Preparation method and application of sea cucumber intestine polysaccharide

Abstract

The invention relates to a preparation method and application of sea cucumber intestine polysaccharide. The preparation method of the sea cucumber intestine polysaccharide comprises the following steps: performing enzymolysis treatment on sea cucumber intestines by using papain, performing enzyme deactivation, performing centrifugation, and taking supernate; and carrying out graded alcohol precipitation on the supernate, centrifuging, taking precipitate, deproteinizing and freeze-drying. The preparation method is simple, green and environment-friendly. The sea cucumber intestine polysaccharide prepared by the method can effectively inhibit the activity of xanthine oxidase; the sea cucumber intestine polysaccharide has no toxic effect on HK-2 cells. The compound has a uric acid reducing effect on HK-2 hyperuricemia cells.

Description

Technical Field

[0001] The invention belongs to the technical field of sea cucumber by-product processing and utilization, and in particular relates to a method for preparing sea cucumber intestinal polysaccharide and application thereof. Background Art

[0002] Sea cucumber (Stichopus japonicus) belongs to the phylum Echinodermata, is a kind of marine invertebrate, widely distributed in benthic areas and deep seas around the world. It is recognized as a tonic and traditional medicine. In addition to being rich in ingredients such as amino acids, fatty acids and trace elements, it also contains various biologically active substances such as polysaccharides, proteins, saponins, etc. However, in the deep processing of sea cucumbers, sea cucumber viscera are often treated arbitrarily as processing byproducts, 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 processing of sea cucumber viscera, thereby causing serious waste of resources and environmental pollution. Among them, sea cucumber intestines are important byproducts produced during sea cucumber processing, with large output and renewable characteristics. However, the utilization rate of sea cucumber intestines in my country is extremely low at present, and it is often discarded arbitrarily as processing byproducts, which cannot be fully utilized, causing huge waste of resources. Nowadays, with the rapid development and increasing maturity of sea cucumber cultivation and production technology, the comprehensive utilization of sea cucumber byproducts has become a problem to be solved urgently.

[0003] With the rapid development of the times, people's material living standards are getting higher and higher, the richness of diet has also greatly improved, and the frequency of intake of high-purine foods has increased significantly, resulting in a particularly high incidence of hyperuricemia in my country. Long-term hyperuricemia will further cause diseases such as gout. The increasing number of patients with hyperuricemia has led scholars to continuously look for substances that lower uric acid. At present, the main treatments for hyperuricemia are allopurinol, colchicine, febuxostat and other drugs, which have significant effects but have certain toxic side effects. Low-toxicity, high-activity natural sources of uric acid-lowering active substances are becoming more and more popular. 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 those of the XO-specific inhibitory drug-allopurinol. The XO inhibition rate IC of natural polysaccharides 50 The value is between 0.83-10.53 mg / mL, and the low activity limits its wide application in the industry. Therefore, it is urgent to provide a method for preparing sea cucumber intestinal polysaccharides with uric acid-lowering activity that is convenient and efficient. 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 intestinal polysaccharide and its application. The method provided by the present invention can be used to prepare sea cucumber intestinal polysaccharide that inhibits xanthine oxidase and reduces uric acid content. The preparation method of the present invention is simple and environmentally friendly. The sea cucumber intestinal polysaccharide can effectively inhibit the activity of xanthine oxidase; the sea cucumber intestinal polysaccharide has no toxic effect on HK-2 cells. It has a uric acid-lowering effect on HK-2 hyperuricemia cells.

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

[0006] The invention provides a method for preparing sea cucumber intestine polysaccharide, comprising the following steps: enzymatically treating the sea cucumber intestine with papain, killing the enzyme, centrifuging and obtaining a supernatant; and graded alcohol precipitation of the supernatant, centrifuging and obtaining a precipitate.

