A multifunctional polysaccharide and a preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology, and provides a multifunctional polysaccharide, its preparation method, and its applications. The preparation method includes the following steps: drying, pulverizing, and defatting Ceylon sea fungus to obtain defatted powder; mixing the defatted powder with water and extracting at room temperature to obtain a cold water extraction residue; mixing the cold water extraction residue with water and heating to obtain a hot water extraction residue; mixing the hot water extraction residue with an alkaline solution to obtain an extract; adjusting the pH of the extract, concentrating, precipitating with alcohol, dialyzing, and lyophilizing to obtain crude polysaccharide; separating the crude polysaccharide using a DEAE Sepharose Fast Flow anion exchange chromatography column to obtain a first component; separating the first component using a Sephacryl S-300 gel column to obtain the multifunctional polysaccharide. The polysaccharide of this invention exhibits antioxidant, anticoagulant, and anti-inflammatory activities.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a multifunctional polysaccharide, its preparation method, and its applications. Background Technology

[0002] Seaweed (Auricularia auricula-judae) belongs to the phylum Rhodophyta, order Cyperales, family Auriculariaceae, and genus Auricularia. It contains over 20 unique nutrients found in marine plants, including abundant seaweed polysaccharides and unsaturated fatty acids. It possesses various pharmacological activities such as anti-inflammatory, lipid-lowering, blood pressure-lowering, blood vessel softening, and anti-cancer effects. In China, this genus contains two species: *Sarcodiaceylonensis* and *Sarcodiaceylanica*. *Sarcodiaceylonensis*, mainly distributed along the southeastern coast of my country, contains over 20 unique nutrients found in marine plants, including abundant seaweed polysaccharides and unsaturated fatty acids, and exhibits multiple effects such as lowering blood lipids, lowering blood pressure, enhancing immunity, and anti-tumor activity.

[0003] Polysaccharides are composed of repetitive structural features linked by glycosidic bonds, exhibiting high structural variability and the ability to preserve ample biological information. Most polysaccharides are relatively non-toxic and do not cause significant side effects.

[0004] Currently, there is limited research on polysaccharides from Ceylon sea fungus. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional polysaccharide, its preparation method, and its applications, so as to help expand the uses of Ceylon sea fungus.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a multifunctional polysaccharide, comprising the following steps: (1) drying, pulverizing and defatting Ceylon sea fungus to obtain defatted powder; (2) mixing the defatted powder with water, extracting at room temperature, separating solid and liquid to obtain cold water extraction residue; (3) mixing the cold water extraction residue with water, heating and extracting, separating solid and liquid to obtain hot water extraction residue; (4) mixing the hot water extraction residue with alkaline solution, heating and extracting, separating solid and liquid to obtain extract; (5) adjusting the pH of the extract, concentrating, precipitating with alcohol, dialyzing and lyophilizing to obtain crude polysaccharide; (6) separating the crude polysaccharide using a DEAE Sepharose FastFlow anion exchange chromatography column to obtain a first component; (7) separating the first component using a Sephacryl S-300 gel column to obtain the multifunctional polysaccharide.

[0007] Preferably, the liquid-to-solid ratio in steps (2)-(4) is independently selected from 40:1 to 80:1 (e.g., 40:1, 50:1, 60:1, 70:1 or 80:1; wherein the amount of liquid is in mL and the amount of solid is in g); in step (2), the extraction time is 2-4 h (e.g., 2 h, 2.5 h, 3 h, 3.5 h or 4 h); in steps (3) and / or (4), the heating temperature is 80-100 °C (e.g., 80 °C, 85 °C, 90 °C, 95 °C or 100 °C), and the extraction time is 4-7 h (e.g., 4 h, 5 h, 6 h or 7 h); in step (4), the alkaline solution is a 3%-6% NaOH solution (e.g., the mass concentration of the NaOH solution is 3%, 4%, 5% or 6%).

[0008] Preferably, 80%-90% ethanol is used to defatt the Ceylon sea fungus powder, wherein the ratio of the Ceylon sea fungus powder to 80%-90% ethanol (e.g., the volume fraction of ethanol is 80%, 82%, 84%, 86%, 88% or 90%) is 50 g / L; the defatting is carried out at 85°C for 3 hours; the operation is repeated once after the first defatting.

[0009] Preferably, in step (6), linear elution is performed using 0-2 mol / L NaCl solution on an ÄKTA-FPLC instrument at a flow rate of 1 mL / min, and 1 mL / tube of eluent is collected. The polysaccharide content of the collected eluent is determined by the phenol-sulfuric acid method, and the absorbance is measured at 490 nm to plot the elution curve. The first component is determined according to the elution curve (based on the peak shape, the first peak in the elution curve corresponds to the first component).

[0010] Preferably, in step (7), a 0.1 mol / L NH4HCO3 solution is used as the mobile phase, and elution is performed at a flow rate of 1 mL / min. The polysaccharide content of the collected eluent is determined by the phenol-sulfuric acid method, and the absorbance is measured at 490 nm to plot the elution curve. The multifunctional polysaccharide is determined according to the elution curve (based on the peak shape, the first peak in the elution curve corresponds to SCA1-1, the second peak corresponds to SCA1-2, the third peak corresponds to SCA1-3, the fourth peak corresponds to SCA1-4, the fifth peak corresponds to SCA1-5, and the sixth peak corresponds to SCA1-6).

[0011] The present invention also provides a multifunctional polysaccharide, which adopts the following technical solution: a multifunctional polysaccharide, wherein the multifunctional polysaccharide is prepared by the method described above.

[0012] Preferably, the monosaccharide composition of the multifunctional polysaccharide includes glucose, galactose, mannose, fucose, rhamnose, and galactosamine, and contains a sulfate group; the infrared spectrum of the multifunctional polysaccharide exhibits absorption peaks for the stretching vibration of the OH group of the sugar ring, the methyl absorption peak of fucose, the asymmetric stretching vibration of -COO-, the symmetric stretching vibration of -COO-, the symmetric stretching vibration of the S=O group of the sulfate ester group, and the absorption peak of 3,6-internal ether-galactose. That is, the multifunctional polysaccharide is at least one of SCA1-1, SCA1-3, SCA1-4, and SCA1-5.

[0013] Preferably, the monosaccharide composition of the multifunctional polysaccharide is: glucose 11.42%, galactose 27.73%, mannose 23.65%, fucose 18.06%, rhamnose 13.17%, and galactosamine 5.97%; the multifunctional polysaccharide has a triple helix structure; and the multifunctional polysaccharide contains a pyranose configuration. That is, the multifunctional polysaccharide is SCA1-1.

