Paeonia suffruticosa polypeptide for reducing uric acid as well as preparation method and application thereof

Abstract

The invention provides peony polypeptide for reducing uric acid as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method comprises the following steps: soaking and cleaning peony seed meal with ethanol, centrifuging at 3000-500r for 30-50min, taking a first precipitate, drying the first precipitate, adding water, adjusting the pH value to 9-10, carrying out alkali extraction at 45-55 DEG C for 70-80min, taking supernate, adjusting the pH value to an isoelectric point, and taking a second precipitate; the second precipitate is subjected to enzymolysis for 2-8 h under the conditions that the temperature is 20-80 DEG C, the pH is 5-9, and the protease addition amount is 1500-9000 U / g, and the peony polypeptide capable of reducing uric acid is obtained. The compound has inhibitory activity on xanthine oxidase, and the active components of the compound have stability and functionality by adopting ultrafiltration grading and simulated digestion model exploration.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a peony polypeptide for lowering uric acid, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Hyperuricemia is a metabolic disease caused by impaired purine metabolism or uric acid excretion. Commonly used medications, such as allopurinol, have a high incidence of hypersensitivity syndrome, while febuxostat can easily cause toxic side effects such as liver dysfunction. Consequently, natural active ingredients with uric acid-lowering activity have attracted widespread attention.

[0004] The main preparation technologies for food-derived uric acid-lowering peptides include enzymatic hydrolysis, microbial fermentation, and chemical synthesis. Enzymatic hydrolysis, with its advantages of strong process controllability, mild reaction conditions, and high product safety, demonstrates significant competitiveness in large-scale production. To enhance the purity of enzymatic hydrolysis products, ultrafiltration is commonly used for graded purification. This method, due to its ease of operation and significant separation efficiency, has become the mainstream technology for active peptide purification.

[0005] Peony seed meal, a major byproduct of peony seed oil processing, boasts a crude protein content of up to 28.54%, making it not only a high-quality plant protein resource but also a potential reservoir of bioactive peptides. The bioactive peptides produced by enzymatic hydrolysis not only possess superior solubility properties compared to the original protein, but also exhibit a low-energy, non-competitive transport mechanism for molecular absorption, resulting in significantly better bioavailability than intact protein. Numerous functional components have been successfully isolated from peony seed meal, including antioxidant peptides, ACE inhibitory peptides, and β-glucosidase inhibitory peptides. However, systematic research on its uric acid-lowering peptides remains underdeveloped. Summary of the Invention

[0006] In view of the current technological status, the purpose of the present invention is to provide a peony polypeptide for lowering uric acid, as well as a preparation method and application thereof. High-quality protein is extracted from peony seed meal by adopting an alkali extraction and acid precipitation method, and the polypeptide prepared by further optimizing the enzymatic hydrolysis process has xanthine oxidase (abbreviated as XOD) inhibitory activity.

[0007] In order to achieve the above objectives, the technical solutions of the present invention are as follows.

[0008] In a first aspect, a method for preparing a uric acid-lowering peony polypeptide comprises the steps of: S1. After peony seed meal is soaked and washed with ethanol, centrifuged at 3000-500r for 30-50min, a first precipitate is obtained, the first precipitate is dried, water is added and the pH is adjusted to 9-10, and alkali extraction is performed at 45-55°C for 70-80min. The supernatant is adjusted to the isoelectric point and a second precipitate is obtained; S2. Enzymatically hydrolyzing the second precipitate at a temperature of 20-80° C., a pH of 5-9, and a protease addition amount of 1500-9000 U / g for 2-8 hours to obtain the uric acid-lowering peony polypeptide.

[0009] Optionally, in S1, after the first precipitate is dried, water is added at a material-liquid ratio of 1 g: (20-25) mL, and the pH is adjusted with a NaOH solution.

[0010] Optionally, in S1, the ethanol is a 95 wt.% ethanol solution, which is soaked and washed multiple times to remove small molecular substances such as flavonoids, and the drying method is vacuum drying at 75-85° C. to completely remove ethanol and water.

[0011] Optionally, in S1, the isoelectric point ranges from pH 3.5 to 4.5, and the protein is extracted using an isoelectric precipitation method.

[0012] Optionally, in S2, the protease is one or more of alkaline protease, composite protease, neutral protease, flavor protease and papain; the composite protease includes papain and acidic protease.

