Soybean peptide with efficacy of preventing and / or treating chronic nephritis, and preparation method and application thereof

Abstract

The application provides a soybean peptide with the efficacy of preventing and / or treating chronic nephritis, and a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The application provides a preparation method of a soybean peptide mixture, adopts a three-step enzymolysis mode, controls the molecular weight of the soybean peptide below 1000 Dalton, mainly uses small molecular oligopeptide, and the prepared soybean peptide mixture has low sodium, phosphorus and potassium contents, and can improve the nutritional status and muscle loss of patients with nephritis. The prepared soybean peptide mixture is further separated and purified, and the purified soybean peptide mixture, single-chain polypeptide S1 and single-chain polypeptide S2 are obtained, the content of uric acid and creatinine can be effectively reduced, inflammation can be inhibited, and the soybean peptide is expected to be developed into a novel polypeptide drug for preventing and / or treating chronic nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a soybean peptide with preventive and / or therapeutic effects on chronic nephritis, its preparation method, and its application. Background Technology

[0002] Chronic kidney disease (CKD) has become a significant public health problem threatening human health worldwide. Most CKD patients are asymptomatic in the early stages, and are often diagnosed at an advanced stage, reducing the opportunity for early intervention. Treatment costs for CKD are high, especially after reaching end-stage renal disease (ESRD), where dialysis and kidney transplantation place a heavy burden on patients' families and the social healthcare system.

[0003] Currently, the main goals of drug intervention for CKD are to control disease progression, delay renal failure, alleviate symptoms, and reduce the occurrence of complications. Drug intervention often proceeds from the following angles: 1. Reducing proteinuria and protecting renal function, using angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) to reduce protein excretion in the urine, lower intraglomerular pressure, and delay the deterioration of renal function. ACEIs / ARBs are first-line drugs for CKD patients with hypertension or proteinuria, but long-term use may cause hyperkalemia. 2. Lowering blood sugar: SGLT-2 inhibitors (such as dapagliflozin and empagliflozin) have been shown to have significant nephroprotective effects, reducing proteinuria and slowing the decline in renal function. SGLT-2 inhibitors are gradually becoming an important option for patients with diabetes and CKD, but some patients may have poor tolerance. 3. Controlling hypertension: ACEIs, ARBs, calcium channel blockers (CCBs), and beta-blockers are used. Blood pressure in CKD patients needs to be controlled below 130 / 80 mmHg (when accompanied by proteinuria). Blood pressure control is an important measure to slow the progression of CKD. Combination therapy is common, but it can easily lead to hypotension or electrolyte disturbances. In addition, other complications of chronic kidney disease include anemia, abnormal bone metabolism, hyperkalemia, and abnormal acid metabolism, which are often treated with different medications. However, drug intervention often has the following limitations: 1. Drug side effects: Many drugs may cause problems such as hyperkalemia, hypotension, and gastrointestinal reactions; 2. Poor long-term adherence: Patients need to use multiple drugs in combination for a long time, resulting in low adherence; 3. Limited etiological treatment: Existing drugs are mostly symptomatic treatments and are difficult to reverse renal function decline; 4. Insufficient individualized treatment: Drug selection needs to be adjusted according to the condition, comorbidities, and patient tolerance, but this is difficult to fully implement in clinical practice; 5. Heavy economic burden: New drugs (such as SGLT-2 inhibitors) are expensive, limiting their widespread use. Therefore, to address the limitations of drug intervention, it is necessary to combine it with non-drug methods such as nutritional intervention and lifestyle modifications to comprehensively manage the condition and quality of life of CKD patients. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of this invention is to provide a soybean peptide mixture with low sodium, phosphorus and potassium content and its preparation method, which has the effect of improving the nutritional status and muscle loss of patients with nephritis.

[0005] The present invention also aims to provide a purified soybean peptide mixture, as well as single-chain polypeptides S1 and S2, obtained by further separation and purification of the soybean peptide mixture, which can effectively reduce uric acid and creatinine levels and inhibit inflammation, and is expected to be developed into a novel polypeptide drug for the prevention and / or treatment of chronic kidney disease.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a soybean peptide mixture, comprising the following steps:

[0008] (1) Soy protein is added to deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%.

[0009] (2) Grind and homogenize the soybean protein solution to obtain a soybean protein homogenate with a particle size of 30-80 μm;

[0010] (3) Keep the soybean protein homogenate at 53-58℃ and stir for 0.5-1h. Adjust the pH to 9.5-10.5, add alkaline protease, and hydrolyze until the pH drops to 7.0-7.5. Then add papain and neutral protease, and hydrolyze for 3.5-4.5h. Add flavor protease and continue hydrolyzing for 2-2.5h to obtain the hydrolysate.

[0011] (4) The enzyme hydrolysate is heated to 108-112℃ and held for 3-10 minutes, then cooled to room temperature and filtered with a ceramic membrane. The clear liquid filtered by the ceramic membrane is filtered with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain the nanofiltration membrane cutoff liquid.

[0012] (5) The nanofiltration membrane intercept liquid is passed through the cation exchange resin and the anion exchange resin in sequence at a flow rate of 48-52 L / h. The material adsorbed by the resin is concentrated, sterilized and dried to obtain a soybean peptide mixture.

[0013] Preferably, based on the mass of the soybean protein homogenate, the amount of alkaline protease added is 1.5 to 2.5 million U / g, the amount of papain added is 700,000 to 900,000 U / g, the amount of neutral protease added is 2.5 to 3.5 million U / g, and the amount of flavor protease added is 400,000 to 600,000 U / g.

[0014] Preferably, the soybean peptide mixture is separated and purified by preparative high performance liquid chromatography (HPLC) using a gradient elution with mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water and mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile to obtain the purified soybean peptide mixture.

[0015] Preferably, the preparation method of the sample solution separated and purified by the preparative high performance liquid chromatograph includes: dissolving the soybean peptide mixture in mobile phase A to prepare a soybean peptide solution, centrifuging, filtering the supernatant using a filter membrane, and using the filtrate as the sample solution.

[0016] Preferably, the elution program for separation and purification using the preparative high-performance liquid chromatography is: 0–5 min 0%–5% B, 5–20 min 5% B–80% B; the elution fraction collected after 11–12 min is the purified soybean peptide mixture.

[0017] The present invention also provides a soybean peptide mixture prepared by the above preparation method.

[0018] Preferably, the soybean peptide mixture includes peptide S1 and peptide S2.

[0019] The present invention also provides a polypeptide S1, wherein the polypeptide S1 is a single-chain polypeptide with the structural sequence YDPSTGIY and a molecular weight of 914.95 Da.

[0020] The present invention also provides a polypeptide S2, wherein the polypeptide S2 is a single-chain polypeptide with the structural sequence VNEPAPGLK and a molecular weight of 924.05 Da.

[0021] The present invention also provides the use of the above-mentioned soybean peptide mixture or the above-mentioned polypeptide S1 or the above-mentioned polypeptide S2 in the preparation of a medicament for the prevention and / or treatment of chronic nephritis.

[0022] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0023] This invention uses a three-step enzymatic hydrolysis method to prepare a soybean peptide mixture with low sodium, phosphorus, and potassium content. The molecular weight of the soybean peptides is controlled below 1000 Daltons, accounting for more than 96%, mainly consisting of small molecule oligopeptides. The sodium content of the obtained soybean peptide mixture is controlled at about 26 mg / 100g, the potassium content is controlled at about 0.12 mg / 100g, and it contains almost no phosphorus. By controlling sodium, phosphorus, and potassium, the burden on the kidneys of patients with chronic nephritis is reduced. Through the intake of soybean peptides, the nutritional status and muscle loss of patients with chronic nephritis are improved.

