Carya illinoensis uric acid reducing active peptide as well as preparation method and application thereof

Abstract

The invention discloses an apocarya uric acid-reducing active peptide as well as a preparation method and application thereof. The invention provides a uric acid reducing active peptide of carya illinoensis, which comprises at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, the amino acid sequence of the FLADAF is SEQ ID No.1, the amino acid sequence of the FFFPGPS is SEQ ID No.2, and the amino acid sequence of the WPFGGDS is SEQ ID No.3. The invention further provides a preparation method of the uric acid reducing active peptide of carya illinoensis. The active peptide from the carya illinoensis not only has the effects of reducing uric acid and resisting uric acid toxicity, but also has the functions of protecting kidneys, livers and intestines, and shows good safety and wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and relates to a thin-shelled pecan uric acid-lowering active peptide, its preparation method, and its application. Background Technology

[0002] Hyperuricemia is a metabolic disease caused by disordered purine metabolism or reduced uric acid excretion, clinically characterized by persistently elevated serum uric acid levels. Long-term hyperuricemia is not only a direct risk factor for gout, but also closely related to various metabolic syndromes such as hypertension, diabetes, chronic kidney disease, and cardiovascular and cerebrovascular diseases, making it a significant public health problem threatening human health. In recent years, with changes in lifestyle and dietary structure, the incidence of hyperuricemia and gout has been increasing year by year, and there is a trend towards younger onset.

[0003] Currently, commonly used uric acid-lowering drugs in clinical practice mainly include xanthine oxidase inhibitors (such as allopurinol and febuxostat) and uricosuric agents (such as benzbromarone). Although these drugs are effective in lowering serum uric acid, long-term use is often accompanied by adverse side effects such as skin rash, liver and kidney toxicity, and gastrointestinal reactions, resulting in poor patient compliance. In addition, some patients have tolerance or contraindications to existing drugs, necessitating the development of safe, natural, and low-side-effect alternative or adjuvant therapies.

[0004] In recent years, food-derived bioactive peptides have attracted widespread attention due to their well-defined structure, high safety, and ease of absorption and utilization. Existing studies have shown that low-molecular-weight peptides derived from dairy products, soybeans, corn, and other foods possess various physiological activities, including antioxidant, blood pressure-lowering, immune-regulating, and metabolic-improving effects. Among these, some short peptides have been reported to have the potential to inhibit xanthine oxidase activity and lower uric acid levels, providing insights for the development of novel uric acid-lowering functional foods and health products. However, the sources of publicly reported uric acid-lowering bioactive peptides are currently limited, and research on their mechanisms of action and applications remains insufficient.

[0005] Pecans (Carya illinoinensis) belong to the genus Carya in the family Juglandaceae. Their kernels are rich in high-quality protein, unsaturated fatty acids, vitamins, and minerals, making them a highly nutritious nut. Current research largely focuses on the antioxidant and cardiovascular protective effects of their lipid components, while studies on their proteins and bioactive peptides are relatively scarce. To date, there are no systematic reports on the effects of bioactive peptides derived from pecans on lowering uric acid or combating gout.

[0006] Therefore, there is an urgent need to discover and identify bioactive peptides with uric acid-lowering effects from thin-shelled pecans, and to develop functional foods or drugs based on natural food-derived components to meet the needs of clinical treatment and health management. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing uric acid-lowering drugs, such as large side effects and limited sources of food-derived functional active peptides, and to provide a uric acid-lowering active peptide derived from thin-shelled pecans and its application, in order to develop safe, natural, and low-side-effect novel uric acid-lowering foods or drugs.

[0008] To achieve the above objectives, the present invention provides a uric acid-lowering active peptide from thin-shelled pecans, the uric acid-lowering active peptide comprising at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No. 3.

[0009] According to the present invention, the above-mentioned antioxidant active peptides are derived from pecan protein.

[0010] According to the present invention, the purity of the above-mentioned uric acid-lowering active peptide is ≥95%.

[0011] According to the present invention, the molecular weight of the above-mentioned uric acid-lowering active peptide is less than 3 kDa.

[0012] The present invention also provides a composition comprising the above-mentioned uric acid-lowering active peptide, and a pharmaceutically acceptable carrier or a food-acceptable excipient.

[0013] This invention also provides a method for preparing uric acid-lowering active peptides from thin-shelled pecans, the method comprising the following steps:

[0014] (1) Enzymatic hydrolysis: Alkaline protease and neutral protease were used to perform double enzymatic hydrolysis on the thin-shelled pecan protein to obtain protein hydrolysate;

[0015] (2) Ultrafiltration: The protein hydrolysate was separated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and small molecule peptides of thin-shelled pecan with a molecular weight of less than 3 kDa were collected.

