Euryale uniform polysaccharide, and preparation method and application thereof

Abstract

The application discloses a gordon euryale uniform polysaccharide and a preparation method and application thereof, and relates to the technical field of gordon euryale polysaccharide, and specifically discloses a gordon euryale uniform polysaccharide, a preparation method and application thereof.

Description

Technical Field

[0001] This invention relates to the field of gorgon fruit application technology, specifically to a homogeneous gorgon fruit polysaccharide, its preparation method, and its application. Background Technology

[0002] Euryale ferox, the dried, mature seed kernel of the Euryale ferox plant (family Nymphaeaceae), is used for its kidney-tonifying, sperm-strengthening, spleen-tonifying, diarrhea-stopping, dampness-removing, and leukorrhea-stopping effects. It is commonly used for spermatorrhea, enuresis, frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and vaginal discharge. The bioactive components of Euryale ferox include saponins, polysaccharides, ketones, and mycotoxins. Research on the chemical composition of Euryale ferox is of great significance to its modern pharmacological research. Studies have shown that the anti-inflammatory activity of Euryale ferox is mainly based on its small-molecule active components, while research on the large-molecule components of its polysaccharides is relatively limited. In recent years, research on Euryale ferox polysaccharides has mainly focused on drug extraction and cultivation, with less emphasis on structural characterization and activity verification.

[0003] Chinese patent CN108424469A discloses a polysaccharide from the kernel of Euryale ferox, its separation and extraction method, and its uses, wherein the molecular weight of the Euryale ferox kernel polysaccharide is 8.75 x 10. 3 Da, the monosaccharide composition and molar ratio are glucuronic acid:mannose:glucose:galactose:arabinose = 0.55:0.6:52.7:1:4.3. Euryale ferox seed polysaccharide can promote glucose uptake by insulin-resistant cells and has the application prospect of developing hypoglycemic health foods and drugs. However, considering that inflammation can aggravate the progression of various diseases, there is very little research on the anti-inflammatory effects of Euryale ferox polysaccharide, and the existing technology is somewhat insufficient. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies, such as limited research on the macromolecular components of Euryale ferox polysaccharides. This application provides a homogeneous Euryale ferox polysaccharide, its preparation method, and its application. Through extraction, separation, purification, chemical structural characterization by sulfation, and functional application of Euryale ferox polysaccharide, this invention utilizes modification methods to enhance the anti-inflammatory effect of Euryale ferox sulfate homogeneous polysaccharide, which is a nutritional polysaccharide from Euryale ferox.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] A method for preparing homogeneous polysaccharide from Euryale ferox includes the following steps:

[0007] a) Take dried Euryale ferox seeds, wash them, extract them, precipitate them, and dry them to obtain crude Euryale ferox polysaccharide;

[0008] b) Dissolve the crude Euryale ferox polysaccharide obtained in step a) in distilled water, load it onto an anionic cellulose chromatography column, and elute with distilled water to obtain the Euryale ferox water-washed polysaccharide fraction.

[0009] c) The polysaccharide of the water-washed component of Euryale ferox obtained in step b) was purified by gel chromatography and eluted with NaCl solution to obtain homogeneous Euryale ferox polysaccharide.

[0010] Furthermore, in step a), the dried fox nuts are first washed and then defatted with ethanol. The defatted fox nuts are then subjected to hot water extraction and overnight ethanol precipitation.

[0011] Further, step a) is as follows: First, the dried Euryale ferox is washed and defatted by ethanol reflux. The defatted Euryale ferox is dried by blowing air and extracted with hot water 2 to 5 times. The extracts are combined, concentrated, centrifuged, and the supernatant is collected for ethanol precipitation and dried to obtain crude Euryale ferox polysaccharide. The centrifugation is carried out at a high speed of 8000 rpm for 10 min.

[0012] Further, in step a), the defatting process uses ethanol with a mass fraction of 50-95%, the material ratio (g / mL) during defatting is 1:3-1:25, and the reflux defatting time is 1-18 hours; the material ratio (g / mL) of the dried gorgon fruit to water is 1:8-1:30, the heating extraction temperature is 60-100℃, and the extraction time for each extraction is 1-8 hours.

[0013] Further, in step a), ethanol is used for precipitation. 2 to 5 times the volume of 90 to 100% ethanol is added, stirred, and stirred until the final ethanol mass fraction is 75 to 85%. The mixture is then allowed to stand for 4 to 48 hours, and the precipitate is collected.

