Hempseed meal protein peptide with uric acid-lowering activity and application thereof

Abstract

The application discloses hempseed meal protein peptides with uric acid reducing activity and application thereof, and belongs to the technical field of plant source protein peptide development. The hempseed meal protein peptides with uric acid reducing activity are screened in vitro, and three hempseed meal protein peptides AASFPILFDMT, GIDFPWPVPNRP and SGDETAPFFGFL with uric acid reducing activity are obtained by combining molecular docking, molecular dynamics simulation and other means. It is found through experiments that the active peptides obtained in the application can significantly reduce the contents of uric acid, creatinine and urea nitrogen, and can simultaneously reduce the expression levels of proinflammatory factors Caspase-3, IL-1beta and NLRP3, and have the dual effects of reducing uric acid and resisting inflammation. The application provides a new raw material source for the development and auxiliary intervention of functional foods for hyperuricemia.

Description

Technical Field

[0001] This invention relates to the field of plant-derived protein peptide development technology, and in particular to a hemp seed meal protein peptide with uric acid-lowering activity and its application. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorders or impaired uric acid excretion. The clinical diagnostic criteria are: serum uric acid (SUA) concentration >7.0 mg / dL (approximately 420 μmol / L) in men and >6.0 mg / dL (approximately 360 μmol / L) in women. HUA is a direct cause of gouty arthritis, uric acid kidney stones, and chronic kidney disease, and is also an independent risk factor for hypertension, cardiovascular disease, diabetes, and metabolic syndrome. Epidemiological data show that long-term hyperuricemia not only significantly increases the risk of gout attacks but is also closely related to various metabolic diseases, seriously threatening human health. The core of HUA pathogenesis is an imbalance between uric acid production and excretion: uric acid is the end product of purine metabolism in the human body, and its production mainly depends on the catalysis of xanthine oxidase (XOD). Genetic factors (such as excessive PRPS activity and HPRT deficiency), high-purine diets, excessive alcohol consumption, and high fructose intake can enhance XOD activity, leading to excessive uric acid production. Approximately 90% of hyperuricemia (HUA) cases originate from renal excretion disorders. Uric acid excretion is precisely regulated by transport proteins; URAT1 and GLUT9 are responsible for reabsorption, while OAT1 and ABCG2 are responsible for secretion. Abnormalities in the function of either lead to uric acid accumulation. Furthermore, abnormal intestinal excretion (accounting for 25% of total excretion) also contributes to the development of HUA. Currently, clinical treatment of hyperuricemia (HUA) primarily relies on drug intervention. Commonly used drugs are mainly divided into two categories: xanthine oxidase inhibitors and uricosuric agents. Representative xanthine oxidase inhibitors include allopurinol and febuxostat, while a representative uricosuric agent is probenecid. As a first-line uric acid-lowering drug, allopurinol specifically inhibits xanthine oxidase activity, reducing uric acid synthesis at its source and thus lowering blood uric acid levels. However, this drug has certain adverse reactions, commonly including gastrointestinal discomfort and skin rashes. In severe cases, it can even cause serious allergic reactions such as Stevens-Johnson syndrome, affecting the safety of medication use. Febuxostat, as a novel xanthine oxidase inhibitor, has the advantage of high uric acid-lowering efficiency, but its clinical application also carries potential risks. Related studies suggest that this drug may be associated with an increased risk of cardiovascular events. Furthermore, long-term use of various chemically synthesized uric acid-lowering drugs may increase the burden on the liver and kidneys, which also limits its safe application in certain specific populations.

[0003] Given the limitations of existing chemical drugs, developing novel uric acid-lowering functional factors that are highly effective, low in toxicity, and derived from natural products has become a crucial research direction in the field of functional food and drug development. Bioactive peptides, as a new type of bioactive factor, exhibit broad application prospects in the field of metabolic regulation due to their small molecular weight, easy absorption by the body, strong targeting, and high safety. In recent years, numerous studies have confirmed that bioactive peptides derived from various animal and plant proteins possess clear anti-hyperuricemia activity. Their mechanisms of action mainly involve inhibiting xanthine oxidase activity, downregulating the expression of key enzymes in purine metabolism, regulating the function of uric acid transport proteins, and improving the structure of the gut microbiota. Among these, bioactive peptides prepared from food-derived proteins such as tuna, bonito, walnuts, rice, milk, and egg white have been validated in in vitro experiments and animal models, achieving uric acid-lowering effects similar to chemical drugs while significantly reducing adverse reactions. These studies have laid a solid theoretical foundation and technical support for the development of functional uric acid-lowering products based on food-derived bioactive peptides.

[0004] Among plant protein resources, hemp, a plant used for both food and medicine, has a long history of consumption and medicinal use. Hemp seeds contain 20%–30% protein, with a balanced amino acid composition, containing all essential amino acids for the human body, and are particularly high in arginine and sulfur-containing amino acids, possessing excellent nutritional and physiological regulatory functions. In recent years, the antioxidant, antihypertensive, anti-inflammatory, and lipid-regulating activities of hemp protein and its hydrolysates have been confirmed. However, systematic research on its uric acid-lowering active peptides remains scarce, and its development potential has not been fully explored. Addressing the clinical pain points of significant side effects and poor long-term patient adherence to existing uric acid-lowering drugs, natural food-derived bioactive peptides have become a research breakthrough. Hemp protein resources are abundant, safe, and possess multiple bioactive bases, making it a high-quality raw material for discovering novel uric acid-lowering peptides. Therefore, it is of great significance to explore novel hemp-derived bioactive peptides or protein substances containing such peptides that have uric acid-lowering functions. Summary of the Invention

[0005] The purpose of this invention is to provide a hemp seed meal protein peptide with uric acid-lowering activity and its application. Based on the pathological mechanism of hyperuricemia, this invention uses hemp seed meal, an inexpensive, readily available, and widely sourced agricultural byproduct, as raw material to prepare a protein peptide with uric acid-lowering activity using biotechnology. This invention not only provides a novel intervention solution with a novel mechanism of action, high safety, and convenient use for people with hyperuricemia, but also helps to realize the high-value utilization of agricultural byproducts, aligning with the concept of green and sustainable development.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a hemp seed meal protein peptide with uric acid-lowering activity, wherein the amino acid sequence of the hemp seed meal protein peptide is AASFPILFDMT, GIDFPWPVPNRP, or SGDETAPFFGFL.

[0007] This invention also provides the use of the hemp seed meal protein peptide in any of the following: (1) Its application in the preparation of drugs for treating hyperuricemia; (2) Application in the preparation of uric acid-lowering drugs; (3) Application in the preparation of products that inhibit xanthine oxidase activity.

[0008] The present invention also provides an alkaline protease hydrolysate of hemp seed meal, wherein the alkaline protease hydrolysate of hemp seed meal contains the aforementioned hemp seed meal protein peptides.

[0009] The present invention also provides a method for preparing the alkaline protease hydrolysate of hemp seed meal, comprising the following steps: Hemp seed meal was mixed with water and extracted to obtain hemp protein; The hemp protein was mixed with water, and alkaline protease was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated and freeze-dried to obtain hemp meal alkaline protease hydrolysate.

[0010] Preferably, the ratio of hemp seed meal to water is 1g:(8-15)mL.

