Dacryopinoid polysaccharide, preparation method and application thereof

Abstract

The application discloses a Dacryopinus spadiceus polysaccharide, a preparation method and application thereof. The Dacryopinus spadiceus polysaccharide is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography and dialysis of Dacryopinus spadiceus fruiting bodies. The Dacryopinus spadiceus polysaccharide is a heteropolysaccharide composed of alpha-D-glucose, beta-D-glucose, alpha-D-galactose and beta-D-mannose, and the molar ratio is 6:3:3:1. The Dacryopinus spadiceus polysaccharide prepared by the application has significant immunoregulation and antitumor activity, and can be applied in medicines, health products or food.

Description

Technical Field

[0001] This invention belongs to the field of fungal polysaccharide application technology, specifically relating to a polysaccharide of Hemorrhoidobacterium tumefaciens, its preparation method, and its application. Background Technology

[0002] Edible fungi fruiting bodies are rich in nutrients such as protein, vitamins, minerals, amino acids, and polysaccharides, while being low in calories and fat. Edible fungi polysaccharides possess excellent bioactivity, including anti-tumor, hypoglycemic, lipid-lowering, and immune-boosting effects. Their safety, non-toxicity, and lack of side effects have made them widely popular in the fields of health foods and biomedicine.

[0003] Trametes sanguinea is a common polypore fungus belonging to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Polyporaceae, and genus Trametes. It is mainly distributed in Jilin, Hebei, Henan, and Fujian provinces of my country. Trametes sanguinea contains highly stable, naturally occurring, non-toxic orange-red pigments, laccase, flavonoids, and other substances, and has been widely researched and applied in food, papermaking, and environmental protection.

[0004] CN112979833A discloses that at concentrations up to 50 μg / mL, total polysaccharide TsLCP from *Heliotropium indicum* has almost no effect on the cell viability of two human triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468. This means that within this concentration range, total polysaccharide TsLCP from *Heliotropium indicum* does not show cytotoxicity to these two cell lines. Based on this, it can be inferred that it may not have a significant inhibitory effect on the proliferation of these cell lines, which may indicate that *Heliotropium indicum* polysaccharide has selectivity in antitumor activity.

[0005] CN114085296A discloses a homogeneous polysaccharide TS2-2A from *Hemibacillus thuringiensis* that exhibits no cytotoxicity against RAW264.7 cells while simultaneously promoting cytokine secretion, thus demonstrating an immune-enhancing effect. However, this homogeneous polysaccharide TS2-2A is a homopolysaccharide composed solely of glucose, specifically a linear dextran composed of →3)-α-Glcp-(1→ and →4)-β-Glcp-(1→) in a molar ratio of 1:4, with a molecular weight of 15 kDa.

[0006] Currently, existing research on Hemoglobin polysaccharides mainly focuses on isopolysaccharides composed of glucose and their immunomodulatory applications. However, research on Hemoglobin polysaccharides with other fine structures and their antitumor and immunomodulatory effects is relatively limited. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, the present invention provides a polysaccharide of Hemoglobinus thrombocytopenia, its preparation method and application.

[0008] In a first aspect, the present invention provides a polysaccharide of Hemoglobinia spp., wherein the polysaccharide is a heteropolysaccharide composed of glucose, galactose and mannose in a molar ratio of 9:3:1.

[0009] Furthermore, the glucose is D-glucose, L-glucose, or D / L-glucose; the galactose is D-galactose, L-galactose, or D / L-galactose; and the mannose is D-mannose, L-mannose, or D / L-mannose.

[0010] Further, the D-glucose is α-D-glucose, β-D-glucose, or α / β-D-glucose; the D-galactose is α-D-galactose, β-D-galactose, or α / β-D-galactose; and the D-mannose is α-D-mannose, β-D-mannose, or α / β-D-mannose.

[0011] Further, the L-glucose is α-L-glucose, β-L-glucose, or α / β-L-glucose; the L-galactose is α-L-galactose, β-L-galactose, or α / β-L-galactose; and the L-mannose is α-L-mannose, β-L-mannose, or α / β-L-mannose.

[0012] Preferably, the Hemoglobin thrombocytosis polysaccharide is a heteropolysaccharide composed of D-glucose, D-galactose, and D-mannose.

