Gorgon fruit homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: taking dried gordon euryale seeds, washing, extracting, precipitating and drying to obtain a gordon euryale seed polysaccharide crude product, dissolving the gordon euryale seed polysaccharide crude product with distilled water, loading a sample on an anionic cellulose chromatographic column, and eluting with distilled water to obtain gordon euryale seed washing component polysaccharide; purifying the gordon euryale seed washing component polysaccharide by using a gel chromatographic column, and eluting by using a Na Cl solution to obtain gordon euryale seed homogeneous polysaccharide; the gordon euryale seed homogeneous polysaccharide does not contain protein or nucleic acid impurities, is high and uniform in purity, can meet the requirements of polysaccharide structure identification and biological activity evaluation, and has a relatively strong anti-inflammatory effect on RAW264.7 cells.

Description

Technical Field

[0001] The invention relates to the technical field of euryale gorgonii application, and in particular to a euryale gorgonii uniform polysaccharide and a preparation method and application thereof. Background Art

[0002] Gorgon fruit is the dried mature seed kernel of Gorgon fruit of Nymphaeaceae family. It has the effects of nourishing kidney and consolidating essence, tonifying spleen and stopping diarrhea, removing dampness and stopping leukorrhea. It is often used for spermatorrhea, enuresis and frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and leukorrhea. The biologically active ingredients of Gorgon fruit are Gorgon saponins, Gorgon polysaccharides, Gorgon ketone, Gorgon mycin, etc. Studying the chemical composition of Gorgon fruit is of great significance for its modern pharmacological research. Studies have shown that the anti-inflammatory activity of Gorgon fruit is mainly based on its small molecule active ingredients, while there is little research on the macromolecular components of Gorgon polysaccharides. In recent years, the research on Gorgon fruit polysaccharides has mainly focused on drug extraction and planting, and there is little research on its structural characterization and activity verification.

[0003] Chinese patent CN108424469A discloses a polysaccharide from Euryale ferox seed kernels and its separation and extraction method and use, wherein the molecular weight of the polysaccharide from Euryale ferox seed kernels 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, and the polysaccharide from Euryale ferox seed kernel can promote the glucose uptake of insulin-resistant cells, and has application prospects in the research and development of blood sugar-lowering health foods and drugs. However, considering that inflammation will increase the progression of various diseases, and there are few studies on the anti-inflammatory effects of Euryale ferox polysaccharides, the existing technology seems to be slightly insufficient. Summary of the invention

[0004] The purpose of the present invention is to address the shortcomings of the prior art, such as the lack of research on the macromolecular components of Gorgon fruit polysaccharides. The present application provides a Gorgon fruit uniform polysaccharide and a preparation method and application thereof. By extracting, separating and purifying the Gorgon fruit polysaccharide, characterizing the chemical structure of sulfation and functional application, Gorgon fruit is a nutritional polysaccharide, and a modification method is used to enhance its anti-inflammatory effect to obtain Gorgon fruit sulfate uniform polysaccharide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] A method for preparing uniform polysaccharide from Euryale ferox comprises the following steps:

[0007] a) taking dried Euryale ferox, washing, extracting, precipitating and drying to obtain a crude Euryale ferox polysaccharide;

[0008] b) dissolving the crude Euryale polysaccharide obtained in step a) with distilled water, applying the solution to an anionic cellulose chromatography column, and eluting with distilled water to obtain Euryale polysaccharide water-washed fraction;

[0009] c) Purifying the polysaccharide of the water-washed fraction of Euryale ferox obtained in step b) by using a gel chromatography column and eluting with a NaCl solution to obtain a uniform polysaccharide of Euryale ferox.

[0010] Furthermore, in the step a), the dried Euryale ferox is firstly washed and then degreased with ethanol, and the degreased Euryale ferox is subjected to hot water extraction and ethanol precipitation overnight.

[0011] Furthermore, the specific steps of step a) are as follows: first, the dried Euryale ferox is washed and then refluxed with ethanol for degreasing, the defatted Euryale ferox is air-dried, extracted with hot water for 2 to 5 times, the extracted liquid is combined, concentrated, centrifuged, the supernatant is collected, ethanol precipitated, and dried to obtain a crude Euryale ferox polysaccharide; the centrifugation is performed at a high speed of 8000 rpm for 10 min.

