Application of holothurian glycosaminoglycan in preparation of medicine for reducing uric acid

Abstract

The present application relates to the application of sea cucumber gut polysaccharide in the preparation of drugs for reducing uric acid. The preparation method of sea cucumber gut polysaccharide comprises the following steps: sea cucumber gut is subjected to enzymatic hydrolysis treatment with papain, and after enzyme inactivation, the supernatant is obtained by centrifugation; the supernatant is subjected to fractional alcohol precipitation, centrifugation, and the precipitate is obtained, and deproteinization and freeze-drying are performed. The preparation method is simple, green and environmentally friendly. The sea cucumber gut polysaccharide prepared by the method can effectively inhibit the activity of xanthine oxidase; the sea cucumber gut polysaccharide has no toxic effect on HK-2 cells. The sea cucumber gut polysaccharide has a uric acid-lowering effect on HK-2 high uric acid cells.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number was 202411847064.5, the application date was December 16, 2024, and the patent title at the time of application was: A method for preparing sea cucumber intestinal polysaccharide and its application. Technical Field

[0002] This invention belongs to the field of sea cucumber by-product processing and utilization technology, and particularly relates to the application of sea cucumber intestinal polysaccharide in the preparation of uric acid-lowering drugs. Background Technology

[0003] Sea cucumbers (Stichopus japonicus) belong to the phylum Echinodermata and are marine invertebrates widely distributed in benthic areas and deep seas worldwide. They are widely recognized as a tonic and traditional medicine, rich in amino acids, fatty acids, and trace elements, as well as polysaccharides, proteins, saponins, and other bioactive substances. However, in the deep processing of sea cucumbers, the viscera are often treated as processing byproducts and disposed of indiscriminately. There is a significant lack of in-depth research on the nutritional components of sea cucumber viscera, and the recycling and processing of these viscera is insufficient, resulting in serious resource waste and environmental pollution. Sea cucumber intestines are an important byproduct generated during sea cucumber processing, characterized by high yield and renewability. Currently, the utilization rate of sea cucumber intestines in my country is extremely low; they are often discarded as processing byproducts, failing to be fully utilized and causing enormous resource waste. Now, with the rapid development and increasing maturity of sea cucumber farming and production technologies, the comprehensive utilization of sea cucumber byproducts has become an urgent problem to be solved.

[0004] With rapid development, people's living standards have improved significantly, and their diets have become much richer. This has led to a marked increase in the frequency of consuming high-purine foods, resulting in a particularly high incidence of hyperuricemia in my country. Prolonged hyperuricemia can further lead to diseases such as gout. The increasing number of hyperuricemia patients has prompted researchers to continuously search for substances that lower uric acid. Current treatments for hyperuricemia mainly rely on drugs such as allopurinol, colchicine, and febuxostat, which are effective but have certain toxic side effects. Low-toxicity, highly active, naturally sourced uric acid-lowering substances are increasingly favored, with natural polysaccharides, characterized by their wide availability and easy absorption, becoming a research hotspot. In recent years, more and more studies have yielded natural polysaccharides with XO inhibitory activity, but their effects are significantly lower than those of the XO-specific inhibitor, allopurinol. The IC50 of natural polysaccharides' XO inhibition rate... 50 The concentration of these polysaccharides ranges from 0.83 to 10.53 mg / mL, and their low activity limits their widespread industrial application. Therefore, it is urgent to provide a convenient and efficient method for preparing sea cucumber intestinal polysaccharides with uric acid-lowering activity and xanthine oxidase-inhibiting activity. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for preparing sea cucumber intestinal polysaccharide and its application. The method provided by the present invention can prepare sea cucumber intestinal polysaccharide that inhibits xanthine oxidase activity and reduces uric acid levels. The preparation method of the present invention is simple, green, and environmentally friendly. The sea cucumber intestinal polysaccharide can effectively inhibit xanthine oxidase activity; the sea cucumber intestinal polysaccharide has no toxic effect on HK-2 cells. It has a uric acid-lowering effect on HK-2 hyperuricemic cells.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a method for preparing sea cucumber intestinal polysaccharide, comprising the following steps: treating sea cucumber intestines with papain, inactivating the enzyme, centrifuging and collecting the supernatant; fractionating the supernatant with alcohol precipitation, centrifuging, and collecting the precipitate.