[0013] More preferably, the Hemoglobin thrombus polysaccharide is a heteropolysaccharide composed of α-D-glucose, β-D-glucose, α-D-galactose, and β-D-mannose, with a molar ratio of 6:3:3:1.

[0014] Furthermore, the chemical structure of the *Hemibarbus thrombectomyces* polysaccharide includes 1,4-linked glucose residues, 1,4,6-linked glucose residues, 1-linked galactose residues, and 4,6-linked mannose residues in a molar ratio of 6:3:3:1. The above-mentioned *Hemibarbus thrombectomyces* polysaccharide incorporates multiple linkage methods.

[0015] Preferably, the chemical structure of the *Hemangiospermum erythrocyte* polysaccharide contains 1,4-linked α-D-glucose residues, 1,4,6-linked β-D-glucose residues, 1-linked α-D-galactose residues, and 4,6-linked β-D-mannose residues in a molar ratio of 6:3:3:1. The above-mentioned *Hemangiospermum erythrocyte* polysaccharide includes multiple linkage methods.

[0016] Furthermore, in the chemical structure of the Hemolymphoidus polysaccharide, the main chain is composed of 1,4-linked glucose residues and 1,4,6-linked glucose residues, and the side chains are composed of 1-linked galactose residues and 4,6-linked mannose residues.

[0017] Preferably, in the chemical structure of the Hemolymphoidus polysaccharide, the main chain is composed of 1,4-linked α-D-glucose residues and 1,4,6-linked β-D-glucose residues, and the side chains are composed of 1-linked α-D-galactose residues and 4,6-linked β-D-mannose residues.

[0018] Furthermore, the Hemoglobin polysaccharide comprises the following structure:

[0019]

[0020] Where n is an integer from 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10).

[0021] Further, the weight-average molecular weight of the *Hemoglobinus thrombectomyces* polysaccharide is 8000-20000 Da (e.g., 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000 Da).

[0022] In one embodiment of the present invention, the weight-average molecular weight of the hemolymphoid polysaccharide is 10955 Da.

[0023] In a second aspect, the present invention provides a method for preparing polysaccharides from Hemolymphoidus, which includes the step of extracting fruiting bodies of Hemolymphoidus.

[0024] Furthermore, the preparation method includes the step of extracting crude polysaccharide by water extraction and alcohol precipitation.

[0025] Furthermore, the preparation method further includes a step of purifying the crude polysaccharide (e.g., by ion exchange column chromatography).

[0026] In some embodiments of the present invention, the preparation method includes the following steps:

[0027] (1) Take the fruiting body powder of Hemolymphoidus and extract it with water (such as cold water or hot water). The resulting water extract is then concentrated, precipitated with alcohol, and deproteinized to obtain crude polysaccharide.

[0028] (2) The crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, eluted, and the eluent is collected;

[0029] (3) Dialyze the eluent obtained in step (2) using a dialysis bag.

[0030] Optionally, (4) freeze-dry the liquid in the dialysis bag after step (3) is completed.

[0031] Further, in step (1), the extraction is performed once or more (e.g., 2, 3, 4, 5 times). In one embodiment of the present invention, the extraction is performed 3 times.

[0032] Further, in step (1), the extraction time for each extraction is 1-10 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours). In one embodiment of the present invention, the extraction time for each extraction is 6 hours.

[0033] Further, in step (1), the extraction temperature is 80-100℃ (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100℃). In one embodiment of the present invention, the extraction temperature is 98℃.

[0034] Further, in step (1), the ratio (w / v, mg / mL) of the *Hemanthophoria rubra* fruiting body powder to water is 1:1-10 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10). In one embodiment of the present invention, the ratio is 1:3.

[0035] In one embodiment of the present invention, the extraction step in step (1) includes: taking Hemoglobinus fruiting body powder, mixing it with water, and extracting it by heating in a water bath.

[0036] Further, in step (1), during the alcohol precipitation step, the volume ratio of alcohol to the concentrated water extract is 1-10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1). In one embodiment of the present invention, the volume ratio is 3:1.

[0037] In one embodiment of the present invention, the alcohol in the alcohol precipitation step is ethanol.

[0038] Furthermore, in step (1), the protein is removed using the Sevage method.

