complex separation and purification." The medium-temperature pretreatment stage precisely controls temperature and time, laying the foundation for subsequent enzymatic hydrolysis. The stepwise enzymatic hydrolysis employs a combination of alkaline and acidic proteases, maximizing the release of target active peptides by controlling pH, temperature, and enzyme dosage. The separation and purification stage uses ultrafiltration to retain active components with a molecular weight less than 5000 Da, combined with activated carbon decolorization and deodorization, improving both product purity and sensory quality. The entire process requires no complex, high-end equipment, is simple and controllable, achieves high active peptide extraction efficiency, and maximizes raw material utilization, meeting the needs of large-scale industrial production. It significantly reduces manufacturing costs and effectively solves the practical problem of existing technologies being difficult to industrialize.

[0018] Addressing the issues of existing uric acid-lowering products relying heavily on chemical synthesis and prone to liver and kidney damage with long-term use, this invention's active ingredient is derived from natural deep-sea long-lived fish. Toxicity prediction and cell experiments have verified that it causes no significant damage to HK-2 cells, exhibiting excellent biocompatibility and higher safety. Furthermore, this precisely targeted active peptide can be widely integrated as a functional factor into various product developments such as functional foods and health supplements. It is suitable for multiple dosage forms, including powders, tablets, and liquids, meeting the needs of different consumer scenarios. This overcomes the limitations of existing products' application scope, providing a safer and more diverse intervention solution for people with high uric acid and related inflammatory issues, further expanding the market application space for uric acid-lowering and anti-inflammatory functional products. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is an analysis of the test results of HK-2 cell viability using different processes and concentrations of the precise active peptides from deep-sea long-lived fish according to the present invention. Figure 2 This is an analysis of the test results of the precise active peptides from deep-sea long-lived fish of the present invention on the UA content in UA-induced HK-2 cells; Figure 3 This is an analysis of the test results of the precise active peptides from deep-sea long-lived fish of the present invention on the ROS content in UA-induced HK-2 cells; Figure 4This is an analysis of the test results of the precise active peptides from deep-sea long-lived fish of the present invention on the IL-1β content in UA-induced HK-2 cells; Figure 5 This is an analysis of the test results of the precise active peptides from deep-sea long-lived fish of the present invention on the NO content in UA-induced HK-2 cells; Figure 6 This is an analysis of the test results of the precise active peptides from deep-sea long-lived fish of the present invention on the expression level of XO mRNA in UA-induced HK-2 cells; Figure 7 This is a graph showing the analysis results of the test on the mRNA expression level of URAT1 in HK-2 cells induced by the precise active peptide of deep-sea long-lived fish of this invention. Figure 8 This is an analysis of the test results of the deep-sea long-lived fish precise active peptide of the present invention on the mRNA expression level of ABCG2 in UA-induced HK-2 cells; Figure 9 This is a graph showing the analysis results of the test on the mRNA expression level of TNF-α in UA-induced HK-2 cells by the precise active peptide of deep-sea long-lived fish of this invention; Figure 10 This is a 3D diagram showing the docking results of the LLL peptide of this invention with the XO receptor protein. Figure 11 This is a 3D diagram showing the docking results of the LLL peptide of this invention with the URAT1 receptor protein. Figure 12 This is a 3D diagram showing the docking results of the LLL peptide of this invention with the ABCG2 receptor protein. Figure 13 This is a 3D diagram showing the docking results of the KLF peptide of this invention with the XO receptor protein. Figure 14 This is a 3D diagram showing the docking results of the KLF peptide of this invention with the URAT1 receptor protein. Figure 15 This is a 3D diagram showing the docking results of the KLF peptide of this invention with the ABCG2 receptor protein. Figure 16 This is a 3D diagram showing the docking results of the peptide ILPL of this invention with the XO receptor protein. Figure 17 This is a 3D diagram showing the docking results of the peptide ILPL of this invention with the URAT1 receptor protein. Figure 18 This is a 3D diagram showing the docking results of the peptide ILPL of this invention with the ABCG2 receptor protein. Figure 19 This is a 3D diagram showing the docking results of the LL peptide of this invention with the XO receptor protein. Figure 20 This is a 3D diagram showing the docking results of the LL peptide of this invention with the URAT1 receptor protein. Figure 21 This is a 3D diagram showing the docking results of the LL peptide of this invention with the ABCG2 receptor protein. Figure 22 This is a 3D diagram showing the docking results of the peptide GIF of this invention with the XO receptor protein; Figure 23 This is a 3D diagram showing the docking results of the peptide GIF of this invention with the URAT1 receptor protein. Figure 24 This is a 3D diagram showing the docking results of the peptide GIF of this invention with the ABCG2 receptor protein; Figure 25 This is a 3D diagram showing the docking results of the LPL peptide of this invention with the XO receptor protein. Figure 26 This is a 3D diagram showing the docking results of the LPL peptide of this invention with the URAT1 receptor protein. Figure 27 This is a 3D diagram showing the docking results of the LPL peptide of this invention with the ABCG2 receptor protein. Figure 28 This is a 3D diagram showing the docking results of the LALPP peptide of this invention with the XO receptor protein. Figure 29 This is a 3D diagram showing the docking results of the LALPP peptide of this invention with the URAT1 receptor protein. Figure 30 This is a 3D diagram showing the docking results of the LALPP peptide of this invention with the ABCG2 receptor protein. Figure 31 This is a 3D diagram showing the docking results of the LLM peptide of this invention with the XO receptor protein. Figure 32 This is a 3D diagram showing the docking results of the LLM peptide of this invention with the URAT1 receptor protein. Figure 33 This is a 3D diagram showing the docking results of the LLM peptide of this invention with the ABCG2 receptor protein. Figure 34 This is a 3D diagram showing the docking results of the LGGVL peptide of this invention with the XO receptor protein. Figure 35 This is a 3D diagram showing the docking results of the LGGVL peptide of this invention with the URAT1 receptor protein. Figure 36 This is a 3D diagram showing the docking results of the LGGVL peptide of this invention with the ABCG2 receptor protein. Figure 37 This is a 3D diagram showing the docking results of the FSL peptide of this invention with the XO receptor protein. Figure 38 This is a 3D diagram showing the docking results of the FSL peptide of this invention with the URAT1 receptor protein. Figure 39This is a 3D diagram showing the docking results of the FSL peptide of this invention with the ABCG2 receptor protein. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and the technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention provides the following Examples 1-3 for preparing a precise active peptide from deep-sea long-lived fish with uric acid-lowering and anti-inflammatory effects. Specific steps are illustrated below: S1. Pretreatment: After removing the gills and internal organs of fresh deep-sea long-lived fish, mince it into fish paste. Mix the fish paste with water at a mass ratio of 1:(2~4) and heat-treat at 54℃~56℃ for 0.5h~1.5h to obtain pre-hydrolyzed solution. S2. First hydrolysis: Adjust the pH of the pre-hydrolysate to 8.0~9.0, add 0.3%~0.7% alkaline protease by weight of fish foam, hydrolyze at 50℃~60℃ for 1h~3h, and inactivate the enzyme at 84℃~86℃ for 15min~20min to obtain the first hydrolysate; S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 2.5~3.5, add 0.5%~1.5% of the fish foam mass of acidic protease, hydrolyze at 45℃~65℃ for 1.5h~2.5h, and inactivate the enzyme at 84℃~86℃ for 15min~20min to obtain the second hydrolysate; S4. Post-treatment: Centrifuge the second hydrolysate at 5000 rpm for 5 min to 10 min, take the supernatant and cool it to 50℃ to 60℃, add 3.0% to 5.0% of the fish foam mass of activated carbon and treat for 0.5 h to 1.5 h, centrifuge at 5000 rpm for 5 min to 10 min, take the supernatant and pass it through diatomaceous earth and a 0.45 nm aqueous membrane, then perform ultrafiltration to retain the deep-sea long-lived fish precise active peptide solution with a molecular weight cutoff of <5000 Da, then desalinate and concentrate it with a nanofiltration machine to obtain a deep-sea long-lived fish precise active peptide concentrate with a mass content of 10% to 15%, and then spray dry it to make powder, which is the deep-sea long-lived fish precise active peptide powder.