[0012] Furthermore, in the step a), the mass fraction of ethanol used for degreasing is 50-95%, the material ratio (g / mL) during degreasing is 1:3-1:25, and the reflux degreasing time is 1-18 hours; the material ratio (g / mL) of the gorgon fruit after air drying to water is 1:8-1:30, the temperature of the heating extraction is 60-100°C, and the time of each extraction is 1-8 hours.

[0013] Furthermore, in the step a), ethanol precipitation is used for precipitation, and 2 to 5 volumes of 90 to 100% ethanol by mass fraction are added, and stirred until the final ethanol mass fraction is 75 to 85%, and then allowed to stand for 4 to 48 hours to collect the precipitate.

[0014] Furthermore, the specific steps of step b) are: taking 10g of the crude euryale polysaccharide obtained in step a), dissolving it in 100mL of distilled water, loading it on a chloride-type anion exchange resin DE-52 chromatography column, eluting it with distilled water, detecting the sugar content using the sulfuric acid-phenol method and detecting it at 490nm on an ELISA reader, obtaining an elution curve, collecting the sugar-containing solution, drying it, dialyzing it with a 6000-10000Da dialysis bag for 24 hours, and freeze-drying it to obtain the euryale water-washed component polysaccharide.

[0015] Furthermore, the crude euryale polysaccharide obtained in step a) is dissolved in water and then applied to an anion exchange cellulose chromatography column, eluted with distilled water, monitored in real time by a phenol-sulfuric acid method, an elution curve is prepared, the sugar solution is collected according to the elution curve, concentrated by a rotary evaporator, dialyzed with flowing water in a 6000-10000Da dialysis bag, and freeze-dried to obtain euryale water-washed component polysaccharide; the chloride-type anion exchange cellulose column is a DE-52 column; the collected water elution component is ultrafiltered with a 10kDa membrane to obtain euryale uniform polysaccharide.

[0016] Furthermore, the specific steps of step c) are: the obtained polysaccharide of the washed component of Euryale ferox is dissolved with a 0.05-0.25 mol / L NaCl solution, centrifuged at 6000 rpm for 5 min, the supernatant is passed through a 0.22 μm filter membrane and then loaded, passed through a gel column, eluted with a 0.05-0.25 mol / L NaCl solution, monitored in real time by a differential detector, collected according to the peak shape, concentrated by a rotary evaporator, dialyzed with a 1000 Da dialysis bag, and freeze-dried in a freeze dryer for 48 hours after concentration to obtain a uniform polysaccharide of Euryale ferox.

[0017] A uniform polysaccharide of Euryale ferox obtained by any of the above preparation methods, wherein the purity of the uniform polysaccharide of Euryale ferox is above 99%, the molecular weight of the uniform polysaccharide is 3.887KDa, the uniform polysaccharide of Euryale ferox is composed of glucose, and the glycosidic bonds are 1-Glcp, 3-Glcp, 4-Glcp, 3,4-Glcp and 4,6-Glcp.

[0018] The present application also discloses the use of Euryale gorse homogeneous polysaccharide in the preparation of anti-inflammatory drugs or Euryale gorse sulfated polysaccharide. The use of Euryale gorse homogeneous polysaccharide in the preparation of Euryale gorse sulfated polysaccharide specifically comprises the following steps:

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

[0020] Step 2: Weigh 100 mg of Euryale ferox homogeneous polysaccharide into a reaction bottle, add 3 mL of formamide, heat until completely dissolved, ice bath to 0°C, add to the sulfuric acid derivatization reagent in the first step, stir to dissolve, react in an ice bath for 2 hours, and react at 20°C for 3 hours;

[0021] Step 3: Add 5 mol / L NaOH under ice bath conditions, adjust pH to 7-8, dialyze for 2 days, and freeze-dry for 2 days to obtain Euryale ferox 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 the present invention does not contain impurities such as protein or nucleic acid, has high purity and uniformity, and can meet the requirements of polysaccharide structure identification and biological activity evaluation;

[0024] 2. This application lays the foundation for exploring the biological activity mechanism of Gorgon fruit polysaccharides and explaining the structure-activity relationship. At the same time, the Gorgon fruit polysaccharide obtained by the Gorgon fruit polysaccharide preparation method of the present invention has a strong anti-inflammatory effect on RAW264.7 cells, and the Gorgon fruit polysaccharide used in the present invention after sulfation modification also shows a strong anti-inflammatory effect on RAW264.7 cells;

