A uniform polysaccharide from polyporus and a preparation method and application thereof

Abstract

The present application relates to a kind of porous fungus uniform polysaccharide and its preparation method and application, belong to polysaccharide technical field.Structure, effective component and quality of natural polysaccharide in prior art for treating ulcerative colitis are difficult to control, and the technical problem of poor treatment effect stability is solved.The chemical structural formula of the uniform polysaccharide of the present application is shown as formula I, and the average molecular weight is 30.557Da.The structure of the uniform polysaccharide is clear, has the exact curative effect for treating ulcerative colitis by relying on the way of intestinal flora, provides new candidate drug for the treatment or prevention of ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide technology, specifically relating to a homogeneous polysaccharide of polypores, its preparation method and application, and particularly to the application of this homogeneous polysaccharide of polypores in the preparation of drugs for treating or preventing ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing, nonspecific inflammatory bowel disease characterized by persistent inflammation and ulceration of the colonic mucosa and submucosa.

[0003] Currently, ulcerative colitis (UC) has become a global public health problem, with its incidence continuing to rise worldwide. Existing clinical treatments, such as aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics, while providing short-term symptom relief, have significant drawbacks, including strong side effects (such as gastrointestinal irritation, metabolic disorders, and immunosuppression), limited long-term efficacy, and a tendency to develop drug resistance.

[0004] In recent years, studies have shown that natural polysaccharides, due to their high safety, low toxicity, and multiple biological activities such as anti-inflammatory, immunomodulatory, and intestinal barrier repair, have become novel candidate drugs for the treatment of ulcerative colitis (UC). For example, studies have reported that bamboo shoot polysaccharides, Panax notoginseng polysaccharides, jellyfish polysaccharides, and fermented wolfberry polysaccharides have ameliorative effects on UC. However, these studies have the following problems: (1) Difficulty in controlling structure, active ingredients and quality: Most natural polysaccharide preparation methods currently produce multi-component mixtures with defects such as low purity, non-uniform molecular weight, difficulty in precise control of structure and difficulty in standardization of active ingredients. This results in large differences in activity between different batches of products, making it difficult to guarantee the stability and reproducibility of therapeutic effects and limiting its further development and application.

[0005] (2) Insufficient stability of therapeutic effect: Existing polysaccharide products lack precise control over the regulation of intestinal microecology, making it difficult to identify the key pathways for exerting therapeutic effects. This results in significant differences in their anti-inflammatory, immune-regulating, and intestinal barrier repair effects, making it difficult to achieve stable and efficient treatment for ulcerative colitis.

[0006] Therefore, there is a need to develop highly effective, low-toxicity drugs from natural sources to treat ulcerative colitis.

[0007] *Daedaleopsis confragosa*, a fungus belonging to the Polyporaceae family, possesses medicinal and nutritional value. Existing literature reports its antioxidant, antiviral, and antitumor activities. However, there are currently no reports, either domestically or internationally, of isolating and purifying homogeneous polysaccharides (polysaccharide components with uniform molecular weight and charge distribution) from *Daedaleopsis confragosa*, nor are there any reports of its use in the treatment of ulcerative colitis. Summary of the Invention

[0008] This invention addresses the limitations of existing technologies in the treatment of ulcerative colitis (UC), such as the limited activity intensity, unstable therapeutic effects, and difficulty in standardizing and controlling the effective components of polysaccharides. Furthermore, the complex origins and significant structural variations of some natural polysaccharides make stable preparation and quality control challenging. This invention provides a homogeneous polysaccharide from polypores, its preparation method, and its application. This homogeneous polysaccharide has a well-defined structure and exhibits definite therapeutic efficacy in treating ulcerative colitis via a gut microbiota-dependent pathway, providing a new candidate drug for the treatment and prevention of UC.

[0009] The present invention addresses the above-mentioned technical problems by adopting the following technical solution.