[0023] Example 1 S1. Pretreatment: Remove the gills and internal organs of fresh deep-sea long-lived fish and mince it into fish paste. Take 500g of fish paste and mix it evenly with 1500g of water. Heat it at 55℃ for 1 hour to obtain a pre-hydrolyzed solution. S2, First hydrolysis: Adjust the pH of the pre-hydrolysate to 8.5, add 2.5g of alkaline protease, hydrolyze at 55℃ for 2h, and inactivate the enzyme at 85℃ for 15min to obtain the first hydrolysate; S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 3.0, add 5.0g of acidic protease, hydrolyze at 55℃ for 2h, and inactivate the enzyme at 85℃ for 15min to obtain the second hydrolysate; S4. Post-processing: Centrifuge the second hydrolysate at 5000 rpm for 5 min, take the supernatant, cool it to 55℃, add 20.0 g of activated carbon and treat for 1 h, centrifuge at 5000 rpm for 5 min, take the supernatant and pass it through diatomaceous earth and a 0.45 nm aqueous membrane, then perform ultrafiltration to retain the deep-sea long-lived fish precise active peptide solution with a molecular weight cutoff of <5000 Da, then desalinate and concentrate it through a nanofiltration machine to obtain a deep-sea long-lived fish precise active peptide concentrate with a mass content of 12.5%, and then spray dry it to make powder, thus obtaining deep-sea long-lived fish precise active peptide powder.

[0024] Example 2 S1. Pretreatment: Remove the gills and internal organs of fresh deep-sea long-lived fish and mince it into fish paste. Take 500g of fish paste and mix it evenly with 1000g of water. Heat it at 54℃ for 0.5h to obtain a pre-hydrolyzed solution. S2, First hydrolysis: Adjust the pH of the pre-hydrolysate to 8.0, add 1.5g of alkaline protease, hydrolyze at 50℃ for 1h, and inactivate the enzyme at 84℃ for 15min to obtain the first hydrolysate; S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 2.5, add 2.5g of acidic protease, hydrolyze at 45℃ for 1.5h, and inactivate the enzyme at 84℃ for 15min to obtain the second hydrolysate. S4. Post-processing: Centrifuge the second hydrolysate at 5000 rpm for 5 min, take the supernatant, cool it to 55℃, add 15.0 g of activated carbon and treat for 0.5 h, centrifuge at 5000 rpm for 5 min, take the supernatant and pass it through diatomaceous earth and a 0.45 nm aqueous membrane, then perform ultrafiltration to retain the deep-sea long-lived fish precise active peptide solution with a molecular weight cutoff of <5000 Da, then desalinate and concentrate it through a nanofiltration machine to obtain a deep-sea long-lived fish precise active peptide concentrate with a mass content of 10%, and then spray dry it to make powder, thus obtaining deep-sea long-lived fish precise active peptide powder.

[0025] Example 3 S1. Pretreatment: Remove the gills and internal organs of fresh deep-sea long-lived fish and mince it into fish paste. Take 500g of fish paste and mix it evenly with 2000g of water. Heat it at 56℃ for 1.5h to obtain a pre-hydrolyzed solution. S2, First hydrolysis: Adjust the pH of the pre-hydrolysate to 9.0, add 3.5g of alkaline protease, hydrolyze at 60℃ for 3h, and inactivate the enzyme at 86℃ for 20min to obtain the first hydrolysate; S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 3.5, add 7.5g of acidic protease, hydrolyze at 65℃ for 2.5h, and inactivate the enzyme at 86℃ for 20min to obtain the second hydrolysate; S4. Post-processing: Centrifuge the second hydrolysate at 5000 rpm for 5 min, take the supernatant, cool it to 55℃, add 25.0 g of activated carbon and treat for 0.5 h, centrifuge at 5000 rpm for 5 min, take the supernatant and pass it through diatomaceous earth and a 0.45 nm aqueous membrane, then perform ultrafiltration to retain the deep-sea long-lived fish precise active peptide solution with a molecular weight cutoff of <5000 Da, then desalinate and concentrate it by nanofiltration to obtain a deep-sea long-lived fish precise active peptide concentrate with a mass content of 15%, and then spray dry it to make powder, thus obtaining deep-sea long-lived fish precise active peptide powder.

[0026] The present invention also provides the following comparative examples 1 to 8: Comparative Example 1 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S3, 5.0g of acidic protease was replaced with 9.4g of neutral protease, and the pH was adjusted to 7.0. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0027] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and neutral protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0028] In Example 1, the amount of alkaline protease added was 2.5g, with an enzyme activity of 400,000 U / g. Converted to per unit weight of deep-sea long-lived fish protein, this corresponds to an enzyme activity of 400,000 U / g × 2.5g / 55g = 18,200 U / g. Similarly, the amount of acidic protease added was 5.0g, with an enzyme activity of 150,000 U / g. Converted to per unit weight of deep-sea long-lived fish protein, this corresponds to an enzyme activity of 150,000 U / g × 5.0g / 55g = 13,600 U / g. Therefore, in Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline and acidic protease used was 31,800 U / g.

[0029] In Comparative Example 1, the amount of alkaline protease added was 2.5g. Based on the protein content of deep-sea long-lived fish, the amount of alkaline protease used was 400,000 U / g × 2.5g / 55g = 1.82 U / g. The amount of neutral protease added was 9.4g, with an enzyme activity of 80,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of neutral protease used was 80,000 U / g × 9.4g / 55g = 1.36 U / g. Therefore, in Comparative Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and neutral protease used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0030] Comparative Example 2 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S2, 2.5g of alkaline protease was replaced with 12.5g of neutral protease, and the pH was adjusted to 7.0; in step S3, 5.0g of acidic protease was replaced with 1.9g of papain, and the pH was adjusted to 6.5. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0031] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of neutral protease and papain used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0032] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0033] In Comparative Example 2, the amount of neutral protease added was 12.5g, with an enzyme activity of 80,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of neutral protease used was 80,000 U / g × 12.5g / 55g = 1.82 U / g. The amount of papain added was 1.9g, with an enzyme activity of 400,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of papain used was 400,000 U / g × 1.9g / 55g = 1.36 U / g. Therefore, in Comparative Example 2, based on the protein content of deep-sea long-lived fish, the total amount of neutral protease and papain used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0034] Comparative Example 3 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S2, 2.5g of alkaline protease was replaced with 10.0g of complex lipase, and the pH was adjusted to 7.0; in step S3, 5.0g of acidic protease was replaced with 2.5g of alkaline protease, and the pH was adjusted to 8.5. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0035] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of compound lipase and alkaline protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0036] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0037] In Comparative Example 3, the amount of compound lipase added was 10.0g, with an enzyme activity of 100,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of compound lipase used was 100,000 U / g × 10.0g / 55g = 1.82 U / g. The amount of alkaline protease added was 1.9g, with an enzyme activity of 400,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of alkaline protease used was 400,000 U / g × 1.9g / 55g = 1.36 U / g. Therefore, in Comparative Example 3, based on the protein content of deep-sea long-lived fish, the total amount of compound lipase and alkaline protease used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0038] Comparative Example 4 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S3, 5.0g of acidic protease was replaced with 1.9g of papain, and the pH was adjusted to 6.5. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0039] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and papain used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0040] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0041] In Comparative Example 4, the amount of alkaline protease added was 2.5g, with an enzyme activity of 400,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of alkaline protease used was 400,000 U / g × 2.5g / 55g = 1.82 U / g. The amount of papain added was 1.9g, with an enzyme activity of 400,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of papain used was 400,000 U / g × 1.9g / 55g = 1.36 U / g. Therefore, in Comparative Example 4, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and papain used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0042] Comparative Example 5 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S2, 2.5g of alkaline protease was replaced with 10.0g of complex lipase, and the pH was adjusted to 7.0. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0043] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of compound lipase and acidic protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0044] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0045] In Comparative Example 5, the amount of compound lipase added was 10.0g, with an enzyme activity of 100,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of compound lipase used was 100,000 U / g × 10.0g / 55g = 1.82 U / g; the amount of acidic protease added was 5.0g, with an enzyme activity of 150,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of acidic protease used was 150,000 U / g × 5.0g / 55g = 1.36 U / g. Therefore, in Comparative Example 5, based on the protein content of deep-sea long-lived fish, the total amount of compound lipase and alkaline protease used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0046] Comparative Example 6 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S2, 2.5g of alkaline protease was replaced with 12.5g of neutral protease, and the pH was adjusted to 7.0. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0047] It should be noted that, based on the protein content of deep-sea long-lived fish, the total amount of neutral protease and acidic protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0048] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0049] In Comparative Example 6, the amount of neutral protease added was 12.5g, with an enzyme activity of 80,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of compound lipase used was 80,000 U / g × 12.5g / 55g = 1.82 U / g. The amount of acidic protease added was 5.0g, with an enzyme activity of 150,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of acidic protease used was 150,000 U / g × 5.0g / 55g = 1.36 U / g. Therefore, in Comparative Example 6, based on the protein content of deep-sea long-lived fish, the total amount of neutral protease and acidic protease used was 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0050] Comparative Example 7 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in step S3, 5.0g of acidic protease was replaced with 1.9g of alkaline protease, and the pH was adjusted to 8.5. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0051] It should be noted that, based on the protein content of deep-sea long-lived fish, the amount of alkaline protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0052] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0053] In Comparative Example 7, the amount of alkaline protease added was 2.5g + 1.9g = 4.4g, and the enzyme activity was 400,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of alkaline protease used was 400,000 U / g × 4.4g / 55g = 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0054] Comparative Example 8 This comparative example prepared a precise active peptide from a deep-sea long-lived fish. The difference from Example 1 of this invention is that in steps S2 and S3, 2.5g of alkaline protease and 5.0g of acidic protease were replaced with 12.5g and 9.4g of neutral protease, respectively, and the pH was adjusted to 7.0. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0055] It should be noted that, based on the protein content of deep-sea long-lived fish, the amount of neutral protease used in this comparative example is equal to the total amount of alkaline protease and acidic protease used in Example 1. The specific calculation process is as follows: The amount of deep-sea long-lived fish added is 500g, with a protein content of 11%, which is 55g.