[0014] This invention also provides an application of a multifunctional polysaccharide, which employs the following technical solution: the application of the multifunctional polysaccharide described above in the preparation of at least one antioxidant, anticoagulant, and anti-inflammatory product. The product includes, but is not limited to, pharmaceuticals and food.

[0015] Beneficial effects:

[0016] The polysaccharide prepared by the method of the present invention has multifunctional activities such as antioxidant, anticoagulant and anti-inflammatory activities, which helps to promote the development and utilization of Ceylon sea fungus polysaccharide. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Wherein:

[0018] Figure 1 Elution curves of SCA via DEAE Sepharose FastFlow;

[0019] Figure 2 Elution curve of SCA1 after passing through Sephacryl S-300;

[0020] Figure 3 High-performance liquid chromatograms of the four purified components;

[0021] Figure 4 This is an infrared spectrum;

[0022] Figure 5 The maximum wavelength variation of the Congo red and polysaccharide mixture under different NaOH concentrations;

[0023] Figure 6 For SCA1-1 1 HNMR spectrum;

[0024] Figure 7 For SCA1-1 13 CNMR spectrum;

[0025] Figure 8 Antioxidant activity of purified polysaccharides from four types of sea fungus: (A) Antioxidant activity against O2 - The ability to scavenge free radicals, (B) the ability to scavenge •OH free radicals, (C) the ability to scavenge DPPH, (D) the ability to scavenge Fe. 2+ chelating ability;

[0026] Figure 9 The effect of purified polysaccharides from four types of sea fungus on APTT;

[0027] Figure 10 The effect of purified polysaccharides from four types of sea fungus on PT;

[0028] Figure 11 The effect of purified polysaccharides from four types of sea fungus on TT;

[0029] Figure 12 The effect of SCA1-1 on the viability of RAW264.7 cells;

[0030] Figure 13 The effects of SCA1-1 on NO and cytokine levels in RAW264.7 cells: (A) NO, (B) TNF-α, (C) IL-1β, (D) IL-6;

[0031] Figure 14 The effect of SCA1-1 on the protein expression of TNF-α and NF-κB: (A) Western Bolt results show, (B) NF-κB protein expression, (C) TNF-α protein expression. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0033] The present invention will now be described in detail with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0034] The following detailed description of the multifunctional polysaccharide of the present invention, its preparation method, and its application are illustrated through specific embodiments.

[0035] 1. The main experimental materials used in the following experiments (all other reagents or instruments not mentioned can be purchased commercially):

[0036] 1.1 Main Reagents: Ceylon sea fungus was purchased from Rongcheng Ningjin Qiuzhen Seafood Store; Coomassie Brilliant Blue G-250 dye was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; Bovine serum albumin (BSA) and carbazole AR were purchased from Beijing Solarbio Science & Technology Co., Ltd.; D-fructose, gelatin, Congo red, dimethyl sulfoxide, disodium EDTA, nitrotetrazole blue chloride, 30% hydrogen peroxide, and trichloroacetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; resorcinol, acetal, TFA, potassium bromide, sodium hydride, and 1,1-diphenyl-2-picrylhydrazine were purchased from Maclean Biochemical Technology Co., Ltd.; PMP was purchased from Tianjin Guangfu Fine Chemical Research Institute; galactose and glucose were also purchased. Aldol, mannose, glucosamine, rhamnose, glucose, xylose, arabinose, and fucose were purchased from Chengdu Manster Biotechnology Co., Ltd.; Tris and Vitamin C were purchased from Beijing Solarbio Technology Co., Ltd.; Potassium ferricyanide was purchased from Tianjin Aopsheng Chemical Co., Ltd.; Activated Partial Thromboplastin Time (APTT) assay kit was purchased from Shenzhen Zike Biotechnology Co., Ltd.; Prothrombin Time (PT) assay kit and Thrombin Time (TT) assay kit were purchased from Beijing Regen Biotechnology Co., Ltd.; DEME medium (high glucose) and Gibco... Fetal bovine serum was purchased from Thermo Fisher Scientific (China) Co., Ltd.; penicillin-streptomycin solution, dimethyl sulfoxide (DMSO) solution, lipopolysaccharide (LPS), glycine, BCA protein assay kit, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), 4× protein buffer, acrylamide (Acr), 30% gelling solution (29:1), 1 M Tris-HCl (pH 6.8), 1.5 M Tris-HCl (pH 8.8), 10% SDS, 10% PAGE gelling agent, PAGE gel accelerator, TEMED, RIPA lysis buffer, and HE staining kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.; iodomethane was purchased from TCI Chemical Industry Development Co., Ltd.; dexamethasone was purchased from Sigma-Aldrich, Inc.; nitric oxide assay kit and TNF-α assay kit were also purchased from Sigma-Aldrich. αThe assay kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.; the IL-1β assay kit and IL-6 assay kit were purchased from Wuhan Boster Biological Engineering Co., Ltd.; the Western blot high-sensitivity chemiluminescence solution was purchased from Nanjing Beyotime Biotechnology Co., Ltd.; the anti-β-Actin monoclonal antibody was purchased from Shanghai Abcam Biotechnology Co., Ltd.; the anti-TNF-α monoclonal antibody and anti-NF-κB monoclonal antibody were purchased from Affinity Bioscience, USA; and Coomassie Brilliant Blue G-250 staining solution was used. 10 mg, 5 mL of 95% ethanol, and 10 mL of 85% phosphoric acid were diluted to 100 mL with ddH2O and stored in a brown bottle at 4°C; 0.1% carbazole solution (solvent: anhydrous ethanol); resorcinol stock solution concentration: 1.5 mg / mL; acetal stock solution concentration: 8.2 mg / mL; resorcinol-acetal solution: 1 mL of acetal stock solution was diluted to 25 mL, and 9 mL of resorcinol stock solution, 100 mL of resorcinol stock solution, and 1 mL of diluted acetal stock solution were mixed to obtain the solution (prepared on the same day and used within 3 hours); gelatin solution: 0.5 g / 100 mL (gelatin dissolved at 60-70°C and then allowed to stand overnight at 4°C); barium chloride-gelatin solution: 0.5 g of BaCl2 and 100 mL of gelatin solution were dissolved and stored at 4°C; 0.5 M PMP, solvent: anhydrous methanol; phosphate buffer: 13.6 g potassium dihydrogen phosphate and 1.8 g NaOH, diluted to 1000 mL with ddH2O; Congo red solution, 80 μmol / L; 50 mM Tris-HCl buffer (pH 8.2); 72 μmol / L nitrotetrazole blue chloride solution: 6 mg nitrotetrazole blue chloride, diluted to 100 mL with 50 mM Tris-HCl buffer (pH 8.2); 30 μmol / L PMS solution: 9 mg PMS, diluted to 1 L with 50 mM Tris-HCl buffer (pH 8.2); 338 μmol / L NADH solution: solvent: Tris-HCl buffer (pH 8.2); 150 mM phosphate buffer (pH 8.2); 7.4); 360 μg / mL saffron T; 2 mM EDTANa2-Fe(II) solution: 11 mg ferrous sulfate was diluted to 10 mL volumetric flask with ddH2O, and 15 mg EDTANa2 was diluted to 10 mL volumetric flask with ddH2O, then mixed 1:1; 5 mM phenoxyazine solution; 0.2 M phosphate buffer (pH 6).6) 1% potassium ferricyanide solution; 10% trichloroacetic acid; 10×Tris-glycine: Tris 30g, glycine 144g, add ddH2O to a final volume of 1L; Electrophoresis buffer: 10×Tris-glycine 100mL, 10% SDS 10mL, add triple-distilled water to a final volume of 1L; Transfer buffer: 10×Tris-glycine 100mL, methanol 100mL, add triple-distilled water to a final volume of 1L; 10×TBST: Tris 24.2g, sodium chloride 80g, add triple-distilled water to a final volume of 1L; TBST: 10×TBST 100mL, Tween-20 1mL, add triple-distilled water to a final volume of 1L; 5% skim milk powder: skim milk powder 5g, TBST 100mL, store at -20℃, can be reused.