[0010] In a first aspect, the present invention provides a homogeneous polysaccharide from a polypore (DCP-A) with an average molecular weight of 30.557 kDa, and its chemical structural formula is shown in Formula I: Formula I.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned homogeneous polysaccharide from polypores, comprising the following steps: S1. Extraction: The dried fruiting bodies of *Schizophyllum commune* were extracted using hot water extraction. The extract was concentrated, precipitated with alcohol, and the precipitate was collected by centrifugation and freeze-dried to obtain crude polysaccharide. S2. Purification: The crude polysaccharide was purified by ion exchange chromatography to obtain homogeneous polysaccharide from polypores.

[0012] Preferably, in S1, the process parameters of the hot water extraction method are: extraction time 2h, extraction temperature 72℃, and material-to-liquid ratio 1g:38mL.

[0013] Preferably, in S1, the concentration is 20-40 times.

[0014] Preferably, in S1, the alcohol precipitation uses ethanol with a final concentration of 75%, a precipitation temperature of 2-8°C, and a precipitation time of 10-16 hours.

[0015] Preferably, in S1, the centrifugation conditions are 8000 rpm / min for 10 min.

[0016] Preferably, in S1, ultrasonic-assisted extraction, microwave-assisted extraction, or enzyme-assisted extraction are used.

[0017] Preferably, in step S2, the crude polysaccharide is dissolved in water and subjected to anion exchange chromatography. Ultrapure water and NaCl aqueous solution are used for elution sequentially. The eluent of the NaCl aqueous solution is collected to obtain the acidic polysaccharide component. After concentration, ultrafiltration and centrifugation are performed, followed by dialysis. The retentate is collected and freeze-dried to obtain homogeneous polysaccharide from polypores.

[0018] More preferably, in S2, the anion exchange chromatography is DEAE-cellulose anion exchange chromatography.

[0019] More preferably, in S2, the concentration of the NaCl aqueous solution is 0.5M.

[0020] More preferably, in S2, the elution flow rates of the ultrapure water and the NaCl aqueous solution are 5 mL / min, respectively.

[0021] Preferably, in S2, the ultrafiltration centrifugation separation uses a 30kDa ultrafiltration centrifuge tube and is centrifuged at 4000 rpm for 15 min.

[0022] More preferably, in S2, the dialysis is performed using a 2000Da dialysis bag.

[0023] Thirdly, the present invention also provides products containing the above-mentioned polysaccharide of the polypore bacteria for the treatment or prevention of ulcerative colitis.

[0024] Preferably, the product is a drug or health food.

[0025] Preferably, the product further includes one or more of fillers, binders, disintegrants, and lubricants.

[0026] Preferably, the dosage form of the product is granules, tablets, capsules, oral liquid, or dry suspension.

[0027] Fourthly, the present invention also provides the use of the above-mentioned polyporous homogeneous polysaccharide in the preparation of products for the treatment or prevention of ulcerative colitis.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The polysaccharide of the present invention, DCP-A, is characterized by its natural origin, high safety and stable biological activity. This polysaccharide can regulate the balance of intestinal microecology, promote the recovery of beneficial flora, and improve the intestinal environment, thereby exerting anti-inflammatory and intestinal protective effects. It has the potential to be used as a low-toxicity, high-efficiency natural-source anti-ulcerative colitis drug or functional preparation.

[0029] This invention prepares a homogeneous acidic polysaccharide derived from *Schizothorax spp.*, which features a homogeneous structure, well-defined molecular weight, and stable composition. This overcomes the problems of complex composition, heterogeneous structure, and difficulty in quality control of existing natural polysaccharides, and is beneficial for the standardized preparation and quality evaluation of polysaccharide products.

[0030] The DCP-A of this invention has a main chain of →6)-β-D-Glcp-(1→), with β-D-glucose residues (β-D-Glcp) linked by β-(1→6) glycosidic bonds. Specifically, the anomeric carbon at the C1 position of one glucose residue is linked to the hydroxyl group at the C6 position of the next glucose residue by a glycosidic bond, forming a continuous repeating structural unit of →6)-β-D-Glcp-(1→). The side chain contains β-D-Glcp-(1→3)-β-D-Glcp-(1→), meaning that two β-D-glucanose (β-D-Glcp) residues are linked by β-( The disaccharide structural unit formed by 1→3) glycosidic bond linkage, β-D-GlcpA-(1→4)-β-D-GlcpA-(1→), that is, the disaccharide structural unit formed by two β-D-glucopyranose (β-D-Glcp) residues linked by β-(1→4) glycosidic bonds, aD-Manp-(1→4)-β-D-Glcp-(1→]9, where α-D-Manp represents α-D-mannopyranose residues, and the C1 position is linked by a glycosidic bond to a β-(1→4)-glucan chain segment composed of 9 β-D-glucopyranose residues.