[0013] Optionally, in S2, the material-liquid ratio of the enzymatic hydrolysis is 1 mg: (30-40) mL, and the liquid component is water.

[0014] Optionally, in S2, after obtaining the peony polypeptide, ultrafiltration is performed to obtain polypeptides with molecular weights below 3 kDa, 3-10 kDa, and above 10 kDa, respectively.

[0015] In a second aspect, the peony polypeptide is prepared by the above-mentioned method for preparing the uric acid-lowering peony polypeptide.

[0016] In a third aspect, the application of the above-mentioned uric acid-lowering peony polypeptide in uric acid-lowering functional foods or medicines.

[0017] In a fourth aspect, a uric acid-lowering functional food or medicine comprising the above-mentioned uric acid-lowering peony polypeptide is provided.

[0018] The beneficial effects of the present invention are: 1. The present invention discovered that peony seed meal protein isolate has in vitro xanthine oxidase inhibitory activity. High-quality protein was extracted from peony seed meal by alkali extraction and acid precipitation. A polypeptide with uric acid-lowering function was prepared by optimizing the enzymatic hydrolysis process. The polypeptide has inhibitory activity against xanthine oxidase, and ultrafiltration fractionation combined with a simulated digestion model was used to explore the stability and functionality of its active components, providing a theoretical basis and technical support for the development of new natural uric acid-lowering preparations.

[0019] 2. This invention not only expands the high-value utilization of peony protein resources but also opens up new avenues for dietary intervention strategies for hyperuricemia. Subsequent integration of molecular docking and structure-activity analysis techniques will further reveal the mechanism of action of the active peptide, accelerating its application in the development of functional foods and pharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The results of a single-factor experiment of alkaline protease in Example 1 of the present invention are shown; wherein (a) is pH, (b) is the amount of enzyme added, (c) is the enzymatic hydrolysis time, and (d) is the enzymatic hydrolysis temperature.

[0022] Figure 2 The results of a single-factor experiment of the composite protease of Example 1 of the present invention are shown in FIG. 1 , wherein (a) is pH, (b) is the amount of enzyme added, (c) is the enzymatic hydrolysis time, and (d) is the enzymatic hydrolysis temperature.

[0023] Figure 3 The results of the single-factor experiment of the sex enzyme in Example 1 of the present invention are shown in FIG. 1 , wherein (a) is pH, (b) is the amount of enzyme added, (c) is the enzymatic hydrolysis time, and (d) is the enzymatic hydrolysis temperature.

[0024] Figure 4 The results of a single-factor experiment of papain in Example 1 of the present invention are shown; wherein (a) is pH, (b) is the amount of enzyme added, (c) is the enzymatic hydrolysis time, and (d) is the enzymatic hydrolysis temperature.

[0025] Figure 5 The results of a single-factor experiment on flavor protease in Example 1 of the present invention are shown in FIG. 1 , wherein (a) is pH, (b) is enzyme addition amount, (c) is enzymolysis time, and (d) is enzymolysis temperature.

[0026] Figure 6 These are the enzymolysis results of the five enzymes in Example 1 of the present invention under their respective optimal conditions.

[0027] Figure 7The results of the interaction analysis between the enzymatic hydrolysis temperature and the enzyme addition amount in Example 2 of the present invention are shown; wherein, (a) is the XOD inhibition rate influence diagram, and (b) is the contour map of the XOD inhibition rate influence.

[0028] Figure 8 The results of the interaction analysis between pH and enzyme addition amount in Example 2 of the present invention are shown; wherein, (a) is the XOD inhibition rate influence diagram, and (b) is the contour map of the XOD inhibition rate influence.

[0029] Figure 9 The results of the interaction analysis between pH and enzymatic hydrolysis temperature in Example 2 of the present invention are shown; (a) is an XOD inhibition rate influence diagram, and (b) is a contour diagram of the XOD inhibition rate influence.

[0030] Figure 10 The XOD inhibition rates of peony polypeptides with different molecular weights according to Example 3 of the present invention are shown.

