Schizophyllan fruiting body polysaccharide having anti-inflammatory activity and uses thereof

Abstract

The present application discloses a schizophyllum commune fruiting body polysaccharide with anti-inflammatory activity and application thereof, the molecular weight of the polysaccharide is 1.87*10 4 Da, the sugar content is 96.41%+ / -2.12, and the molar ratio of monosaccharide composition is mannose:glucose:galactose:arabinose:fucose=9.057:9.633:9.631:1.561:1. The schizophyllum commune fruiting body polysaccharide of the present application shows excellent anti-inflammatory activity, has good development and utilization prospect, and can be widely applied in the fields of health-care food and medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of schizophyllum commune fruiting body polysaccharide with anti-inflammatory activity and its preparation method and use, belong to food and medical technology field. BACKGROUND

[0002] When the body is attacked by heterogenous material, the immune system is activated to identify and attack non-self foreign substances, induce immune response, to achieve the purpose of clearance. However, the immune response is too intense to cause inflammation, usually accompanied by vasodilation, increased permeability and other phenomena, leading to local tissue and cell damage, immune substances and coagulation factor outflow, cyclooxygenase COX2 is activated, inflammation is intensified, further accelerate cell aging and apoptosis.

[0003] Clinically used anti-inflammatory drugs include non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. Non-steroidal anti-inflammatory drugs include non-selective cyclooxygenase inhibitors and selective cyclooxygenase-2 inhibitors, and steroidal anti-inflammatory drugs are mainly glucocorticoid drugs. However, these anti-inflammatory drugs usually have some side effects, such as gastrointestinal discomfort and ulcer, allergic reaction, central obesity, water and sodium retention, etc., which increase the metabolic burden of liver and kidney, and long-term use can cause organ damage. Therefore, it is particularly important to find a natural anti-inflammatory active substance with less side effects and toxic side effects. Studies have shown that edible fungi are one of the main sources of polysaccharides, and natural fungal polysaccharides have various biological activities, such as anti-inflammatory, antioxidant and antitumor activities, etc. Because of low toxicity and good biocompatibility, active polysaccharides have become a research hotspot in the field of natural medicines and health products.

[0004] Schizophyllum commune Fr., also known as Bai Shen, tree flower, white flower, etc., is a large fungus of Schizophyllaceae and Schizophyllum. Schizophyllum commune is a fungus with both food and medicinal value. Its flesh is soft and tender, rich in amino acids and essential trace elements for the human body, and has important food and medicinal value. According to traditional Chinese medicine theory, it is flat, and has tonifying and sedative effects. Schizophyllan (SPG) obtained by liquid deep fermentation technology has immunomodulatory and antitumor effects, and has realized commercial production. The schizophyllum commune fruiting body polysaccharide discovered for the first time in the present application has excellent anti-inflammatory activity, and there is no report so far, which has very important development value. SUMMARY

[0005] The present application aims to provide a kind of schizophyllum commune fruiting body polysaccharide with anti-inflammatory activity and its preparation method and use.

[0006] To achieve the purpose, the present application adopts the following technical solutions:

[0007] The present application first provides a kind of schizophyllum commune fruiting body polysaccharide with anti-inflammatory activity, and its molecular weight is 1.84 x 10 4Da, the sugar content is 96.41%±2.12, the ultraviolet full wavelength scanning shows that it does not contain protein, the infrared spectrum shows that it has significant polysaccharide infrared characteristic absorption peak, and the molar ratio of monosaccharide composition is mannose: glucose: galactose: arabinose: fucose = 9.057: 9.633: 9.631: 1.561: 1.