[0039] In one embodiment of the present invention, step (1) includes: taking the fruiting body powder of Hemolymphoides, extracting it with hot water, collecting the supernatant, concentrating it, adding anhydrous ethanol, collecting the precipitate, drying it, removing the protein therein, and obtaining crude polysaccharide.

[0040] Further, in step (2), the ion exchange column is a cellulose column, and the packing material of the cellulose column is DEAE-cellulose.

[0041] Further, in step (2), the elution is gradient elution. Specifically, the concentration of the eluent in the gradient elution is selected from two or more of the following: 0.001-0.30 mol / L (e.g., 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30 mol / L).

[0042] Further, in step (2), the eluent used for elution is a NaCl solution. Specifically, the concentration of the NaCl solution is 0.001-0.30 mol / L (e.g., 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30 mol / L).

[0043] In one embodiment of the present invention, step (2) includes: passing the aqueous solution of crude polysaccharide obtained in step (1) through a cellulose column, eluting by gradient, collecting the eluent, and concentrating it.

[0044] Further, in step (3), the molecular weight cutoff of the dialysis bag is 5000-10000 Da (e.g., 5000, 6000, 7000, 8000, 9000, 10000 Da). In one embodiment of the present invention, the molecular weight cutoff is 7000 Da.

[0045] In one embodiment of the present invention, step (3) includes: placing the eluent obtained in step (2) into a dialysis bag for dialysis for 2 days.

[0046] The present invention also provides a crude polysaccharide obtained by the above preparation method.

[0047] The preparation method of the present invention can prepare the hemolymph polysaccharide described in the first aspect.

[0048] A third aspect of the present invention provides a composition comprising Hemoglobinosporium polysaccharide.

[0049] Furthermore, the composition also includes excipients, such as pharmaceutically acceptable excipients, or food or health product acceptable excipients.

[0050] Furthermore, the pharmaceutically acceptable excipients are selected from: stabilizers, gliding agents, pressure enhancers, flavoring agents, preservatives, suspending agents, coating agents, flavoring agents, anti-adhesion agents, antioxidants, antioxidant synergists, chelating agents, skin penetration enhancers, air substitutes, pH adjusters, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, adsorbents, plasticizers, surfactants, lyophilized fillers, lyophilized protectants, empty capsules, colloidal stabilizers, vaccine adjuvants, excipients for processing traditional Chinese medicine, foaming agents, defoamers, thickeners, encapsulating agents, protectants, humectants, softeners, absorbents, diluents, flocculants, filter aids, inks, and pressure-sensitive adhesives.

[0051] Furthermore, the acceptable excipients for the food or health product are selected from: colorants, color protectants, emulsifiers, enzyme preparations, flavor enhancers, food flavorings, processing aids for the food industry, flour treatment agents, coating agents, moisture retainers, nutritional fortifiers, preservatives, acidity regulators, anti-caking agents, defoamers, antioxidants, bleaching agents, leavening agents, chewing gum base agents, stabilizers and coagulants, sweeteners, and thickeners.

[0052] Furthermore, the composition is a pharmaceutical composition, a food composition, or a health product composition.

[0053] Furthermore, the pharmaceutical composition may be an antitumor drug, an immune enhancer, an immune adjuvant, an antiviral drug (such as an anti-HCMV drug), an antibacterial drug (studies have shown that the extract of *Rhizoctonia solani* has a significant inhibitory effect on *Escherichia coli*, *Staphylococcus aureus*, *Streptococcus*, and *Salmonella*), a biological response modifier, or an anti-inflammatory drug (which can be used to treat toothache, pharyngitis, bacillary dysentery, arthritis, gout, for hemostasis, sore throat, ulcers, fever, inflammatory bowel disease), etc.

[0054] Furthermore, the hemolymphoid polysaccharide can be used alone or in combination with other active ingredients.

[0055] Furthermore, the dosage form of the pharmaceutical composition is an oral dosage form or an injectable dosage form, preferably an oral dosage form.

[0056] Furthermore, the dosage form of the food composition or health product composition is an oral dosage form.

[0057] Furthermore, the oral dosage form is selected from: solutions, mixtures, syrups, powders, pellets, pills, tinctures, decoctions, medicated wines, emulsions, tablets, capsules, granules, and suspensions.