[0016] (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: After the small molecule peptides were reduced, alkylated and desalted, they were analyzed by liquid chromatography-mass spectrometry system to obtain raw mass spectrometry data, and de novo analysis was performed by PEAKS Studio 10.6 software to obtain the amino acid sequence information of the small molecule peptides;

[0017] (4) Screening: The xanthine oxidase (XOD) binding activity of the small molecule peptides was evaluated based on molecular simulation docking, and bioinformatics prediction scores were combined to screen candidate uric acid-lowering active peptides with high biological activity and no toxicity.

[0018] (5) Cellular and in vitro enzymatic validation: In the HK-2 cell model and in vitro enzymatic system, the inhibitory effects of the above-mentioned candidate uric acid-lowering active peptides on XOD were determined and the inhibition rate / IC50 ratio was established. 50 Based on indicators, combined with cell viability and safety assessments, uric acid-lowering active peptides that meet preset thresholds are screened out.

[0019] (6) In vivo functional verification: The drug was evaluated in a mouse model of hyperuricemia induced by PO+AD, and serum uric acid and related metabolic / histochemical indicators were detected to confirm the uric acid-lowering active peptide uric acid regulation effect of the preset threshold, thereby finally obtaining the uric acid-lowering active peptide of the present invention.

[0020] According to the present invention, the conditions for enzymatic hydrolysis in step (1) above are pH 7.0-8.5, temperature 50-60℃, and enzymatic hydrolysis time 120-180min.

[0021] The present invention also provides the use of urate-lowering active peptides in the preparation of urate-lowering drugs, wherein the urate-lowering active peptides include at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No. 3.

[0022] The present invention also provides the use of a urate-lowering active peptide in the preparation of a medicament for the treatment or prevention of hyperuricemia, the urate-lowering active peptide comprising at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No. 3.

[0023] The present invention also provides the use of uric acid-lowering active peptides in the preparation of functional foods or health products, said foods or health products for lowering serum uric acid levels, wherein the uric acid-lowering active peptides comprise at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No. 3.

[0024] The uric acid-lowering active peptides from thin-shelled pecans disclosed in this invention exhibit significant uric acid-lowering and cytoprotective effects both in vitro and in vivo. In an HK-2 cell hyperuricemia model, FLADAF, FFFPGPS, and WPFGGDS significantly reduced uric acid levels in cell culture medium in a dose-dependent manner; under medium and high doses, their uric acid-lowering effects were comparable to the positive control allopurinol. Simultaneously, in vitro enzyme activity experiments confirmed that these peptides can inhibit xanthine oxidase (XOD) activity, reducing uric acid production at its source, and mitigating cytotoxicity in an exogenous uric acid-induced damage model, significantly maintaining HK-2 cell viability and demonstrating good anti-uric acid toxicity function. Animal experiments further showed that the above-mentioned active peptides significantly reduced serum uric acid, blood urea nitrogen, and creatinine levels in a hyperuricemic mouse model, improved the trend of weight loss, and protected the structure of the kidney, liver, and colon at the histological level. Specifically, it repairs the morphology of glomeruli and renal tubules, reduces inflammatory cell infiltration and fibrosis, alleviates disordered dilation and cell necrosis of hepatic sinusoids, and improves intestinal villus length and crypt structure abnormalities. In summary, the bioactive peptides derived from thin-shelled pecans not only have uric acid-lowering and anti-uric acid toxicity effects, but also possess kidney-protecting, liver-protecting, and intestinal-protecting functions, demonstrating good safety and broad application prospects.

[0025] The uric acid-lowering active peptides from thin-shelled pecans disclosed in this invention significantly reduced uric acid levels in cell culture medium in an HK-2 cell hyperuricemia model in a dose-dependent manner. FLADAF, under high-dose conditions, showed uric acid-lowering effects comparable to the positive control allopurinol. Simultaneously, it inhibited xanthine oxidase activity, directly reducing uric acid production. In animal studies, the uric acid-lowering active peptides of this invention significantly reduced serum uric acid, blood urea nitrogen, and creatinine levels in a mouse model of hyperuricemia, improving body weight changes. They also protected the structure of the kidneys, liver, and colon, repaired glomerular and tubular morphology, reduced inflammatory cell infiltration and fibrosis, alleviated disordered dilation of hepatic sinusoids, and improved the morphology of intestinal villi and crypts.

[0026] Furthermore, the uric acid-lowering active peptides of this invention can alleviate uric acid-induced cytotoxicity, improve cell survival rate, and reduce inflammatory responses. These peptides are simple to prepare and easy to mass-produce, and can be used as raw materials for functional foods, health products, or pharmaceuticals, demonstrating good application value and industrialization prospects. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0028] In the following figure, NC represents the normal group, MC represents the model control group, AL and BZ represent the two positive drug control groups, and FLADAF, FFFPGPS, and WPFGGDS represent the three peptide treatment groups. The ### above each bar indicates p < 0.001 compared to the control group; *** indicates p < 0.001 compared to the hyperuricemia model group. Sample size N > 12. See the detailed figure below:

[0029] Figure 1 The results of mass spectrometry analysis of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS are as follows:

[0030] Figure 2 The results of molecular docking simulations of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS are shown below.