[0037] 1.2 Major Instruments: Shodex OHpak SB-804HQ column, Showa Denko Scientific Instruments (Shanghai) Co., Ltd., Japan; Shimadzu LC20AT high-performance liquid chromatograph, Shimadzu Corporation, Japan; UltiMate™ 3000 high-performance liquid chromatograph, Thermo Fisher Scientific (China) Co., Ltd., Shanghai; Diamonsil C18 column, Dima Technology Co., Ltd., Beijing; Agilent 7890B-5977A gas chromatography-mass spectrometry system and Agilent HP-5mscapillary column, Agilent Technologies, Inc., USA; DEAE Sepharose FastFlow column, Sephacryl S-300 column and ÄKTAavant 150 protein purification liquid chromatography system, General Electric, Inc., USA.

[0038] 1.3 Data Analysis: All data are expressed as mean ± standard deviation and statistical analysis was performed using IBM SPSS Statistics 26.0. A p-value ≤ 0.05 was considered statistically significant.

[0039] 2. Extraction of multifunctional polysaccharides according to the present invention

[0040] 2.1 Extraction of crude polysaccharides from Ceylon sea fungus

[0041] 2.1.1 Raw material pretreatment: The dried Ceylon sea fungus was crushed using a pulverizer and passed through a 40-mesh sieve, and then stored in a sealed container at room temperature.

[0042] 2.1.2 Extraction of crude polysaccharides from Ceylon sea fungus: (1) Weigh 100g of Ceylon sea fungus powder, add 2L of 85% ethanol, defatt and decolorize in a water bath at 85℃ for 3h, repeat once, centrifuge at 6000rpm for 10min, discard the supernatant to obtain the precipitate, and dry in an oven at 35℃ to obtain defatted powder; (2) Add the defatted powder to distilled water at a liquid-to-solid ratio of 60:1, stir and extract at room temperature for 3h, centrifuge for 10min (6000rpm) to obtain cold water extraction residue of Ceylon sea fungus; (3) Add to the cold water extraction residue of Ceylon sea fungus at a liquid-to-solid ratio of 60:1. Add distilled water and stir in a water bath at 90°C for 6 hours, centrifuge for 10 minutes (6000 rpm) to obtain the hot water extraction residue of Ceylon sea fungus; (4) Continue to extract the hot water residue of Ceylon sea fungus with 4% NaOH solution at a liquid-to-material ratio of 60:1 at 90°C for 6 hours. After centrifugation, take the supernatant, adjust the pH value to 7 with 2% HCl solution, concentrate, precipitate with alcohol (add 4 times the volume of 95% ethanol, stand overnight at 4°C, centrifuge, discard the supernatant, dissolve the precipitate with an appropriate amount of distilled water), dialyze (with a molecular weight cutoff of 7 kDa) for 1 hour, freeze dry to obtain alkaline extracted polysaccharide (SCA).

[0043] 2.2 Isolation and purification of crude polysaccharide (SCA) from Ceylon sea fungus

[0044] 2.2.1 Separation using a DEAE Sepharose FastFlow anion exchange column

[0045] Weigh an appropriate amount of SCA sample, dissolve it in 3 mL of distilled water, and load the sample onto a DEAE Sepharose FastFlow anion exchange column (3 cm × 23 cm). Perform linear elution with 0.2 mol / L NaCl solution (elution volume = 10 column volumes) on an ÄKTA-FPLC instrument at a flow rate of 1 mL / min. Collect 1 mL of eluent per tube. Determine the polysaccharide content of the collected eluent using the phenol-sulfuric acid method, measuring the absorbance at 490 nm, and plot the elution curve. Results are as follows: Figure 1 As shown. Two main components, SCA1 and SCA2, were obtained, and SCA1 was further purified.

[0046] 2.2.2 Gel permeation chromatography (Sephacryl S-300 gel column separation)

[0047] The eluent was collected according to the elution curve. SCA1 was concentrated to an appropriate volume, dialyzed, and then loaded onto a Sephacryl S-300 gel column (2.5 cm × 90 cm). 0.1 mol / L NH4HCO3 solution was used as the mobile phase, and elution was performed at a flow rate of 1 mL / min. 2 mL was collected from each tube. The polysaccharide content of the collected eluent was determined using the phenol-sulfuric acid method, and the elution curve was plotted.

[0048] SCA1 was processed with Sephacryl S-300 to obtain 6 components ( Figure 2 (Sorted according to the order of effluent components), named SCA1-1, SCA1-2, SCA1-3, SCA1-4, SCA1-5, and SCA1-6 respectively, with yields of 9.30%, 4.56%, 7.39%, 9.44%, 8.49%, and 8.05% respectively.

[0049] 3. Physicochemical Properties of Purified Polysaccharides from Ceylon Sea Fungus

[0050] 3.1 Method for determining total sugar content: The total sugar content was determined by the sulfuric acid-phenol method.

