Trametes lactinea polysaccharide TLHP-1 with immunoregulatory activity and preparation method thereof

Abstract

This invention relates to the preparation, structural characterization, and application in the fields of biomedicine and health food technologies of active polysaccharides from edible and medicinal fungi. The invention discloses a white matsutake mushroom polysaccharide TLHP-1 with immunomodulatory activity. The weight-average molecular weight (Mw) of TLHP-1 is 1.726 × 10⁴–1.908 × 10⁴ Da, and the molecular weight distribution coefficient (Mw / Mn) is 1.137–1.257. TLHP-1 is a heteropolysaccharide composed of fucose, galactose, glucose, mannose, and xylose. Sugars, rhamnose; This invention yields a polysaccharide TLHP-1 from *Matsutake* mushroom with a uniform structure and stable physicochemical properties. Its molecular weight, monosaccharide composition, glycosidic bonds, and sugar residue structure are clearly defined. It exhibits high purity and few impurities, solving the problems of mixed components and batch instability in traditional crude polysaccharides. The process parameters are precise and highly reproducible, making it suitable for both laboratory preparation and industrial production. This polysaccharide can effectively repair cyclophosphamide-induced immunosuppressive damage, regulate immune factors, gut microbiota, and key metabolic pathways, and can be used to prepare immunomodulatory drugs and health foods.

Description

Technical Field

[0001] This invention relates to the fields of preparation, structural characterization, and biomedical and health food technologies of active polysaccharides from edible and medicinal fungi, specifically to a white matsutake polysaccharide TLHP-1 with immunomodulatory activity and its preparation method. Background Technology

[0002] White matsutake (Tricholoma lobayense Heim.) is a rare edible and medicinal fungus with high nutritional value. It is rich in polysaccharides, polyphenols, proteins, and various monosaccharide active components, showing promising application prospects in the fields of dietary therapy and the development of natural immunomodulators. Fungal polysaccharides are the core active substances of white matsutake, possessing multiple physiological functions such as immunomodulation, intestinal flora regulation, anti-inflammation, and improvement of metabolism, and have become a research hotspot in the fields of functional foods and medicine.

[0003] Existing research largely focuses on the conventional extraction and preliminary exploration of basic activities of crude polysaccharides from *Matsutake* mushrooms, lacking homogeneous polysaccharide components with well-defined structures, uniform molecular weights, and clear monosaccharide compositions. Traditional extraction and purification processes are crude, resulting in polysaccharides with low purity, mixed structures, wide molecular weight distributions, weak immunomodulatory activity, and poor batch-to-batch stability. Furthermore, there is a lack of comprehensive research on the fine structural characterization, systemic immunosuppressive repair function, and industrial-scale preparation process of the homogeneous polysaccharide TLHP-1 from *Matsutake* mushrooms, limiting its development and application in immunomodulatory drugs, functional foods, and health foods. Therefore, we propose a polysaccharide TLHP-1 from *Matsutake* mushrooms with immunomodulatory activity and its preparation method to address the aforementioned problems. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a white matsutake polysaccharide TLHP-1 with immunomodulatory activity and its preparation method. This solves the problems that existing white matsutake polysaccharides are mostly crude extracts, which have unclear structures, poor uniformity, low purity, crude preparation processes, batch instability, and lack of systematic research on fine structures and immunomodulatory mechanisms.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a white matsutake polysaccharide TLHP-1 with immunomodulatory activity, wherein the weight-average molecular weight (Mw) of the white matsutake polysaccharide TLHP-1 is 1.726 × 10⁻⁶. 4 -1.908×10 4 Da, the molecular weight distribution coefficient Mw / Mn is 1.137-1.257;

[0006] The white matsutake polysaccharide TLHP-1 is a heteropolysaccharide composed of fucose, galactose, glucose, mannose, xylose, rhamnose, galactosamine, glucosamine, galacturonic acid, and glucuronic acid.

[0007] The content of each monosaccharide, by molar percentage, is as follows: galactose 62.5%-69.1%, fucose 16.2%-18.0%, mannose 11.0%-12.2%, glucose 3.4%-3.8%, xylose 0.85%-0.95%, rhamnose 0.19%-0.21%, galactosamine 0.095%-0.105%, glucosamine 0.285%-0.315%, galacturonic acid 0.285%-0.315%, and glucuronic acid 0.095%-0.105%.

[0008] Preferably, the weight-average molecular weight (Mw) of the white matsutake polysaccharide TLHP-1 is 1.817 × 10⁻⁶. 4 Da, with a number-average molecular weight Mn of 1.518 × 10⁻⁶. 4 Da, the molecular weight distribution coefficient Mw / Mn is 1.197;

[0009] The specific content of each monosaccharide, in molar percentage, is as follows: galactose 65.8%, fucose 17.1%, mannose 11.6%, glucose 3.6%, xylose 0.9%, rhamnose 0.2%, galactosamine 0.1%, glucosamine 0.3%, galacturonic acid 0.3%, and glucuronic acid 0.1%.

[0010] Preferably, the total sugar content of the white matsutake polysaccharide TLHP-1 is 85.23±0.91%, the total protein content is 0.43±0.1%, and the total phenol content is 2.15±0.08%.