[0011] Preferably, the water extraction method includes: mixing the hemp seed meal with the water evenly, adjusting the pH value to alkaline, stirring and extracting at room temperature for 1-2 hours, filtering and centrifuging after extraction, collecting the supernatant, adjusting the pH value to acidic, letting it stand at room temperature for 1-2 hours, centrifuging again, discarding the supernatant, resolving the obtained precipitate in water, adjusting the pH value to neutral, and freeze-drying to obtain the hemp protein.

[0012] Preferably, the ratio of hemp protein to water is 1g:(8-15)mL.

[0013] Preferably, the enzymatic hydrolysis conditions are: pH value of 8.0, temperature of 55-60℃, and time of 2-4h.

[0014] This invention also provides the use of the aforementioned hemp seed meal alkaline protease hydrolysate in any of the following: (1) Its application in the preparation of drugs for treating hyperuricemia; (2) Application in the preparation of uric acid-lowering drugs; (3) Application in the preparation of products that inhibit xanthine oxidase activity.

[0015] The present invention also provides a medicament for treating hyperuricemia, comprising the hemp seed meal protein peptides described above or the hemp seed meal alkaline protease hydrolysate described in claim 3.

[0016] The present invention discloses the following beneficial effects: The hemp seed meal protein peptides (AASFPILFDMT, SGDETAPFFGFL, and GIDFPWPVPNRP) prepared by this invention exhibit good in vitro xanthine oxidase inhibitory activity.

[0017] The hemp seed meal protein peptide prepared by this invention has the significant characteristic of a molecular weight of less than 1500 Da, which allows it to be directly absorbed and utilized by the human body, with a bioavailability far exceeding that of natural hemp protein. More importantly, this peptide not only lowers uric acid by improving metabolic disorders but also possesses certain anti-inflammatory effects, which can help improve hyperuricemia-related symptoms from multiple perspectives. Furthermore, this peptide is derived from a byproduct of hemp processing, achieving high-value utilization of agricultural waste and aligning with the concept of green and sustainable development. Simultaneously, it contains no chemical drug components, avoids the toxic side effects of chemical drugs, and has high safety, making it widely applicable in the development of functional foods or adjuvant therapeutic agents. It can effectively meet the clinical intervention and health needs of sub-healthy individuals and those with hyperuricemia.

[0018] The hemp seed meal protein peptide prepared in this invention exhibits significant uric acid-lowering activity, and its mechanism of action is achieved by inhibiting xanthine oxidase (XOD) activity. Animal experiments further confirmed that the hemp seed meal protein peptide can effectively reduce the levels of uric acid, creatinine, and blood urea nitrogen in hyperuricemia model animals, while significantly downregulating the expression levels of pro-inflammatory factors Caspase-3, IL-1β, and NLRP3, thus possessing both uric acid-lowering and anti-inflammatory effects. 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 embodiments will be briefly introduced below. Obviously, the drawings described below are only 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 The characteristics of hemp seed meal protein hydrolysates are shown in Figure 1; A represents XOD inhibition rate, B represents peptide yield, C represents peptide content, and D represents the IC50 value for determining the XOD inhibitory activity of different hydrolysates. 50 Values; AC on the horizontal axis of the figure represent papain hydrolysate, alkaline hydrolysate, and complex hydrolysate of hemp seed meal, respectively; different lowercase letters indicate significant differences, p < 0.05; Figure 2The results show the stability evaluation of alkaline protease hydrolysates; A represents the inhibition rate of alkaline protease on XOD at different temperatures, B represents the inhibition rate of alkaline protease on XOD at different pH values, C represents the inhibition rate of alkaline protease on XOD in simulated gastrointestinal digestive fluid in vitro, and D represents the inhibition rate of alkaline protease on XOD under different metal ion environments. Figure 3 The following are the results of peptide sequence identification of hemp seed meal enzymatic hydrolysate: A shows the distribution of peptides in hemp seed meal protein analyzed by bioinformatics methods; B shows the number of peptides based on the number of amino acids; C shows the number of peptides based on molecular weight distribution; and D shows the number of peptides with a bioactivity score > 0.8 based on molecular weight. Figure 4 The following are the docking results of peptides with xanthine oxidase: A is the docking result of the active peptide with the amino acid sequence SYPTLPGWFPSP, B is the docking result of the active peptide with the amino acid sequence SGDETAPFFGFL, C is the docking result of the active peptide with the amino acid sequence FWDWDLPRT, D is the docking result of the active peptide with the amino acid sequence AASFPILFDMT, and E is the docking result of the active peptide with the amino acid sequence GIDFPWPVPNRP. Figure 5 Fourier transform infrared spectra and secondary structure analysis of different bioactive peptides; A is the bioactive peptide with amino acid sequence AASFPILFDMT, B is the bioactive peptide with amino acid sequence GIDFPWPVPNRP, C is the bioactive peptide with amino acid sequence SGDETAPFFGFL, and D is the secondary structure analysis of different bioactive peptides. Figure 6The results are from molecular dynamics simulations; A shows the RMSD curves of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes; B shows the RMSD values ​​of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes; C shows the RMSF values ​​of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes; D shows the Rg values ​​of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes; E shows the Rg values ​​of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes. Fluctuations in RP and XOD / SGDETAPFFGFL complexes: F represents the SASA value analysis of XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes; G represents the residue energy decomposition results of the XOD / AASFPILFDMT complex; H represents the residue energy decomposition results of the XOD / GIDFPWPVPNRP complex; I represents the residue energy decomposition results of the XOD / SGDETAPFFGFL complex; J represents the 2D and 3D spectra of the Gibbs free energy of the XOD / AASFPILFDMT complex; K represents the 2D and 3D spectra of the Gibbs free energy of the XOD / GIDFPWPVPNRP complex; and L represents the 2D and 3D spectra of the Gibbs free energy of the XOD / SGDETAPFFGFL complex. Figure 7 The effects of hemp seed meal alkaline protein hydrolysate on the physiological and biochemical properties of mice; A is body weight, B is liver index, C is kidney index, D is uric acid content, E is serum creatinine content, F is serum urea nitrogen content, G is liver XOD activity, and H is kidney XOD activity. Figure 8 The effect of alkaline protein hydrolysate of hemp seed meal on the levels of Casp-3, IL-1β, and NLRP3 in mouse kidney and liver; A, C, and E represent the expression levels of Casp-3, IL-1β, and NLRP3 in liver, respectively; B, D, and F represent the expression levels of Casp-3, IL-1β, and NLRP3 in kidney, respectively. Figure 9 Results of H&E staining of kidney sections; Figure 10 The results of H&E staining of liver sections. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1 1. Extraction of hemp protein Hemp seed meal and deionized water were mixed evenly at a material-to-liquid ratio of 1:10 (g / mL). The pH of the system was adjusted to 9 using 0.1M NaOH solution, and the mixture was extracted magnetically at room temperature for 1 h. After filtration through a mesh screen, the filtrate was centrifuged at 4000 r / min for 25 min at 4℃, and the supernatant was collected. Subsequently, the pH of the supernatant was adjusted to 4.5 with 1.0 mol / L HCl solution, and the mixture was allowed to stand at room temperature for 2 h before centrifugation again, discarding the supernatant. The resulting precipitate was reconstituted with a small amount of deionized water, and the pH was adjusted to 7. After freeze-drying, hemp protein was obtained.