[0008] Furthermore, the following steps are included:

[0009] (1) Mix sea cucumber intestines with water, hydrolyze with papain to inactivate the enzyme; after cooling, centrifuge and take the supernatant 1;

[0010] (2) The supernatant 1 was precipitated with 25% ethanol solution and centrifuged to obtain precipitate 1 and supernatant 2;

[0011] (3) After concentrating the supernatant 2, precipitate it with 55% ethanol solution, centrifuge, and obtain precipitate 2 and supernatant 3;

[0012] (4) After concentrating the supernatant 3, precipitate it with 75% ethanol solution, centrifuge, and take the precipitate 3.

[0013] The beneficial effects of adopting the above technical solution include: during the research process, the above operations were performed using compound protease, neutral protease, and papain respectively, and the results showed that the yield was highest when using papain.

[0014] Furthermore, in step (1), the amount of papain added is 2%.

[0015] Furthermore, in step (1), the enzymatic hydrolysis conditions include: hydrolysis time of 3 h, hydrolysis temperature of 54℃, and pH=7.3.

[0016] Furthermore, in step (1), the enzyme inactivation conditions include: boiling water bath for 20 min.

[0017] Furthermore, in step (1), the centrifugation conditions are 8000 r / min for 10 min.

[0018] Furthermore, in step (1), the ratio of sea cucumber intestines to water is 1 g: 3 mL.

[0019] Furthermore, it also includes the step of freeze-drying the precipitate.

[0020] Furthermore, it also includes a step of removing proteins from the precipitate.

[0021] Furthermore, the deproteinization includes the following steps: preparing the precipitate into a solution, adding an equal volume of 10% trichloroacetic acid solution, stirring, incubating overnight at 4°C, centrifuging, taking the supernatant, and dialyzing the supernatant under a 3500 Da dialysis bag for 48 h.

[0022] In the above steps, the precipitate can be prepared into a 0.01 g / mL solution before deproteinization.

[0023] The advantages of adopting the above technical solution include: the above parameters have the advantages of high yield and simple operation.

[0024] This invention provides a sea cucumber intestinal polysaccharide, which is prepared using the method described above.

[0025] The beneficial effects of the above approach include: sea cucumber intestinal polysaccharide is a heteropolysaccharide mainly composed of glucose, with a molecular weight of 618.1 kDa. Sea cucumber intestinal polysaccharide has no effect on the proliferation rate of HK-2 cells, but it can reduce the uric acid content in the supernatant of HK-2 cells.

[0026] This invention provides the application of the above-mentioned sea cucumber intestinal polysaccharide in lowering uric acid and / or inhibiting xanthine oxidase.

[0027] The sea cucumber intestinal polysaccharide provided by this invention can be used to prepare uric acid-lowering drugs.

[0028] This invention provides the application of sea cucumber intestinal polysaccharide in the preparation of uric acid-lowering drugs. The sea cucumber intestinal polysaccharide is prepared by the following method:

[0029] (1) Mix sea cucumber intestines with water, with a material-to-liquid ratio of 1 g: 3 mL; use papain for enzymatic hydrolysis, with an addition amount of 2% and hydrolysis conditions including: hydrolysis time of 3 h, hydrolysis temperature of 54℃, and pH=7.3; inactivate enzyme, with conditions including boiling water bath for 20 min; after cooling, centrifuge and take 1 g of supernatant.

[0030] (2) The supernatant 1 was precipitated with 3 times the volume of 25% ethanol solution, and centrifuged to obtain precipitate 1 and supernatant 2;

[0031] (3) After concentrating the supernatant 2, precipitate it with 3 times the volume of 55% ethanol solution, centrifuge, and obtain precipitate 2 and supernatant 3;

[0032] (4) Deproteinization: Precipitate 2 was prepared into a solution, and an equal volume of 10% trichloroacetic acid solution was added. The solution was stirred and incubated overnight at 4°C. The supernatant was centrifuged and dialyzed with a 3500 Da dialysis bag for 48 h. The supernatant was then freeze-dried to obtain sea cucumber intestinal polysaccharide SCP55.

[0033] This invention provides the application of sea cucumber intestinal polysaccharide in the preparation of drugs that inhibit xanthine oxidase. The sea cucumber intestinal polysaccharide is prepared by the following method:

[0034] (1) Mix sea cucumber intestines with water, with a material-to-liquid ratio of 1 g: 3 mL; use papain for enzymatic hydrolysis, with an addition amount of 2% and hydrolysis conditions including: hydrolysis time of 3 h, hydrolysis temperature of 54℃, and pH=7.3; inactivate enzyme, with conditions including boiling water bath for 20 min; after cooling, centrifuge and take 1 g of supernatant.

