A preparation method and application of sea cucumber intestine polysaccharide

Abstract

The present invention relates to a preparation method and application of sea cucumber intestine polysaccharide. The preparation method of sea cucumber intestine polysaccharide comprises the following steps: subjecting sea cucumber intestine to enzymatic hydrolysis treatment with papain, inactivating the enzyme, centrifuging and taking the supernatant; subjecting the supernatant to fractional alcohol precipitation, centrifuging, taking the precipitate, deproteinizing and freeze-drying. The preparation method of the present invention is simple, green and environment-friendly. The sea cucumber intestine polysaccharide prepared by the method of the present invention can effectively inhibit the activity of xanthine oxidase; the sea cucumber intestine polysaccharide has no toxic effect on HK-2 cells. It has the effect of reducing uric acid on HK-2 hyperuricemic cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of processing and utilization of sea cucumber by-products, and particularly relates to a preparation method and application of sea cucumber intestine polysaccharide. Background Art

[0002] Sea cucumbers (Stichopus japonicus) belong to the phylum Echinodermata and are a type of marine invertebrate widely distributed in benthic zones and deep seas around the world. They are recognized as a tonic and traditional medicine, and in addition to being rich in amino acids, fatty acids, trace elements and other components, they also contain various bioactive substances such as polysaccharides, proteins, and saponins. However, during the deep processing of sea cucumbers, sea cucumber viscera are often randomly disposed of as processing by-products, and there is still a large gap in the in-depth study of the nutritional components of sea cucumber viscera, and there is a lack of recycling and processing of sea cucumber viscera, resulting in serious waste of resources and environmental pollution. Among them, sea cucumber intestines are important by-products generated during sea cucumber processing, with the characteristics of large yield and renewable. At present, the utilization rate of sea cucumber intestines in China is extremely low, often being randomly discarded as processing by-products and not being fully utilized, causing huge waste of resources. Nowadays, with the rapid development and increasing maturity of sea cucumber breeding and production technologies, the comprehensive utilization of sea cucumber by-products has become an urgent problem to be solved.

[0003] With the rapid development of the times, people's material living standards are getting higher and higher, the richness of diet has also been greatly improved, and the frequency of ingesting high-purine foods has increased significantly, resulting in a particularly high incidence of hyperuricemia in China. And prolonged hyperuricemia will further trigger diseases such as gout. The increasing number of hyperuricemia patients has led scholars to continuously search for substances that can reduce uric acid. Currently, the main drugs for treating hyperuricemia are allopurinol, colchicine, febuxostat, etc., which have significant effects but have certain toxic side effects. And natural-source hypouricemic active substances with low toxicity and high activity are increasingly favored by people. Among them, natural polysaccharides have become a research hotspot due to their wide sources and easy absorption. In recent years, more and more studies have obtained natural polysaccharides with XO inhibitory activity, but their effects are significantly lower than that of the XO-specific inhibitory drug - allopurinol. The XO inhibition rate IC 50 value of natural polysaccharides is between 0.83 - 10.53 mg / mL, and the low activity limits their wide application in the industry. Therefore, it is extremely urgent to provide a convenient and efficient preparation method of sea cucumber intestine polysaccharide with hypouricemic activity. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing sea cucumber intestine polysaccharide and its application. The method provided by the present invention can prepare sea cucumber intestine polysaccharide with the ability to inhibit xanthine oxidase and reduce uric acid content. The preparation method of the present invention is simple, green and environmentally friendly. The sea cucumber intestine polysaccharide can effectively inhibit the activity of xanthine oxidase; the sea cucumber intestine polysaccharide has no toxic effect on HK-2 cells. It has the effect of reducing uric acid on HK-2 hyperuricemic cells.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a method for preparing sea cucumber intestine polysaccharide, comprising the following steps: enzymatically hydrolyzing sea cucumber intestine with papain, inactivating the enzyme, and taking the supernatant after centrifugation; subjecting the supernatant to fractional ethanol precipitation, centrifuging, and taking the precipitate.

[0007] Further, it comprises the following steps:

[0008] (1) Mix sea cucumber intestine with water, enzymatically hydrolyze with papain, and inactivate the enzyme; after cooling, centrifuge and take supernatant 1;

[0009] (2) Subject supernatant 1 to ethanol precipitation with 25% ethanol solution, centrifuge to obtain precipitate 1 and supernatant 2;

[0010] (3) After concentrating supernatant 2, subject it to ethanol precipitation with 55% ethanol solution, centrifuge to obtain precipitate 2 and supernatant 3;

[0011] (4) After concentrating supernatant 3, subject it to ethanol precipitation with 75% ethanol solution, centrifuge and take precipitate 3.