[0031] Figure 11 This is the XOD inhibition rate of the peony polypeptides of different molecular weights after gastrointestinal digestion in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] The reagents involved in the specific embodiment of the present invention include: Peony seed meal, Heze Guyu Peony Biotechnology Co., Ltd.; Anhydrous ethanol, analytical grade AR, Tianjin Damao Chemical Reagent Factory; Sodium hydroxide, analytical grade AR, Xilong Scientific Co., Ltd.; Hydrochloric acid, high-grade GR, Xilong Scientific Co., Ltd.; Neutral protease, 50,000 U / g, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. Flavor protease, 20,000 U / g, Nanning Pangbo Bioengineering Co., Ltd. Papain, 100,000 u / g, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. Compound protease, 100,000 U / g, Henan Wansheng Industrial Co., Ltd.; Alkaline protease, 200,000 U / g, Shandong Longkote Enzyme Preparation Co., Ltd.; Xanthine, high purity 98%, Shanghai Yuanye Biotechnology Co., Ltd.; Xanthine oxidase, 9.5 U / mg, Shanghai Yuanye Biotechnology Co., Ltd.; Pepsin (porcine gastric mucosa), 30,000 U / g, Shanghai Yuanye Biotechnology Co., Ltd.; Porcine bile salt, Shandong Keyuan Biochemical Co., Ltd.; α-amylase (porcine pancreas), 8 U / mg, Shanghai Yuanye Biotechnology Co., Ltd.; Trypsin (porcine pancreas), 250 U / g, Shanghai Yuanye Biotechnology Co., Ltd.; PBS phosphate buffer solution, 0.05 mol / L, pH 8.5, Shanghai Yuanye Biotechnology Co., Ltd.; Sodium hydrogen phosphate (AR), analytical grade, Xilong Scientific Co., Ltd. Sodium chloride, analytical grade AR, Tianjin Kemeiou Chemical Reagent Co., Ltd.; Sodium bicarbonate, analytical grade AR, Tianjin Kemeiou Chemical Reagent Co., Ltd.; Potassium dihydrogen phosphate, analytical grade AR, Sinopharm Chemical Reagent Co., Ltd.; Sodium tetraborate, Xilong Scientific Co., Ltd.; Sodium lauryl sulfate, Tianjin Zhonglian Chemical Reagent Co., Ltd.; 1-4-Dithiothreitol, 99% biotechnology grade, Shanghai MacLean Biochemical Technology Co., Ltd.; DL-serine, Shanghai MacLean Biochemical Technology Co., Ltd.; Phthalaldehyde, Shanghai MacLean Biochemical Technology Co., Ltd.

[0035] The instruments and equipment involved in the specific implementation of the present invention include: Digital constant temperature water bath, Changzhou Noki Instrument Co., Ltd. SCIENTZ-12N freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; Centrifuge, Changsha Xiangzhi Centrifuge Instrument Co., Ltd.; Polysulfone hollow fiber filter column, Ruiplegin (Shanghai) Biotechnology Co., Ltd.; SepctraMax i3x microplate reader, Meigu Molecular Instruments (Shanghai) Co., Ltd.

[0036] Example 1, single factor analysis test A peony polypeptide for lowering uric acid, the preparation method of which comprises the steps of: The peony seed meal was soaked and washed with 95wt.% ethanol solution, centrifuged at 3000r for 30min, and the precipitate was separated as the first precipitate. The first precipitate was vacuum dried at 80℃, and then distilled water was added at a solid-liquid ratio of 1:20 g / mL. The pH was adjusted to 9 with 1mol / L NaOH solution, and alkaline extraction was performed at 50℃ for 70min. The supernatant was taken and the pH of the supernatant was adjusted to 4.0 with hydrochloric acid. Protein was extracted by isoelectric precipitation method to obtain the second precipitate. The pH of the second precipitate was adjusted to neutral and freeze-dried for later use.

[0037] The obtained second precipitate is mixed with different types of proteases and enzymatically hydrolyzed under set enzymatic conditions to obtain an enzymatic hydrolyzate sample containing peony polypeptide.

[0038] The enzymatic hydrolysis conditions include: a four-factor test for each protease at a solid-liquid ratio of 1:40 (mg / mL) (preliminary experiments have shown that this is the optimal solid-liquid ratio); the four factors are enzymatic hydrolysis temperature, enzymatic hydrolysis time, pH, and enzyme addition amount; the enzymatic hydrolysis temperature includes 20℃, 40℃, 50℃, 60℃ and 80℃; the enzymatic hydrolysis time is 2h, 4h, 6h and 8h; the pH includes 5, 6, 7, 8 and 9; the enzyme addition amount includes 1500U / g, 2500U / g, 5000U / g, 7500U / g and 9000U / g.