[0031] Figure 3 The results of cytotoxicity assays of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS are presented in this invention.

[0032] Figure 4 This study aims to verify the in vitro uric acid-lowering function of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention.

[0033] Figure 5 This is a dose-response diagram showing the XOD inhibitory activity of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, WPFGGDS and the positive control drug allopurinol in this invention.

[0034] Figure 6 Figure 1 shows the cell experiment results verifying the anti-uric acid toxicity function of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS from this invention.

[0035] Figure 7 This is a graph showing the changes in mouse body weight during the establishment of the hyperuricemia mouse model according to the present invention;

[0036] Figure 8 The figure shows the effects of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention on serum uric acid content, urea nitrogen content, and serum creatinine content in mice.

[0037] Figure 9 Figure showing the effect of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention on the appearance of mouse kidneys;

[0038] Figure 10Figure showing the effects of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention on mouse kidney tissue;

[0039] Figure 11 Figure showing the effects of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention on mouse liver tissue;

[0040] Figure 12 This diagram illustrates the effects of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of the present invention on mouse colon tissue. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Example 1

[0045] The steps for extracting protein from thin-shelled pecans are as follows:

[0046] 1. Raw material preparation: Select defatted meal of thin-shelled pecans, add it to distilled water at a ratio of 1:50 (w / v), and disperse it fully.

[0047] 2. Dissolving and adjusting pH: After heating the mixture to 55°C, adjust the pH to 11.0 using a 1 mol / L sodium hydroxide solution, and continue stirring for 90 minutes to ensure complete dissolution of the protein.

[0048] 3. Centrifugation: Centrifuge at 8000 rpm for 10 minutes, collect the supernatant and remove impurities.

[0049] 4. Precipitate protein: Adjust the pH of the supernatant to 4.0 with 1 mol / L hydrochloric acid, let it stand to precipitate, and finally take the precipitate as thin-shelled pecan protein.

[0050] Example 2

[0051] The following steps are used to illustrate the preparation of thin-shelled pecan peptides:

[0052] Disperse pecan protein at a 1:50 (w / v) ratio in distilled water and heat to 90°C for 15 min. Cool to the enzymatic hydrolysis temperature of 53°C, adjust the protein solution to pH 7.2 with 1.0 mol / L NaOH, add 9000 U / g of neutral protease, and hydrolyze for 120 min, continuously adjusting the pH with NaOH to maintain a stable pH of 7.2. Place in a 90°C water bath for 15 min to inactivate the enzyme. Cool to the enzymatic hydrolysis temperature of 56°C, adjust the protein solution to pH 8.4 with 1.0 mol / L NaOH, add 7000 U / g of alkaline protease, and hydrolyze for 180 min, continuously adjusting the pH with NaOH to maintain a stable pH of 8.4. Place in a 90°C water bath for 15 min to inactivate the enzyme, adjust the pH to 7.0, centrifuge at 8000 rpm, 4°C for 10 min, and the supernatant is the pecan peptide.

[0053] The thin-shelled pecan peptide solution was subjected to ultrafiltration separation using a 3kDa ultrafiltration membrane and a peristaltic pump at 120 rpm. The ultrafiltration fraction with a molecular weight <3kDa was collected, freeze-dried under vacuum, and the thin-shelled pecan peptide powder was prepared.

[0054] Example 3

[0055] The steps for identifying the uric acid-lowering active peptide from thin-shelled pecans are as follows:

[0056] (1) Sample pretreatment: Weigh 1 mg of thin-shelled pecan peptide powder, add an appropriate amount of 50 mmol / L ammonium bicarbonate solution to dissolve the sample, add dithiothreitol solution to make the final concentration 10 mmol / L, and reduce in a 56℃ water bath for 1 h. Add iodoacetamide solution to make the final concentration 55 mmol / L, react in the dark for 40 min, desalt using a self-packed desalting column, and evaporate the solvent in a vacuum centrifuge at 45℃.

[0057] (2) LC-MS / MS detection: Liquid chromatography conditions: Pre-column: 150 μm idx 5 cm, packing: Reprosil-Pur 120C18-AQ 3 μm; Analytical column: 100 μm idx 180 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; Flow rate: 600 nL / min; Analysis time for each component: 60 min.