[0008] The application further provides a preparation method of the Schizophyllum commune fruiting body polysaccharide with anti-inflammatory activity, comprising the following steps:

[0009] (1) crushing the dried Schizophyllum commune fruiting body through a pulverizer and then screening the crushed product through an 80-mesh screen to obtain a powder;

[0010] (2) extracting the obtained powder by hot water at a temperature of 60-100 DEG C and a material-liquid ratio of 1:10-50 for 1-4 h, repeating 2-3 times, and combining the extraction solutions;

[0011] (3) concentrating the extraction solution obtained in step (2) by rotary evaporation to 1 / 4-1 / 10 of the volume, adding ethanol to the obtained concentrated solution until the final volume concentration of ethanol is 60-90%, and then alcohol-sedimenting at 4 DEG C for 8-12 h, and collecting the precipitate;

[0012] (4) dissolving the precipitate obtained in step (3) in water, removing ethanol by rotary evaporation and continuing to concentrate by rotary evaporation to obtain a sugar solution; removing the protein in the sugar solution by using the Sevag method: uniformly mixing the sugar solution, n-butanol and trichloromethane at a volume ratio of 5:1~4:1~4, shaking, standing to separate the layers, and removing the protein layer, and repeating until no protein layer appears;

[0013] packing the remaining sugar solution into a dialysis bag with a molecular weight cut-off of 3-5 kDa, dialyzing for 24-48 h with flowing water and 48 h with static water, concentrating and freeze-drying to obtain the crude Schizophyllum commune fruiting body polysaccharide;

[0014] (5) preparing a solution of the crude Schizophyllum commune fruiting body polysaccharide obtained in step (4) in deionized water at a concentration of 20-40 mg / mL, removing insoluble impurities by centrifugation, filtering through a 0.22-μm filter membrane, and then eluting the filtrate in a DEAE-cellulose 52 ion exchange column with deionized water as the mobile phase at a flow rate of 0.5-1.2 mL / min, collecting the product and freeze-drying;

[0015] (6) preparing a solution of the dried product obtained in step (5) in deionized water at a concentration of 50-80 mg / mL, removing insoluble impurities by centrifugation, filtering through a 0.22-μm filter membrane, and then eluting in a CL-4B molecular sieve, collecting the eluate in tubes by using an automatic collector at a flow rate of 0.5-1.2 mL / min and 8 min / tube, collecting the product and freeze-drying to obtain a multi-tube dried product;

[0016] (7) The multi-tube dry product obtained in step (6) is respectively prepared into a solution of 3-5 mg / mL with deionized water, filtered through a 0.22 μm filter membrane, and then detected by high performance liquid TSK-6000, with water as the mobile phase, a flow rate of 0.6 mL / min, and a sample injection amount of 20 μL, to obtain the target product Schizophyllum commune fruiting body polysaccharide with a uniform and symmetrical peak type.

[0017] The application further discloses a use of the Schizophyllum commune fruiting body polysaccharide, which is characterized by being used as an anti-inflammatory active agent to prepare an anti-inflammatory health food or an anti-inflammatory drug.

[0018] The application has the following beneficial effects:

[0019] 1. The Schizophyllum commune fruiting body polysaccharide prepared in the application has a uniform and symmetrical peak type, a molecular weight of 1.84 x 10 4 Da, a sugar content of 96.41% ± 2.12, and a molar ratio of monosaccharide composition of mannose: glucose: galactose: arabinose: fucose = 9.057: 9.633: 9.631: 1.561: 1, and is a natural active polysaccharide with high purity.

[0020] 2. The Schizophyllum commune fruiting body polysaccharide discovered in the application has excellent anti-inflammatory activity: in-vitro anti-inflammatory tests show that the Schizophyllum commune fruiting body polysaccharide has a relieving effect on cell inflammation of LPS-induced RAW264.7 macrophages, has no toxicity to normal cells, and the anti-inflammatory effect is enhanced and presents a dose-dependent manner with the increase of the polysaccharide concentration, indicating that the Schizophyllum commune fruiting body polysaccharide has an important relieving effect on cell inflammation. Meanwhile, in-vivo tests prove that the Schizophyllum commune fruiting body polysaccharide has a significant treatment effect on DSS-induced UC mice. At present, there is no report about the anti-inflammatory activity of the Schizophyllum commune fruiting body polysaccharide, and therefore the in-vitro and in-vivo anti-inflammatory properties of the Schizophyllum commune fruiting body polysaccharide have important development and utilization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a high performance liquid chromatogram of SCP-1.

[0022] Figure 2 It is a monosaccharide composition standard liquid chromatogram (see (A) in Figure 2 ) and a SCP-1 monosaccharide composition liquid chromatogram (see (B) in Figure 2 ).

