A high-molecular-weight polysaccharide from *Rhizopus rubrum* and its applications

Abstract

The application discloses a blood red hammerhead mushroom high-molecular polysaccharide and application thereof, and belongs to the field of biology. The main chain structure of the blood red hammerhead mushroom high-molecular polysaccharide is beta-(1→6)-Man p . The blood red hammerhead mushroom high-molecular polysaccharide comprises five branched chains. The five branched chains comprise three Gals and two Fucs at the end. The blood red hammerhead mushroom high-molecular polysaccharide forms a repeating unit with the main chain and the branched chains. The application of the blood red hammerhead mushroom high-molecular polysaccharide is also disclosed. The blood red hammerhead mushroom high-molecular polysaccharide can be used for preparing products for regulating immune function, in particular, products for improving or treating intestinal flora disorder caused by immune damage, such as a pharmaceutical composition and the like.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a high-molecular-weight polysaccharide from *Rhizopus rubrum* and its applications. Background Technology

[0002] Chemotherapy drugs such as cyclophosphamide (CTX) are widely used clinically, but one of their serious side effects is damage to the immune system, manifested as atrophy of immune organs, decreased lymphocyte count, and suppressed function. Therefore, finding natural protective agents that can effectively alleviate chemotherapy-induced immune damage is of great significance. Recent studies have shown that the immunomodulatory effects of polysaccharides are closely related to their regulation of the gut microbiota. As the human body's "second genome," the gut microbiota's structural disorder is associated with the occurrence and development of various diseases. Many active polysaccharides cannot be directly digested and absorbed by the human body, but they can be fermented and utilized by gut microbes as prebiotics, regulating the microbiota structure and producing beneficial metabolites such as short-chain fatty acids, thereby affecting the overall immune status through the "gut-immune axis."

[0003] Polysaccharides are important biomolecules in organisms with a wide range of biological activities, such as immunomodulation, antitumor activity, antioxidant activity, and regulation of gut microbiota. Edible and medicinal fungi are an important source of bioactive polysaccharides; for example, lentinan from shiitake mushrooms and polysaccharides from Ganoderma lucidum have been extensively studied and applied in the medical and health fields. These fungal polysaccharides exert their immune-enhancing effects by activating immune cells and secreting cytokines, and are considered potential natural immunomodulators.

[0004] *Chroogomphus rutilus* is a valuable edible fungus, rich in various nutrients and bioactive substances. Current research on *Chroogomphus rutilus* mainly focuses on the identification of its chemical components and the activity screening of crude extracts. Although some studies have mentioned its polysaccharide components, a clear and definitive characterization of the precise primary structure of its polysaccharides (such as the monosaccharide composition ratio, glycosidic bond type, and molecular weight) is still lacking. More importantly, whether its polysaccharides exert their immunoprotective effects through the specific pathway of the "gut microbiota-metabolite-immunity" axis has not yet been reported in depth and systematically. Therefore, there is a need to provide a structurally well-defined, highly immunomodulatory water-extracted polysaccharide from *Chroogomphus rutilus*, and to elucidate its mechanism of action, providing a scientific basis for its application in the pharmaceutical field. Summary of the Invention

[0005] This invention provides a high-molecular-weight polysaccharide from *Rhizopus hemlockenspidium*, clarifies its specific chemical structure and molecular weight, and provides its application in immunomodulation, particularly in repairing cyclophosphamide-induced immune damage, thus providing a solid theoretical basis for its application in the pharmaceutical field.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high-molecular-weight polysaccharide from *Rhizopus rubrum*, wherein the main chain structure of the *Rhizopus rubrum* high-molecular-weight polysaccharide is β-(1→6)-Man. p The *Rhizopus rubiginosa* polymer polysaccharide comprises five branches; the ends of the five branches contain three Gal and two Fuc; the *Rhizopus rubiginosa* polymer polysaccharide forms repeating units with the main chain and branches.

[0007] Preferably, the side chains of the *Rhizopus rubiginosa* polymer polysaccharide further contain β-(1→6)-Glc. p and α-(1→6)-Gal p and α-(1→3)-Ara f .

[0008] Preferably, the monosaccharide molar ratio of the *Rhizopus rubiginii* polymer polysaccharide is Fuc:Ara:Man:Glc:Gal = 1.04:0.56:2.58:2.12:1.00.

