CN117088991A - Phellinus linteus polysaccharide, and preparation method and application thereof in preparation of immunomodulatory drugs - Google Patents
Phellinus linteus polysaccharide, and preparation method and application thereof in preparation of immunomodulatory drugs Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Public Health (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Sustainable Development (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application discloses a preparation method of a phellinus linteus polysaccharide IHP-1aa, which comprises the following steps: removing fat by ethanol, extracting with water, precipitating with ethanol to obtain the final product, removing protein by Sevage method, separating neutral polysaccharide component by DEAE-52 cellulose column and DEAE agarose gel column, and purifying by gel G-100. The polysaccharide of the Inonotus obliquus has uniform purity and definite composition, is a heteropolysaccharide composed of glucose, galactose, mannose, fucose and methyl galactose, and has a molecular weight of 26.9kDa. In vitro experiments show that: the recombinant human macrophage RAW264.7 has NO adverse effect on normal cells, can obviously stimulate the macrophage RAW264.7 to secrete NO, IL-6 and TNT-alpha, has good immunoregulatory activity, and can be used for developing immunoregulatory products.
Description
Technical Field
The application belongs to the technical field of polysaccharides, and particularly relates to a phellinus linteus polysaccharide IHP-1aa with immunoregulatory activity, a preparation method thereof and application thereof in preparing immunoregulatory medicines.
Background
Polysaccharides are a further important class of macromolecules in organisms, in addition to proteins and nucleic acids, and are widely found in animals, plants and microorganisms. In recent years, polysaccharides have attracted a great deal of attention due to their good physiological activity and safety and non-toxicity characteristics, and fungal polysaccharides have gradually become a research hotspot as a broad-spectrum immune activator. The biological activity of polysaccharides is closely related to its structural features, including molecular weight, monosaccharide and glycosidic bond composition, degree of branching, chain conformation, and the like.
Fomitopsis pinicola (Inonotus hispidus) is a relatively high value, both edible and medicinal saprophytic fungus that has been found to be parasitic on various broad-leaved trees in China and Europe, including mulberry, ash, aspen, elm, sorbitol and maple. In the axsu region of Xinjiang and the Shandong Xiajin county of China, phellinus linteus on mulberry is used as a traditional Chinese medicine of Phellinus linteus for treating dyspepsia, cancer, diabetes, gastropathy and the like. Modern researches have found that the extract of the trephine hirsutum has good anti-tumor, antioxidant, antibacterial, antiviral, neuroprotective and other activities. Among them, polysaccharide is one of its main active ingredients, but there has been no study on the structure and immunomodulatory activity of the polysaccharide of Fomitopsis pinicola. Therefore, the active polysaccharide in the trephine is prepared by separation and purification, the structure of the active polysaccharide is characterized, the efficacy and the application of the active polysaccharide are researched, and the method has important significance for the development and the utilization of trephine resources.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide the polysaccharide of the phellinus linteus with immunoregulation activity, and the prepared polysaccharide HP-1aa has uniform purity and definite structure, and in vitro experiments show that the polysaccharide HP-1aa can obviously stimulate macrophage RAW264.7 to secrete NO, IL-6 and TNT-alpha, has good immunoregulation activity, and can be used for enhancing the development and utilization of immunocompetence products.
The application also provides a preparation method of the phellinus linteus polysaccharide IHP-1aa and application of the phellinus linteus polysaccharide IHP-1aa in preparation of immunoregulation medicines.