[0024] This invention further separates and purifies a mixture of soybean peptides to obtain a purified mixture of soybean peptides. The structure of the purified mixture of soybean peptides is then identified, revealing a single-chain polypeptide S1 with the structure YDPSTGIY and a single-chain polypeptide S2 with the structure VNEPAPGLK. Polypeptides S1 and S2 are not degraded by digestive proteases and can effectively reduce uric acid and creatinine levels and inhibit inflammation. They are expected to be developed into novel polypeptide drugs for the prevention and / or treatment of chronic kidney disease. Attached Figure Description

[0025] Figure 1 Standard curve for creatinine detection;

[0026] Figure 2 Chromatogram of the soybean peptide mixture prepared in Example 1;

[0027] Figure 3 Chromatogram of component 12 obtained in Example 2;

[0028] Figure 4 : Secondary mass spectrum of single-chain polypeptide S1;

[0029] Figure 5 : Secondary mass spectrum of single-chain polypeptide S2;

[0030] Figure 6 Typical fluorescence images of zebrafish nephritis cells in each group;

[0031] Figure 7 : Pathological examination of liver tissue of experimental mice (HE staining method, 20×);

[0032] Figure 8 : Pathological examination of kidney tissue in experimental mice (HE staining method, 20×). Detailed Implementation

[0033] This invention provides a method for preparing a soybean peptide mixture, comprising the following steps:

[0034] (1) Soy protein is added to deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%.

[0035] (2) Grind and homogenize the soybean protein solution to obtain a soybean protein homogenate with a particle size of 30-80 μm;

[0036] (3) Keep the soybean protein homogenate at 53-58℃ and stir for 0.5-1h. Adjust the pH to 9.5-10.5, add alkaline protease, and hydrolyze until the pH drops to 7.0-7.5. Then add papain and neutral protease, and hydrolyze for 3.5-4.5h. Add flavor protease and continue hydrolyzing for 2-2.5h to obtain the hydrolysate.

[0037] (4) The enzyme hydrolysate is heated to 108-112℃ and held for 3-10 minutes, then cooled to room temperature and filtered with a ceramic membrane. The clear liquid filtered by the ceramic membrane is filtered with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain the nanofiltration membrane cutoff liquid.

[0038] (5) The nanofiltration membrane intercept liquid is passed through the cation exchange resin and the anion exchange resin in sequence at a flow rate of 48-52 L / h. The material adsorbed by the resin is concentrated, sterilized and dried to obtain a soybean peptide mixture.

[0039] In step (1) of this invention, the soybean protein is commercially available soybean protein; the deionized water with a pH of 9.5 to 10.5 is obtained by adjusting the deionized water with 30% NaOH, and the pH value of the deionized water is preferably 10; the water temperature of the deionized water is 45 to 55°C, preferably 50°C; and the concentration of the soybean protein solution is preferably 5%.

[0040] In step (2) of the present invention, the grinding is preferably carried out by a colloid mill, and the grinding time is 1 to 2 hours, preferably 1.5 hours; the homogenization is preferably carried out by a high-pressure homogenizer, and the pressure is 45 to 55 MPa, preferably 50 MPa.

[0041] In step (3) of this invention, the soybean protein homogenate is preferably kept at 55°C, and the pH value is preferably adjusted to 10; the pH is adjusted using 30% NaOH; based on the mass of the soybean protein homogenate, the amount of alkaline protease added is 1.5 million to 2.5 million U / g, preferably 2 million U / g, the amount of papain added is 700,000 to 900,000 U / g, preferably 800,000 U / g, the amount of neutral protease added is 2.5 million to 3.5 million U / g, preferably 3 million U / g, and the amount of flavor protease added is 400,000 to 600,000 U / g, preferably 500,000 U / g.

[0042] In step (4) of this invention, the enzymatic hydrolysate is preferably heated to 110°C and held for 5 minutes; the ceramic membrane has a pore size of 45-55 nm, preferably 50 nm. This invention can filter out large molecular proteins that have not been completely hydrolyzed through the ceramic membrane, and filter out small molecular substances such as free amino acids and sodium chloride in the enzymatic hydrolysate through the nanofiltration membrane.

[0043] In step (5) of this invention, cation exchange resin and anion exchange resin are used to adsorb heavy metals in the enzymatic hydrolysate. The nanofiltration membrane intercepting solution is preferably passed sequentially through the cation exchange resin and anion exchange resin at a flow rate of 50 L / h. As an optional embodiment, the cation exchange resin is pretreated before use. The pretreatment steps include: passing a 4% sodium hydroxide solution (4 times the resin volume) through the resin column at a flow rate of 1-2 times the resin volume per hour; rinsing the resin with pure water after the process is complete; stopping the rinsing when the outlet pH is neutral; then passing a 4% hydrochloric acid solution (4 times the resin volume) through the resin column at a flow rate of 1-2 times the resin volume per hour; rinsing the resin with pure water after the process is complete; stopping the rinsing when the outlet pH is neutral; and then preparing the solution for use. As an optional implementation, the anion exchange resin is pretreated before use. The pretreatment steps include: passing a 4% sodium hydroxide solution (3 times the resin volume) through the resin column at a flow rate of twice the resin volume per hour; washing the resin with pure water until the outlet pH is <9; passing a 4% hydrochloric acid solution (3 times the resin volume) through the resin column at a flow rate of twice the resin volume per hour; washing the resin with pure water until the outlet pH is >5; and then setting it aside for use.

[0044] In step (5) of this invention, the concentration is preferably carried out by double-effect falling film concentration to increase the material concentration to 35% to 40%; the sterilization is preferably carried out by high-temperature sterilization machine, and the sterilization conditions are 115°C and 3s; the drying is preferably carried out by spray drying, using a spray drying tower, and the drying conditions are: air inlet temperature 180 to 190°C, air outlet temperature 70 to 80°C, and atomization pressure 150 to 160 Bar.

[0045] The soybean peptide mixture prepared by this invention has high protein and peptide content and low sodium, phosphorus and potassium content, which can effectively reduce the uric acid and creatinine content in zebrafish nephritis models and has an auxiliary effect in improving nephritis.

[0046] The soybean peptide mixture of the present invention is separated and purified using preparative high-performance liquid chromatography (HPLC). The separation and purification involves gradient elution using mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water, and mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile to obtain the purified soybean peptide mixture. The preparation of the sample loading solution of the present invention includes: dissolving the soybean peptide mixture in mobile phase A to prepare a soybean peptide solution, centrifuging, and filtering the supernatant using a filter membrane; the filtrate is the sample loading solution. The concentration of the soybean peptide solution is preferably 8–12 mg / mL, more preferably 10 mg / mL; the centrifugation conditions are preferably 9000–11000 r / min for 3–8 min, more preferably 10000 r / min for 5 min; the filter membrane is preferably a 0.22 μm filter membrane. The elution procedure of the present invention is as follows: 0-5 min 0%-5% B, 5-20 min 5% B-80% B; the elution fraction collected after 11-12 min is a purified soybean peptide mixture; the sample loading volume during elution is preferably 15 mL.