[0009] Preferably, the molecular weight of the *Rhizopus rubiginosa* high-molecular-weight polysaccharide is (2.6±2)×10⁻⁶. 6 Da.

[0010] Preferably, the repeating unit of the blood-red rosary mushroom high molecular weight polysaccharide contains sixteen Man, nine Glc, five Gal, two Fuc, and one Ara.

[0011] This invention also provides the application of Heliotropium indicum high molecular weight polysaccharide in the preparation of products for regulating immune function.

[0012] Preferably, the application is in the preparation of products for improving or treating intestinal flora imbalance caused by immune damage.

[0013] The present invention also provides a pharmaceutical composition comprising the aforementioned high-molecular-weight polysaccharide from *Rhizopus heliotropium*.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 1. Well-defined structure and strong innovation: This invention is the first to isolate and purify a well-defined polysaccharide from Rhizopus rubrum, Rhizopus rubrum high molecular weight polysaccharide (CRWP), and accurately characterizes its monosaccharide composition molar ratio, molecular weight and glycosidic bond linkage mode. This polysaccharide with a specific structure is reported for the first time and is a newly discovered active substance.

[0016] 2. Significant activity and clear function: In vivo experiments have confirmed that CRWP (200 mg / kg) has excellent immune repair function in a mouse model of cyclophosphamide-induced immune damage, and can effectively increase the proportion of lymphocyte subsets and restore immune balance.

[0017] 3. Novel Mechanism, In-depth Elucidation: This invention goes beyond simple phenotypic observation and deeply elucidates the mechanism of action of CRWP. It is the first to discover and confirm that CRWP exerts its immunomodulatory effect by regulating the gut microbiota structure (especially increasing the abundance of Lactobacillus) and influencing the gut metabolic profile (such as promoting plant hormone signal transduction pathways), ultimately through the systemic pathway of the "gut microbiota-metabolite-immune axis," providing solid theoretical support for its application.

[0018] 4. Broad Application Prospects: The CRWP preparation method provided by this invention is simple, reliable, and easy to scale up for production. As a natural immunomodulator, this polysaccharide has enormous application potential in the development of adjuvant immunotherapy drugs, adjuvant drugs for tumor chemotherapy, or health products for enhancing immunity.

[0019] Other features and advantages of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments or related technologies of this disclosure, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 This is a size exclusion chromatogram of liquid chromatography used to determine the average molecular weight of the high molecular weight polysaccharide from *Rhizopus rubrum* according to the present invention.

[0022] Figure 2 This is a graph showing the results of gas chromatography-mass spectrometry (GC-MS) analysis of the monosaccharide composition of the high molecular weight polysaccharide from *Rhizopus rubrum* according to the present invention.

[0023] Figure 3 This is an infrared spectrum analysis of the characteristic functional groups of the *Rhizopus rubrum* polymer polysaccharide of this invention.

[0024] Figure 4 Nuclear magnetic resonance imaging of the high molecular weight polysaccharide of *Rhizopus rubrum* in this invention. 1 H spectrum;

[0025] Figure 5 The image shows the methylation detection results of the high molecular weight polysaccharide from *Rhizopus rubrum* according to this invention.

[0026] Figure 6 This is a structural formula of the high molecular weight polysaccharide of *Rhizopus rubrum* according to the present invention;

[0027] Figure 7This is the second structural formula of the high molecular weight polysaccharide of *Rhizopus rubrum* in this invention;

[0028] Figure 8 This is the third structural formula of the high molecular weight polysaccharide of *Rhizopus rubrum* in this invention;

[0029] Figure 9 The Venn diagram shows the effect of the high molecular weight polysaccharide of *Rhizopus heliotropium* on the diversity of intestinal flora in immune-damaged mice.

[0030] Figure 10 This is a diagram showing the effect of the high molecular weight polysaccharide of *Rhizopus rubrum* on the genus-level distribution of intestinal flora in immune-damaged mice.

[0031] Figure 11 This is a ternary phase diagram illustrating the effect of the high molecular weight polysaccharide of *Rhizopus hemlockenspi* on the intestinal flora of immune-damaged mice.

[0032] Figure 12 This is a heatmap showing the effect of the high molecular weight polysaccharide of *Rhizopus rubrum* on the content of intestinal flora metabolites in mice with immune damage.

