Extraction method and application of ganoderma sub-flexural-stem fruiting body polysaccharide

Abstract

The invention discloses an extraction method and application of ganoderma subflexuosus sporocarp polysaccharide, and belongs to the technical field of microorganisms, the ganoderma subflexuosus sporocarp polysaccharide obtained through the extraction method is high in purity and strong in activity, and in-vitro cell experiments prove that the sporocarp polysaccharide has a remarkable protection effect on HepG2 cell damage induced by carbon tetrachloride. The traditional Chinese medicine composition can be used for preparing liver-protecting medicines to achieve the effect of preventing or treating hepatocyte injury.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to the extraction and application of polysaccharide from Ganoderma lucidum fruiting body. Background Art

[0002] In recent years, bioactive substances from natural products have been discovered and used for health care and treatment of diseases. Among them, polysaccharides have attracted more and more scientific researchers' interest and attention due to their high activity, non-toxicity and no adverse effects. As medicinal fungi, Ganoderma fungi have the effects of calming the nerves, helping sleep, protecting the liver, anti-aging, and prolonging life. Ganoderma subflexipes, commonly known as "bamboo Ganoderma", is a new species of Ganoderma that has just been published in recent years. So far, there has been no literature reporting on the chemical composition and biological activity of Ganoderma subflexipes. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for extracting polysaccharide from Ganoderma lucidum fruiting body and application thereof.

[0004] The technical solution of the present invention is: use of Ganoderma subflexipes fruiting body polysaccharide in preparing liver protection medicine.

[0005] Furthermore, the liver protection drug refers to a drug for preventing or treating liver cell damage.

[0006] A method for preparing the above-mentioned Ganoderma subflexipes fruiting body polysaccharide comprises the following steps:

[0007] (1) crushing the dried Ganoderma lucidum fruiting body to obtain fruiting body powder;

[0008] (2) soaking the fruiting body powder in ethanol, removing the supernatant and drying the residue;

[0009] (3) washing the filter residue treated in step (2) with hot water, collecting the filtrate and centrifuging it to obtain the supernatant;

[0010] (4) Concentrating the supernatant of step (3) by rotary evaporation to obtain a concentrated solution, adding ethanol to the concentrated solution, and standing at 4° C. overnight;

[0011] (5) centrifuging the liquid treated in step (4), removing the supernatant, and freeze-drying the precipitate to obtain a crude fruiting body polysaccharide;

[0012] (6) dissolving the crude polysaccharide from the fruiting body in water, and adding calcium chloride and papain to remove the protein;

[0013] (7) Rotate evaporate and concentrate the solution processed in step (6), and then freeze-dry it to obtain Ganoderma subamboinense fruit body polysaccharide.

[0014] Further, the ethanol is ethanol with a volume concentration of 95%.

[0015] Further, in step (3), the temperature of the hot water is 85 °C.

[0016] Further, the temperature of the rotary evaporation and concentration is 60 °C.

[0017] Further, the centrifugation conditions are: rotation speed 4000 r / min, 10 min.

[0018] Further, the specific steps are as follows:

[0019] (1) Crush the dried Ganoderma subamboinense fruit body with a pulverizer;

[0020] (2) Weigh the fruit body powder, add 20 times the volume of 95% ethanol, stir properly and soak overnight, filter with double-layer gauze, discard the supernatant, obtain the filter residue, and dry the filter residue;

[0021] (3) Take out after drying, and then under the condition of stirring in a water bath at 85 °C, extract the filter residue in 20 times the volume of distilled water for 2 h, filter and collect the first filtrate and the filter residue, repeat the extraction 3 times in this way, combine the three filtrates, centrifuge at 4000 r / min for 10 min, and take the supernatant;

[0022] (4) Then rotate evaporate and concentrate the filtrate at 60 °C to one-tenth of the original volume, and then pour the concentrated filtrate into 4 times the volume of 95% ethanol solution under the condition of sufficient stirring, and place it in the refrigerator at 4 °C for 24 h;

[0023] (5) Then use a centrifuge to centrifuge the alcohol precipitation solution at 4000 r / min for 10 min, finally remove the supernatant, leave the precipitate, and freeze-dry to obtain the crude fruit body polysaccharide;

[0024] (6) Dissolve the crude polysaccharide in distilled water, stir evenly to fully dissolve the polysaccharide, and use the calcium chloride combined with papain method to remove proteins from the solution;