[0027] 2. Enzymatic hydrolysis of hemp seed meal Hemp protein was dissolved in deionized water at a material-to-liquid ratio of 1:10 (g / mL), and the pH was adjusted to the optimal level using 0.1M NaOH and HCl solutions. Each protease was added at a 4% (w / w) enzyme-to-protein ratio for hydrolysis. The specific hydrolysis conditions were: alkaline protease: pH 8.0, 50℃, 3h; papain: pH 6.0, 40℃, 3h; complex protease (Angel Enzyme Preparations Yichang Co., Ltd., composed of flavor protease and alkaline protease): pH 7.0, 60℃, 3h. During hydrolysis, 0.1M NaOH or HCl solution was added dropwise as needed to maintain a stable pH. After hydrolysis, the reaction solution was heated at 95℃ for 10min to inactivate the enzymes. After cooling to room temperature, the pH was adjusted to neutral, and the solution was centrifuged at 4000 rpm for 15min. The supernatant was collected and freeze-dried to obtain the lyophilized enzyme hydrolysate.

[0028] 3. Peptide content determination Glutathione at concentrations of 0.5, 1, 2, 3, 4, and 5 mg / mL was used as a standard to construct a standard curve for the determination of peptide content in samples. Reagent preparation: ① Weigh 120 mg of o-phthalaldehyde (OPA) and dissolve it completely in 3 mL of methanol; ② Prepare a 0.1 mol / L sodium tetraborate solution; ③ Prepare a 5% SDS solution; ④ After preparing the above three reagents, take 10 mL of sodium tetraborate solution, 4 mL of 5% SDS solution, 6.78 mL of ultrapure water, 40 μL of dimercaptoethanol, and 400 μL of OPA solution, and mix well to obtain the working reagent.

[0029] Sample preparation: Weigh 30 mg of each ultrafiltration fraction with different molecular weights and dissolve it in 1 mL of ultrapure water to prepare a 30 mg / mL sample solution. Add 200 μL of working reagent and 5 μL of sample solution to a 96-well plate, with three replicates for each sample. Peptide content determination was performed using the standard curve equation: y = 0.553x + 0.0049 (R²). 2 =0.999), and the peptide content in each sample was calculated based on the standard curve.

[0030] ; In the above formula (1), A1 and A2 represent the actual measured peptide content and the sample mass, respectively, and the unit of peptide content is _____.

[0031] 4. Determination of xanthine oxidase (XOD) inhibition rate Using 50mM, pH 7.4 phosphate buffer, 0.06mg / mL xanthine solution and 0.05U / mL xanthine oxidase solution were prepared respectively. 50μL of sample solution (or blank buffer) and 50μL of xanthine oxidase solution were mixed to form a reaction system and incubated at 37℃ for 30min. Then, 150μL of 0.06mg / mL xanthine solution was added to start the enzymatic reaction. Immediately after the sample addition was completed, the absorbance was measured at 292nm wavelength using a multi-functional microplate reader. The experiment was set up with sample group, sample blank group, control group, and blank group. The sample addition of each group is shown in Table 1. The xanthine oxidase inhibition rate was calculated according to formula (2):

[0032]

[0033] 5. Stability Evaluation The stability of hemp protein-derived XOD inhibitory peptides was determined under different pH, temperature, and ionic strength conditions, as well as after simulated gastrointestinal digestion. The lyophilized enzyme hydrolysate was reconstituted with distilled water to a concentration of 1 mg / mL and subjected to various treatments to evaluate its stability under different pH (3, 5, 7, 9, and 12), temperatures (RT, 20, 40, 60, 80, and 100 °C), and metal ion concentrations (0, 20, 40, 60, 80, and 100 mmol / L). + Ca + Na + Inhibitory activity in solution.

[0034] The procedure for simulating gastrointestinal digestion is as follows: For pepsin digestion, the peptide solution was adjusted to pH 2 using 1 mol / L hydrochloric acid, and 0.2 mg of pepsin was added. The mixture was incubated at 37°C and 200 rpm for 2 hours, followed by the addition of 0.2 mg of pepsin inhibitor to terminate the reaction.

[0035] For trypsin digestion, the peptide solution was adjusted to pH 7.5 using 1 mol / L sodium hydroxide, and 0.2 mg of trypsin was added. The mixture was incubated at 37 °C and 200 rpm for 2 h, and then the reaction was terminated using 0.2 mg of trypsin inhibitor.

[0036] For sequential digestion with pepsin and trypsin, the peptide solution was adjusted to pH 2 using 1 mol / L hydrochloric acid, and 0.2 mg of pepsin was added. The mixture was then incubated at 37°C and 200 rpm for 1 hour, followed by termination of the reaction with 0.2 mg of pepsin inhibitor. Finally, the pH of the mixture was adjusted to 7.5 using 1 mol / L sodium hydroxide. Then, 0.2 mg of trypsin inhibitor was added, and the mixture was incubated at 37°C and 200 rpm for 1 hour. 0.2 mg of trypsin inhibitor was added at the end of the reaction.

[0037] After each treatment, xanthine oxidase inhibitory activity was measured to assess the effect of simulated digestion on peptide stability.

[0038] 6. Results and Analysis 6.1 Characterization of enzymatic hydrolysates of hemp protein and evaluation of xanthine oxidase inhibitory activity Papain, alkaline protease, and a complex protease were used to enzymatically hydrolyze hemp seed protein to obtain papain hydrolysate (peptide A), alkaline protease hydrolysate (peptide B), and a complex protease hydrolysate (peptide C) from hemp seed meal. The peptide content, peptide yield, and xanthine oxidase inhibition rate were determined. Figure 1 As shown in Figure A, the compound protease hydrolysate exhibited the best xanthine oxidase inhibition rate (84.05±4.86%), followed by the alkaline protease hydrolysate (69.61±0.08%), and lastly papain (60.17±2.74%). The peptide content of the three protease hydrolysates was determined by the OPA method. Figure 1 As can be seen from Figure B, the complex protease hydrolysate has the highest peptide content (57.88±0.68%), followed by the alkaline protease hydrolysate (52.52±0.17%) and the papain hydrolysate (50.95±1.03%). Considering the overall peptide content, peptide yield, and IC50... 50 IC 50 The smaller the value, the stronger the inhibitory activity.

[0039] The optimal enzymatic hydrolysate (alkaline protease hydrolysate) was finally selected. Figure 1 The peptide yield of papain hydrolysate was 86.48%, that of alkaline hydrolysate was 60.05%, and that of complex hydrolysate was 29.38%. The inhibitory effects of papain, alkaline protease, and complex protease on xanthine oxidase activity were determined. Figure 1 As shown in Figure D, the results indicate that all three proteases exhibited good inhibitory activity against xanthine oxidase, with IC50 values ​​for peptides A, B, and C being [missing data]. 50 The IC50 values ​​were 1.27±0.13 mg / mL, 0.59±0.03 mg / mL, and 0.72±0.07 mg / mL, respectively. The IC50 values ​​of alkaline protease compared to complex protease and papain were... 50There was a significant decrease. This may be because alkaline proteases can specifically hydrolyze to produce small peptides with smaller molecular weights, moderate hydrophobicity, and rich amino acid residues. Furthermore, the products have a higher proportion of flexible conformations such as random coils, which are more likely to intercalate into the XOD active site and bind stably through hydrogen bonds and hydrophobic interactions, thus exhibiting a stronger in vitro xanthine oxidase inhibitory effect.