[0047] The soybean peptide mixture obtained by separation and purification using preparative high-performance liquid chromatography can effectively reduce the uric acid and creatinine levels in a zebrafish nephritis model, and has an auxiliary effect in improving nephritis.

[0048] The soybean peptide mixture prepared by this invention was structurally identified by nano-level liquid chromatography-Q EXACTIVE mass spectrometry, and two peptides, including peptide S1 and peptide S2, were identified.

[0049] The polypeptide S1 described in this invention is a single-chain polypeptide with the structural sequence YDPSTGIY(H-Tyr-Asp-Pro-Ser-Thr-Gly-Ile-Tyr-OH) and a molecular weight of 914.95 Da. The polypeptide S2 is also a single-chain polypeptide with the structural sequence VNEPAPGLK(H-Val-Asn-Glu-Pro-Ala-Pro-Gly-Leu-Lys-OH) and a molecular weight of 924.05 Da. The polypeptides S1 and S2 prepared in this invention exhibit significant uric acid-lowering, creatinine-lowering, and anti-inflammatory effects, while also possessing good antioxidant activity. Polypeptide S1 shows superior effects compared to polypeptide S2, demonstrating a significant renal protective effect.

[0050] The present invention also provides the use of the above-mentioned soybean peptide mixture or the above-mentioned polypeptide S1 or the above-mentioned polypeptide S2 in the preparation of a drug for the prevention and / or treatment of chronic nephritis, which is expected to be developed into a novel polypeptide drug for improving or treating chronic kidney disease.

[0051] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0052] In the specific embodiments and comparative examples of this invention, soybean protein was purchased from Shandong Yuwang Ecological Food Industry Co., Ltd.; alkaline protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; papain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.; neutral protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; flavor protease was purchased from Novozymes (China) Biotechnology Co., Ltd.; potassium oxonate (PO) was purchased from Beijing Solarbio Technology Co., Ltd., with a purity ≥98%; allopurinol tablets were purchased from Hefei Jiulian Pharmaceutical Co., Ltd.; sodium carboxymethyl cellulose (CMC-Na) was purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; uric acid, xanthine oxidase, and adenosine deaminase kits were purchased from Nanjing Jiancheng Bioengineering Institute; urea nitrogen and creatinine kits were purchased from Ningbo Meikang Biotechnology Co., Ltd.; and all other reagents were of analytical grade.

[0053] Unless otherwise specified, the following embodiments are all conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] Example 1

[0056] Preparation of soybean peptide mixture:

[0057] 1. Prepare 1000L of deionized water (conductivity less than 20 μS / cm) and heat it to 50℃. Adjust the pH of the deionized water to 10.0 using 30% NaOH. Take 50kg of soybean protein for slurry preparation to obtain a 5% soybean protein solution.

[0058] 2. Stir the soybean protein solution evenly, process it with a colloid mill for 1.5 hours, and then process it with a high-pressure homogenizer at 50 MPa for 2 hours to obtain a homogenate. The particle size of the protein particles in the homogenate is 30-80 μm.

[0059] 3. Heat the homogenate to 55℃ and keep it at that temperature while stirring for 0.5 to 1 hour.

[0060] 4. The pH of the homogenate after heat preservation treatment is adjusted to 10.0 with 30% NaOH solution, and the temperature is kept constant.

[0061] 5. Add alkaline protease (2 million U / g) to the homogenate from step 4. During enzymatic hydrolysis, continuously monitor the pH. When the pH drops to 7.0-7.5, add papain (800,000 U / g) and neutral protease (3 million U / g). Continue enzymatic hydrolysis for 4 hours, then add flavor protease (500,000 U / g) and continue enzymatic hydrolysis for 2-2.5 hours.

[0062] 6. Heat the enzymatic hydrolysate obtained in step 5 to 110°C and hold for 5 minutes to inactivate the protease.

[0063] 7. After cooling the enzyme-inactivated hydrolysate to room temperature, pass it through a ceramic membrane with a pore size of 50 nm.

[0064] 8. Collect the clear liquid from the ceramic membrane filtration and filter it using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain the nanofiltration membrane cutoff liquid.

[0065] 9. Use anion exchange resin and cation exchange resin to adsorb heavy metals in the enzymatic hydrolysate.

[0066] 9.1 Pretreatment of cation exchange resin

[0067] (1) Resin column packing: Weigh 50 kg of cation exchange resin (Langfang Jinan Resin Co., Ltd. D113 model) using an electronic scale, compact it, and transfer it to the resin column with deionized water, leaving 3-5 cm of water at the top of the resin.

[0068] (2) Resin pretreatment: Pass 4 times the resin volume of 4% sodium hydroxide solution through the resin column at a flow rate of 1 to 2 times the resin volume per hour. After the pretreatment, rinse the resin with pure water and stop rinsing when the outlet pH is neutral. Then pass 4 times the resin volume of 4% hydrochloric acid solution through the resin column at a flow rate of 1 to 2 times the resin volume per hour. After the pretreatment, rinse the resin with deionized water and stop rinsing when the outlet pH is neutral. The resin is then ready for use.

[0069] 9.2 Anion Exchange Resin Pretreatment

[0070] (1) Resin column packing: Weigh 50kg of anion exchange resin (D301 model from Langfang Jinan Resin Co., Ltd.) using an electronic scale, compact it, and transfer it to the resin column with deionized water, leaving 3-5cm of water at the top of the resin column.

[0071] (2) Resin pretreatment: Pass 3 times the resin volume of 4% sodium hydroxide solution through the resin column at a flow rate of 2 times the resin volume per hour. After the process, wash the resin with pure water until the outlet pH is <9. Then pass 3 times the resin volume of 4% hydrochloric acid solution through the resin column at a flow rate of 2 times the resin volume per hour. After the process, wash the resin with deionized water until the outlet pH is >5. The resin is ready for use.

[0072] 9.3 Column Passage: The feed solution to be processed (the nanofiltration membrane effluent obtained in step 8) is passed through the resin column at a flow rate of 50 L / h. The feed solution first passes through the cation exchange resin and then through the anion exchange resin. The conductivity of the effluent is detected. When the feed solution concentration is 5%, the conductivity is higher than 500 μSiemens per centimeter (µs / cm). If the requirement is not met, the feed solution passage is stopped.

[0073] 9.4 Water washing: Replace the residual liquid in the resin column with 1 to 2 times the volume of pure water.

[0074] 10. The material after resin adsorption treatment is concentrated using a double-effect falling film method to increase the material concentration to 35% to 40%.

[0075] 11. The concentrated material is sterilized using a high-temperature sterilizer at 115℃ for 3 seconds.

[0076] 12. After sterilization, the material is dried using a spray drying tower. The drying conditions are: inlet air temperature 180-190℃, outlet air temperature 70-80℃, and atomization pressure 150-160 Bar. Finally, 25 kg of soybean peptide mixture is obtained after spray drying.

[0077] The basic physicochemical properties of the soybean peptide mixture were determined according to the national standard for soybean peptides GB / T 22492-2008. The results are shown in Table 1.

[0078] Table 1. Basic physicochemical properties of the soybean peptide mixture prepared in Example 1.

[0079] 95.6 4.2 0.11 Carbohydrate content (%) Fat content (%) Peptide content (%) 0.05 0.04 96.3 Sodium (mg / 100g) Potassium (mg / 100g) Phosphorus (mg / 100g) 26.2 0.12 0

[0080] As shown in Table 1, the present invention prepares a soybean peptide mixture with low sodium, phosphorus, and potassium content.