[0025] (7) Concentrate the obtained solution through a rotary evaporator, and perform freeze-drying on the concentrated liquid to obtain the fruit body polysaccharide.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The polysaccharide obtained from the fruiting body of Ganoderma subflexipes by the method of the present invention has high purity and strong activity. In vitro cell experiments have proved that this polysaccharide from the fruiting body has a significant protective effect on carbon tetrachloride-induced HepG2 cell damage. It can be used to prepare liver-protecting drugs to achieve the effect of preventing or treating liver cell damage. Description of the Drawings

[0028] Figure 1 Standard curve of glucose solution;

[0029] Figure 2 Results of GSB cell activity experiment;

[0030] Figure 3 Results of the experiment on the activity of HepG2 cells with different concentrations of carbon tetrachloride (*P < 0.05 indicates comparison with the normal group, ***P < 0.001 indicates comparison with the normal group);

[0031] Figure 4 Results of the effect of polysaccharide from the fruiting body of Ganoderma subflexipes on the content of ALT in the culture medium of HepG2 cells ( ### P < 0.01 indicates comparison with the normal group, *P < 0.05 indicates comparison with the model group, **P < 0.01 indicates comparison with the model group);

[0032] Figure 5 Results of the effect of polysaccharide from the fruiting body of Ganoderma subflexipes on the content of AST in the culture medium of HepG2 cells ( ### P < 0.01 indicates comparison with the normal group, *P < 0.05 indicates comparison with the model group, **P < 0.01 indicates comparison with the model group, ***P < 0.001 indicates comparison with the model group). Detailed Embodiments

[0033] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0034] The fruiting body of Ganoderma subflexipes used in this experiment is an artificially cultivated fruiting body in the laboratory. The HepG2 cell line is provided by Wuhan Punosai Life Science Co., Ltd.

[0035] Example 1 Isolation and Extraction of Polysaccharide from the Fruiting Body of Ganoderma subflexipes

[0036] The dried Ganoderma subamboinense fruit bodies were pulverized with a pulverizer. 10 g of the powder was weighed, 20 times the volume of 95% ethanol was added, and after proper stirring, it was soaked overnight. It was filtered through double-layer gauze, the supernatant was discarded (defatted), and the filter residue was obtained, which was then dried. After drying, under the condition of stirring in an 85°C water bath, the filter residue was extracted with 20 times the volume of distilled water for 2 h, and the first filtrate and the filter residue were collected by filtration. The extraction was repeated 3 times, and the three filtrates were combined. The filtrate was centrifuged at 4000 r / min for 10 min, and the supernatant was taken. Subsequently, the filtrate was rotary evaporated and concentrated to one-tenth of the original volume at 60°C, and 4 times the volume of 95% ethanol solution was added to the concentrated solution under sufficient stirring conditions, and it was left to stand in a 4°C refrigerator for 24 h. The alcohol-precipitated solution was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the precipitate was retained. After freeze-drying, the crude polysaccharide of the fruit body was obtained.

[0037] The crude polysaccharide was dissolved in an appropriate amount of distilled water and stirred evenly to fully dissolve the polysaccharide. The protein was removed by the calcium chloride combined with papain method (Zhang Fengsen, Shui Jinman, Zhang Shoude. Study on the method for removing protein during the preparation of Cynomorium songaricum polysaccharide [J]. Qinghai Science and Technology, 2023, 30(02): 112–118). The solution after protein removal was concentrated by a rotary evaporator, and finally the concentrated solution was freeze-dried to obtain a polysaccharide powder with most of the protein removed. The obtained fruit body polysaccharide was named GSB.

[0038] Example 2 Determination of the polysaccharide content in the fruit body

[0039] (1) Preparation of the glucose standard solution: 10 mg of the glucose standard was weighed with an analytical balance and placed in a 10 mL weighing bottle, and it was prepared into a solution with a concentration of 0.1 mg / mL using distilled water.