[0056] In Example 1, based on the protein content of deep-sea long-lived fish, the total amount of alkaline protease and acidic protease used was 31,800 U / g.

[0057] In Comparative Example 8, the amount of neutral protease added was 12.5g + 9.4g = 21.9g, and the enzyme activity was 80,000 U / g. Based on the protein content of deep-sea long-lived fish, the amount of neutral protease used was 80,000 U / g × 21.9g / 55g = 3.18 U / g, which is the same as the total amount of alkaline protease and acidic protease used in Example 1.

[0058] Based on the above embodiments and comparative examples, the present invention also conducted the following experiments: Experiment 1: Determination of in vitro antioxidant activity XO, as a key enzyme in purine metabolism, catalyzes the production of uric acid (UA) while simultaneously generating a large amount of ROS, primarily superoxide anions (O2·4O3 ... - O2 is the common source connecting high uric acid and oxidative stress; superoxide dismutase (SOD), as a core antioxidant enzyme, is responsible for scavenging O2· - It converts it into H2O2 and O2.

[0059] In a state of high uric acid, excessive ROS will deplete SOD, weaken its defense capabilities, and lead to O2 ·- Accumulation; hydroxyl radicals (·OH) are the most damaging ROS, mainly generated from H₂O₂ via the Fenton reaction catalyzed by metal ions. Impaired SOD function leads to O₂· - The accumulation of H2O2 provides conditions for the large-scale generation of ·OH, which in turn leads to severe oxidative damage.

[0060] Elevated uric acid (UA) activates xanthine (XO), promoting the conversion of hypoxanthine and xanthine into uric acid, accompanied by the generation of large amounts of reactive oxygen species (ROS). ROS is converted into ·OH via the Fenton reaction, causing its concentration to surge. At the same time, high uric acid inhibits SOD activity, weakening its clearance capacity, forming a vicious cycle that exacerbates high uric acid and complications.

[0061] The deep-sea long-lived fish precise active peptides provided by this invention can effectively inhibit XO activity, activate SOD activity, and remove ·OH, thereby achieving the purpose of lowering uric acid and anti-inflammation.

[0062] 1.1 Determination of the inhibition rate of XO by precise bioactive peptides from deep-sea long-lived fish For details on the determination of the inhibitory effect of the precise bioactive peptides of deep-sea long-lived fish on XO activity, please refer to "Enzymatic Preparation and Mechanism of Action of Wakame Protein Xanthine Oxidase Inhibiting Peptide", as follows: The precise active peptides of deep-sea long-lived fish were dissolved in 50 mmol / L phosphate buffer solution with pH 7.5 to prepare a peptide solution with a mass concentration of 1 mg / mL.

[0063] Prepare the following reaction systems, react them at 37℃ for 60 min, then add 150 μL of 1 mol / L hydrochloric acid to terminate the reaction, and measure the absorbance of each system at 290 nm.

[0064] Experimental group: Mix 50 μL of the peptide solution with 150 μL of 0.05 U / mL XO solution, and then add 150 μL of 0.48 mmol / L xanthine solution; Blank group: XO solution was replaced with 150 μL of deionized water; Negative control group: 50 μL of deionized water was used instead of the peptide solution; Negative control group: Replace XO and peptide solution with 200 μL of deionized water.

[0065] The XO inhibition rate was calculated according to the following formula [1], and the results are summarized in Table 1.

[0066]

[0067] In the formula, Y XO XO inhibition rate, % A1: Absorbance of the experimental group at 290nm; A2: Absorbance of the blank group at 290nm; A3: Absorbance of the negative control group at 290 nm; A4: Absorbance of the negative control group at 290nm.

[0068] Table 1. Determination of the inhibition rate of XO by precise bioactive peptides from deep-sea long-lived fish.

[0069] As can be seen from Table 1, the XO inhibition rate in Examples 1 to 3 can all reach over 89%, indicating that the deep-sea long-lived fish precise active peptides prepared in the embodiments of the present invention are effective in inhibiting XO, and the difference in inhibition effect among the various examples is small.

[0070] The XO inhibition rates of Comparative Examples 1 to 8 varied considerably. Among them, the XO inhibition rate of Comparative Example 2 was 6.27%, which was significantly lower than the clearance rate of the Example. The XO inhibition rates of Comparative Examples 5 and 6 were over 90%, which was higher than the inhibition rates of the other comparative examples and slightly higher than that of Example 1, but the difference was small.

[0071] In summary, the deep-sea long-lived fish precise active peptides prepared in Example 1, Comparative Example 5, and Comparative Example 6 all have a good inhibitory effect on XO.

[0072] 1.2 Determination of the effect of precise bioactive peptides from deep-sea long-lived fish on superoxide dismutase (SOD) activity For the determination of SOD activity by the precise bioactive peptides from deep-sea long-lived fish, please refer to GB / T5009.171-2003 "Determination of Superoxide Dismutase (SOD) Activity in Health Foods", as detailed below: (1) Determination of the auto-oxidation rate of pyrogallol: At 25℃, 2.35 mL of solution A, 2 mL of distilled water, and 0.15 mL of solution B were added sequentially to a 10 mL colorimetric tube. The mixture was immediately stirred after adding solution B and poured into a cuvette. The absorbance values ​​at 325 nm were measured at the initial time and after 1 min. The difference between the two values ​​is the auto-oxidation rate of pyrogallol ΔA. 325 This experiment determined ΔA 325 It is 0.060.

[0073] (2) Determination of the rate of pyrogallol autoxidation inhibited by sample solution and SOD enzyme solution: 2.35 mL of solution A, 1.8 mL of distilled water, 20 μL of polypeptide solution and 0.15 mL of solution B were used as sample solutions. 20 μL of distilled water was used as a blank control instead of polypeptide solution, and 20 μL of SOD solution was used as the SOD enzyme solution instead of polypeptide solution. Following the above steps, a certain amount of sample solution or SOD enzyme solution was added to inhibit the rate of pyrogallol autoxidation to approximately 1 / 2ΔA. 325 That is, ΔA 325 'It is 0.030.

[0074] In this experiment, solution A was a 0.1 mol / L Tris-hydrochloric acid buffer solution with a pH of 8.2, and solution B was a 4.5 mmol / L pyrogallol hydrochloric acid solution. The total volume of both solutions was 4.5 mL.

[0075] SOD enzyme activity was calculated according to the following formula [2], and the results are summarized in Table 2.

[0076]

[0077] In the formula, Y SOD SOD enzyme activity, U / mL; ΔA 325 : The auto-oxidation rate of pyrogallol; ΔA 325 ': Sample solution or SOD enzyme solution inhibits the auto-oxidation rate of pyrogallol;' V: Volume of added enzyme solution or sample solution, mL; D: The dilution factor of the enzyme solution or sample solution.

[0078] Table 2. Determination of the effect of precise bioactive peptides from deep-sea long-lived fish on SOD activity.

[0079] As can be seen from Table 2, the SOD activity in Examples 1-3 can all reach above 540 U / mL, indicating that the deep-sea long-lived fish precise active peptides prepared in the examples can activate SOD activity, and the difference in effect between the examples is small.

[0080] The effects of comparative examples 1 to 8 on SOD activity varied considerably. The SOD activity of the sample in comparative example 8 was 211.9 U / mL, which was significantly lower than that in the example. The SOD activities of comparative examples 1 and 4 were better than those of the other comparative examples, and were the same as those in example 1.

[0081] In summary, the deep-sea long-lived fish precise active peptides prepared in Example 1, Comparative Example 1, and Comparative Example 4 can all effectively activate SOD activity.

[0082] 1.3 Determination of the ·OH scavenging rate of precise bioactive peptides from deep-sea long-lived fish For details on the determination of the ·OH scavenging rate of the precise active peptides from deep-sea long-lived fish, please refer to "Study on the Antioxidant Activity of Panax notoginseng Polysaccharides", as follows: Prepare the following reaction systems, ensuring that the H2O2 solution is added last. Start the reaction and react at 37°C for 30 min. Using distilled water as a reference, measure the absorbance of each group at 510 nm.