[0011] Preferably, the main glycosidic bond type of the white matsutake polysaccharide TLHP-1 is selected from at least one of the following: Xylp-(1→, Fucp-(1→, →2)-Fucp-(1→, Glcp-(1→, Galp-(1→, →2)-Manf-(1→, →4)-Galp-(1→, →6)-Manp-(1→, →6)-Galp-(1→, →2,4)-Manp-(1→, →4,6)-Manp-(1→, →4,6)-Glcp-(1→, →3,6)-Galp-(1→).

[0012] Preferably, the sugar residues of the white matsutake polysaccharide TLHP-1 are mainly composed of the following seven types: α-L-Fucp-(1→, α-D-Glcp-(1→, →4)-α-D-Galp-(1→, →6)-Manp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→).

[0013] This invention provides a method for preparing TLHP-1, a polysaccharide from *Matsutake* mushrooms with immunomodulatory activity, comprising the following steps:

[0014] S1. Extract the dried white matsutake mushroom powder by reflux with 75% volume fraction ethanol at a material-to-liquid ratio of 1:4, and collect the ethanol-insoluble residue.

[0015] S2. Add water to the ethanol-insoluble residue and gently reflux hot water at 85-95℃ for 2 hours. Repeat the extraction 3 times, combine the extracts, filter and concentrate to a relative density of 1.1 g / cm³.

[0016] S3. Add 95% volume fraction ethanol to the concentrate to adjust the final ethanol concentration of the system to 75%, let it stand at room temperature for 24 hours to precipitate, collect the precipitate, dry it, and obtain white matsutake crude polysaccharide.

[0017] S4. The crude polysaccharide of white matsutake mushroom is repeatedly deproteinized using the Sevag method to obtain deproteinized polysaccharide;

[0018] S5. The deproteinized polysaccharide was separated and purified sequentially by DEAE-52 anion exchange chromatography column and Sephadex G-300 gel filtration chromatography column. The single symmetrical elution peak fraction was collected and dried to obtain white matsutake polysaccharide TLHP-1.

[0019] Preferably, in step S5, the DEAE-52 anion exchange chromatography column is eluted with a gradient of 0-1 mol / L NaCl solution, and the Sephadex G-300 gel filtration chromatography column is eluted with distilled water as the mobile phase.

[0020] The white matsutake polysaccharide TLHP-1 specified above in this invention, or the white matsutake polysaccharide TLHP-1 prepared by the above preparation method, can be used to prepare drugs, functional foods or health foods with immunomodulatory functions.

[0021] Preferably, the immunomodulatory function is to improve cyclophosphamide-induced immunosuppressive damage in the body, including at least one of the following:

[0022] Increase body weight and thymus index in immunosuppressed individuals, and decrease spleen index;

[0023] Restoring colorectal length in immunosuppressed individuals;

[0024] It upregulates serum IFN-γ, IL-2, IgA, and IgG levels, and downregulates serum IL-6 and TNF-α levels;

[0025] It regulates the structure of the gut microbiota, increases the abundance of beneficial bacteria, and inhibits the abundance of pathogenic bacteria;

[0026] It regulates arachidonic acid metabolism, riboflavin metabolism, vitamin digestion and absorption, mTOR signaling pathway, and arginine biosynthesis and metabolism pathway.

[0027] Beneficial effects

[0028] This invention provides a polysaccharide TLHP-1 from *Matsutake* mushrooms with immunomodulatory activity and its preparation method. Compared with the prior art, it has the following advantages:

[0029] The immunomodulatory polysaccharide TLHP-1 from white matsutake mushroom and its preparation method yielded a homogeneous polysaccharide TLHP-1 from white matsutake mushroom with uniform structure and stable physicochemical properties. The molecular weight distribution, molar composition of ten monosaccharides, glycosidic bond type and sugar residue configuration were precisely defined. The product has high total sugar purity and extremely low protein and impurity residues, which solves the industry shortcomings of traditional white matsutake mushroom crude polysaccharide, such as mixed components, unclear structure, poor uniformity and unstable batch quality.

[0030] A standardized and reproducible preparation process was established, with clearly defined process parameters for ethanol defatting, constant temperature hot water extraction, precise alcohol precipitation, Sevag deproteinization, and chromatography purification. The process is controllable and reproducible, suitable for both small-scale laboratory preparation and large-scale industrial production, overcoming the shortcomings of traditional processes such as crudeness, weak activity, and poor stability.

[0031] Multidimensional experiments have confirmed that TLHP-1 can significantly improve cyclophosphamide-induced immunosuppression damage, increase body weight and thymus index in immunosuppressed individuals, repair immune organs and intestinal tissue structure, regulate serum cytokine and immunoglobulin levels, reshape intestinal flora homeostasis, and regulate key pathways such as arachidonic acid metabolism, riboflavin metabolism, and mTOR signaling pathway, thereby regulating the body's immune balance at multiple levels from organ, body fluid, microecology and metabolic perspectives.