[0040] Pipette the 0.1 mg / mL glucose standard solution, with the sampling range being 0–2.0 mL and the gradient being 0.4 mL; place it in 6 graduated test tubes, first make up to 2.0 mL with distilled water; then add 1.0 mL of 5% phenol solution, and then quickly pipette 5.0 mL of concentrated sulfuric acid into it with a pipette, and then shake it up and down until the solution is mixed evenly. Then leave it to stand at room temperature for 10 min, and finally place it in a 100°C water bath and heat it for 15 min. After quickly cooling to room temperature, pour the solution into a cuvette and measure its ultraviolet absorbance at a wavelength of 490 nm. Repeat the experiment 3 times. With the standard solution concentration as the abscissa and A 490 as the ordinate, draw the standard curve.

[0041] The glucose standard curve is as Figure 1 shown. The abscissa is the concentration of the glucose standard solution, and the ordinate is the absorbance value (OD value) of the glucose standard solution at 490 nm. The regression equation of the obtained standard curve is: y = 7.35x – 0.005, R2 = 0.9991, indicating a good linear relationship within the concentration range of 0 - 0.25 mg / mL.

[0042] (2) Determination of polysaccharide content in polysaccharide: Weigh 10 mg of polysaccharide sample GSB into a volumetric flask, add 10 mL of distilled water to make the concentration 1 mg / mL. Pipette 0.1 mL of the sample solution into a test tube, make up to 2 mL with distilled water, and then measure the ultraviolet absorbance of the solution at 490 nm wavelength according to the steps in (1). Then substitute the measured absorbance into the glucose standard curve prepared above and calculate the polysaccharide content of Ganoderma curtisii polysaccharide.

[0043] The phenol - sulfuric acid method was used to determine the polysaccharide content, and the content of the crude polysaccharide was 80.1%.

[0044] Example 3 In vitro hepatoprotective activity of fruiting body polysaccharide

[0045] (1) Preparation of samples

[0046] ① Preparation of DMEM complete medium: Slowly pour 45 mL of DMEM cell basal medium into a 50 mL sterile centrifuge tube, then add 5 mL of fetal bovine serum and 500 μL of double antibodies, and finally filter and sterilize through a 0.22 μm microporous membrane and store at 4 °C for later use.

[0047] ② Preparation of DMEM low - serum medium: Place 49 mL of DMEM cell basal medium in a 50 mL sterile centrifuge tube, add 1 mL of fetal bovine serum, then filter and sterilize through a 0.22 μm microporous membrane and store at 4 °C for later use.

[0048] ③ Preparation of saturated carbon tetrachloride solution: Add an excessive amount of carbon tetrachloride solution to a centrifuge tube containing DMEM serum medium, then seal the centrifuge tube and shake for 10 min. Subsequently, place the centrifuge tube in a 37 °C incubator for 24 h. At this time, carbon tetrachloride crystals precipitate at the bottom of the centrifuge tube, and the upper layer above the carbon tetrachloride crystals is the saturated carbon tetrachloride solution at 37 °C. Aspirate the upper layer solution and store it at 37 °C for later use. Subsequently, mix the saturated carbon tetrachloride solution and normal DMEM low - serum culture medium in ratios of 1:3, 1:1, 3:1, and 1:0 respectively to prepare 25%, 50%, 75%, and 100% carbon tetrachloride solutions.

[0049] ④ Preparation and treatment of polysaccharide sample solution: Accurately weigh 8 mg of polysaccharide, dissolve it in 10 mL of medium, mix well, and then filter and sterilize through a 0.22 μm microporous membrane to obtain a polysaccharide solution with a concentration of 800 μg / mL. Subsequently, perform gradient serial dilution on the solution to prepare polysaccharide solutions with concentrations of 400, 200, 100, and 50 μg / mL respectively, and store for later use.

[0050] (2) Cell culture

[0051] ① Cell resuscitation: Place the cryopreservation tube containing HepG2 cells in a 37°C sterile water bath for rapid thawing. After thawing, quickly transfer the cell suspension to a centrifuge tube pre-filled with 9 mL of complete DMEM medium in a laminar flow hood and gently mix. Centrifuge at 1000 r / min for 5 min and discard the supernatant. Add 4 mL of complete medium to the cell pellet, gently pipette up and down (avoiding the formation of bubbles), pipette at least 120 times until no cell clumps or aggregates are observed under an inverted microscope. Subsequently, transfer the cell suspension to a T25 culture flask and gently shake the flask using the cross-cross method to evenly distribute the cells on the bottom of the flask. Finally, place the culture flask in an incubator at 37°C and 5% CO2 for culturing. During cell growth, if the color of the medium changes, fresh medium needs to be replaced in a timely manner.