[0040] 6.3 Stability Evaluation like Figure 2 As shown, the stability of xanthine oxidase inhibitory peptides from hemp seed meal protein hydrolysates was evaluated under different environmental and physiological conditions (temperature, pH, simulated gastrointestinal digestion, sodium ion concentration). Thermal stability analysis showed that the inhibitory activity of this xanthine oxidase inhibitory peptide decreased slowly with increasing temperature, indicating good overall thermal stability. Within the temperature range of room temperature to 80℃, its inhibitory activity remained relatively stable; however, when the temperature was raised to 100℃, the inhibitory activity decreased significantly. Figure 2 (A) pH is a key factor affecting peptide activity. Xanthine oxidase inhibition rates are relatively stable in both strongly acidic and strongly alkaline environments, with the strongest inhibition rate observed under neutral conditions. This may be due to conformational changes in the peptide structure. Figure 2 (B) From Figure 2 As shown in the C-cell analysis, the gastrointestinal digestive stability experiment revealed that the inhibition rate remained stable after 2 hours of treatment with pepsin and trypsin, indicating that this uric acid-lowering peptide may possess good stable activity in vivo. Conversely, Ca... 2+ K + Na + Increased concentration leads to a decrease in inhibition rate, which may be attributed to changes in peptide structure. Figure 2 (D) Overall, these peptides exhibit good stability during processing and digestion. However, to maintain optimal xanthine oxidase inhibitory activity, exposure to highly acidic or high-salt environments should be avoided as much as possible.

[0041] Example 2: Peptide sequence identification, screening, and structural characterization of alkaline protease hydrolysates 1. Identification of target peptide sequence The bioactive peptides in the alkaline protease hydrolysate of hemp seed meal were analyzed and identified using LC-MS / MS. 38 mg of sample was accurately weighed and dissolved in 0.25 mL of 0.1% (m / m) trifluoroacetic acid solution. After vortexing and mixing, the solution was centrifuged at 4 °C and 14000 × g for 5 min. The supernatant was transferred to an ultrafiltration tube and centrifuged for another 15 min at the same temperature and speed. The filter membrane was discarded, and the filtrate was collected and freeze-dried. For reconstitution, 30 μL of 0.1% trifluoroacetic acid solution was added, and the solution was vortexed for 1 min and then centrifuged. The supernatant was used for sample analysis. The liquid chromatography conditions were as follows: a 0.15 mm × 150 mm, 3 μm column was used; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-acetonitrile solution; the gradient elution program was set as follows: 0–50 min, the concentration of phase B increased from 4% to 50%; 50–54 min, the concentration of phase B increased from 50% to 100%; 54–60 min, the concentration of phase B remained constant at 100%. Mass spectrometry was performed in positive ion mode. Ten sets of secondary fragment spectra were acquired after a full scan, and the mass-to-charge ratio information of the peptide and its fragments was obtained through spectral analysis.

[0042] 2. Activity prediction and molecular docking Activity was predicted using the Peptides Bioactive database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ); the activity of the screened peptides was scored using the Peptide Activity Database (http: / / distilldeep.ucd.ie / PeptideRanker / ); peptide sequence toxicity was predicted using the Virtual database (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php); and peptide sequence solubility was calculated using the peptide parameter calculator (https: / / www.allpeptide.com / canshu.html).

[0043] 3. Infrared spectroscopy determination of secondary structure in solid state Accurately weigh 1 mg of hemp seed meal protein and alkaline protease hydrolysate powder, mix them separately with 100 mg of potassium bromide, and grind them thoroughly in a mortar for 15-20 minutes. After grinding until uniform, compress the mixture into tablets for 1 minute. Then, perform infrared spectroscopy. The measurement conditions are set as follows: scan wavenumber range 400–4000 cm⁻¹. -1 The resolution is 4cm. -1 The wavenumber accuracy is 0.01cm. -1Under these conditions, 64 consecutive scans were performed. The obtained infrared spectra were analyzed and processed using Peakfit Version software to calculate the relative content of each secondary structure.

[0044] 4. Molecular docking Molecular docking of XOD (PDB: 1FIQ) was performed using AutoDock Vina. Combining affinity and interacting residues were visualized in PyMOL and Discovery Studio.

[0045] 5. Molecular dynamics simulation Using the peptide-protein complex obtained through molecular docking as the initial structure, all-atom molecular dynamics simulations were performed using AMBER 24 software. The preprocessing steps before simulation were as follows: both peptides and proteins were described using the ff14SB protein force field; hydrogen atoms were added to each system using the LEaP module of the software, and the distance from the system was... Add a truncated octahedral TIP3P solvent box to the system, and simultaneously add Na to the system. + / Cl - After balancing the system charge and completing the preprocessing, the topology and parameter files are output for subsequent molecular dynamics simulations.

[0046] 6. Results and Analysis 6.1 To further analyze the active components in the fraction with a molecular weight <3 kDa, LC-MS / MS identification was performed. Bioinformatics analysis of the total peptides in the alkaline enzymatic hydrolysate of hemp seed meal identified 2040 peptides, of which 924 had a score >100 and 28 had a PeptideRanker score >0.8. Detailed statistical analysis was conducted based on molecular weight distribution, types of active peptides, peptide bioactivity scores, and peptide lengths to gain a deeper understanding of the functional peptides in hemp seed meal. The peptides identified in the hemp seed hydrolysate mainly originated from 11S seed storage protein and Midasin, accounting for 23.3% and 22.6%, respectively (see...). Figure 3 (A) For example Figure 3As shown in Figure B, peptides mainly consist of 3-4 amino acids, totaling 1411 peptide sequences, accounting for 57.33% of the total peptides. The next largest group consists of 9-14 amino acid peptides, totaling 723 sequences, accounting for 29.38% of the total peptides. The number of peptides corresponding to each residue number is 92, 135, 154, 140, 124, and 78, respectively. The number of peptides within this range initially increases and then decreases with increasing amino acid residue number, reaching a peak at 11 amino acid residues. This distribution characteristic is directly related to the enzymatic hydrolysis reaction characteristics. Enzymes exhibit site preference and randomness in the hydrolysis of peptide bonds, more readily generating peptides of this amino acid length. These peptides also exhibit high stability in the enzymatic hydrolysis system and are not easily further hydrolyzed. Conversely, as the peptide chain length continues to increase, longer peptides are more easily recognized and hydrolyzed by the enzyme, thus the number of remaining peptides gradually decreases. Figure 3 As shown in section C, there are 1103 peptides with a molecular weight <0.5kDa, accounting for 44.82% of the total peptides; 394 peptides with a molecular weight of 0.5~1.0kDa, accounting for 16% of the total peptides; 604 peptides with a molecular weight of 1.0~1.5kDa, accounting for 24.54% of the total peptides; and 255 peptides with a molecular weight of 1.5~2kDa, accounting for 10.36% of the total peptides. Figure 3 As shown in Table D, the bioactivity of peptides was scored using the Peptide Ranker database, with 28 peptides having a score >0.8. These peptides with bioactivity scores >0.8 were mainly concentrated in the 1.0–1.5 kDa range. Furthermore, based on the characteristics of the urate-lowering peptides and comparison with unreported bioactive peptides in the BIOPEP-UWM database, five novel peptides with potential xanthine oxidase activity were selected (Table 2).

[0047]

[0048] 6.2 Structural characterization of hemp seed meal protein hydrolysate Fourier transform infrared spectroscopy can be used to resolve the secondary structures of peptides SGDETAPFFGFL, AASFPILFDMT, and GIDFPWPVPNRP. This is based on the differences in absorption peaks at characteristic wavenumbers for different functional groups. Specifically, the 1600–1700 cm⁻¹ peak value is... -1 The amide I band within the range is the core region reflecting the secondary structure and is directly related to the degree of order in the polypeptide backbone conformation. Generally, the higher the wavenumber of the absorption peak, the stronger the structural order.