[0039] The method for determining the xanthine oxidase inhibition rate includes: mixing 40 μL of the enzymatic hydrolyzate sample and 40 μL of 0.05 U / mL xanthine oxidase solution, incubating at 25°C for 5 minutes to obtain a sample product; then adding 120 μL of 0.48 mM xanthine solution and mixing evenly to obtain a product containing a buffer solution and XOD, and incubating at 25°C for 25 minutes to obtain a product containing a buffer solution; measuring the liquid absorbance at 290 nm at different stages, and calculating the xanthine oxidase inhibition rate using the following formula: ； Wherein: D is the xanthine oxidase inhibition rate, %; A1 is the absorbance of the sample and xanthine oxidase; A2 is the absorbance of the product containing the sample; A3 is the absorbance of the buffer solution and xanthine oxidase; A4 is the absorbance of the product containing the buffer solution.

[0040] The degree of hydrolysis is determined using the o-phthalaldehyde (OPA) method for quantitative analysis. Using 0.9516 mmol of serine as the standard and purified water as the blank control, the enzymatic hydrolyzate sample is diluted to 0.5 mg / mL and 40 μL of each is added to a 96-well microtiter plate. 300 μL of OPA reagent is then added, and the absorbance of the product containing the buffer solution is measured at 340 nm. The degree of hydrolysis is calculated according to the following formula: ; Wherein, the degree of hydrolysis (DH) refers to the percentage of broken peptide bonds, htot depends on the type of raw material, and h is a function of the number of millimoles of serine amino groups; mmol / g protein; ; Wherein, C is the protein concentration of the sample, mg / mL; , and htot are taken as 1, 0.4 and 9.5198 respectively according to the characteristics of the raw materials.

[0041] Figure 1 The results of the single-factor experiment of alkaline protease showed that the optimal conditions for alkaline protease hydrolysis of peony protein were pH 8, enzyme addition 5000U / g, hydrolysis time 6h, and hydrolysis temperature 60℃. At this time, the hydrolysis degrees could reach 56.10857%±1.1%, 56.0963%±0.366%, 55.7695%±0.294%, and 55.64102%±0.795%, respectively, and the XOD inhibition rates could reach 37.7232%±1.08%, 38.9490%±1.04%, 38.0216%±1.24%, and 35.8578%±1.28%, respectively.

[0042] Figure 2 The results of the single-factor experiment of composite protease showed that the optimal conditions for the enzymatic hydrolysis of peony seed meal protein by composite protease were pH 6, enzyme addition 5000U / g, enzymatic hydrolysis time 6h, and enzymatic hydrolysis temperature 40℃. At this time, the hydrolysis degree and XOD inhibition rate were the highest, with the XOD inhibition rates reaching 53.7678%±0.48%, 52.5581%±0.45%, 54.1861%±1.38%, and 52.5581%±1.23%, respectively. The hydrolysis degrees were 50.6083%±0.275%, 51.7196%±0.227, 50.90648%±0.168%, and 51.25887%±0.24%, respectively.

[0043] Figure 3The results of the single-factor experiment with neutral protease showed that the optimal conditions for the enzymatic hydrolysis of peony protein by neutral protease were pH 6, enzyme addition 2500U / g, enzymatic hydrolysis time 6h, and enzymatic hydrolysis temperature 40℃. At this time, the hydrolysis degree and XOD inhibition rate were the highest. The XOD inhibition rates were 38.7052%±0.552%, 45.8678%±0.53%, 27.135%±0.413%, and 43.38843%±0.69%, respectively. The hydrolysis degrees could reach 51.15014%±0.014%, 47.65529%±0.957%, 51.33595%±0.218%, and 51.20295%±0.504%, respectively.

[0044] Figure 4 The results of the papain single-factor experiment showed that the optimal conditions for papain hydrolysis of peony protein were pH 7, enzyme addition 2500U / g, hydrolysis time 6h, and hydrolysis temperature 60℃. At this time, the hydrolysis degree and XOD inhibition rate were the highest. The XOD inhibition rates were 37.8655%±0.585%, 38.7427%±0.761%, 39.0351%±0.578%, and 41.0819%±0.731%, respectively. The hydrolysis degrees were 51.3538%±0.236%, 51.89306%±0.688%, 51.48374%±0.727%, and 51.3538%±0.163%, respectively.