[0052] ② Subculture of cells: When the HepG2 cells reach 80%–90% confluence during adherent growth, subculture can be carried out. First, aspirate the medium from the T25 culture flask and gently wash the cells twice with PBS buffer to remove residual medium. Subsequently, add 1 mL of trypsin solution using a pipette, shake the flask gently from side to side several times to evenly cover the cell layer, and place the culture flask in a cell incubator at 37°C and 5% CO2 for 2 min 30 s. After taking it out, gently pipette the cell layer to completely detach the adherent cells and transfer the cell suspension to a 15 mL centrifuge tube. Rinse the bottom of the culture flask with a small amount of PBS buffer and transfer the rinse solution to the centrifuge tube. Centrifuge at 1000 r / min for 5 min, discard the supernatant, and retain the cell pellet. Add 3 mL of fresh complete DMEM medium to the cell pellet and pipette up and down at least 120 times until no obvious cell clumps or aggregates are observed in the cell suspension under an inverted microscope. Finally, inoculate the cell suspension into a new T25 culture flask at a subculture ratio of 1:3, gently shake the flask to evenly distribute the cells, and then place the culture flask in an incubator at 37°C and 5% CO2 for continued culturing.

[0053] ③ Cryopreservation of cells: After the cells adhered and grew to confluence, aspirate the culture medium in the T25 culture flask, gently wash the cells twice with PBS buffer to remove the residual culture medium. Subsequently, add 1 mL of trypsin solution, shake horizontally several times to evenly cover the cell layer, and incubate the culture flask in a cell culture incubator at 37 °C and 5% CO2 for 2 min 30 s. After taking it out, gently pipette the cell layer with a sterile pipette to completely detach the adherent cells. Transfer the cell suspension to a centrifuge tube, centrifuge at a speed of 1000 r / min for 5 min, and discard the supernatant. Add 1800 μL of fetal bovine serum to the cell pellet, gently pipette several times to evenly suspend the cells. Quickly aliquot the cell suspension into 2 pre-prepared cryotubes, add 100 μL of dimethyl sulfoxide (DMSO) to each tube, and gently mix. Transfer the cryotubes to a programmable freezer box, and then place the programmable freezer box in an -80 °C refrigerator for programmed cooling for several days to ensure a gradual decrease in the sample temperature. After the cooling program is completed, take out the cryotubes from the programmable freezer box and quickly transfer them to a liquid nitrogen tank to achieve long-term stable preservation of the cells.

[0054] (3) Effect of Ganoderma subamboinense polysaccharide on the viability of HepG2 cells

[0055] When the cells adhered and grew to 80% confluence, aspirate the culture medium in the T25 culture flask, then wash twice with PBS solution, subsequently add 1 mL of trypsin, shake horizontally several times, and then place it in a cell culture incubator (37 °C, 5% CO2) for 2 min 30 s. After taking it out, gently pipette the adherent HepG2 cells with a sterile pipette to make them detach. Centrifuge at a speed of 1000 r / min for 5 min and discard the supernatant. Subsequently, add the prepared complete DMEM medium, pipette evenly, calculate the cell density with a cell counter, and adjust the cell density to 1×10 5 / mL, inoculate 100 μL per well in a 96-well plate, and place it in a cell culture incubator (37 °C, 5% CO2) for 24 h. After the cells adhered, aspirate the old complete culture medium, add 100 μL of DMEM low-serum medium to each well, and place it in the cell culture incubator for 24 h to achieve cell synchronization. Subsequently, aspirate the culture solution, add polysaccharide solutions at different concentrations (50, 100, 200, 400, 800 μg / mL respectively), and at the same time set up a blank control group (without cells) and a normal control group (with cells but without polysaccharide). After continuing to culture for 24 h, remove the old culture medium, add 10 μL of CCK-8 and 100 μL of complete DMEM medium to each well, then gently mix, continue to incubate in the incubator for 1 h, and then measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader. Then calculate the cell viability according to the steps in the instruction manual.