[0058] Furthermore, the injectable dosage form is selected from: injection solution, sterile powder for injection (including lyophilized powder for injection).

[0059] In a fourth aspect, the present invention provides the use of Heme Cortex polysaccharide in the preparation of antitumor drugs.

[0060] Furthermore, the tumor is selected from: gastric cancer, cardia cancer, esophageal cancer, cervical cancer, sarcoma, Ehrlich ascites carcinoma, colon cancer, breast cancer, cervical cancer, and liver cancer.

[0061] In some embodiments of the present invention, the tumor is gastric cancer.

[0062] Furthermore, the antitumor drug also includes pharmaceutically acceptable excipients.

[0063] Furthermore, the hemolymphoid polysaccharide can be used alone or in combination with other active ingredients.

[0064] In a fifth aspect, the present invention provides the use of Heme thrombocytopenia polysaccharide in the preparation of immunomodulatory drugs or immunoadjuvants.

[0065] Furthermore, the *Hemibacillus thrombocytogeneticus* polysaccharide can enhance the body's immune function (such as non-specific or specific immune function) by activating immune cells (such as lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, platelets, etc.) or immune-active substances (such as antibodies, lysozyme, complement, immunoglobulins, interferon, interleukins, tumor necrosis factor, etc.), thereby restoring the weakened immune function to normal. The *Hemibacillus thrombocytogeneticus* polysaccharide can also act as an adjuvant to enhance the immunogenicity of co-administered antigens and accelerate the induction of immune responses.

[0066] Furthermore, the immune enhancer or immune adjuvant also includes pharmaceutically acceptable excipients.

[0067] Furthermore, the hemolymphoid polysaccharide can be used alone or in combination with other active ingredients.

[0068] In a sixth aspect, the present invention provides the use of Heme thrombocytogen polysaccharide in the preparation of foods or health products that enhance immunity.

[0069] Furthermore, the hemolymphoid polysaccharide can enhance the body's immune function (such as non-specific or specific immune function) by activating immune cells (such as lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, platelets, etc.) or immune-active substances (such as antibodies, lysozyme, complement, immunoglobulins, interferon, interleukins, tumor necrosis factor, etc. cytokines), thereby achieving the purpose of enhancing immunity.

[0070] Furthermore, the immune-boosting food or health product also includes food or health product-acceptable excipients.

[0071] Furthermore, the hemolymphoid polysaccharide can be used alone or in combination with other active ingredients.

[0072] In a seventh aspect, the present invention provides the use of Heme Typhimurium polysaccharide in the preparation of a drug for inhibiting tumor cells (such as inhibiting the growth, proliferation, and differentiation of tumor cells).

[0073] Further, the concentration of Hemoglobin polysaccharide in the drug is 0.01-20 μg / mL (e.g., 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20 μg / mL), preferably 1-15 μg / mL, more preferably 5-10 μg / mL.

[0074] Furthermore, the tumor is selected from: gastric cancer, cardia cancer, esophageal cancer, cervical cancer, sarcoma, Ehrlich ascites carcinoma, colon cancer, breast cancer, cervical cancer, and liver cancer.

[0075] In some embodiments of the present invention, the tumor is gastric cancer.

[0076] In some embodiments of the present invention, the tumor cells are gastric cancer cells (such as MFC cells).

[0077] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0078] Furthermore, the hemolymphoid polysaccharide can be used alone or in combination with other active ingredients.

[0079] This invention discloses a polysaccharide derived from *Hemanthotomyces* through isolation and purification. The polysaccharide's molecular weight, monosaccharide composition, and chemical structure were analyzed and identified, determining its weight-average molecular weight and structural composition. Cell experiments showed that this polysaccharide possesses significant immunomodulatory and antitumor activities, particularly exhibiting the highest proliferation rate in RAW264.7 cells at a concentration of 20 μg / mL, and the highest inhibition rate in MFC cells at a concentration of 10 μg / mL. Based on these findings, this polysaccharide can be used to prepare pharmaceuticals, as well as health products and foods that enhance immunity, demonstrating promising application prospects and commercial value. Attached Figure Description

[0080] Figure 1 The image shown is the HPGPC spectrum of TS-P.

[0081] Figure 2 The image shown is the infrared spectrum of TS-P.