[0032] The physicochemical, structural characterization, and pharmacodynamic experimental data of the polysaccharide have been disclosed. The technology is fully disclosed, the product is safe without chemical modification, has good thermal stability, and high bioavailability. It can be widely used in the development of immunomodulatory drugs, functional foods, and health foods, and has good scientific research value and industrial application prospects. Attached Figure Description

[0033] Figure 1 This is a chromatogram showing the separation, purification, and chromatographic analysis of TLHP-1 in this invention;

[0034] Figure 2 The spectrum and thermal stability characterization of TLHP-1 of the present invention are shown below;

[0035] Figure 3 This is a scanning electron microscope image of the TLHP-1 of the present invention;

[0036] Figure 4 The structure diagram is shown below, which is the analytical and derivation diagram of the nuclear magnetic resonance structure of TLHP-1 of the present invention.

[0037] Figure 5 The figure shows the experimental results of the mouse immunosuppression model of the present invention;

[0038] Figure 6 This is a bar chart showing the detection of mouse serum cytokines and immunoglobulins according to the present invention.

[0039] Figure 7 This is a diagram illustrating the gut microbiota composition analysis of this invention.

[0040] Figure 8 This is a diagram illustrating the non-targeted metabolomics analysis of feces according to the present invention. Detailed Implementation

[0041] 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. 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.

[0042] like Figure 1-8 As shown:

[0043] A polysaccharide from *Matsutake* mushroom, TLHP-1, with immunomodulatory activity, has a weight-average molecular weight (Mw) of 1.726 × 10⁻⁶. 4 -1.908×10 4 Da, with a molecular weight distribution coefficient Mw / Mn of 1.137-1.257; the white matsutake polysaccharide TLHP-1 is a heteropolysaccharide composed of ten monosaccharides: fucose, galactose, glucose, mannose, xylose, rhamnose, galactosamine, glucosamine, galacturonic acid and glucuronic acid.

[0044] The content of each monosaccharide, by molar percentage, is as follows: galactose 62.5%-69.1%, fucose 16.2%-18.0%, mannose 11.0%-12.2%, glucose 3.4%-3.8%, xylose 0.85%-0.95%, rhamnose 0.19%-0.21%, galactosamine 0.095%-0.105%, glucosamine 0.285%-0.315%, galacturonic acid 0.285%-0.315%, and glucuronic acid 0.095%-0.105%.

[0045] In the preferred embodiment, the weight-average molecular weight (Mw) of the white matsutake polysaccharide TLHP-1 is 1.817 × 10⁻⁶. 4 Da, with a number-average molecular weight Mn of 1.518 × 10⁻⁶. 4Da has a molecular weight distribution coefficient (Mw / Mn) of 1.197. The specific content of each monosaccharide in molar percentage is as follows: galactose 65.8%, fucose 17.1%, mannose 11.6%, glucose 3.6%, xylose 0.9%, rhamnose 0.2%, galactosamine 0.1%, glucosamine 0.3%, galacturonic acid 0.3%, and glucuronic acid 0.1%.

[0046] Further optimization revealed that the total sugar content of the white matsutake polysaccharide TLHP-1 was 85.23±0.91%, the total protein content was 0.43±0.1%, and the total phenol content was 2.15±0.08%.

[0047] The main glycosidic bond type of white matsutake polysaccharide TLHP-1 is selected from at least one of the following: Xylp-(1→, Fucp-(1→, →2)-Fucp-(1→, Glcp-(1→, Galp-(1→, →2)-Manf-(1→, →4)-Galp-(1→, →6)-Manp-(1→, →6)-Galp-(1→, →2,4)-Manp-(1→, →4,6)-Manp-(1→, →4,6)-Glcp-(1→, →3,6)-Galp-(1→).

[0048] The sugar residues of white matsutake polysaccharide TLHP-1 are mainly composed of the following seven configurations: α-L-Fucp-(1→, α-D-Glcp-(1→, →4)-α-D-Galp-(1→, →6)-Manp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→).

[0049] Example 1

[0050] Standardized preparation process of white matsutake polysaccharide TLHP-1:

[0051] Select dried white matsutake fruiting bodies that are free from mold and insect damage, crush them with a pulverizer, pass them through a 40-mesh standard sieve to obtain uniform dried white matsutake powder, and seal and store them in a cool place for later use.

[0052] Add 75% ethanol solution at a material-to-liquid ratio of 1:4 (g:mL), reflux in a water bath for 2 hours, filter after extraction, discard the ethanol filtrate containing pigments, lipids, and small molecule impurities, and collect the filter residue, which is the ethanol-insoluble residue.

[0053] Add the defatted residue to deionized water, keep the solid-liquid ratio fixed, control the temperature in the water bath at 85-95℃, and gently reflux for 2 hours; after extraction, filter, and repeat the extraction under the same conditions for a total of 3 times with the remaining filter residue; combine all 3 water extracts, and filter through four layers of gauze to remove suspended insoluble matter.

[0054] The combined aqueous extracts were placed in a rotary evaporator and concentrated under vacuum. The relative density at the concentration endpoint was controlled to be 1.1 g / cm³ to obtain a viscous polysaccharide concentrate.