[0056] The results are as Figure 2As shown, compared with the normal control group, when the GSB concentration was 50–800 μg / mL, the cell survival rate was relatively high, overall approaching or exceeding 100%, indicating that GSB was non-toxic within the range of 50–800 μg / mL and had the effect of promoting the proliferation of HepG2 cells; when the GSB concentration was 800 μg / mL, the cell survival rate was 96%, and there was no significant difference compared with the normal control group (P>0.05). Therefore, we selected GSB concentrations of 200, 400, and 800 μg / mL as the safe dosage range for subsequent studies on the protective effect of polysaccharides on carbon tetrachloride-induced hepatocyte injury.

[0057] (4) Establishment of a carbon tetrachloride-induced injury model of HepG2 cells in vitro

[0058] When the cells in the T25 flask adhered and grew to 80%, aspirate the medium in the T25 culture flask, then wash it twice with PBS solution. Subsequently, add 1 mL of trypsin, shake it horizontally several times, and then place it in the cell culture incubator (37 °C, 5% CO2) for 2 min 30 s. After taking it out, gently pipette the adherent cells with a pipette to make them detached, centrifuge at 1000 r / min for 5 min and discard the supernatant. Subsequently, add DMEM complete medium, pipette it evenly, and adjust the cell density to 1×10 5 / mL, inoculate 100 μL per well in a 96-well plate, and incubate it in the incubator for 24 h. After the cells adhered, aspirate the supernatant, wash the cell layer with PBS solution, and then replace it with DMEM low-serum medium for starvation treatment for 24 h. Aspirate the old medium with a sterile pipette and wash it twice with PBS buffer solution. Add 100 μL of carbon tetrachloride saturated solution with different concentrations (25%, 50%, 75%, 100%) to each well, and at the same time set a normal control group (without saturated carbon tetrachloride solution), and incubate it in the incubator for 24 h. Subsequently, aspirate the old medium, and wash each well with PBS solution. Add 10 μL and 100 μL of CCK-8 and DMEM to each well respectively. Shake the above mixed solution horizontally to make it uniform and incubate it in the incubator for 1 h. Then, measure the optical absorption value at 450 nm with an enzyme label. Finally, calculate the cell survival rate according to the instructions.

[0059] SPSS 17.0 and GraphPad prism 8.0.2 software were used to process the data, and the results were expressed as "mean ± standard deviation" ", where *P<0.05 represents a significant difference, and **P<0.01 or ***P<0.001 represents a highly significant difference.

[0060] The results are as Figure 3As shown, after the saturated carbon tetrachloride solutions at concentrations of 25%, 50%, 75% and 100% acted on HepG2 cells for 24 h, compared with the control group, the cell survival rates were 83.55%, 73.68%, 68.57% and 59.4% respectively. After the saturated carbon tetrachloride solutions at concentrations of 25% and 50% acted on HepG2 cells for 24 h, there was no significant difference in absorbance (P>0.05); while after the saturated carbon tetrachloride solution at a concentration of 75% acted on HepG2 cells for 24 h, its cell survival rate showed a significant difference compared with the control group (P<0.05). After the saturated carbon tetrachloride solution at a concentration of 100% acted on HepG2 cells for 24 h, its cell survival rate showed a highly significant difference compared with the control group (P<0.001). After the saturated carbon tetrachloride solution at a concentration of 100% acted on the cells, its cell survival rate was approximately half of that of the control group, approaching the median lethal dose. Therefore, the saturated carbon tetrachloride solution at a concentration of 100% was selected for subsequent cell experiment modeling.

[0061] (5) Protective effect of polysaccharide on carbon tetrachloride-induced HepG2 cell injury

[0062] Use a cell counter to adjust the cell density to 1×10 5 / mL, inoculate 100 μL per well in a 96-well plate, and culture for 24 h under the conditions of 37 °C and 5% CO2. Inoculate the cells on a 96-well plate, 100 μL per well, and place them in a cell incubator (37 °C, 5% CO2) for 24 h. After the cells adhered, aspirate and discard the old complete medium, wash the cell layer with PBS, and then replace it with DMEM low-serum medium for starvation treatment for 24 h. Aspirate and discard the medium, and wash it twice with PBS buffer. Add 3 different concentrations of polysaccharide solutions (200, 400, 800 μg / mL respectively) to each well, and culture for 24 h under the conditions of 37 °C and 5% CO2. Then aspirate and discard the culture solution. Except for the normal control group which is replaced with complete medium, the other groups are replaced with a saturated carbon tetrachloride solution at a concentration of 100%, and culture for 24 h under the conditions of 37 °C and 5% CO2. After incubation for 24 h, detect the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant of the cell culture medium according to the instructions.