In order to achieve the above purpose, the application adopts the following technical scheme:
a method for preparing a polysaccharide of a trephine, comprising the following steps:
1) Raw material degreasing treatment: cutting dried fruiting body of Phellinus linteus, soaking in ethanol water solution for removing fat to obtain crude fruiting body extract;
2) Water extraction and alcohol precipitation: according to the feed liquid ratio of 1g:8-30ml of distilled water is added into the fruiting body crude extract, the fruiting body crude extract is heated to 70-90 ℃ for leaching for 2-5h, ethanol is added into the concentrated extract until the final concentration of the ethanol is 75%, the mixture is stood for ethanol precipitation, and the precipitate is collected and dried in the shade;
3) Removing protein: removing protein by adopting a Sevage method, precipitating with ethanol again, collecting precipitate, and freeze-drying to obtain crude polysaccharide of the trephine crude polysaccharide after removing protein;
4) And (3) separating and purifying: separating and eluting crude polysaccharide of the crude phellinus linteus after removing protein by using a DEAE cellulose-52 and DEAE-Sepharose Fast Flow ion exchange chromatographic column, dialyzing, freeze-drying, purifying and eluting by using a Sephadex G-100 chromatographic column, collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain the crude phellinus linteus polysaccharide named IHP-1aa.
Specifically, in the step 1), the volume concentration of the ethanol water solution is 70-80%, and the soaking time is 60-84 hours.
Specifically, in the step 2), standing and alcohol precipitating are carried out for 20-28h at the temperature of 2-6 ℃.
Specifically, in step 4), the separation and elution are performed through a DEAE cellulose-52 ion exchange chromatography column, specifically: dissolving crude polysaccharide of Fomitopsis pinicola after removing protein in distilled water to obtain 15-25mg/ml solution, centrifuging, collecting supernatant, filtering, loading, eluting with distilled water, collecting eluate rich in polysaccharide (eluate rich in polysaccharide refers to colored tube number after developing with phenol-sulfuric acid method), and concentrating under reduced pressure.
Further, in the step 4), the separation elution and dialysis by a DEAE-Sepharose Fast Flow ion exchange chromatography column are specifically: and (3) loading the polysaccharide solution purified by the DEAE-52 ion exchange chromatography column onto the DEAE-Sepharose Fast Flow ion exchange chromatography column, eluting by using distilled water, collecting eluent rich in polysaccharide, and concentrating under reduced pressure.
Specifically, in the step 4), sephadex G-100 chromatographic column is used for purification and elution, specifically: the polysaccharide solution purified by the DEAE-Sepharose Fast Flow ion exchange column was applied to a Sephadex G-100 column and eluted with distilled water.
Further, in the step 4), the molecular weight cut-off of the dialysis bag selected during dialysis is 8000-12000 Da.
The application provides the phellinus linteus polysaccharide IHP-1aa prepared by the preparation method. The Inonotus obliquus polysaccharide IHP-1aa is a heteropolysaccharide composed of glucose, galactose, mannose, fucose and methyl galactose, and has a molecular weight of 26.9kDa and a molecular structure shown below.
The application also provides application of the phellinus linteus polysaccharide in preparing immunoregulation medicines.
The application aims to solve the defects in the existing research, and provides a crude downy mildew polysaccharide IHP-1aa with immunoregulatory activity and a preparation method thereof (the crude downy mildew polysaccharide IHP-1aa is obtained by ethanol degreasing, water extraction and ethanol precipitation, then protein removal by a Sevage method, separation of neutral polysaccharide components by a DEAE-52 cellulose column and a DEAE agarose gel column and final purification by gel G-100), and the prepared crude downy mildew polysaccharide HP-1aa has uniform purity and definite structure, and in vitro experiments show that the crude downy mildew polysaccharide IHP-1aa can obviously stimulate macrophages RAW264.7 to secrete NO, IL-6 and TNT-alpha, has good immunoregulatory activity and can be used for development and utilization of immunocompetence enhancement products. Compared with the prior art, the application has the following beneficial effects:
the application obtains macromolecular polysaccharide HP-1aa with biological activity for the first time through extracting and separating the trepang, the neutral sugar content of the polysaccharide is detected to be more than 95%, and the polysaccharide is a heteropolysaccharide composed of glucose, galactose, mannose, fucose and methyl galactose through monosaccharide composition identification. High performance gel permeation chromatography demonstrated uniform purity and a molecular weight distribution of 26.9kDa. The nuclear magnetic resonance spectrum determines the connection mode of the glycosidic bond and the polysaccharide structure.