[0012] The beneficial effects of adopting the above technical solutions include: during the research process, the above operations were respectively carried out using compound protease, neutral protease, and papain, and it was found that the yield was the highest when using papain.

[0013] Further, in step (1), the addition amount of papain is 2%.

[0014] Further, in step (1), the enzymatic hydrolysis conditions include: enzymatic hydrolysis time 3h, enzymatic hydrolysis temperature 54 °C, pH = 7.3.

[0015] Further, in step (1), the enzyme inactivation conditions include: boiling water bath for 20 min.

[0016] Further, in step (1), the centrifugation conditions are centrifugation at 8000 r / min for 10 min.

[0017] Further, in step (1), the material-liquid ratio of sea cucumber intestine to water is 1 g: 3 mL.

[0018] Further, it also includes the step of freeze-drying the precipitate.

[0019] Furthermore, it further includes a step of precipitating and deproteinizing.

[0020] Furthermore, the deproteinization includes the following steps: preparing the precipitate into a solution, adding an equal volume of 10% trichloroacetic acid solution, stirring, overnight at 4°C, centrifuging, taking the supernatant, and dialyzing the supernatant under a 3500Da dialysis bag for 48h.

[0021] In the above steps, the precipitate can be prepared into a 0.01g / mL solution and then deproteinized.

[0022] The beneficial effects of adopting the above technical solutions include: adopting the above parameters has the advantages of high yield and simple operation.

[0023] The present invention provides a sea cucumber intestine polysaccharide, which is prepared by the above method.

[0024] The beneficial effects of adopting the above scheme include: the sea cucumber intestine polysaccharide is mainly a heteropolysaccharide composed of glucose, with a molecular weight of 618.1kDa. The sea cucumber intestine polysaccharide has no effect on the proliferation rate of HK-2 cells and can reduce the uric acid content in the supernatant of HK-2 cells.

[0025] The present invention provides the application of the above sea cucumber intestine polysaccharide in reducing uric acid and / or inhibiting xanthine oxidase.

[0026] The present invention provides that the above sea cucumber intestine polysaccharide can be used to prepare products for reducing uric acid; it can also be used to prepare products for inhibiting xanthine oxidase; the products are not limited to foods, health products, drugs, etc.

[0027] In view of the low utilization rate of existing sea cucumber intestines, the present invention provides a method for obtaining sea cucumber intestine polysaccharide with uric acid-lowering activity based on papain enzymolysis and then through fractional alcohol precipitation. The present invention verifies the xanthine oxidase inhibitory activity of the sea cucumber intestine polysaccharide and its uric acid-lowering effect on HK-2 cells. The results show that the three sea cucumber intestine polysaccharides screened by the present invention all have the activity of inhibiting xanthine oxidase, among which SCP55 has the best effect, and subsequent cell experiments further verify its biological activity.

[0028] The present invention has the following beneficial effects: Sea cucumbers have high edible value, and the low utilization rate of by-products is an urgent problem to be solved in the current sea cucumber aquaculture industry. The present invention prepares and isolates sea cucumber intestine uric acid-lowering polysaccharide, which can improve 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 the present invention is simple, fast, has good reproducibility, is suitable for industrial production, has good safety, has no toxic and side effects on the human body, and conforms to the modern health concept.

[0029] The present invention separates and purifies polysaccharides according to molecular weight by means of fractional alcohol precipitation, greatly reducing the operation time. Only simple instruments and reagents are required, and the operation is simple and efficient. Trichloroacetic acid is used in the present invention to effectively remove proteins from sea cucumber intestine polysaccharides, greatly improving the purity of sea cucumber intestine polysaccharides.