[0031] (2) This invention not only demonstrates the in vivo efficacy of DCP-A, but also, through antibiotic clearance and fecal microbiota transplantation experiments, confirms for the first time that DCP-A relies on gut microbiota to repair the intestinal barrier, inhibit inflammation and oxidative stress, and exert its anti-UC effect. This complete "phenotype-mechanism" evidence chain is not found in most existing studies. DCP-A can effectively alleviate colonic inflammatory damage, reduce inflammatory factor levels, improve intestinal barrier function, and reduce colonic tissue pathological damage, showing good therapeutic effects on ulcerative colitis.

[0032] (3) This invention optimizes the polysaccharide extraction process, ensuring the stability of extraction efficiency; the DCP-A preparation method provided by this invention has the characteristics of strong operability and good reproducibility. The DCP-A obtained through separation and purification has high purity and the preparation process is stable, which can provide technical support for subsequent drug development, functional food development and industrial application of related products. Attached Figure Description

[0033] Figure 1 This is a high-performance gel permeation chromatography of DCP-A prepared in Example 1 of the present invention.

[0034] Figure 2The results of PMP pre-column derivatization-HPLC determination of DCP-A prepared in Example 1 of this invention are shown in the figure. In the figure, Man: mannose; GlcN: glucosamine hydrochloride; Rha: rhamnose; GlcA: glucuronic acid; GalA: galacturonic acid; Glc: glucose; Gal: galactose; Xyl: xylose; Ara: arabinose; Fuc: fucose; Standard Sample is the standard sample.

[0035] Figure 3 The ultraviolet spectrum of DCP-A prepared in Example 1 of this invention.

[0036] Figure 4 The infrared spectrum of DCP-A prepared in Example 1 of this invention.

[0037] Figure 5 The DCP-A prepared in Example 1 of this invention 1 H-NMR nuclear magnetic resonance spectrum; Figure 6 The DCP-A prepared in Example 1 of this invention 13 C-NMR nuclear magnetic resonance spectrum; Figure 7 The heteronuclear single quantum coherence spectrum (HSQC) of DCP-A prepared in Example 1 of this invention; Figure 8 The correlated magnetic resonance spectrum (COSY) of DCP-A prepared in Example 1 of this invention; Figure 9 HSQC-COSY of DCP-A prepared in Example 1 of this invention; Figure 10 The heteronuclear multiple bond coherence spectrum (HMBC) of DCP-A prepared in Example 1 of this invention; Figure 11 The nuclear Overhaus effect spectrum (NOESY) of DCP-A prepared in Example 1 of this invention; Figure 5-11 In the diagram, A represents →3)-β-D-Glcp-(1-sugar residue), B represents →3,6)-β-D-Glcp-(1-sugar residue), C represents →6)-β-D-Glcp-(1-sugar residue), D represents β-D-Glcp-(1-sugar residue), E represents →4)-β-D-Glcp-(1-sugar residue), F represents →4,6)-β-D-Glcp-(1-sugar residue), G represents aD-Manp-(1-sugar residue), H represents →4)-β-D-GIcpA-(1-sugar residue), I represents β-D-GIcpA-(1-sugar residue), & represents &, and f1 and f2 represent chemical shifts.

[0038] Figure 12The effect of DCP-A prepared in Example 1 of this invention on the clinical symptoms of DSS-induced colitis mice is shown in the figure. In the figure, A represents the change in colon length, B represents the change in body weight, C represents the change in disease activity index (DAI), and D represents the change in spleen index. Indicates significance.