[0007] Further, the following steps are included:

[0008] (1) mixing sea cucumber intestines with water, enzymolyzing with papain to inactivate the enzyme; cooling and centrifuging, and taking supernatant 1;

[0009] (2) precipitating the supernatant 1 with a 25% ethanol solution and centrifuging to obtain a precipitate 1 and a supernatant 2;

[0010] (3) After concentrating the supernatant 2, precipitating the supernatant with 55% ethanol solution, and centrifuging to obtain precipitate 2 and supernatant 3;

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

[0012] The beneficial effects of adopting the above technical solution include: during the research process, composite protease, neutral protease and papain were used to perform the above operations respectively, and the results showed that papain had the highest yield.

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

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

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

[0016] Furthermore, in step (1), the centrifugation condition is 8000 r / min for 10 min.

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

[0018] Furthermore, the method further comprises the step of freeze-drying the precipitate.

[0019] Furthermore, the method further comprises the step of deproteinizing the precipitate.

[0020] Furthermore, the deproteinization comprises 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 in a 3500Da dialysis bag for 48 hours.

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

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

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

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

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

[0026] The sea cucumber intestinal polysaccharide provided by the present invention can be used to prepare uric acid-lowering products; it can also be used to prepare xanthine oxidase-inhibiting products; the products are not limited to food, health products, medicines, 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 polysaccharides with uric acid lowering activity based on papain enzymolysis and then graded alcohol precipitation. The present invention verifies the xanthine oxidase inhibitory activity of sea cucumber intestine polysaccharides and the 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 ability to inhibit xanthine oxidase activity, 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: the edible value of sea cucumbers is high, and the low utilization rate of by-products is a problem that needs to be solved urgently in the current sea cucumber breeding industry. The sea cucumber intestinal uric acid-lowering polysaccharide prepared and separated by the present invention 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 sea cucumber deep processing industry chain. The preparation method provided by the present invention is simple, fast, reproducible, suitable for industrial production, safe, non-toxic and side effects to the human body, and in line with the health concept of modern people.

[0029] The invention adopts the method of graded alcohol precipitation to separate and purify polysaccharides according to molecular weight, which greatly reduces the operation time, requires only simple instruments and reagents, is simple to operate, and has high efficiency. The invention adopts trichloroacetic acid to effectively remove protein from sea cucumber intestinal polysaccharides, greatly improving the purity of sea cucumber intestinal polysaccharides.

[0030] The sea cucumber intestinal 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 high uric acid cell model. The three sea cucumber polysaccharides with uric acid-lowering activity isolated by the present invention can effectively inhibit the activity of xanthine oxidase, among which SCP55 has the best effect in inhibiting xanthine oxidase, IC 50 It is 0.248 mg / mL and has high application development value. It can be used as a functional ingredient in food, health care products and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the xanthine oxidase inhibition rate of the sea cucumber intestinal polysaccharide of the present invention; wherein the abscissa is the mass concentration of the three sea cucumber intestinal polysaccharides, and the ordinate is the xanthine oxidase inhibition rate.

[0032] Figure 2 UV spectral analysis of sea cucumber intestinal polysaccharide components.

[0033] Figure 3 The effect of the sea cucumber intestinal 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 intestinal polysaccharide, and the ordinate is the relative survival rate of the cells.

[0034] Figure 4 This is a graph showing the effect of the sea cucumber intestinal polysaccharide prepared by the present invention on the uric acid content in the supernatant of the HK-2 hyperuricemia cell model; wherein the abscissa is the mass concentration of the sea cucumber intestinal polysaccharide, and the ordinate is the uric acid content in the supernatant of the HK-2 hyperuricemia cell model. DETAILED DESCRIPTION

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

[0036] The present invention uses sea cucumber as raw material, freeze-drying, and adopts an enzymatic method to prepare sea cucumber intestinal polysaccharide, and then uses ethanol graded alcohol precipitation, centrifugation, deproteinization, and freeze-drying to obtain uric acid-lowering polysaccharide. In view of the low resource utilization rate of the existing sea cucumber byproduct-sea cucumber intestine, the present invention provides an efficient and convenient preparation method of sea cucumber intestinal polysaccharide with the ability to inhibit xanthine oxidase and reduce the uric acid content of HK-2 cell supernatant.