[0058] Mass spectrometry conditions: Primary mass spectrometry parameters: Resolution: 70,000; AGC target: 3e6; Maximum IT: 60 ms; Scan range: 100 to 1500 m / z. Secondary mass spectrometry parameters: Resolution: 17,500; AGC target: 5e4; Maximum IT: 80 ms; NCE / stepped NCE: 27.

[0059] (3) Database Search: Peptide sequences were analyzed using PEAKS Studio 10.6 De novo software on the raw mass spectrometry files. The search parameters were as follows: Fixed modifications; Variable modifications; Enzyme; Maximum Missed Cleavages; Peptide Mass Tolerance (PMT); Fragment Mass Tolerance (FMT). Results of FLADAF, FFFPGPS, and WPFGGDS peptide mass spectrometry analyses are shown below. Figure 1 This refers to the thin-shelled pecan uric acid-lowering active peptide of the present invention.

[0060] Example 4

[0061] The bioinformatics test used to demonstrate the uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of thin-shelled pecans is performed as follows:

[0062] (1) Molecular simulation docking: 2D structures of small molecule ligands were obtained from the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ), and their 3D structures were created using Chem Office software and saved as mol2 files. Then, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen for high-resolution crystal structures of protein targets as molecular docking acceptors. PyMOL software was used to perform operations such as dehydration and dephosphateation on the proteins, and the results were saved as PDB files. AutoDock Vina 1.5.6 software was used for molecular docking to explore the interactions between protein and ligands. AutoDock was used to process the structures of proteins and small molecules, including hydrogenation and dehydration of proteins, hydrogenation of small molecule ligands, and determination of torsional forces. The docking box coordinates were then determined, and the optimal conformation of the molecular simulation was obtained by comparing the docking scores. The interaction between the test compound and key residues was visualized using Discovery Studio 2019 software, resulting in 2D and 3D analysis diagrams. The docking results of FLADAF, FFFPGPS, and WPFGGDS peptides with their receptors are shown below. Figure 2 .

[0063] (2) Prediction of potential biological activity of peptides: The potential biological activity of the three obtained peptide sequences was analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). Peptides were ranked according to their predicted biological activity probabilities. The preset threshold of the PeptideRanker prediction model was 0.5; a threshold greater than 0.5 indicated biological activity. The predicted values ​​for the three novel peptides screened in this application, FLADAF, FFFPGPS, and WPFGGDS, were 0.832085, 0.929779, and 0.738948, respectively. Bioinformatics prediction analysis results indicate that FLADAF, FFFPGPS, and WPFGGDS have high potential biological activity.

[0064] (3) Prediction of potential peptide toxicity: using Toixinpred ( https: / / webs.iiitd.edu.in / raghava / toxinpred / index.h-tml The platform, based on the SVM (Swiss-Port) algorithm, predicts the potential toxicity of novel peptides. According to the potential toxicity analysis, peptides FLADAF, FFFPGPS, and WPFGGDS are all non-toxic.

[0065] Example 5

[0066] The following steps were taken for the cellular and in vitro enzymatic verification of the uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS from thin-shelled pecans:

[0067] In the corresponding cytotoxicity assessment, HK-2 showed a result of 1×10⁻⁶. 5 Candidate peptides were seeded per well in 96-well plates and allowed to adhere for 24 hours. After pre-culturing in serum-free medium for 24 hours, candidate peptides (20-100 μL / mL or 50-1600 μM) were added and incubated for 24 hours. Then, 10 μL of CCK-8 working solution was added per well and incubated for 4 hours. The results were read at 450 nm and the survival rate was calculated as: [(OD sample - OD blank) / (OD control - OD blank)] × 100. To avoid interference from indicators and serum, all CCK-8 assays were performed under serum-free conditions with blank well correction; weak background absorption from phenol red-containing medium was excluded if necessary. The analytical results are as follows: Figure 3 The three small peptides showed reduced cell activity at a concentration of 3200 μM, and this reduction was dose-dependent.

[0068] To systematically evaluate the uric acid-lowering effects of pecan-derived peptides (FLADAF, FFFPGPS, WPFGGDS), we constructed an exogenous uric acid (UA) stimulation model and an adenosine + exogenous XOD purine production model in HK-2 human renal proximal tubular epithelial cells. Furthermore, we used HK-2 lysis buffer as an endogenous enzyme source to detect the inhibitory activity against xanthine oxidase (XOD). Cells were routinely cultured in MEM basal medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C and 5% CO2. All HK-2 cells used in the experiments were in the logarithmic growth phase, with passage numbers controlled within 5-20 generations, and were confirmed to be morphologically normal and free of mycoplasma contamination before the experiments.

