Multifunctional polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a multifunctional polysaccharide as well as a preparation method and application thereof. The preparation method comprises the following steps: drying, crushing and degreasing the sarcodia stannanensis to obtain degreased powder; mixing the degreased powder with water, and extracting at room temperature to obtain cold water extraction residues; mixing the cold water extraction residues with water, heating and extracting to obtain hot water extraction residues; mixing hot water extraction residues with alkali liquor to obtain an extracting solution; adjusting the pH value of the extracting solution, concentrating, carrying out alcohol precipitation, dialyzing and freeze-drying to obtain crude polysaccharide; separating the crude polysaccharide by adopting a DEAE (Diethylaminoethyl) Sepharose Fast Flow anion exchange chromatographic column, so as to obtain a first component; and separating the first component by adopting a Sephacryl S-300 gel column, so as to obtain the polysaccharide with multiple functions. The polysaccharide disclosed by the invention has antioxidant, anticoagulant and anti-inflammatory activity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a multifunctional polysaccharide and a preparation method and application thereof. Background Art

[0002] Sarcodiaceylonensis, a member of the Rhodophyta, Gigarticales, Sarcodiaceae, and Sarcodiaceae genus, is rich in over 20 nutrients unique to marine plants, including seaweed polysaccharides and unsaturated fatty acids. It exhibits a variety of pharmacological activities, including anti-inflammatory, lipid-lowering, blood pressure-lowering, vascular softening, and anti-cancer properties. In China, there are two species of this genus: Sarcodiaceylonensis and Sarcodiaceylanica. Sarcodiaceylonensis, primarily found along my country's southeastern coast, is rich in over 20 nutrients unique to marine plants, including seaweed polysaccharides and unsaturated fatty acids. It has a variety of benefits, including anti-lipid and blood pressure-lowering, immune-boosting, and anti-tumor properties.

[0003] Polysaccharides are composed of repetitive structural features linked by glycosidic bonds, with a high degree of structural variability that preserves ample biological information. Most polysaccharides are relatively non-toxic and do not cause significant side effects.

[0004] Currently, there are few studies on Auricularia auricularia polysaccharides. Summary of the Invention

[0005] The purpose of the present invention is to provide a multifunctional polysaccharide and a preparation method and application thereof, so as to help expand the use of Ceylon sea fungus.

[0006] In order to achieve the above-mentioned object, the present invention provides the following technical scheme: A method for preparing a multifunctional polysaccharide, comprising the following steps: (1) drying, crushing and defatting Ceylon sea fungus to obtain defatted powder; (2) mixing the defatted powder with water, extracting at room temperature, and separating the solid and liquid to obtain a cold water extraction residue; (3) mixing the cold water extraction residue with water, heating extraction, and separating the solid and liquid to obtain a hot water extraction residue; (4) mixing the hot water extraction residue with alkaline solution, heating extraction, and separating the solid and liquid to obtain an extract; (5) adjusting the pH of the extract, concentrating, precipitating with alcohol, dialyzing and freeze-drying to obtain a crude polysaccharide; (6) separating the crude polysaccharide using a DEAE Sepharose Fast Flow anion exchange chromatography column to obtain a first component; and (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) to (4) is independently selected from 40:1-80:1 (for example, 40:1, 50:1, 60:1, 70:1 or 80:1; wherein the amount of liquid is measured in mL and the amount of solid is measured in g); in step (2), the extraction time is 2-4 h (for example, 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 (for example, 80 ° C, 85 ° C, 90 ° C, 95 ° C or 100 ° C), and the extraction time is 4-7 h (for example, 4 h, 5 h, 6 h or 7 h); in step (4), the alkali solution is 3%-6% NaOH solution (for example, the mass concentration of NaOH solution is 3%, 4%, 5% or 6%).

[0008] Preferably, the Ceylon fungus powder is degreased using 80%-90% ethanol, and the ratio of the Ceylon fungus powder to 80%-90% ethanol (for example, the volume fraction of ethanol is 80%, 82%, 84%, 86%, 88% or 90%) is 50 g / L; the degreasing is carried out at 85° C. for 3 hours; and the operation is repeated once after the first degreasing is completed.

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

[0010] Preferably, in step (7), 0.1 mol / L NH4HCO3 solution is selected as the mobile phase, and elution is performed at a flow rate of 1 mL / min. The collected eluate is used to determine the polysaccharide content by the phenol-sulfuric acid method, the absorbance is measured at 490 nm, and an elution curve is drawn; the multifunctional polysaccharide is determined according to the elution curve (based on the peak type, 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, which is prepared by the method described above.

[0012] Preferably, the multifunctional polysaccharide comprises monosaccharides including glucose, galactose, mannose, fucose, rhamnose, and galactosamine, and contains sulfate groups. The infrared spectrum of the multifunctional polysaccharide exhibits a stretching vibration absorption peak of the sugar ring OH, a fucose methyl absorption peak, an asymmetric stretching vibration of -COO-, a symmetric stretching vibration of -COO-, a characteristic peak of the symmetric stretching vibration of the sulfate group S=O, and a 3,6-ether-galactose absorption peak. In other words, the multifunctional polysaccharide is at least one of SCA1-1, SCA1-3, SCA1-4, and SCA1-5.

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

[0014] The present invention also provides an application of a multifunctional polysaccharide, which employs the following technical solution: application of the multifunctional polysaccharide described above to the preparation of at least one of antioxidant, anticoagulant, and anti-inflammatory products, including but not limited to pharmaceuticals and foods.