[0037] The present invention provides a method for preparing sea cucumber intestinal polysaccharide, comprising the following steps: using papain to hydrolyze to obtain sea cucumber intestinal hydrolysate; separating and purifying the obtained sea cucumber intestinal hydrolysate by graded alcohol precipitation with an ethanol solution, determining the xanthine oxidase inhibition rate of each separated component, freeze drying, and obtaining a high uric acid lowering activity sea cucumber intestinal polysaccharide freeze-dried powder; using high performance liquid chromatography technology to obtain the monosaccharide and molecular weight composition of the sea cucumber intestinal polysaccharide. HK-2 cell experiments are used to verify the sea cucumber intestinal polysaccharide, and sea cucumber intestinal polysaccharide with potential high inhibition of xanthine oxidase activity and reduction of uric acid content in HK-2 cell supernatant is obtained.

[0038] Furthermore, the preparation method may include the following steps: extracting sea cucumber intestinal polysaccharides: cleaning the sea cucumber intestine, freeze-drying and then crushing it, adding deionized water, adjusting the pH and adding papain for enzymolysis, inactivating it in a boiling water bath after the enzymolysis is completed, centrifuging, and taking the supernatant; subjecting the supernatant to graded alcohol precipitation with a solution prepared with 3 times the volume of anhydrous ethanol in different proportions, centrifuging, taking the precipitate, and freeze-drying; configuring the freeze-dried sea cucumber intestinal polysaccharide into a sea cucumber intestinal polysaccharide solution, adding an equal volume of trichloroacetic acid solution, stirring at room temperature, keeping it in a refrigerator at 4°C overnight, centrifuging, taking the supernatant, dialyzing and freeze-drying to obtain three types of sea cucumber polysaccharides; verifying the xanthine oxidase inhibitory activity of the three types of sea cucumber intestinal polysaccharides in vitro, and screening out the component with the best xanthine oxidase inhibitory effect; using HK-2 cells to determine the uric acid content in the supernatant of the screened active components, and further verifying whether the sea cucumber intestinal polysaccharide has uric acid-lowering activity.

[0039] Furthermore, the preparation method of the above-mentioned sea cucumber intestinal polysaccharide has the following specific operation process:

[0040] (1) Preparation of sea cucumber intestine powder: Wash the sea cucumber intestine to remove the mud and sand, freeze-dry and then crush to obtain sea cucumber intestine powder, which is stored for later use.

[0041] (2) Extraction of sea cucumber polysaccharides: Accurately weigh the sea cucumber intestine powder, mix the sea cucumber intestine powder with ultrapure water at a solid-liquid ratio of 1 g:3 mL, add papain (enzyme addition amount is 2%), adjust the pH to 7.3, stir the reaction at 54°C for 3 h, heat in a boiling water bath at 98°C for 20 min to inactivate the enzyme, cool to room temperature, centrifuge at 8000 r / min for 10 min, and take the supernatant 1.

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

[0043] (4) Deproteinization of crude sea cucumber intestinal polysaccharide: The three crude sea cucumber intestinal polysaccharides in step (3) were respectively prepared into 0.01 g / mL crude sea cucumber intestinal polysaccharide solutions, and an equal volume of 10% trichloroacetic acid solution was added respectively, stirred at room temperature for 30 minutes, kept at 4°C overnight, centrifuged at 8000 r / min for 10 minutes, and the supernatant was taken, dialyzed under a 3500Da dialysis bag for 48 hours, and freeze-dried to obtain three types of sea cucumber intestinal polysaccharides. The xanthine oxidase inhibition rates of the three components were determined respectively, and the most active component was screened out, and the components were freeze-dried to obtain sea cucumber intestinal polysaccharides with high uric acid lowering activity, namely SCP25, SCP55 and SCP75.

[0044] Afterwards, the monosaccharide composition of sea cucumber intestinal polysaccharide was identified: high performance liquid chromatography was used to identify the monosaccharide composition of sea cucumber intestinal polysaccharide. The mobile phase was potassium dihydrogen phosphate solution-acetonitrile (volume ratio of 83:17), the potassium dihydrogen phosphate solution was adjusted to pH 6.7 with sodium hydroxide solution, the potassium dihydrogen phosphate concentration in the potassium dihydrogen phosphate solution was 0.05M, the sample volume was 250μL, and the column flow rate was 1.0mL / min.