[0023] Figure 3 It is a toxicity test of SCP-1 on RAW264.7 macrophages.

[0024] Figure 4 It is a detection of the NO release amount of SCP-1 on LPS-induced RAW264.7 macrophages.

[0025] Figure 5The release amount of inflammatory factors IL-1β (see (A) in Figure 5 ), IL-6 (see (B) in Figure 5 ) and TNF-α (see (C) in Figure 5 ) of LPS-induced RAW264.7 macrophages was detected for SCP-1.

[0026] Figure 6 The changes of body weight (see (A) in Figure 6 ), colon length (see (B) in Figure 6 ), disease activity index (DAI) (see (C) in Figure 6 ) and spleen weight (see (D) in Figure 6 ) of UC mice after administration of SCP were determined.

[0027] Figure 7 The H&E staining pictures and histological scores (see (A) in Figure 7 ) and AB-PAS staining pictures and the number of goblet cells (see (B) in Figure 7 ) of colon tissues of mice were determined.

[0028] Figure 8 The contents of inflammatory factors IL-6 (see (A) in Figure 8 ), IL-1β (see (B) in Figure 8 ), TNF-α (see (C) in Figure 8 ) and IL-10 (see (D) in Figure 8 ) in serum of mice were determined. DETAILED DESCRIPTION

[0029] The following examples can make the person skilled in the art more fully understand the present application, but do not limit the present application in any way.

[0030] Example 1: Extraction, separation and purification of Schizophyllum commune fruiting body polysaccharide from Schizophyllum commune fruiting body

[0031] (1) The dried Schizophyllum commune fruiting body was crushed by a crusher and then passed through an 80-mesh sieve to obtain a powder.

[0032] (2) The obtained powder was extracted by hot water at a temperature of 85°C for 2h with a solid-liquid ratio of 1:20, and the extraction was repeated for 3 times, and the extraction solutions were combined.

[0033] (3) The extraction solution obtained in step (2) was concentrated by rotary evaporation to 1 / 4 of the volume, and ethanol was added to the obtained concentrated solution to a final volume concentration of 80%, and then alcohol precipitation was carried out at 4°C, and the precipitate was collected after standing for 12h.

[0034] (4) The precipitate obtained in step (3) is dissolved in water, ethanol is removed by rotary evaporation, and the concentration is continued by rotary evaporation to obtain a sugar solution; the Sevag method is used to remove the protein in the sugar solution: the sugar solution, n-butanol and chloroform are mixed uniformly according to a volume ratio of 5:1:4, shaken for 10 min, and after the protein layer is removed by layering, the operation is repeated until no protein layer appears.

[0035] The remaining sugar solution is loaded into a 3.5 kDa dialysis bag and dialyzed for 24 h with flowing water and 48 h with static water. After concentration, freeze-drying is performed to obtain the crude Schizophyllum commune fruiting body polysaccharide (named SCP).

[0036] (5) The crude Schizophyllum commune fruiting body polysaccharide obtained in step (4) is prepared into a 40 mg / mL solution with deionized water, insoluble impurities are removed by centrifugation, and after filtration through a 0.22 μm filter membrane, the filtrate is subjected to elution on a DEAE-cellulose 52 ion exchange column using deionized water as the mobile phase at a flow rate of 1 mL / min. The product is collected and freeze-dried.

[0037] (6) The dried product obtained in step (5) is prepared into an 80 mg / mL solution with deionized water, insoluble impurities are removed by centrifugation, and after filtration through a 0.22 μm filter membrane, the filtrate is subjected to elution on a CL-4B molecular sieve. The product is collected and freeze-dried to obtain a multi-tube dried product.

[0038] (7) The multi-tube dried product obtained in step (6) is prepared into a 4 mg / mL solution with deionized water, filtered through a 0.22 μm filter membrane, and subjected to detection by high performance liquid chromatography (HPLC) using water as the mobile phase at a flow rate of 0.6 mL / min and a sample injection amount of 20 μL. The target product Schizophyllum commune fruiting body polysaccharide (SCP-1) with a symmetrical peak type is detected at a peak time of 18.627 min.