[0082] Figure 3 The HPLC chromatogram of TS-P hydrolysate is shown, where peak 1 is mannose, peak 2 is glucose, and peak 3 is galactose.

[0083] Figure 4 The image shown is of TS-P. 1 H NMR spectrum.

[0084] Figure 5 The image shown is of TS-P. 13 C10 NMR spectrum.

[0085] Figure 6 The image shown is of TS-P. 1 H- 1 H-COSY spectrum.

[0086] Figure 7 The image shows the HMQC spectrum of TS-P.

[0087] Figure 8 The image shows the HMBC spectrum of TS-P.

[0088] Figure 9 The chemical structure of TS-P is shown.

[0089] Figure 10 The results show the effects of TS-P on the proliferation of RAW264.7 cells.

[0090] Figure 11 The results show the effects of TS-P on MFC cell proliferation. Detailed Implementation

[0091] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0092] In this invention, "Trametes sanguinea" belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, family Polyporaceae, and genus Trametes.

[0093] The term "tumor" refers to an abnormal mass of tissue that grows beyond and out of harmony with the growth of normal tissue. Tumors can be "benign" or "malignant," depending on characteristics such as the degree of cell differentiation (including morphology and function), growth rate, local invasion, and metastasis. "Benign tumors" are typically well-differentiated, characterized by slower growth than malignant tumors, and remain confined to their site of origin. Furthermore, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors may later develop into malignant tumors, possibly due to additional genetic alterations in a subset of the tumor's proliferative cells, and these tumors are called "precancerous tumors." "Malignant tumors" are typically poorly differentiated (anaplastic) and characterized by rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissues. Furthermore, malignant tumors often have the ability to metastasize to distant sites.

[0094] The term “cancer” refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyled.; Williams & Wilkins: Philadelphia, 1990).

[0095] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0096] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0097] Example 1: Isolation and extraction of Hemorrhage polysaccharide TS-P

[0098] 1. Isolation and extraction of Hemoglobin thrombocytopenia polysaccharide TS-P

[0099] 1.1 Extraction of crude polysaccharides from Hemoglobinobacterium using water extraction and alcohol precipitation method

[0100] Weigh 200g of dried *Heliotropium indicum* fruiting bodies and pulverize them. Add the pulverized fruiting bodies and distilled water to a beaker at a ratio of 1:3 (material to liquid). Incubate at 98℃ for 6 hours. Collect the supernatant and concentrate. Repeat this process three times, finally concentrating all the supernatant to 200mL. Add three times the volume of anhydrous ethanol to precipitate the precipitate. Collect the precipitate and dry it to remove proteins from the extract, thus obtaining crude polysaccharide from *Heliotropium indicum*.

[0101] 1.2. DEAE-cellulose column chromatography for the separation and purification of crude polysaccharides from Hemorrhage bacteria.

[0102] Accurately weigh 50g of DEAE cellulose and dissolve it in 1L of ultrapure water. Stir thoroughly until no cellulose particles are visible to the naked eye, then stop stirring. Let stand for 24 hours, discard the supernatant, and set aside for later use. Prepare 0.5mol / L NaOH and soak the cellulose for 6 hours. Wash with ultrapure water until neutral, discard the supernatant, then add 0.5mol / L HCl and soak for 6 hours. Wash with distilled water until neutral, discard the supernatant, then add 0.5mol / L NaOH again and soak for 6 hours. Wash with distilled water until neutral, then set aside for later use.

[0103] After packing the activated cellulose into a column, the crude polysaccharide was purified by column equilibration with distilled water for 24 hours. The supernatant (5 mL) of the diluted crude polysaccharide was added to a DEAE cellulose column, and eluted with different concentrations of NaCl (0.01 mol / L, 0.05 mol / L, 0.1 mol / L). The polysaccharide was determined using the sulfuric acid-phenol method. The eluent was concentrated to 5 mL, and the sample was purified on a cellulose column. Dialysis was performed using a dialysis bag (Mw ≥ 7 kDa) for 48 hours, followed by freeze-drying to obtain the *Strombus haematobium* polysaccharide, named TS-P.

[0104] 2. Structural identification of Hemoglobin thrombus polysaccharide TS-P

[0105] The structure of Hemoglobin thrombocytosis polysaccharide (TS-P) was determined using acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance.