The preparation method of the application has simple operation and good repeatability, can obtain macromolecules with definite structures, and provides research value for further researching the structure-activity relationship. The polysaccharide prepared by the method can better retain the natural structure, has lower equipment requirement, and is beneficial to large-scale popularization, development and use in industrial production.
The prepared Inonotus obliquus polysaccharide IHP-1aa has high purity, can obviously stimulate macrophage RAW264.7 to secrete NO, IL-6 and TNT-alpha, has good immunoregulatory activity, can be used for developing immunopotentiator products, and is helpful for improving the application value of Inonotus obliquus fruiting bodies.
Drawings
In FIG. 1, A is an elution profile of a DEAE cellucose-52 ion exchange chromatography column; b is an elution curve graph of the DEAE-Sepharose Fast Flow ion exchange chromatographic column; c is the elution profile of a Sephadex G-100 column (Absorbance at 490nm is the Absorbance at 490nm and tube number is the tube number).
In FIG. 2, A is a purity detection HPGPC chart of IHP-1aa and dextran standard curves; b is the monosaccharide composition of IHP-1aa.
FIG. 3 is an NMR spectrum of IHP-1aa, respectively 1 H-NMR spectrum (A), 13 c atlas (B), 1 H- 1 H COSY(C)， 1 H- 13 C HMBC(D)，HSQC(E)，TOSY(F)；
FIG. 4 is the effect of IHP-1aa on RAW264.7 cell viability (A); IHP-1aa affects NO (B), IL-6 (C), TNF- α (D) in RAW 264.7.
Detailed Description
The following describes the technical scheme of the present application in further detail with reference to examples, but the scope of the present application is not limited thereto.
Hymenochaetaria crassipes (Fr.) KummerInonotus hispidus) Fruit body is purchased fromNanyang Phellinus linteus agricultural development Co. In the following examples, the ethanol concentrations refer to the volume percent concentrations unless otherwise specified.
Example 1:
a preparation method of a phellinus linteus polysaccharide IHP-1aa comprises the following steps:
1) Raw material degreasing treatment: drying and cutting Phellinus linteus, soaking in 75% ethanol water solution (10 ml of 75% ethanol water solution, w/v) for twice for degreasing, soaking for 72 hr each time, and filtering to obtain residue as crude extract of fruiting body.
2) Water extraction and alcohol precipitation: extracting the residue with distilled water (feed-liquid ratio, 1:20, g/ml) at 80deg.C for 3 hr, filtering, collecting filtrate, and repeating the operation three times. Mixing the extractive solutions, concentrating under reduced pressure at 55deg.C to obtain solution with density of 1.2g/cm 3 Adding 95% ethanol under stirring to adjust the final concentration of ethanol to 75%, standing at 4deg.C for 24 hr, filtering, collecting precipitate, and drying in the shade.
3) Removing protein: protein removal using Sevage reagent (chloroform: n-butanol=4:1, v/v) (which is a conventional technique in the art and is not an innovation of the present application and is not described in detail), re-alcohol precipitation (i.e., adding 95% ethanol with stirring to an ethanol final concentration of 75%, standing at 4 ℃ for 24 hours), filtration and collection of the precipitate, vacuum freezing at-60 ℃ for 12 hours to complete drying, obtaining crude polysaccharide of the crude trephine after removal of the protein for further purification.
4) Taking 300mg of crude polysaccharide of the crude polyporus crude polysaccharide after removing protein, dissolving in 15mL of distilled water to prepare 20mg/mL solution, centrifuging (at 8000rpm,5 min), taking supernatant, filtering by a 0.45 mu m filter membrane, loading 15mL to a DEAE cellucose-52 ion exchange chromatographic column (2.6X40 cm), purifying, sequentially carrying out gradient elution (with a flow rate of 1mL/min and 8 min/tube) by using distilled water, 0.05, 0.1 and 0.2mol/L NaCl solution, collecting eluent by using an automatic collector, carrying out color development detection by using a phenol-sulfuric acid method, measuring absorbance (OD value) at 490nm, taking the elution tube number as an abscissa and the absorbance as an ordinate, drawing a polysaccharide elution curve (see FIG. 1A), combining polysaccharide samples of the same elution peak, concentrating under reduced pressure at 55 ℃, dialyzing for 24h (dialysis bag cut-off molecular weight 8000-12000 Da, 3 times) at 4 ℃, and carrying out vacuum freeze drying for 12h at 60 ℃, wherein the elution peak of distilled water is a group of P-1 and the IH peak of P-0.05 mol/L NaCl is a group of P-P2.