[0030] The sea cucumber intestine polysaccharide provided by the present invention is non-toxic to HK-2 cells and can significantly reduce the uric acid content in the supernatant of the hyperuricemic cell model. Three sea cucumber polysaccharides with uric acid-lowering activity isolated by the present invention can effectively inhibit the activity of xanthine oxidase. Among them, SCP55 has the best inhibitory effect on xanthine oxidase, and the I C 50 is 0.248 mg / mL, which has high application and development value and can be used as a functional ingredient in the fields of food, health products, etc. Brief Description of the Drawings

[0031] Figure 1 It is a graph for measuring the inhibitory rate of xanthine oxidase of the sea cucumber intestine polysaccharide of the present invention; wherein, the abscissa is the mass concentration of three kinds of sea cucumber intestine polysaccharides, and the ordinate is the inhibitory rate of xanthine oxidase.

[0032] Figure 2 It is the ultraviolet spectrum analysis of the sea cucumber intestine polysaccharide component.

[0033] Figure 3 It is the influence of the sea cucumber intestine uric acid-lowering polysaccharide prepared by the present invention on the proliferation activity of HK-2 cells; wherein, the abscissa is the mass concentration of the sea cucumber intestine polysaccharide, and the ordinate is the relative cell survival rate.

[0034] Figure 4 It is a graph for measuring the influence of the sea cucumber intestine polysaccharide prepared by the present invention on the uric acid content in the supernatant of the HK-2 hyperuricemic cell model; wherein, the abscissa is the mass concentration of the sea cucumber intestine polysaccharide, and the ordinate is the uric acid content in the supernatant of the HK-2 hyperuricemic cell model. Detailed Embodiments

[0035] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0036] The present invention uses sea cucumbers as raw materials, undergoes freeze-drying, and prepares sea cucumber intestine polysaccharides by an enzymatic method. Then, uric acid-lowering polysaccharides are obtained by ethanol fractional alcohol precipitation, centrifugation, deproteinization, and freeze-drying. Aiming at the phenomenon of low resource utilization rate of the existing sea cucumber by-product - sea cucumber intestine, the present invention provides a method for preparing sea cucumber intestine polysaccharides with high efficiency and convenience, which can inhibit xanthine oxidase and reduce the uric acid content in the supernatant of HK-2 cells.

[0037] The present invention provides a preparation method of sea cucumber intestine polysaccharide, comprising the following steps: enzymatically hydrolyzing with papain to obtain a sea cucumber intestine hydrolysate; separating and purifying the obtained sea cucumber intestine hydrolysate by fractional ethanol precipitation with an ethanol solution, measuring the xanthine oxidase inhibition rate of each separated component, and freeze-drying to obtain a freeze-dried powder of sea cucumber intestine polysaccharide with high uric acid-lowering activity; subjecting the obtained sea cucumber intestine polysaccharide to high performance liquid chromatography technology to obtain the monosaccharide and molecular weight composition of the sea cucumber intestine polysaccharide. Conducting cell verification on the sea cucumber intestine polysaccharide by using HK-2 cell experiments to obtain a sea cucumber intestine polysaccharide with potentially high xanthine oxidase inhibitory activity and capable of reducing the uric acid content in the supernatant of HK-2 cells.

[0038] Further, the above preparation method may include the following steps: extracting sea cucumber intestine polysaccharide: cleaning the sea cucumber intestine, freeze-drying and pulverizing it, adding deionized water, adjusting the pH and adding papain for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivating it in a boiling water bath, centrifuging, and taking the supernatant; subjecting the above supernatant to fractional ethanol precipitation with solutions prepared with different proportions of absolute ethanol at 3 times the volume, centrifuging, taking the precipitate, and freeze-drying; dissolving the freeze-dried sea cucumber intestine polysaccharide into a sea cucumber intestine polysaccharide solution, adding an equal volume of trichloroacetic acid solution, stirring at room temperature, leaving it overnight in a refrigerator at 4 °C, centrifuging, taking the supernatant, dialyzing and then freeze-drying to obtain three kinds of sea cucumber polysaccharides; verifying the xanthine oxidase inhibitory activity of the obtained three kinds of sea cucumber intestine polysaccharides in vitro, and screening out the component with the best xanthine oxidase inhibitory effect; measuring the uric acid content in the supernatant by using HK-2 cells for the screened active component to further verify whether the sea cucumber intestine polysaccharide has uric acid-lowering activity.

[0039] Even further, the preparation method of the above sea cucumber intestine polysaccharide has the following specific operation process:

[0040] (1) Preparing sea cucumber intestine powder: washing the sand off the sea cucumber intestine, freeze-drying and pulverizing it to obtain sea cucumber intestine powder, and storing it for later use.