[0049] like Figure 5 As shown in the spectrum, peak fitting of the amide I band in this invention yielded four characteristic sub-peaks: β-fold (1600~1640 cm⁻¹). -1 Irregular curls (1640~1650cm) -1α-helix (1650~1660cm) -1 ), β-turn (1660~1700cm) -1 The absorption characteristics of the amide I band can directly reflect the ordered arrangement of the polypeptide backbone, further corroborating its secondary structure composition.

[0050] By analyzing the absorption peak areas at different wavelengths, the secondary structure of peptide AASFPILFDMT was determined to consist of β-sheets (13.46%), random coils (36.12%), α-helices (29.29%), and β-turns (21.14%). The secondary structure of SGDETAPFFGFL consisted of β-sheets (14.1%), random coils (36.28%), α-helices (29.38%), and β-turns (20.24%). The secondary structure of GIDFPWPVPNRP consisted of β-sheets (55.59%) and β-turns (44.41%). Secondary structure analysis of GIDFPWPVPNRP showed that β-sheets and β-turns were its predominant conformations. Previous studies have confirmed that the β-sheet conformation is closely related to XOD inhibitory activity, while hydrogen bonds formed in the β-turn region can further enhance structural stability. The active site of XOD is shaped like a hydrophobic pocket, in which the β-turn of the polypeptide can be flexibly inserted. It binds to key residues through hydrogen bonds and hydrophobic interactions, occupies substrate binding sites, and thus inhibits enzyme activity. The rigid sheet structure of β-sheet can enhance the stability of the polypeptide-XOD complex, hindering the enzyme's catalytic process by occupying space and reducing uric acid production.

[0051] The peptides AASFPILFDMT and SGDETAPFFGFL are mainly composed of random coils and α-helices. From the perspective of their uric acid-lowering mechanism, the random coil structure is highly flexible and conformationally adaptable, allowing it to adaptively enter the hydrophobic pocket of the XOD active site. Through hydrogen bonds and hydrophobic interactions, it fully binds to the enzyme, thereby occupying the substrate binding site and inhibiting XOD catalytic activity. The α-helix, with its regular spatial conformation, enhances the stability of the peptide's binding to XOD, reduces dissociation caused by conformational changes, and improves the inhibitory effect. Previous studies have shown that small peptides rich in random coils and α-helices can also exert XOD inhibitory effects through flexible binding, spatial occupancy, and hydrophobic interactions, thereby reducing uric acid production. Therefore, the secondary structures of AASFPILFDMT and SGDETAPFFGFL, primarily composed of random coils and α-helices, exhibit a good structure-activity relationship with their uric acid-lowering activity. In summary, peptides AASFPILFDMT, SGDETAPFFGFL, and GIDFPWPVPNRP all possess regular and typical secondary structures, consistent with the structure-activity relationships reported in existing literature.

[0052] 6.3 Investigating the interaction between hemp protein peptides and xanthine oxidase based on molecular docking Molecular docking technology can precisely identify potential ligand binding sites on the surface of receptor proteins, analyze intermolecular binding strength and interaction modes, and provide an important theoretical basis for the rapid and efficient screening and evaluation of bioactive substances by simulating ligand-receptor interactions at the molecular level. Xanthine oxidase (XOD) is a key molybdenum-containing flavonoid protein, with its molybdenum cofactor domain as the core catalytic region. The substrate xanthine must enter this active site through a narrow hydrophobic channel to complete the catalytic reaction and generate uric acid. For example... Figure 4 As shown, molecular docking results revealed that all five peptides could form numerous hydrogen bonds and hydrophobic interactions with xanthine oxidase (XOD). Hydrogen bonds, with their strong binding energy and directionality, are key forces for the specific molecular recognition of XOD by the peptides; while hydrophobic interactions facilitate the stable binding of the peptides to the XOD active site in an aqueous environment, promoting a tighter binding. These interactions collectively mediate the effective binding of the peptides to XOD, providing an important molecular basis for their inhibition of XOD activity and reduction of uric acid production. Figure 4 It can be seen that the docking scores of SYPTLPGWFPSP, SGDETAPFFGFL, FWDWDLPRT, AASFPILFDMT, and GIDFPWPVPNRP with 1fiq are -7.852, -7.54, -7.418, -7.335, and -7.313 kcal / mol, respectively. Generally, the lower the receptor-ligand binding score, the stronger the binding affinity. Figure 4 As shown in section A, SYPTLPGWFPSP forms hydrogen bonds with amino acid residues Lys771, Ser876, Asn650, and Gln1016, and hydrophobic interactions with amino acid residues Leu648, Val1011, and Leu1014. It also forms π–σ interactions and π–π stacking with Leu1014 and Phe1013, thereby enhancing the stability of the binding. Figure 4As shown in section B, based on molecular docking SGDETAPFFGFL, hydrogen bonds are formed with the target proteins Ser774, Lys713, and Tyr1010 of ligand. Hydrophobic interactions are formed with Leu1014, Val1011, Pro1012, Phe1013, Pro1076, Ala1078, and Ala1079. Two salt bridges exist with Met770 and Glu879, thereby enhancing binding stability. Furthermore, an interaction π–σ is formed with Leu1014, filling the hydrophobic region of the XOD active pocket and reducing ligand interaction. The hydrophobic interface of the enzyme enhances the positioning accuracy of the ligand in the pocket and helps maintain conformation. FWDWDLPRT forms 5 hydrogen bonds with amino acids Lys713, Tyr1140, Ser876, Phe1013, and Phe1142, 2 hydrophobic interactions with Pro1076 and Phe1013, 2 salt bridges with Glu1143 and His875, and π–π stacking and π–σ interactions with Phe1013 and His875, forming docking results. The results indicate that multiple amino acid residues in the peptide chain can form a hydrogen bond network with the target protein. Figure 4 (C) Its aromatic side chains can generate π–π stacking and π–σ interactions with the hydrophobic pockets of proteins; the positively charged residues in the peptide chain can also form electrostatic attraction with the negatively charged regions of proteins. This peptide achieves highly efficient inhibition of target protein function through structural complementarity and synergistic effects of multiple types of non-covalent interactions. Figure 4 As shown in D, AASFPILFDMT forms three hydrogen bonds with amino acids Asn650, Phe1142, and Lys771, and five hydrophobic interactions with Phe1013, Val784, Val1011, Lys778, and Ala777. It also has a salt bridge with Lys771. The aromatic ring of the ligand forms π–π stacking with the aromatic residues (Phe649 and His875) in the XOD hydrophobic pocket, which stabilizes the spatial structure of the ligand in the active site and enhances the competitive inhibition type against the substrate. GIDFPWPVPNRP primarily interacts with XOD through hydrogen bonds (Lys771, His875, Ser1141) and hydrophobic interactions (Leu648, Leu873, Leu1014, Phe649, His875, Pro1012, Phe1142), forms salt bridges with ligand's target protein Glu1143 residues, and forms π–π stacking and π–σ interactions with ligand's target proteins Leu648 and Phe1013. Figure 4 (E). Multiple amino acid residues in the polypeptide chain can form a hydrogen bond network with the carboxyl group of the target protein, while its C–H bonds form specific weak hydrogen bonds and π–alkyl interactions with the protein backbone. This polypeptide can achieve highly efficient inhibition of target protein function through multi-level and multi-site synergy by blocking substrate entry channels and inducing conformational changes in the target protein.