[0069] In the purine production model, after cells adhered for 48 h, they were washed three times with PBS and then cultured in serum-free medium to reduce the background of exogenous proteins and endogenous purines. They were then incubated for 30 h with adenosine at 2.5 mmol / L, followed by incubation for another 8 h with exogenous XOD (0.005 U / mL) to obtain the "adenosine + XOD" group; the controls were "no adenosine / no XOD" (blank) and "no adenosine / plus XOD" (XO). Candidate peptides or AL were added at the beginning of the above timeline and maintained until the endpoint; peptide doses were expressed as 200 / 800 / 1600 μM. If alignment with the aforementioned μM system was required, the volume fraction could be converted to the final concentration (μM) based on the respective stock solution concentration. The endpoint treatment and uric acid quantification method were the same as above. This model increases extracellular uric acid flux in a serum-free environment through exogenous substrates and enzymes, facilitating comparison of the reduction in different treatments under low background conditions; all groups received an equal volume of solvent when XOD or adenosine was added to maintain consistent volume and osmotic pressure. Analysis Figure 4It was found that the three peptides could effectively reduce the uric acid content in the cell culture supernatant at all three concentrations. Among them, FL6, FF7, and WP7 peptides showed comparable uric acid-lowering effects to the positive control drug allopurinol at treatment concentrations of 800 μM and 1600 μM, exhibiting excellent activity characteristics.

[0070] Through analysis Figure 5 It was found that in the in vitro chemical assay, the three peptides FLADAF, WPFGGDS, and FFFPGPS, as well as allopurinol, all inhibited XOD activity. Furthermore, XOD activity gradually decreased with increasing peptide concentration, indicating a dose-response relationship that these peptides could inhibit XOD activity. Simultaneously, the in vitro inhibitory activity of FLADAF, WPFGGDS, and FFFPGPS on XOD was comparable to that obtained from cellular activity studies, suggesting good consistency in the uric acid-lowering function of these three peptides.

[0071] Example 6

[0072] The steps for verifying the anti-uric acid toxicity function of the uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS from thin-shelled pecans are as follows:

[0073] Materials and Methods: In the exogenous uric acid model, HK-2 was used at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 mL per well in 24-well plates and allowed to adhere for 24 hours. Candidate peptides were pretreated in basal medium for 12 hours, followed by incubation for 24 hours with uric acid working solution (dissolved in 1N NaOH and adjusted to pH 7.2-7.4) to achieve a final uric acid concentration of 40 mg / dL (or a gradient of 30-50 mg / dL). The peptide concentration was set at 800 μM. Groups included a blank control (without uric acid or drug) and a model control (with uric acid and an equal volume of solvent). The uric acid stock solution was briefly incubated at 37°C after dissolution, and then sterilized by passing it through a 0.22 μm filter before use to minimize low-temperature crystallization. If necessary, a rapid microscopic examination was performed before adding cells to check for crystal deposition. At the endpoint, the culture supernatant was collected, centrifuged at 1000×g for 5 min to remove cell debris, and uric acid was quantified using the urate oxidase-peroxidase colorimetric method. A standard curve and quality control wells were set up within the same plate. The relative effect was expressed as a decrease rate: decrease rate (%) = [1 - (sample group uric acid / model control uric acid)] × 100. To avoid interference from the formulation background, both colorimetric and spectral measurements included "substrate-free" or "enzyme-free" controls with baseline subtraction. If microplate readings were used, effective optical path correction was performed according to the manufacturer's instructions, or the molar extinction coefficient was replaced with a standard curve for conversion.

[0074] analyze Figure 6It can be obtained that through the pre-experiment of the simple uric acid concentration gradient, the effect of uric acid on cell viability and the applicable concentration range for subsequent experiments were clarified; then, by comparing the control group and the uric acid + peptide group, cell viability was detected. The results showed that under all concentration gradients, the peptides FLADAF, WPFGGDS, and FFFPGPS could resist or mitigate uric acid-induced cell damage and maintain cell survival.

[0075] Example 7

[0076] To illustrate the uric acid-lowering effect of the pecan protein peptides FLADAF, FFFPGPS, and WPFGGDS in a hyperuricemia mouse model, the specific details are as follows:

[0077] 1 Materials and methods:

[0078] 1.1 Peptide synthesis

[0079] Using solid-phase amino acid synthesis technology, small peptides were chemically synthesized. The synthetic peptides were chemically solid-phase synthesized by Jiangsu Jitai Peptide Industry Technology Co., Ltd., and the purity of the synthetic peptides was >95%.

[0080] 1.2 Experimental subjects

[0081] A total of 84 specific pathogen-free (SPF) male KunMing mice with a body weight of (22 ± 2) g were randomly selected and provided by Hangzhou Qizhen Laboratory Animal Technology Co., Ltd. The production license number of experimental animals: SCXK(Zhe)2022-0005. The animal experiments were approved by the Animal Ethics Committee of Zhejiang A&F University (approval number: ZAFUAC202541) to ensure that all animal experiments complied with relevant regulations. All mice were placed in a stable environment with experimental feeding room conditions (temperature (25 ± 1)°C, humidity 50 ± 5%, and light cycle of 12 h (9:00 - 21:00)) for adaptive cultivation for seven days, during which they could freely access water and food.