[0083] Experimental group: 1 mL of 9 mmol / L FeSO4 solution, 1 mL of 9 mmol / L salicylic acid-ethanol solution, 1 mL of 8.8 mmol / L H2O2 solution, and 1 mL of deep-sea long-lived fish precise active peptide solution prepared by different processes; Blank group: 1 mL of distilled water was used instead of the deep-sea long-lived fish precise active peptide solution; Negative control group: 1 mL of distilled water was used instead of H2O2 solution; Negative control group: 1 mL of H2O2 solution was used to replace the deep-sea long-lived fish precise active peptide solution.

[0084] The activity of ·OH enzyme was calculated according to the following formula [3], and the results are summarized in Table 3.

[0085]

[0086] In the formula, Y ·OH OH removal rate, % A1: Absorbance of the experimental group at 510nm; A2: Absorbance of the blank group at 510nm; A3: Absorbance of the negative control group at 510 nm; A4: Absorbance of the negative control group at 510nm.

[0087] Table 3. Determination of the ·OH scavenging rate by precise bioactive peptides from deep-sea long-lived fish.

[0088] As can be seen from Table 3, the scavenging rate of ·OH in Examples 1-3 can all reach over 90%, indicating that the deep-sea long-lived fish precise active peptides prepared in the examples are effective in scavenging ·OH, and the difference in effect between the examples is small.

[0089] The ·OH scavenging rates of Comparative Examples 1 to 8 varied considerably. The scavenging rate of Comparative Example 8 was 35.35%, which was significantly lower than that of the Example. The scavenging rates of Comparative Examples 5 and 6 were better than those of the other comparative examples, but slightly lower than that of Example 1.

[0090] In summary, the precise active peptides from deep-sea long-lived fish can effectively inhibit XO activity, activate SOD activity, and scavenge ·OH, thereby inhibiting uric acid production and eliminating free radicals in the body to jointly lower uric acid levels. While the indicator data of Example 1 were not entirely better than other comparative examples, the superiority or inferiority of a single indicator data point cannot judge the progress of the patent application; a comprehensive evaluation of the indicator data is necessary. Therefore, Example 1, Comparative Example 5, and Comparative Example 6 were selected for further cell experiments.

[0091] Experiment 2: Construction of HK-2 cytotoxicity and oxidative damage model HK-2 cells are an immortalized cell line derived from human renal proximal tubular epithelial cells. They possess the typical morphology and functional characteristics of renal epithelial cells and actively participate in the reabsorption and secretion of uric acid, making them an ideal cell model for studying renal physiological function and the pathogenesis of kidney diseases. Hyperuricemia, a common metabolic disease, is closely related to abnormal renal tubular uric acid excretion. Many studies have used HK-2 cells to establish hyperuricemia models to explore the mechanisms of oxidative stress, inflammatory responses, and cell damage caused by uric acid metabolism disorders.

[0092] The HK-2 cell injury assay is commonly used to assess the effects of exogenous compounds on the viability, proliferation, and function of renal tubular epithelial cells, thereby providing a preliminary assessment of renal injury or biocompatibility. WST-8 in the CCK-8 reagent can be reduced by intracellular dehydrogenases in HK-2 cells in the presence of an electron carrier to produce a water-soluble, orange-yellow formazan product, the yield of which is directly proportional to the number of viable cells. By detecting the absorbance at 450 nm, the activity of HK-2 cells and their injury response to the compound can be effectively reflected.

[0093] 2.1 Effects of Precision Active Peptides and Positive Drugs from Deep-Sea Long-lived Fish on HK-2 Cell Viability The effect of precise bioactive peptides from deep-sea long-lived fish on the proliferation activity of HK-2 cells was determined using the CCK-8 assay. HK-2 cells in the logarithmic growth phase were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a density of 1×10⁻⁶ cells per 100 μL. 4 HK-2 cells were seeded at 10 cells / mL into 96-well plates and incubated at 37°C for 24 hours in a 5% CO2 incubator to allow the HK-2 cells to adhere to the wells. The 96-well plates were then removed and the culture medium was aspirated.

[0094] Prepare the following reaction systems, incubate them in a 5% CO2 incubator at 37°C for 24 hours, then aspirate the culture medium and add 10 μL of CCK-8 solution to each well under dark conditions. Return the wells to the 5% CO2 incubator and continue incubating at 37°C for 20 minutes. After incubation, measure the absorbance of each well at 450 nm using a microplate reader.

[0095] Preferably, the concentration of CCK-8 reagent is 1 mg / mL to 2 mg / mL. To ensure that the CCK-8 reagent is fully mixed with HK-2 cells, the culture plate can be placed on a shaker and gently shaken at low speed for 1 to 2 minutes, taking care to avoid generating air bubbles.

[0096] Experimental group: Complete culture medium containing 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL of deep-sea long-lived fish precise active peptide solution, respectively; Positive control group: complete culture medium containing benzbromarone at concentrations of 1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, and 100 μmol / L, respectively; Control group: Complete culture medium with fresh culture medium; Control group: Complete culture medium without HK-2 cells.

[0097] Cell viability was calculated according to the following formula [4], and the results of the experimental groups were summarized to Figure 1 The results of the positive control group are summarized in Table 4.

[0098]

[0099] In the formula, Y HK-2 HK-2 cell viability, % A1: Absorbance of the experimental group or positive control group at 450nm; A1': Absorbance of the control group at 450nm; A0: Absorbance of the blank group at 450nm.

[0100] Table 4. Results of the effect of different concentrations of benzbromarone on HK-2 cell viability.

[0101] Figure 1 The graph shows the results of testing the effect of different concentrations of the deep-sea long-lived fish precise active peptides prepared in Example 1, Comparative Example 5, and Comparative Example 6 on the viability of HK-2 cells. Among them, the viability of HK-2 cells in the control group is 100%. In Example 1, Comparative Example 5 and Comparative Example 6, the samples with peptide concentrations of 0.5 mg / mL and below showed HK-2 cell viability of over 100%, indicating that the above samples did not damage HK-2 cells. In Example 1, HK-2 cells maintained high activity levels in samples with various peptide concentrations, all exceeding 100%, indicating that the deep-sea long-lived fish precise active peptides prepared in Example 1 did not damage HK-2 cells at peptide concentrations of 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL. In Comparative Example 5, the HK-2 cell viability was less than 50% in samples with peptide concentrations of 1.0 mg / mL and above, indicating that the deep-sea long-lived fish precise active peptides prepared in Comparative Example 5 damaged HK-2 cells at concentrations of 1.0 mg / mL and above. In Comparative Example 6, the HK-2 cells maintained relatively high activity, all above 80%, in samples with peptide concentrations of 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL. However, the activity of HK-2 cells gradually decreased, indicating that as the concentration of the deep-sea long-lived fish precise active peptide prepared in Comparative Example 6 increased, it caused slight damage to HK-2 cells.

[0102] As shown in Table 4, in the experiment on the viability of HK-2 cells at different concentrations of benzbromarone, the HK-2 cells were not damaged in the samples with a concentration of less than 40 μmol / L, but the viability of HK-2 cells was less than 80% in the samples with a concentration of more than 60 μmol / L, indicating that benzbromarone at a concentration of more than 60 μmol / L damaged HK-2 cells.

[0103] In summary, the high bioactivity of the deep-sea long-lived fish precise bioactive peptide at 0.5 mg / mL was observed in HK-2 cells, while the positive control benzbromarone at 40 μmol / L showed the highest bioactivity in HK-2 cells. Therefore, this concentration will be used for subsequent experiments.

[0104] 2.2 Construction of a uric acid-induced oxidative damage model in HK-2 cells Adenosine and XO together act as modeling agents, specifically inducing the production of large amounts of uric acid by mimicking the metabolic pathway of "adenosine-xanthine-uric acid" in the body, thereby constructing oxidative damage models, such as cellular oxidative damage and inflammation-related oxidative stress models.

[0105] This experiment investigated the effects of adenosine and XO on the oxidative stress-induced damage activity of HK-2 cells, as detailed below: The steps for HK-2 cell culture and cell viability assay are the same as in Experiment 2.1 above. The results of the effect of adenosine on HK-2 cell viability are summarized in Table 5, and the results of the effect of XO on HK-2 cell viability are summarized in Table 6.

[0106] Adenosine experimental groups: complete culture media containing 0.1 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 0.8 mmol / L, 1.0 mmol / L, 2.5 mmol / L, 5.0 mmol / L, and 10.0 mmol / L adenosine, respectively; XO experimental groups: complete culture media containing 0.001 U / mL, 0.005 U / mL, 0.025 U / mL, and 0.125 U / mL XO, respectively; Control group: Complete culture medium with fresh culture medium; Control group: Complete culture medium without HK-2 cells.