[0039] Figure 1 The high performance liquid chromatogram of SCP-1 is shown in FIG. 1. Figure 1 As can be seen from FIG. 1, the SCP-1 polysaccharide has a single symmetrical absorption peak with a retention time of 18.627 min. The molecular weight of SCP-1 is calculated to be 1.84 × 10 4 Da.

[0040] Figure 2 The standard monosaccharide composition diagram and the SCP-1 monosaccharide composition diagram are shown in FIGS. 2 and 3, respectively. The molar ratio of the monosaccharide composition of SCP-1 is mannose: glucose: galactose: arabinose: fucose = 9.057: 9.633: 9.631: 1.561: 1.

[0041] Example 2: Extraction, separation and purification of Schizophyllum commune fruiting body polysaccharide from Schizophyllum commune fruiting body

[0042] (1) The dried fruiting bodies of the schizophyllum commune are crushed by a pulverizer and then passed through an 80-mesh sieve to obtain powder.

[0043] (2) The obtained powder was extracted with hot water at 60°C for 3 hours at a material-to-liquid ratio of 1:10. The extraction was repeated 3 times, and the extracts were combined.

[0044] (3) The extract obtained in step (2) is concentrated by rotary evaporation to 1 / 4 of its volume. Ethanol is added to the concentrate until the final volume concentration of ethanol is 80%. Then, the precipitate is precipitated at 4°C and allowed to stand for 8 hours. The precipitate is then collected.

[0045] (4) Dissolve the precipitate obtained in step (3) in water, remove the ethanol by rotary evaporation and continue to concentrate by rotary evaporation to obtain a sugar solution; remove the protein in the sugar solution using the Sevag method: mix the sugar solution, n-butanol and chloroform in a volume ratio of 5:2:3, shake for 10 min, let stand for layering and remove the protein layer, repeat until no protein layer appears.

[0046] The remaining sugar solution was placed in a 4kDa dialysis bag and dialyzed. The solution was dialyzed with running water for 24 hours and with static water for 48 hours. After concentration, it was freeze-dried to obtain crude polysaccharide from the fruiting bodies of Schizophyllum commune.

[0047] (5) The crude polysaccharide of the fruiting body of Schizophyllum commune obtained in step (4) was prepared into a solution of 20 mg / mL with deionized water. The solution was centrifuged to remove insoluble impurities, filtered through a 0.22 μm filter membrane, and the retentate solution was placed in a DEAE-cellulose 52 ion exchange column for elution. Deionized water was used as the mobile phase at a flow rate of 1 mL / min. The product was collected and freeze-dried.

[0048] (6) The dried product obtained in step (5) was prepared into a solution of 70 mg / mL with deionized water, centrifuged to remove insoluble impurities, filtered through a 0.22 μm filter membrane, and then eluted in a CL-4B molecular sieve. The product was collected in separate tubes using an automatic collector with a flow rate of 1 mL / min and 8 min / tube. The product was collected and freeze-dried to obtain multiple tubes of dried product.

[0049] (7) The dried product from the multi-tube sample in step (6) was prepared into a 5 mg / mL solution with deionized water. After filtration through a 0.22 μm filter membrane, it was detected by high performance liquid chromatography (HPLC). The mobile phase was water, the flow rate was 0.6 mL / min, and the injection volume was 20 μL. The target product, polysaccharide of Schizophyllum commune fruiting body, with a uniform and symmetrical peak shape was detected (peak elution time was 18.627 min).

[0050] Example 3: Extraction, separation and purification of polysaccharides from Schizophyllum commune fruiting bodies.

[0051] (1) The dried fruiting bodies of the schizophyllum commune are crushed by a pulverizer and then passed through an 80-mesh sieve to obtain powder.

[0052] (2) The obtained powder was extracted with hot water at 75°C for 1 hour at a material-to-liquid ratio of 1:50. This process was repeated 3 times, and the extracts were combined.

[0053] (3) Concentrate the extract obtained in step (2) to 1 / 4 of its volume, add ethanol to the concentrate until the final volume concentration of ethanol is 80%, then precipitate at 4°C, let stand for 12 hours, and collect the precipitate.