[0015] Beneficial effects: The multifunctional polysaccharide prepared by the method of the present invention has anti-oxidation, anti-coagulation and anti-inflammatory activities, and is helpful to promote the development and utilization of Ceylon auricularia auricularia polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. Among them: Figure 1 is the elution curve of SCA through DEAE Sepharose Fast Flow; Figure 2 is the elution curve of SCA1 through Sephacryl S-300; Figure 3 HPLC chromatograms of four purified components; Figure 4 is the infrared spectrum; Figure 5 The maximum wavelength change of Congo red and polysaccharide mixture under different NaOH concentrations; Figure 6 For SCA1-1 1 HNMR spectrum; Figure 7For SCA1-1 13 CNMR spectrum; Figure 8 The antioxidant activity of four purified polysaccharides from Auricularia auricula: (A) - Free radical scavenging ability, (B) scavenging ability for •OH free radicals, (C) scavenging ability for DPPH, (D) scavenging ability for Fe 2+ chelating capacity; Figure 9 The effects of four kinds of purified polysaccharides from Auricularia auriculariae on APTT; Figure 10 The effects of four types of purified polysaccharides from Auricularia auriculariae on PT were investigated. Figure 11 The effects of four types of purified polysaccharides from Auricularia auriculariae on TT; Figure 12 The effect of SCA1-1 on the viability of RAW264.7 cells; Figure 13 Effects of SCA1-1 on NO and cytokine levels in RAW264.7 cells: (A) NO, (B) TNF-α, (C) IL-1β, (D) IL-6; Figure 14 Effects of SCA1-1 on the protein expressions of TNF-α and NF-κB: (A) Western Bolt results, (B) NF-κB protein expression, (C) TNF-α protein expression. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection of the present invention.

[0018] The present invention will be described in detail below with reference to the embodiments. It should be noted that the embodiments and features of the embodiments of the present invention can be combined with each other without conflict.

[0019] The multifunctional polysaccharide of the present invention, its preparation method and application are described in detail below through specific examples.

[0020] 1. The main experimental materials used in the following experiments (other reagents or instruments not mentioned can be purchased commercially): 1.1 Main reagents: Ceylon fungus was purchased from Ningjin Qiuzhen Seafood Trading Company, Rongcheng City; 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 Solaibao Technology Co., Ltd.; D-fructose, gelatin, Congo red, dimethyl sulfoxide, ethylenediaminetetraacetic acid disodium salt, nitro tetrazolium blue chloride, 30% hydrogen peroxide, and trichloroacetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; m-diphenol, acetal, TFA, potassium bromide, sodium hydride, and 1,1-diphenyl-2-picrylhydrazyl were purchased from MacLean Biochemical Technology Co., Ltd.; PMP was purchased from Tianjin Guangfu Fine Chemical Research Institute; galactose and glucose Aldehydic acid, mannose, glucosamine, rhamnose, glucose, xylose, arabinose, and fucose were purchased from Chengdu Munster Biotechnology Co., Ltd.; Tris and vitamin C were purchased from Beijing Solaibao Technology Co., Ltd.; potassium ferrocyanide was purchased from Tianjin Aopusheng 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), 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 detection kit, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), 4× protein buffer, acrylamide (Acr), 30% gel preparation solution (29:1), 1 M Tris-HCl (pH 6.8), 1.5 M Tris-HCl (pH 8.8), 10% SDS, 10% PAGE gel coagulant, PAGE gel accelerator, TEMED, RIPA lysis buffer, and HE staining kit were purchased from Beijing Solebao Technology Co., Ltd.; iodomethane was purchased from TCI Chemical Industry Development Co., Ltd.; dexamethasone was purchased from Sigma-Aldrich; nitric oxide detection kit and TNF-α were purchased from Sigma-Aldrich. αThe assay kits were purchased from Nanjing Jiancheng Biological Co., Ltd.; the IL-1β assay kit and IL-6 assay kit were purchased from Wuhan Boster Biotechnology Co., Ltd.; the Western blot ultrasensitive luminescent solution was purchased from Nanjing Biyuntian Biological Co., Ltd.; the anti-β-Actin monoclonal antibody was purchased from Shanghai Abcam Biological Co., Ltd.; the anti-TNF-α monoclonal antibody and the anti-NF-κB monoclonal antibody were purchased from Affinity Bioscience, USA; the Coomassie Brilliant Blue G-250 staining solution was purchased from Coomassie Brilliant Blue G-250. 10 mg of hydroxybenzoic acid, 5 mL of 95% ethanol, and 10 mL of 85% phosphoric acid were added to ddH2O to make the volume 100 mL, and the mixture was placed in a brown bottle and stored at 4°C. 0.1% carbazole solution (the solvent is anhydrous ethanol); the concentration of the resorcinol stock solution is 1.5 mg / mL; the concentration of the acetal stock solution is 8.2 mg / mL; resorcinol-acetal solution: 1 mL of the acetal stock solution was diluted to 25 mL, and 9 mL of the resorcinol stock solution, 100 mL of the resorcinol stock solution, and 1 mL of the diluted acetal stock solution were mixed to prepare the solution (prepare on the same day and use within 3 hours); gelatin solution: 0.5 g / 100 mL (gelatin was dissolved at 60-70°C and then allowed to stand at 4°C overnight); barium chloride-gelatin solution: 0.5 g of BaCl2 and 100 mL of the 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, add ddH2O to 1000 mL; Congo red solution, 80 μmol / L; 50 mM Tris-HCl buffer (pH 8.2); 72 μmol / L nitro blue tetrazolium chloride solution: 6 mg nitro blue tetrazolium chloride, add 50 mM Tris-HCl buffer (pH 8.2) to 100 mL volumetric flask; 30 μmol / L PMS solution: 9 mg PMS, add 50 mM Tris-HCl buffer (pH 8.2) to 1 L volumetric flask; 338 μmol / L NADH solution: solvent: Tris-HCl buffer (pH 8.2); 150 mM phosphate buffer (pH 7.4); 360 μg / mL Crocus T; 2 mM EDTANa2-Fe(II) solution: 11 mg of ferrous sulfate was added to ddH2O in a 10 mL volumetric flask, and 15 mg of EDTANa2 was added to ddH2O in a 10 mL volumetric flask, followed by a 1:1 mixture; 5 mM ferrozine solution; 0.2 M phosphate buffer (pH 6.6); 1% potassium ferricyanide solution; 10% trichloroacetic acid; 10× Tris-glycine: 30 g Tris, 144 g glycine, dilute to 1 L with ddH2O; electrophoresis buffer: 100 mL 10× Tris-glycine, 10 mL 10% SDS, dilute to 1 L with triple-distilled water; transfer buffer: 100 mL 10× Tris-glycine, 100 mL methanol, dilute to 1 L with triple-distilled water; 10× TBST: 24.2 g Tris, 80 g sodium chloride, dilute to 1 L with triple-distilled water; TBST: 100 mL 10× TBST, 1 mL Tween-20, dilute to 1 L with triple-distilled water; 5% skim milk powder: 5 g skim milk powder, 100 mL TBST, store at -20°C, can be reused.