[0014] Further, step b) specifically involves: dissolving 10g of crude Euryale ferox polysaccharide obtained in step a) in 100mL of distilled water, loading the solution onto a DE-52 chloride anion exchange resin column, eluting with distilled water, detecting the sugar content using the sulfuric acid-phenol method at 490nm using an ELISA reader, obtaining the elution curve, collecting the sugar-containing solution, drying it, dialyzing it with a 6000-10000Da dialysis bag for 24 hours, and then freeze-drying it to obtain the water-washed Euryale ferox polysaccharide fraction.

[0015] Further, the crude Euryale ferox polysaccharide obtained in step a) is dissolved in water and then loaded onto an anion exchange cellulose chromatography column. It is eluted with distilled water, monitored in real time using the phenol-sulfuric acid method, and an elution curve is generated. The sugar solution is collected based on the elution curve, concentrated using a rotary evaporator, dialyzed with running water using a 6000-10000 Da dialysis bag, and freeze-dried to obtain the water-washed Euryale ferox polysaccharide fraction. The chloride-type anion exchange cellulose column is a DE-52 column. The collected water-eluted fraction is subjected to 10 kDa membrane ultrafiltration to obtain homogeneous Euryale ferox polysaccharide.

[0016] Further, step c) specifically involves dissolving the obtained water-washed polysaccharide fraction of Euryale ferox in a 0.05–0.25 mol / L NaCl solution, centrifuging at 6000 rpm for 5 min, taking the supernatant and filtering it through a 0.22 μm filter membrane before loading it onto a gel column, eluting it with a 0.05–0.25 mol / L NaCl solution, monitoring in real time using a differential detector, collecting the peaks based on their shape, concentrating the polysaccharide using a rotary evaporator, dialyzing it with running water using a 1000 Da dialysis bag, and then freeze-drying it for 48 h to obtain homogeneous Euryale ferox polysaccharide.

[0017] A homogeneous polysaccharide of Euryale ferox prepared by any of the above preparation methods, wherein the homogeneous polysaccharide has a purity of 99% or higher and a molecular weight of 3.887 kDa, wherein the homogeneous polysaccharide is composed of glucose and the glycosidic bonds are 1-Glcp, 3-Glcp, 4-Glcp, 3,4-Glcp and 4,6-Glcp.

[0018] This application also discloses the application of homogeneous gorgon fruit polysaccharide in the preparation of anti-inflammatory drugs or sulfated gorgon fruit polysaccharide. The application of homogeneous gorgon fruit polysaccharide in the preparation of sulfated gorgon fruit polysaccharide specifically includes the following steps:

[0019] Step 1: Add 1 mL of pyridine to a 100 mL round-bottom flask, bring to 0 °C in an ice bath, and add 1-2 mL of chlorosulfonic acid dropwise while stirring. Stir at room temperature for 20 min, and it will gradually melt into a pale yellow clear liquid, which is the sulfuric acid derivatization reagent.

[0020] Step 2: Weigh 100mg of Euryale ferox homogeneous polysaccharide and place it in a reaction flask. Add 3mL of formamide, heat until completely dissolved, place in an ice bath at 0℃, add to the sulfuric acid derivatization reagent from Step 1 above, stir to dissolve, react in an ice bath for 2 hours, and then react at 20℃ for 3 hours.

[0021] Step 3: Add 5 mol / L NaOH under ice bath conditions, adjust pH to 7-8, dialyze for 2 days, freeze dry for 2 days to obtain euryale sulfated polysaccharide.

[0022] Compared with the prior art, the beneficial effects of the present invention include:

[0023] 1. The homogeneous polysaccharide of Euryale ferox prepared by this invention is free of impurities such as proteins or nucleic acids, has high purity and uniformity, and can meet the requirements for polysaccharide structure identification and bioactivity evaluation.

[0024] 2. This application lays the foundation for exploring the bioactive mechanism of Euryale ferox polysaccharides and elucidating the structure-activity relationship. At the same time, the Euryale ferox polysaccharides obtained by the Euryale ferox polysaccharides preparation method described in this invention have a strong anti-inflammatory effect on RAW264.7 cells. The Euryale ferox polysaccharides used in this invention, after being modified by sulfation, also show a strong anti-inflammatory effect on RAW264.7 cells.