[0081] Comparative Example 1

[0082] Preparation of soybean peptide mixture:

[0083] Steps 1 to 8 are the same as in Example 1.

[0084] 9. The nanofiltration membrane effluent obtained in step 8 is concentrated using a double-effect falling membrane to increase the material concentration to 35%–40%.

[0085] 10. The concentrated material is sterilized using a high-temperature sterilizer at 115°C for 3 seconds.

[0086] 11. After sterilization, the material is dried using a spray drying tower. The drying conditions are: inlet air temperature 180-190℃, outlet air temperature 70-80℃, and atomization pressure 150-160 Bar. Finally, 25 kg of soybean peptide mixture is obtained after spray drying.

[0087] The basic physicochemical properties of the soybean peptide mixture were determined according to the national standard for soybean peptides GB / T 22492-2008. The results are shown in Table 2.

[0088] Table 2. Basic physicochemical properties of the soybean peptide mixture prepared in Comparative Example 1.

[0089] 89.79 4.1 5.9 Carbohydrate content (%) Fat content (%) Peptide content (%) 0.12 0.09 83.9 Sodium (mg / 100g) Potassium (mg / 100g) Phosphorus (mg / 100g) 2126.1 365.3 86.9

[0090] As shown in Table 2, the soybean peptide mixture prepared in Comparative Example 1 has higher sodium, phosphorus, potassium and ash contents, and slightly lower peptide and protein contents.

[0091] Experimental Example 1

[0092] The efficacy of the soybean peptide mixture prepared in Example 1 and Comparative Example 1 in assisting the improvement of nephritis in zebrafish was evaluated.

[0093] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in six-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Nephritis models were established in the experimental, positive control, and model control groups by inducing nephritis in zebrafish using potassium oxonate combined with aristolochic acid. The experimental groups were administered a water-soluble mixture of soybean peptides (prepared in Example 1 and Comparative Example 1) at concentrations of 50, 250, and 1250 μg / mL, respectively. The positive control group was administered allopurinol at a concentration of 136 μg / mL. The normal control group and the model control group were treated with fish tank water. Uric acid and creatinine levels in the zebrafish were measured after 1 day of treatment in each group.

[0094] 1. Sample preparation: Collect zebrafish from each treatment group, rinse thoroughly with ultrapure water, transfer to 2mL centrifuge tubes, remove as much water as possible, add 500μL of deionized water to each tube, grind evenly with a tissue homogenizer under ice bath conditions to prepare homogenate, centrifuge the homogenate at 10000rpm for 10min, and take the supernatant for later use.

[0095] 2. Uric acid content detection: The supernatant obtained in step 1 was analyzed by high-performance liquid chromatography (HPLC). Chromatographic conditions: detector wavelength: γ = 288 nm; column: Shimadzu C18 (5 μm 4.6 × 250 mm); mobile phase: water (0.2% acetic acid): methanol (V:V) = 94:6; flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 10 μL. The efficacy of soybean peptide components in improving nephritis in zebrafish was evaluated based on statistical analysis of uric acid content. Statistical results are expressed as mean ± SE. The formula for calculating the uric acid-lowering effect is as follows:

[0096]

[0097] Statistical analysis was performed using ANOVA and Dunnett's T-test. P < 0.05 indicated a significant difference. The results are shown in Table 3.

[0098] Table 3. Experimental results on the uric acid-lowering effect of soybean peptide treatment.

[0099]

[0100] The uric acid content in zebrafish treated with soybean peptide mixture was significantly lower than that in the model control group (*P<0.05); compared with the normal control group, the uric acid content in different groups was significantly lower (#P<0.05, ##P<0.01).

[0101] The results showed that the uric acid content in the zebrafish of the model control group was significantly lower than that of the normal control group (P<0.01), indicating successful model establishment. The uric acid content in the zebrafish of the positive control group was significantly lower than that of the model control group (P<0.05), with a 21% reduction in uric acid levels, indicating that allopurinol has a uric acid-lowering effect on zebrafish. In Example 1, compared with the model control group, the 250 μg / mL and 1250 μg / mL concentration groups showed significant differences (P<0.05), suggesting that soybean peptides at concentrations of 250 μg / mL and 1250 μg / mL have a uric acid-lowering effect on zebrafish. In Comparative Example 1, the uric acid content in the soybean peptide mixture group was significantly different from that in the normal control group. Compared with the model control group, the uric acid reduction effects of the 250 μg / mL and 1250 μg / mL concentration groups were 5.3% and 6.3%, respectively. The results indicate that the uric acid-lowering effect of the soybean peptide mixture in Example 1 is superior to that in Comparative Example 1.

[0102] 3. Creatinine Level Detection: Changes in creatinine levels are primarily determined by the kidney's filtration capacity. When kidney function is impaired, creatinine accumulates in the body, leading to elevated levels. A creatinine assay kit was used to detect creatinine levels in different groups of zebrafish.

[0103] Standard curve establishment: Prepare creatinine standard solutions of different concentrations (range: 0, 10, 20, 50, 100, 200 μmol / L). Add reagents to each standard solution according to the kit procedure. After the reaction is complete, measure the absorbance at 570 nm using a microplate reader and record the absorbance values ​​for each concentration. Establish the relationship between concentration and absorbance using the standard curve formula y = kx + b. The standard curve is shown below. Figure 1 As shown.

[0104] Sample testing: Take the supernatant obtained in step 1, add reagents according to the kit instructions and react. After the reaction, measure the absorbance at 570 nm using an ELISA reader and calculate the creatinine content. The results are shown in Table 4.

[0105] Table 4. Experimental results on the effect of soybean peptide treatment on reducing creatinine.

[0106]

[0107]

[0108] The creatinine content of zebrafish treated with soybean peptide mixture was significantly lower than that of the model control group (*P<0.05); compared with the normal control group, the creatinine content of different groups was significantly lower (#P<0.05, ##P<0.01).

[0109] The results showed that the creatinine content of zebrafish in the model control group was significantly lower than that in the normal control group (P<0.01), indicating successful model establishment. The creatinine content of zebrafish in the positive control group was also significantly lower than that in the model control group (P<0.01), indicating that allopurinol had a creatinine-lowering effect on zebrafish. In Example 1, the creatinine content of zebrafish in the soybean peptide mixture group was significantly lower than that in the model control group at concentrations of 250 μg / mL and 1250 μg / mL (P<0.01), suggesting that soybean peptides at these concentrations had a creatinine-lowering effect on zebrafish. In Comparative Example 1, the creatinine content of zebrafish in the soybean peptide mixture group was significantly lower than that in the normal control group. The uric acid content in different groups showed significant differences compared to the model control group. The uric acid reduction effects at concentrations of 250 μg / mL and 1250 μg / mL were 7.4% and 9.5%, respectively, indicating that the uric acid-lowering effect of the soybean peptide mixture in Example 1 was superior to that in Comparative Example 1.

[0110] Example 2

[0111] The soybean peptide mixture prepared in Example 1 was separated and purified using preparative high-performance liquid chromatography (HPLC).

[0112] The separation and purification parameters for preparative high-performance liquid chromatography are as follows:

[0113] Mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water; Mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile;

[0114] Detection wavelength: 214nm; detection time: 30min; column temperature: 21℃~25℃.

[0115] Six column volumes were equilibrated using mobile phase A at a rate of 10 ml / min until the baseline stabilized.