[0039] Figure 13 The effect of DCP-A prepared in Example 1 of this invention on the intestinal barrier integrity and colonic mucus layer of DSS-induced colitis mice is shown in Figure 1. A represents H&E staining, B represents AB-PAS staining, and C represents immunofluorescence staining (corresponding to the results of incubation with primary antibodies against tight junction protein ZO-1, Occludin, and mucin MUC-2, respectively).

[0040] Figure 14 This invention relates to the effects of DCP-A prepared in Example 1 on inflammation and oxidative stress in mice with DSS-induced colitis. In the figure, A represents the IL-6 level in colonic tissue, B represents the TNF-α level in colonic tissue, C represents the IL-1β level in colonic tissue, D represents the IL-17A level in colonic tissue, E represents MPO activity and oxidative stress markers, F represents MDA activity and oxidative stress markers, G represents GSH activity and oxidative stress markers, and H represents T-AOC level. In the figure, IL-6 represents interleukin-6, TNF-α represents tumor necrosis factor-α, IL-1β represents interleukin-1β, IL-17A represents interleukin-17A, MPO represents myeloperoxidase, MDA represents malondialdehyde, GSH represents reduced glutathione, and T-AOC represents total antioxidant capacity. Indicates significance.

[0041] Figure 15 The figure shows the effect of DCP-A prepared in Example 1 of this invention on the composition of mouse gut microbiota after intervention. In the figure, A is principal component analysis (PCA), B is principal coordinate analysis (PCoA), C is nonmetric multidimensional scaling analysis (NMDS), and D is partial least squares discriminant analysis (PLS-DA). In the figure, PC1, NMDS1, and COMP1 are the first principal components, and PC2, NMDS2, and COMP2 are the second principal components.

[0042] Figure 16 The microbial composition of the posterior fossa of the mouse gut prepared by DCP-A intervention in Example 1 of this invention.

[0043] Figure 17 The relative abundance of primary gut microbiota in mice after DCP-A intervention prepared in Example 1 of this invention.

[0044] Figure 18The effect of DCP-A prepared in Example 1 of this invention on colitis symptoms in antibiotic-treated colitis mice is shown in the figure. In the figure, A represents change in colon length, B represents change in body weight, C represents change in disease activity index (DAI), and D represents change in spleen index. Indicates significance.

[0045] Figure 19 The effect of DCP-A prepared in Example 1 of this invention on the intestinal barrier and colonic mucus of antibiotic-treated colitis mice is shown in Figure 1. A represents H&E staining, B represents AB-PAS staining, and C represents immunofluorescence staining (corresponding to the results of incubation with primary antibodies against tight junction protein ZO-1, Occludin, and mucin MUC-2, respectively).

[0046] Figure 20 The figure shows the effect of FMT after DCP-A intervention on colitis mice prepared in Example 1 of this invention. In the figure, A represents the change in colon length, B represents the change in body weight, C represents the change in disease activity index (DAI), and D represents the change in spleen index. Indicates significance.

[0047] Figure 21 The effect of FMT after DCP-A intervention prepared in Example 1 of this invention on the intestinal barrier and colonic mucus of colitis mice is shown in Figure A, where A is H&E staining, B is AB-PAS staining, and C is immunofluorescence staining (corresponding to the results of incubation with primary antibodies against tight junction protein ZO-1, Occludin, and mucin MUC-2, respectively).

[0048] Figure 22 This figure illustrates the effect of DCP-A intervention followed by FMT on colitis mice in Example 1 of this invention. In the figure, A represents the IL-6 level in colonic tissue, B represents the TNF-α level in colonic tissue, C represents the IL-1β level in colonic tissue, D represents the IL-17A level in colonic tissue, E represents MPO activity and oxidative stress markers, F represents MDA activity and oxidative stress markers, G represents GSH activity and oxidative stress markers, and H represents T-AOC level. In the figure, IL-6 represents interleukin-6, TNF-α represents tumor necrosis factor-α, IL-1β represents interleukin-1β, IL-17A represents interleukin-17A, MPO represents myeloperoxidase, MDA represents malondialdehyde, GSH represents reduced glutathione, and T-AOC represents total antioxidant capacity. Indicates significance. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0050] The technical solutions disclosed in the various embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0051] Example 1 Preparation of homogeneous polysaccharides from polypores: (1) Extraction: Take dried fruiting bodies of *Pterygomyces spp.* and add them to water at a ratio of 1 g: 38 mL. Extract at 72°C for 2 h to obtain the extract (polysaccharide extraction rate can reach 3.12%). After concentrating the extract 30 times, add 75% ethanol and precipitate at 4°C for 12 h. Centrifuge at 8000 rpm / min for 10 min to collect the precipitate. Freeze dry to obtain crude polysaccharide (DCP).