[0021] 1.2 Main instruments: Shodex OHpak SB-804HQ chromatographic 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 chromatographic column, Beijing Dima Technology Co., Ltd.; Agilent 7890B-5977A gas chromatography-mass spectrometer and Agilent HP-5m Scapillary chromatographic column, Agilent Corporation, USA; DEAE Sepharose Fast Flow column, Sephacryl S-300 column and ÄKTA avant 150 protein purification liquid chromatography system, General Electric Company, USA.

[0022] 1.3 Data Analysis: All data are expressed as mean ± standard deviation and analyzed using IBM SPSS Statistics 26.0. P ≤ 0.05 was considered statistically significant.

[0023] 2. Extraction of the multifunctional polysaccharide of the present invention 2.1 Extraction of Crude Polysaccharides from Auricularia auricularia 2.1.1 Raw material pretreatment: Crush the dried Ceylon fungus with a grinder and pass it through a 40-mesh sieve, then store it in a sealed container at room temperature.

[0024] 2.1.2 Extraction of crude polysaccharide from Auricularia auricula: (1) Weigh 100 g of Auricularia auricula powder, add 2 L of 85% ethanol, degrease and decolorize in a water bath at 85 °C for 3 h, repeat the operation once, centrifuge at 6000 rpm for 10 min, discard the supernatant to obtain the precipitate, and dry in an oven at 35 °C to obtain defatted powder; (2) Add distilled water to the defatted powder at a liquid-to-solid ratio of 60:1, stir and extract at room temperature for 3 h, centrifuge for 10 min (6000 rpm) to obtain the Ceylon Auricularia auricula cold water extraction residue; (3) Add ethanol to the Ceylon Auricularia auricula cold water extraction residue at a liquid-to-solid ratio of 60:1. Add distilled water and stir in a water bath at 90℃ for 6h, centrifuge for 10min (6000rpm) to obtain the Ceylon Auricularia auricularia hot water extraction residue; (4) The Ceylon Auricularia auricularia hot water extraction residue was further extracted with 4% NaOH solution at a liquid-to-solid ratio of 60:1 at 90℃ for 6h, centrifuged and the supernatant was taken, and the pH value was adjusted to 7 with 2% HCl solution. After concentration, alcohol precipitation (add 4 times the volume of 95% ethanol, stand at 4℃ overnight, centrifuge, discard the supernatant, and dissolve the precipitate with appropriate amount of distilled water), dialyzed (molecular weight cutoff 7kDa) for 1h, and freeze-dried, alkali-extracted polysaccharide (SCA) was obtained.

[0025] 2.2 Isolation and purification of Ceylon Auricularia auriculariae crude polysaccharide (SCA) 2.2.1 DEAE Sepharose Fast Flow anion exchange chromatography column separation Weigh an appropriate amount of SCA sample, dissolve it in 3 mL of distilled water, load it onto a DEAE Sepharose Fast Flow anion exchange chromatography column (3 cm × 23 cm), and perform linear elution (elution volume is 10 column volumes) with 0-2 mol / L NaCl solution on an ÄKTA-FPLC instrument at a flow rate of 1 mL / min. Collect the eluate in 1 mL / tube. The collected eluate was used to determine the polysaccharide content using the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and the elution curve was plotted. The results are shown in Figure 2. Figure 1 As shown, two major components, SCA1 and SCA2, were obtained, and SCA1 was further purified.

[0026] 2.2.2 Gel Permeation Chromatography (Sephacryl S-300 Gel Column Separation) The eluate was collected according to the elution curve. SCA1 was concentrated to an appropriate amount and then dialyzed before loading onto a Sephacryl S-300 gel column (2.5 cm × 90 cm). Elution was performed at a flow rate of 1 mL / min using a 0.1 mol / L NH₄HCO₃ solution as the mobile phase, collecting 2 mL per tube. The collected polysaccharide eluate was analyzed for polysaccharide content using the phenol-sulfuric acid method, and an elution curve was plotted.

[0027] SCA1 was purified by Sephacryl S-300 to obtain 6 components ( Figure 2; sorted by the order of elution components), named SCA1-1, SCA1-2, SCA1-3, SCA1-4, SCA1-5, and SCA1-6, with yields of 9.30%, 4.56%, 7.39%, 9.44%, 8.49%, and 8.05%, respectively.

[0028] 3 Study on the physicochemical properties of purified polysaccharides from Auricularia auricularia 3.1 Total sugar content determination method: The total sugar content was determined by sulfuric acid-phenol method.

[0029] The order of total sugar content is: SCA1-4>SCA1-1>SCA1-3>SCA1-5>SCA1-2>SCA1-6 (Table 1).

[0030] 3.2 Protein content determination method: Coomassie brilliant blue method.

[0031] The protein content measured 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 were 7.78%, 10.98%, 11.71%, 10.34%, 12.87%, and 11.80%, respectively.