[0033] Figure 13 Volcano diagram showing the effect of the high molecular weight polysaccharide of *Rhizopus rubrum* on the content of intestinal flora metabolites in immune-damaged mice;

[0034] Figure 14 The effect of the high molecular weight polysaccharide of *Rhizopus hemlockii* on the intestinal flora metabolism of immune-damaged mice is shown in the KEGG enrichment bubble diagram.

[0035] Figure 15 This is a diagram showing the effect of the high molecular weight polysaccharide of *Rhizopus rubrum* on the distribution of peripheral blood lymphocyte subsets in immune-damaged mice.

[0036] Figure 16 This figure shows the effect of the high molecular weight polysaccharide of *Rhizopus heliotropium* on the proportion of peripheral blood lymphocyte subsets in immune-damaged mice. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0039] For ease of description, spatial relative terms such as "above," "on top of," "on the upper surface of," "above," etc., are used herein to describe the spatial positional relationship of a device or feature as shown in the figures to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation beyond the orientation of the device as described in the figures. For example, if the device in the figures were inverted, a device described as "above" or "on top of" other devices or structures would subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0040] Please see Figures 1-16 The present invention provides a technical solution:

[0041] A high-molecular-weight polysaccharide (CRWP) from dried *Rhizopus rubrum* fruiting bodies was prepared by hot water extraction, ethanol precipitation, dialysis purification, rotary evaporation concentration, and freeze-drying. This polysaccharide possesses the following defined characteristics:

[0042] 1. Monosaccharide composition: Composed of five monosaccharides: fucose (Fuc), arabinose (Ara), mannose (Man), glucose (Glc), and galactose (Gal) in a molar ratio of 1.04:0.56:2.58:2.12:1.00;

[0043] 2. Molecular weight: Its average molecular weight (Mw) is (2.6±2)×10 6 Da;

[0044] 3. Structural characteristics: Its main chain consists of β-(1→6)-Manp, and its branches consist of β-(1→6)-Glcp, α-(1→6)-Galp, and α-(1→3)-Ara. f constitute;

[0045] The above-mentioned method for preparing CRWP includes the following steps:

[0046] a) Hot water extraction: Using dried blood-red rosary mushroom fruiting bodies as raw materials, hot water was used for extraction to obtain crude polysaccharide extract;

[0047] b) Ethanol precipitation: After concentrating the above crude polysaccharide extract, add ethanol to a certain final concentration, let it stand to precipitate, and collect the polysaccharide precipitate by centrifugation.

[0048] c) Dialysis purification: After redissolving the obtained polysaccharide precipitate, place it in a dialysis bag with a molecular weight cutoff and dialyze it with running water or deionized water to remove small molecule impurities and salts.

[0049] d) Concentration and drying: The polysaccharide solution after dialysis was concentrated by rotary evaporation and then freeze-dried to obtain purified CRWP.

[0050] The effective dosage of CRWP in the preparation of drugs for regulating immune function, especially repairing immune damage, is 200 mg / kg body weight, administered by gavage.

[0051] CRWP exerts its immunomodulatory effect through the following mechanisms:

[0052] Significantly increases the abundance of beneficial bacteria, Lactobacillus, in the gut microbiota;

[0053] Promotes metabolic pathways such as plant hormone signal transduction;

[0054] Mediating the gut microbiota-metabolite-immune axis regulatory pathway, ultimately effectively enhancing lymphocyte subsets (such as CD3). + T cells and CD19 + This increases the proportion of B cells, thereby systematically enhancing the body's immune function.

[0055] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0056] Example 1: Preparation of high molecular weight polysaccharide (CRWP) from Rhizopus rubrum.

[0057] 1.1 Experimental Materials

[0058] Raw material: Dried fruiting bodies of Chroogomphus rutilus.

[0059] Reagents: distilled water, anhydrous ethanol.

[0060] 1.2 Preparation steps

[0061] Raw material pretreatment: Take 1.0 kg of dried *Rhizophora heliotropium* fruiting bodies, add 10 L of distilled water (solid-to-liquid ratio 1:10, g / mL), place in a constant temperature water bath, and extract at 90℃ for 3 h to ensure complete extraction. After extraction, transfer the mixture to a high-speed centrifuge, centrifuge at 4000 rpm for 15 min, and collect the supernatant; add 8 L of distilled water to the residue, repeat the above extraction and centrifugation steps, and combine the two supernatants.