[0045] Figure 5 The results of the single-factor experiment of flavor protease showed that the optimal conditions for flavor protease hydrolysis of peony protein were pH 7, enzyme addition 5000U / g, hydrolysis time 6h, and hydrolysis temperature 50℃. At this time, the hydrolysis degree and XOD inhibition rate were the highest. The XOD inhibition rates were 73.0355%±0.592%, 73.9011%±0.589%, 73.4505%±0.296%, and 74.72527%±0.888%, respectively. The hydrolysis degrees could reach 63.08685%±0.463%, 62.81109%±0.256%, 61.31772%±0.122%, and 61.73016%±0.225%, respectively.

[0046] As can be seen, the XOD inhibition rate of the enzymatic hydrolysis product first increases and then decreases with changes in pH. This may be because different enzymes have different optimal pH ranges, and excessive acidity or alkalinity will affect enzyme activity. The XOD inhibition rate of the enzymatic hydrolysis product first increases and then decreases with increasing enzyme addition. This is because when the enzyme addition amount is small, the enzyme and substrate have not yet fully reacted. When the enzyme addition amount is too high, saturation is reached, and the excess enzyme will also decompose the active peptide obtained after enzymatic hydrolysis, causing its function to change. The XOD inhibition rate of different enzymes first increases and then tends to decrease gently with the extension of reaction time. It is speculated that excessive reaction may occur after a long time, causing the functional changes of the active peptides and a slight decrease in the XOD inhibition rate. The XOD inhibition rate of different enzymes first increases and then decreases with increasing temperature. This is related to enzyme activity. Different enzymes have their own optimal reaction temperature range. Within this range, increasing temperature promotes the reaction between the enzyme and substrate, while excessively high temperature destroys enzyme activity and reduces the enzymatic hydrolysis effect. The optimal enzymatic hydrolysis conditions of the five enzymes are shown in Table 1.

[0047] Table 1

[0048] According to Table 1, enzymolysis was performed under the optimal conditions of the five enzymes. The results were as follows: Figure 6 As shown by Figure 6 As can be seen, under their respective optimal reaction conditions, flavor protease had the highest XOD inhibition rate and hydrolysis degree. Its XOD inhibition rate was nearly 17% higher than that of the second-highest composite protease, while the XOD inhibition rates of alkaline protease, neutral protease, and papain were similar. Therefore, flavor protease was selected to prepare peony peptides for lowering uric acid.

[0049] Example 2, response surface analysis test The flavor protease selected in the examples was used for response surface analysis. The enzymatic hydrolysis conditions in Example 1 were changed to the response surface analysis test including enzyme addition amount, enzymatic hydrolysis temperature and pH according to Table 2, and the XOD inhibition rate was obtained.

[0050] Table 2

[0051] The data in Table 3 were fitted with quadratic regression using Design-expert software, and the quadratic polynomial regression equation for the three influencing factors, enzyme addition amount (A), enzymatic hydrolysis temperature (B), and pH (C), and the XOD inhibition rate (Y) was obtained: Y=79.53637-0.3747A+2.1283B+4.03177C+1.34892AB-0.419664AC+3.20743BC-6.94444A 2 -6.19504B2 -5.98521C 2 ; The total coefficient of determination (R2) of the regression polynomial equation was 0.9704, indicating that the quadratic polynomial regression equation was highly reliable and could well predict and analyze the XOD inhibition rate. A variance analysis was performed on the above regression model, and the results are shown in Table 3.

[0052] Table 3

[0053] *** indicates P < 0.001, extremely significant difference; ** indicates P < 0.01, highly significant difference; * indicates P < 0.05, significant difference.

[0054] As shown in Table 3, the model's F value is 18.21 and its P value is 0.0026, indicating statistical significance. The lack-of-fit term, P = 0.3821, is greater than 0.05, indicating high confidence and good prediction and analysis performance. The P and F values in Table 3 indicate that the order of influence of the three factors on the XOD inhibition rate of peony enzyme hydrolysate is: pH > temperature > enzyme addition amount.

[0055] The results of model confidence analysis are shown in Table 4.

[0056] Table 4

[0057] Combining Tables 4 and 5, it can be seen that the quadratic polynomial model fits the experimental data well and can better predict and analyze the XOD inhibition rate of peony enzyme hydrolysate.