[0055] 95% ethanol was slowly added dropwise to the concentrate with stirring to adjust the final ethanol concentration to 75%. After stirring evenly, the mixture was sealed and allowed to stand at room temperature for 24 hours to allow the water-soluble polysaccharide to fully flocculate and precipitate. The bottom flocculent precipitate was collected by centrifugation and dried under vacuum at a constant temperature to obtain crude polysaccharide from white matsutake mushroom.

[0056] Prepare an aqueous solution of crude polysaccharide at a suitable concentration, add an equal volume of Sevag reagent (chloroform: n-butanol = 4:1), shake vigorously, let stand to separate into layers, discard the lower organic phase and the interfacial protein flocculents, repeat the operation 5-6 times until there is no obvious protein precipitation at the interface, and collect the upper aqueous polysaccharide solution.

[0057] Chromatographic separation and purification:

[0058] The deproteinized polysaccharide solution was loaded onto a DEAE-52 anion exchange cellulose column and eluted using a linear gradient of 0-1 mol / L NaCl. The samples were automatically collected in separate tubes, and the main active components were combined according to the elution curve.

[0059] After dialysis to remove salt from the combined components, the samples were loaded onto a Sephadex G-300 gel filtration chromatography column and eluted with distilled water as the mobile phase. Elution curves were plotted and the eluent from the single symmetrical elution peak interval was collected.

[0060] The eluent was freeze-dried to obtain pure white, powdery, homogeneous white matsutake polysaccharide TLHP-1.

[0061] Example 2

[0062] The physicochemical and component analysis of TLHP-1 prepared in Example 1 was performed, and the results are shown in the table below:

[0063] Table 1 Physicochemical characteristics and molecular weight parameters of TLHP-1:

[0064]

[0065] The results showed that TLHP-1 had high total sugar purity, extremely low protein residue, narrow molecular weight distribution, and excellent uniformity. It was a heteropolysaccharide composed of ten monosaccharides, and the component ratios were fixed and controllable.

[0066] Example 3

[0067] TLHP-1 methylation analysis and glycosidic bond type identification

[0068] Thirteen glycosidic bond linkages were identified using methylation derivatization and GC-MS determination. The results are shown in Table 2 below.

[0069]

[0070] Example 4

[0071] Spectroscopic, microscopic and thermal stability characterization of TLHP-1;

[0072] Ultraviolet spectroscopy: No characteristic absorption peaks were observed at 260 nm and 280 nm, indicating the absence of nucleic acids and a large amount of residual proteins, and the sample has high purity.

[0073] Infrared spectrum: Polysaccharide characteristic absorption peaks appear at 3380.1 cm⁻¹, 2929.5 cm⁻¹, 1644.5 cm⁻¹, 1401.3 cm⁻¹, 1081.8 cm⁻¹, 1024.6 cm⁻¹, 869.6 cm⁻¹, and 810 cm⁻¹, which are consistent with the infrared fingerprint characteristics of fungal polysaccharides;

[0074] Congo red conformation experiment: No significant red shift was observed in the NaOH concentration range of 0.0-0.5 mol / L, indicating that TLHP-1 does not possess a typical triple helix conformation;

[0075] X-ray diffraction (XRD): A sharp diffraction peak appears at 17.99°, along with a broad, diffuse peak, indicating that the polysaccharide has both crystalline and amorphous structures.

[0076] Thermogravimetric analysis (TG-DTG): First stage of weight loss: 40-200℃, maximum weight loss temperature 84.61℃; Second stage: 200-400℃, maximum weight loss temperature 294.50℃; Total weight loss rate at 600℃: 84.61%, good thermal stability, suitable for processing and storage.

[0077] SEM (Scanning Electron Microscopy): TLHP-1 is composed of a stack of sheet-like structures and a small number of irregular rod-like structures; under high magnification, the surface is rough, porous, and multi-branched, and the loose porous structure is conducive to in vivo dissolution, adsorption and absorption.

[0078] Example 5

[0079] TLHP-1 NMR structural analysis of sugar residues:

[0080] Analysis of ¹H NMR, ¹³CNMR, HSQC, HCOSY, and HMBC spectra confirmed that TLHP-1 contains seven characteristic sugar residues, with chemical shifts assigned as follows:

[0081] Table 3. ¹³C / ¹H NMR chemical shifts of sugar residues in TLHP-1:

[0082]

[0083] Example 6

[0084] Validation of the activity of animal immunosuppression models:

[0085] Laboratory animals: SPF grade BALB / c male mice, 6-8 weeks old, weighing 18-22g, acclimatized for 7 days;

[0086] Grouping settings: Randomly divide into 5 groups, with 10 animals in each group;

[0087] Normal control group (Control);

[0088] Model control group (CTX);

[0089] Low-dose TLHP-1 group: 100 mg / kg;

[0090] High-dose TLHP-1 group: 200 mg / kg;

[0091] Positive control group LM: Levamisole 40 mg / kg;

[0092] Modeling and drug administration:

[0093] Days 1-3: Except for the normal group, all other groups were intraperitoneally injected with cyclophosphamide 80 mg / kg / day to establish an immunosuppressive model; Days 4-17: The drug was administered by gavage for 14 consecutive days, and the normal group and the model group were administered the same amount of physiological saline by gavage.