[0025] 3. The preparation method of the present invention is simple and can quickly obtain high-purity uniform polysaccharides, which is suitable for large-scale production and provides certain ideas for in-depth research on the anti-inflammatory effects of Euryale polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The polysaccharides extracted from Euryale ferox in this application are shown in Figure 1, wherein (A) is a chromatogram eluted from a DE-52 column, (B) is a graph of the average relative molecular mass of polysaccharides determined by high performance gel permeation chromatography (HPGPC), and (C) is an image of the monosaccharide composition of a mixed standard and Euryale ferox polysaccharides obtained by HPAEC;

[0027] Figure 2 This is the GC spectrum of the PMAA derivative of Gorgon fruit polysaccharide of this application;

[0028] Figure 3 For this application, Euryale polysaccharide 1 HNMR spectrum;

[0029] Figure 4 For this application, Euryale polysaccharide 13 C NMR spectrum;

[0030] Figure 5 This is the HSQC spectrum of Gorgon polysaccharide of this application;

[0031] Figure 6 This is the HMBC spectrum of Gorgon polysaccharide of this application;

[0032] Figure 7 This is the HHCOSY spectrum of Gorgon fruit polysaccharide of this application;

[0033] Figure 8 is the chemical structural formula of Gorgon fruit polysaccharide of this application;

[0034] Fig. 9 is the reaction formula of sulfation of Euryale polysaccharide in this application;

[0035] Fig.10 The molecular weight and infrared spectrum of sulfated Euryale ferox polysaccharide of the present application, wherein (A) is the molecular weight diagram and (B) is the infrared spectrum diagram;

[0036] Fig.11 This is the electron micrograph of the sulfated Euryale polysaccharide of this application;

[0037] Fig.12 This is the energy spectrum of sulfation of Euryale polysaccharide in this application;

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

[0039] Fig.14 The graphs of the effect of Euryale polysaccharide on the expression of inflammatory factors in Raw264.7 cells of the present application, wherein (A) is the iNOS graph, (B) is the IL-1β graph, (C) is the IL-6 graph, and (D) is the TNF-α graph; DETAILED DESCRIPTION

[0040] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0041] Example 1

[0042] A method for preparing uniform polysaccharide from Euryale ferox comprises the following steps:

[0043] a) Take 2 kg of dried Euryale ferox, wash, and defatting with ethanol having a mass fraction of 50 to 95%, the material ratio (g / mL) during defatting is 1:3 to 1:25, and the reflux degreasing time is 1 to 18 hours; the defatted Euryale ferox is air-dried; the material ratio (g / mL) of Euryale ferox to water after air drying is 1:8 to 1:30, 10 L of water is added, and under boiling conditions, extraction is performed 3 times, each extraction time is 1 to 8 hours, the extraction liquid is combined, concentrated, and high-speed centrifuged at 8000 rpm for 10 minutes. After collecting the supernatant, 4 times the volume of ethanol having a mass fraction of 90 to 100% is added, and after stirring, until the final mass fraction of ethanol is 75 to 85%, it is allowed to stand for 24 hours, filtered, and the precipitate is collected and dried to remove the residual ethanol to obtain a crude Euryale ferox polysaccharide;

[0044] b) dissolving 10 g of the crude polysaccharide of Euryale ferox obtained in step a) in 100 mL of distilled water, loading the mixture onto a chloride-type anion exchange resin DE-52 chromatography column, eluting the mixture with distilled water, detecting the sugar content using a sulfuric acid-phenol method and detecting the mixture at 490 nm using an ELISA reader to obtain an elution curve, collecting the sugar-containing solution, drying the solution, dialyzing the solution with an 8000 Da dialysis bag for 24 hours, and freeze-drying the solution to obtain a polysaccharide in a water-washed fraction of Euryale ferox;

[0045] c) dissolving the polysaccharide of the washed Euryale ferox obtained in step b) in a 0.2 mol / L NaCl solution, centrifuging at 6000 rpm for 5 min, taking the supernatant and filtering it through a 0.22 μm filter membrane, applying the sample to a gel chromatography column for purification, eluting with a 0.2 mol / L NaCl solution, and detecting with a differential detector. According to the peak shape, Figure 1 The extract (A) was collected and concentrated, and dialyzed using a 1000Da dialysis bag to obtain uniform polysaccharide from Euryale ferox.