[0053]

[0054] 6.4 Molecular Dynamics Simulation To verify the molecular docking results and eliminate false positives, this invention performed 100 ns molecular dynamics simulations on the screened peptide-enzyme complexes and evaluated the overall conformational stability of the complexes based on root mean square deviation (RMSD) analysis. Figure 6 As shown in Figure A, the RMSD curves of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes entered a relatively stable phase after 10–20 ns, with average RMSD values ​​of 0.287±0.052 nm, 0.383±0.097 nm, and 0.326±0.090 nm, respectively. The XOD / AASFPILFDMT complex exhibited the least overall fluctuation, indicating that the complex formed after the peptide binds to XOD has the best conformational stability. RMSD represents the conformational fluctuation of the peptide molecule at each time step compared to the initial structure. Figure 6 As shown in Figure B, the RMSD values ​​of the ligands (peptides) of the XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL complexes are 0.084±0.034 nm, 0.081±0.033 nm, and 0.085±0.036 nm, respectively. The overall fluctuations are mainly concentrated between 0.05–0.12 nm, with only a few residues showing higher peak values ​​(approximately 0.3–0.5 nm), lower than the 0.18 nm of XOD, indicating a change in XOD structure. Furthermore, molecular docking results show that the ligands AASFPILFDMT, GIDFPWPVPNRP, and SGDETAPFFGFL affect the XOD receptor, possibly because they occupy more active pockets in XOD, establishing more interaction forces, thereby leading to induced conformational changes in the active residues of XOD. Molecular dynamics simulations show that AASFPILFDMT, GIDFPWPVPNRP, and SGDETAPFFGFL can all interact with XOD, and the resulting complexes reach a relatively stable state within 100 ns of the simulation. Figure 6 As shown in Figure C, the overall variation of amino acid residues in the XOD-peptide complex is significantly stronger than that of XOD. This indicates that AASFPILFDMT, GIDFPWPVPNRP, and SGDETAPFFGFL can bind to XOD to form a protein-peptide complex.

[0055] The deviations in RMSF values ​​mainly originated from three regions: region A, region B, and region C (residues 497-503, 529-535, and 564-569). In region A, AASFPILFDMT-XOD and XOD exhibited the highest RMSF values, indicating a higher level of flexibility. Under simulated conditions, the largest RMSF values ​​for all ligands were also less than 1, suggesting that the XOD-peptide is relatively stable during complex formation.

[0056] In molecular dynamics simulations, Rg and SASA values ​​can be used to determine the compactness of protein-peptide complexes. Generally, lower Rg and SASA values ​​indicate complexes with a more compact molecular structure. During MD simulations, the Rg value of the XOD / SGDETAPFFGFL complex fluctuated the least (3.150±0.014 nm), indicating a relatively more compact structure. The Rg values ​​of the non-complex XOD and the XOD / AASFPILFDMT and XOD / GIDFPWPVPNRP complexes were 3.163±0.010 nm and 3.166±0.009 nm, respectively, showing slightly higher fluctuations. Figure 6 The overall fluctuations were small, with no sustained expansion or collapse observed, indicating that the system maintained a stable folded state during the simulation. The dynamic changes in hydrogen bonding between peptides and proteins were also observed. The XOD / SGDETAPFFGFL complex had the highest average number of hydrogen bonds (7.25±1.95), significantly higher than XOD / AASFPILFDMT (4.62±1.88) and XOD / GIDFPWPVPNRP (3.97±1.65), indicating a richer and more persistent hydrogen bond network during binding. Although all three systems showed some fluctuations, XOD / SGDETAPFFGFL maintained a high level throughout the simulation, suggesting that hydrogen bonds play a dominant role in its binding stability. Figure 6 (E).

[0057] In MD simulations, consistent with the observed Rg value trend, the SASA values ​​of the XOD / GIDFPWPVPNRP and XOD / SGDETAPFFGFL complexes were 423.02 ± 8.24 nm. 2 and 424.94±10.50nm 2 The SASA value was lower than that of the non-complexed XOD, indicating that the binding of GIDFPWPVPNRP and SGDETAPFFGF to XOD resulted in less structural change, thus demonstrating the stability of the protein-peptide complex. Figure 6 (F).

[0058]

[0059] As shown in Table 4, the binding energies of XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL are -30.91±2.45, -49.23±4.12, and -52.75±6.50 kcal / mol, respectively. Negative values ​​indicate that these two molecules have a strong affinity for the target protein; lower values ​​indicate stronger binding. The results show that XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL have very strong binding affinity. Energy decomposition reveals that the main contributing factor to the binding of XOD-AASFPILFDMT, XOD-GIDFPWPVPNRP, and XOD-SGDETAPFFGFL is electrostatic energy, followed by van der Waals energy, and then nonpolar solvation free energy.

[0060] Figure 6 G represents the residue energy decomposition results of the XOD / AASFPILFDMT complex. The main contributing residues are concentrated in hydrophobic or aromatic residues such as PHE1218, PHE1222, and LEU1221, with PHE1218 contributing the most (approximately -4 kcal / mol or more). PHE548, ASN549, and LEU547 also provide stable negative energy contributions, indicating that the peptide mainly relies on hydrophobic interactions and aromatic stacking for stable binding, while polar residues provide auxiliary stabilizing effects, and the overall energy distribution is relatively uniform. Figure 6 In the middle H, the XOD / GIDFPWPVPNRP complex, the binding energy contribution is more significant. The key residues ILE1216, PHE1218, and TRP1220 have lower contribution values ​​(about -7 to -8 kcal / mol), showing strong hydrophobic and π-π interactions dominating the binding. The others, such as SER609, PHE912, and MET669, provide moderate stabilizing effects, indicating that the system forms a strong and relatively concentrated core of key interactions. Figure 6 In the first system, the XOD / SGDETAPFFGFL complex showed the most significant contribution from residues. The contributions from THR909 and ARG779 were the lowest (close to -10 kcal / mol), which were much higher than those from other systems, indicating that they formed strong polar or charge interactions. Meanwhile, PHE908, SER775, and PHE1223 also provided stable negative energy contributions. The overall binding energy distribution was wide and the amplitude was large, suggesting that the peptide formed a stronger and more stable interaction network at the binding interface.

[0061] Gibbs free energy maps can be used to evaluate the binding stability of receptor-ligand pairs. The blue to purple regions correspond to low free energy states, representing stable conformational binding. A single, smooth potential well in the energy landscape usually indicates strong and stable binding; multiple rough potential wells suggest weaker binding or poorer conformational stability. Figure 6 As shown in Figures J~L, for the 2D and 3D Gibbs free energy morphology diagrams of XOD / AASFPILFDMT, XOD / GIDFPWPVPNRP, and XOD / SGDETAPFFGFL, a comparison of the free energy landscape analysis from molecular dynamics simulations reveals that the free energy landscape (FEL) of all three systems exhibits a single, deep energy well, indicating that the system is mainly distributed in a stable low-energy conformation region during the simulation. GIDFPWPVPNRP has an even deeper energy well and a very compact morphology, indicating that its conformational distribution is highly concentrated in a single main cluster, and the conformational transition path is smooth. AASFPILFDMT and SGDETAPFFGFL also have concentrated and clear main energy wells and smooth convergence gradients, but their lowest energy regions are relatively wider, and the core fluctuation range of the stable conformation is slightly wider. Overall, both systems are dominated by a single dominant conformation, with GIDFPWPVPNRP exhibiting a more concentrated conformational cluster, potentially indicating higher conformational rigidity. Molecular dynamics analysis based on molecular docking results showed that peptides AASFPILFDMT, GIDFPWPVPNRP, and SGDETAPFFGFL can all form stable complexes with XOD, thereby exerting an inhibitory effect.