[0062] Concentration and ethanol precipitation: The combined supernatant was transferred to a rotary evaporator and concentrated to 1 / 5 of its original volume under vacuum. Under stirring at room temperature, 4 times the volume of anhydrous ethanol was slowly added to the concentrate to bring the final ethanol concentration to 80% (v / v). After stirring for 10 min, the mixture was transferred to a refrigerator at 4°C and left to stand overnight (12 h).

[0063] Purification and Drying: The next day, the mixture after standing was centrifuged at 5000 rpm for 20 min using a high-speed centrifuge, and the polysaccharide precipitate at the bottom was collected. The precipitate was redissolved in 500 mL of distilled water, transferred to a pretreated dialysis bag, the bag was sealed tightly, and placed in 5 L of distilled water. Dialysis was performed at 4 °C for 48 h (the distilled water was changed every 12 h), followed by dialysis in static distilled water for 24 h (the distilled water was changed twice) to remove small molecule impurities (such as monosaccharides and salts). After dialysis, the solution in the bag was transferred to a rotary evaporator and concentrated to approximately 200 mL at 60 °C. It was then pre-frozen in an ultra-low temperature freezer at -80 °C for 4 h, and finally freeze-dried in a freeze dryer at -50 °C under a vacuum of 0.1 Pa for 48 h to obtain the CRWP sample.

[0064] Example 2: Structural identification of CRWP.

[0065] 2.1 Molecular weight analysis

[0066] The average molecular weight of CRWP was determined, and the results are shown in [Figure number missing]. Figure 1 A single, narrow, and symmetrical peak appeared on the chromatogram detected by size exclusion chromatography, proving that CRWP has a uniform molecular weight distribution and high purity after purification. Substituting the retention time of 5.995 min into the standard curve (y = -0.4601x + 9.1698, R...),... 2 = 0.9984, where y represents the logarithm of the molecular weight and x represents the corresponding retention time. Therefore, the average molecular weight of CRWP is approximately (2.6 ± 2) × 10⁻⁶. 6 Da.

[0067] 2.2 Monosaccharide Composition Analysis

[0068] The monosaccharide composition of CRWP was determined, and the results are shown in [the table below]. Figure 2 In the figure, peaks 1, 2, 3, 4, and 5 represent fucose, arabinose, mannose, glucose, and galactose, respectively. The results show that CRWP is composed of fucose, arabinose, mannose, glucose, and galactose, with a molar ratio of approximately 1.04:0.56:2.58:2.12:1.00.

[0069] 2.3 Characteristic Functional Group Analysis

[0070] The main characteristic functional groups of CRWP were determined, and the results are shown in [Figure number missing]. Figure 3 The characteristic absorption peaks of the polysaccharides were at 3408 cm⁻¹. -1 2926 cm -1 and 1408 cm -1 , representing the stretching vibration of OH, the stretching vibration of CH, and the deformation vibration of CH, respectively. 1647cm -1 The strong absorption peak at 1000-1200 cm⁻¹ is attributed to the bending vibration absorption of OH groups, while the strong absorption peak at 1000-1200 cm⁻¹ is attributed to the bending vibration absorption of OH groups. -1 The absorption peak at that point is due to the presence of COC glycosidic bonds in the polysaccharide.

[0071] 2.4 Polysaccharide 1H NMR Spectroscopy Analysis

[0072] Figure 4 For CRWP 1 H spectrum. The chemical shift at 4.79 ppm can be attributed to the presence of a proton signal from D2O. The signal range of 4.839–4.868 ppm indicates that CRWP contains an anomeric hydrogen signal from an α-glycosidic bond, while the signal value of 4.749–4.765 ppm indicates that it mainly contains anomeric hydrogen signals from a β-glycosidic bond. Due to the large molecular weight of the polysaccharide, the H2–H6 signals in these sugar residues are not obvious.

[0073] 2.5 Methylation Analysis

[0074] Table 1 shows the methylation detection and analysis results of CRWP, and the corresponding total ion chromatogram is shown in Table 1. Figure 5 The numbers in the diagram are the codes in Table 1.

[0075] Table 1 shows the methylation detection and analysis results of CRWP.