[0041] (2) Extracting sea cucumber polysaccharide: accurately weighing the sea cucumber intestine powder, mixing the sea cucumber intestine powder and ultrapure water at a material-liquid ratio of 1 g: 3 mL, adding papain (the enzyme addition amount is 2%), adjusting the pH to 7.3, stirring and reacting at 54 °C for 3 h, heating in a boiling water bath at 98 °C for 20 min to inactivate the enzyme, cooling to room temperature, centrifuging at 8000 r / min for 10 min, and taking the supernatant 1.

[0042] (3) Preparation of sea cucumber intestine polysaccharide: The supernatant 1 in step (2) was precipitated with 3 volumes of 25% ethanol solution, left overnight at 4°C, and centrifuged at 8000 r / min for 10 min to obtain precipitate 1 and supernatant 2. Precipitate 1 was the crude sea cucumber intestine polysaccharide 1; the supernatant 2 was concentrated to a certain volume with a rotary evaporator and then precipitated with 3 volumes of 55% ethanol solution. The above operation was repeated to obtain precipitate 2 and supernatant 3. Precipitate 2 was the crude sea cucumber intestine polysaccharide 2; the supernatant 3 was continuously concentrated and then precipitated with 3 volumes of 75% ethanol solution, left overnight at 4°C, centrifuged at 8000 r / min for 10 min, and freeze-dried to obtain precipitate 3, which was the crude sea cucumber intestine polysaccharide 3. Three crude sea cucumber intestine polysaccharides with different molecular weights could be obtained by the above method.

[0043] (4) Deproteinization of the crude sea cucumber intestine polysaccharide: The three crude sea cucumber intestine polysaccharides in step (3) were respectively formulated into crude sea cucumber intestine polysaccharide solutions with a concentration of 0.01 g / mL, and equal volumes of 10% trichloroacetic acid solution were added. Stir at room temperature for 30 min, leave overnight at 4°C, centrifuge at 8000 r / min for 10 min, take the supernatant, dialyze under a 3500 Da dialysis bag for 48 h, and freeze-dry to obtain three sea cucumber intestine polysaccharides. The xanthine oxidase inhibition rates of the three components were measured respectively, and the component with the strongest activity was screened out. The component was freeze-dried to obtain sea cucumber intestine polysaccharides with relatively high uric acid-lowering activity, namely SCP25, SCP55, and SCP75.

[0044] After that, the monosaccharide composition of the sea cucumber intestine polysaccharide was identified: High performance liquid chromatography was used to identify the monosaccharide composition of the sea cucumber intestine polysaccharide. 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. The concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate solution was 0.05 M, the sample injection volume was 250 μL, and the column flow rate was 1.0 mL / min.

[0045] Verification of the activity of the sea cucumber intestine polysaccharide: The sea cucumber intestine polysaccharide obtained by fractional precipitation was verified for its biological activity through the xanthine oxidase inhibition rate, and further verified by HK-2 cells.

[0046] The preparation method provided by the present invention is an efficient and rapid method for obtaining polysaccharides. By combining the hot water method and the fractional precipitation method, it saves the time for separation and purification, improves efficiency, and obtains components with high inhibitory activity against xanthine oxidase. The present invention provides hypouricemic sea cucumber intestine polysaccharides with inhibitory activity against xanthine oxidase and the ability to reduce the uric acid content in the supernatant of HK-2 cells. The sea cucumber intestine polysaccharides include SCP25, SCP55, and SCP75. The present invention extends the industrial chain for the processing and utilization of sea cucumber by-products, realizing the high-value utilization of sea cucumber by-products. All three components, SCP25, SCP55, and SCP75, can effectively inhibit xanthine oxidase, and SCP55 has a better effect. The method provided by the present invention is an efficient and convenient method for preparing sea cucumber intestine polysaccharides, with good solubility and no toxicity to HK-2 cells. Sea cucumber intestine polysaccharides can be applied to the fields of food, health products, medicine, etc.

[0047] The present invention provides a method for preparing hypouricemic sea cucumber intestine polysaccharides with inhibitory activity against xanthine oxidase and the ability to reduce the uric acid content in the supernatant of HK-2 cells.

[0048] The present invention provides three sea cucumber intestine polysaccharides with hypouricemic activity, and the sea cucumber intestine polysaccharides are SCP25, SCP55, and SCP75. The above-mentioned sea cucumber intestine polysaccharides have the effects of inhibiting xanthine oxidase activity and reducing the uric acid content in the supernatant of HK-2 cells.