[0094] Detection indicators and results:

[0095] Growth and immune organs: CTX group mice showed significantly decreased body weight and thymus index, abnormally increased spleen index and shortened colon and rectum; high dose of TLHP-1 significantly restored body weight, increased thymus index, decreased spleen index and restored colon and rectum length.

[0096] Serum immune factors: CTX group significantly downregulated IFN-γ, IL-2, IgA, and IgG, and upregulated pro-inflammatory factors IL-6 and TNF-α; high dose of TLHP-1 can significantly reverse the above indicators and balance immune and inflammatory levels.

[0097] Splenic pathology: In the CTX group, the spleen white pulp was atrophied, lymphocytes were sparse, and the boundary between red and white pulp was blurred; after TLHP-1 intervention, the spleen tissue structure was significantly repaired, lymphocytes were dense, and the white pulp morphology was restored to completeness.

[0098] Intestinal flora: In the model group, the abundance of pathogenic bacteria such as Deferobacterium and Myxospirillum increased, while the abundance of beneficial bacteria such as Lactobacillus decreased; TLHP-1 can reshape the flora structure, enrich Akkermansia, Bifidobacterium, and beneficial bacteria of the Family of Herba Cytobacteriales, and inhibit the proliferation of pathogenic bacteria.

[0099] Metabolomics and pathways: TLHP-1 can revert differential metabolites and regulate arachidonic acid metabolism, riboflavin metabolism, vitamin digestion and absorption, mTOR signaling pathway, and arginine biosynthesis, thereby regulating immune homeostasis at the metabolic level.

[0100] This method yielded a homogeneous polysaccharide TLHP-1 from white matsutake mushrooms with a uniform structure and stable physicochemical properties. It precisely defined the molecular weight distribution, molar composition of ten monosaccharides, glycosidic bond type, and sugar residue configuration. The product has high total sugar purity and extremely low protein and impurity residues, solving the industry shortcomings of traditional white matsutake crude polysaccharides, such as mixed components, unclear structure, poor uniformity, and unstable batch quality.

[0101] A standardized and reproducible preparation process was established, with clearly defined process parameters for ethanol defatting, constant temperature hot water extraction, precise alcohol precipitation, Sevag deproteinization, and chromatography purification. The process is controllable and reproducible, suitable for both small-scale laboratory preparation and large-scale industrial production, overcoming the shortcomings of traditional processes such as crudeness, weak activity, and poor stability.

[0102] Multidimensional experiments have confirmed that TLHP-1 can significantly improve cyclophosphamide-induced immunosuppression damage, increase body weight and thymus index in immunosuppressed individuals, repair immune organs and intestinal tissue structure, regulate serum cytokine and immunoglobulin levels, reshape intestinal flora homeostasis, and regulate key pathways such as arachidonic acid metabolism, riboflavin metabolism, and mTOR signaling pathway, thereby regulating the body's immune balance at multiple levels from organ, body fluid, microecology and metabolic perspectives.

[0103] The physicochemical, structural characterization, and pharmacodynamic experimental data of the polysaccharide have been disclosed. The technology is fully disclosed, the product is safe without chemical modification, has good thermal stability, and high bioavailability. It can be widely used in the development of immunomodulatory drugs, functional foods, and health foods, and has good scientific research value and industrial application prospects.

[0104] It should be noted that:

[0105] like Figure 1 The following are chromatograms: A. Elution curve of TLHP-1 on a DEAE-52 ion column; B. Elution curve of TLHP-1 on a Sephacryl S-300 gel filtration column; C. HPGPC chromatogram; D. Ion chromatogram of TLHP-1.

[0106] The crude polysaccharide extracted from white matsutake mushrooms was first purified by anion exchange chromatography using a DEAE-52 column to obtain two main components, TLHP-1 and TLHP-2. The elution curve of TLHP-1 after purification by Sephadex G-300 gel filtration column showed a single peak. Figure 1AB). Subsequently, the total sugar content of TLHP-1 was measured to be 85.23±0.91 (%), the protein content was 0.43±0.1 (%), and the total phenol content was 2.15±0.08 (%).

[0107] like Figure 2 As shown: A. Ultraviolet scanning spectrum; B. Infrared scanning spectrum; C. Triple helix conformation test - Congo red test; DX diffraction pattern; E. Thermogravimetric / microquotient thermogravimetric scanning curve.

[0108] like Figure 3 As shown, SEM images of TLHP-1 at different magnifications reveal that this polysaccharide consists of various morphological plates and a small number of irregularly stacked rod-like structures. At 20,000x magnification, the surface of TLHP-1 is observed to be rough and exhibits significant porosity; this porous structure displays numerous branches. Figure 1 SEM scan images (A. 500×; B. 1000×; C. 5000×; D. 20000×).

[0109] like Figure 4 The NMR scanning spectra are shown in Table 3. A. 1H NMR; B. 13C NMR; C. HSQC; D. HCOSY; E. HMBC; F. TLHP-1 deduced structure. The TLHP-1 polysaccharide was analyzed by NMR, and a total of seven sugar residues were obtained (Table 3).