[0032] 3.3.3,6-Ether-galactose content determination method: resorcinol colorimetric method.

[0033] The content of 3,6-endo-Gal was determined by the m-diphenol method. The results are shown in Table 1. Among them, SCA1-3 had the highest 3,6-endo-Gal content (5.14%).

[0034] 3.4 Determination of sulfate content: ion chromatography.

[0035] The sulfate content of the six polysaccharide components was determined as shown in Table 1. The order of sulfate content is: SCA1-1 > SCA1-5 > SCA1-3 > SCA1-4 > SCA1-2 > SCA1-6.

[0036] 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 the carbazole reagent in sulfuric acid to produce a purple-red compound. The color intensity is proportional to the uronic acid content, with a maximum absorption value at 530 nm, allowing for colorimetric quantification.

[0037] (1) Preparation of the standard curve: Weigh 10.0 mg of galacturonic acid reference substance and dilute to 10 mL. Take 0, 2, 4, 6, 8, and 10 μL of galacturonic acid standard solution, add water to 1 mL, and shake well. Add different concentrations of galacturonic acid standard solution to 6 mL of concentrated sulfuric acid in stoppered test tubes, shake well, incubate at 85°C for 20 min, cool to room temperature, add 0.2 mL of carbazole ethanol solution to each test tube, shake well, incubate at 85°C for 40 min, and measure the absorbance at 530 nm.

[0038] (2) Sample determination: First add 6 mL of concentrated H2SO4 into the test tube, then take 1 mL of 1.0 mg / mL sample solution into the test tube. According to the above steps, calculate the uronic acid content in the sample using the standard curve.

[0039] Test results: The uronic acid content of the six components was determined by carbazole colorimetry as 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%).

[0040] Table 1 Physicochemical properties of the six purified fractions

[0041] Due to 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 after comprehensive consideration.

[0042] 4 Structural characterization and analysis of purified polysaccharides from Auricularia auricularia 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.

[0043] Using high performance gel permeation chromatography, the molecular weights of SCA1-1, SCA1-3, SCA1-4 and SCA1-5 were measured to be 107.51 kDa, 70.26 kDa, 40.51 kDa and 23.18 kDa, respectively.

[0044] 4.2 Monosaccharide Composition Analysis Method: The monosaccharide composition of polysaccharide samples was determined using high-performance liquid chromatography with pre-column derivatization using an Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of 0.1 mol / L phosphate buffer-acetonitrile (volume ratio 83:17). The column temperature was 30°C, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and detection was performed online with a UV detector (DAD, 245 nm).

[0045] The monosaccharide composition of SCA1-1, SCA1-3, SCA1-4 and SCA1-5 was analyzed by PMP pre-column derivatization HPLC. Figure 3 As shown in Table 2, the four purified fractions contained similar monosaccharide compositions, consisting of Glc (glucose), Gal (galactose), Man (mannose), Fuc (fucose), Rha (rhamnose), and GalN (galactosamine), but the relative amounts of these monosaccharides varied. As shown in Table 2, SCA1-1 had the highest Gal content (27.73%), with a molar ratio of Gal:Man:Fuc:Rha:Glc:GalN of 4.6:4.0:3.0:2.2:1.9:1.0. SCA1-3 had the highest Glc content (37.74%), followed by Gal (18.69%), and the lowest Fuc content (6.07%), with a molar ratio of Glc:Gal:Man:GalN:Rha:Fuc of 6.2:3.1:2.8:1.8:1.5:1.0. SCA1-4 had the highest Glc content (50.18%), with a molar ratio of Glc:Man:Gal:Fuc:GalN:Rha:GalA:Xyl of 44.0:12.2:11.4:8.5:7.0:2.3:1.4:1.0. SCA1-5 had the highest Fuc content (24.15%) and the lowest GalA content (6.69%), with a molar ratio of Fuc:Man:Glc:Gal:GalN:Rha:GalA of 3.6:2.8:2.6:2.3:1.4:1.34:1.00.

[0046] Table 2 Monosaccharide composition of the four purified fractions

[0047] 4.3 Infrared spectroscopy analysis method: Weigh about 1 mg of dry polysaccharide sample, mix it with an appropriate amount of KBr, grind it evenly, press it into tablets, and use an infrared spectrometer at 400~4000cm- 1 Infrared spectrum scanning was performed within the range.

[0048] In order to preliminarily characterize the structures of SCA1-1, SCA1-3, SCA1-4 and SCA1-5, infrared spectroscopy was performed on them. The results are as follows Figure 4 As shown. After analysis, the four purified components were found to have a wavelength of 3600-3200 cm -1 There are absorption peaks at 2934cm, which are the stretching vibration absorption peaks of sugar ring OH; -1 、2936cm -1 and 2963cm -1 There is a weak absorption peak at 1616cm, 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 1404cm, which is the asymmetric stretching vibration of -COO-; -1 、1409cm -1 、1406cm -1 and 1404cm -1 The -COO- symmetrical stretching vibration is at 1258cm, indicating that all four components contain a certain amount of uronic acid, which is consistent with the results of carbazole colorimetry. The monosaccharide results may not be seen due to the low content. -1 、1256cm -1 、1231cm -1 and 1252cm -1 It is the characteristic peak of the symmetrical stretching vibration of sulfate ester S=O, indicating that all four components contain a small amount of sulfate groups; at 818cm -1 The absorption peak near the C6 position indicates that it is a sulfate group; 950-940cm -1 The absorption peak at 1010-1100 indicates the presence of pyranose rings in all four components.

[0049] 4.4 Congo Red Assay: Take 1 mL of a 1.0 mg / mL polysaccharide sample solution, add 1 mL of an 80 μmol / L Congo Red solution, and mix thoroughly. Sequentially add 1 mol / L NaOH to adjust the final NaOH concentrations to 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. Incubate at room temperature for 5 min. Perform a full UV wavelength scan in the range of 400-800 nm, and record the maximum wavelength.