[0116] The obtained soybean peptide mixture was dissolved in mobile phase A to prepare a soybean peptide concentration of 10 mg / mL. The mixture was centrifuged at 10000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The sample loading volume was 15 mL. Gradient elution was performed using a mixture of mobile phase A and mobile phase B, and the elution gradient is shown in Table 5.

[0117] Table 5 Analytical Liquid Mobile Phase Gradient

[0118]

[0119]

[0120] From 0 to 5 min, the concentration of mobile phase B was gradually increased from 0% to 5%; from 5 to 20 min, the concentration of mobile phase B was gradually increased from 5% to 80%; from 20 to 25 min, the concentration of mobile phase B was gradually decreased from 80% to 5%; from 25 to 30 min, the concentration of mobile phase B was maintained at 5% for elution for 5 min.

[0121] Different components were collected every 1 minute, resulting in 16 components. The peak times of the 16 components are shown in Table 6.

[0122] Table 6 shows the peak times of 16 components.

[0123] 1 0~1min 9 8-9 min 2 1-2 min 10 9–10 min 3 2-3 min 11 10-11 min 4 3-4 min 12 11-12 min 5 4-5 minutes 13 12-13 min 6 5-6 min 14 13-14 min 7 6-7 min 15 14-15 min 8 7-8 minutes 16 15-16 min

[0124] The chromatogram of the soybean peptide mixture prepared in Example 1 is shown below. Figure 2 As shown, the chromatogram for the separation and purification of component 12 is as follows. Figure 3 As shown. According to Figure 2 It can be seen that component 12 has a larger peak area in the soybean peptide mixture, indicating that component 12 accounts for a higher proportion in the soybean peptide mixture. Figure 3 It can be seen that the purity of fraction 12 obtained through separation and purification is relatively high. Further analysis of the purity and structure of fraction 12 was performed using a nano-scale liquid chromatography-Q EXACTIVE mass spectrometry system. Detection conditions:

[0125] Mobile phase: Phase A: 100% purified water + 0.1% formic acid; Phase B: 100% acetonitrile + 0.1% formic acid;

[0126] Mobile phase flow rate: 300 nL / min; injection volume: 1 μL supernatant; mobile phase gradient program is shown in Table 7.

[0127] Table 7 Mobile Phase Gradient Program

[0128] A(％) 97 97 63 10 10 97 97 B(％) 3 3 37 90 90 3 3

[0129] The structure of component 12 was identified using nano-level liquid chromatography-Q EXACTIVE mass spectrometry. Two polypeptides were identified. The amino acid composition of S1 is YDPSTGIY(H-Tyr-Asp-Pro-Ser-Thr-Gly-Ile-Tyr-OH), with a molecular weight of 914.95 Da. The secondary mass spectrum is shown below. Figure 4As shown; the amino acid composition of S2 is VNEPAPGLK (H-Val-Asn-Glu-Pro-Ala-Pro-Gly-Leu-Lys-OH), with a molecular weight of 924.05 Da. The secondary mass spectrum is shown below. Figure 5 As shown in the figure. Further analysis using liquid chromatography-mass spectrometry (LC-MS) determined the mass percentages of these two single-chain polypeptides in soybean peptides to be 1.26% and 0.35%, respectively.

[0130] Experimental Example 2

[0131] The efficacy of the soybean peptide mixture components 1-16 purified in Example 2 in assisting the improvement of nephritis in zebrafish was evaluated.

[0132] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in six-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Nephritis models were established in the experimental and model control groups by inducing zebrafish nephritis using potassium oxonate combined with aristolochic acid. The experimental groups were administered water-soluble components 1–16 of a soybean peptide mixture at a concentration of 50 μg / mL. The normal control group and the model control group were treated with fish tank water. Uric acid and creatinine levels in the zebrafish were measured after one day of treatment in each group. The detection methods were the same as in Experiment 1, and the results are shown in Table 8.

[0133] Table 8. Experimental results on the effect of soybean peptide mixture components 1-16 on reducing creatinine and uric acid.

[0134]

[0135] The results showed that component 12 of the soybean peptide mixture had a significantly higher effect on reducing creatinine and uric acid than other components.

[0136] Experimental Example 3

[0137] In vitro digestion experiments were conducted on peptides S1 and S2:

[0138] Preparation of artificial gastric juice: Take 240 mL of HCl solution (pH=2), 1.8 g of pepsin (3500 U / g), shake well, dilute with water to 300 mL, and store in a refrigerator at 4℃ for later use.

[0139] Preparation of artificial small intestinal fluid: Take 2g of potassium dihydrogen phosphate, add 150mL of water to dissolve it, adjust the pH value to 6.8 with 0.1mol / L NaOH solution, take 3g of pancreatic enzyme (3000U / g), add an appropriate amount of water to dissolve it, mix the above prepared solutions, dilute with water to 300mL, and store in a refrigerator at 4℃ for later use.

[0140] Using simulated gastric and intestinal fluids as solvents, peptides S1 and S2 were added respectively at a concentration of 4 mg / mL. 1 mg of sample was added to 20 mL of simulated gastric fluid, incubated at 37°C with shaking at 100 rpm for 2 hours, pH adjusted to 6.8, and then transferred to 20 mL of simulated small intestinal fluid. The mixture was incubated at 37°C with shaking at 100 rpm for 3 hours, and finally, enzyme inactivation was performed at 85°C for 10 minutes. After cooling to room temperature, ultrafiltration and centrifugation were performed, and the filtrate was retained. Mass spectrometry analysis of the filtrate revealed no change in the molecular weights of peptides S1 and S2, indicating that peptides S1 and S2 were not degraded by digestive proteases.

[0141] Test Example 4

[0142] Based on the structures of polypeptides S1 and S2 obtained in Example 2, Shanghai Newp Biotechnology Co., Ltd. was commissioned to synthesize the two single-chain polypeptides for evaluation of their efficacy in assisting the improvement of nephritis in zebrafish.

[0143] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in six-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Nephritis models were established in the experimental group, positive control group, and model control group by inducing nephritis in zebrafish using potassium oxonate combined with aristolochic acid. The experimental group was administered water-soluble soybean peptide S1 and soybean peptide S2 at concentrations of 5, 25, and 125 μg / mL, respectively. The positive control group was administered allopurinol at a concentration of 136 μg / mL. The normal control group and the model control group were treated with fish tank water.

[0144] 1. The uric acid content in zebrafish was measured 1 day after treatment in each group. The detection method was the same as in Experiment 1. The results are shown in Table 9.

[0145] Table 9. Experimental results of uric acid reduction effect after treatment with soybean peptides S1 and S2.

[0146]

[0147] Compared with the model control group, the uric acid content of zebrafish treated with soybean peptides was *P<0.05; compared with the normal control group, the uric acid content of different groups was #P<0.05, and ##P<0.01.

[0148] The results showed that the uric acid content in the zebrafish of the model control group was significantly lower than that of the normal control group (P<0.01), indicating successful model establishment. The uric acid content in the zebrafish of the positive control group was significantly lower than that of the model control group (P<0.01), with a 28.2% reduction in uric acid levels, indicating that allopurinol has a uric acid-lowering effect on zebrafish. Compared with the model control group, the uric acid reduction effects of the soybean peptide S1 group at concentrations of 5 μg / mL and 25 μg / mL were 11.4% and 14.5% respectively (P<0.05), and at the 125 μg / mL concentration group, the reduction was 24.7% (P<0.01), suggesting that soybean peptide S1 has a uric acid-lowering effect on zebrafish. Compared with the model control group, the uric acid reduction effects of the soybean peptide S2 group at concentrations of 25 μg / mL and 125 μg / mL were 11.3% and 19.3% respectively (P<0.05), indicating that soybean peptide S1 has a better uric acid-lowering effect than soybean peptide S2.