[0035] (2) The supernatant 1 was precipitated with 3 times the volume of 25% ethanol solution, and centrifuged to obtain precipitate 1 and supernatant 2;

[0036] (3) After concentrating the supernatant 2, precipitate it with 3 times the volume of 55% ethanol solution, centrifuge, and obtain precipitate 2 and supernatant 3;

[0037] (4) Deproteinization: Precipitate 2 was prepared into a solution, and an equal volume of 10% trichloroacetic acid solution was added. The solution was stirred and incubated overnight at 4°C. The supernatant was centrifuged and dialyzed with a 3500 Da dialysis bag for 48 h. The supernatant was then freeze-dried to obtain sea cucumber intestinal polysaccharide SCP55.

[0038] This invention addresses the low utilization rate of sea cucumber intestines in existing treatments by providing a method for obtaining sea cucumber intestinal polysaccharides with uric acid-lowering activity through papain enzymatic hydrolysis followed by fractional alcohol precipitation. This invention verifies the xanthine oxidase inhibitory activity of the sea cucumber intestinal polysaccharides and their uric acid-lowering effect on HK-2 cells. Results show that the three sea cucumber intestinal polysaccharides screened in this invention all inhibit xanthine oxidase activity, with SCP55 exhibiting the best effect. Subsequent cell experiments further validated its biological activity.

[0039] The beneficial effects of this invention include:

[0040] Sea cucumbers have high nutritional value, but the low utilization rate of by-products is a pressing issue in the sea cucumber farming industry. This invention prepares and isolates sea cucumber intestinal uric acid-lowering polysaccharide, which can increase the added value of sea cucumber by-products, realize the high-value utilization of sea cucumber resources, reduce environmental pollution, and extend the deep processing industrial chain of sea cucumbers. The preparation method provided by this invention is simple, rapid, and reproducible, suitable for industrial production, and has good safety, with no toxic side effects on the human body, aligning with modern health concepts.

[0041] This invention employs a fractional alcohol precipitation method to separate and purify polysaccharides according to their molecular weight, significantly reducing operation time. It requires only simple instruments and reagents, and is easy to operate with high efficiency. Furthermore, this invention utilizes trichloroacetic acid to effectively remove proteins from sea cucumber intestinal polysaccharides, greatly improving their purity.

[0042] The sea cucumber intestinal polysaccharide provided by this invention is non-toxic to HK-2 cells and can significantly reduce the uric acid content in the supernatant of a hyperuricemic cell model. This invention isolates three sea cucumber polysaccharides with uric acid-lowering activity that can effectively inhibit xanthine oxidase activity, among which SCP55 shows the best inhibitory effect on xanthine oxidase, IC50. 50 With a concentration of 0.248 mg / mL, it has high application development value and can be used as a functional ingredient in pharmaceuticals and other fields. Attached Figure Description

[0043] Figure 1 This is a graph showing the determination of xanthine oxidase inhibition rate of sea cucumber intestinal polysaccharides according to the present invention; wherein, the horizontal axis represents the mass concentration of the three sea cucumber intestinal polysaccharides, and the vertical axis represents the xanthine oxidase inhibition rate.

[0044] Figure 2 Ultraviolet spectral analysis of the intestinal polysaccharide components of sea cucumber.

[0045] Figure 3 The effect of the sea cucumber intestinal uric acid-lowering polysaccharide prepared in this invention on the proliferation activity of HK-2 cells; wherein, the horizontal axis is the mass concentration of sea cucumber intestinal polysaccharide, and the vertical axis is the relative cell survival rate.

[0046] Figure 4 The graph shows the effect of the sea cucumber intestinal polysaccharide prepared in this invention on the uric acid content in the supernatant of the HK-2 hyperuricemia cell model; where the horizontal axis represents the mass concentration of the sea cucumber intestinal polysaccharide and the vertical axis represents the uric acid content in the supernatant of the HK-2 hyperuricemia cell model. Detailed Implementation

[0047] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0048] This invention uses sea cucumber as raw material, and prepares sea cucumber intestinal polysaccharide through freeze-drying and enzymatic method. Then, it is fractionated with ethanol, centrifuged, deproteinized, and freeze-dried to obtain uric acid-lowering polysaccharide. Addressing the low utilization rate of sea cucumber intestine resources—a by-product of sea cucumber—this invention provides a highly efficient and convenient method for preparing sea cucumber intestinal polysaccharide that inhibits xanthine oxidase and reduces uric acid content in HK-2 cell supernatant.