[0025] 3. The preparation method of this invention is simple and can quickly obtain high-purity homogeneous polysaccharides, which is suitable for large-scale production and provides some ideas for in-depth research on the anti-inflammatory effects of Euryale ferox polysaccharides. Attached Figure Description

[0026] Figure 1 The images show the polysaccharides extracted from Euryale ferox in this application, where (A) is a chromatogram eluted from a DE-52 column, (B) is a chromatogram of the average relative molecular mass of the polysaccharides determined by high performance gel permeation chromatography (HPGPC), and (C) is an image of the monosaccharide composition of a mixture of standards and Euryale ferox polysaccharides obtained by HPAEC.

[0027] Figure 2 GC spectrum of PMAA derivative of Euryale ferox polysaccharide in this application;

[0028] Figure 3 For the application of Euryale ferox polysaccharide 1 HNMR spectrum;

[0029] Figure 4 For the application of Euryale ferox polysaccharide 13 C NMR spectrum;

[0030] Figure 5 This is the HSQC spectrum of the Euryale ferox polysaccharide in this application;

[0031] Figure 6 This is the HMBC spectrum of the Euryale ferox polysaccharide in this application;

[0032] Figure 7 The HHCOSY spectrum of the Euryale ferox polysaccharide in this application;

[0033] Figure 8 The chemical structural formula of the Euryale ferox polysaccharide in this application is shown below.

[0034] Figure 9 This is the reaction structure of the sulfation of Euryale ferox polysaccharide in this application;

[0035] Figure 10 The images show the molecular weight and infrared spectrum of the sulfated Euryale ferox polysaccharide of this application, where (A) is the molecular weight spectrum and (B) is the infrared spectrum.

[0036] Figure 11 This is an electron microscope image of the sulfated euryale polysaccharide from this application;

[0037] Figure 12 This is the energy spectrum of the sulfated euryale polysaccharide from this application;

[0038] Figure 13This is a map showing the effects of the Euryale ferox polysaccharide of this application on cell proliferation, phagocytic activity, nitric oxide production, and inflammatory factor expression in Raw264.7 cells. (A) is a cell proliferation map, (B) is a phagocytic activity map, and (C) is a nitric oxide production map.

[0039] Figure 14 This is a map showing the effect of Euryale ferox polysaccharide on the expression of inflammatory factors in Raw264.7 cells, where (A) is the iNOS map, (B) is the IL-1β map, (C) is the IL-6 map, and (D) is the TNF-α map. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0041] Example 1

[0042] A method for preparing homogeneous polysaccharide from Euryale ferox includes the following steps:

[0043] a) Take 2 kg of dried Euryale ferox seeds, wash them, and defatt them with ethanol at a mass fraction of 50-95%. The material ratio (g / mL) during defatting is 1:3-1:25, and the defatting time is 1-18 hours. After defatting, the Euryale ferox seeds are dried by blowing. The material ratio (g / mL) of the dried Euryale ferox seeds to water is 1:8-1:30. Add 10 L of water and extract three times under boiling conditions. The extraction time is 1-8 hours each time. Combine the extracts, concentrate them, and centrifuge them at 8000 rpm for 10 min. After collecting the supernatant, add 4 times the volume of ethanol at a mass fraction of 90-100%. Stir until the final ethanol mass fraction is 75-85%. Let it stand for 24 hours, filter it, collect the precipitate, and dry it to remove residual ethanol to obtain crude Euryale ferox polysaccharide.

[0044] b) Take 10g of crude Euryale ferox polysaccharide obtained in step a) and dissolve it in 100mL of distilled water. Then, load the sample onto a DE-52 chloride anion exchange resin column and elute with distilled water. Detect the sugar content using the sulfuric acid-phenol method and detect it at 490nm using an ELISA reader to obtain the elution curve. Collect the sugar-containing solution, dry it, dialyze it with an 8000Da dialysis bag for 24 hours, and then freeze-dry it to obtain the water-washed Euryale ferox polysaccharide fraction.

[0045] c) Dissolve the polysaccharide from the water-washed Euryale ferox fraction obtained in step b) in 0.2 mol / L NaCl solution, centrifuge at 6000 rpm for 5 min, collect the supernatant and filter through a 0.22 μm filter membrane, load the sample and purify using a gel chromatography column, eluting with 0.2 mol / L NaCl solution, and detect using a differential detector. Based on the peak shape, see... Figure 1 The contents of the sample (A) were collected and concentrated, and then dialyzed using a 1000 Da dialysis bag to obtain homogeneous polysaccharide from Euryale ferox.