[0149] 2. The creatinine content in zebrafish was measured 1 day after treatment in each group. The detection method was the same as in Experiment 1. The results are shown in Table 10.

[0150] Table 10. Experimental results on the creatinine reduction effect after treatment with soybean peptides S1 and S2.

[0151]

[0152] The creatinine content of zebrafish treated with soybean peptides was significantly lower than that of the model control group (*P<0.05); compared with the normal control group, the creatinine content of different groups was significantly lower (#P<0.05, ##P<0.01).

[0153] The results showed that the creatinine content in the model control group was significantly lower than that in the normal control group (P<0.01), indicating successful model establishment. The creatinine content in the positive control group was significantly lower than that in the model control group (P<0.01), with a creatinine reduction effect of 18.5%, indicating that allopurinol has a creatinine-reducing effect on zebrafish. Compared with the model control group, the creatinine reduction effects of the soybean peptide S1 group at concentrations of 25 μg / mL and 125 μg / mL were 11.7% and 20.1%, respectively (P<0.01), suggesting that soybean peptide S1 has a creatinine-reducing effect on zebrafish at concentrations of 25 μg / mL and 125 μg / mL. Compared with the normal control group, the uric acid content in the soybean peptide S2 group showed significant differences in all groups. Compared with the model control group, the uric acid reduction effects of the 25 μg / mL and 125 μg / mL concentration groups were 7.7% and 14.6%, respectively, indicating that soybean peptide S1 had a better creatinine-reducing effect than soybean peptide S2.

[0154] 3. Kidney Inflammation Detection: After 18 hours of treatment, 10 zebrafish from each group were randomly selected and photographed under a fluorescence microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity (H) of inflammatory cells in the zebrafish kidneys was analyzed. The statistical analysis results of this index were used to evaluate the inhibitory efficacy of the samples against kidney inflammation. Statistical results are expressed as mean ± SE. The formula for calculating the inhibitory efficacy of the samples against kidney inflammation is as follows:

[0155]

[0156] The results are as follows Figure 6 As shown in Table 11. Figure 6 In the image, the red fluorescent cells represent zebrafish nephritis cells.

[0157] Table 11. Experimental results evaluating the efficacy of each group in inhibiting renal inflammatory cells (n=10)

[0158]

[0159] Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0160] The results showed that the fluorescence intensity of zebrafish kidney inflammatory cells at the 5 μg / mL soybean peptide S1 concentration was 295,055 pixels, which was significantly lower than that of the model control group (P < 0.01), indicating that soybean peptide S1 had a significant inhibitory effect on the growth of zebrafish kidney inflammation at this concentration. The fluorescence intensities of zebrafish kidney inflammatory cells at the 25 μg / mL and 125 μg / mL soybean peptide S1 concentrations were 250,933 and 219,110 pixels, respectively, which were significantly lower than those of the model control group (P < 0.001), with inflammation inhibition rates of 29.7% and 38.61%, respectively. This indicates that soybean peptide S1 has an inhibitory effect on zebrafish kidney inflammation. The fluorescence intensities of zebrafish kidney inflammatory cells at the 5 μg / mL and 125 μg / mL soybean peptide S2 concentrations were 302,772 pixels and 278,807 pixels, respectively, which were significantly lower than those of the model control group (P < 0.01), indicating that soybean peptide S2 had a significant inhibitory effect on zebrafish kidney inflammation at this concentration. The fluorescence intensity of inflammatory cells in zebrafish kidneys at a concentration of 125 μg / mL of soybean peptide S2 was 253213 pixels, which was significantly lower than that in the model control group (P < 0.001), indicating an inflammatory inhibitory effect of 29.06%. This demonstrates that soybean peptide S2 also has an inhibitory effect on kidney inflammation in zebrafish.

[0161] 4. The supernatant was obtained using the sample preparation steps in Example 1. The MDA content, CAT activity, and GSH content were detected using MDA, CAT, and GSH kits. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; P < 0.05 indicated statistical significance. The results are shown in Table 12.

[0162] Table 12 Evaluation results of MDA content, CAT activity and GSH content in each group (n=3)

[0163]

[0164]

[0165] Compared with the model control group, **P<0.01, ***P<0.001.

[0166] The results showed that compared with the model control group, the normal control group and the zebrafish showed significantly lower levels of MDA (P<0.001), CAT activity (P<0.01), and GSH content (P<0.01), indicating successful model establishment. The positive control group showed significantly lower levels of MDA, CAT activity, and GSH compared with the model control group (P<0.01), indicating that allopurinol has good antioxidant activity. Compared with the model control group, the soybean peptide S1 group showed significantly lower levels of MDA, CAT activity, and GSH at both the 25 μg / mL and 125 μg / mL concentrations. Compared with the model control group, the soybean peptide S2 group at a concentration of 25 μg / mL showed significantly lower MDA content (P<0.01), CAT activity (P<0.01), and GSH content (P<0.01), while the group at a concentration of 125 μg / mL showed significantly lower MDA content (P<0.01), CAT activity (P<0.001), and GSH content (P<0.01). This suggests that soybean peptides S1 and S2 exhibit excellent antioxidant activity at concentrations of 25 μg / mL and 125 μg / mL, specifically by reducing MDA content, increasing GSH content in zebrafish, and enhancing CAT activity.

[0167] Experimental Example 5

[0168] Based on the structure of polypeptide S1 obtained in Example 2, it was synthesized by Shanghai Newp Biotechnology Co., Ltd., and its efficacy in assisting the improvement of nephritis in rats was evaluated.

[0169] Experimental animals: Ninety 5-week-old male C57BL / 6J mice of SPF grade, with body weight of (20.0±2) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The production license number of experimental animals is SCXK (Jing) 2021-0006. The use license number of the experimental institution is SYXK (Lu) 20190005. The animals were housed in an animal room with constant temperature (22±2)°C, constant humidity (55±5)%, and a 12h / 12h light-dark alternation cycle. The conventional feed was purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and the mice had free access to food and drinking water. According to the principle of equal body weight distribution, the mice were randomly divided into 9 groups according to the test items, with 10 mice in each group.

[0170] Test substance dose setting: The recommended human intake of the test sample, the soybean peptide (the soybean peptide mixture prepared in Example 1), is 10 g / 60 kg.bw per person per day, which is equivalent to 0.1667 mg / g.bw per day. The low-, medium- and high-dose groups for mice are determined as 5 times, 10 times and 30 times the recommended human intake, respectively. That is, the daily intake of mice in each dose group of the sample is 0.8335 mg / g.bw, 1.667 mg / g.bw and 5 mg / g.bw, respectively. The recommended human intake of soybean peptide S1 is 1 g / 60 kg.bw per person per day, which is equivalent to 0.01667 mg / g.bw per day. The low-, medium- and high-dose groups for mice are determined as 5 times, 10 times and 30 times the recommended human intake, respectively. That is, the daily intake of mice in each dose group of the sample is 0.08335 mg / g.bw, 0.1667 mg / g.bw and 0.5 mg / g.bw, respectively. The recommended human intake of allopurinol tablets for the positive control group is 0.2 g / 60 kg.bw, which is equivalent to 3.3 mg / kg.bw. Calculated according to the equivalent dose ratio converted by body surface area between humans and animals specified in *Pharmacological Experimental Methodology Appendix*, the daily intake for mice is 46.2 mg / kg.bw.