[0050] Test results: Congo red is an acidic dye that can react with the triple helix structure of polysaccharides when the concentration of NaOH solution is low. As the concentration of NaOH solution increases, the maximum wavelength λ maxIt will shift to the long-wave direction and red-shift. When the NaOH concentration reaches a certain value, the complex will hydrolyze and the maximum wavelength will decrease. Figure 5 As can be seen, after mixing SCA1-1, SCA1-3, and SCA1-4 with Congo red solution, the wavelengths of the mixed solutions exhibited a significant red shift compared to the maximum wavelength of the Congo red solution within the 0-0.4 mmol / L concentration range, indicating that SCA1-1, SCA1-3, and SCA1-4 possess triple helical structures. The maximum wavelength of SCA1-5 decreased with increasing NaOH concentration, indicating that SCA1-5 does not have a triple helical structure.

[0051] 4.5 Nuclear Magnetic Resonance (NMR) Analysis: Dissolve 50 mg of SCA1-1 sample in 0.5 mL of heavy water and freeze-dry. Repeat this process three times to complete the heavy water exchange. Redissolve the sample in 0.5 mL of D2O and transfer it to an NMR tube for analysis. Measure at 25°C and 600 MHz. 1 H-NMR, 13 C-NMR, 1 H- 1 HCOSY), HSQC and HMBC.

[0052] The detailed structural information of SCA1-1 was further elucidated by NMR spectroscopy: 1 In the HNMR spectrum, the chemical shift of the terminal hydrogen is concentrated in the range of δ5.5-4.4ppm. 1 HNMR spectrum ( Figure 6 The peak at δ1.2-1.4 ppm is the rhamnose methyl group signal, indicating the possible presence of rhamnose in SCA1-1, which is consistent with the monosaccharide composition results. The peak at around δ2.0 ppm is generally the methyl group signal of the acetyl group, indicating the possible presence of acetylamino, which is consistent with the monosaccharide composition results.

[0053] The terminal carbon signals of polysaccharides are generally concentrated in the range of δ110-90ppm ( 13 CNMR spectrum). For example, SCA1-1 13 CNMR spectrum ( Figure 7 ) shows that the signals are mainly concentrated in δ100-60ppm, with two terminal carbons (δ101.16, 99.96ppm). C3 or C5 of furanose has signals between δ84-82ppm, while C3 and C5 of pyranose are generally less than δ80ppm. Figure 7A clear signal is observed below δ80 ppm, but no signal is observed between δ84-82 ppm, indicating that SCA1-1 contains only pyranose and no furanose. The peak at δ24.42 ppm is likely the methyl signal of the acetylamino group. The absence of a rhamnose carbonyl signal may be related to the low rhamnose content.

[0054] Evaluation of the in vitro antioxidant activity of purified polysaccharides from Auricularia auricularia 5.1 to O2 - Free radical scavenging method: using phenazine methyl sulfate (PMS)-reduced coenzyme (NADH) system to generate O2 - To 300 μL of polysaccharide sample solutions with different mass concentrations, 50 μL of 72 μmol / L nitro blue tetrazolium chloride (NBT), 30 μmol / L PMS and 338 μmol / L NADH prepared with Tris-HCl buffer (50 mmol / L, pH = 8.2) were added in sequence, mixed evenly, reacted at 25°C for 5 min, and the absorbance of the reaction solution was measured at a wavelength of 560 nm. Vitamin C was used as a positive control, and O2 - The clearance rate is calculated as follows: Clearance rate (%) = [1-(A 样品 -A 空白 ) / A 对照 ]×100%.

[0055] Where A 样品 is the OD560 of the polysaccharide sample; A 空白 The OD560 was determined by using Tris-HCl buffer instead of NBT; A 对照 The OD560 was measured using deionized water instead of the polysaccharide sample.

[0056] Test results: Effects of different concentrations of purified Auricularia auricularia polysaccharides (SCA1-1, SCA1-3, SCA1-4, SCA1-5) and Vc on O2 - Free radical scavenging ability Figure 8 As shown in A. With the increase of polysaccharide concentration, the effect of purified polysaccharide from Auricularia auricula on O2 - The free radical scavenging ability is enhanced. When the concentration is 10mg / mL, its O2 - The order of free radical scavenging effect is SCA1-1>SCA1-5>SCA1-3>SCA1-4.

[0057] 5.2 Scavenging effect on •OH free radicals Method: •OH was generated using the EDTANa2-Fe(II)-H2O2 system. 200μL of phosphate buffer (150mmol / L, pH=7.4), 200μL of 360μg / mL saffron T, 100μL of 2mmol / L EDTANa2-Fe(II) solution, and 200μL of 30% H2O2 solution were added to 200μL of polysaccharide solutions of different mass concentrations, and the absorbance of the reaction solution was measured at a wavelength of 520nm. Using vitamin C as a positive control, the scavenging rate of •OH was calculated as follows: Scavenging rate (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )×100%.

[0058] Where A 样品 is the OD of the polysaccharide sample 520 ; A 空白 The OD was determined by replacing the polysaccharide sample with deionized water. 520 ; A control is to replace the polysaccharide sample and H2O2 with deionized water to measure OD 520 .

[0059] Test results: Figure 8 As shown in Figure B, the four purified Auricularia auricularia polysaccharides exhibited a concentration-dependent ability to scavenge •OH free radicals. This ability increased with increasing polysaccharide concentration. SCA1-1 exhibited a strong •OH free radical scavenging ability, approaching that of Vc at a concentration of 10 mg / mL.

[0060] 5.3 DPPH Radical Scavenging Method: Add 200 μL of 0.04 mg / mL DPPH ethanol solution to 200 μL of polysaccharide sample solutions of different mass concentrations, mix well, react at 25°C for 30 minutes, and measure the absorbance at 517 nm. Using vitamin C as a positive control, the DPPH scavenging rate was calculated as follows: Clearance rate (%) = [1-(A 样品 -A 空白 ) / A 对照 ]×100%.