[0046] Purity and molecular weight determination:

[0047] High performance liquid chromatography-gel chromatography (HPGPC) was used: the column was BRT105-104-102, the detector was a differential detector, the mobile phase was 0.05 mol / L sodium chloride solution, the column temperature was 40℃, the flow rate was 0.6 mL / min, and the injection volume was 20 μL.

[0048] Accurately weigh the sample and standards. Prepare a 5 mg / mL solution of the sample, centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm microporous membrane, and then transfer the sample to a 1.8 mL vial with an injection volume of 20 μL. The mobile phase of the instrument is 0.05 M sodium chloride solution, and the flow rate is 0.6 mL / min. Use dextran with different relative molecular masses (Mw1152, 11600, 23800, 48600, 80900, 148000, 273000, 409800) as standards to construct a standard curve and determine the purity and relative molecular mass of the polysaccharides.

[0049] Based on the standard curve, a calculation formula is derived to calculate the molecular weight of each sample. For example... Figure 1 Image (B) shows the high-performance liquid chromatography-gel chromatography (HPGPC) chromatogram of Euryale ferox polysaccharide. The single symmetrical peak indicates that it is a homogeneous polysaccharide. The relative molecular weight of Euryale ferox polysaccharide was calculated to be 3.887 kDa using the formula.

[0050] Monosaccharide composition analysis:

[0051] Accurately weigh 3 mg of homogeneous polysaccharide from Euryale ferox seeds, then add 2 mL of trifluoroacetic acid. Hydrolyze in a metal bath at 120 °C for 2 h, evaporate by rotary evaporation until no sour taste remains, then add sodium borohydride as a reducing agent for reduction overnight. Neutralize excess sodium borohydride with acetic acid. Evaporate using a rotary evaporator until a viscous consistency is reached, then add 3-5 mL of methanol solution, repeating 3 times. Then add 1 mL of acetic anhydride, and react at 101 °C for 1 h. Terminate the reaction by adding water, and extract with dichloromethane. Wash 3 times with 10 mL of distilled water each time. Finally, collect the dichloromethane layer, dry it with anhydrous sodium sulfate, and analyze it using a Shimadzu GCMS-QP2010.

[0052] GC-MS conditions: RXI-5SIL MS column (30*0.25*0.25); temperature program: initial temperature 120℃, ramped at 3℃ / min to 250℃ / min; hold for 5 min; injector temperature 250℃, detector temperature 250℃ / min; carrier gas: helium, flow rate 1 mL / min. Standards were selected in the following order: fucose, galactose, rhamnose, arabinose, glucosamine, galactopyranose, glucose, acetylglucosamine, xylose, mannose, and fructose.

[0053] Detection results: GC-MS analysis of the homogeneous polysaccharide from Euryale ferox showed one peak, which, after comparison with standard monosaccharides, was confirmed to be glucose. Figure 1 As shown in (C).

[0054] Polysaccharide methylation analysis:

[0055] Weigh 10 mg of freeze-dried Euryale ferox polysaccharide, add 2 mL of dimethyl sulfoxide, then add NaOH powder. Wrap the test tube with aluminum foil and operate in the dark. Then add iodomethane reagent and react under ice bath conditions. Finally, add water to terminate the reaction, dialyze and freeze dry. Take 1-2 mg of fully methylated polysaccharide, add 2 mL of 2M trifluoroacetic acid and hydrolyze for 2 h. Evaporate to dryness until no sour taste remains, then add an appropriate amount of sodium borohydride for reduction overnight, evaporate to a viscous state, then add 2-5 mL of methanol, and repeat evaporation three times. Then add acetic anhydride, acetylate, and neutralize with water. Finally, analyze using gas chromatography-mass spectrometry (Shimadzu GCMS-QP2010).

[0056] GC-MS conditions: RXI-5SIL MS column 30*0.25*0.25; temperature program conditions: initial temperature 120℃, increase to 280℃ / min at 4℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1mL / min.

[0057] Methylation analysis revealed that the primary polysaccharide sequence in Euryale ferox polysaccharide consists of 5 linked glucose residues and a terminal glucose group. 4-D-Glcp is the main glucan structure of the polysaccharide, meaning that the primary polysaccharide sequence is a 1-4-linked glucan.

[0058] Carbon NMR spectroscopy analysis:

[0059] Accurately weigh 45 mg of Euryale ferox polysaccharide, dissolve it in deuterated water, and then freeze-dry it. Add 500 μL of deuterated water again, and then perform spectral acquisition using a nuclear magnetic resonance (NMR) instrument at 500 MHz.