[0049] This invention provides a method for preparing sea cucumber intestinal polysaccharide, comprising the following steps: obtaining sea cucumber intestinal hydrolysate by papain enzymatic hydrolysis; separating and purifying the obtained sea cucumber intestinal hydrolysate by fractional ethanol precipitation, determining the xanthine oxidase inhibition rate of each separated component, and freeze-drying to obtain a lyophilized sea cucumber intestinal polysaccharide powder with high uric acid-lowering activity; and obtaining the monosaccharide and molecular weight composition of the obtained sea cucumber intestinal polysaccharide by high performance liquid chromatography. The sea cucumber intestinal polysaccharide was validated using HK-2 cell experiments, yielding a sea cucumber intestinal polysaccharide with potential high xanthine oxidase inhibition activity and reduced uric acid content in HK-2 cell supernatant.

[0050] Furthermore, the above preparation method may include the following steps: Extraction of sea cucumber intestinal polysaccharides: Clean the sea cucumber intestines, freeze-dry and pulverize them, add deionized water, adjust the pH and add papain for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate in a boiling water bath, centrifuge and collect the supernatant; perform fractional alcohol precipitation with solutions prepared with three times the volume of anhydrous ethanol in different proportions, centrifuge, collect the precipitate and freeze-dry; prepare a sea cucumber intestinal polysaccharide solution after freeze-drying, add an equal volume of trichloroacetic acid solution, stir at room temperature, incubate overnight at 4°C, centrifuge, collect the supernatant, dialyze and freeze-dry to obtain three types of sea cucumber polysaccharides; verify the xanthine oxidase inhibitory activity of the three types of sea cucumber intestinal polysaccharides in vitro, and screen out the component with the best xanthine oxidase inhibitory effect; use HK-2 cells to determine the uric acid content of the supernatant of the screened active component to further verify whether the sea cucumber intestinal polysaccharides have uric acid-lowering activity.

[0051] Furthermore, the specific operation process for preparing the above-mentioned sea cucumber intestinal polysaccharide is as follows:

[0052] (1) Preparation of sea cucumber intestine powder: Wash the sea cucumber intestine to remove mud and sand, freeze dry and then pulverize to obtain sea cucumber intestine powder, and store it for later use.

[0053] (2) Extraction of sea cucumber polysaccharides: Accurately weigh the sea cucumber intestine powder, mix the sea cucumber intestine powder with ultrapure water at a ratio of 1 g: 3 mL, add papain (enzyme addition amount of 2%), adjust the pH to 7.3, stir and react at 54℃ for 3 h, heat in a boiling water bath at 98℃ for 20 min to inactivate the enzyme, cool to room temperature, centrifuge at 8000 r / min for 10 min, and take the supernatant 1.

[0054] (3) Preparation of sea cucumber intestinal polysaccharide: The supernatant 1 from step (2) was precipitated with 3 times its volume of 25% ethanol solution, incubated overnight at 4°C, and centrifuged at 8000 r / min for 10 min to obtain precipitate 1 and supernatant 2. Precipitate 1 is sea cucumber intestinal crude polysaccharide 1. The supernatant 2 was concentrated to a certain volume using a rotary evaporator, and then precipitated with 3 times its volume of 55% ethanol solution. The above operation was repeated to obtain precipitate 2 and supernatant 3. Precipitate 2 is sea cucumber intestinal crude polysaccharide 2. The supernatant 3 was further concentrated and then precipitated with 3 times its volume of 75% ethanol solution, incubated overnight at 4°C, centrifuged at 8000 r / min for 10 min, and freeze-dried to obtain precipitate 3, which is sea cucumber intestinal crude polysaccharide 3. Three different molecular weights of sea cucumber intestinal crude polysaccharides can be obtained through the above method.

[0055] (4) Deproteinization of sea cucumber intestinal crude polysaccharides: The three types of sea cucumber intestinal crude polysaccharides from step (3) were prepared into 0.01 g / mL solutions. An equal volume of 10% trichloroacetic acid solution was added to each solution. The solutions were stirred at room temperature for 30 min, incubated overnight at 4°C, centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The solutions were dialyzed under a 3500 Da dialysis bag for 48 h and then freeze-dried to obtain the three types of sea cucumber intestinal polysaccharides. The xanthine oxidase inhibition rate of the three components was measured, and the component with the strongest activity was screened. The components were freeze-dried to obtain sea cucumber intestinal polysaccharides with high uric acid-lowering activity, namely SCP25, SCP55 and SCP75.

[0056] Next, the monosaccharide composition of sea cucumber intestinal polysaccharides was identified: High performance liquid chromatography (HPLC) was used to identify the monosaccharide composition of sea cucumber intestinal polysaccharides. The mobile phase was potassium dihydrogen phosphate solution-acetonitrile (volume ratio 83:17). The pH of the potassium dihydrogen phosphate solution was adjusted to 6.7 with sodium hydroxide solution, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution was 0.05 M. The sample loading volume was 250 μL, and the column flow rate was 1.0 mL / min.