[0106] 2.1 Determination of molecular weight

[0107] Dissolve 10 mg of Hemoglobin thrombocytosis polysaccharide TS-P sample in 1 mL ddH2O, sonicate for 5 min, and perform HPGPC analysis.

[0108] 2.2 Infrared Spectroscopic Analysis of Hemoglobin Thymosin TS-P

[0109] 2 mg TS-P was mixed with KBr and compressed into tablets. The tablets were then scanned at 4000 cm⁻¹ using an infrared spectrophotometer. -1 -500cm -1 scope.

[0110] 2.3 Monosaccharide composition analysis of Hemlock polysaccharide TS-P

[0111] Six standards (fructose, rhamnose, arabinose, mannose, glucose, and galactose) (purchased from Chengdu Manster Biotechnology Co., Ltd.) and the TS-P sample after TFA acid hydrolysis were dissolved in the mobile phase (75% acetonitrile) and then analyzed by HPLC.

[0112] 2.4 Nuclear Magnetic Resonance Analysis of Hemoglobin thrombocytopenia polysaccharide TS-P

[0113] Dissolve 50 mg of TS-P sample in 0.6 mL of heavy water (D2O), place it in an NMR tube, and detect it on an NMR spectrometer.

[0114] 2.5. GC-MS analysis of methylation and silanization derivatization of *Strombum haematobium* polysaccharide TS-P.

[0115] Weigh 20 mg of TS-P sample, seal the beaker, add 2 mL of DMSO (dimethyl sulfoxide) to the sealed beaker, and gently shake the beaker to fully dissolve the TS-P. Then add 200 mg of NaOH until the NaOH just stops dissolving, and place the beaker in a shaker at room temperature for 1 hour. After shaking, add 1.5 mL of iodomethane, and react in the dark for 1 hour. The reaction is terminated by adding water. Extract the product with chloroform, and dry to obtain methylated polysaccharide. The methylated polysaccharide is then completely acid-hydrolyzed by TFA, washed three times with water to obtain the fully acid-hydrolyzed methylated product.

[0116] The above sample was reacted with 2 mL of hexamethyldisilamide, 1 mL of trimethylchlorosilane and 2 mL of anhydrous pyridine, and then incubated in a water bath at 50 °C for 20 min. The mixture was then centrifuged at 12,000 rpm / min and 4 °C for 10 min using a low-temperature high-speed centrifuge. The precipitate was discarded, and the mixture was filtered through a 0.22 μm filter. The supernatant was used for GC-MS analysis.

[0117] 3. Results

[0118] 3.1 Results of basic properties of Hemoglobin thrombocytopenia polysaccharide TS-P

[0119] The HPGPC spectrum of TS-P is as follows Figure 1 As shown, the weight-average molecular weight of TS-P is 10955 Da.

[0120] 3.2 FTIR spectral analysis of Hemorrhage Pulmonary Polysaccharide TS-P

[0121] The primary structure of TS-P was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 2 As shown, the wavenumber is at 3389 cm⁻¹ -1 2934cm -1 1679cm-1 and 1400-1200cm -1 The presence of typical polysaccharide absorption peaks and the absence of other impurity peaks indicates that the isolated and purified TS-P is a polysaccharide. The wavenumber is 3389 cm⁻¹. -1 The broad absorption peak is the stretching vibration peak of OH, at 2934 cm⁻¹. -1 The absorption peak within the range is the stretching vibration peak of CH, at 1679 cm⁻¹. -1The peak is the C=O stretching vibration peak, 1405 cm⁻¹. -1 The peak represents the in-plane bending vibration of -CHO at 1200-1000 cm⁻¹. -1 The absorption peaks within the range are due to the absorption of pyranose ring lactones and hydroxyl groups, indicating that TS-P has a pyran ring. At 806 cm⁻¹ -1 The presence of absorption peaks confirms the existence of a pyranose ring structure in TS-P. Furthermore, at 1730 cm⁻¹... -1 The absence of absorption peaks nearby indicates that TS-P does not contain uronic acid.