[0049] The following is an introduction through specific examples.

[0050] The experimental methods used in the following examples are all conventional methods in the art unless otherwise specified. The materials, reagents, methods, and instruments used, unless otherwise specified, are all conventional materials, reagents, methods, and instruments in the art, and those skilled in the art can obtain them through commercial channels or prepare them by conventional methods. The solutions involved in the present invention are prepared with water as the solvent unless otherwise specified.

[0051] Sea cucumber intestines were purchased from Linghai Dalian Seafood Breeding 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 Procell Life Science & Technology Co., Ltd.; neutral protease (ZN7232) was purchased from Hefei Bomei Biotechnology Co., Ltd.; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; reagents such as penicillin-streptomycin mixture, MEM (containing NEAA), CCK-8, and phosphate buffer solution (PBS) 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 Bio-Technology Co., Ltd.; and the uric acid kit was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd.

[0052] Example 1

[0053] Extract crude polysaccharide from sea cucumber intestine, including the following steps:

[0054] (1) Mix the sea cucumber intestine powder with ultrapure water at a material-liquid ratio of 1 g: 3 mL, add papain, and the addition amount of papain is 2% of the mass of the sea cucumber intestine powder. Adjust the pH to 7.3 and extract in a water bath at 54 °C for 3 h. Inactivate the enzyme in a boiling water bath for 20 min. After cooling to room temperature, centrifuge at 8000 r / min for 10 min, and take the supernatant 1.

[0055] (2) Add 3 volumes of 25% ethanol solution (in the 25% ethanol solution, the volume concentration of absolute ethanol is 25%, prepared with water) to the supernatant 1, leave it overnight in a 4 °C refrigerator, centrifuge at 8000 r / min for 10 min, and obtain precipitate 1 and supernatant 2.

[0056] (3) Concentrate the supernatant 2 to the same volume as the supernatant 1 using a rotary evaporator, add 3 volumes of 55% ethanol solution (in the 55% ethanol solution, the volume concentration of absolute ethanol is 55%, prepared with water), leave it overnight in a 4 °C refrigerator, centrifuge at 8000 r / min for 10 min, and take precipitate 2 and supernatant 3.

[0057] (4) Concentrate the supernatant 3 to the same volume as the supernatant 2 using a rotary evaporator, then add the concentrated supernatant 3 to 3 volumes of 75% ethanol solution (in the 75% ethanol solution, the volume concentration of absolute ethanol is 75%, prepared with water), leave it overnight in a 4 °C refrigerator, centrifuge at 8000 r / min for 10 min, and take precipitate 3.

[0058] (5) Freeze-dry precipitate 1, precipitate 2, and precipitate 3 to obtain three kinds of crude polysaccharides from sea cucumber intestine (i.e., crude polysaccharide 1, crude polysaccharide 2, and crude polysaccharide 3).

[0059] Comparative Example 1

[0060] On the basis of Example 1, in Comparative Example 1, when extracting sea cucumber intestine polysaccharide, compound protease is used to replace papain in Example 1, and the remaining steps are the same as those in Example 1.

[0061] Comparative Example 2

[0062] On the basis of Example 1, in Comparative Example 2, when extracting sea cucumber intestine polysaccharide, neutral protease is used to replace papain in Example 1, and the remaining steps are the same as those in Example 1.

[0063] Effect Example 1

[0064] The yields of sea cucumber intestine polysaccharides prepared in Example 1, Comparative Example 1, and Comparative Example 2 were measured. It was found that the yield of polysaccharides after hydrolysis by papain was 13%, while the yield after hydrolysis by compound protease was 8%, and the yield after hydrolysis by neutral protease was 11%. It can be seen that the yield of sea cucumber intestine polysaccharides extracted by papain is the highest, which is better than that of sea cucumber intestine polysaccharides prepared by other enzymatic hydrolysis methods. Therefore, papain was selected for the experiment.