5) Dissolving the main component (IHP-1) obtained in the step 4) in 10mL of distilled water, loading the solution into a DEAE-Sepharose Fast Flow chromatographic column (2.6X40 cm) for purification, eluting with distilled water (the flow rate is 1mL/min,8 min/tube), developing by using a phenol-sulfuric acid method, drawing a polysaccharide elution curve (see B in figure 1) according to absorbance (OD value) at 490nm to obtain a single elution peak, concentrating the polysaccharide sample of the elution peak under reduced pressure at 55 ℃, dialyzing for 24 hours (dialysis bag cut-off molecular weight 8000-12000 Da at 4 ℃ for 3 times, changing water), and freeze-drying at 60 ℃ in vacuum for 12 hours to obtain a distilled water eluting component (IHP-1 a).
6) Dissolving the distilled water eluting component (IHP-1 a) obtained in the step 5) in 5ml of distilled water, loading the distilled water into a Sephadex G-100 chromatographic column for further purification, eluting with distilled water (the flow rate is 0.1 mL/min,25 min/tube), developing by using a phenol-sulfuric acid method, drawing a polysaccharide eluting curve according to absorbance (OD value) at 490nm, displaying the eluting curve as a group of uniform eluting peaks (see C in figure 1), merging, concentrating under reduced pressure at 55 ℃, dialyzing for 24 hours at 4 ℃ (the molecular weight cutoff of a dialysis bag is 8000-12000 Da, changing water for 3 times), and performing vacuum freeze drying at 60 ℃ to obtain the high-purity refined crude downy porus polysaccharide (IHP-1 aa).
The obtained crude downy mildew polysaccharide IHP-1aa is white flocculent powder at normal temperature, and is preserved at room temperature (below 25 ℃) in dark and dry conditions.
The purified Inonotus obliquus polysaccharide (IHP-1 aa) prepared in example 1 was subjected to the relevant detection and cell assay.
Example 2 purity detection:
FIG. 2 shows a purity detection HPGPC chart of the Indomycete polysaccharide IHP-1aa and a dextran standard curve (A) and monosaccharide composition (B) of IHP-1aa.
As shown in FIG. 2, the purity and molecular weight of the Ind. Crude polysaccharide IHP-1aa obtained in example 1 were measured by HPGPC method, and the measurement was performed by using a Shimadzu LC-20A HPLC system (Shimadzu Co., ltd.) as a Shodex OHPak SB-806 HQ column (10 μm,8 mm X300 mm) equipped with a 20A Refractive Index Detector (RID). The result shows a single symmetrical peak, which proves that the Indomycete polysaccharide IHP-1aa is a uniform polysaccharide. IHP-1aa has a molecular weight of 26.9kDa as obtained by the dextran standard curve.
Example 3 monosaccharide composition detection:
the Indomycosin IHP-1aa prepared in example 1 was assayed by PMP-HPLC. The mass percentage (%, w/w) of each monosaccharide was calculated using an internal standard method, and thus, the obtained Inonotus obliquus polysaccharide IHP-1aa was mainly composed of glucose (51.9%) and galactose (18.9%), which contained a small amount of fucose (9.9%), mannose (5%), methylgalactose and a trace amount of glucuronic acid (0.8%).