[0082] 1.3 Experimental grouping

[0083] The mice were divided into a normal group (Normal Control, NC), a model control group (Model Control, MC), a positive drug control group I (allopurinol), a positive drug treatment group II (benzbromarone), and three peptide treatment groups (hexapeptide FLADAF, heptapeptide FFFPGPS, heptapeptide WPFGGDS), with 12 mice in each group.

[0084] 1.4 Modeling and functional verification

[0085] Mice in the NC group were administered 0.2 mL of 0.5% CMC-Na solution via gavage and injection. Mice in all other groups were administered 150 mg / kg adenine via gavage and 300 mg / kg potassium oxonate via intraperitoneal injection, once daily for three weeks. During the modeling period, the MC group was administered 0.2 mL of 0.5% CMC-Na via gavage. Positive drug treatment groups I and II were administered 10 mg / kg allopurinol and benzbromarone via gavage, respectively. The three peptide treatment groups were administered 60 mg / kg FLADAF, FFFPGPS, and WPFGGDS peptide solutions via gavage, respectively. The health status of the mice was checked daily, and their weight, food intake, and water intake were recorded regularly.

[0086] 1.5 Sample Collection

[0087] Mouse serum sample collection: Mice were intraperitoneally injected with 50 mg / kg of 1% sodium pentobarbital. After the mice lost their corneal reflex and limbs showed no spontaneous movement, blood samples were collected using the retro-orbital venous plexus method via capillary tube. Approximately 0.8-1.0 mL of blood was collected from each mouse and transferred to centrifuge tubes. The centrifuge tubes containing the blood samples were placed in a 4°C freezer for 30 min, and then centrifuged in a high-speed refrigerated centrifuge at 4°C and 3000×g for 15 min. After centrifugation, the supernatant serum was aspirated and transferred to sterile EP tubes, which were stored at -80°C for subsequent serum marker detection. Tissue sample collection and processing: After serum collection, mice were immediately euthanized by cervical dislocation, and the mice were quickly dissected. The kidneys, liver, and colon tissues were separated and removed, and the surface bloodstains were washed off with PBS. The dissected kidney, liver, and colon tissues were placed in light-proof EP tubes pre-filled with 4% paraformaldehyde fixative (tissue to fixative volume ratio of 1:10) and fixed at room temperature for 24 hours. They were then embedded in paraffin, sectioned, and used for subsequent HE staining and pathological observation.

[0088] 2 Experimental Results

[0089] like Figure 7 As shown, the body weight of mice after modeling and treatment varied. The MC group mice showed a significant decrease in body weight, while the positive drug groups I and II, and the three peptide treatment groups all showed a recovery trend. Serum uric acid levels were measured in each group of mice, and the results are as follows: Figure 8As shown in Figure A, the serum uric acid level in the MC group mice was significantly increased. The serum uric acid levels in the two positive drug groups and the three peptide treatment groups were all lower than those in the MC group, and the serum uric acid level in the hexapeptide FLADAF treatment group was similar to that in the NC group, indicating that all three uric acid-lowering peptides have good uric acid-lowering effects. In the assessment system of hyperuricemia, serum creatinine and blood urea nitrogen are important indicators for evaluating renal function, and serum creatinine and blood urea nitrogen are closely related to serum uric acid levels. Elevated serum creatinine and blood urea nitrogen levels indicate abnormal renal function, suggesting pathological damage to the kidneys. The blood urea nitrogen levels in each group of mice were measured, and the results are as follows: Figure 8 As shown in Figure B. Analysis revealed that compared to the MC group mice, the blood urea nitrogen levels in all three peptide-treated groups were significantly reduced, with effects similar to the two positive drug-treated groups, indicating that all three peptides possess excellent renal protective capabilities. Serum creatinine levels in each group of mice were measured, and the results are shown below. Figure 8 As shown in Figure C. Analysis revealed that the serum creatinine level of mice in the MC group was significantly increased, while the serum uric acid levels of hyperuricemic mice treated with FLADAF, FFFPGPS, and WPFGGDS were significantly decreased compared to the MC group. This indicates that the three peptides can improve the renal metabolic capacity under hyperuricemia, enhance glomerular filtration capacity, and thus lower uric acid levels.