[0107] Table 5. Results of HK-2 cell viability assay under different concentrations of adenosine.

[0108] Table 6. Results of HK-2 cell viability assay under different concentrations of XO

[0109] According to the results in Table 5, in the experiment on the viability of HK-2 cells with different concentrations of adenosine, the viability of HK-2 cells gradually decreased in samples with adenosine concentrations of 0.8 mmol / L and above, indicating that increasing adenosine concentration would cause certain damage to HK-2 cells; among them, when the adenosine concentration was 2.5 mmol / L, the viability of HK-2 cells was 73.75%, which was already below 80%.

[0110] According to the results in Table 6, in the experiment on the viability of HK-2 cells with different concentrations of XO, the viability of HK-2 cells was greater than 100% for samples with XO concentrations between 0 U / mL and 0.125 U / mL, indicating that XO did not cause damage to HK-2 cells; among them, the HK-2 cell viability was the highest when the XO concentration was 0.005 U / mL, reaching 108.82%.

[0111] Based on the above results, adenosine concentrations of 0.8 mmol / L, 1.0 mmol / L, 1.5 mmol / L, and 2.5 mmol / L and XO of 0.005 U / mL were selected to explore the modeling concentration. The results are summarized in Table 7.

[0112] Table 7. Uric acid content under the combined effects of different concentrations of adenosine and 0.005 U / mL XO

[0113] According to the results in Table 7, among the experiments on uric acid content under the combined effects of different concentrations of adenosine and 0.005 U / mL XO, the uric acid content was highest when 2.5 mmol / L adenosine and 0.005 U / mL XO were used together. Therefore, this concentration was selected for cell modeling to obtain the UA-induced HK-2 cell model.

[0114] 2.3 Effects of Precision Active Peptides and Positive Drugs from Deep-Sea Long-lived Fish on UA-Induced HK-2 Cell Viability Based on Experiments 2.1 and 2.2 above, experiments were conducted to investigate the effects of precise bioactive peptides and positive control drugs from deep-sea long-lived fish on the viability of UA-induced HK-2 cells. The results are summarized in Table 8, as follows: The procedures for HK-2 cell culture and cell viability assay are the same as those in Experiment 2.1 above.

[0115] Blank control group: Complete culture medium containing 2.5 mmol / L adenosine and 0.005 U / mL XO; Positive control group: The blank control group was supplemented with 40 mmol / L benzbromarone complete culture medium; Experimental group: Based on the blank control group, 0.3 mol / L of the complete culture medium containing the deep-sea long-lived fish precise active peptides prepared in Example 1, Comparative Example 5, or Comparative Example 6 was added.

[0116] Table 8. Results of tests on the effects of precise bioactive peptides and positive control drugs from deep-sea long-lived fish on uric acid-induced HK-2 cell viability.

[0117] According to the results in Table 8, in the experiment on the effect of deep-sea long-lived fish precise active peptides and positive drugs on the viability of UA-induced HK-2 cells, the deep-sea long-lived fish precise active peptides and positive drugs caused very little damage to the cells, and the cell viability was more than 90% of that of the blank control group. This indicates that the deep-sea long-lived fish precise active peptides and positive drugs do not synergistically enhance the damage to HK-2 cells with the modeling agent.

[0118] Experiment 3. Determination of the effects of precise bioactive peptides from deep-sea long-lived fish on uric acid (UA), reactive oxygen species (ROS), interleukin-1β (IL-1β), and nitric oxide (NO) levels induced by UA in HK-2 cells. Increased UA activates XO, promoting the conversion of hypoxanthine and xanthine into UA, accompanied by the generation of large amounts of ROS. ROS is converted into ·OH through the "Fenton reaction," causing its concentration to surge. At the same time, HUA inhibits SOD activity, weakening its clearance capacity, forming a vicious cycle and triggering severe oxidative damage. In addition, both ROS and UA can activate inflammatory pathways, promoting the release of inflammatory factors such as IL-1β, exacerbating the inflammatory response. NO metabolism disorder also interacts with oxidative stress, leading to endothelial dysfunction and vascular damage.

[0119] This experiment explores how the precise bioactive peptides from deep-sea long-lived fish can prevent and treat hyperuricemia by reducing the combined effects of UA, ROS, IL-1β, and NO levels in HK-2 cells. The specific steps are as follows: (1) Cell seeding: HK-2 cells with good growth were seeded at a ratio of 1×10⁻⁶ cells / plate. 4 Planted at a density of cells / well in 96-well plates and incubated at 37°C, 5% CO2 for 24 hours. After observing adhesion, the upper culture medium was discarded. (2) The following experimental groups were placed in a 5% CO2 incubator and incubated at 37°C for 24 hours.

[0120] Control group: Added complete culture medium; Model group: Complete culture medium containing the corresponding concentration of adenosine; Experimental group: Complete culture medium containing corresponding concentrations of adenosine and corresponding concentrations of deep-sea long-lived fish precise active peptides; Positive control group: Complete culture medium containing the corresponding concentrations of adenosine and benzbromarone was added.

[0121] (3) Add 0.005 U / mL XO to the model group, positive control group and experimental group respectively, and continue the intervention for 4 hours; (4) After the intervention, UA, ROS, IL-1β and NO were measured according to the instructions.

[0122] 3.1 Effects of Precision Bioactive Peptides from Deep-Sea Long-lived Fish on UA ​​Content in UA-Induced HK-2 Cells Figure 2 The graph shows the analysis results of the test on the effect of UA on UA ​​content in HK-2 cells by each experimental group in this experiment. It can be seen that: Compared with the control group, the UA content in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the UA content in UA-induced HK-2 cells in the positive control group. Compared with the model group, the experimental group, Example 1, Comparative Example 5 and Comparative Example 6 can effectively reduce the UA content in UA-induced HK-2 cells. Among them, Example 1 has the best effect in reducing UA.

[0123] 3.2 Effects of Precision Bioactive Peptides from Deep-Sea Long-lived Fish on ROS Content in UA-Induced HK-2 Cells Figure 3 The graph shows the results of the test on the effect of each experimental group on ROS content induced by UA in HK-2 cells. It can be seen that: Compared with the control group, the ROS content in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the ROS content in UA-induced HK-2 cells in the positive control group. Compared with the model group, the experimental group, Example 1, Comparative Example 5 and Comparative Example 6 can effectively reduce the ROS content in UA-induced HK-2 cells. Among them, Example 1 has the best effect in reducing ROS.

[0124] 3.3 Effects of Precision Bioactive Peptides from Deep-Sea Long-lived Fish on IL-1β Levels in UA-Induced HK-2 Cells Figure 4 The graph shows the results of the test on the effect of each experimental group on UA-induced IL-1β levels in HK-2 cells. It can be seen that: Compared with the control group, the IL-1β content in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the IL-1β content in UA-induced HK-2 cells in the positive control group. Compared with the model group, the experimental group, Example 1, Comparative Example 5 and Comparative Example 6 can effectively reduce the IL-1β content in UA-induced HK-2 cells. Among them, Comparative Example 6 has the best effect in reducing IL-1β content, but the effect of Example 1 and Comparative Example 6 in reducing IL-1β content is not much different.

[0125] 3.4 Effects of Precision Bioactive Peptides from Deep-Sea Long-lived Fish on NO Content in UA-Induced HK-2 Cells Figure 5 The graph shows the results of the test on the effect of each experimental group on NO content induced by UA in HK-2 cells. It can be seen that: Compared with the control group, the NO content in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the NO content in UA-induced HK-2 cells in the positive control group. Compared with the model group, the experimental group, Example 1, Comparative Example 5 and Comparative Example 6 can effectively reduce the NO content in UA-induced HK-2 cells. Among them, Comparative Example 1 has the best effect in reducing NO content.

[0126] Experiment 4. Determination of the effect of precise bioactive peptides from deep-sea long-lived fish on the mRNA expression levels of XO, URAT1, ABCG2, and TNF-α in UA-induced HK-2 cells. XO is a key rate-limiting enzyme in purine metabolism, directly catalyzing the formation of hypoxanthine → xanthine → uric acid. Increased mRNA expression of XO leads to excessive uric acid production and is the core pathogenic factor of hyperuricemia with excessive production.

[0127] URAT1 is an important uric acid reabsorption transporter located on the apical membrane of renal tubular epithelial cells. It is responsible for reabsorbing more than 90% of uric acid in the primary urine back into the blood. Overexpression of URAT1 mRNA leads to increased uric acid reabsorption and decreased excretion, which is the primary cause of hyperuricemia with reduced excretion.