[0171] Experimental grouping: After 7 days of observation with maintenance feed for mice in a barrier system, the mice were randomly divided into 9 groups according to body weight, with 10 mice in each group. The groups were respectively normal control group, model control group, positive control group, low-dose soybean peptide group, medium-dose soybean peptide group, high-dose soybean peptide group, low-dose soybean peptide S1 group, medium-dose soybean peptide S1 group, and high-dose soybean peptide S1 group.

[0172] Model Construction and Sample Intervention: At 9:00 AM after the adaptation period, except for the normal control group which was administered 0.50% CMC-Na solution by gavage, all other groups were administered 250 mg / kg of PO (suspended in 0.50% CMC-Na solution) by gavage for model construction, with a gavage volume of 0.1 mL / 10 g bw. At 2:00 PM, mice were administered the test substance by gavage. The concentrations of the test substance aqueous solution in the low, medium, and high dose groups of soybean peptide were 83.35 mg / mL, 166.7 mg / mL, and 500 mg / mL, respectively. The concentrations of the test substance aqueous solution in the low, medium, and high dose groups of soybean peptide S1 were 8.335 mg / mL, 16.67 mg / mL, and 50 mg / mL, respectively. The positive control group received 4.62 mg / mL. The normal control group and the model control group were administered the same volume of deionized water instead of the test substance solution by gavage, with a gavage volume of 0.1 mL / 10 g bw. On day 33, urine was collected over 24 hours using a metabolic cage; on day 35, after each group was administered the drug via gavage for 30 minutes, blood was collected by enucleation, and liver and kidney tissues were dissected to measure various indicators of the auxiliary function in lowering uric acid.

[0173] 1. Measurement of mouse body weight and organ index: During the feeding process, the body weight of the experimental animals was measured weekly. After enucleation and blood collection, the experimental animals were dissected, and the liver and kidneys were removed and weighed wetly. The organ index was calculated as follows: Organ index (%) = Organ weight (g) / Experimental animal body weight (g) × 100%. The results are shown in Table 13.

[0174] Table 13 Body weight and organ index of mice in each group

[0175] normal control group 10 49.05±3.21 4.51±0.36 1.45±0.19 Model control group 10 46.12±5.35 4.01±0.41 1.25±0.35 Positive control group 10 48.91±4.12 4.39±0.61 1.41±0.13 Low-dose soybean peptide group 10 47.32±4.20 4.44±0.83 1.26±0.10 Medium-dose group of soybean peptides 10 47.85±3.99 4.21±0.65 1.29±0.23 High-dose soybean peptide group 10 48.59±4.30 4.49±0.72 1.35±0.31 Low-dose group of soybean peptide S1 10 46.55±4.33 4.19±0.58 1.26±0.15 Medium-dose group of soybean peptide S1 10 47.61±3.12 4.34±0.66 1.25±0.22 High-dose group of soybean peptide S1 10 47.72±4.68 4.39±0.28 1.33±0.15

[0176] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0177] The results showed that, compared with the normal control group, the body weight of all model groups decreased, but there was no significant difference in final body weight (P>0.05). Compared with the model control group, there were no statistically significant differences in body weight, liver index, and kidney index among the groups (P>0.05).

[0178] 2. Determination of serum uric acid, blood urea nitrogen, and serum creatinine levels: After blood collection, let it stand at room temperature for 1 hour, centrifuge at 3000 r / min for 10 min, collect serum, and determine the levels of SUA, BUN, and SCr according to the kit. The results are shown in Table 14.

[0179] Table 14. Serum uric acid, blood urea nitrogen, and serum creatinine levels in mice of each group

[0180]

[0181]

[0182] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0183] Compared with the normal control group, the model control group showed a significant increase in serum uric acid levels (P<0.01), indicating successful establishment of the nephritis mouse model. The model control group also showed significant increases in serum urea nitrogen and creatinine levels (P<0.01), indicating successful establishment of the renal function impairment model. Compared with the model control group, the positive control group and all dose groups of soybean peptide S1 showed decreases (P<0.05, P<0.01). Compared with the model control group, the positive control group showed significantly lower levels of uric acid, creatinine, and urea nitrogen, indicating that the positive control drug has a good protective effect on renal function, approaching that of the normal control group. The soybean peptide groups showed a dose-dependent gradient decrease in uric acid, creatinine, and urea nitrogen levels. The medium and high dose groups of soybean peptide showed significant and highly significant differences (P<0.05, P<0.01). The high-dose group's effect in reducing urea nitrogen was similar to that of the positive control group, indicating that soybean peptide has a strong protective effect on renal function. All dose groups of soybean peptide S1 were superior to the ordinary soybean peptide group in reducing uric acid and improving creatinine and urea nitrogen levels. In particular, the high-dose group showed significant improvement, with its effect in reducing uric acid and improving creatinine and urea nitrogen levels approaching that of the positive control group. The creatinine and urea nitrogen levels in the high-dose soybean peptide S1 group were close to those in the positive control group, demonstrating its excellent renal function protection effect.

[0184] 3. Determination of xanthine oxidase (XOD) and adenosine deaminase (ADA) activities in liver tissue: Liver tissue was mixed with pre-cooled physiological saline at a ratio of 1g:9mL in an ice-water bath, and then homogenized into a 10% homogenate using a tissue homogenizer. The homogenate was centrifuged at 2500 rpm for 10 min, and the lipid layer was carefully removed. The supernatant was then used to determine the XOD and ADA activities according to the kit. The results are shown in Table 15.

[0185] Table 15 XOD and ADA values ​​of mice in each group

[0186] normal control group 10 <![CDATA[12.56±2.67 ** ]]> <![CDATA[0.63±0.04 ** ]]> Model control group 10 21.71±3.31 1.41±0.14 Positive control group 10 <![CDATA[14.06±2.16 ** ]]> <![CDATA[0.81±0.09 ** ]]> Low-dose soybean peptide group 10 19.41±3.72 1.31±0.13 Medium-dose group of soybean peptides 10 <![CDATA[17.02±2.61 * ]]> <![CDATA[1.03±0.10 * ]]> High-dose soybean peptide group 10 <![CDATA[15.34±2.81 ** ]]> <![CDATA[0.93±0.12 ** ]]> Low-dose group of soybean peptide S1 10 18.79±3.13 1.23±0.16 Medium-dose group of soybean peptide S1 10 <![CDATA[16.41±2.69 ** ]]> <![CDATA[1.13±0.08 * ]]> High-dose group of soybean peptide S1 10 <![CDATA[14.73±1.65 ** ]]> <![CDATA[0.94±0.10 ** ]]>

[0187] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0188] The results showed that the XOD and ADA activities in the model control group were significantly higher than those in the normal control group, indicating that the model group mice experienced oxidative stress and abnormal adenosine metabolism, suggesting that the model was successfully established. The XOD and ADA activities in the positive control group were significantly reduced, especially the reduction in XOD, showing a highly significant difference compared to the model control group (P<0.01), indicating that the positive control drug had good antioxidant and anti-inflammatory effects. In the low-dose soybean peptide group, the reduction in XOD and ADA activities was smaller and did not reach a significant difference (P>0.05). The medium-dose soybean peptide group showed a significant decrease in XOD and ADA activities, especially the medium-dose group (P<0.05). The high-dose soybean peptide group showed a significant decrease in XOD and ADA activities, with a larger reduction, approaching the level of the positive control group, showing a highly significant difference (P<0.01), indicating that the high-dose group had stronger antioxidant and anti-inflammatory effects. The low-dose soybean peptide S1 group showed a decrease in XOD and ADA activities, but the effect was slightly weaker than that of the ordinary low-dose soybean peptide group. In the medium-dose group of soybean peptide S1, the activities of XOD and ADA decreased significantly, and the effect was weaker than that of the ordinary medium-dose group of soybean peptide. In the high-dose group of soybean peptide S1, the activities of OD and ADA decreased significantly, which was close to that of the positive control group, and the decrease was close to that of the ordinary high-dose group of soybean peptide, showing a highly significant difference (P<0.01).