[0062] Example 3: Animal Experiment 1. Establishment of a mouse model of hyperuricemia (HUA) Mice were purchased from Yunnan Besttech Biotechnology Co., Ltd. Seventy-two ICR mice (5 weeks old, weighing 18-22g) were used, with the room temperature controlled at 24±2℃ and the environmental humidity at 50%-60%. Before the formal experiment began, all mice were given an acclimatization diet for one week to adapt to the surrounding environment. Throughout the experiment, all mice had free access to food and water and were periodically housed under a standard 12h light / 12h dark cycle.

[0063] After one week of acclimatization, mice were randomly divided into six groups of 12 mice each: normal control (NC), model control (Model), positive control (allopurinol, PO), low-dose hemp seed meal alkaline protein hydrolysate group (HE-L, 50 mg / kg / day), medium-dose hemp seed meal alkaline protein hydrolysate group (HE-M, 150 mg / kg / day), and high-dose hemp seed meal alkaline protein hydrolysate group (HE-H, 450 mg / kg / day). Administered via gavage at a volume of 10 mL / kg. The dosage and concentration of the modeling drug and HE were determined by reviewing relevant literature and previous experimental results. Every morning at 9:00 AM, the other five groups (excluding the NC group) were administered the modeling drugs via gavage: hypoxanthine (200 mg / kg / day) and potassium oxonate (200 mg / kg / day). The modeling drugs were dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution for 21 consecutive days. One hour after administering the modeling drug via gavage, the positive control group was administered 10 mg / kg / day of allopurinol via gavage, while the HE-L, HE-M, and HE-H groups were administered 50, 150, and 450 mg / kg, respectively, via gavage, and the NC group was administered physiological saline via gavage. Throughout the experiment, the mice's mental state, coat luster, and other characteristics were monitored, and their weight was recorded daily.

[0064] 2. The histopathological changes of mouse kidney tissue were observed using hematoxylin-eosin (HE) staining. Mouse kidney and liver tissues were taken, rinsed with physiological saline, and fixed in 4% paraformaldehyde solution. After dehydration with graded ethanol and clearing with xylene, they were routinely embedded in paraffin and sections with a thickness of 4 μm were prepared. HE staining was performed, and the histopathological morphological changes of mouse kidney tissue in each group were observed and analyzed under an optical microscope.

[0065] 3. Results and Analysis 3.1 Mouse body weight can serve as a direct indicator of health status; the body weight of normal mice generally increases steadily and slightly over time. For example... Figure 7 As shown in Figure A, the body weight of mice in all experimental groups increased steadily, indicating that the peptide had no significant toxicity or adverse effects on mice, and the experiment was safe. During the 3-week gavage intervention with hypoxanthine and potassium oxonate, the body weight of rats in all groups continued to increase, with the NC group showing the fastest growth, followed by the HE-H, HE-M, and HE-L groups, while the Model and PO groups showed the slowest growth. These results indicate that gavage with hypoxanthine combined with potassium oxonate can inhibit body weight gain in rats to some extent; meanwhile, the HE-H, HE-M, and HE-L groups significantly promoted body weight gain in hyperuricemic mice.

[0066] exist Figure 7In the B group, there were no significant differences in liver indices among the groups (p>0.05), indicating that hypoxanthine and potassium oxonate did not cause significant liver damage in the model group, and HE-H, HE-M, and HE-L showed no hepatotoxicity. Figure 7 As shown in Figure C, the kidney index of the model group rats was slightly higher than that of the normal control group. After gavage administration of HE-H, kidney damage in rats was significantly improved. In conclusion, HE-H, HE-M, and HE-L have no adverse effects on the liver.

[0067] To investigate the effects of hemp seed meal alkaline protease hydrolysate on hyperuricemia (HUA), a mouse model of HUA induced by hypoxanthine and potassium oxonate was established. Mice were treated with 50 mg / kg, 150 mg / kg, and 450 mg / kg of hemp seed meal alkaline protease hydrolysate, respectively. Compared with the NC group (80.37±7.25 μmol / L), the serum uric acid (UA) level in the Model group (210.15±9.53 μmol / L) was significantly higher (P<0.01), confirming the successful establishment of HUA. The serum UA level in the PO group (87.86±14.19 μmol / L) (P<0.01) was significantly lower than that in the Model group. After oral administration of 50 mg / kg, 150 mg / kg, and 450 mg / kg of HE, UA decreased by 50.08%, 52.35%, and 54.35%, respectively, in a dose-dependent manner. Serum creatinine and blood urea nitrogen (BUN) are important indicators reflecting renal function, and elevated levels are positively correlated with the degree of renal impairment. Figure 7 (D-F). Figure 7 E and F show that the levels of creatinine and urea nitrogen in the Model group were significantly higher than those in the NC group, indicating that hypoxanthine combined with potassium oxonate caused kidney damage in mice. The high, medium and low dose peptide groups and the PO group could significantly reduce the levels of creatinine and urea nitrogen. Among them, the HE-H group had a similar or even better effect on reducing creatinine and urea nitrogen than the positive control group (PO), showing a good kidney protective effect.

[0068] XOD is a key enzyme in purine metabolism, catalyzing the oxidation of xanthine and hypoxanthine to uric acid. Inhibiting XOD activity can effectively reduce uric acid synthesis. Uric acid is mainly synthesized in the liver and excreted through the kidneys; therefore, this invention simultaneously measures the activity of XOD in the liver and kidneys. Figure 7 As observed in the G-H assay, compared with the NC group, the XOD activity in the Model group was significantly increased (p < 0.05), while the XOD activity in the HE-L, HE-M, HE-H, and PO groups was significantly decreased. All three doses of hemp seed meal alkaline protein hydrolysate significantly reduced XOD activity, and the effect was comparable to that in the PO group. Figure 7 In the G group, XOD activity decreased by 25.61%, 30.73%, and 46.81% in the HE-L, HE-M, and HE-H groups, respectively. Figure 7The XOD activity in the HE-L, HE-M, and HE-H groups decreased by 22.57%, 32.32%, and 37.41%, respectively, while that in the PO group decreased by 42.59%. This indicates that the alkaline protein hydrolysate of hemp seed meal can effectively inhibit XOD activity in vivo, thereby reducing uric acid synthesis and lowering the serum uric acid level in hyperuricemic rats.

[0069] 3.2 Regulatory Effects of Hemp Seed Meal Protein Peptides on Inflammatory Factors Hyperuricemia is often accompanied by kidney inflammation. High uric acid can activate caspase-associated inflammasomes, leading to cell membrane rupture and the release of large amounts of inflammatory factors, thereby inducing chronic inflammation. The caspase family is a core molecule regulating apoptosis, among which caspase-3 (abbreviated Casp-3) activation can initiate downstream apoptotic proteases. The NLRP3 inflammasome, as an important component of innate immunity, can be activated by various danger signals and participates in immune responses and the progression of various diseases. IL-1β, as a key pro-inflammatory cytokine, participates in autoimmune inflammatory responses, cell proliferation, differentiation, and apoptosis, and mediates neuroprotection and tissue repair; its overexpression is associated with pathophysiological changes in various diseases and plays an important role in acute and chronic inflammation and autoimmune diseases.