[0054] (4) Dissolve the precipitate obtained in step (3) in water, remove the ethanol by rotary evaporation and continue to concentrate by rotary evaporation to obtain a sugar solution; remove the protein in the sugar solution using the Sevag method: mix the sugar solution, n-butanol and chloroform in a volume ratio of 5:4:1, shake for 10 min, let stand for layering and remove the protein layer, repeat until no protein layer appears.

[0055] The remaining sugar solution was placed in a 5kDa dialysis bag and dialyzed. The solution was dialyzed with running water for 48 hours and with static water for 48 hours. After concentration, it was freeze-dried to obtain crude polysaccharide from the fruiting body of Schizophyllum commune.

[0056] (5) The crude polysaccharide of the fruiting body of Schizophyllum commune obtained in step (4) was prepared into a solution of 20 mg / mL with deionized water. The insoluble impurities were removed by centrifugation. After filtration through a 0.22 μm filter membrane, the retentate solution was placed in a DEAE-cellulose 52 ion exchange column for elution. Deionized water was used as the mobile phase at a flow rate of 1.2 mL / min. The product was collected and freeze-dried.

[0057] (6) The dried product obtained in step (5) was prepared into a 50 mg / mL solution with deionized water, centrifuged to remove insoluble impurities, filtered through a 0.22 μm filter membrane, and then eluted in a CL-4B molecular sieve. The product was collected in separate tubes using an automatic collector with a flow rate of 0.5 mL / min and 8 min / tube. The product was collected and freeze-dried to obtain a multi-tube dried product.

[0058] (7) The dried products from step (6) were prepared into 4 mg / mL solutions with deionized water, filtered through a 0.22 μm filter membrane, and detected by high performance liquid chromatography (HPLC). The mobile phase was water, the flow rate was 0.6 mL / min, and the injection volume was 20 μL. The polysaccharides from the fruiting bodies of *Schizophyllum commune* with uniform and symmetrical peaks were detected (elution time was 18.627 min).

[0059] Example 4: In vitro anti-inflammatory activity assay of SCP-1

[0060] 1. Cytotoxicity assay

[0061] RAW 264.7 macrophages in good growth condition were seeded into 96-well plates (1×10⁶ cells / well). 5 Cells were cultured in 96-well plates at 37°C in a CO2 incubator. The effect of the samples on cell viability was assessed using the MTT assay. 1 μg / mL LPS was used as a positive control, and fresh culture medium was added to the blank control group. The sample groups were treated with SCP-1 (50-600 μg / mL) and incubated for 24 h. After incubation, the cell supernatant was removed, and 100 μL of MTT solution (0.5 mg / mL) was added to each well for 4 h. The 96-well plates were then removed from the incubator, the supernatant was discarded, 100 μL of LDMSO was added, and the plates were shaken for 10 min. The absorbance at 570 nm was measured using a microplate reader. The results of the SCP-1 cytotoxicity on macrophages are as follows: Figure 3 As shown, SCP-1 has no toxic effect on 264.7 macrophages at concentrations ranging from 50 μg / mL to 600 μg / mL.

[0062] 2. Detection of NO release from LPS-induced inflammation in RAW264.7 macrophages using SCP-1

[0063] NO production was detected using the Griess method. RAW 264.7 macrophages in good growth condition (1×10⁻⁶ cells) were used. 5 Cells (1 cell / well) were placed in 96-well plates and incubated in a CO2 incubator at 37°C for 24 h. The supernatant was removed, and cells were treated with SCP-1 (50-600 μg / mL) in medium containing LPS (4 μg / mL). After incubation for 24 h, the cell supernatant was collected and mixed with an equal volume of Griess reagent. The mixture was incubated at room temperature for 30 min and measured at 540 nm using a microplate reader. Figure 4 The results show that after 24 hours of treatment with different concentrations of SCP-1, the release of NO from inflammatory cells gradually decreased. When the concentration of SCP-1 reached 600 μg / mL, the NO production was reduced by 51.24% compared to the LPS group. The experimental results indicate that SCP-1 has an inhibitory effect on LPS-induced cellular inflammation and NO production.