[0110] like Figure 5 As shown, 50 BALB / c mice were randomly divided into 5 groups of 10 mice each: a normal control group (Control), a model control group (CTX), a low-dose TLHP-1 group (LTLHP-1), a high-dose TLHP-1 group (HTLHP-1), and a positive control group (LM). The experiment adopted a "modeling + treatment" model. From day 1 to day 3, except for the Control group, all other groups received daily intraperitoneal injections of CTX (80 mg / kg / day) to establish an immunosuppressive model; the Control group received an equal volume of saline. From day 4 to day 17 (14 days in total), the LTLHP-1 and HTLHP-1 groups were administered different doses of the target polysaccharide solution by gavage; the LM group was administered levamisole (40 mg / kg) by gavage; and the Control and CTX groups were administered an equal volume of sterile saline by gavage. During the experiment, the mice's body weight, food intake, and mental status were monitored daily. Figure 5 A);

[0111] After the experiment, the mice in each group were dissected, and the colon, rectum, and spleen were photographed and arranged. It was observed that the colon and rectum of the CTX group mice were shorter than those in the Control, LTLHP-1, HTLHP-1, and LM groups, and the spleen length was longer than that of the other groups. Figure 5B). Statistical analysis was then performed on the body weight, thymus index, spleen index, and colon / rectum length of all groups of mice. Compared with the control group, the CTX group showed a significantly lower final body weight, a decreased thymus index, an increased spleen index, and a decreased colon / rectum length (p<0.0001). Compared with the CTX group, the low-dose group (LTLHP-1) showed an increased final body weight (p<0.001), a decreased spleen index (p<0.05), and no statistically significant difference in thymus index and colon / rectum length. The high-dose group (HTLHP-1) showed a significantly increased final body weight, an increased thymus index, a decreased spleen index (p<0.01), and an increased colon / rectum length (p<0.0001). The LM group showed a decreased spleen index (p<0.001), an increased colon / rectum length (p<0.001), and no statistically significant difference in body weight and thymus index. Figure 5 CF);

[0112] Observed after HE staining of mouse spleen sections ( Figure 5 In the G) and Control groups, the spleen tissue capsule was intact, and the overall structure was normal. The boundary between the white and red pulp was extremely clear. The white pulp exhibited a typical nodular or cylindrical structure, with extremely dense and abundant lymphocytes and deeply stained nuclei. In the CTX group, the spleen tissue structure was severely damaged. The most significant feature was that the boundary between the white and red pulp became extremely blurred, even difficult to identify. The white pulp area significantly atrophied, and the number of lymphocytes decreased sharply, with sparse and scattered arrangement. Congestion or increased hemosiderin deposition was observed in the red pulp area, accompanied by nuclear pyknosis or fragmentation of some cells. Compared with the CTX group, the white pulp area in the LM group was significantly restored and expanded, and the boundary between the white and red pulp became clearer again. The density of lymphocytes increased significantly, and the degree of aggregation increased. Compared with the CTX group, the spleen tissue morphology in the LTLHP-1 group showed some reduction in white pulp atrophy, and the number of lymphocytes began to recover and aggregate, but the boundary between the white and red pulp was still not as clear as in the normal group. In the HTLHP-1 group, the white pulp volume was significantly increased, the shape was fuller, and the boundary with the red pulp became very clear again. The lymphocytes inside the white pulp were densely packed and extremely abundant, and the germinal centers reappeared;

[0113] It should be noted that: Figure 2 A. TLHP-1 dietary intervention and CTX-induced immunosuppression in mice; B. Representative appearance of colorectal and spleen images; C. Final body weight of mice; D. Thymus index; E. Spleen index; F. Colorectal length; G. Spleen H&E staining, scale bar, 100 micrometers (Values ​​are expressed as mean ± standard deviation, n=4-6, representing p<0.0001; representing p<0.001; representing p<0.01; representing p<0.05; ns indicates no significant difference.)

[0114] like Figure 6As shown in Figure X, the levels of immune-related cytokines (IFN-γ, IL-6, TNF-α, and IL-2) and immunoglobulins (IgA and IgG) in the serum of mice in each group were measured using ELISA. The results showed that compared with the normal control group (Control), the CTX group significantly downregulated the levels of IFN-γ, IL-2, IgA, and IgG, and significantly upregulated the levels of IL-6 and TNF-α (p<0.0001). Compared with the CTX group, the low-dose polysaccharide LTLHP1 group downregulated the levels of IL-6 and TNF-α (p<0.05), while showing no significant changes in the levels of IFN-γ, IL-2, IgA, and IgG (no statistical significance). Compared with the CTX group, the high-dose polysaccharide HTLHP1 group significantly increased the levels of IFN-γ, IL-2, IgA, and IgG (p<0.001), and decreased the levels of IL-6 and TNF-α (p<0.01). Compared with the CTX group, the levels of IFN-γ, IL-2, IgA and IgG in the positive drug LM group were significantly increased (p<0.0001), while the levels of IL-6 and TNF-α were decreased (p<0.001).