[0045] Verification of the activity of sea cucumber intestinal polysaccharides: The biological activity of the sea cucumber intestinal polysaccharides obtained by graded alcohol precipitation was verified by 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, which uses a hot water method and a graded precipitation method in combination to save separation and purification time, improve efficiency, and obtain components with high inhibition of xanthine oxidase activity. The present invention provides uric acid-lowering sea cucumber intestinal polysaccharides that inhibit xanthine oxidase and reduce the uric acid content in the supernatant of HK-2 cells, and the sea cucumber intestinal polysaccharides include SCP25, SCP55, and SCP75. The present invention extends the industrial chain of processing and utilization of sea cucumber by-products and realizes the high-value utilization of sea cucumber by-products. The three components of SCP25, SCP55, and SCP75 can all effectively inhibit xanthine oxidase, among which SCP55 has a better effect. The method provided by the present invention is an efficient and convenient method for preparing sea cucumber intestinal polysaccharides, which has good solubility and is non-toxic to HK-2 cells. Sea cucumber intestinal polysaccharides can be used in the fields of food, health products, and medicine.

[0047] The invention provides a method for preparing uric acid-reducing sea cucumber intestinal polysaccharide capable of inhibiting xanthine oxidase activity and reducing uric acid content in HK-2 cell supernatant.

[0048] The present invention provides three sea cucumber intestinal polysaccharides with uric acid lowering activity, wherein the sea cucumber intestinal polysaccharides are SCP25, SCP55 and SCP75. The above sea cucumber intestinal 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 embodiments.

[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 are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels or prepared by conventional methods by those skilled in the art. The solutions involved in the present invention are all prepared with water as the solvent unless otherwise specified.

[0051] Sea cucumber intestines were purchased from Linghai Dalian Seafood Breeding Co., Ltd.; composite protease (C8800), 1-phenyl-3-methyl-5-pyrazolone (PMP), methanol, xanthine and xanthine oxidase (XO) were purchased from Beijing Solebow Technology Co., Ltd.; HK-2 cells were purchased from Wuhan Pronosai Life Science 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 buffer solution (PBS) and other reagents were 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 kit was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd.

[0052] Example 1

[0053] Extracting crude polysaccharide from sea cucumber intestines comprises the following steps:

[0054] (1) The sea cucumber intestine powder was mixed with ultrapure water at a solid-liquid ratio of 1 g:3 mL, and papain was added. The amount of papain added was 2% of the mass of the sea cucumber intestine powder. The pH was adjusted to 7.3 and the mixture was immersed in a 54°C water bath for 3 h. The enzyme was inactivated in a boiling water bath for 20 min. After cooling to room temperature, the mixture was centrifuged at 8000 r / min for 10 min, and the supernatant 1 was taken.

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

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

[0057] (4) The supernatant 3 was concentrated to the same volume as the supernatant 2 using a rotary evaporator, and then the concentrated supernatant 3 was added with 3 times the volume of 75% ethanol solution (in the 75% ethanol solution, the volume concentration of anhydrous ethanol is 75%, prepared with water), and placed in a 4°C refrigerator overnight, centrifuged at 8000 r / min for 10 min, and the precipitate 3 was taken.

[0058] (5) Precipitate 1, Precipitate 2 and Precipitate 3 are freeze-dried to obtain three types of sea cucumber intestinal crude polysaccharides (i.e., crude polysaccharide 1, crude polysaccharide 2 and crude polysaccharide 3), respectively.

[0059] Comparative Example 1

[0060] On the basis of Example 1, in Comparative Example 1, composite protease was used to replace papain in Example 1 when extracting sea cucumber intestinal polysaccharide, and the remaining steps were the same as those in Example 1.