[0064] 3. Detection of inflammatory factor release from LPS-induced inflammation in RAW264.7 macrophages by SCP-1

[0065] The release levels of IL-1β, IL-6, and TNF-α were detected using a kit. RAW 264.7 macrophages in good growth condition (1×10⁻⁶ cells) were used. 5Cells (1 cell / well) were placed in a 96-well plate and incubated in a CO2 incubator at 37°C for 24 h. The supernatant was removed, and cells were treated with SCP-1 (50-600 μg / mL) in medium containing LPS (4 μg / mL). After incubation for 24 h, the cell supernatant was collected. Following the kit instructions, samples and standards of different concentrations were added to the corresponding wells at 100 μL / well. For the blank control group, 100 μL of diluent was added. The plate was sealed and incubated at 37°C for 1 h. After incubation, the plate was removed, the liquid was discarded, and 100 μL of biotinylated antibody working solution was added to each well. The plate was sealed again and incubated at 37°C for 1 h. After the above steps, the liquid was discarded, and 300 μL of washing buffer was added to each well. The plate was allowed to stand for 1 min, the washing buffer was discarded, and this process was repeated 3 times. Finally, 100 μL of enzyme conjugate working solution was added to each well, the plate was sealed, and incubated at 37°C for 30 min. Wash the plate 3-5 times again, add 90 μL of substrate (TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Measure the OD value of each well at 450 nm. Results are as follows: Figure 5 As shown, SCP-1 treatment of LPS-induced inflammatory cells inhibited the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in a dose-dependent manner, indicating that SCP-1 has a certain anti-inflammatory effect.

[0066] Example 5: In vivo anti-inflammatory activity assay of SCP

[0067] The ulcerative colitis (UC) mouse model was induced using 2.5% DSS to investigate the alleviating effect of SCP on ulcerative colitis. Sixty C57BL / 6 mice were randomly divided into six groups (control group CTRL, model group DSS, Mes group, L-SCP group, M-SCP group, and H-SCP group). After one week of acclimatization, starting on day 8, the control group had free access to distilled water, the DSS model group had free access to 2.5% DSS, and the experimental groups had free access to 2.5% DSS and were administered the positive control drug mesalazine (Mes) (100 mg / kg) or low (L-SCP, 100 mg / kg), medium (M-SCP, 200 mg / kg), and high (H-SCP, 400 mg / kg) crude polysaccharides from *Schizophyllum commune* fruiting bodies via gavage. The administration lasted for seven days.

[0068] 1. Measurement of colitis-related indicators

[0069] During the experiment, the mice's fur and activity were observed daily. Mice were weighed to monitor weight changes and the Disease Activity Index (DAI). Blood was collected from one eye of each mouse for subsequent experiments. Mice were euthanized, and the colon and spleen were harvested to measure colon length and spleen weight. The DAI was scored according to the criteria in Table 1. Results are as follows: Figure 6As shown, after administration of SCP, the symptoms of weight loss and colon shortening in mice were significantly improved, the disease activity index increased, and the spleen weight was increased, indicating that SCP has a relieving effect on DSS-induced ulcerative colitis in mice.

[0070] Table 1

[0071]

[0072] 2. The alleviating effect of SCP on tissue damage in UC mice

[0073] After 24 hours of fixation, colon tissue was dehydrated, cleared, and embedded in paraffin. It was then cut into 2-4 μm sections, dried, and dewaxed to obtain tissue sections. Hematoxylin was used for primary staining, followed by eosin counterstaining. After drying, the sections were mounted to obtain H&E-stained sections. Simultaneously, AB-PAS staining was used to assess the presence of mucin and goblet cells in the tissue (scoring criteria are shown in Table 2). Microscopic observation and photography were performed. Results are as follows: Figure 7 As shown, SCP can maintain the normal crypt structure of the intestine, reduce goblet cell loss, alleviate the degree of inflammatory infiltration, and protect epithelial cells.