[0061] Where A 样品 is the OD of the polysaccharide sample 517 ; A 空白 OD was determined by using anhydrous ethanol instead of DPPH 517 ; A 对照 The OD was determined by replacing the polysaccharide sample with deionized water. 517 .

[0062] Test results: Figure 8 As shown in Figure C, within the 0-10 mg / mL concentration range, the four purified Auricularia auricularia polysaccharides exhibited some DPPH radical scavenging activity, which increased with increasing polysaccharide concentration. At a concentration of 1 mg / mL, the DPPH radical scavenging abilities of SCA1-1 and SCA1-5 approached those of Vc. At a concentration of 10 mg / mL, SCA1-1 exhibited the strongest DPPH radical scavenging activity, while SCA1-4 exhibited the weakest.

[0063] 5.4 Fe 2+ Free radical chelating ability method: add 20 μL 2mmol / L FeCl2 and 40 μL 5mmol / L ferrozine solution to 200 μL polysaccharide sample solution of different mass concentrations, mix well, react at 25℃ for 10 minutes, and measure the absorbance at 562nm. EDTANa2 was used as positive control. 2+ The chelation rate is calculated as follows:

[0064] Chelation rate (%) = [1-(A 样品 -A 空白 ) / A 对照 ]×100%.

[0065] Where A 样品 is the OD of the polysaccharide sample 562 ; A 空白 OD was determined by replacing FeCl2 with deionized water. 562 ; A 对照 The OD was determined by replacing the polysaccharide sample with deionized water. 562 .

[0066] Test results: Figure 8 As shown in D, within the experimental range, the four purified polysaccharides from Auricularia auriculariae showed strong Fe 2+ With the increase of polysaccharide concentration, the chelating ability of purified polysaccharide from Auricularia auricula to Fe 2+ When the concentration was 10 mg / mL, SCA1-1 and SCA1-5 had a strong chelating ability on Fe 2+ The chelating ability of EDTA is strong, close to that of EDTA.

[0067] 6 Evaluation of the anticoagulant activity of purified polysaccharides from Auricularia auricularia ceylon 6.1APTT assay method: The activated partial thromboplastin time (APTT) assay kit was used for determination.

[0068] Test results: Figure 9As shown, SCA1-1, SCA1-3, SCA1-4, and SCA1-5 effectively prolonged the APTT time in a dose-dependent manner, indicating that all of these components exert anticoagulant activity by inhibiting the intrinsic coagulation pathway. The anticoagulant activity of the four purified Auricularia auricularia polysaccharides was lower than that of heparin. Within the 2-5 mg / mL range, SCA1-1 exhibited higher anticoagulant activity than the other three purified polysaccharides.

[0069] 6.2 PT determination method: Prothrombin time (PT) detection kit is used for determination.

[0070] like Figure 10 As shown, all four purified polysaccharides from Auricularia auriculariae exhibited a dose-dependent effect in prolonging the PT coagulation time, indicating that they all have anticoagulant effects by inhibiting the extrinsic coagulation pathway. Within the 0-5 mg / mL range, SCA1-4 exhibited the strongest PT prolongation, approaching that of heparin.

[0071] 6.3 TT determination method: Thrombin time (TT) detection kit is used for determination.

[0072] The test results are as follows Figure 11 As shown, all four purified Auricularia auricularia polysaccharides were able to prolong TT, demonstrating their ability to inhibit fibrin formation and exert an anticoagulant effect. Within the 3-5 mg / mL concentration range, SCA1-1 exhibited higher anticoagulant activity than the other three purified polysaccharides and was comparable to that of heparin.

[0073] In summary, the results of APTT, PT and TT assays showed that SCA1-1 had the strongest anticoagulant activity and could effectively inhibit the intrinsic coagulation pathway as well as the common coagulation pathway, but its effect in inhibiting the extrinsic coagulation pathway was weaker than that of SCA1-4.

[0074] Study on the anti-inflammatory activity of purified polysaccharide SCA1-1 from Auricularia auricularia auricula in vitro 7.1 Sample Solution Preparation: Prepare SCA1-1 in distilled water to a concentration of 4 mg / mL, filter through a 0.22 μm filter, and store at 4°C. Dilute the sample solution sequentially with DEME medium to 200, 100, 50, 25, 12.5, and 6.25 μg / mL. Prepare dexamethasone in anhydrous ethanol to a concentration of 1 mg / mL, filter through a 0.22 μm filter, and store at -20°C.

[0075] 7.2Cultivation of Raw264.7 cells: (1) Cell recovery: Preheat the water bath to 37°C, take out the cryovials from the -150°C freezer, and thaw them in a 37°C water bath. After complete thawing, add 10 mL of complete culture medium (DMEM basal culture medium: fetal bovine serum: penicillin-streptomycin double antibody solution = 89:10:1), centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove DMSO. Pipette 1 mL of culture medium and blow the cells. After blowing and mixing, inoculate the cells into a 10 cm plate containing 10 mL of culture medium. 2 Place the culture dish in a 37°C, 5% CO2 cell culture incubator and culture. Observe the cell status and density regularly. When the cells adhere to the wall and the density reaches about 80%, the cells are passaged.

[0076] (2) Cell passaging: Use a disposable dropper to remove the old culture medium from the culture dish, add 3-5 mL of PBS to wash the cells three times, then add PBS and use a 1 mL pipette to blow the cells off the bottom of the culture dish. Centrifuge at 1000 rpm for 5 minutes. Use 1 mL of fresh culture medium to blow the cells to evenly disperse them. Transfer the cell suspension to a new culture dish (containing 10 mL of fresh culture medium) at a ratio of 1 to 2, and place it in an incubator for culture.