[0076]

[0077] As shown in Table 1, the glycosidic bonds in CRWP are mainly composed of (1→6)-Manp (33.11%), (1→2, 6)-Manp (12.95%) and (1→6)-Glcp (29.74%).

[0078] like Figure 6 , Figure 7 and Figure 8 As shown, based on the above results, the possible structural formula of CRWP is deduced. Its main chain consists of β-(1→6)-Manp, and its branches consist of β-(1→6)-Glcp, α-(1→6)-Galp, and α-(1→3)-Ara. f constitute.

[0079] Example 3: Evaluation of the effect of CRWP on gut microbiota metabolism in immune-damaged mice

[0080] 3.1 Animal Experiment Design

[0081] SPF-grade female Kunming mice, 6-8 weeks old and weighing 20±2g, were procured. After one week of acclimatization, they were randomly divided into four groups of 10 mice each using a completely randomized design: a control group, a model group (cyclophosphamide modeling group), a low-dose CRWP (100 mg / kg) intervention group, and a high-dose CRWP (200 mg / kg) intervention group. The intervention protocol was as follows: On days 1-3, the control group received an intraperitoneal injection of 0.2 mL of physiological saline, while the model group and polysaccharide intervention group received an equal volume of cyclophosphamide solution (30 mg / kg) intraperitoneally; on days 4-19, the control group and model group received deionized water by gavage, while the polysaccharide intervention group received an equal volume of CRWP aqueous solution by gavage according to the dosage design; on day 20, at the end of the experimental period, samples were collected for data analysis.

[0082] 3.2 Analysis of gut microbiota distribution

[0083] like Figure 9 As shown, each circle represents a group, and the numbers in the overlapping (non-overlapping) parts of the ellipses indicate the number of shared (unique) OTUs. As mentioned above, the blank group, model group, and high-dose CRWP group have a total of 67 OTUs, while the blank group, model group, and high-dose CRWP group have 123, 144, and 67 unique OTUs, respectively. This indicates that these experimental groups exhibit extensive gut microbiota diversity, which may be the reason for the differences in immune capacity among different groups.

[0084] Figure 10 The relative abundance of the top 10 gut microbiota in each group of mice is shown. Compared with the model group, the abundance of Lactobacillus was significantly increased in the blank group and the CRWP group, indicating that it is closely related to the immune regulation capacity of mice. This may be the main reason why the immune capacity of immune-damaged mice was enhanced after CRWP intervention.

[0085] Figure 11 This is a ternary phase diagram of the gut microbiota at the genus level in the control group, model group, and high-dose CRWP group. The results showed that compared with the model group, the abundance of Lactobacillus genus in the gut of the CRWP group and the control group was significantly increased, consistent with the previous study results, indicating that Lactobacillus is a key gut microbiota for CRWP to regulate the immune capacity of immune-damaged mice.

[0086] 3.3 Analysis of intestinal metabolite distribution

[0087] Non-targeted metabolomics was used to detect metabolites with significantly different expression between the model group and the high-dose CRWP group, and the results are as follows: Figure 12 As shown. Colors from blue to red indicate an increase in the relative content of metabolites, and the horizontal direction represents sample information (3 parallel samples). Nineteen metabolites in the high-dose CRWP group mice showed significantly different expression levels compared to the model group (p<0.05), with 10 downregulated and 9 upregulated. However, the function of these metabolites in immune-damaged mice requires further analysis.

[0088] exist Figure 13 In the volcano plot, each point represents a metabolite. Metabolites that are significantly upregulated (downregulated) compared to the model group are represented by red (blue) dots, while metabolites with no significant difference are represented by gray. The results are compared with... Figure 12 Consistent.

[0089] Differential metabolites obtained from statistical analysis were enriched using the KEGG database, and pathway analysis was performed, such as... Figure 14 As shown in the diagram, each bubble represents a metabolic pathway, and differences in metabolites may be related to changes in cellular pathways. Compared to the model group, the high-dose CRWP group showed the most significant effect on intestinal metabolism through the plant hormone signaling pathway. Although auxin primarily functions in plants, studies have found that plant-derived auxin analogs or their metabolites may enter mice and affect intestinal immunity. From an evolutionary perspective, some signaling pathways in organisms exhibit a degree of conservation, and mouse intestinal cells may contain potential targets that can interact with auxin or its analogs. Furthermore, studies have shown that certain key protein domains in the auxin signaling pathway are similar to some protein domains involved in signal transduction in mammalian cells, providing a structural basis for the association between the auxin signaling pathway and mouse intestinal immune regulation. Although the specific mechanism of action is not yet clear, this structural similarity suggests that the auxin signaling pathway may affect the function of intestinal immune cells by influencing the activity of certain signaling molecules in the mouse gut.