[0128] ABCG2 is an important uric acid excretion transporter located in renal tubules and intestinal epithelial cells. It is responsible for secreting intracellular uric acid into the kidneys or intestines. Decreased mRNA expression of ABCG2 leads to reduced uric acid excretion and is the second most important pathogenic factor for hyperuricemia with reduced excretion after URAT1.

[0129] TNF-α is a pro-inflammatory cytokine that does not directly regulate uric acid metabolism. However, high uric acid can induce an increase in TNF-α expression. Crystalline uric acid activates inflammatory pathways, and TNF-α, in turn, aggravates kidney damage and reduces uric acid excretion, forming a vicious cycle of "high uric acid → inflammation → more severe high uric acid".

[0130] This experiment investigated the effects of precise bioactive peptides from deep-sea long-lived fish on the mRNA expression levels of XO, URAT1, ABCG2, and TNF-α in UA-induced HK-2 cells. The effects of these peptides on the expression levels of genes related to uric acid oxidative damage in HK-2 cells were determined using RT-qPCR. The specific steps are as follows: (1) Cell seeding: HK-2 cells with good growth were seeded at a ratio of 1×10⁻⁶ cells / plate. 4 Planted at a density of cells / well in 96-well plates and incubated at 37°C, 5% CO2 for 24 hours. After observing adhesion, the upper culture medium was discarded. (2) Add 100 μL of complete culture medium containing 0.3 mg / mL polypeptide solution and incubate at 37°C and 5% CO2 for 24 h. The polypeptide solution is the solution of deep-sea long-lived fish precise active peptide prepared in Example 1, Comparative Example 5, or Comparative Example 6. After the culture is completed, discard the upper culture medium and wash HK-2 cells three times with PBS to remove residual culture medium and drugs. Then add 2.5 mmol / L adenosine and incubate for 24 h. After the incubation, add XO solution containing 0.005 U / mL to the model group, positive control group, and experimental group for 4 h of further intervention. After the culture is completed, discard the culture medium and wash HK-2 cells three times with PBS to remove residual culture medium and drugs.

[0131] (3) RNA extraction: Following the instructions of the RNA extraction kit, add an appropriate amount of lysis buffer to each culture dish to fully lyse HK-2 cells, extract total RNA, and use a micro-nucleic acid analyzer to detect the concentration and purity of RNA to ensure that the quality of RNA meets the requirements of subsequent experiments, such as OD. 260 / OD 280 The ratio is between 1.8 and 2.0.

[0132] (4) Reverse transcription to obtain cDNA: Take an appropriate amount of total RNA and reverse transcribe it into cDNA according to the operation steps of the reverse transcription kit. The reverse transcription reaction conditions are usually: incubate at 42℃ for 60 min to 90 min, and then heat at 70℃ for 10 min to 15 min to terminate the reaction. The obtained cDNA can be stored at -20℃ for later use.

[0133] (5) PCR: Prepare the PCR reaction system, including SYBR Green fluorescent dye, upstream and downstream primers, cDNA template, PCR buffer, dNTPs and Taq enzyme, etc.; add the PCR reaction system to a 96-well plate or a 384-well plate, and set up 3 replicates for each sample; the PCR reaction program is generally as follows: 95℃ pre-denaturation for 3 min to 5 min; then 95℃ denaturation for 15 s to 30 s, 60℃ annealing for 30 s to 60 s, 72℃ extension for 30 s to 60 s, for a total of 40 to 50 cycles.

[0134] (6) Melting curve analysis: GAPDH was used as an internal reference gene as a control. The relative expression level of the target gene was calculated by comparing the Ct value (cycle threshold), and the data was analyzed by the 2-ΔΔCt method.

[0135] The primer information for HK-2 cell-related genes is shown in Table 9.

[0136] Table 9 Primer information for HK-2 cell-related genes

[0137] 4.1 Effects of precise bioactive peptides from deep-sea long-lived fish on XO mRNA expression levels in UA-induced HK-2 cells Figure 6 This is a graph showing the results of the test on the effect of each group on the mRNA expression level of XO induced by UA in HK-2 cells in this experiment. It can be seen that: Compared with the control group, the mRNA expression level of XO in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the mRNA expression level of XO in HK-2 cells induced by UA. Compared with the model group, Example 1 effectively reduced the mRNA expression level of XO in HK-2 cells induced by UA, while Comparative Examples 5 and 6 promoted the mRNA expression level of XO in HK-2 cells induced by UA. This may be because HK-2 cells sensed the reduction of uric acid, initiated feedback compensation, and upregulated the gene expression of XO.

[0138] 4.2 Effects of precise bioactive peptides from deep-sea long-lived fish on UA-induced URAT1 mRNA expression in HK-2 cells Figure 7 This is a graph showing the results of the test on the effect of each group on the expression level of URAT1 in HK-2 cells induced by UA in this experiment. It can be seen that: Compared with the control group, the mRNA expression level of URAT1 in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the mRNA expression level of URAT1 in UA-induced HK-2 cells in the positive control group. Compared with the model group, both Example 1 and Comparative Example 6 effectively reduced the mRNA expression level of URAT1 in HK-2 cells induced by UA. Among them, Example 1 had the best effect in reducing the mRNA expression level of URAT1, while Comparative Example 5 promoted the mRNA expression level of URAT1 in HK-2 cells induced by UA.

[0139] 4.3 Effects of precise bioactive peptides from deep-sea long-lived fish on ABCG2 mRNA expression levels in UA-induced HK-2 cells Figure 8 This is a graph showing the effect of each group on the mRNA expression level of ABCG2 induced by UA in HK-2 cells in this experiment. It can be seen that: Compared with the model group, the mRNA expression level of ABCG2 in HK-2 cells induced by UA in the model group was significantly lower than that in the control group. Compared with the model group, benzbromarone effectively increased the mRNA expression level of ABCG2 in UA-induced HK-2 cells; Compared with the model group, the experimental group, Example 1, Comparative Example 5 and Comparative Example 6 can effectively increase the mRNA expression level of ABCG2 in UA-induced HK-2 cells. Among them, Example 1 has the best effect in increasing the mRNA expression level of ABCG2.

[0140] 4.4 Effects of precise bioactive peptides from deep-sea long-lived fish on TNF-α mRNA expression levels in UA-induced HK-2 cells Figure 9 This is a graph showing the effect of each group on the mRNA expression level of TNF-α induced by UA in HK-2 cells in this experiment. It can be seen that: Compared with the model group, the mRNA expression level of TNF-α in HK-2 cells induced by UA in the model group was significantly higher than that in the control group. Compared with the model group, benzbromarone effectively reduced the mRNA expression of TNF-α in UA-induced HK-2 cells in the positive control group. Compared with the model group, both Example 1 and Comparative Example 6 effectively reduced the mRNA expression level of TNF-α induced by UA in HK-2 cells. Among them, Example 1 had the best effect in reducing the mRNA expression level of TNF-α, while Comparative Example 5 promoted the mRNA expression level of TNF-α induced by UA in HK-2 cells.

[0141] Experiment 5 Precise docking of bioactive peptides from deep-sea long-lived fish 5.1 Preprocessing (1) Precision dissolution of active peptides from deep-sea long-lived fish Weigh 6 mg of the deep-sea long-lived fish precise active peptide powder prepared in Example 1, add it to a 1.5 mL centrifuge tube, and then use a pipette to transfer 600 µL of 50 mmol / L NH4HCO3 solution into the deep-sea long-lived fish precise active peptide to dissolve it, so as to obtain a peptide solution with a concentration of 10 μg / μL.

[0142] (2) Ultrafiltration 10kDa 1) Accurately transfer the peptide solution into a 10 kDa ultrafiltration tube using a pipette, and centrifuge at 4°C and 12,000 rpm for 10 min; 2) Collect the filtrate with less than 10 kDa in a 1.5 mL centrifuge tube for later use.

[0143] (3) Nanodrop concentration measurement 1) Rinse the detection head twice with 2μL of distilled water; 2) Load the blank control solution with a sample volume of 2 μL, measure the data at 205 nm, and deduct the result after measurement; 3) Test for precise bioactive peptides from deep-sea long-lived fish, with a sample loading volume of 2 μL, and read the data after testing; 4) Rinse the detection head twice with 2μL of distilled water.

[0144] (4) Reductive alkylation 1) Transfer 100 μg of sample to a 1.5 mL centrifuge tube, and use a pipette to add 50 mmol / L NH4HCO3 to make up the volume to 100 μL for subsequent experiments; 2) Use a pipette to transfer 1 μL of 1 mmol / L DTT solution into the deep-sea long-lived fish precise active peptide to make the final DTT concentration 10 mmol / L, and reduce it in a water bath at 56℃ for 1 h; 3) Use a pipette to transfer 2 μL of 1 mmol / L TAM solution into the deep-sea long-lived fish precise active peptide to make the final TAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min. 4) Use a pipette to transfer 1 μL of 1 mmol / L DTT solution into the deep-sea long-lived fish precise active peptide to make the final DTT concentration 10 mmol / L, so as to neutralize the unreacted TAM.