[0051] The total sugar content is ranked from highest to lowest as follows: SCA1-4 > SCA1-1 > SCA1-3 > SCA1-5 > SCA1-2 > SCA1-6 (Table 1).

[0052] 3.2 Protein content determination method: Coomassie brilliant blue method.

[0053] The protein content determined by the Coomassie Brilliant Blue method is shown in Table 1. The protein contents of SCA1-1, SCA1-2, SCA1-3, SCA1-4, SCA1-5, and SCA1-6 are 7.78%, 10.98%, 11.71%, 10.34%, 12.87%, and 11.80%, respectively.

[0054] 3.33,6-Laminated ether-galactose content determination method: resorcinol colorimetric method.

[0055] The content of 3,6-endo-Gal was determined by the m-diphenol method, and the results are shown in Table 1. Among them, SCA1-3 had the highest content of 3,6-endo-Gal (5.14%).

[0056] 3.4 Determination of sulfate content: Ion chromatography.

[0057] The sulfate content of the six polysaccharide components was determined and is shown in Table 1. The sulfate content was ranked from highest to lowest as follows: SCA1-1 > SCA1-5 > SCA1-3 > SCA1-4 > SCA1-2 > SCA1-6.

[0058] 3.5 Determination of uronic acid content: The uronic acid content was determined using the carbazole-sulfuric acid colorimetric method. Polysaccharides are hydrolyzed to produce uronic acid, which undergoes a condensation reaction with carbazole reagent in sulfuric acid to form a purple-red compound. The color intensity is directly proportional to the uronic acid content, with a maximum absorbance at 530 nm, allowing for quantitative colorimetric determination.

[0059] (1) Construction of standard curve: Weigh 10.0 mg of galacturonic acid reference standard, dilute to 10 mL, and take 0, 2, 4, 6, 8, and 10 μL of galacturonic acid standard solution respectively, add water to make up to 1 mL, and shake well. Add galacturonic acid standard solution of different concentrations to stoppered test tubes containing 6 mL of concentrated sulfuric acid, shake well, incubate at 85℃ for 20 min, cool to room temperature, add 0.2 mL of carbazole ethanol solution to each test tube, shake well, incubate at 85℃ for 40 min, and measure the absorbance at 530 nm.

[0060] (2) Sample determination: First, add 6 mL of concentrated H2SO4 to the test tube, then take 1 mL of the 1.0 mg / mL sample solution to be tested into the test tube. Calculate the uronic acid content in the sample using the standard curve according to the above steps.

[0061] Test results: The uronic acid content of the six components was determined by the carbazole colorimetric method and is shown in Table 1. Among them, SCA1-5 had the highest uronic acid content (12.87%), and SCA1-1 had the lowest uronic acid content (7.78%).

[0062] Table 1 Physicochemical properties of the six purified components

[0063]

[0064] Considering the high total sugar content, sulfate content, and yield of SCA1-1, SCA1-3, SCA1-4, and SCA1-5, SCA1-1, SCA1-3, SCA1-4, and SCA1-5 were selected for structural characterization analysis.

[0065] 4. Structural Characterization Analysis of Purified Polysaccharides from Ceylon Sea Fungus

[0066] 4.1 Molecular weight determination method: High performance gel permeation chromatography was used for determination; chromatographic column: Shodex OHpak SB-804HQ (8.0 mm × 300 mm, 6 μm); mobile phase: 0.1 mol / L Na2SO4; detector: differential detector.

[0067] The molecular weights of SCA1-1, SCA1-3, SCA1-4, and SCA1-5 were determined to be 107.51 kDa, 70.26 kDa, 40.51 kDa, and 23.18 kDa, respectively, by high-performance gel permeation chromatography.

[0068] 4.2 Monosaccharide Composition Analysis Method: The monosaccharide composition of polysaccharide samples was determined by pre-column derivatization high-performance liquid chromatography (PMP). An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used. The mobile phase was 0.1 mol / L phosphate buffer-acetonitrile (83:17 v / v). The column temperature was 30 °C, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and online detection was performed using a digital ultraviolet (DAD, 245 nm) detector.

[0069] Monosaccharide composition analysis of SCA1-1, SCA1-3, SCA1-4, and SCA1-5 was performed using PMP pre-column derivatization high-performance liquid chromatography. The results are as follows: Figure 3 As shown in Table 2, the four purified fractions contain similar types of monosaccharides, consisting of Glc (glucose), Gal (galactose), Man (mannose), Fuc (fucose), Rha (rhamnose), and GalN (galactosamine), but the relative contents of each monosaccharide differ. In SCA1-1, the Gal content is the highest (27.73%), with a molar ratio of Gal:Man:Fuc:Rha:Glc:GalN = 4.6:4.0:3.0:2.2:1.9:1.0. In SCA1-3, the Glc content is the highest (37.74%), followed by the Gal content (18.69%), and the Fuc content is the lowest (6.07%), with a molar ratio of Glc:Gal:Man:GalN:Rha:Fuc = 6.2:3.1:2.8:1.8:1.5:1.0. In SCA1-4, Glc (50.18%) had the highest content, with a molar ratio of Glc:Man:Gal:Fuc:GalN:Rha:GalA:Xyl = 44.0:12.2:11.4:8.5:7.0:2.3:1.4:1.0. In SCA1-5, Fuc had the highest content (24.15%) and GalA had the lowest content (6.69%), with a molar ratio of Fuc:Man:Glc:Gal:GalN:Rha:GalA = 3.6:2.8:2.6:2.3:1.4:1.34:1.00.

[0070] Table 2 Monosaccharide composition of the four purified components

[0071]

[0072] 4.3 Infrared Spectroscopy Analysis Method: Weigh approximately 1 mg of dried polysaccharide sample, mix with an appropriate amount of KBr, grind evenly, compress into a tablet, and analyze using an infrared spectrometer at 400~4000 cm⁻¹. 1 Infrared spectral scanning was performed within the range.