[0057] Verification of the activity of sea cucumber intestinal polysaccharide: The bioactivity of sea cucumber intestinal polysaccharide obtained by fractional alcohol precipitation was verified by the xanthine oxidase inhibition rate, and further verified by HK-2 cells.

[0058] This invention provides a highly efficient and rapid method for obtaining polysaccharides. It combines hot water extraction and fractional precipitation, saving separation and purification time, improving efficiency, and yielding components with high xanthine oxidase inhibition activity. This invention provides uric acid-lowering sea cucumber intestinal polysaccharides that inhibit xanthine oxidase and reduce uric acid content in HK-2 cell supernatant. The sea cucumber intestinal polysaccharides include SCP25, SCP55, and SCP75. This invention extends the industrial chain of sea cucumber by-product processing and utilization, realizing the high-value utilization of sea cucumber by-products. All three components, SCP25, SCP55, and SCP75, can effectively inhibit xanthine oxidase, with SCP55 showing better efficacy. The method provided by this invention is a highly efficient and convenient method for preparing sea cucumber intestinal polysaccharides, exhibiting good solubility and non-toxicity to HK-2 cells. Sea cucumber intestinal polysaccharides can be applied in medicine and other fields.

[0059] This invention provides a method for preparing uric acid-lowering sea cucumber intestinal polysaccharide that inhibits xanthine oxidase activity and reduces uric acid content in HK-2 cell supernatant.

[0060] This invention provides three sea cucumber intestinal polysaccharides with uric acid-lowering activity, namely SCP25, SCP55, and SCP75. These sea cucumber intestinal polysaccharides inhibit xanthine oxidase activity and reduce the uric acid content in the supernatant of HK-2 cells.

[0061] The following is a description through specific embodiments.

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. The materials, reagents, methods, and instruments used, unless otherwise specified, are conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially or prepared using conventional methods by those skilled in the art. Unless otherwise specified, the solutions involved in this invention are prepared using water as the solvent.

[0063] Sea cucumber intestines were purchased from Linghai Dalian Marine Products Aquaculture Co., Ltd.; compound protease (C8800), 1-phenyl-3-methyl-5-pyrazolone (PMP), methanol, xanthine, and xanthine oxidase (XO) were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; HK-2 cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; neutral protease (ZN7232) was purchased from Hefei Bomei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; penicillin-streptomycin mixture, MEM (containing NEAA), CCK-8, phosphate buffered solution (PBS), and other reagents were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.; allopurinol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; adenosine was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and uric acid reagent kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0064] Example 1

[0065] Extracting crude polysaccharides from sea cucumber intestines includes the following steps:

[0066] (1) Mix sea cucumber intestine powder with ultrapure water at a ratio of 1 g: 3 mL, add papain, the amount of papain added is 2% of the mass of sea cucumber intestine powder, adjust the pH to 7.3, extract in a water bath at 54℃ for 3 h, inactivate the enzyme in a boiling water bath for 20 min, cool to room temperature, centrifuge at 8000 r / min for 10 min, and take 1 supernatant.

[0067] (2) Add supernatant 1 to 3 times the volume of 25% ethanol solution (the volume concentration of anhydrous ethanol in the 25% ethanol solution is 25%, prepared with water), incubate overnight at 4℃, and centrifuge at 8000 r / min for 10 min to obtain precipitate 1 and supernatant 2.

[0068] (3) Concentrate the supernatant 2 to the same volume as the supernatant 1 using a rotary evaporator, add 3 times the volume of 55% ethanol solution (the volume concentration of anhydrous ethanol in the 55% ethanol solution is 55%, prepared with water), incubate overnight at 4℃, centrifuge at 8000 r / min for 10 min, and take precipitate 2 and supernatant 3.

[0069] (4) Concentrate the supernatant 3 to the same volume as the supernatant 2 using a rotary evaporator, then add 3 times the volume of 75% ethanol solution (the volume concentration of anhydrous ethanol in the 75% ethanol solution is 75%, prepared with water), incubate overnight at 4℃, centrifuge at 8000 r / min for 10 min, and take the precipitate 3.

[0070] (5) Precipitates 1, 2 and 3 were freeze-dried to obtain three kinds of crude polysaccharides from sea cucumber intestines (i.e., crude polysaccharide 1, crude polysaccharide 2 and crude polysaccharide 3).

[0071] Comparative Example 1

[0072] Based on Example 1, in Comparative Example 1, a complex protease was used to replace the papain in Example 1 when extracting sea cucumber intestinal polysaccharides, and the remaining steps were the same as in Example 1.