[0145] (5) SP2 desalination 1) After the deep-sea long-lived fish precise active peptides were reduced and alkylated, they were dried, reconstituted with 10 μL of 0.1% formic acid, and 10 μL of magnetic beads were accurately transferred into the deep-sea long-lived fish precise active peptides with a pipette and mixed well. 2) Add 380 μL of acetonitrile to make the final concentration 95%. Mix by vertical transfer to ensure uniform dispersion of the magnetic beads. Then let stand for 5 min, place on a magnetic rack for 2 min, and remove the supernatant. 3) Add 200 μL of 100% acetonitrile to completely cover the magnetic beads. Mix by vertical transfer to ensure uniform dispersion of the magnetic beads. Let stand for 2 minutes, then place in a magnetic rack and let stand for 1 minute before removing the supernatant. 4) Repeat the cleaning process 3 times; 5) Add 54 μL of 2% acetonitrile, shake the magnetic beads thoroughly for 15 min, and transfer the supernatant to a new centrifuge tube; 6) Repeat the process of merging the supernatant once, drain it, and wait for testing.

[0146] (6) Nanodrop concentration measurement 1) Rinse the detection head twice with 2μL of distilled water; 2) Load the blank control solution with a sample volume of 2 μL, measure the data at 205 nm, and deduct the result after measurement; 3) Test for precise bioactive peptides from deep-sea long-lived fish, with a sample loading volume of 2 μL, and read the data after testing; 4) Rinse the detection head twice with 2μL of distilled water.

[0147] 5.2 Hands-on Practice The processed deep-sea long-lived fish precise bioactive peptides were detected on a liquid chromatography-mass spectrometry instrument.

[0148] (1) Liquid chromatography conditions 1) Pre-column: 150 μm d. × 50 mm, packing: Reprosil-Pur120C18-AQ 3 μm; Analytical column: 150 μm d. × 170 mm, packing: Reprosil-Pur120C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% FA; 3) Mobile phase B: 0.1% FA, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are listed in Table 10: Table 7 Specific chromatographic conditions for liquid chromatography

[0149] (2) Mass spectrometry conditions Level 1 mass spectrometry parameters: 1) Resolution: 70,000; 2) AGCtarget: 3e6; 3) MaximumIT: 100 ms; 4) Scanrange: 100to1500 m / z.

[0150] Secondary mass spectrometry parameters: 1) Resolution: 17,500; 2) AGCtarget: 1e5; 3) MaximumIT: 50 ms; 4) TopN: 20; 5) NCE / steppedNCE: 28.

[0151] Raw data was obtained through mass spectrometry.

[0152] (3) Search conditions The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: 1) Fixed modifications: Carbamidomethyl(C); 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term); 3) Enzyme: Nonspecific; 4) Database: uniprotkb_Acipenser(genus)_2024_11_13; uniprotkb_Oreochromis(genus)_2024_12_05; 5) Peptide Mass Tolerance: 20 ppm; 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.03 Da.

[0153] 5.3 Peptide Screening The raw files acquired by mass spectrometry contained a total of 341 peptides ranging from 2 to 9. First, peptides with 2 or more peptides were selected based on peak area (abundance), with a relative content >1.5%, and 13 peptides containing 2 or more peptides. Activity scoring was performed using PeptideRanker data, selecting 10 peptides with a score >0.50 and containing 2 or more peptides. Further searching the BIOPEP database of precise bioactive peptides from deep-sea long-lived fish revealed 10 previously unreported peptides. ToxinPred toxicity predictions for these 10 peptides showed they were all non-toxic. Relevant information for these 10 peptides is listed in Table 11. Table 11 Peptide Information

[0154] 5.4 Molecular docking Molecular docking software was used to perform molecular docking of the above 10 peptides with XO, URAT1, and ABCG2 receptors. The three-dimensional structures of XO (ID: 3NRZ), URAT1 (ID: 9JDV), and ABCG2 (ID: 6ETI) were downloaded from the PDB database (http: / / www.rcsb.org / ). The docking binding energies of the 10 peptides with different receptors are shown in Table 12.

[0155] Table 12. Binding energy between peptides and receptors

[0156] CDOCKERENERGY is an energy value calculated during CDOCKER docking. It mainly reflects the overall binding energy of the ligand and receptor during docking. This energy comprehensively considers various interactions between the ligand and receptor, such as van der Waals forces and electrostatic interactions. Physically speaking, the lower the CDOCKERENERGY value, the tighter and more stable the binding between the ligand and receptor.

[0157] XO, as a core oxidoreductase in the purine metabolism pathway, is mainly expressed in the cytoplasm of liver cells and on the surface of capillary endothelial cells. It can specifically catalyze the oxidation process of hypoxanthine to xanthine and finally to uric acid, and is the final key molecule in the synthesis of uric acid in the body.

[0158] Excessive activation of xanthocyanin (XO) can significantly lead to hyperuricemia, through mechanisms including: accelerating the formation of purine catabolism end products; increasing oxidative stress and generating reactive oxygen species; and promoting the release of inflammatory factors. Simultaneously, XO directly regulates serum uric acid levels, promoting gouty arthritis by increasing urate saturation and promoting urate crystal precipitation. Deep-sea long-lived fish precise bioactive peptides can competitively inhibit XO activity, reducing uric acid synthesis at its source.

[0159] URAT1 belongs to the family of organic anion transporters and is highly expressed in the apical membrane of proximal tubular epithelial cells of the kidney. As a hub for uric acid reabsorption, it can specifically recognize anion molecules such as urate and lactate.

[0160] After urate binds to URAT1, its conformational change drives the outflow of intracellular anions, initiating the transmembrane influx of urate. The physiological significance of URAT1 lies in mediating the reabsorption of nearly 90% of filtered uric acid and maintaining serum uric acid homeostasis. Hyperfunction of URAT1 in hyperuricemia leads to impaired renal uric acid excretion, increasing the risk of primary gout. Precision bioactive peptides from deep-sea long-lived fish can regulate the expression of the renal URAT1 transporter, inhibiting reabsorption and promoting secretion, thus excreting uric acid through urine.

[0161] ABCG2 is a key transporter protein regulating uric acid excretion in the human body. It uses ATP hydrolysis to generate energy and actively excretes uric acid from the body as a "molecular pump." ABCG2 is expressed in the intestine, where it pumps uric acid from the blood into the intestinal lumen for excretion in feces. It is also expressed in renal tubular cells, where it "reverse-pumps" uric acid that is about to be reabsorbed back into the blood into the urine, thus greatly enhancing the efficiency of uric acid excretion by the kidneys. Therefore, loss-of-function mutations in ABCG2 directly lead to a decrease in uric acid excretion capacity.

[0162] By inhibiting XO activity to reduce uric acid production and suppressing URAT1 reabsorption, ABCG2 hydrolyzes ATP to provide energy for uric acid excretion, thus inhibiting uric acid reabsorption and promoting uric acid secretion. In the proximal tubule of the kidney, ABCG2 pumps uric acid into the lumen, while the adjacent URAT1 undergoes reabsorption. This dynamic antagonism precisely regulates the final excretion of uric acid. Based on this, the body's purine metabolism balance is achieved, effectively reducing the concentration of uric acid in the body. This synergistic approach of "reducing uric acid at its source" and "unblocking its exit" synergistically lowers blood uric acid levels, thereby preventing and alleviating hyperuricemia and related problems such as gout.