[0073] To preliminarily characterize the structures of SCA1-1, SCA1-3, SCA1-4, and SCA1-5, infrared spectroscopy analysis was performed. The results are as follows: Figure 4 As shown. Analysis of the four purified components at 3600-3200 cm⁻¹ -1 Absorption peaks appear at all locations, which are absorption peaks due to the stretching vibration of the OH group in the sugar ring; at 2934 cm⁻¹... -1 2936cm -1 and 2963cm -1 There is a weak absorption peak at 1616 cm⁻¹, which is the CH stretching vibration peak and the fucose methyl absorption peak; -1 1624cm -1 1641cm -1 and 1629cm -1 There is a strong absorption peak at 1404 cm⁻¹, which is the asymmetric stretching vibration of -COO-; -1 1409cm -1 1406cm -1 and 1404cm -1 The -COO- symmetric stretching vibration indicates that all four components contain a certain amount of uronic acid, consistent with the results of the carbazole colorimetric method. The monosaccharide results may not be visible due to the low content. At 1258 cm⁻¹ -1 1256cm -1 1231cm -1 and 1252cm -1 The characteristic peak of the S=O symmetric stretching vibration of the sulfate ester group indicates that all four components contain a small amount of sulfate groups; at 818 cm⁻¹ -1 The presence of absorption peaks nearby indicates that the sulfate group is substituted at the C6 position; 950-940 cm⁻¹ -1 The absorption at 3,6-lactone is a 3,6-lactone-galactose absorption peak, indicating that all four components contain a small amount of 3,6-lactone-galactose, consistent with the results determined by the resorcinol method. The absorption peaks at 1010-1100 indicate the presence of a pyran ring in all four components.

[0074] 4.4 Congo Red Experiment: Take 1 mL of a 1.0 mg / mL polysaccharide sample solution, add 1 mL of 80 μmol / L Congo red solution, and mix well. Then, add 1 mol / L NaOH sequentially to achieve final NaOH concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. Incubate at room temperature for 5 min, and perform a full-wavelength UV scan in the 400-800 nm range, recording the maximum wavelength.

[0075] Test results: Congo red is an acidic dye that can undergo a complexation reaction with the triple helix structure of polysaccharides at low NaOH solution concentrations. The maximum wavelength λ increases with increasing NaOH solution concentration. maxIt will shift towards longer wavelengths, resulting in a redshift. When the NaOH concentration reaches a certain value, the complex hydrolyzes, and the maximum wavelength decreases accordingly. Figure 5 As can be seen, when SCA1-1, SCA1-3, and SCA1-4 are mixed with Congo red solution, within the concentration range of 0-0.4 mmol / L, the wavelengths of the three components in the mixed solution with Congo red show a significant red shift compared to the maximum wavelength of the Congo red solution, indicating that SCA1-1, SCA1-3, and SCA1-4 possess a triple helix structure. The maximum wavelength of SCA1-5 decreases with increasing NaOH concentration, indicating that SCA1-5 does not possess a triple helix structure.

[0076] 4.5 Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis: 50 mg of SCA1-1 sample was dissolved in 0.5 mL of heavy water and lyophilized. This process was repeated three times to complete the heavy water exchange. The sample was then redissolved in 0.5 mL of L₂O and transferred to an NMR tube for testing. Measurements were taken at 25 °C and 600 MHz. 1 H-NMR, 13 C-NMR, 1 H- 1 HCOSY), HSQC, and HMBC.

[0077] Detailed structural information of SCA1-1 was further elucidated using NMR spectroscopy: 1 In the HNMR spectrum, the chemical shifts of the terminal hydrogens are concentrated in the range of δ 5.5–4.4 ppm. According to SCA1-1... 1 HNMR spectrum ( Figure 6 The signal peak appearing at δ 1.2-1.4 ppm is a rhamnose methyl signal peak, indicating that SCA1-1 may contain rhamnose, which is consistent with the monosaccharide composition results. The signal peak appearing at around δ 2.0 ppm is generally a methyl signal peak of acetyl, indicating that acetylamino may be present, which is consistent with the monosaccharide composition results.

[0078] The terminal carbon signals of polysaccharides are generally concentrated in the range of δ 110-90 ppm. 13 CNMR spectrum). For example, SCA1-1 13 CNMR spectrum ( Figure 7 As shown, the signal is mainly concentrated in the δ100-60ppm range, with two terminal carbons (δ101.16, 99.96ppm). Furanose C3 or C5 has a signal between δ84-82ppm, while pyranose C3 and C5 are generally less than δ80ppm. Figure 7It can be seen that there is a significant signal near δ80 ppm, but no signal in the δ84-82 ppm range, indicating that SCA1-1 does not contain the furanose configuration, but only the pyranose configuration. The peak at δ24.42 ppm may be the methyl signal of the acetamino group. The absence of a rhamnose carbonyl signal may be related to the low rhamnose content.

[0079] Evaluation of the in vitro antioxidant activity of purified polysaccharides from Ceylon sea fungus

[0080] 5.1 Regarding O2 - Free radical scavenging method: O2 is generated using a phenazine methyl sulfate (PMS)-reduced coenzyme (NADH) system. - Add 50 μL of 72 μmol / L nitrotetrazolium chloride (NBT), 30 μmol / L LPMS, and 338 μmol / L NADH, respectively, prepared in Tris-HCl buffer (50 mmol / L, pH=8.2), to 300 μL of polysaccharide sample solutions of different mass concentrations. Mix thoroughly and react at 25℃ for 5 min. Measure the absorbance of the reaction solution at 560 nm. Use vitamin C as a positive control. - The clearance rate is calculated using the following formula:

[0081] Clearance rate (%) = [1 - (A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0082] In the formula, A 样品 It is the OD560 of the polysaccharide sample; A 空白 OD560 was measured using Tris-HCl buffer instead of NBT; A 对照 The OD560 was determined using deionized water instead of polysaccharide samples.

[0083] Test results: Different concentrations of purified polysaccharides (SCA1-1, SCA1-3, SCA1-4, SCA1-5) and vitamin C on O2 - Free radical scavenging ability, such as Figure 8 As shown in Figure A, with increasing polysaccharide concentration, the purified polysaccharide from seaweed showed an effect on O2. - The ability to scavenge free radicals is enhanced. At a concentration of 10 mg / mL, its O2... - The order of free radical scavenging strength is SCA1-1 > SCA1-5 > SCA1-3 > SCA1-4.

[0084] 5.2 Method for scavenging •OH free radicals: •OH was generated using an EDTANa2-Fe(II)-H2O2 system. 200 μL of phosphate buffer (150 mmol / L, pH=7.4), 200 μL of 360 μg / mL saffron T, 100 μL of 2 mmol / L EDTANa2-Fe(II) solution, and 200 μL of 30% H2O2 solution were added sequentially to 200 μL of polysaccharide solutions of different mass concentrations, respectively. The absorbance of the reaction solutions was measured at 520 nm. Using vitamin C as a positive control, the scavenging rate of •OH was calculated using the following formula: Scavenging rate (%) = (A... 样品 -A 空白 ) / (A 对照 -A 空白 ) × 100%.