[0058] The results of the interaction analysis between enzymatic hydrolysis temperature and enzyme addition amount are shown in Figure 2. Figure 7 The results of the interaction analysis between pH and enzyme addition amount are shown in Figure 8 The results of the interaction analysis between pH and enzymatic hydrolysis temperature are shown in Figure 9 As shown in the figure, the three-dimensional response surface plots for all three factors open downward, with maximum values, and the contour plots are elliptical, indicating interactions between the factors. The figure shows that with increasing enzyme addition, the XOD inhibition rate first increases and then decreases. When the pH is fixed at 7, XOD inhibition first increases and then decreases with increasing enzyme addition and temperature, showing a clear trend.

[0059] As can be seen from the present embodiment, the best extraction process for preparing peony protein hydrolysate is: pH 7.4, temperature 43 ° C, enzyme addition 4968.66 U / g, under which the XOD inhibition rate predicted value is 79.5346%; Under laboratory conditions, the extraction conditions are corrected to pH 7.5, temperature 43 ° C, enzyme addition 4970 U / g, solid-liquid ratio 1: 40, and enzymolysis time 6 hours. In order to investigate the stability and feasibility of the optimized process, under these process conditions, repeatability tests were carried out 3 times, and the mean value of the XOD inhibition rate was 78.7267%, which is less than 1% from the theoretical predicted value of 79.5346%, indicating that the process is stable and feasible.

[0060] Example 3: In vitro uric acid-lowering effect of ultrafiltration fractions and simulated digestion evaluation Peony seed meal protein was enzymatically hydrolyzed with flavor protease under optimal reaction conditions. The hydrolyzed solution was then ultrafiltered using hollow cellulose membranes of specified specifications to retain peptides with molecular weights less than 3 kDa, 3-10 kDa, and greater than 10 kDa, which were then freeze-dried for later use. The XOD inhibition rates of the freeze-dried peptides with different molecular weights were determined and simulated in vitro digestion was performed. The XOD inhibition rate was used as an indicator to evaluate their uric acid-lowering effects.

[0061] The in vitro simulated digestion test was conducted based on the technical document “Guo Yanmei, Master of Engineering. Preparation of bonito roe XOD inhibitory peptide, study of inhibitory activity and its molecular docking mechanism [D]. Shihezi University [2025-04-07]”.

[0062] The results of XOD inhibition rate determination are as follows Figure 10 As shown, peptides with a molecular weight of less than 3 kDa had a higher XOD inhibition rate, reaching 84.0115% ± 1.83%, which was better than the 78.7267% XOD inhibition rate of the enzymatic hydrolysate. Peptides with a molecular weight of 3-10 kDa had an XOD inhibition rate of 68.3668% ± 3.78%, also showing a considerable in vitro uric acid-lowering effect. Peptides with a molecular weight greater than 3 kDa had a lower uric acid-lowering effect, at only 39.914% ± 3.16%. This indicates that the highly active components of peony protein peptides are concentrated in peptides with a molecular weight of less than 3 kDa.

[0063] In simulated gastrointestinal digestion, the stability differences of the active peptides were further revealed: during gastric digestion, the fraction with a molecular weight less than 3 kDa (small peptide) maintained an inhibition rate of 54.06% ± 0.5%, significantly higher than the fractions with a molecular weight of 3-10 kDa (30.18% ± 1.15%) and greater than 10 kDa (42.02% ± 0.08%). After entering the intestinal digestion phase, the activity of the fraction with a molecular weight less than 3 kDa recovered to 73.50% ± 1.35%, showing a dose-dependent release pattern, while the fractions with a molecular weight of 3-10 kDa and greater than 10 kDa decreased to 44.31% ± 1.64% and 36.50% ± 0.4%, respectively. This phenomenon suggests that the peony small peptide not only has potent XOD inhibition ability but also exhibits good digestive stability and targeted release characteristics.

[0064] The XOD inhibition rates of peptides with a molecular weight less than 10 kDa were all improved during intestinal digestion. The possible reason was that trypsin cleaved some peptide segments, thereby enhancing the inhibitory effect. However, components with a molecular weight greater than 10 kDa were probably mostly protein residues that could not be enzymatically hydrolyzed or were completely hydrolyzed, enzymes that did not participate in the reaction or remained after participating, and a small amount of small molecular weight peptides that were not ultrafiltered. Therefore, the uric acid-lowering effect decreased after simulated intestinal digestion.