Example 4 nuclear magnetic resonance analysis:
30mg of the Inonotus obliquus polysaccharide IHP-1aa prepared in example 1 was dissolved in 0.6mL of 99.9% D 2 O, data were collected in a Bruker Avanced III MHz NMR spectrometer (Brucker, switzerland) and recorded at 298K 1 H-NMR、 13 C-NMR、 1 H- 1 H COSY, TOSY, HSQC and HMBC spectra. Based on monosaccharide composition results, nuclear magnetic analysis and related literature alignment, the chemical shift of the sugar residues of IHP-1aa is shown in Table 1 below, all of which are indicated in FIG. 3 (A-F), and the partial structure of this polysaccharide is predicted to be:
TABLE 1 chemical shift assignment of IHP-1aa sugar residues
Example 5 in vitro cell experiments:
in vitro immunization experiments were performed on the Fomitopsis crude polysaccharide IHP-1aa prepared in example 1
1. Materials and reagents: example 1 isolated and purified Inonotus crassa polysaccharide IHP-1aa; mouse macrophage RAW264.7 (purchased from the national academy of sciences typical culture collection committee cell bank); penicillin and streptomycin mixed solution (Beijing Soy Bao technology Co., ltd.); fetal bovine serum (FBS, gibco BRL, usa), ELISA kit (beggar biotech ltd, beijing); NO kit (Shanghai Biyun biotechnology Co., ltd.).
2. Instrument: a carbon dioxide incubator (Thermo scientific, model 3111); FRESCO 21 centrifuge (Thermo); multiskanGO full wavelength microplate reader (Thermo); 96-well, 24-well cell culture plates (Corning); DMEM medium (prinocetary).
3. Experimental method
(1) Determination of cell viability by MTT method
RAW264.7 mouse macrophages were cultured in DMEM medium (hereinafter referred to as DMEM complete medium) containing penicillin (100U/mL), streptomycin (100. Mu.g/mL) and FBS (10%) at 37℃and 5% C0 2 Culturing under the condition. The MTT assay detects the effect of IHP-1aa on RAW264.7 cell viability. Briefly, cells were grown at 1.5X10 4 The number of individual wells/well was inoculated in 96-well plates and placed at 37℃in 5% CO 2 After 24 hours incubation with different concentrations (50-800. Mu.g/mL) of HP-1aa (100. Mu.L) for 24 hours with DMEM complete medium as negative control. Thereafter, 10 μl of MTT solution (5 mg/mL in PBS) was pipetted into each well and incubation was continued for 4 hours, the supernatant was discarded, then dissolved with 100 μl of DMSO and absorbance values were measured at 490nm using a microplate reader.
Cell viability was calculated as follows: cell viability (%) = a/b×100%, where a is absorbance of the experimental group and B is absorbance of the negative control group.
(2) Detection of NO, IL-6 and TNF-alpha content released by RAW264.7 cells
RAW264.7 cells were grown at 1.5X10 5 Individual cells/wells were seeded in 24-well plates at 37℃with 5% CO 2 Incubation for 24 hours In Incubator (II) with LPS (1. Mu.g/mL, positive group) and DMEM, respectivelyWhole medium (control) and IHP-1aa (200, 400, 800. Mu.g/mL) at various concentrations at 37℃with 5% CO 2 The lower treatment was carried out for 24 hours. RAW264.7 cell culture supernatants were collected and assayed for production of NO and cytokines IL-6, TNF- α using Griess and ELISA kits, respectively.
4. Data processing
Results were expressed as mean ± SD values, and data statistics were compared for significant differences using Graphpad prism 9.5 software one-way anova.