[0122] 3.3 Monosaccharide composition analysis of Hemorrhage Cytomegalovirus polysaccharide TS-P

[0123] After complete hydrolysis of TS-P, its monosaccharide composition was analyzed by HPLC, and the results are as follows: Figure 3 As shown, peak 1 is mannose (Man), with a retention time of 8.335 min; peak 2 is glucose (Glc), with a retention time of 9.239 min; and peak 3 is galactose (Gal), with a retention time of 9.735 min. The ratio of mannose, glucose, and galactose is 1:9:3.

[0124] 3.4 NMR spectral analysis of TS-P polysaccharide from Hemangiospermum erythrocytes

[0125] TS-P 1 The H NMR results are as follows Figure 4 As shown in the figure. The results show that TS-P has four anomeric hydrogen signals: δ4.98ppm, δ4.96ppm, δ4.95ppm and δ4.87ppm, with an integrated area ratio of 1.0:3.07:3.32:5.92. The signals between δ3.0 and 4.2ppm are attributed to hydrogen signals from C2-C6 of the sugar residues.

[0126] TS-P 13 The C NMR results are as follows Figure 5 As shown, TS-P exhibits four anodic carbon signals at δ102.33ppm, δ101.50ppm, δ97.87ppm, and δ97.87ppm. The signals between δ60 and 85ppm are attributed to C2-C6 carbon signals in sugar residues.

[0127] TS-P 1 H- 1 H-COSY spectrum as shown Figure 6 As shown, the coupling relationship between adjacent hydrogen nuclei can be identified. The H1 / H2 signals for part A are δ4.98 / 3.99, for part B they are δ4.96 / 3.72, for part C they are δ4.95 / 3.70, and for part D they are δ4.87 / 3.76.

[0128] All the chemical shifts of hydrogen are summarized in Table 1.

[0129] The HMQC spectrum of TS-P is as follows: Figure 7 As shown, it can identify short-range related factors. 1 H and 13 The coupling relationship between C. The signal of H1 / C1 in part A is δ4.98 / 102.33, the signal of H1 / C1 in part B is δ4.96 / 101.50, the signal of H1 / C1 in part C is δ4.95 / 97.87, and the signal of H1 / C1 in part D is δ4.87 / 97.87.

[0130] The HMBC spectrum of TS-P is as follows Figure 8 As shown, it can identify remote related... 1 H and 13 The coupling relationships between residues C and C are as follows: H4 / C6 signal of residue A is δ3.77 / 68.21, H1 / C4 signal of residue B is δ4.96 / 67.20, H4 / C2 signal of residue C is δ3.70 / 69.49, and H1 / C5 signal of residue D is δ4.87 / 69.52.

[0131] All the chemical shifts of carbon are summarized in Table 2.

[0132] Table 1 TS-P 1 Chemical shift of H

[0133]

[0134] Table 2 TS-P 13 Chemical shift of C

[0135]

[0136] 3.5 Gas Chromatography and Mass Spectrometry Analysis of Hemoglobin Thunb. Polysaccharide TS-P

[0137] The methylation results are shown in Table 3. Due to steric hindrance at C2 and C3 in the sugar ring, methylation was incomplete. The main repeating structural unit of TS-P consists of 1,4-linked α-D-glucose residues and 1,4,6-linked β-D-glucose residues, while the side chains consist of 1-linked α-D-galactose residues and 4,6-linked β-D-mannose residues. Based on these findings, the structure of TS-P can be preliminarily deduced as follows: Figure 9 As shown.

[0138] Table 3 Analysis of TS-P methylation results

[0139]

[0140] Example 2: Study on the antitumor and immunomodulatory activities of Hemorrhage Cytomegalovirus polysaccharide TS-P

[0141] The antitumor and immunomodulatory activities of Hemoglobin TS-P were determined in vitro using two CCK-8 assays.

[0142] 1. Reagents

[0143] The CCK-8 kit, RPIM1640, FBS, DMSO, and bispecific antibodies were all commercially available products. Man (mannan peptide) was purchased from Chengdu Lier Pharmaceutical Co., Ltd.

[0144] 2. Instruments

[0145] ELISA reader; cell culture incubator.