[0052] (2) Purification: The crude polysaccharide was dissolved in water and loaded onto a DEAE-Cellulose anion exchange chromatography column (26 mm × 50 cm). It was eluted sequentially with ultrapure water and 0.5 M NaCl aqueous solution at a flow rate of 5 mL / min. The acidic polysaccharide fraction eluted by NaCl aqueous solution was collected and centrifuged at 4000 rpm for 15 min using a 30 kDa ultrafiltration centrifuge tube. The sample was dialyzed using a 2000 Da dialysis bag, and the retentate was collected and freeze-dried to obtain a homogeneous polysaccharide, named Polyporus homogeneous polysaccharide, denoted as DCP-A.

[0053] 1. The structure of DCP-A prepared in Example 1 was analyzed.

[0054] 1.1 The molecular weight and homogeneity of DCP-A were determined by high-performance gel permeation chromatography (HPGPC), and the results are as follows: Figure 1 As shown in the figure, DCP-A has a single symmetrical peak and a molecular weight of 30.557 kDa.

[0055] 1.2 The monosaccharide composition of DCP-A was determined using the PMP pre-column derivatization-HPLC method, and the results are as follows: Figure 2 As shown in the figure, DCP-A is mainly composed of glucose (Glc), glucuronic acid (GlcA) and mannose (Man), with a molar ratio of 68.346:9.707:6.679, and contains a small amount of other monosaccharides.

[0056] 1.3 Ultraviolet spectroscopy was performed on DCP-A, and the results are as follows: Figure 3As shown in the figure, there are no absorption peaks at 260nm and 280nm, indicating that there are no nucleic acid and protein impurities.

[0057] 1.4 Infrared spectroscopy was performed on DCP-A, and the results are as follows: Figure 4 As shown in the figure, at 3394 cm -1 (OH), 2928 cm -1 (CH), 1609 cm -1 (-COO - Typical polysaccharide characteristic absorption peaks are found at locations such as [insert locations here], confirming that it is an acidic polysaccharide.

[0058] 1.5 Methylation of DCP-A and analysis by gas chromatography-mass spectrometry (GC-MS) were performed to determine its glycosidic bond linkage mode. The results are shown in Table 1.

[0059] Table 1. Types and proportions of DCP-A glycosidic bonds

[0060] Continued from Table 1: Types and proportions of DCP-A glycosidic bonds

[0061] Continued from Table 1: Types and proportions of DCP-A glycosidic bonds

[0062] As shown in Table 1, the main glycosidic bond types of DCP-A include →6)-Glcp-(1→(32.84%), t-Glcp-(1→(17.88%), →4)-Glcp-(1→(14.36%), →3)-Glcp-(1→(7.35%), and →3,6)-Glcp-(1→(6.54%).

[0063] 1.6 Nuclear magnetic resonance (NMR) analysis was performed on DCP-A, including... 1 H-NMR nuclear magnetic resonance analysis, 13 C-NMR nuclear magnetic resonance analysis, heteronuclear single quantum coherence spectroscopy (HSQC) analysis, correlated magnetic resonance spectrum (COSY) analysis, HSQC-COSY analysis, heteronuclear multibonded coherence spectroscopy (HMBC), and nuclear Overhaus effect spectroscopy (NOESY) results are as follows: Figure 5-11 As shown, a comprehensive one-dimensional ( 1 H-NMR, 13Using C-NMR and two-dimensional (HSQC, HMBC, COSY, NOESY) data, the sugar residue signals of DCP-A were precisely assigned, ultimately determining its main chain and side chain structures. Its main chain consists of →6)-β-D-Glcp-(1→), with side chain branching points mainly at the O-3 and O-6 positions of the →3,6)-β-D-Glcp-(1→ residues. Connected side chain fragments include β-D-Glcp-(1→, β-D-GlcpA-(1→, →4)-β-D-Glcp-(1→, →3)-β-D-Glcp-(1→, →4)-β-D-GlcpA-(1→, α-D-Manp-(1→), etc., thus determining its chemical structural formula.