[0061] Comparative Example 2

[0062] On the basis of Example 1, in Comparative Example 2, neutral protease was used to replace papain in Example 1 when extracting sea cucumber intestinal polysaccharides, and the remaining steps were the same as those in Example 1.

[0063] Effect Example 1

[0064] The yield of sea cucumber intestinal polysaccharide prepared in Example 1, Comparative Example 1 and Comparative Example 2 was determined. The result showed that the yield of polysaccharide after enzymatic hydrolysis by papain was 13%, while the yield after enzymatic hydrolysis by composite protease was 8%, and the yield after enzymatic hydrolysis by neutral protease was 11%. It can be seen that the yield of sea cucumber intestinal polysaccharide extracted by papain is the highest, which is better than that of sea cucumber intestinal polysaccharide prepared by other enzymatic hydrolysis methods. Therefore, papain was selected for the experiment.

[0065] Example 2

[0066] The deproteinization of the crude sea cucumber intestinal polysaccharide comprises the following steps: the three crude sea cucumber intestinal polysaccharides obtained in Example 1 are respectively prepared into 0.01 g / mL crude sea cucumber intestinal polysaccharide solutions, and equal volumes of trichloroacetic acid solution (in the trichloroacetic acid solution, the mass concentration of trichloroacetic acid is 10%) are respectively added, stirred at room temperature for 30 minutes, transferred to a 4°C refrigerator overnight, centrifuged at 8000r / min for 10 minutes, and the supernatant is taken, and the supernatant is placed in a 3500Da dialysis bag for dialysis for 48 hours, the liquid outside the dialysis bag is pure water, and the liquid in the dialysis bag after dialysis is freeze-dried to obtain three sea cucumber intestinal polysaccharides with different molecular weights, namely SCP25, SCP55 and SCP75. SCP25 is a sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 1 using the above method, SCP55 is a sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 2 using the above method, and SCP75 is a sea cucumber intestinal polysaccharide obtained by deproteinizing crude polysaccharide 3 using the above method.

[0067] Example 3

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

[0069] Ultrapure water was used to prepare a 0.02U / mL XO solution. PBS buffer solution (pH 7.4) was used to prepare a 0.48mmol / L xanthine substrate solution. The sample concentrations were set to 2, 1, 0.5, 0.25, and 0.125mg / mL, respectively.

[0070] Sample group with or without XO: 50 μL of XO solution and 50 μL of PBS were added to the 96-well plate;

[0071] Sample group without XO: 100 μL of PBS was added to the 96-well plate;

[0072] XO plus sample group: add 50 μL of XO solution and 50 μL of sample to the 96-well plate;

[0073] For the sample group without XO, 50 μL of sample and 50 μL of PBS were added to the 96-well plate;

[0074] Each group was incubated at 25°C for 5 min, 150 μL of xanthine substrate solution was added, and incubated at 25°C for 30 min, and the absorbance was measured at a wavelength of 290 nm.

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

[0076]

[0077] Where: A represents the OD with XO but without sample;

[0078] B represents the OD without XO and sample;

[0079] C represents the OD of the sample with XO added;

[0080] D represents the OD of the sample without XO addition.

[0081] The results of xanthine oxidase inhibition rate test of sea cucumber intestinal polysaccharide are as follows Figure 1 As shown in the figure, it can be seen that SCP25, SCP55 and SCP75 all exhibit different xanthine oxidase inhibition effects. Compared with SCP25 and SCP75, SCP55 has a higher xanthine oxidase inhibition ability, IC 50 0.248mg / mL; followed by SCP75. Although SCP75 also has a significant effect, its IC 50 The inhibition rate of SCP25 at 2 mg / mL was less than 50%. Therefore, SCP55 was selected for subsequent experiments. 50 The value is 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] The monosaccharide composition of SCP55 (prepared by the method of Example 2) was determined by using Shimadzu LC-20AD with a chromatographic column of Xtimate C18 4.6*200mm 5um.

[0084] Prepare mixed control solution: accurately weigh appropriate amount of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose, and fucose reference substances, dissolve and dilute each reference substance in water to obtain a mixed control solution. The concentration of each control in the mixed control solution is 50ug / mL.