[0085] In the formula, A 样品 OD of polysaccharide samples 520 A 空白 OD was determined using deionized water instead of polysaccharide samples. 520 Control A was obtained by measuring OD using deionized water instead of polysaccharide samples and H2O2. 520 .

[0086] Test results: such as Figure 8 As shown in B, the four purified polysaccharides from *Auricularia auricula-judae* exhibited certain abilities to scavenge •OH free radicals, which were concentration-dependent. The ability to scavenge hydroxyl free radicals increased with increasing polysaccharide concentration. SCA1-1 showed the strongest •OH free radical scavenging ability; at a concentration of 10 mg / mL, its scavenging ability was close to that of vitamin C.

[0087] 5.3 Method for scavenging DPPH free radicals: Add 200 μL of 0.04 mg / mL DPPH ethanol solution to 200 μL of polysaccharide sample solutions of different mass concentrations, mix thoroughly, react at 25℃ for 30 min, and measure the absorbance at 517 nm. Using vitamin C as a positive control, the DPPH scavenging rate was calculated using the following formula:

[0088] Clearance rate (%) = [1 - (A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0089] In the formula, A 样品 OD of polysaccharide samples 517 A 空白 OD was measured using anhydrous ethanol instead of DPPH. 517 A 对照 OD was determined using deionized water instead of polysaccharide samples. 517 .

[0090] Test results: such as Figure 8 As shown in Figure C, within the concentration range of 0-10 mg / mL, the four purified polysaccharides from *Auricularia auricula-judae* exhibited a certain scavenging ability against DPPH free radicals, and the scavenging ability increased with increasing polysaccharide concentration. When the concentration reached 1 mg / mL, the scavenging abilities of SCA1-1 and SCA1-5 against DPPH free radicals were close to those of Vc. At a concentration of 10 mg / mL, SCA1-1 showed the strongest scavenging effect against DPPH free radicals, while SCA1-4 showed the weakest.

[0091] 5.4 Fe 2+ Free radical chelating ability method: Add 20 μL of 2 mmol / L FeCl2 and 40 μL of 5 mmol / L phenazine solution sequentially to 200 μL of polysaccharide sample solutions of different mass concentrations, mix thoroughly, react at 25℃ for 10 min, and measure the absorbance at 562 nm. Use EDTANa2 as a positive control to measure the chelating ability of Fe... 2+ The chelation rate is calculated using the following formula:

[0092] Chelation rate (%) = [1 - (A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0093] In the formula, A 样品 OD of polysaccharide samples 562 A 空白 OD was measured using deionized water instead of FeCl2. 562 A 对照 OD was determined using deionized water instead of polysaccharide samples. 562 .

[0094] Test results: such as Figure 8 As shown in D, within the experimental range, the four purified polysaccharides from seaweed exhibited strong resistance to Fe. 2+ The chelating ability of the purified polysaccharide from seaweed increased with increasing polysaccharide concentration, showing a greater effect on Fe. 2+ The chelating ability of SCA1-1 and SCA1-5 is enhanced at a concentration of 10 mg / mL. 2+ It has a strong chelating ability, close to that of EDTA.

[0095] Evaluation of the in vitro anticoagulant activity of purified polysaccharides from Ceylon sea fungus

[0096] 6.1 APTT assay method: The activated partial thromboplastin time (APTT) assay kit was used for the assay.

[0097] Test results: such as Figure 9As shown, SCA1-1, SCA1-3, SCA1-4, and SCA1-5 effectively prolonged APTT time in a dose-dependent manner, indicating that all the above components can exert anticoagulant activity by inhibiting the intrinsic coagulation pathway. The anticoagulant activity of the four purified polysaccharides from *Auricularia auricula-judae* was lower than that of heparin. Within the range of 2-5 mg / mL, the anticoagulant activity of SCA1-1 was higher than that of the other three purified polysaccharides.

[0098] 6.2 PT Measurement Method: The prothrombin time (PT) test kit was used for measurement.

[0099] like Figure 10 As shown, all four purified polysaccharides from *Auricularia auricula-judae* exhibited a prolonging effect on prostatic clotting time (PT) in a significant dose-dependent manner, indicating that they all possess anticoagulant effects by inhibiting the extrinsic coagulation pathway. Within the range of 0-5 mg / mL, SCA1-4 showed the strongest PT-prolonging effect, approaching that of heparin.

[0100] 6.3 TT assay method: Thrombin time (TT) was measured using a thrombin time (TT) assay kit.

[0101] Test results are as follows Figure 11 As shown, all four purified polysaccharides from *Auricularia auricula-judae* prolonged the time to blood viscosity (TT), indicating that they can inhibit fibrin formation and exert an anticoagulant effect. Within the concentration range of 3-5 mg / mL, SCA1-1 exhibited higher anticoagulant activity than the other three purified polysaccharides and was comparable to the anticoagulant activity of heparin.

[0102] In summary, the results of APTT, PT, and TT measurements showed that SCA1-1 had the strongest anticoagulant activity, effectively inhibiting the intrinsic coagulation pathway and the common coagulation pathway, but its effect on inhibiting the extrinsic coagulation pathway was weaker than that of SCA1-4.

[0103] 7. In vitro anti-inflammatory activity study of purified polysaccharide SCA1-1 from Ceylon sea fungus

[0104] 7.1 Sample solution preparation: Dissolve SCA1-1 in distilled water to a concentration of 4 mg / mL, filter through a 0.22 μm filter, and store at 4℃. Dilute the sample solution sequentially with DEME medium to concentrations of 200, 100, 50, 25, 12.5, and 6.25 μg / mL. Dissolve dexamethasone in anhydrous ethanol to a concentration of 1 mg / mL, filter through a 0.22 μm filter, and store at -20℃.

[0105] 7.2 Raw264.7 cell culture: (1) Cell resuscitation: Preheat the water bath to 37°C, take the cryovials out of the -150°C freezer, and thaw them in the 37°C water bath. After complete thawing, add 10 mL of complete culture medium (DMEM basal medium: fetal bovine serum: penicillin-streptomycin solution = 89:10:1), centrifuge at 1000 rpm for 5 min, and discard the supernatant to remove DMSO. Take 1 mL of culture medium and pipette the cells to mix them. Then seed the cells into a 10 cm medium containing 10 mL of culture medium. 2 Gently shake the culture dish and place it in a 37℃, 5% CO2 cell culture incubator. Observe the cell status and density regularly. When the cells adhere to the wall and the density reaches about 80%, pass the cells.