[0074] Table 2

[0075]

[0076] 3. Effects of SCP on the levels of inflammatory factors in UC mice

[0077] The release of IL-1β, IL-6, TNF-α, and IL-10 from mouse serum was detected using a kit. Following the kit instructions, samples and standards of different concentrations were added to the corresponding wells at 100 μL / well, with 100 μL of diluent added to the blank control group. The plates were sealed and incubated at 37°C for 1 h. After incubation, the plates were removed, the liquid was discarded, and 100 μL of biotinylated antibody working solution was added to each well. The plates were sealed again and incubated at 37°C for 1 h. After the above steps, the liquid was discarded, and 300 μL of washing buffer was added to each well, allowing it to stand for 1 min. The washing buffer was then discarded, and this process was repeated 3 times. 100 μL of enzyme conjugate working solution was added to each well, and the plates were sealed and incubated at 37°C for 30 min. The plates were washed 3-5 times, and 90 μL of substrate (TMB) was added to each well. After incubation at 37°C in the dark for 15 min, 50 μL of stop solution was added to each well. The OD values ​​of each well were measured at 450 nm. The results are shown below. Figure 8 As shown, SCP inhibited the production of pro-inflammatory factors IL-6, IL-1β, and TNF-α in mice and promoted the release of anti-inflammatory factor IL-10 in a dose-dependent manner, indicating that SCP has a certain anti-inflammatory effect.

[0078] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A polysaccharide from the fruiting body of *Schizophyllum commune* with anti-inflammatory activity, characterized in that: The polysaccharide from the fruiting body of *Schizophyllum commune* has a molecular weight of 1.87 × 10⁻⁶. 4 Da, with a sugar content of 96.41% ± 2.12, and a monosaccharide molar ratio of mannose: glucose: galactose: arabinose: fucose = 9.057: 9.633: 9.631: 1.561:

1.

2. A method for preparing the anti-inflammatory polysaccharide from the fruiting body of *Schizophyllum commune* as described in claim 1, characterized in that, Includes the following steps: (1) The dried fruiting bodies of the schizophyllum commune are crushed by a pulverizer and then passed through an 80-mesh sieve to obtain powder; (2) The obtained powder was extracted with hot water at 85°C for 2 hours at a material-to-liquid ratio of 1:20, and the extraction was repeated 3 times. The extracts were then combined. (3) The extract obtained in step (2) is concentrated by rotary evaporation to 1 / 4 of its volume. Ethanol is added to the concentrate until the final volume concentration of ethanol is 80%. Then, the precipitate is precipitated at 4°C and allowed to stand for 12 h. The precipitate is then collected. (4) Dissolve the precipitate obtained in step (3) in water, remove the ethanol by rotary evaporation and continue to concentrate by rotary evaporation to obtain a sugar solution; remove the protein in the sugar solution using the Sevag method: mix the sugar solution, n-butanol and chloroform in a volume ratio of 5:1:4, shake for 10 min, let stand for layering and remove the protein layer, repeat until no protein layer appears. The remaining sugar solution was placed in a 3.5 kDa dialysis bag for dialysis. The solution was dialyzed with running water for 24 h and with static water for 48 h. After concentration, it was freeze-dried to obtain crude polysaccharide from the fruiting body of Schizophyllum commune. (5) The crude polysaccharide of the fruiting body of Schizophyllum commune obtained in step (4) was prepared into a solution of 40 mg / mL with deionized water. The insoluble impurities were removed by centrifugation and filtered through a 0.22 μm filter membrane. The retentate solution was placed in a DEAE-cellulose 52 ion exchange column for elution with deionized water as the mobile phase at a flow rate of 1 mL / min. The product was collected and freeze-dried. (6) The dried product obtained in step (5) was prepared into a solution of 80 mg / mL with deionized water, centrifuged to remove insoluble impurities, filtered through a 0.22 μm filter membrane, and then placed in a CL-4B molecular sieve for elution. The product was collected in separate tubes using an automatic collector with a flow rate of 1 mL / min and 8 min / tube. The product was collected and freeze-dried to obtain multiple tubes of dried product. (7) The dried products obtained in step (6) were prepared into 4 mg / mL solutions with deionized water. After filtration through a 0.22 μm filter membrane, they were detected by high performance liquid chromatography TSK-6000 with water as the mobile phase, a flow rate of 0.6 mL / min, and an injection volume of 20 μL. The target product, polysaccharide of Schizophyllum commune fruiting body, with uniform and symmetrical peak shape was detected.

3. The use of the anti-inflammatory polysaccharide from the fruiting body of *Schizophyllum commune* as described in claim 1, characterized in that: Used to prepare anti-inflammatory active agents for the treatment of colitis.

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