[0065] Example 2

[0066] The crude sea cucumber intestine polysaccharides were deproteinized, including the following steps: The three crude sea cucumber intestine polysaccharides obtained in Example 1 were respectively prepared into crude sea cucumber intestine polysaccharide solutions with a concentration of 0.01 g / mL, and equal volumes of trichloroacetic acid solutions (in the trichloroacetic acid solution, the mass concentration of trichloroacetic acid was 10%) were added. Stir at room temperature for 30 min, transfer to a refrigerator at 4°C overnight, centrifuge at 8000 r / min for 10 min, take the supernatant, load the supernatant into a 3500 Da dialysis bag for dialysis for 48 h, the liquid outside the dialysis bag was pure water, and the liquid inside the dialysis bag after dialysis was freeze-dried to obtain three sea cucumber intestine polysaccharides with different molecular weights, namely SCP25, SCP55, and SCP75. SCP25 was the sea cucumber intestine polysaccharide obtained by deproteinizing crude polysaccharide 1 using the above method, SCP55 was the sea cucumber intestine polysaccharide obtained by deproteinizing crude polysaccharide 2 using the above method, and SCP75 was the sea cucumber intestine polysaccharide obtained by deproteinizing crude polysaccharide 3 using the above method.

[0067] Example 3

[0068] The xanthine oxidase inhibitory abilities of different components (the sea cucumber intestine polysaccharides obtained in Example 2, namely SCP25, SCP55, and SCP75) were measured.

[0069] An XO solution with a concentration of 0.02 U / mL was prepared with ultrapure water. A xanthine substrate solution with a concentration of 0.48 mmol / L was prepared with PBS buffer solution (pH 7.4). The sample concentrations were set to 2, 1, 0.5, 0.25, and 0.125 mg / mL respectively.

[0070] XO without sample group: Add 50 μL of XO solution and 50 μL of PBS to a 96-well plate;

[0071] No XO and no sample group: Add 100 μL of PBS to a 96-well plate;

[0072] XO with sample group: Add 50 μL of XO solution and 50 μL of sample to a 96-well plate;

[0073] No XO with sample group: Add 50 μL of sample and 50 μL of PBS to a 96-well plate respectively;

[0074] Incubate each group at 25 °C for 5 min, add 150 μL of xanthine substrate solution, incubate at 25 °C for 30 min, and measure the absorbance at a wavelength of 290 nm.

[0075] The calculation formula for the xanthine oxidase inhibition rate is as follows:

[0076]

[0077] In the formula: A represents the OD value when adding XO but not the sample;

[0078] B represents the OD value when neither XO nor the sample is added;

[0079] C represents the OD value when adding XO and the sample;

[0080] D represents the OD value when not adding XO but adding the sample.

[0081] The detection results of the xanthine oxidase inhibition rate of sea cucumber intestine polysaccharide are as Figure 1 shown. It can be seen that SCP25, SCP55, and SCP75 all show different effects of inhibiting xanthine oxidase. Compared with SCP25 and SCP75, the xanthine oxidase inhibition ability of the SCP55 component is higher, and the IC 50 is 0.248 mg / mL; followed by SCP75. Although the effect of SCP75 is also significant, its IC 50 is 0.39 mg / mL; finally, it is SCP25, and the inhibition rate of SCP25 is less than 50% at 2 mg / mL. Therefore, SCP55 is selected for subsequent experiments. The IC 50 values of the existing natural polysaccharides for XO inhibition rate are between 0.83 - 10.53 mg / mL. It can be seen that the components prepared by the present invention have a better effect on the XO inhibition rate.

[0082] Example 4

[0083] Using Shimadzu LC-20AD, with the chromatographic column being Xtimate C18 4.6 * 200 mm 5 μm, determine the monosaccharide composition of SCP55 (prepared by the method of Example 2).

[0084] Prepare a mixed control solution: Weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose, and fucose reference substances respectively. Dissolve and dilute each reference substance with water to obtain a mixed control solution. In the mixed control solution, the concentration of each control is 50 μg / mL.

[0085] Hydrolysis of SCP55: Weigh 10 mg of the sample precisely into a 10-mL ampoule. Add 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) into the 10-mL ampoule, fill it with nitrogen, seal the tube, and hydrolyze it with acid at 120 °C for 4 h. Take out 1.0 mL and blow dry the TFA with nitrogen using methanol. Re-dissolve it with 1.0 mL of water to obtain the solution after the hydrolysis of SCP55.