[0090] Hyperuricemia causes changes in the appearance and morphology of the kidneys and liver in mice, reflecting the damage and recovery caused by hyperuricemia. From Figure 9 As can be seen, the kidneys of mice in the NC group were normal in morphology, rosy in color, and smooth in surface, while the kidneys of mice in the MC group were significantly shrunken, pale in color, and extremely rough in surface, proving that mice treated with adenine and potassium oxonate in combination exhibited obvious hyperuricemia, as expected. The kidneys of mice treated with the positive control group showed significantly improved appearance compared to the MC group. The kidneys of mice treated with the three peptides also showed varying degrees of improvement compared to the MC group, all exhibiting a rosy and smooth appearance.

[0091] Paraffin sections of the kidneys of mice in each group were stained with hematoxylin and eosin (HE), and the kidney sections were observed under 1x and 20x optical microscopes. The results are as follows: Figure 10As shown in the figure. Analysis revealed that the kidneys in the NC group had intact structure, with normal morphology of glomeruli, tubules, and interstitium, and no inflammation or pathological changes were observed. The kidneys in the MC group were severely damaged, with surface depression, significant glomerular proliferation, tubular dilation, and obvious inflammatory infiltration, fibrosis, and hyalinous lesions. The groups treated with the positive control drugs allopurinol and benzbromarone showed significantly less kidney tissue damage than the MC group. The kidney tissue damage in the FLADAF group was similar to that in the positive control group, with only a few tubular dilations and interstitial edema. Kidney tissue sections from mice treated with FFFPGPS and WPFGGDS also showed some recovery, with less damage than the MC group, and smoother kidney edges. In addition, no obvious inflammatory cell infiltration was observed in the FLADAF-treated group, indicating that this peptide has excellent anti-inflammatory capabilities.

[0092] from Figure 11 As can be seen, the liver tissue structure of mice in the NC group was clear, the hepatocytes were of normal size and neatly arranged, and no obvious pathological changes were observed. Compared with the NC group, the liver damage in the MC group was severe, with disordered dilation of the sinusoids, chaotic cell arrangement, partial cell necrosis, and obvious inflammatory cell infiltration. The liver morphology of the AL group was abnormal, with obvious dilation of the sinusoids accompanied by congestion and mild inflammatory cell infiltration. The liver tissue structure of the BZ group was slightly abnormal, with slight dilation of the sinusoids and orderly cell arrangement. The liver tissue of the FL6 group was slightly abnormal, with slight dilation of a few sinusoids, orderly cell arrangement, and no obvious inflammatory cell infiltration. The liver structure of the FF7 group was slightly abnormal, with slight dilation of a small number of sinusoids, no obvious inflammatory response, and relatively orderly cell arrangement. The liver of the WP7 group showed more obvious damage, with disordered dilation of the sinusoids and mild inflammatory cell infiltration, but it was still better than the MC group. This indicates that the combined modeling of adenine and potassium oxonate can induce hyperuricemia in mice and cause liver damage. Oral administration of three uric acid-lowering peptides derived from thin-shelled pecans can alleviate liver damage to some extent, reduce intrahepatic inflammation, and improve the recovery ability of hyperuricemia.

[0093] Colonic lesions are also a complication of hyperuricemia, generally manifesting as changes in intestinal structure, damage to intestinal villi, and alterations to intestinal crypts. According to... Figure 12Analysis revealed that the NC group mice exhibited good intestinal structure, with normal villus length and structure, no pathological changes, and crypts arranged in a "test tube rack" pattern. The MC group mice showed significant intestinal damage, with significantly shortened villus length, severe villus edema, extensive epithelial cell shedding and necrosis, disordered crypt structure, increased plasma cells at the base, and significant inflammatory cell infiltration. Compared to the MC group, the AL positive drug treatment group showed significant improvement in intestinal damage, with slight villus atrophy, a small amount of epithelial cell shedding, no significant crypt structural disorder, and only a small amount of inflammatory cell infiltration. The BZ positive drug treatment group also showed significant improvement in intestinal damage, with abnormal villus length and structure, no significant crypt structural disorder, and only a small amount of inflammatory cell infiltration. The FL6 treatment group showed significant improvement in intestinal damage, with normal villus length and structure, no significant crypt structural disorder, and only a small amount of inflammatory cell infiltration. The intestinal damage of mice treated with FF7 was significantly improved, with slight atrophy of villi, normal structure, increased crypt spacing, and a small amount of inflammatory cell infiltration. The intestinal damage of mice treated with WP7 was improved compared with that of the MC group, with slight atrophy of villi and a small amount of epithelial cell shedding.