[0073] Comparative Example 2

[0074] Based on Example 1, in Comparative Example 2, a neutral protease was used to replace the papain in Example 1 when extracting sea cucumber enteropolysaccharides, and the remaining steps were the same as in Example 1.

[0075] Example 1

[0076] The yields of sea cucumber intestinal polysaccharides prepared in Example 1, Comparative Example 1, and Comparative Example 2 were determined. The results showed that the polysaccharide yield after papain hydrolysis was 13%, while the yield after hydrolysis with a compound protease was 8%, and the yield after hydrolysis with a neutral protease was 11%. It can be seen that the sea cucumber intestinal polysaccharide extracted using papain had the highest yield, superior to that prepared by other enzymatic hydrolysis methods. Therefore, papain was selected for the experiment.

[0077] Example 2

[0078] The deproteinization of sea cucumber intestinal crude polysaccharides includes the following steps: The three types of sea cucumber intestinal crude polysaccharides obtained in Example 1 were each prepared into 0.01 g / mL solutions. An equal volume of trichloroacetic acid solution (10% by mass) was added to each solution. The solutions were stirred at room temperature for 30 min, transferred to a 4°C refrigerator overnight, centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The supernatant was then placed in a 3500 Da dialysis bag and dialyzed for 48 h. The liquid outside the dialysis bag was pure water. The liquid inside the dialysis bag was freeze-dried to obtain three sea cucumber intestinal polysaccharides with different molecular weights: SCP25, SCP55, and SCP75. SCP25 is the sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 1 using the above method; SCP55 is the sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 2 using the above method; and SCP75 is the sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 3 using the above method.

[0079] Example 3

[0080] The xanthine oxidase inhibitory capacity of different components (sea cucumber intestinal polysaccharides obtained in Example 2, namely SCP25, SCP55 and SCP75) was determined.

[0081] An XO solution with a concentration of 0.02 U / mL was prepared using ultrapure water. A xanthine substrate solution with a concentration of 0.48 mmol / L was prepared using PBS buffer (pH 7.4). Sample concentrations were set at 2, 1, 0.5, 0.25, and 0.125 mg / mL, respectively.

[0082] XO-added but sample-free group: Add 50 μL of XO solution and 50 μL of PBS to a 96-well plate;

[0083] No XO, no sample group: Add 100 μL of PBS to the 96-well plate;

[0084] Add XO and sample: Add 50 μL of XO solution and 50 μL of sample to a 96-well plate;

[0085] Sample group without XO: Add 50 μL of sample and 50 μL of PBS to each well of a 96-well plate;

[0086] Each group was incubated at 25℃ for 5 min, then 150 μL of xanthine substrate solution was added, and the mixture was incubated at 25℃ for 30 min. The absorbance was then measured at a wavelength of 290 nm.

[0087] The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0088]

[0089] In the formula: A represents the OD with XO but without the sample;

[0090] B indicates the OD without XO and without sample;

[0091] C indicates the addition of XO to the sample's OD;

[0092] D indicates the OD of the sample without XO.

[0093] The results of the xanthine oxidase inhibition rate detection of sea cucumber intestinal polysaccharides are as follows: Figure 1 As shown, SCP25, SCP55, and SCP75 all exhibit varying degrees of inhibition of xanthine oxidase. Compared to SCP25 and SCP75, the SCP55 fraction demonstrates a higher inhibitory capacity for xanthine oxidase, with an IC50 value of [missing value]. 50 The concentration was 0.248 mg / mL; followed by SCP75. Although SCP75 also had a significant effect, its IC50 concentration was 0.248 mg / mL. 50 The concentration was 0.39 mg / mL; finally, SCP25 showed an inhibition rate of less than 50% at 2 mg / mL. Therefore, SCP55 was selected for subsequent experiments. The existing XO inhibition rate IC50 of natural polysaccharides... 50 The values ​​ranged from 0.83 to 10.53 mg / mL, indicating that the component prepared in this invention has a better inhibitory effect on XO.

[0094] Example 4

[0095] The monosaccharide composition of SCP55 (prepared using the method in Example 2) was determined using a Shimadzu LC-20AD chromatographic column of Ultimate C18 4.6*200mm 5um.

[0096] Preparation of mixed control solution: Accurately weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, and fucose reference standards, respectively. Dissolve and dilute each reference standard with water to obtain a mixed control solution. The concentration of each reference standard in the mixed control solution is 50 ug / mL.