[0064] 2. Study on the ameliorative effect and mechanism of DCP-A on ulcerative colitis (UC).

[0065] Fifty mice were randomly divided into five groups (n=10 per group): control group (Con), model group (Mod), mesalazine treatment group (5-ASA), low-dose DCP-A group (L-DCP-A), and high-dose DCP-A group (H-DCP-A). Except for the Con group, which received sterile water, the other groups received 3% (w / v) sodium dextran sulfate (DSS) in their drinking water for seven consecutive days to induce acute colitis. During the experiment, mice in the Con and Mod groups were administered physiological saline (0.1 mL / 10 g body weight) by gavage. The 5-ASA group received mesalazine 200 mg / kg / day. Mice in the L-DCP-A and H-DCP-A groups received DCP-A at doses of 200 and 400 mg / kg / day, respectively. Fresh fecal samples were collected during the treatment period and immediately stored at -80°C. Mouse body weight, fecal occult blood disease activity index (DAI), and spleen index were monitored daily. At the end of the experiment, the mice were euthanized, the length of the colon was measured, and tissue from the distal end of the colon was collected.

[0066] 2.1 Effects of DCP-A on colon length, mouse body weight, DAI, and spleen index, as follows Figure 12 As shown in the AD diagram, the disease symptoms improved: the high-dose DCP-A group significantly reversed the weight loss, shortened colon length, increased DAI and splenomegaly caused by DSS (p<0.001).

[0067] 2.2 Distal colon tissue was collected, rinsed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4 μm thick sections. For histological evaluation, the sections were stained with H&E and AB-PAS and examined under a light microscope. The results are as follows: Figure 13As shown in Figures A and B. After dewaxing and replenishing the solution, the antigen was recovered in 0.01 mol / L MEDTA buffer (pH 8.0), stored at 95-100℃ for 15 min, cooled to room temperature, and then blocked with 5% bovine serum albumin solution containing 0.3% Triton X-100 for 1 h. The cells were then incubated with ZO-1, Occludin, and MUC2 primary antibodies at 4℃, washed with PBST, and incubated with Cy3-labeled secondary antibody at room temperature in the dark for 1 h. Cell nuclei were reverse stained with DAPI, and fluorescence images were captured under a fluorescence microscope. The results are shown in Figures A and B. Figure 13 As shown in Figure C. H&E and AB-PAS staining showed that DCP-A significantly reduced pathological damage in colon tissue and increased the number of goblet cells. Immunofluorescence results showed that DCP-A significantly upregulated the expression of ZO-1, Occludin, and MUC-2 in colon tissue (p<0.001).

[0068] 2.3 Determination of inflammatory cytokines and oxidative stress markers: Distal colon tissue was collected, homogenized in the appropriate extraction buffer, and centrifuged to obtain the supernatant. The levels of TNF-α, IL-1β, IL-6, MPO, T-AOC, SOD, GSH, and MDA were quantified using commercial ELISA kits according to the manufacturer's instructions. The results are shown below. Figure 14 As shown in the figure. The results showed that DCP-A significantly reduced the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β, IL-17A) and myeloperoxidase (MPO) in colon tissue, while increasing antioxidant markers (T-AOC, GSH) and decreasing malondialdehyde (MDA) levels (p<0.001).