[0060] The results are shown in the table below:

[0061]

[0062] Example 2

[0063] The application of a homogeneous polysaccharide from Euryale ferox in the preparation of sulfated polysaccharides from Euryale ferox includes the following steps:

[0064] Step 1: Add 1 mL of pyridine to a 100 mL round-bottom flask, bring to 0 °C in an ice bath, and add 1-2 mL of chlorosulfonic acid dropwise while stirring. Stir at room temperature for 20 min, and it will gradually melt into a pale yellow clear liquid, which is the sulfuric acid derivatization reagent.

[0065] Step 2: Weigh 100mg of polysaccharide and place it in a reaction flask. Add 3mL of formamide, heat until completely dissolved, place in an ice bath at 0℃, add to the sulfuric acid derivatization reagent from Step 1 above, stir to dissolve, react in an ice bath for 2 hours, and then react at 20℃ for 3 hours.

[0066] Step 3: Add 5 mol / L NaOH under ice bath conditions, adjust pH to 7-8, dialyze for 2 days, freeze dry for 2 days to obtain sulfated polysaccharide.

[0067] Characterization of sulfated Euryale ferox polysaccharides:

[0068] Accurately weigh 2 mg of sulfated Euryale ferox polysaccharide and 200 mg of KBr, press them together, and scan in transmission mode. A KBr pellet was used as a blank control. The scan was performed at 4000-400 cm⁻¹. -1 The spectrum was obtained within the specified range. The sample was scanned and recorded using an FT-IR 650.

[0069] SEM images of the surface morphology of EFSP-S were obtained using a GeminiSEM 300 Zeiss field emission scanning electron microscope at an accelerating voltage of 2 kV. The composition of C, O, and S in EFSP-S was then analyzed by EDS, utilizing the different characteristic energies of X-ray photons from different elements.

[0070] Example 3

[0071] The role of a homogeneous polysaccharide from Euryale ferox in an anti-inflammatory drug specifically includes the following steps:

[0072] Step (1): The anti-inflammatory activity of Euryale ferox polysaccharide was screened by applying different concentrations of Euryale ferox polysaccharide to Raw264.7 cells. Specifically, Raw264.7 cells were revived, cultured, and passaged in DMEM medium and seeded in 96-well plates. After 24 hours of cell culture and adherence, different concentrations of Euryale ferox polysaccharide extract were added, and the cells were incubated at 37°C for 24 hours. Subsequently, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation for 4 hours, the supernatant was discarded, and then 100 μL of dimethyl sulfoxide solution was added. Under light-protected conditions, the cells were shaken on a shaker for 15 minutes, and the absorbance was measured at 570 nm using a microplate reader to calculate cell viability. The results showed that Euryale ferox polysaccharide and its sulfated modified polysaccharide did not affect cell proliferation.

[0073] Step (2): RAW 264.7 macrophages were cultured using the same grouping strategy as described above. The supernatant was then transferred to another 96-well plate for NO testing. After removing the supernatant, 100 μL of 0.1% neutral red solution was added to each well. After further incubation for one hour, the supernatant was removed, and cell lysis buffer was added. The absorbance at 540 nm was calculated for each well using a microplate reader. The results indicate that Euryale ferox polysaccharides and their sulfated modified polysaccharides did not affect phagocytosis.

[0074] In step (3), the Griess reaction, NO 2- First, the cell culture supernatant was reacted with sulfanilamide in an acidic environment to form a diazonium salt. This intermediate reacted with NED to form a stable purple azo compound. The cell culture supernatant was mixed with 100 μL of Griess reagent. After incubation at room temperature for 10 minutes, the absorbance was measured at 492 nm. The results were calculated using NaNO2 as a standard curve.

[0075] Step (4): Seed Raw264.7 macrophages into 6-well plates overnight, 10 cells per well. 6 Cells were cultured for 8 hours, and then 50 μg / mL of Euryale ferox polysaccharide and its sulfated modified polysaccharide were added, respectively. The blank control group was given the same dose of complete culture medium. After 8 hours of culture, RNA was extracted from the cells using TRIzol reagent according to the manufacturer's instructions, and the mRNA expression of cytokines and TLRs was quantitatively analyzed using a one-step QRT-PCR kit. -ΔΔCt The relative expression of the target genes was calculated. The results showed that Euryale ferox polysaccharides and their sulfated modified polysaccharides exhibited good anti-inflammatory effects.