[0065] Example 4, Animal Experiment The operation process of this example was carried out using the polypeptide with a molecular weight of less than 3 kDa in Example 3 as the uric acid-lowering peony small molecule peptide.

[0066] Healthy male mice (SD strain, weighing 18-22 g) were randomly divided into three groups: control group, model group and dose group, with 10 mice in each group. They were fed with standard experimental animal feed and completed the adaptation period of feeding and management.

[0067] The control group was gavaged with the same dose of normal saline; the model group and the dose group were gavaged with 250 mg / kg of potassium oxonate and 250 mg / kg of hypoxanthine every day for 14 days; the drugs were administered at a fixed time every day (3 pm); the serum uric acid index was tested 3 hours after the end of the drug administration on the 14th day. The serum uric acid values of the model group and the dose group reached 110 μmol or above, indicating that the model was successful; at this time, the uric acid value of the control group was normal.

[0068] The dose group was then given standard and uric acid-lowering peony small molecule peptides (400 mg / kg body weight, dissolved in 1 mL of normal saline) daily by gavage, while the control and model groups were gavaged with 1 mL of sterile distilled water daily. The three groups continued to be gavaged with potassium oxonate and hypoxanthine daily for 21 days.

[0069] After the experiment, the mice were fasted for 12 h and anesthetized with ether. Blood was collected by removing the eyeballs, and about 1 mL of blood sample was collected. The blood sample was centrifuged at 3000 rpm for 10 min to separate the serum. The serum uric acid content was determined using a uric acid detection kit according to the instructions. The results are shown in Table 5.

[0070] Table 5

[0071] Among them, dose group 1, dose group 2 and dose group 3 represent the measurement values of a single mouse in the dose group, respectively. Dose group 1 and dose group 3 represent the best effects, dose group 2 represents the worst effect, and the average serum uric acid level of mice in the other dose groups is 62.08 umol / L. The results in Table 5 show that the peony uric acid-lowering small molecule peptide has a good uric acid-lowering effect.

[0072] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a peony polypeptide for lowering uric acid, characterized in that: Including steps: S1. After peony seed meal is soaked and washed with ethanol, centrifuged at 3000-500r for 30-50min, a first precipitate is obtained, the first precipitate is dried, water is added and the pH is adjusted to 9-10, and alkali extraction is performed at 45-55°C for 70-80min. The supernatant is adjusted to the isoelectric point and a second precipitate is obtained; S2. Enzymatically hydrolyzing the second precipitate at a temperature of 20-80° C., a pH of 5-9, and a protease addition amount of 1500-9000 U / g for 2-8 hours to obtain the uric acid-lowering peony polypeptide.

2. The method for preparing the uric acid-lowering peony polypeptide according to claim 1, wherein: In S1, after the first precipitate is dried, water is added at a material-liquid ratio of 1 g: (20-25) mL, and the pH is adjusted with NaOH solution.

3. The method for preparing the uric acid-lowering peony polypeptide according to claim 1, wherein: In S1, the drying method is vacuum drying at 75-85°C.

4. The method for preparing the uric acid-lowering peony polypeptide according to claim 1, wherein The isoelectric point ranges from pH 3.5 to 4.

5.

5. The method for preparing the uric acid-lowering peony polypeptide according to claim 1, wherein: In S2, the protease is one or more of alkaline protease, composite protease, neutral protease, flavor protease and papain.

6. The method for preparing the uric acid-lowering peony polypeptide according to claim 1, wherein: In S2, the material-liquid ratio of enzymatic hydrolysis was 1 mg: (30~40) mL.

7. The method for preparing the peony polypeptide according to claim 1, wherein: In S2, the peony polypeptides were obtained and then ultrafiltered to obtain polypeptides with molecular weights below 3 kDa, 3-10 kDa, and above 10 kDa, respectively.

8. A uric acid-lowering peony polypeptide prepared according to the method for preparing a uric acid-lowering peony polypeptide according to any one of claims 1 to 7.

9. Use of the uric acid-lowering peony polypeptide according to claim 8 in uric acid-lowering functional foods or medicines.

10. A uric acid-lowering functional food or medicine comprising the uric acid-lowering peony polypeptide according to claim 8.

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