[0090] 3.4 Effects of CRWP on the distribution of peripheral blood lymphocyte subsets in immune-injured mice

[0091] Figure 15 The peripheral blood CD3 count of mice in the blank group, model group, and high-dose CRWP group is shown. + T cells and CD19 + The distribution of B cells showed that each cell subpopulation was well-clustered, indicating that the results have good reliability.

[0092] Figure 16 The peripheral blood CD3 of mice in the blank group, model group, and high-dose CRWP group were displayed. + T cells and CD19 + B cell proportion and significant differences. Compared with the control group, the model group had higher CD3 counts. + T cells and CD19 + The proportion of B cells was significantly reduced (p<0.05), while the CD3 ratio in the CRWP group was significantly lower. + T cells and CD19 + The proportion of B cells was significantly higher than that in the model group (p<0.05), with more pronounced changes in T cells, suggesting that CRWP may have a stronger immunomodulatory effect on T cells. Combined with the previous results, it can be inferred that CTX exhibited inhibitory activity against all lymphocyte subsets, while CRWP plays an important regulatory role in lymphocyte immunity by modulating gut microbiota metabolism.

[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high molecular weight polysaccharide from Russula sanguinaria, characterized by: The blood red pin fungus high molecular polysaccharide is prepared by the following steps: Raw material pretreatment: take 1.0 kg of dried blood red pin fungus fruiting body, add 10 L of distilled water, the ratio of material to liquid is 1:10 g / mL, place in a constant temperature water bath, extract at 90 DEG C constant temperature for 3 h, ensure sufficient extraction, after extraction, transfer the mixed solution to a high speed centrifuge, centrifuge at 4000 rpm for 15 min, collect the supernatant; add 8 L of distilled water to the residue, repeat the above extraction and centrifugation steps, combine the two supernatants; Concentration and ethanol precipitation: transfer the combined supernatant to a rotary evaporator, concentrate under vacuum to 1 / 5 of the original volume, slowly add 4 times the volume of anhydrous ethanol to the concentrated solution under room temperature stirring conditions, so that the final concentration of ethanol is 80% v / v, continue stirring for 10 min, then transfer the mixed solution to a 4 DEG C refrigerator and stand overnight for 12 h; Purification and drying: the next day, centrifuge the standing mixed solution at 5000 rpm for 20 min, collect the polysaccharide precipitate, redissolve the precipitate with 500 mL of distilled water, transfer to a pretreated dialysis bag, tighten the bag opening, place in 5 L of distilled water at 4 DEG C, change the distilled water every 12 h for 48 h, then transfer to static distilled water dialysis for 24 h, change the distilled water twice, remove monosaccharide, salt and small molecule impurities, after dialysis, transfer the solution in the bag to a rotary evaporator, concentrate to 200 mL at 60 DEG C, then pre-freeze in a-80 DEG C ultra-low temperature refrigerator for 4 h, finally put into a freeze dryer under the conditions of-50 DEG C and 0.1 Pa vacuum for 48 h, to obtain the blood red pin fungus high molecular polysaccharide product.

2. The high molecular weight polysaccharide of claim 1, wherein: The molecular weight of the blood red pin mushroom polysaccharide is (2.6±2) x 10 6 Da.

3. Use of the blood red pin fungus high molecular polysaccharide of any one of claims 1-2 in the preparation of a medicament for regulating immune function damage induced by cyclophosphamide.

4. The use of a high molecular weight polysaccharide of Rhodotus palmivorus in the preparation of a medicament for regulating the immune function damage induced by cyclophosphamide according to claim 3, characterized in that: The application is the use in the preparation of a medicament for improving or treating intestinal flora disorder caused by immune damage.

5. A pharmaceutical composition, characterized by: A blood red pin fungus high molecular polysaccharide according to any one of claims 1-2.

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