[0046] Purity and molecular weight determination:

[0047] High performance liquid gel chromatography (HPGPC) was used: the chromatographic 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°C, the flow rate was 0.6 mL / min, and the injection volume was 20 uL.

[0048] Weigh the sample and standard accurately, prepare the sample into 5mg / ml solution, centrifuge at 12000rpm for 10min, filter the supernatant with 0.22μm microporous filter membrane, and then transfer the sample to 1.8ml injection vial with injection volume of 20μl. The mobile phase of the instrument is 0.05M sodium chloride solution with a flow rate of 0.6mL / min. Use dextran of different relative molecular weights (Mw1152, 11600, 23800, 48600, 80900, 148000, 273000, 409800) as standard, make a standard curve, and determine the purity and relative molecular weight of polysaccharide.

[0049] According to the standard curve, the calculation formula is obtained to calculate the molecular weight of each sample. Figure 1 (B) The high performance liquid gel chromatography (HPGPC) of Euryale polysaccharide was obtained, which showed a single symmetrical peak, indicating that it was a uniform polysaccharide. The relative molecular weight of Euryale polysaccharide was calculated to be 3.887 kDa by the formula.

[0050] Monosaccharide composition analysis:

[0051] Weigh 3 mg of Euryale ferox homogeneous polysaccharide accurately, then add 2 mL of trifluoroacetic acid, hydrolyze in a metal bath at 120 ° C for 2 hours, evaporate until there is no sour taste, then add reducing agent sodium borohydride for reduction overnight, and add acetic acid to neutralize the excess sodium borohydride. Use a rotary evaporator to evaporate until it becomes viscous, add 3-5 mL of methanol solution, and repeat 3 times. Then add 1 mL of acetic anhydride and react at 101 ° C for 1 hour. Then add water to terminate the reaction and extract with dichloromethane. Wash with water 3 times, each time with 10 mL of distilled water. Finally, absorb the dichloromethane layer, dry it with anhydrous sodium sulfate, and analyze it using Shimadzu GCMS-QP2010.

[0052] GC-MS conditions: RXI-5SIL MS column 30*0.25*0.25; program temperature conditions: starting temperature 120℃, heating at 3℃ / min to 250℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1mL / min. The order of the standards is: fucose, galactose, rhamnose, arabinose, glucosamine, galactopyranose, glucose, acetylglucosamine, xylose, mannose, fructose.

[0053] Test results: The GC-MS analysis of the homogeneous polysaccharide of Euryale ferox showed a peak, which was proved to be glucose after comparison with the standard monosaccharide. Figure 1 As shown in (C).

[0054] Polysaccharide methylation analysis:

[0055] Weigh 10 mg of freeze-dried Euryale polysaccharide, add 2 mL of dimethyl sulfoxide, then add NaOH powder, wrap the test tube with tin foil, and operate in dark. Then add iodomethane reagent and react under ice bath conditions. Finally, add water to terminate the reaction and dialyze and freeze-dry. Take 1-2 mg of fully methylated polysaccharide and add 2 mL of 2M trifluoroacetic acid to hydrolyze for 2 hours. Rotary evaporate until there is no sour taste, then add an appropriate amount of sodium borohydride to reduce overnight, rotary evaporate until viscous, then add 2-5 mL of methanol, and evaporate repeatedly 3 times. Then add acetic anhydride, acetylate, and neutralize in water. Finally, analyze using gas chromatography-mass spectrometry (Shimadzu GCMS-QP2010).

[0056] GC-MS conditions: RXI-5SIL MS column 30*0.25*0.25; programmed temperature conditions: starting temperature 120°C, heating to 280°C / min at 4°C / min; maintaining for 5 min; injection port temperature 250°C, detector temperature 250°C / min, carrier gas helium, flow rate 1 mL / min.

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

[0058] NMR carbon spectrum analysis:

[0059] 45 mg of Euryale polysaccharide was accurately weighed, dissolved in deuterated water, and then freeze-dried. 500 ul of deuterated water was added again, and then a 500 MHz nuclear magnetic resonance (NMR) instrument was used to collect spectra.