[0097] SCP55 hydrolysis: Accurately weigh 10 mg of sample into a 10 mL ampoule, add 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) to the 10 mL ampoule, fill with nitrogen, seal the ampoule, and acid hydrolyze at 120 °C for 4 h. Remove 1.0 mL of the solution, add methanol and nitrogen to evaporate the TFA, and redissolve in 1.0 mL of water to obtain the hydrolyzed SCP55 solution.

[0098] Derivatization: Accurately pipette 250 μL of the mixed control solution into a 5 mL EP tube, add 250 μL of 0.6 mol / L NaOH and 500 μL of 0.4 mol / L PMP-methanol, and react at 70 °C for 1 h. Cool in cold water for 10 min; neutralize with 500 μL of 0.3 mol / L hydrochloric acid (HCl), then add 1 mL of chloroform, vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, extract three times, and use the extracted supernatant for HPLC analysis. Derivatize the SCP55 sample solution using the above method to obtain the supernatant for subsequent HPLC analysis.

[0099] Determination: The supernatants obtained from the derivatization of the mixed control solution and the SCP55 sample solution were respectively used for monosaccharide composition identification by HPLC. The mobile phase was 0.05 M potassium dihydrogen phosphate solution-acetonitrile (v / v ratio 83:17). The pH of the 0.05 M potassium dihydrogen phosphate solution was adjusted to 6.70 with sodium hydroxide solution. The sample loading volume was 250 μL, and the column flow rate was 1.0 mL / min.

[0100] The results of the monosaccharide composition analysis are shown in Table 1. It can be seen that SCP55 is a heteropolysaccharide mainly containing glucose, and in addition to glucose, it also contains small amounts of mannose, ribose, glucuronic acid, galacturonic acid, galactose, xylose, arabinose and fucose.

[0101] Table 1 Monosaccharide composition analysis of sea cucumber intestinal polysaccharides

[0102]

[0103] Note: The percentages in Table 1 are molar percentages.

[0104] Example 5

[0105] Accurately weigh SCP55 to prepare a 5 mg / mL polysaccharide solution, filter it through a 0.22 µm microporous membrane, and determine its molecular weight using a Shimadzu GPC-20A gel permeation chromatograph with a TOSOH (TSK) TSKgel GMPWXL aqueous gel column. Pullulan from Shiodex Corporation of Japan was used as a standard. The mobile phase was 0.1 mol / L NaNO3 + 0.06% NaN3 aqueous solution, and the flow rate was 0.6 mL / min.

[0106] The molecular weight analysis results are shown in Table 2. The molecular weight of SCP55 is 618.12 kDa.

[0107] Table 2 Molecular weight analysis of sea cucumber intestinal polysaccharide components

[0108]

[0109] Example 6

[0110] Accurately weigh SCP55 to prepare a 0.5 mg / mL polysaccharide solution, and scan it in a UV spectrophotometer with a scanning wavelength range of 200-400 nm.

[0111] The results of ultraviolet spectroscopy analysis are as follows Figure 2 As shown, nucleic acids and proteins have absorption peaks at 260 nm and 280 nm, respectively. The detection results show that SCP55 did not have a significant absorption peak in the range of 200-400 nm. Therefore, it can be concluded that SCP55 contains neither nucleic acids nor proteins.

[0112] Example 7: Detection of the effect of SCP55 on the proliferation rate of HK-2 cells

[0113] When HK-2 cells reached 80%-90% confluence, they were digested with 1 mL of 0.25% trypsin and then resuspended in complete culture medium (MEM containing a mixture of 10% serum and 1% penicillin-streptomycin). The cells were then cultured at a concentration of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 cells / well into 96-well plates. After 24 h, the original culture medium in the wells was discarded, and the cells were washed once with PBS. A control group, different concentrations of sea cucumber enteropolysaccharide groups (2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL), and a blank group were set up, with 6 replicates in each group.

[0114] Control group: 100 μL of MEM (containing NEAA) was added to the well containing cells.

[0115] Sea cucumber intestinal polysaccharide groups at different concentrations (experimental group): SCP55 was dissolved in MEM (containing NEAA) to prepare solutions of different concentrations (2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, respectively), and 100 μL was added to the wells containing cells.

[0116] Blank group: Add 100 μL of MEM (containing NEAA) to wells without cells.

[0117] After incubating the 96-well plate in a 37°C CO2 incubator for 24 h, the culture medium was discarded, and each well was washed once with PBS. Then, 90 μL of incomplete culture medium (MEM containing NEAA) and 10 μL of CCK-8 reagent were added to each well, and the plate was incubated at 37°C for another 1 h. The absorbance at 450 nm was measured using a microplate reader. The calculation formula is as follows:

[0118]

[0119] In the formula: A1 represents the absorbance value at 450 nm in the experimental group;

[0120] A2 represents the absorbance value at 450 nm for the blank group, which contains only culture medium and no cells.