[0189] 4. Pathological observation of kidney tissue: Liver and kidney tissues were taken from the same location, fixed in 4% neutral formaldehyde fixative, dehydrated, embedded, and prepared into paraffin sections. They were then stained with hematoxylin and eosin (HE) and observed under a microscope.

[0190] Histopathological examination of liver tissue in laboratory mice (HE staining, 20×) as follows: Figure 7 As shown in the figure. The results showed that the liver tissue cells in each group had normal morphology and were arranged neatly and orderly.

[0191] Histopathological examination of mouse kidney tissue (HE staining, 20×) Figure 8 As shown in the figure. The results showed that, compared with the normal control group, the model group had obvious lesions in the kidney tissue, including cell atrophy, renal tubular dilation, and inflammatory cell infiltration. The test sample group showed improvement compared with the model group.

[0192] 5. Determination of urinary uric acid content: Fresh urine was analyzed at 3000 r·min -1 Centrifuge for 15 min, remove sediment, collect supernatant, and determine UUA content according to the kit. Results are shown in Table 16.

[0193] Table 16 Uric acid content in mice of each experimental group

[0194] normal control group 10 60.10±16.03 Model control group 10 169.50±25.81 Positive control group 10 <![CDATA[86.29±31.26 ** ]]> Low-dose soybean peptide group 10 139.51±27.92 Medium-dose group of soybean peptides 10 <![CDATA[105.08±28.30 * ]]> High-dose soybean peptide group 10 <![CDATA[96.21±20.38 ** ]]> Low-dose group of soybean peptide S1 10 <![CDATA[126.82±22.13 * ]]> Medium-dose group of soybean peptide S1 10 <![CDATA[95.31±23.63 ** ]]> High-dose group of soybean peptide S1 10 <![CDATA[86.07±29.06 ** ]]>

[0195] *Compared with the model control group, P<0.05; **Compared with the model control group, P<0.01.

[0196] The results showed that the uric acid level in the model control group was significantly higher than that in the normal control group (approximately 1.6 times), indicating that the model successfully induced a hyperuricemic state. The uric acid level in the positive control group was significantly lower than that in the model control group (P<0.01), showing that the positive control group had a good uric acid-lowering effect, approaching the level of the normal control group. The low-dose soybean peptide group did not significantly reduce uric acid levels, showing no significant difference from the model control group (P>0.05). The medium-dose soybean peptide group significantly reduced uric acid levels (P<0.05), showing a significant uric acid-lowering effect. The high-dose soybean peptide group further reduced uric acid levels, approaching the level of the positive control group, showing a highly significant difference (P<0.01). The low-dose soybean peptide S1 group showed a slightly greater reduction in uric acid levels than the ordinary low-dose soybean peptide group, with a significant difference. The medium-dose soybean peptide S1 group significantly reduced uric acid levels, with a better uric acid-lowering effect than the ordinary medium-dose soybean peptide group, indicating that soybean peptide S1 has higher biological activity at the same dosage. The uric acid level in the high-dose soybean peptide S1 group was close to that in the positive control group, and further decreased compared with the high-dose soybean peptide group, showing a highly significant difference (P<0.01).

[0197] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a mixture of soybean peptides, characterized in that, Includes the following steps: (1) Soy protein is added to deionized water with a pH of 9.5 to 10.5 to prepare a soy protein solution with a concentration of 4.5 to 5.5%. (2) Grind and homogenize the soybean protein solution to obtain a soybean protein homogenate with a particle size of 30-80 μm; (3) Keep the soybean protein homogenate at 53-58℃ and stir for 0.5-1h. Adjust the pH to 9.5-10.5, add alkaline protease, and hydrolyze until the pH drops to 7.0-7.

5. Then add papain and neutral protease, and hydrolyze for 3.5-4.5h. Add flavor protease and continue hydrolyzing for 2-2.5h to obtain the hydrolysate. (4) The enzyme hydrolysate is heated to 108-112℃ and held for 3-10 minutes, then cooled to room temperature and filtered with a ceramic membrane. The clear liquid filtered by the ceramic membrane is filtered with a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to obtain the nanofiltration membrane cutoff liquid. (5) The nanofiltration membrane intercepted liquid is passed through the cation exchange resin and the anion exchange resin in sequence at a flow rate of 48-52 L / h. The material adsorbed by the resin is concentrated, sterilized and dried to obtain a soybean peptide mixture. The soybean peptide mixture includes peptide S1 and peptide S2; peptide S1 is a single-chain peptide with the structural sequence YDPSTGIY and a molecular weight of 914.95 Da; peptide S2 is a single-chain peptide with the structural sequence VNEPAPGLK and a molecular weight of 924.05 Da.

2. The preparation method according to claim 1, characterized in that, Based on the mass of the soybean protein homogenate, the amount of alkaline protease added is 1.5 to 2.5 million U / g, the amount of papain added is 700,000 to 900,000 U / g, the amount of neutral protease added is 2.5 to 3.5 million U / g, and the amount of flavor protease added is 400,000 to 600,000 U / g.

3. The preparation method according to claim 1, characterized in that, The soybean peptide mixture was separated and purified using preparative high-performance liquid chromatography (HPLC). The purified soybean peptide mixture was obtained by gradient elution with mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water and mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile.

4. The preparation method according to claim 3, characterized in that, The preparation method of the sample solution separated and purified by the preparative high performance liquid chromatograph includes: dissolving the soybean peptide mixture in mobile phase A to prepare a soybean peptide solution, centrifuging, filtering the supernatant through a filter membrane, and using the filtrate as the sample solution.

5. The preparation method according to claim 3, characterized in that, The elution program for separation and purification using the preparative high-performance liquid chromatography is as follows: 0–5 min 0%–5% B, 5–20 min 5% B–80% B; the elution fraction collected after 11–12 min is the purified soybean peptide mixture.

6. The soybean peptide mixture prepared by the preparation method according to any one of claims 1 to 5.

7. The soybean peptide mixture according to claim 6, characterized in that, The soybean peptide mixture includes peptide S1 and peptide S2.

8. Polypeptide S2, characterized in that, The polypeptide S2 is a single-chain polypeptide with the structural sequence VNEPAPGLK and a molecular weight of 924.05 Da.

9. The use of the soybean peptide mixture according to any one of claims 6 to 7 or the polypeptide S2 according to claim 8 in the preparation of a medicament for treating chronic nephritis.

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