[0070] As shown in Figures A–B of 8, Casp-3 levels in the kidneys and liver of the Model group were significantly elevated. After peptide administration, the Casp-3 level in the HE–H group was significantly lower than that in the Model group, reaching the same level as the NC group. The HE–L and HE–M groups showed slight but insignificant reductions, and no decrease in liver Casp-3 was observed, indicating that HE-M had no inhibitory effect on liver Casp-3. However, the HE–H and PO groups showed similarly excellent levels. Due to cell membrane rupture, a large number of inflammatory factors are produced. Figure 8 As can be seen from C–D, the IL–1β level in the Model group was significantly higher than that in the NC group. After administration, the IL–1β level was significantly reduced in all different doses of HE, with HE–H showing better inhibitory effects than HE–L and HE–M.

[0071] Depend on Figure 8 As can be seen from E–F, the level of the inflammatory factor NLRP3 in the Model group was significantly higher than that in the NC group. After administration, the NLRP3 levels in the HE–H, HE–M, and PO groups were significantly reduced, with HE–H showing the most significant reduction. HE–L was consistent with the Model group, indicating that HE–L had no inhibitory effect, while high doses had a significant inhibitory effect on NLRP3.

[0072] The detection of three inflammatory factors revealed that the expression levels of Caspase-3, IL-1β, and NLRP3 in hyperuricemia model mice were significantly higher than those in normal mice, indicating that the model mice had significant kidney inflammation. Different doses of HE showed varying inhibitory effects on each inflammatory factor: only high-dose HE-H showed the most significant inhibitory effect on NLRP3; while low, medium, and high doses of hemp seed meal alkaline protease all showed good inhibitory effects on Caspase-3 and IL-1β, with the high dose showing the best effect. Although the commonly used clinical drug PO can be used for related treatments, its anti-inflammatory effect is limited and it has certain toxic side effects. Therefore, the development of safe and non-toxic polypeptide substances with both uric acid-lowering and anti-inflammatory activities is of great significance. In summary, hemp seed meal alkaline protease hydrolysate exhibits excellent uric acid-lowering and anti-inflammatory potential, possessing good prospects for development and application. This invention can provide a theoretical basis and experimental evidence for its subsequent development and utilization.

[0073] 4. Effects of hemp seed meal alkaline protease on the kidneys and liver H&E staining is a commonly used method in histological and pathological studies for observing cell morphology, tissue structure, and pathological damage. Hyperuricemia can induce various kidney diseases, including asymptomatic uric acid nephropathy, uric acid stones, acute / chronic uric acid nephropathy, and chronic renal failure. Kidney tissue biopsy reveals inflammatory cell infiltration; as the disease progresses, changes such as glomerular basement membrane thickening, glomerular fibrosis, tubular atrophy and degeneration, and interstitial vascular dilation may occur. Figure 9 As can be seen, the renal tissue in the NC group was clear and intact, with cells arranged in a tight and orderly manner. Compared with the NC group, the Model group showed significant damage to the renal tubular structure, manifested as inflammatory cell infiltration in the renal interstitium. Severe chronic tubulointerstitial nephritis with glomerular sclerosis (reduced peripheral space) exacerbated the damage, with significant interstitial fibrosis and inflammation, tubular dilation, and vacuolar formation. Since the renal reabsorption capacity has a significant impact on serum uric acid levels, tubular damage may lead to increased uric acid levels, causing gout and kidney stones. After drug intervention, the HE-L, HE-M, and HE-H groups all showed varying degrees of improvement in renal tissue pathological damage. Among them, the HE-M and HE-H groups showed no obvious inflammatory cell infiltration, intact glomerular basement membrane structure, no shedding of renal tubular epithelial cells, and basically normal morphological structure of the glomeruli and tubules; while the HE-L group still showed lesions such as vacuolar degeneration, inflammatory infiltration, and glomerular atrophy. Compared with the Model group, the PO group showed reduced glomerular atrophy, but still exhibited some degree of tubular dilation and inflammatory cell infiltration, suggesting that while allopurinol significantly improved kidney injury induced by hypoxanthine combined with potassium oxonate, it also damaged kidney tissue to some extent. In conclusion, hemp seed meal alkaline protease hydrolysate can effectively improve hyperuricemia-related kidney injury and is a potentially promising anti-hyperuricemia functional factor.

[0074] In addition, increased UA levels in the body can cause oxidative stress damage to the liver. Figure 10 In the NC group, the livers of mice were normal, with neatly arranged cells and abundant cytoplasm. In the Model group, hepatic sinusoids were dilated, with round vacuoles in the cytoplasm, inflammatory cells around the veins, and indistinct intercellular spaces. The PO group still exhibited vacuolar degeneration and inflammatory cell infiltration, but to a significantly reduced degree. The HE-L group showed significant improvement in hepatic sinusoidal dilation, but some vacuoles remained. Furthermore, high-dose HE-H significantly improved the marked dilation and vacuolation of hepatic sinusoids, indicating that hemp seed peptides can alleviate liver damage in hyperuricemic mice.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A hemp seed meal protein peptide with uric acid-lowering activity, characterized in that, The amino acid sequence of the hemp seed meal protein peptide is AASFPILFDMT, GIDFPWPVPNRP, or SGDETAPFFGFL.

2. The use of the hemp seed meal protein peptide as described in claim 1 in any of the following: (1) Its application in the preparation of drugs for treating hyperuricemia; (2) Application in the preparation of uric acid-lowering drugs; (3) Application in the preparation of products that inhibit xanthine oxidase activity.

3. A hemp seed meal alkaline protease hydrolysate, characterized in that, The alkaline protease hydrolysate of hemp seed meal contains the hemp seed meal protein peptides as described in claim 1.

4. A method for preparing the alkaline protease hydrolysate of hemp seed meal according to claim 3, characterized in that, Includes the following steps: Hemp seed meal was mixed with water and extracted to obtain hemp protein; The hemp protein was mixed with water, and alkaline protease was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated and freeze-dried to obtain hemp meal alkaline protease hydrolysate.

5. The preparation method according to claim 4, characterized in that, The ratio of hemp seed meal to water is 1g:(8-15)mL.

6. The preparation method according to claim 4, characterized in that, The water extraction method includes: mixing the hemp seed meal with the water evenly, adjusting the pH value to alkaline, stirring and extracting at room temperature for 1-2 hours, filtering and centrifuging after extraction, collecting the supernatant, adjusting the pH value to acidic, letting it stand at room temperature for 1-2 hours, centrifuging again, discarding the supernatant, resolving the obtained precipitate in water, adjusting the pH value to neutral, and freeze-drying to obtain the hemp protein.

7. The preparation method according to claim 4, characterized in that, The ratio of hemp protein to water is 1g:(8-15)mL.

8. The preparation method according to claim 4, characterized in that, The enzymatic hydrolysis conditions are: pH 8.0, temperature 55-60℃, and time 2-4h.

9. The use of the hemp seed meal alkaline protease hydrolysate as described in claim 3 in any of the following: (1) Its application in the preparation of drugs for treating hyperuricemia; (2) Application in the preparation of uric acid-lowering drugs; (3) Application in the preparation of products that inhibit xanthine oxidase activity.

10. A drug for treating hyperuricemia, characterized in that, It includes the hemp seed meal protein peptides of claim 1 or the hemp seed meal alkaline protease hydrolysate of claim 3.

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