[0121] A3 represents the absorbance value at 450 nm for the control group, which contains culture medium and cells.

[0122] The results of SCP55's effect on the proliferation activity of HK-2 cells are as follows: Figure 3 As shown, from Figure 3 It is known that different concentrations of SCP55 have no effect on the survival rate of HK-2 cells, indicating that the preparation of this invention has no toxic effect on HK-2 cells.

[0123] Example 8: Effect of SCP55 on uric acid content in the supernatant of HK-2 hyperuricemic cells

[0124] The study included a blank control group (NC), a model group (MC), an SCP55 group, and a positive control group (allopurinol, AP). Each group was administered 1×10⁻⁶ mg / L. 6HK-2 cells were seeded in 24-well plates at a density of cells / mL (total volume 1.0 mL / well) and incubated at 37°C for 30 h. The complete culture medium was then aspirated, and the cells were washed twice with PBS. The control and model groups were then replaced with serum-free incomplete culture medium MEM (containing NEAA). The positive control group received allopurinol (AP, final concentration 15.0 μg / mL), and the SCP55 groups received different concentrations (1, 0.5, 0.25, 0.125 mg / mL) of SCP55 solution, all at a total volume of 1.0 mL / well. Cells were cultured for another 24 h. The culture medium was aspirated, and the cells were washed twice with PBS. Afterward, the HK-2 cells in the control group were cultured in incomplete culture medium MEM (containing NEAA) for 28 h. The HK-2 cells in the model, positive control, and SCP55 groups were cultured in incomplete culture medium induced with adenosine (2.5 mmol / L) for 24 h, followed by the addition of 1 mL of 0.005 U / mL XO, and cultured for another 4 h.

[0125] Centrifuge the supernatant from each group and strictly follow the instructions in the uric acid kit to detect the uric acid content in the cell culture supernatant.

[0126] Figure 4 The figure shows the effect of sea cucumber intestinal polysaccharide SCP55 on the uric acid content in the supernatant of the HK-2 hyperuricemia cell model. It can be seen that different concentrations of sea cucumber intestinal uric acid-lowering polysaccharide (1, 0.5, 0.25, and 0.125 mg / mL) reduced the uric acid content in the supernatant of HK-2 cells. Furthermore, the uric acid content decreased with increasing concentration; the effect of 1 mg / mL sea cucumber intestinal uric acid-lowering polysaccharide was similar to that of allopurinol.

[0127] Allopurinol is a drug for treating hyperuricemia, but it has strong side effects and is quite harmful to the body. Few bioactive substances or natural polysaccharides have a similar uric acid-lowering effect to allopurinol. However, the SCP55 component provided by this invention is a bioactive substance with advantages such as safety and non-toxicity to cells.

[0128] Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the present invention. Those skilled in the art can modify and improve it without departing from the scope of the present invention. Therefore, the scope of protection of the present invention should be determined according to the contents defined in the claims.

Claims

1. Use of a sea cucumber gut polysaccharide in the preparation of a drug for reducing uric acid, characterized in that, The sea cucumber intestine polysaccharide is prepared by the following method: (1) sea cucumber intestine is mixed with water, and the ratio of sea cucumber intestine to water is 1 g:3 mL; papain is used for enzymolysis, and the addition amount of papain is 2%; the enzymolysis conditions include an enzymolysis time of 3 h, an enzymolysis temperature of 54 DEG C, and pH=7.3; enzyme is inactivated, and the inactivation conditions include a boiling water bath for 20 min; after cooling, centrifugation is performed, and the supernatant 1 is taken; (2) the supernatant 1 is alcohol precipitated by using 3 times the volume of 25% ethanol solution, centrifugation is performed, and the precipitate 1 and the supernatant 2 are obtained; (3) after the supernatant 2 is concentrated, alcohol precipitation is performed by using 3 times the volume of 55% ethanol solution, centrifugation is performed, and the precipitate 2 and the supernatant 3 are obtained; (4) deproteinization: the precipitate 2 is prepared into a 0.01 g / mL solution, an equal volume of 10% trichloroacetic acid solution is added, stirring is performed, and the solution is placed at 4 DEG C overnight; centrifugation is performed, the supernatant is taken, the supernatant is dialyzed by using a 3500 Da dialysis bag for 48 h, and freeze-drying is performed, to obtain sea cucumber intestine polysaccharide SCP55.

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