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

[0061]

[0062] Example 2

[0063] Application of Euryale gorgoniensis homogeneous polysaccharide in the preparation of Euryale gorgoniensis sulfated polysaccharide specifically comprises the following steps:

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

[0065] Step 2: Weigh 100 mg of polysaccharide into a reaction bottle, add 3 mL of formamide, heat until completely dissolved, ice bath to 0°C, add to the sulfuric acid derivatization reagent in the first step, stir to dissolve, react in an ice bath for 2 h, and react at 20°C for 3 h;

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

[0067] Characterization of Sulfated Euryale Polysaccharide:

[0068] Sulfated Euryale polysaccharide (2 mg) and KBr (200 mg) were accurately weighed and pressed together and scanned in transmission mode. The KBr pressed pellet was used as a blank control. -1 The spectra were obtained in the range of 1.40 nm and 1.50 nm. The samples were scanned and recorded by FT-IR 650.

[0069] GeminiSEM 300 Zeiss field emission scanning electron microscope was used to obtain SEM images of the surface morphology of EFSP-S at an accelerating voltage of 2kV. EDS was used to analyze the composition of C, O, and S in EFSP-S using the different characteristic energies of X-ray photons of different elements.

[0070] Example 3

[0071] The effect of a uniform polysaccharide of Euryale ferox in an anti-inflammatory drug comprises the following steps:

[0072] Step (1), the anti-inflammatory activity of Gorgon fruit polysaccharide was screened by acting on Raw264.7 cells with different concentrations of Gorgon fruit uniform polysaccharide. Specifically as follows: Raw264.7 cells were revived, cultured, and passaged, cultured with DMEM medium, and the cells were planted in a 96-well plate. After the cells were cultured for 24 hours and adhered to the wall, different concentrations of Gorgon fruit polysaccharide extract were added and placed in a 37-degree incubator for 24 hours. Subsequently, 20ul of MTT solution (5mg / ml) was added to each well. Incubate for 4 hours, discard the supernatant, and then add 100ul of dimethyl sulfoxide solution. Shake on a shaker for 15 minutes under light-proof conditions, measure the absorbance at 570nm using an enzyme reader, and calculate the cell survival rate. The results show that Gorgon fruit polysaccharide and its sulfated modified polysaccharide have no effect on cell proliferation;

[0073] Step (2), the culture of RAW 264.7 macrophages adopts the same grouping strategy as above. The supernatant is then placed in another 96-well plate for NO test. After removing the supernatant, 100 μL of a 0.1% neutral red solution is added to each well. After further incubation for one hour, the supernatant is removed and then cell lysis solution is added. Using a microplate reader, the absorbance of each well at 540 nm is calculated. The results show that Euryale polysaccharide and its sulfated modified polysaccharide have no effect on cell phagocytosis;

[0074] Step (3), in the Griess reaction, NO 2- First, react with the diazonium agent sulfonamide in an acidic environment to form a diazonium salt. This intermediate reacts with NED to form a purple stable azo compound. Mix the cell culture supernatant with 100 μL of Griess reagent. After incubation at room temperature for 10 minutes, measure the absorbance at 492 nm. Calculate the results using NaNO2 as the standard curve;

[0075] Step (4), Raw264.7 macrophages were seeded into 6-well plates overnight, with 10 cells per well. 6 cells, and then 50 μg / mL of Euryale polysaccharide and its sulfated modified polysaccharide were added respectively. The blank control group was given the same dose of complete culture medium. After 8 h of culture, TRIzol reagent was used to extract cell RNA 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 Methods The relative expression of the tested genes was calculated. The results showed that Euryale polysaccharide and its sulfated modified polysaccharide exhibited good anti-inflammatory effects.

[0076] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a uniform polysaccharide from Euryale ferox, characterized in that: The steps include: a) taking dried Euryale ferox, washing, extracting, precipitating and drying to obtain a crude Euryale ferox polysaccharide; b) dissolving the crude Euryale polysaccharide obtained in step a) with distilled water, applying the solution to an anionic cellulose chromatography column, and eluting with distilled water to obtain Euryale polysaccharide water-washed fraction; c) Purifying the polysaccharide of the water-washed fraction of Euryale ferox obtained in step b) by using a gel chromatography column and eluting with a NaCl solution to obtain a uniform polysaccharide of Euryale ferox.

2. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 1, characterized in that: In the step a), the dried Euryale ferox is firstly washed and then subjected to ethanol degreasing operation, and the defatted Euryale ferox is subjected to hot water extraction and ethanol precipitation operation overnight.

3. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 2, characterized in that: The specific steps of step a) are as follows: first, the dried Euryale ferox is washed and then refluxed with ethanol for degreasing, the defatted Euryale ferox is air-dried, extracted with hot water for 2 to 5 times, the extracted liquid is combined, concentrated, centrifuged, the supernatant is collected, ethanol precipitated, and dried to obtain a crude Euryale ferox polysaccharide; the centrifugation is performed at a high speed of 8000 rpm for 10 minutes.

4. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 3, characterized in that: In the step a), the mass fraction of ethanol used for degreasing is 50-95%, the material ratio (g / mL) during degreasing is 1:3-1:25, and the reflux degreasing time is 1-18 hours; the material ratio (g / mL) of the euryale ferox after blast drying and water is 1:8-1:30, the temperature of heating extraction is 60-100°C, and the time of each extraction is 1-8 hours.

5. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 1, characterized in that: In the step a), ethanol precipitation is used for precipitation, and 2 to 5 volumes of 90 to 100% ethanol by mass fraction are added, and stirred until the final ethanol mass fraction is 75 to 85%, and then allowed to stand for 4 to 48 hours to collect the precipitate.

6. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 1, characterized in that: The specific steps of step b) are: taking 10 g of the crude polysaccharide of Euryale ferox obtained in step a), dissolving it in 100 mL of distilled water, loading it on a chloride-type anion exchange resin DE-52 chromatography column, eluting it with distilled water, detecting the sugar content by sulfuric acid-phenol method and detecting it at 490 nm with an ELISA reader, obtaining an elution curve, collecting the sugar-containing solution, drying it, dialyzing it with a 6000-10000Da dialysis bag for 24 hours, and freeze-drying it to obtain the polysaccharide of the water-washed component of Euryale ferox.

7. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 1, characterized in that: The crude polysaccharide of Euryale ferox obtained in step a) is dissolved in water and then applied to an anion exchange cellulose chromatography column, eluted with distilled water, monitored in real time by phenol-sulfuric acid method, an elution curve is prepared, and the sugar solution is collected according to the elution curve, concentrated by a rotary evaporator, dialyzed by running water in a 6000-10000Da dialysis bag, and freeze-dried to obtain polysaccharide of Euryale ferox water-washed component; The chloride-type anion exchange cellulose column is a DE-52 column; the collected water elution fractions are subjected to 10 kDa membrane ultrafiltration to obtain uniform polysaccharides from Euryale ferox.

8. The method for preparing a uniform polysaccharide from Euryale ferox according to claim 1, characterized in that: The specific steps of step c) are: dissolving the obtained polysaccharide of the washed Euryale ferox with 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, loading it with a sample, passing it through a gel column, eluting it with a 0.05-0.25 mol / L NaCl solution, monitoring it in real time with a differential detector, collecting it according to the peak shape, concentrating it with a rotary evaporator, dialyzing it with flowing water with a 1000 Da dialysis bag, and freeze-drying it in a freeze dryer for 48 hours after concentration to obtain a uniform polysaccharide of Euryale ferox.

9. A homogeneous polysaccharide of Euryale ferox obtained by the preparation method of any one of claims 1 to 8, characterized in that: The purity of the euryale gorgon fruit uniform polysaccharide is above 99%, the molecular weight of the uniform polysaccharide is 3.887KDa, the euryale gorgon fruit uniform polysaccharide is composed of glucose, and the glycosidic bonds are 1-Glcp, 3-Glcp, 4-Glcp, 3,4-Glcp and 4,6-Glcp.

10. The use of the Euryale gorgonii homogeneous polysaccharide according to claim 9 in the preparation of anti-inflammatory drugs or Euryale gorgonii sulfated polysaccharides, characterized in that: The application of the Euryale gorgoniensis homogeneous polysaccharide in the preparation of Euryale gorgoniensis sulfated polysaccharide specifically comprises the following steps: Step 1: Add 1 mL of pyridine to a 100 mL round-bottom flask, cool to 0°C in an ice bath, add 1-2 mL of chlorosulfonic acid dropwise while stirring, stir at room temperature for 20 min, and gradually melt to become a light yellow clear liquid, which is the sulfuric acid derivatization reagent; Step 2: Weigh 100 mg of Euryale ferox homogeneous polysaccharide into a reaction bottle, add 3 mL of formamide, heat until completely dissolved, ice bath to 0°C, add to the sulfuric acid derivatization reagent in the first step, stir to dissolve, react in an ice bath for 2 hours, and react 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, and freeze-dry for 2 days to obtain Euryale ferox sulfated polysaccharide.

Images