[0163] Figure 10 The results show that there are van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions between LLL and the XO receptor protein. Figure 11 The results show that there are van der Waals forces, conventional hydrogen bonds, π-cations, π-donor hydrogen bonds, alkyl groups, and π-alkyl groups as the main interactions between LLL and the URAT1 receptor protein. Figure 12 The results show that there are interactions between LLL and the ABCG2 receptor protein, mainly van der Waals forces, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 13 The results show that there are interactions between KLF and XO receptor protein, mainly van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 14 The results show that there are van der Waals forces, conventional hydrogen bonds, π-cations, π-anions, π-π stacking, alkyl groups, and π-alkyl groups as the main interactions between KLF and the URAT1 receptor protein; Figure 15 The results show that there are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, and alkyl-based interactions between KLF and the ABCG2 receptor protein. Figure 16 The results show that there are interactions between ILPL and XO receptor protein, mainly van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 17 The results show that there are interactions between ILPL and the URAT1 receptor protein, mainly involving van der Waals forces, conventional hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 18 The results show that there are interactions between ILPL and the ABCG2 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 19The results show that there are interactions between LL and XO receptor proteins, mainly van der Waals forces, attractive charges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 20 The results show that there are interactions between LL and the URAT1 receptor protein, mainly involving van der Waals forces, salt bridges, π-cations, alkyl groups, and π-alkyl groups. Figure 21 The results show that there are van der Waals forces, π-cations, alkyl groups, and π-alkyl groups as the main interactions between LL and the ABCG2 receptor protein; Figure 22 The results show that there are van der Waals forces, salt bridges, conventional hydrogen bonds, conventional hydrogen bonds, and alkyl-based interactions between GIF and the XO receptor protein. Figure 23 The results show that there are van der Waals forces, salt bridges, conventional hydrogen bonds, π-donor hydrogen bonds, and π-alkyl-based interactions between GIF and the URAT1 receptor protein. Figure 24 The results show that there are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, and alkyl-based interactions between GIF and the ABCG2 receptor protein. Figure 25 The results show that there are van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, and alkyl-based interactions between LPL and the XO receptor protein. Figure 26 The results show that there are interactions between LPL and the URAT1 receptor protein, mainly involving van der Waals forces, conventional hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 27 The results show that there are interactions between LPL and the ABCG2 receptor protein, mainly involving van der Waals forces, C-H bonds, alkyl groups, and π-alkyl groups. Figure 28 The results show that LALPP interacts with the XO receptor protein primarily through van der Waals forces, salt bridges, attractive charges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 29 The results show that there are van der Waals forces, attractive charges, conventional hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups as the main interactions between LALPP and the URAT1 receptor protein. Figure 30 The results show that there are interactions between LALPP and the ABCG2 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 31 The results show that there are interactions between LLM and XO receptor protein, mainly van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 32The results show that there are interactions between LLM and the URAT1 receptor protein, mainly involving van der Waals forces, conventional hydrogen bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 33 The results show that there are interactions between LLM and the ABCG2 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 34 The results show that there are van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds and alkyl-based interactions between LGGVL and the XO receptor protein; Figure 35 The results show that there are interactions between LGGVL and the URAT1 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, C-H bonds, π-cations, alkyl groups, and π-alkyl groups. Figure 36 The results show that there are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, π-anions, π-alkyls and alkyl-based interactions between LGGVL and the ABCG2 receptor protein; Figure 37 The results show that there are interactions between FSL and XO receptor protein, mainly van der Waals forces, salt bridges, conventional hydrogen bonds, C-H bonds, alkyl groups, and π-alkyl groups. Figure 38 The results show that there are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-cations, π-π stacking, and alkyl-based interactions between FSL and the URAT1 receptor protein; Figure 39 The results show that there are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, π-π stacking, amide-π stacking, and alkyl-based interactions between FSL and the ABCG2 receptor protein.

[0164] According to the data in Table 9, all 10 peptides have certain binding energies with purine oxidase, URAT1, and ABCG2 receptors. This indicates that the 10 peptides Leu-Leu-Leu (LLL), Lys-Leu-Phe (KLF), Ile-Leu-Pro-Leu (ILPL), Leu-Leu (LL), Gly-Ile-Phe (GIF), Leu-Pro-Leu (LPL), Leu-Ala-Leu-Pro-Pro (LALPP), Leu-Leu-Met (LLM), Leu-Gly-Gly-Val-Leu (LGGVL), and Phe-Ser-Leu (FSL) are active fragments of precise active peptides from deep-sea long-lived fish with uric acid-lowering and anti-inflammatory functions.

[0165] It should be noted that the terms used in this invention, such as ultrafiltration, centrifugation, spray drying, mass spectrometry, molecular docking, and RNA extraction, are names of processing steps commonly used by those skilled in the art, and their names can accurately describe the processing procedures, so they will not be repeated in this invention.

[0166] The raw materials used in this invention, such as alkaline protease (model 37071, enzyme activity 400,000 U / g, Novozymes (China) Biotechnology Co., Ltd.), acidic protease (enzyme activity 150,000 U / g, Shandong Longket Enzyme Preparation Co., Ltd.), neutral protease (model 0.8L, enzyme activity 80,000 U / g, Novozymes (China) Biotechnology Co., Ltd.), papain (enzyme activity 400,000 U / g, Nanning Pangbo Bioengineering Co., Ltd.), and complex lipase (enzyme activity 100,000 U / g, Dongheng Huadao Biotechnology Co., Ltd.), are all commercially available enzymes that can be purchased and obtained by those skilled in the art. In addition, unless otherwise specified, other raw materials used in this invention can also be conventional commercial products in the field or prepared by conventional methods in the field. That is, reagents, instruments, consumables used in this invention whose manufacturers are not specified can also be purchased from the market.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing precisely active peptides from deep-sea long-lived fish, characterized in that, Includes the following steps: S1. Pretreatment: Remove the gills and internal organs of fresh deep-sea long-lived fish, mince it into fish paste, add water and mix, and heat treat at 54℃~56℃ for 0.5h~1.5h to obtain pre-hydrolysate. S2. First hydrolysis: Adjust the pH of the pre-hydrolysate to 8.0~9.0, add alkaline protease, and hydrolyze at 50℃~60℃ for 1h~3h. After inactivating the enzyme, the first hydrolysate is obtained. S3. Second hydrolysis: Adjust the pH of the first hydrolysate to 2.5~3.5, add acidic protease, and hydrolyze at 45℃~65℃ for 1.5h~2.5h. After enzyme inactivation, the second hydrolysate is obtained. S4. Post-processing: The second hydrolysate is centrifuged, decolorized and deodorized with activated carbon, filtered with diatomaceous earth, ultrafiltered, nanofiltered, desalinated and concentrated, and spray-dried to obtain deep-sea long-lived fish precise active peptide powder. Among them, the molecular weight cutoff of ultrafiltration is <5000Da; the content of precise active peptides of deep-sea long-lived fish obtained by concentration is 10%~15%.

2. The method for preparing precise bioactive peptides from deep-sea long-lived fish according to claim 1, characterized in that: In step S1, the minced fish paste is mixed with water at a mass ratio of 1:(2~4); In step S2, the amount of alkaline protease added is 0.3% to 0.7% of the mass of the fish foam; In step S3, the amount of acidic protease added is 0.5% to 1.5% of the mass of fish foam.

3. The method for preparing precise bioactive peptides from deep-sea long-lived fish according to claim 2, characterized in that: The alkaline protease has an enzyme activity of 0.73 million U / g to 2.55 million U / g, and the acidic protease has an enzyme activity of 0.68 million U / g to 2.05 million U / g.

4. The method for preparing precise active peptides from deep-sea long-lived fish according to claim 2, characterized in that, In steps S2 and S3, the enzyme inactivation specifically involves inactivating the enzyme at 84℃~86℃ for 15min~20min.

5. The method for preparing precise active peptides from deep-sea long-lived fish according to claim 2, characterized in that, In step S4, the activated carbon decolorization and deodorization specifically involves: centrifuging the second hydrolysate to obtain the supernatant, heating it to 50℃~60℃, adding activated carbon for treatment for 0.5h~1.5h, centrifuging again to obtain the supernatant, and passing it through a diatomaceous earth and 0.45nm aqueous membrane. The activated carbon is added at a rate of 3.0% to 5.0% of the mass of the fish foam.

6. The method for preparing precise bioactive peptides from deep-sea long-lived fish according to claim 5, characterized in that, The centrifugation is specifically performed at 5000 rpm for 5 to 10 minutes.

7. A precise active peptide from deep-sea long-lived fish, characterized in that: The deep-sea long-lived fish precise active peptide was prepared according to any one of claims 1 to 6.

8. A functional product, characterized in that: Its components include the deep-sea long-lived fish precise active peptides as described in claim 7.

9. The functional product according to claim 8, characterized in that, Includes at least one of the following functions: (1) Antioxidant; (2) Inhibits xanthine oxidase activity; (3) Inhibit the secretion of inflammatory factors; (4) Inhibits uric acid synthesis and reabsorption; (5) Promotes uric acid excretion.

10. The use of the deep-sea long-lived fish precise active peptide composition as described in claim 7 in the preparation of a product for significantly scavenging hydroxyl radicals, activating SOD activity, inhibiting xanthine oxidase activity in vitro, reducing the content of reactive oxygen species (ROS) in HK-2 cells, reducing the content of uric acid in HK-2 cells, reducing the content of interleukin-1β (IL-1β) in HK-2 cells, reducing the content of nitric oxide (NO) in HK-2 cells, inhibiting the mRNA expression of xanthine oxidase (XO), urea transporter 1 (URAT1), and tumor necrosis factor-α (TNF-α), and promoting the mRNA expression of ATP binding cassette subfamily G Member 2 (ABCG2).

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