[0115] It is important to note that: A. Serum IFN-γ; B. Serum IL-6; C. Serum TNF-α; D. Serum IL-2; E. Serum immunoglobulin IgA; F. Serum immunoglobulin IgG (values ​​are expressed as mean ± standard deviation, n=4-6, representing p<0.0001; represents p<0.001; represents p<0.01; represents p<0.05; ns indicates no significant difference).

[0116] like Figure 7 As shown, gut microbiota analysis was performed on mice in the control group, model group (CTX), low-dose group (LTLHP-1), high-dose group (HTLHP-1), and treatment group (LM). Significant differences were observed between the CTX group and the control group in the Shannon and Simpson alpha diversity indices (p<0.05), while no statistically significant differences were found between the other three groups and the CTX group. Figure 7 A). Principal Component Analysis (PCA) Figure 7 B) shows that the samples from each experimental group overlap to a certain extent in spatial coordinates.

[0117] The species composition distribution map visually illustrates the proportional composition of species in each treatment group. At the phylum level, Firmicutes and Bacteroidota were the most prevalent dominant phyla across all groups. Notably, compared to the Control group, CTX intervention led to an increase in the relative abundance of Deferribacterota and a decrease in the relative abundance of Bacteroidota. After administration of low-dose (LTLHP1) and high-dose (HTLHP1) polysaccharides, as well as LM intervention, the abundance of Deferribacterota and Bacteroidota in mice was alleviated. Figure 7 C). At the genus level, compared with the Control group, the relative abundance of *Mucispirillum* (belonging to the phylum *Fructomyces*) increased in the CTX group; meanwhile, the relative abundance of the probiotic *Lactobacillus* decreased. However, HTLHP-1 polysaccharide intervention reversed this trend, not only significantly reducing the proportion of *Mucispirillum*, but also promoting an increase in the abundance of *Lactobacillus* and other potentially beneficial bacteria. Figure 7 D).

[0118] LEfSe analysis further investigated the effect of HTLHP-1 on CTX-induced changes in gut microbiota function. The linear discriminant analysis (LEfSe) score threshold >3 was used as the criterion. Pathogenic bacteria *Mucispirillum*, *Enterobacter*, and unclassified *Oscillospiraceae* were dominant in the CTX group. Conversely, in the HTLHP-1 group, beneficial bacteria were enriched in unclassified *Muribaculaceae*, *Muribaculum*, *Akkermansia*, *Bifidobacterium*, and *Odoribacter*. Figure 7 E);

[0119] Note the following: A. Alpha diversity (Shannon diversity index, Simpson diversity index); B. Principal component analysis; C. Fecal microbial composition at the phylum level; D. Fecal microbial composition at the genus level; E. LEfSe analysis results based on the genus level (LDA value > 3); F. LEFSe branch structure analysis.

[0120] like Figure 8As shown, non-targeted metabolomics analysis was performed on the feces of mice in the control group (Control), model group (CTX), and high-dose group (HTLHP-1). Results showed that between the Control and CTX groups, 11 differentially expressed metabolites were identified, with 1 significantly upregulated and 10 significantly downregulated. Between the CTX and HTLHP-1 groups, 109 differentially expressed metabolites were identified, with 23 significantly upregulated and 86 significantly downregulated. The two groups shared 4 core differentially expressed metabolites. These 4 metabolites were significantly downregulated under CTX induction but upregulated after HTLHP-1 intervention. The CTX group downregulated 10 metabolites, including Tributyrylglycerol, Leukotriene F4, Norvaline, METHACHOLINE, Acetaldehyde butyl phenethyl acetal, and Sabinene hydrate. The HTLHP1 group upregulated 23 metabolites, including Tributyrylglycerol, Norvaline, Acetaldehyde butylphenethyl acetal, Sabinene hydrate, Riboflavin, Stearamide, and 13Z-Docosenamide. Four core, commonly differentiated metabolites were identified as Tributyrylglycerol, Norvaline, Acetaldehyde butylphenethyl acetal, and Sabinene hydrate. Furthermore, pathway enrichment analysis revealed that CTX downregulated the arachidonic acid metabolism pathway, while HTLHP-1 upregulated the riboflavin metabolism and vitamin digestion and absorption pathways. Meanwhile, pathways such as the mTOR signaling pathway and arginine biosynthesis showed significant downregulation.

[0121] The following should be noted: A. VENN plot of differentially metabolites; B. Principal component analysis (PCA); C. Volcano plot of differentially metabolites between the Control group and the CTX group; D. Volcano plot of differentially metabolites between the CTX group and the HTLHP-1 group; E. Differential abundance score plot of differentially metabolites between the Control group and the CTX group; F. Differential abundance score plot of differentially metabolites between the CTX group and the HTLHP-1 group.