[0069] 2.4 Gut Microbiota Analysis: Total genomic DNA was extracted from fecal samples of the control group, model group, and high-dose DCP-A group using the FastPure fecal DNA isolation kit, according to the manufacturer's instructions. DNA quality was assessed by 1% agarose gel electrophoresis and quantified using a NanoDrop2000 spectrophotometer (Thermo Scientific, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 338F and 806R. The purified PCR products were used to construct libraries using the NEXTFLEX rapid DNA-Seq kit, and sequencing was performed on the Illumina NextSeq2000 platform by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. Beta diversity analysis, including PCA, PCoA, NMDS, and PLS-DA, was performed as follows: Figure 15 The figure shows that there are obvious clusters among the control group, model group, and high-dose DCP-A group. The microbial community structure of the high-dose DCP-A group has significantly shifted towards that of the control group, indicating that the DSS-induced microbial ecological imbalance has been substantially restored. Figure 16 As shown, at the phylum level, Bacteroidetes, Bacteroidetes, and Verrucomicrobiota were identified as the dominant taxa. DSS treatment disrupted the microbial composition by decreasing the relative abundance of Bacteroidetes and increasing the relative abundance of Bacteroidetes, while DCP-A intervention partially reversed these changes, promoting a more balanced microbial community. Figure 17 As shown, at the genus level, norank_f__Muribaculaceae, Akkermansia, and Bacteroides represent the dominant bacterial taxa.

[0070] 3. Antibiotic-mediated microbial depletion experiment.

[0071] To investigate whether the protective effect of DCP-A depends on the gut microbiota, we established an antibiotic depletion model. Thirty mice were randomly divided into three groups (n=10): the antibiotic treatment group (ABX), the DSS group (AD), and the DCP-A treatment group (ADP). The experimental protocol lasted for 18 days. From day 1 to day 10, all mice were orally administered once daily an antibiotic consisting of ampicillin (2 mg), neomycin sulfate (2 mg), metronidazole (2 mg), and vancomycin (1 mg) to deplete the gut microbiota. From day 11 to day 17, mice in the AD and ADP groups received drinking water supplemented with 3% DSS, while mice in the ABX group received drinking water supplemented with distilled water. Simultaneously, mice in the ABX and AD groups were administered physiological saline by gavage, while mice in the ADP group were administered DCP-A (400 mg / kg / day) by gavage. Mouse body weight, DAI (digestive acid intake), and spleen index were monitored daily. At the end of the experiment, the mice were euthanized, colon length was measured, and distal colon tissue was collected.

[0072] Effects on colon length, mouse body weight, DAI, and spleen index, such as Figure 18 As shown in the figure, under conditions of microbiome depletion, the therapeutic effect of DCP-A was largely negated, with no significant improvement in weight loss, DAI, colon length reduction, and spleen index.

[0073] The distal colon tissue was subjected to H&E staining, AB-PAS staining, and immunofluorescence detection (ZO-1, Occludin, and MUC-2) according to the method described in section 2.2. The results are as follows: Figure 19 As shown in AC, it can be seen from the figure that, with Figure 18 Consistently, DCP-A failed to improve DSS-induced intestinal histological damage or tight junction protein expression in antibiotic-treated mice.

[0074] 4. Fecal microbiota transplantation (FMT).

[0075] Fresh fecal samples were collected from donor mice in the DCP-A treatment group (ADP) in an antibiotic-mediated microbiota depletion experiment. Fecal microbiota suspensions were prepared according to a previously reported procedure (Zhang S, Zhou R, Xie X, Xiong S, Li L, Li Y. Polysaccharides from Lycium barbarum, yam, and sunflower ameliorate colitis in a structure and intrinsic flora-dependent manner. Carbohydr Polym. 2025;349(Pt A):122905.). Thirty recipient mice were randomly assigned to three groups (n=10): a healthy control group (F-Con), a DSS model group (F-Mod), and an FMT treatment group (F-DCP-A). During the experiment, mice in the F-Con group received sterile drinking water, while mice in the F-Mod and F-DCP-A groups received 3% DSS solution to induce colitis for 7 consecutive days. During the experiment, mice in the F-Con and F-Mod groups were given physiological saline (0.1 mL / 10 g body weight), while mice in the F-DCP-A group were administered 200 μL of freshly prepared fecal suspension by gavage daily. Mouse body weight, DAI (digestive acidity), and spleen index were monitored daily. At the end of the experiment, mice were euthanized, colon length was measured, and distal colon tissue was collected.

[0076] Effects on colon length, mouse body weight, DAI, and spleen index, such as Figure 20 As shown in the AD diagram, there were significant improvements in weight loss, DAI, colon length reduction, and spleen index.