[0077] (3) Cell freezing: Prepare cell freezing solution at a ratio of fetal bovine serum to DMSO = 9:1. Select cells in the logarithmic growth phase that have not been activated. Discard the old culture medium first, rinse with PBS three times, and then gently pipette the cells with a 1 mL pipette. After centrifugation, discard the supernatant and add 1 mL of the prepared cell freezing solution. Pipet and mix thoroughly. Transfer to a labeled cryotube, then place in a gradient cooling box, freeze in a -80°C refrigerator overnight, and finally transfer to a -150°C refrigerator.

[0078] 7.3 Cytotoxicity test: Observe the cell status under a microscope, select cells with good growth status, and prepare cell suspension using the above-mentioned passaging method. Count the cells using a hemocytometer, and calculate the number of cells per 5×10 5 Cells were seeded at a density of 100 μg / mL in a 96-well plate, and PBS was added to the outermost circle of the plate. After 24 hours of incubation, the culture medium was aspirated, and 100 μL of culture medium was added to each well of the control group. 100 μL of SCA1-1 solution at 200, 100, 50, 25, 12.5, or 6.25 μg / mL was added to each well of the SCA1-1 group. Six replicates of each concentration were added and incubated for 24 hours. The supernatant was aspirated, and 20 μL of 5 mg / mL MTT was added to each well. The cells were incubated in the incubator for another 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken on a shaker for 10 minutes until the formazan was completely dissolved. The absorbance of each well at 490 nm was measured using a microplate reader.

[0079] The MTT assay was used to detect the toxicity of SCA1-1 to Raw264.7 cells to avoid unnatural cell death under the action of polysaccharides. Figure 12 As shown in the results, SCA1-1 showed no cytotoxicity to RAW264.7 cells within the range of 6.25-50 μg / mL, and had relatively little effect on cell viability within the range of 6.25-25 μg / mL. Therefore, the following SCA1-1 concentrations were selected in subsequent experiments: 6.25, 12.5, and 25 μg / mL.

[0080] 7.4 Determination of NO and Cellular Inflammatory Factors: Following the seeding method described above for the toxicity experiment, cells were cultured for 24 h, and the supernatant discarded. The control group was treated with 100 μL of culture medium, the LPS group with 100 μL of LPS solution, and the DEX (dexamethasone) group with 100 μL of DEX solution containing 1 μg / mL LPS (final concentration of 12.5 μg / mL). The SCA1-1 polysaccharide group (containing 1 μg / mL LPS at final concentrations of 6.25, 12.5, and 25 μg / mL) was treated with 100 μL of polysaccharide solutions of varying concentrations. After 24 h of culture, the cell supernatant was collected and assayed for NO and inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) using the nitrate reductase method and ELISA, respectively, according to the manufacturer's instructions.

[0081] The production of NO in the body is catalyzed by nitric oxide synthase. During inflammatory reactions, inflammatory factors can activate iNOS to produce excessive NO, thereby making the inflammatory reaction more intense. Therefore, the effect of SCA1-1 on LPS-induced NO production was studied. The results are as follows: Figure 13 A. LPS significantly promoted NO production in Raw264.7 cells compared with the control group. Compared with the LPS group, SCA1-1 significantly inhibited LPS-induced NO levels in Raw264.7 cells in a concentration-dependent manner.

[0082] LPS stimulates macrophages to produce TNF-α, IL-1β, and IL-6, which are considered to be one of the most important cytokines that promote inflammatory responses and can lead to acute inflammatory diseases and tissue damage. Therefore, the present invention studies the use of these cytokines as inflammatory markers to test the anti-inflammatory effect of SCA1-1. The results are as follows Figure 13BD. Compared with the LPS group, SCA1-1 significantly inhibited LPS-induced TNF-α and IL-6 secretion 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 suggest that SCA1-1, at appropriate concentrations, has a potent anti-inflammatory potential against LPS-stimulated inflammation by suppressing NO, TNF-α, IL-1β, and IL-6 secretion.

[0083] 7.5 Western Blot: (1) Protein extraction was performed at 2×10 6 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours. The supernatant was removed and the cells were treated according to group. After 24 hours of treatment, the cells were washed three times with PBS, harvested by pipetting, placed in a sterile EP tube, and centrifuged (1000 rpm for 5 minutes). The supernatant was discarded, and the cells were lysed with RIPA buffer for 30 minutes and centrifuged at 12000 rpm for 20 minutes at 4°C (the entire process should be performed on ice). Protein concentration was determined using a BCA protein assay kit. Lysates were denatured at 100°C for Western blotting.

[0084] (2) Glue preparation: Place a clean glass plate on a horizontal laboratory table, align it, and clamp it on 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 recipe, vortex and shake to mix, and quickly use a pipette to pour the gel into the gap between the two glass plates until the liquid level is about 1 cm away from the highest point of the short glass plate. Stop pouring the gel, then slowly add anhydrous ethanol to seal the liquid. Let it stand at room temperature for 20 minutes. When a clear dividing line appears at the junction of anhydrous ethanol and separation gel, pour out the anhydrous ethanol. Prepare the top layer of gel according to the recipe, shake and mix, then add the top layer of gel to the solidified separation gel until it is flush with the top of the short plate. Immediately insert a clean comb straightly to avoid bubbles. Let it stand for 15 minutes. After the gel solidifies, slowly and carefully remove the comb.

[0085] Table 3 Preparation formula of separation gel and stacking gel

[0086] (3) Electrophoresis: Place the gel plate in the electrophoresis tank, add electrophoresis solution, rinse the sample wells with electrophoresis solution, and straighten the gel teeth between the sample wells with a needle. Add the Maker and protein sample to the sample wells and cover the electrophoresis tank electrodes (positive to positive, negative to negative). First, adjust the voltage to 90V and perform constant voltage electrophoresis until the bromophenol blue indicator migrates to the junction of the lower and upper gel layers and the marker begins to separate. Then, increase the voltage to 120V and stop the electrophoresis when the dye moves to 1 cm from the bottom of the gel plate.