[0146] 3. Detection method for the effect of TS-P on the proliferation of RAW264.7 cells and MFC cells

[0147] The effect of *Strombum rubrum* polysaccharide (TS-P) on the proliferation of RAW264.7 and MFC cells was determined using a cell counting kit (CCK-8). RAW264.7 and MFC cells were cultured in vitro to the logarithmic growth phase. After cell counting using a cell counting chamber, the cell suspension was diluted to 1 × 10⁻⁶ cells / mL with fresh culture medium. 5 Cell suspension was added to 96-well plates at a concentration of 100 μL / mL, and the plates were incubated in a CO2 incubator for 24 h. After 24 h, different concentrations of TS-P solution (5, 10, and 20 μg / mL) were added to the experimental groups. The positive control group (LPS group) for RAW264.7 cell proliferation was treated with 100 μL of LPS (10 μg / mL), the positive control group (Man group) for MFC cell proliferation was treated with 100 μL of Man (10 μg / mL), and the blank group was treated with 100 μL of cell culture medium. After 24 h of incubation in a CO2 incubator, 5 μL of CCK-8 was added to each well, and the plates were incubated in a CO2 incubator for 3 h. The absorbance was then measured (450 nm) using a microplate reader, and images were captured. Finally, the cell proliferation rate or inhibition rate was calculated using the following equation:

[0148] A1-A2

[0149] p = A2 - A0

[0150] In the formula, p is the cell proliferation rate (or inhibition rate), A0 is the average absorbance value of the culture medium; A2 is the average absorbance value of the blank group; and A1 is the average absorbance value of the drug group or the positive group.

[0151] 4. Results

[0152] 4.1 Effect of TS-P on the proliferation of RAW264.7 cells

[0153] The results are as follows Figure 10 As shown, compared with the control group, the LPS group significantly (P<0.01) promoted the proliferation of RAW264.7 cells, with a proliferation rate of 476.70%. When the mass concentration of TS-P was 5, 10, and 20 μg / mL, there was a trend that the proliferation-promoting effect of TS-P on RAW264.7 cells was stronger with increasing concentration. Moreover, when the mass concentration of TS-P was 20 μg / mL, the proliferation effect of TS-P on RAW264.7 cells was the most obvious, with a maximum proliferation rate of 143.40%.

[0154] 4.2 Effect of TS-P on the proliferation of MFC cells

[0155] The results are as follows Figure 11 As shown, compared with the blank group, the Man group significantly (P<0.05) inhibited the proliferation of MFC cells, with an inhibition rate of 49.38%. When the mass concentration of TS-P was 5, 10 and 20 μg / mL, it could significantly (P<0.01) inhibit the proliferation of MFC cells. When the mass concentration of TS-P was 10 μg / mL, the inhibitory effect of TS-P on MFC cells was the most obvious, with a maximum inhibition rate of 76.00%.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0157] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0158] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A polysaccharide from *Hemiglossum*, wherein the polysaccharide is... α - D -glucose, β - D -glucose, α - D -galactose, β - D -A heteropolysaccharide composed of mannose in a molar ratio of 6:3:3:1, the Hemoglobinococcus polysaccharide comprises the following structure: ， n is an integer between 3 and 10; The weight-average molecular weight of the hemoglobin polysaccharide is 8000-20000 Da.

2. A method for preparing the polysaccharide of *Hemiberlesia lataniae* as described in claim 1, the method comprising the following steps: (1) Take the fruiting body powder of Hemolymphoidus, extract it with water, and then concentrate, precipitate with alcohol and remove protein to obtain crude polysaccharide. (2) The crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, eluted, and the eluent is collected; (3) Dialyze the eluent obtained in step (2) using a dialysis bag; In step (1), the extraction is performed 1-5 times, and each extraction lasts 1-10 hours; In step (1), the extraction temperature is 80-100℃; In step (1), the ratio of the powder of the fruiting body of the red blood fungus to water is 1:1-10; In step (1), the volume ratio of alcohol to concentrated water extract in the alcohol precipitation step is 1-10:1; In step (2), the ion exchange column is a cellulose column, and the packing material of the cellulose column is DEAE-cellulose; In step (2), the elution is gradient elution.

3. A composition comprising the hemolymphoid polysaccharide as described in claim 1.

4. The use of the Heme Pleurotus polysaccharide as described in claim 1 in the preparation of antitumor drugs, immune enhancers, immune adjuvants, and immune-enhancing foods or health products.

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