[0077] The distal colon tissue was subjected to H&E staining, AB-PAS staining, and immunofluorescence detection (ZO-1, Occludin, and MUC-2) according to the method described in section 2.2. The results are as follows: Figure 21 As shown in AC, DCP-A significantly reduces pathological damage to colon tissue, increases the number of goblet cells, and significantly upregulates the expression of tight junction proteins ZO-1, Occludin, and mucin MUC-2 in colon tissue (p<0.001).

[0078] The inflammatory cytokines and oxidative stress markers were measured according to the method in section 2.3, and the results are as follows: Figure 22 As shown in Figure AH, this demonstrates that FMT from the DCP-A-treated donor effectively reconstructed its protective effect, leading to significant improvements in inflammatory status, oxidative stress, and intestinal barrier integrity in recipient mice.

[0079] The above results indicate that the beneficial effects of DCP-A heavily depend on a complete and well-functioning gut microbiota, highlighting the importance of the microbiota as a key medium for its treatment of DSS-induced colitis.

[0080] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A homogeneous polysaccharide from polypores, characterized in that, The average molecular weight is 30.557 kDa, and the chemical structure is shown in Formula I: ； Formula I.

2. The method for preparing homogeneous polysaccharide from polypores according to claim 1, characterized in that, Includes the following steps: S1. Extraction: The dried fruiting bodies of *Schizophyllum commune* were extracted using hot water extraction. The extract was concentrated, precipitated with alcohol, and the precipitate was collected by centrifugation and freeze-dried to obtain crude polysaccharide. S2. Purification: The crude polysaccharide was purified by ion exchange chromatography to obtain homogeneous polysaccharide from polypores.

3. The method for preparing homogeneous polysaccharide from polypores according to claim 2, characterized in that, S1 possesses one or more of the following characteristics: The process parameters for the hot water extraction method are: extraction time 2h, extraction temperature 72℃, and material-to-liquid ratio 1g:38mL. The concentration is 20-40 times concentrated; The alcohol precipitation uses ethanol with a final concentration of 75%, a precipitation temperature of 2-8℃, and a precipitation time of 10-16h. The centrifugation conditions were 8000 rpm / min for 10 min; Ultrasonic-assisted extraction, microwave-assisted extraction, or enzyme-assisted extraction are employed.

4. The method for preparing homogeneous polysaccharide from polypores according to claim 2, characterized in that, In S2, the crude polysaccharide was dissolved in water and subjected to anion exchange chromatography. Ultrapure water and NaCl aqueous solution were used for elution sequentially. The eluent of NaCl aqueous solution was collected to obtain the acidic polysaccharide component. After concentration, ultrafiltration and centrifugation were performed, followed by dialysis. The retentate was collected and freeze-dried to obtain homogeneous polysaccharide from polypores.

5. The method for preparing homogeneous polysaccharide from polypores according to claim 4, characterized in that, S2 possesses one or more of the following characteristics: The anion exchange chromatography is DEAE-cellulose anion exchange chromatography; The concentration of the NaCl aqueous solution is 0.5M; The elution flow rates for the ultrapure water and NaCl aqueous solution were 5 mL / min, respectively. The ultrafiltration centrifugation separation was performed using 30kDa ultrafiltration centrifuge tubes, centrifuged at 4000rpm for 15min; The dialysis was performed using a 2000Da dialysis bag.

6. A product containing the homogeneous polysaccharide of the polypores as described in claim 1 for the treatment or prevention of ulcerative colitis.

7. The product for treating or preventing ulcerative colitis according to claim 6, characterized in that, The product is a medicine or health food.

8. The product for treating or preventing ulcerative colitis according to claim 6, characterized in that, It also includes one or more of fillers, adhesives, disintegrants, and lubricants.

9. The product for treating or preventing ulcerative colitis according to claim 6, characterized in that, The dosage form of the product is granules, tablets, capsules, oral liquid, or dry suspension.

10. The use of the polyporous homogeneous polysaccharide according to claim 1 in the preparation of products for the treatment or prevention of ulcerative colitis.

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