[0094] The above results all demonstrate that the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of this invention possess excellent uric acid-lowering capabilities. Serum uric acid, blood urea nitrogen, and creatinine results all indicate the outstanding uric acid-lowering effects of these peptides. Kidney histological observation further confirmed that the kidneys of mice in the peptide-treated group appeared rosy and smooth, with significantly improved tissue structure, normalized glomerular and tubular morphology, and significantly reduced inflammatory infiltration and fibrosis. Liver histological analysis showed that all three peptides effectively alleviated liver damage caused by hyperuricemia. Simultaneously, colon histopathological observation showed that all three peptides effectively improved intestinal damage caused by hyperuricemia. The intestinal villus length and structure of the treated mice were restored, epithelial cell shedding was reduced, crypt structure was normalized, and inflammatory cell infiltration was significantly reduced, indicating that these peptides have good intestinal protective and repair functions.

[0095] The discovery of the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS in this invention opens up new avenues for the treatment of hyperuricemia, gout, and related metabolic diseases. They have great potential to be developed into functional food additives, health products, or new drugs, and have broad market application prospects.

[0096] In summary, the thin-shelled pecan uric acid-lowering active peptides FLADAF, FFFPGPS, and WPFGGDS of this invention have multiple effects such as lowering uric acid, protecting the kidneys, liver, intestines, and anti-inflammation, with good safety. They provide an innovative solution for the treatment of hyperuricemia and have important scientific value and broad industrialization prospects.

[0097] 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 technical principles 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 thin-shelled pecan uric acid-lowering active peptide, characterized in that, The uric acid-lowering active peptide comprises at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No.

3.

2. The uric acid-lowering active peptide according to claim 1, characterized in that, The uric acid-lowering active peptide is derived from thin-shelled pecan protein.

3. The uric acid-lowering active peptide according to claim 1, characterized in that, The purity of the uric acid-lowering active peptide is ≥95%.

4. The uric acid-lowering active peptide according to claim 1, characterized in that, The molecular weight of the uric acid-lowering active peptide is less than 3 kDa.

5. A composition, characterized in that, The composition comprises the uric acid-lowering active peptide of any one of claims 1-4, and a pharmaceutically acceptable carrier or a food-acceptable excipient.

6. A method for preparing the uric acid-lowering active peptide from thin-shelled pecans according to claim 1, characterized in that, The method includes the following steps: (1) Enzymatic hydrolysis: Alkaline protease and neutral protease were used to perform double enzymatic hydrolysis on the thin-shelled pecan protein to obtain protein hydrolysate; (2) Ultrafiltration: The protein hydrolysate was separated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and small molecule peptides of thin-shelled pecan with a molecular weight of less than 3 kDa were collected. (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: After the small molecule peptides were reduced, alkylated and desalted, they were analyzed by liquid chromatography-mass spectrometry system to obtain raw mass spectrometry data, and Denovo analysis was performed using PEAKS Studio 10.6 software to obtain the amino acid sequence information of the small molecule peptides; (4) Screening: The xanthine oxidase (XOD) binding activity of the small molecule peptides was evaluated based on molecular simulation docking, and bioinformatics prediction scores were combined to screen candidate uric acid-lowering active peptides with high biological activity and no toxicity. (5) Cellular and in vitro enzymatic validation: In the HK-2 cell model and in vitro enzymatic system, the inhibitory effect of the candidate uric acid-lowering active peptide on XOD was determined and the inhibition rate / IC50 ratio was established. 50 Based on indicators, combined with cell viability and safety assessments, uric acid-lowering active peptides that meet preset thresholds are screened out. (6) In vivo functional verification: The drug was evaluated in a mouse model of hyperuricemia induced by PO+AD, and serum uric acid and related metabolic / histochemical indicators were detected to confirm the uric acid-lowering active peptide uric acid regulation effect of the preset threshold, thereby finally obtaining the uric acid-lowering active peptide as described in claim 1.

7. The method according to claim 6, characterized in that, The conditions for enzymatic hydrolysis in step (1) are pH 7.0-8.5, temperature 50-60℃, and hydrolysis time 120-180min.

8. The use of the uric acid-lowering active peptide according to any one of claims 1-4 in the preparation of uric acid-lowering drugs, characterized in that, The uric acid-lowering active peptide comprises at least one of the following amino acid sequences: FLADAF, FFFPGPS, and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No.

3.

9. The use of the uric acid-lowering active peptide according to any one of claims 1-4 in the preparation of a medicament for treating or preventing hyperuricemia is characterized in that... The uric acid-lowering active peptide comprises at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No.

3.

10. The use of the uric acid-lowering active peptide according to any one of claims 1-4 in the preparation of functional foods or health products, wherein the food or health product is used to lower serum uric acid levels, characterized in that, The uric acid-lowering active peptide comprises at least one of the following amino acid sequences: FLADAF, FFFPGPS and / or WPFGGDS, wherein the amino acid sequence of FLADAF is SEQ ID No. 1, the amino acid sequence of FFFPGPS is SEQ ID No. 2, and the amino acid sequence of WPFGGDS is SEQ ID No. 3.

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