[0063] Use SPSS 17.0 and GraphPad prism 8.0.2 software to process the data, and express the results as "mean ± standard deviation ". Among them, *P<0.05 represents a significant difference, and **P<0.01 or ***P<0.001 represents a highly significant difference.

[0064] From Figure 4It can be seen that, compared with the normal group, the ALT content in the cell culture medium of the model group increased, and the result had a highly significant difference (P<0.001), indicating that the carbon tetrachloride-induced hepatocyte injury model was successfully induced; compared with the model group, when the GSB concentration was 400 μg / mL and 800 μg / mL, the ALT content in the cell culture medium decreased, and there was a significant difference (P<0.05).

[0065] It can be seen from Figure 5 that, compared with the normal group, the AST content in the cell culture medium of the model group increased, and the result had a highly significant difference (P<0.001), indicating that the carbon tetrachloride-induced hepatocyte injury model was successfully induced; compared with the model group, when the GSB concentration was 200 μg / mL, 400 μg / mL and 800 μg / mL, the AST content in the cell culture medium decreased, and there was a significant difference (P<0.05).

Claims

1. Use of polysaccharides from the fruiting body of Ganoderma subflexipes in the preparation of liver protection drugs.

2. The use according to claim 1, characterized in that The liver protection drug refers to a drug for preventing or treating liver cell damage.

3. A method for preparing the Ganoderma subflexipes fruiting body polysaccharide as claimed in claim 1 or 2, characterized in that: The steps include: (1) crushing the dried Ganoderma lucidum fruiting body to obtain fruiting body powder; (2) soaking the fruiting body powder in ethanol, removing the supernatant and drying the residue; (3) washing the filter residue treated in step (2) with hot water, collecting the filtrate and centrifuging it to obtain the supernatant; (4) Concentrating the supernatant of step (3) by rotary evaporation to obtain a concentrated solution, adding ethanol to the concentrated solution, and standing at 4° C. overnight; (5) centrifuging the liquid treated in step (4), removing the supernatant, and freeze-drying the precipitate to obtain a crude fruiting body polysaccharide; (6) dissolving the crude polysaccharide from the fruiting body in water, and adding calcium chloride and papain to remove the protein; (7) Concentrating the solution after treatment in step (6) by rotary evaporation, and freeze-drying to obtain Ganoderma lucidum fruiting body polysaccharide.

4. The method according to claim 3, characterized in that The ethanol is 95% ethanol by volume.

5. The method according to claim 3, characterized in that: In step (3), the hot water temperature is 85°C.

6. The method according to claim 3, characterized in that: The temperature of the rotary evaporation concentration is 60°C.

7. The method according to claim 3, characterized in that The centrifugal conditions are: rotation speed 4000r / min, 10min.

8. The method according to claim 3, characterized in that The specific steps are as follows: (1) crushing the dried Ganoderma lucidum fruiting body with a crusher; (2) Weigh the fruiting body powder, add 20 times the volume of 95% ethanol, stir appropriately, soak overnight, filter through double-layer gauze, discard the supernatant, obtain the filter residue, and dry the filter residue; (3) After drying, the residue was extracted with 20 times the volume of distilled water for 2 h under stirring at 85°C in a water bath, and the first filtrate and residue were collected by filtration. This extraction was repeated three times, and the three filtrates were combined and centrifuged at 4000 r / min for 10 min to obtain the supernatant; (4) The filtrate was then concentrated to one tenth of its original volume by rotary evaporation at 60°C, and then poured into 4 times the volume of 95% ethanol solution under sufficient stirring, and placed in a refrigerator at 4°C for 24 h; (5) The alcohol precipitation solution was then centrifuged at 4000 r / min for 10 min, and the supernatant was removed, and the precipitate was retained and freeze-dried to obtain a crude fruiting body polysaccharide; (6) dissolving the crude polysaccharide in distilled water and stirring evenly to fully dissolve the polysaccharide, and removing protein from the solution using calcium chloride combined with papain; (7) The obtained solution is concentrated by a rotary evaporator, and the liquid obtained after concentration is freeze-dried to obtain the fruiting body polysaccharide.

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