[0087] (4) Transfer: Soak the sponge pad and filter paper in transfer buffer and set aside. Use a gel cutter to cut off the concentrated gel and excess gel, and keep it moist with transfer buffer. Cut the PVDF membrane to the same size as the gel and activate it in methanol for 30 seconds. Assemble in the order of cathode plastic clip, sponge pad, filter paper, PVDF membrane, gel, filter paper, sponge pad, and anode plastic clip. Exclude bubbles between each layer and keep the gel and membrane moist. Avoid drying. Place in the transfer tank (positive to positive, negative to negative), pour in 4°C transfer buffer, cover with transfer cap, ice bath, 150mA constant current for 90-120min, and adjust the transfer time according to the molecular weight of the target protein.

[0088] (5) Blocking: Remove the PVDF membrane, wash it with TBST for 5 minutes, place it in 5% skim milk powder, and block it on a shaker at room temperature for 2 hours.

[0089] (6) After blocking with primary antibody, wash the membrane three times with TBST, 10 min each time. Cut the membrane into the appropriate molecular weight size according to the Maker, place it in the primary antibody incubation box, and incubate at 4°C overnight.

[0090] (7) Secondary Antibody Incubation: Wash the membrane three times, washing once every 10 minutes, and incubate with the secondary antibody for 1 hour. Wash once every 10 minutes, for a total of three washes.

[0091] (8) Exposure: Prepare solution A and solution B in a 1:1 ratio using the ECL kit (prepare immediately before use, in the dark). Evenly drop the mixture onto the PVDF membrane and use a chemiluminescence imaging system to detect the bands. Use ImageJ software to quantitatively analyze the grayscale values of the protein bands.

[0092] WesternBolt result analysis: NF-κB is an important transcription factor that positively regulates the expression of inflammatory mediators such as TNF-α and IL-1β, and plays a key role in the development of inflammation. Its expression was detected by Western blotting. The results of Western blotting showed that ( 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, suggesting that SCA1-1 can exert anti-inflammatory effects through the NF-κB signaling pathway.

[0093] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional polysaccharide, characterized in that: The steps include: (1) drying, crushing and defatting the Ceylon sea fungus to obtain defatted powder; (2) mixing the defatted powder with water, extracting at room temperature, and separating the solid and liquid to obtain a cold water extraction residue; (3) mixing the cold water extraction residue with water, performing heating extraction, and performing solid-liquid separation to obtain a hot water extraction residue; (4) Mixing the hot water extraction residue with alkaline solution, heating extraction, and solid-liquid separation to obtain an extract; (5) adjusting the pH of the extract, concentrating, precipitating with alcohol, dialyzing, and freeze-drying to obtain crude polysaccharide; (6) separating the crude polysaccharide using a DEAE Sepharose Fast Flow anion exchange chromatography column to obtain a first fraction; (7) Sephacryl S-300 gel column is used to separate the first component to obtain the multifunctional polysaccharide.

2. The method for preparing a multifunctional polysaccharide according to claim 1, wherein: The liquid-to-solid ratios in steps (2) to (4) are each independently selected from 40:1 to 80:1, the amount of liquid used is in mL, and the amount of solid used is 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 alkali solution is a 3wt%-5wt% NaOH solution.

3. The method for preparing a multifunctional polysaccharide according to claim 1, wherein: In step (1), the Ceylon fungus powder is defatted using 80%-90% ethanol by volume, and the ratio of the Ceylon fungus powder to the 80%-90% ethanol is 50 g / L; The degreasing was carried out at 85° C. for 3 h; the operation was repeated once after the first degreasing was completed.

4. The method for preparing a multifunctional polysaccharide according to claim 1, wherein: In step (6), linear elution was performed on an ÄKTA-FPLC instrument using a 0-2 mol / L NaCl solution at a flow rate of 1 mL / min. The collected eluate was used to determine the polysaccharide content using the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and an elution curve was drawn. The first component is determined based on the elution curve.

5. The method for preparing a multifunctional polysaccharide according to claim 1, wherein: In step (7), 0.1 mol / L NH4HCO3 solution was selected as the mobile phase, and elution was performed at a flow rate of 1 mL / min. The collected eluate was used to determine the polysaccharide content by the phenol-sulfuric acid method, and the absorbance was measured at 490 nm to draw an elution curve; The multifunctional polysaccharide is determined based on the elution curve.

6. A multifunctional polysaccharide, characterized in that: The multifunctional polysaccharide is prepared by the method according to any one of claims 1 to 5.

7. The multifunctional polysaccharide according to claim 6, wherein The monosaccharide composition of the multifunctional polysaccharide includes glucose, galactose, mannose, fucose, rhamnose and galactosamine, and contains sulfate groups; The infrared spectrum of the multifunctional polysaccharide has a stretching vibration absorption peak of the sugar ring OH, a fucose methyl absorption peak, an asymmetric stretching vibration of -COO-, a symmetric stretching vibration of -COO-, a sulfate group S=O symmetric stretching vibration characteristic peak and a 3,6-ether-galactose absorption peak.

8. The multifunctional polysaccharide according to claim 7, wherein The monosaccharide composition of the multifunctional polysaccharide is as follows: 11.42 wt% glucose, 27.73 wt% galactose, 23.65 wt% mannose, 18.06 wt% fucose, 13.17 wt% rhamnose and 5.97 wt% galactosamine; The multifunctional polysaccharide has a triple helical structure; The multifunctional polysaccharide contains a pyranose structure.

9. Use of the multifunctional polysaccharide according to claim 7 or 8 in the preparation of at least one of antioxidant, anticoagulant and anti-inflammatory products.

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