[0085] SCP55 hydrolysis: accurately weigh 10 mg of sample into a 10 mL ampoule, add 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) into the 10 mL ampoule, fill with nitrogen, seal the tube, acid hydrolyze at 120°C for 4 h, remove 1.0 mL, add methanol and nitrogen to evaporate TFA, add 1.0 mL of water to re-dissolve, and obtain the hydrolyzed solution of SCP55.

[0086] Derivatization: Accurately pipette 250uL of mixed control solution into a 5mL EP tube, add 250uL 0.6mol / L NaOH, 500uL 0.4mol / L PMP-methanol, and react at 70℃ for 1h. Cool in cold water for 10min; add 500uL 0.3mol / L hydrochloric acid (HCl) to neutralize, then add 1mL chloroform and vortex for 1min, centrifuge at 3000r / min for 10min, carefully take the supernatant, extract 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: The supernatant obtained by derivatization of the mixed control solution and the supernatant obtained by derivatization of the SCP55 sample solution were taken separately, and the monosaccharide composition was identified by HPLC. The mobile phase was 0.05M potassium dihydrogen phosphate solution-acetonitrile (volume ratio was 83:17), and the pH of 0.05M potassium dihydrogen phosphate solution was adjusted to 6.70 with sodium hydroxide solution. The sample volume was 250μL, and the column flow rate was 1.0mL / min.

[0088] The results of 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 Analysis of monosaccharide composition of sea cucumber intestinal polysaccharides

[0090]

[0091]

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

[0093] Example 5

[0094] Accurately weigh SCP55 to prepare a 5 mg / mL polysaccharide solution, pass it through a 0.22 μm microporous filter membrane, and use a Shimadzu GPC-20A gel permeation chromatograph, a TSKgelGMPWXL aqueous gel chromatography column from TOSOH (TSK) of Japan, pullulan from Shodex of Japan as the standard, a mobile phase of 0.1 mol / L NaNO3+0.06% NaN3 aqueous solution, and a flow rate of 0.6 mL / min to determine its molecular weight.

[0095] The molecular weight analysis results are shown in Table 2. The molecular weight of SCP55 is 618.12 kDa, and the polydispersity coefficient is 432.33. The low polydispersity coefficient indicates that SCP55 has a high degree of uniformity and high solubility and will not form large aggregates.

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

[0097]

[0098] Example 6

[0099] SCP55 was accurately weighed to prepare a 0.5 mg / mL polysaccharide solution, which was placed in an ultraviolet spectrophotometer for scanning and detection, with a scanning wavelength range of 200-400 nm.

[0100] The results of UV spectroscopy analysis are as follows Figure 2 As shown, nucleic acids and proteins have absorption peaks at 260nm and 280nm, and the test results show that SCP55 has no obvious absorption peak in the range of 200-400nm. Therefore, it is shown 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 grew to 80%-90%, they were digested with 1 mL of 0.25% trypsin and then added with complete culture medium (MEM containing 10% serum and 1% penicillin-streptomycin mixture) to resuspend the cells. 4 The cells were inoculated into a 96-well plate at a density of 10 cells / well. After 24 hours, the original culture medium in the wells was discarded and the wells were washed once with PBS. A control group, groups with different concentrations of sea cucumber intestinal polysaccharides (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.

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

[0104] Different concentrations of sea cucumber intestinal polysaccharide groups (experimental groups): 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, respectively.

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

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

[0107]

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

[0109] A2 represents the absorbance value at 450 nm of the blank group, which contains no cells and only culture medium;

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

[0111] The results of SCP55 on the proliferation activity of HK-2 cells are as follows Figure 3 As shown, from Figure 3 It can be seen that different concentrations of SCP55 have 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 Uric Acid Content in Supernatant of HK-2 Hyperuricemic Cells