5. Experimental results
NO is a non-specific molecule secreted by activated macrophages that kills or inhibits the growth of a variety of pathogenic microorganisms. Activated macrophages can produce a variety of cytokines (e.g., TNF- α and IL-6) to regulate cellular and humoral immune responses. FIG. 4A demonstrates that IHP-1aa is non-cytotoxic and FIG. 4 (B-D) shows the effect of IHP-1aa on the production of immunocompetent factors. FIG. 4B shows that the production of NO is significantly increased over the control group at IHP-1aa concentrations of 200, 400 and 800. Mu.g/mL. After 24h cells were stimulated with 800. Mu.g/mL IHP-1aa, the NO secretion reached 23.47.+ -. 0.52. Mu.M, an increase of 10.9. Mu.M compared to the blank group, was 56.24% of the LPS group, and the differences were all significant (P < 0.001). Meanwhile, IHP-1aa at different concentrations (200-800 μg/mL) significantly promoted the secretion of IL-6 by RAW264.7 cells compared to the placebo group, and exhibited a dose-response effect (C in FIG. 4). As shown in FIG. 4D, TNF-. Alpha.was significantly elevated at 400. Mu.g/mL (p < 0.001) compared to the blank. In conclusion, a significant increase in NO production suggests that the Indomycosin IHP-1aa may have good immunostimulatory effect and the ability to inhibit tumor cell proliferation. The release of cytokines TNF-alpha and IL-6 shows that IHP-1aa has good immunopotentiation effect, which indicates that the cytokine TNF-alpha and IL-6 can be used for preparing immunopotentiator.
Claims (9)
1. The preparation method of the trephine crude polysaccharide is characterized by comprising the following steps:
1) Raw material degreasing treatment: cutting dried fruiting body of Phellinus linteus, soaking in ethanol water solution for removing fat to obtain crude fruiting body extract;
2) Water extraction and alcohol precipitation: adding distilled water into fruiting body crude extract, heating to 70-90deg.C, leaching, concentrating the extractive solution, adding ethanol until the final concentration of ethanol is 75%, standing for alcohol precipitation, collecting precipitate, and drying in the shade;
3) Removing protein: removing protein by adopting a Sevage method, precipitating with ethanol again, collecting precipitate, and freeze-drying to obtain crude polysaccharide of the trephine crude polysaccharide after removing protein;
4) And (3) separating and purifying: separating and eluting crude polysaccharide of the crude Maoshima after removing protein by using DEAE cellulose-52 and DEAE-Sepharose Fast Flow ion exchange chromatography columns, dialyzing, purifying and eluting by using Sephadex G-100 chromatography column, collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying.
2. The method for preparing a polysaccharide of Phellinus linteus as set forth in claim 1, wherein in step 1), the volume concentration of the aqueous ethanol solution is 70-80% and the soaking time is 60-84 hours.
3. The method for preparing a polysaccharide of Phellinus linteus as set forth in claim 1, wherein in step 2), the solution is left to stand at 2 to 6℃for 20 to 28 hours.
4. The method for preparing the polysaccharide of the phellinus linteus as set forth in claim 1, wherein in the step 4), the separation and elution are carried out by a DEAE cellulose-52 ion exchange chromatography column, specifically: dissolving crude polysaccharide of Fomitopsis pinicola after removing protein in distilled water, centrifuging, collecting supernatant, filtering, loading, eluting with distilled water, collecting eluate, and concentrating under reduced pressure.
5. The method for preparing the polysaccharide of the phellinus linteus as set forth in claim 1, wherein in the step 4), the polysaccharide is eluted by DEAE-Sepharose Fast Flow ion exchange chromatography column separation and dialysis, specifically: and (3) loading the polysaccharide solution purified by the DEAE cellulose-52 ion exchange chromatography column onto the DEAE-Sepharose Fast Flow ion exchange chromatography column, eluting by using distilled water, collecting the eluent, and concentrating under reduced pressure.
6. The method for preparing the polysaccharide of the trephine crude fiber according to claim 1, wherein in the step 4), purification elution is performed by using a Sephadex G-100 chromatographic column, specifically: the polysaccharide solution purified by the DEAE-Sepharose Fast Flow ion exchange column was applied to a Sephadex G-100 column and eluted with distilled water.
7. The method for producing a polysaccharide of Phellinus linteus as defined in claim 1, wherein the dialysis bag used in the dialysis has a molecular weight cut-off of 8000-12000 Da.
8. A polysaccharide of phellinus linteus prepared by the preparation method of any one of claims 1 to 7.
9. Use of the trepang polysaccharide of claim 8 in the preparation of an immunomodulatory drug.
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