[0076] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox in the preparation of anti-inflammatory drugs, characterized in that, The preparation method of the homogeneous polysaccharide from Euryale ferox includes the following steps: a) Take dried Euryale ferox seeds, wash them, extract them, precipitate them, and dry them to obtain crude Euryale ferox polysaccharide; b) Dissolve the crude Euryale ferox polysaccharide obtained in step a) in distilled water, load it onto an anionic cellulose chromatography column, and elute with distilled water to obtain the Euryale ferox water-washed polysaccharide fraction. c) The polysaccharide of the water-washed fraction of Euryale ferox obtained in step b) was purified by gel chromatography column elution with NaCl solution to obtain homogeneous Euryale ferox polysaccharide; The preparation method of the sulfated polysaccharide of Euryale ferox includes the following steps: Step 1: Add 1 mL of pyridine to a 100 mL round-bottom flask, bring to 0°C in an ice bath, and add 1-2 mL of chlorosulfonic acid dropwise while stirring. Stir at room temperature for 20 min, and it will gradually melt into a pale yellow clear liquid, which is the sulfuric acid derivatization reagent. Step 2: Weigh 100 mg of the homogeneous polysaccharide of Euryale ferox and place it in a reaction flask. Add 3 mL of formamide, heat until completely dissolved, place in an ice bath to 0°C, add to the sulfuric acid derivatization reagent in Step 1 above and stir to dissolve. React in an ice bath for 2 hours and then at 20°C for 3 hours. Step 3: Add 5 mol / L NaOH under ice bath conditions, adjust pH to 7-8, dialyze for 2 days, freeze dry for 2 days to obtain euryale ferox sulfated polysaccharide.

2. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that: In step a), the dried fox nuts are first washed and then defatted with ethanol. The defatted fox nuts are then subjected to hot water extraction and overnight ethanol precipitation.

3. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 2 in the preparation of anti-inflammatory drugs, characterized in that, The specific steps of step a) are as follows: First, wash the dried Euryale ferox seeds and then defatt them by reflux with ethanol. After defatting, dry the Euryale ferox seeds by blowing air and extract them with hot water 2 to 5 times. Combine the extracts, concentrate them, centrifuge them, collect the supernatant, precipitate them with ethanol, and dry them to obtain crude Euryale ferox polysaccharide. The centrifugation is carried out at a high speed of 8000 rpm for 10 min.

4. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 3 in the preparation of anti-inflammatory drugs, characterized in that: In step a), the defatting process uses ethanol with a mass fraction of 50-95%, the material ratio during defatting is 1:3-1:25 g / mL, and the reflux defatting time is 1-18 hours; the material ratio of the dried foxnut to water is 1:8-1:30 g / mL, the extraction temperature is 60-100℃, and the extraction time is 1-8 hours each time.

5. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that: In step a), ethanol is used for precipitation. 2 to 5 times the volume of 90 to 100% ethanol is added, stirred, and stirred until the final ethanol mass fraction is 75 to 85%. The mixture is then allowed to stand for 4 to 48 hours, and the precipitate is collected.

6. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that, The specific steps of step b) are as follows: Take 10g of crude Euryale ferox polysaccharide obtained in step a), dissolve it in 100mL of distilled water, load it onto a DE-52 chloride anion exchange resin column, elute with distilled water, detect the sugar content using the sulfuric acid-phenol method and detect it at 490nm using an ELISA reader, obtain the elution curve, collect the sugar-containing solution, dry it, dialyze it with a 6000-10000Da dialysis bag for 24 hours, freeze-dry it, and obtain the water-washed Euryale ferox polysaccharide fraction.

7. The application of a homogeneous polysaccharide or sulfated polysaccharide of Euryale ferox according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that, Step c) is as follows: The obtained water-washed polysaccharide fraction of Euryale ferox is dissolved in 0.05-0.25 mol / L NaCl solution, centrifuged at 6000 rpm for 5 min, and the supernatant is filtered through a 0.22 μm filter membrane and loaded onto a gel chromatography column. Elution is performed with 0.05-0.25 mol / L NaCl solution, and real-time monitoring is carried out using a differential detector. The sample is collected according to the peak shape, concentrated using a rotary evaporator, dialyzed with running water using a 1000 Da dialysis bag, and then freeze-dried for 48 h to obtain homogeneous Euryale ferox polysaccharide.

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