[0113] A blank group (NC), a model group (MC), a SCP55 group, and a positive control group (allopurinol, AP) were set up. Each group was treated with 1×10 6HK-2 cells were inoculated in a 24-well plate at a density of 1.0 mL / well (total volume 1.0 mL / well). After incubation at 37°C for 30 hours, the complete culture medium was aspirated and washed twice with PBS. Then the blank group and model group were replaced with serum-free incomplete culture medium MEM (containing NEAA), the positive control group was added with allopurinol (AP, final concentration 15.0 μg / mL), and the SCP55 group was added with different concentrations (1, 0.5, 0.25, 0.125 mg / mL) of SCP55 solution, the total volume was 1.0 mL / well, and continued to be cultured for 24 hours. The culture medium was aspirated and washed twice with PBS. After that, the HK-2 cells in the blank group were cultured in incomplete culture medium MEM (containing NEAA) for 28 hours; the model group, positive control group and SCP55 group: HK-2 cells were cultured in incomplete culture medium induced by adenosine (2.5 mmol / L) for 24 hours, and then 1 mL of 0.005 U / mL XO was added and cultured for 4 hours.

[0114] The supernatant of each group was centrifuged, and the uric acid content in the cell culture supernatant was detected in strict accordance with the instructions of the uric acid kit.

[0115] Figure 4 This is a graph showing the effect of sea cucumber intestinal polysaccharide SCP55 on the uric acid content in the supernatant of the HK-2 high uric acid cell model. It can be seen that different concentrations of sea cucumber intestinal uric acid-lowering polysaccharides (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 decreases, and 1 mg / mL sea cucumber intestinal uric acid-lowering polysaccharide has a similar effect to 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 that have similar uric acid-lowering effects as allopurinol. The SCP55 component provided by the present invention is a bioactive substance and has the advantages of being safe and non-toxic to cells.

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

Claims

1. A method for preparing sea cucumber intestinal polysaccharide, characterized in that: The following steps are involved: The sea cucumber intestines are enzymatically treated with papain, the enzyme is inactivated, and the supernatant is collected after centrifugation; The supernatant was graded and precipitated with alcohol, centrifuged and the precipitate was taken.

2. The method for preparing sea cucumber intestinal polysaccharide according to claim 1, characterized in that: The following steps are involved: (1) mixing sea cucumber intestines with water, enzymolyzing with papain to inactivate the enzyme; cooling and centrifuging, and taking supernatant 1; (2) precipitating the supernatant 1 with a 25% ethanol solution and centrifuging to obtain a precipitate 1 and a supernatant 2; (3) After concentrating the supernatant 2, precipitating the supernatant with 55% ethanol solution, and centrifuging to obtain precipitate 2 and supernatant 3; (4) After concentrating the supernatant 3, precipitate it with 75% ethanol solution, centrifuge it, and take the precipitate 3.

3. The method for preparing sea cucumber intestinal polysaccharide according to claim 2, characterized in that: In step (1), the amount of papain added is 2%.

4. The method for preparing sea cucumber intestinal polysaccharide according to claim 2, characterized in that: In step (1), the enzymatic hydrolysis conditions include: enzymatic hydrolysis time 3 hours, enzymatic hydrolysis temperature 54° C., pH=7.3; and / or, the enzyme inactivation conditions include: boiling water bath for 20 minutes.

5. The method for preparing sea cucumber intestinal polysaccharide according to claim 2, characterized in that: In step (1), the liquid ratio of sea cucumber intestine to water is 1 g:3 mL.

6. The method for preparing sea cucumber intestinal polysaccharide according to any one of claims 1 to 5, characterized in that: The method also includes the step of freeze-drying the precipitate.

7. The method for preparing sea cucumber intestinal polysaccharide according to any one of claims 1 to 5, characterized in that: The method also includes a step of deproteinizing the precipitate.

8. The method for preparing sea cucumber intestinal polysaccharide according to claim 7, characterized in that: The deproteinization comprises 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 in a 3500Da dialysis bag for 48 hours.

9. A sea cucumber intestinal polysaccharide, characterized in that: The sea cucumber intestinal polysaccharide is prepared by the method according to any one of claims 1 to 8.

10. Use of the sea cucumber intestinal polysaccharide according to claim 9 in lowering uric acid and / or inhibiting xanthine oxidase.

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