[0106] (2) Cell passage: Use a disposable dropper to aspirate the old culture medium from the culture dish, add 3-5 mL of PBS to wash the cells 3 times, then add PBS and pipette 1 mL of the solution to detach the cells from the bottom of the culture dish. Centrifuge at 1000 rpm for 5 min. Pipe the cells with 1 mL of fresh culture medium to disperse them evenly. Transfer the cell suspension to a new culture dish (containing 10 mL of fresh culture medium) at a 1:2 ratio and incubate in an incubator.

[0107] (3) Cell cryopreservation: Prepare cell cryopreservation solution according to the ratio of fetal bovine serum:DMSO=9:1. Select cells in the logarithmic growth phase that have not been activated, discard the old culture medium, wash 3 times with PBS, gently pipette the cells with 1mL pipette, centrifuge, discard the supernatant, add 1mL of the prepared cell cryopreservation solution, mix well by pipetting, transfer to the labeled cryopreservation tube, then place in a gradient cooling box, freeze overnight at -80℃, and finally transfer to -150℃ freezer.

[0108] 7.3 Cytotoxicity Assay: Observe cell state under a microscope, select cells in good growth condition, and prepare cell suspension using the passage method described above. Count cells using a hemocytometer at 5 × 10⁻⁶. 5 Seeds were inoculated into 96-well plates at a density of [number] cells / mL, with PBS added to the outermost ring. After 24 h of incubation, the culture medium was aspirated from the wells. 100 μL of medium was added to each well of the Control group, and 100 μL of SCA1-1 solution was added to each well of the SCA1-1 groups (200, 100, 50, 25, 12.5, and 6.25 μg / mL), with 6 replicates per concentration. The plates were incubated for 24 h. The supernatant was aspirated, and 20 μL of 5 mg / mL MTT was added to each well. The plates were then incubated for another 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min until the formazan was completely dissolved. The absorbance at 490 nm was measured using a microplate reader.

[0109] The MTT assay was used to detect the toxicity of SCA1-1 to Raw264.7 cells to avoid unnatural cell death caused by polysaccharides. Figure 12 As shown, SCA1-1 exhibited no cytotoxicity against RAW264.7 cells within the range of 6.25–50 μg / mL; and its effect on cell viability was relatively small within the range of 6.25–25 μg / mL. Therefore, the following SCA1-1 concentrations were selected for subsequent experiments: 6.25, 12.5, and 25 μg / mL.

[0110] 7.4 Determination of NO and cellular inflammatory factors: Using the seeding method described in the above toxicity experiment, cells were cultured for 24 h, and the supernatant was discarded. 100 μL of culture medium was added to the Control group, 100 μL of LPS solution was added to the LPS group, and 100 μL of DEX (dexamethasone) solution containing 1 μg / mL LPS (final concentration 12.5 μg / mL) was added to the DEX (dexamethasone) group. 100 μL of polysaccharide solution of different concentrations was added to the SCA1-1 polysaccharide groups (polysaccharide solutions containing 1 μg / mL LPS, final concentrations of 6.25, 12.5, and 25 μg / mL). After culturing for 24 h, the cell supernatant was collected, and the NO content and the levels of cellular inflammatory factors (TNF-α, IL-1β, and IL-6) were determined according to the instructions using the nitrate reductase method and ELISA.

[0111] The production of NO in the body is catalyzed by nitric oxide synthase. In inflammatory responses, inflammatory factors can activate iNOS to produce excessive NO, thereby intensifying the inflammatory response. Therefore, this study investigated the effect of SCA1-1 on LPS-induced NO production, and the results are as follows: Figure 13 A. Compared with the control group, LPS significantly promoted NO production in Raw264.7 cells. Compared with the LPS group, SCA1-1 significantly inhibited LPS-induced NO levels in Raw264.7 cells in a concentration-dependent manner.

[0112] LPS-stimulated macrophages produce TNF-α, IL-1β, and IL-6, which are considered among the most important cytokines promoting inflammatory responses and can lead to acute inflammatory diseases and tissue damage. Therefore, this invention investigates the use of these cytokines as inflammatory markers to test the anti-inflammatory effect of SCA1-1, and the results are as follows: Figure 13BD. Compared with the LPS group, SCA1-1 significantly inhibited LPS-induced TNF-α and IL-6 secretion levels in Raw264.7 cells in a concentration-dependent manner. High-dose SCA1-1 inhibited LPS-induced IL-1β levels in Raw264.7 cells. These results indicate that SCA1-1, at appropriate concentrations, possesses strong anti-inflammatory potential against LPS-stimulated inflammation by inhibiting NO, TNF-α, IL-1β, and IL-6 secretion levels.

[0113] 7.5 Western Blot:

[0114] (1) Protein extraction at 2×10 6 Cells were seeded at a density of cells / well in 6-well plates and cultured for 24 h. The supernatant was removed, and cells were treated according to their assigned groups. After 24 h of treatment, cells were washed three times with PBS, collected by pipetting, and placed in sterile EP tubes. Centrifugation was performed (1000 rpm, 5 min). The supernatant was discarded, and cells were lysed with RIPA buffer for 30 min, followed by centrifugation at 12000 rpm for 20 min at 4 °C (all operations must be performed on ice). Protein concentration was determined using a BCA protein assay kit. The lysates were denatured at 100 °C for Western blotting.

[0115] (2) Gel preparation: Place a clean glass plate on a horizontal experimental table, align it, and clamp it firmly onto the gel preparation rack. Add distilled water and let it stand for 10 minutes to check for leaks. Prepare 10% SDS-PAGE gel according to the formula, vortex to mix, and quickly pour the gel into the gap between the two glass plates using a pipette until the liquid level is about 1 cm from the highest point of the shorter glass plate. Then, slowly add anhydrous ethanol for liquid sealing and let it stand at room temperature for 20 minutes. When a clear boundary line appears at the interface between the anhydrous ethanol and the separating gel, pour off the anhydrous ethanol. Prepare the upper layer gel according to the formula, vortex to mix, and then add the upper layer gel to the solidified separating gel until it is level with the top of the shorter plate. Immediately insert a clean comb straight in place to avoid air bubbles. Let it stand for 15 minutes. After the gel solidifies, slowly and carefully remove the comb.