[0122] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polysaccharide TLHP-1 from Tricholoma lobayense having immunomodulatory activity, characterized in that: The weight-average molecular weight (Mw) of the white matsutake polysaccharide TLHP-1 is 1.726 × 10⁻⁶. 4 -1.908×10 4 Da, the molecular weight distribution coefficient Mw / Mn is 1.137-1.257; The white matsutake polysaccharide TLHP-1 is a heteropolysaccharide composed of fucose, galactose, glucose, mannose, xylose, rhamnose, galactosamine, glucosamine, galacturonic acid, and glucuronic acid. The content of each monosaccharide, by molar percentage, is as follows: galactose 62.5%-69.1%, fucose 16.2%-18.0%, mannose 11.0%-12.2%, glucose 3.4%-3.8%, xylose 0.85%-0.95%, rhamnose 0.19%-0.21%, galactosamine 0.095%-0.105%, glucosamine 0.285%-0.315%, galacturonic acid 0.285%-0.315%, and glucuronic acid 0.095%-0.105%.

2. The *Matsutake* polysaccharide TLHP-1 with immunomodulatory activity according to claim 1, characterized in that: The weight-average molecular weight (Mw) of the white matsutake polysaccharide TLHP-1 is 1.817 × 10⁻⁶. 4 Da, with a number-average molecular weight Mn of 1.518 × 10⁻⁶. 4 Da, the molecular weight distribution coefficient Mw / Mn is 1.197; The specific content of each monosaccharide, in molar percentage, is as follows: galactose 65.8%, fucose 17.1%, mannose 11.6%, glucose 3.6%, xylose 0.9%, rhamnose 0.2%, galactosamine 0.1%, glucosamine 0.3%, galacturonic acid 0.3%, and glucuronic acid 0.1%.

3. The Trametes versicolor polysaccharide TLHP-1 with immunoregulatory activity according to claim 2, characterized in that: The total sugar content of the white matsutake polysaccharide TLHP-1 was 85.23±0.91%, the total protein content was 0.43±0.1%, and the total phenol content was 2.15±0.08%.

4. The Trametes versicolor polysaccharide TLHP-1 with immunoregulatory activity according to claim 1, characterized in that: The main glycosidic bond type of the white matsutake polysaccharide TLHP-1 is selected from at least one of the following: Xylp-(1→, Fucp-(1→, →2)-Fucp-(1→, Glcp-(1→, Galp-(1→, →2)-Manf-(1→, →4)-Galp-(1→, →6)-Manp-(1→, →6)-Galp-(1→, →2,4)-Manp-(1→, →4,6)-Manp-(1→, →4,6)-Glcp-(1→, →3,6)-Galp-(1→).

5. The *Matsutake* polysaccharide TLHP-1 with immunomodulatory activity according to claim 1, characterized in that: The sugar residues of the white matsutake polysaccharide TLHP-1 are mainly composed of the following seven types: α-L-Fucp-(1→, α-D-Glcp-(1→, →4)-α-D-Galp-(1→, →6)-Manp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→).

6. A method for preparing the immunomodulatory polysaccharide TLHP-1 of Tricholoma lobatum according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Extract the dried white matsutake mushroom powder by reflux with 75% volume fraction ethanol at a material-to-liquid ratio of 1:4, and collect the ethanol-insoluble residue. S2. Add water to the ethanol-insoluble residue and gently reflux hot water at 85-95℃ for 2 hours. Repeat the extraction 3 times, combine the extracts, filter and concentrate to a relative density of 1.1 g / cm³. S3. Add 95% volume fraction ethanol to the concentrate to adjust the final ethanol concentration of the system to 75%, let it stand at room temperature for 24 hours to precipitate, collect the precipitate, dry it, and obtain white matsutake crude polysaccharide. S4. The crude polysaccharide of white matsutake mushroom is repeatedly deproteinized using the Sevag method to obtain deproteinized polysaccharide; S5. The deproteinized polysaccharide was separated and purified sequentially by DEAE-52 anion exchange chromatography column and Sephadex G-300 gel filtration chromatography column. The single symmetrical elution peak fraction was collected and dried to obtain white matsutake polysaccharide TLHP-1.

7. The method for preparing the Tricholoma lobatum polysaccharide TLHP-1 with immunoregulatory activity according to claim 6, characterized in that: In step S5, the DEAE-52 anion exchange chromatography column is eluted with a gradient of 0-1 mol / L NaCl solution, and the Sephadex G-300 gel filtration chromatography column is eluted with distilled water as the mobile phase.

8. The use of the white matsutake polysaccharide TLHP-1 according to any one of claims 1-5, or the white matsutake polysaccharide TLHP-1 prepared by the preparation method according to any one of claims 6-7, in the preparation of drugs, functional foods or health foods with immunomodulatory functions.

9. Use according to claim 8, characterized in that, The immunomodulatory function is to improve cyclophosphamide-induced immunosuppressive damage in the body, including at least one of the following: Increase body weight and thymus index in immunosuppressed individuals, and decrease spleen index; Restoring colorectal length in immunosuppressed individuals; It upregulates serum IFN-γ, IL-2, IgA, and IgG levels, and downregulates serum IL-6 and TNF-α levels; It regulates the structure of the gut microbiota, increases the abundance of beneficial bacteria, and inhibits the abundance of pathogenic bacteria; It regulates arachidonic acid metabolism, riboflavin metabolism, vitamin digestion and absorption, mTOR signaling pathway, and arginine biosynthesis and metabolism pathway.

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