[0086] Derivatization: Precisely pipette 250 μL of the mixed control solution into a 5-mL EP tube. Add 250 μL of 0.6 mol / L NaOH, 500 μL of 0.4 mol / L PMP-methanol, and react at 70 °C for 1 h. Cool it in cold water for 10 min; add 500 μL of 0.3 mol / L hydrochloric acid (HCl) for neutralization, then add 1 mL of chloroform and vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully take the supernatant, extract it 3 times, and use the extracted supernatant for HPLC determination. Derivatize the SCP55 sample solution according to the above method to obtain the supernatant for subsequent HPLC determination.

[0087] Determination: Take the supernatant obtained by derivatizing the mixed control solution and the supernatant obtained by derivatizing the SCP55 sample solution respectively, and identify the monosaccharide composition by HPLC. The mobile phase is 0.05 M potassium dihydrogen phosphate solution - acetonitrile (volume ratio 83:17). Adjust the pH of the 0.05 M potassium dihydrogen phosphate solution to 6.70 with sodium hydroxide solution. The sample injection volume is 250 μL, and the column flow rate is 1.0 mL / min.

[0088] 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 a small amount of mannose, ribose, glucuronic acid, galacturonic acid, galactose, xylose, arabinose, and fucose.

[0089] Table 1 Monosaccharide composition analysis of the polysaccharide from sea cucumber intestine

[0090]

[0091]

[0092] Note: The % in Table 1 is the molar percentage.

[0093] Example 5

[0094] Accurately weigh SCP55 to prepare a polysaccharide solution with a concentration of 5 mg / mL. After passing through a 0.22 μm microporous filter membrane, use a Shimadzu GPC-20A gel permeation chromatograph in Japan and a TSKgel GMPWXL aqueous gel chromatographic column from TOSOH (TSK Tosoh) Corporation in Japan. Use pullulan polysaccharide from Shodex Corporation in Japan as the standard product. The mobile phase is an aqueous solution of 0.1 mol / L NaNO3 + 0.06% NaN3, and the flow rate is 0.6 mL / min to measure its molecular weight.

[0095] The results of molecular weight analysis are shown in Table 2. The molecular weight of SCP55 is 618.12 kDa, and the polydispersity coefficient is 432.33. The lower polydispersity coefficient indicates a higher degree of homogeneity of SCP55, high solubility, and no formation of large aggregates.

[0096] Table 2 Molecular weight analysis of polysaccharide components in sea cucumber intestine

[0097]

[0098] Example 6

[0099] Accurately weigh SCP55 to prepare a polysaccharide solution with a concentration of 0.5 mg / mL, place it in a UV spectrophotometer for scanning detection, and the scanning wavelength range is 200 - 400 nm.

[0100] The results of UV spectral analysis are as Figure 2 shown. Nucleic acids and proteins have absorption peaks at 260 nm and 280 nm. The detection results show that no obvious absorption peaks are found in the range of 200 - 400 nm for SCP55. Therefore, it indicates that SCP55 contains neither nucleic acids nor proteins.

[0101] Example 7 Detection of the effect of SCP55 on the proliferation rate of HK-2 cells

[0102] When HK-2 cells grow to 80% - 90%, use 1 mL of 0.25% trypsin to digest and then add complete culture medium (MEM containing 10% serum and 1% penicillin-streptomycin mixture) to resuspend the cells. Inoculate the cells into a 96-well plate at a density of 1×10 4 cells / well. After 24 hours, discard the original culture medium in the wells and wash once with PBS. Set up a control group, groups of sea cucumber intestine polysaccharides with different concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL), and a blank group, with 6 replicates in each group.

[0103] Control group: Add 100 μL of MEM (containing NEAA) to the wells containing cells.

[0104] Experimental groups with different concentrations of sea cucumber intestinal polysaccharide (SCP55): Dissolve SCP55 in MEM (containing non-essential amino acids, NEAA) to prepare solutions with different concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL), and add 100 μL to each well containing cells.

[0105] Blank group: Add 100 μL of MEM (containing NEAA) to wells without cells.

[0106] After culturing the 96-well plates in a 37 °C carbon dioxide incubator for 24 h, discard the culture medium, wash each well once with PBS, then add 90 μL of incomplete medium and 10 μL of CCK-8 reagent to each well. The incomplete medium is MEM (containing NEAA), and continue to incubate at 37 °C for 1 h. Measure the absorbance at 450 nm using a microplate reader. The calculation formula is as follows:

[0107]

[0108] In the formula: A1 represents the absorbance value at 450 nm of the experimental group;

[0109] A2 represents the absorbance value at 450 nm of the blank group, with only medium without cells;

[0110] A3 represents the absorbance value at 450 nm of the control group, containing medium and cells.