[0116] Table 3 Formulations for separating gel and stacking gel

[0117]

[0118] (3) Electrophoresis: Place the gel plate into the electrophoresis tank, pour in the electrophoresis buffer, rinse the sample wells with the buffer, and straighten the gel teeth between the wells with a needle. Add the marker and protein sample to the sample wells, and cover the electrophoresis tank electrodes (positive electrode to positive electrode, negative electrode to negative electrode). First, adjust the voltage to 90V and perform constant voltage electrophoresis until the bromophenol blue indicator migrates to the interface between the lower and upper gels, and the markers begin to separate. Then, increase the voltage to 120V and stop electrophoresis when the dye reaches 1cm from the bottom of the gel plate.

[0119] (4) Transfer: Immerse the sponge pad and filter paper in transfer buffer and set aside. Use a gel cutter to remove the stacking gel and excess gel, and keep them moist with transfer buffer. Cut a PVDF membrane to the same size as the gel and activate it in methanol for 30 seconds. Assemble the membrane in the following order: cathode plastic clip, sponge pad, filter paper, PVDF membrane, gel, filter paper, sponge pad, and anode plastic clip. Air bubbles should be removed between each layer, and the gel and membrane should be kept moist. Avoid drying. Place the membrane in the transfer tank (positive electrode to positive electrode, negative electrode to negative electrode), pour in the transfer solution at 4°C, cover with the transfer cap, incubate on ice, and run at a constant current of 150mA for 90-120 minutes. Adjust the transfer time according to the molecular weight of the target protein.

[0120] (5) After sealing, remove the PVDF membrane, wash with TBST for 5 min, place it in 5% skim milk powder, and seal it in a shaker at room temperature for 2 h.

[0121] (6) After primary antibody incubation and blocking, the membrane was washed three times with TBST for 10 min each time. The membrane was cut to the appropriate molecular weight according to the Maker and placed in the primary antibody incubation box and incubated overnight at 4°C.

[0122] (7) Secondary antibody incubation: Wash the membrane 3 times, once every 10 minutes, and incubate with secondary antibody for 1 hour. Wash once every 10 minutes, for a total of 3 washes.

[0123] (8) Exposure: According to the ECL kit, prepare solution A and solution B in a 1:1 ratio (prepare fresh and use in the dark), and drop them evenly onto the PVDF membrane. Use a chemiluminescence imaging system to detect the bands. Use ImageJ software to quantitatively analyze the gray values ​​of the protein bands.

[0124] Western Bolt Results Analysis:

[0125] NF-κB is an important transcription factor that positively regulates the expression of inflammatory mediators such as TNF-α and IL-1β, playing a crucial role in the development of inflammation. Its expression was detected using Western blotting. The immunoblotting results showed ( Figure 14 Compared with the control group, LPS significantly upregulated the expression levels of p65 and TNF-α. Compared with the LPS group, SCA1-1 treatment significantly reduced the LPS-induced expression levels of p65 and TNF-α, indicating that SCA1-1 can exert anti-inflammatory effects through the NF-κB signaling pathway.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional polysaccharide, characterized in that, Includes the following steps: (1) The Ceylon sea fungus was dried, pulverized and degreased to obtain a defatted powder; (2) The defatted powder is mixed with water, extracted at room temperature, and the solid and liquid are separated to obtain cold water extraction residue; (3) The cold water extraction residue is mixed with water, heated for extraction, and then separated into solid and liquid components to obtain the hot water extraction residue; (4) Mix the hot water extraction residue with alkaline solution, heat to extract, and separate the solid and liquid to obtain the extract; (5) The pH of the extract was adjusted, concentrated, precipitated with alcohol, dialyzed and lyophilized to obtain crude polysaccharide; (6) The crude polysaccharide was separated using a DEAE Sepharose Fast Flow anion exchange column to obtain the first component; (7) The first component was separated using a Sephacryl S-300 gel column to obtain the multifunctional polysaccharide; In step (6), linear elution was performed using 0-2 mol / L NaCl solution on an ÄKTA-FPLC instrument at a flow rate of 1 mL / min. The polysaccharide content of the collected eluent was determined by the phenol-sulfuric acid method, and the absorbance was measured at 490 nm to plot the elution curve. The first component is determined based on the elution curve; the first component is the component corresponding to the first peak in the elution curve obtained in step (6); In step (7), 0.1 mol / L NH4HCO3 solution was selected as the mobile phase and eluted at a flow rate of 1 mL / min. The polysaccharide content of the collected eluent was determined by the phenol-sulfuric acid method, and the absorbance was measured at 490 nm to plot the elution curve. The multifunctional polysaccharide is determined based on the elution curve, wherein the multifunctional polysaccharide is the component corresponding to the first peak in the elution curve obtained in step (7).

2. The method for preparing a multifunctional polysaccharide as described in claim 1, characterized in that, The liquid-to-solid ratios in steps (2)-(4) are each independently selected from 40:1 to 80:1, with the amount of liquid in mL and the amount of solid in g; In step (2), the extraction time is 2-4 hours; In steps (3) and / or (4), the heating temperature is 80-100 °C and the extraction time is 4-7 h; In step (4), the alkaline solution is a 3wt%-5wt% NaOH solution.

3. The method for preparing a multifunctional polysaccharide as described in claim 1, characterized in that, In step (1), Ceylon sea fungus powder is degreased with ethanol of 80%-90% by volume, and the ratio of Ceylon sea fungus powder to 80%-90% ethanol is 50g / L. The degreasing was carried out at 85 °C for 3 hours; the process was repeated once after the first degreasing.

4. A multifunctional polysaccharide, characterized in that, The multifunctional polysaccharide is prepared by the method described in any one of claims 1-3.

5. The multifunctional polysaccharide as described in claim 4, characterized in that, The infrared spectrum of the multifunctional polysaccharide exhibits absorption peaks for the stretching vibration of the OH group of the sugar ring, the methyl absorption peak of fucose, the asymmetric stretching vibration of -COO-, the symmetric stretching vibration of -COO-, the symmetric stretching vibration of the S=O group of the sulfate ester group, and the absorption peak of 3,6-endoether-galactose.

6. The multifunctional polysaccharide as described in claim 5, characterized in that, The monosaccharide composition of the multifunctional polysaccharide is as follows: glucose 11.42 wt%, galactose 27.73 wt%, mannose 23.65 wt%, fucose 18.06 wt%, rhamnose 13.17 wt%, and galactosamine 5.97 wt%. The multifunctional polysaccharide has a triple helix structure; The multifunctional polysaccharide contains a pyranose configuration.

7. The use of the multifunctional polysaccharide as described in claim 5 or 6 in the preparation of at least one antioxidant, anticoagulant and anti-inflammatory product.

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