[0111] The results of the effect of SCP55 on the proliferation activity of HK-2 cells are as Figure 3 shown. It can be seen from Figure 3 that SCP55 at different concentrations has no effect on the survival rate of HK-2 cells, indicating that the preparation of the present invention has no toxic effect on HK-2 cells.

[0112] Example 8 Effect of SCP55 on the uric acid content in the supernatant of HK-2 hyperuricemic cells

[0113] Set up a blank group (NC), a model group (MC), an SCP55 group, and a positive control group (allopurinol, AP). Each group has 1×10 6Inoculate HK-2 cells into 24-well plates at a density of cells / mL (total volume 1.0 mL / well). After incubation at 37 °C for 30 h, aspirate the complete culture medium and wash twice with PBS. Then, replace the medium in the blank group and the model group with serum-free incomplete medium MEM (containing NEAA), add allopurinol (AP, final concentration 15.0 μg / mL) to the positive control group, and add SCP55 solutions at different concentrations (1, 0.5, 0.25, 0.125 mg / mL) to the SCP55 groups. The total volume is 1.0 mL / well for all groups, and continue to culture for 24 h. Aspirate the culture medium and wash twice with PBS. After that, the HK-2 cells in the blank group are continuously cultured in incomplete medium MEM (containing NEAA) for 28 h; in the model group, positive control group, and SCP55 groups: the HK-2 cells are continuously cultured in incomplete culture medium induced with adenosine (2.5 mmol / L) for 24 h, and then add 1 mL of XO at 0.005 U / mL and continue to culture for 4 h.

[0114] Centrifuge the supernatant of each group and strictly operate according to the instructions in the uric acid kit to detect the uric acid content in the supernatant of the cell culture medium.

[0115] Figure 4 It is a graph showing the effect of sea cucumber intestine polysaccharide SCP55 on the uric acid content in the supernatant of the HK-2 hyperuricemic cell model. It can be seen that sea cucumber intestine uric acid-lowering polysaccharides at different concentrations (1, 0.5, 0.25, 0.125 mg / mL) have a reducing effect on the uric acid content in the supernatant of HK-2 cells. And as the concentration increases, the uric acid content becomes lower. The effect of 1 mg / mL sea cucumber intestine uric acid-lowering polysaccharide is close to that of allopurinol.

[0116] Allopurinol is a drug for treating hyperuricemia, which has strong side effects and causes great harm to the body. There are few bioactive substances or natural polysaccharides with a uric acid-lowering effect similar to that of allopurinol. The SCP55 component provided by the present invention belongs to bioactive substances and has advantages such as safety and no toxic effect on cells.

[0117] Although the present invention has been disclosed in the above preferred embodiments, it is not used to limit the present invention. Without violating the scope requirements of the present invention, those familiar with the technology can modify and improve it. Therefore, the protection scope of the present invention should be determined according to the content defined in the claims.

Claims

1. A preparation method of sea cucumber intestine polysaccharide, characterized in that, Comprising the following steps: (1) Mix sea cucumber intestine with water at a material-liquid ratio of 1 g:3 mL; enzymatically hydrolyze with papain at an addition amount of 2%. The enzymatic hydrolysis conditions include: enzymatic hydrolysis time of 3 h, enzymatic hydrolysis temperature of 54 °C, and pH = 7.3; inactivate the enzyme, and the enzyme inactivation conditions include boiling water bath for 20 min; After cooling, centrifuge and take the supernatant 1; (2) Precipitate the supernatant 1 with 3 times the volume of 25% ethanol solution, centrifuge to obtain precipitate 1 and supernatant 2; (3) After concentrating the supernatant 2, precipitate it with 3 times the volume of 55% ethanol solution, centrifuge to obtain precipitate 2 and supernatant 3; (4) Deproteinize: Dissolve precipitate 2 into a solution, add an equal volume of 10% trichloroacetic acid solution, stir, leave overnight at 4 °C, centrifuge, take the supernatant, dialyze the supernatant with a 3500 Da dialysis bag for 48 h, and freeze-dry to obtain sea cucumber intestine polysaccharide SCP55.

2. A sea cucumber intestine polysaccharide, characterized in that, The sea cucumber intestine polysaccharide is prepared by the method described in claim 1.

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