A russula ericaea polysaccharide, and a preparation method and application thereof

Abstract

The application discloses a kind of Russula nigra polysaccharides and its preparation method and application.The Russula nigra polysaccharides is composed of glucose, galactose heteropolysaccharide, which includes 1,6-connected galactose residue, 1,4-connected D-glucose residue, 1,4-connected D-galactose residue, 1,4,6-connected glucose residue and 1-connected galactose residue, and the molar ratio of 1-Galp, 1,6-Galp, 1,4,6-Glcp, 1,4-D-Galp and 1,4-D-Glcp residue is about 2:1:2:1:4.The Russula nigra polysaccharides is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography, dialysis and concentration.The Russula nigra polysaccharides has significant immunomodulatory activity and antitumor activity, and can be applied in medicine, health care products or food.

Description

Technical Field

[0001] This invention relates to the field of fungal polysaccharide application technology, and in particular to a polysaccharide from *Russula smokyans*, its preparation method, and its application. Background Technology

[0002] Edible fungi, commonly known as mushrooms, are large fungi whose fruiting bodies are rich in nutrients such as protein, vitamins, minerals, amino acids, and polysaccharides. Edible fungal polysaccharides possess a variety of biological activities, including antiviral, antioxidant, antitumor, lipid-lowering, immune cell proliferation and differentiation promotion, lymphokine secretion, and complement activation, among others. They are safe and non-toxic, and have attracted widespread attention in the fields of health foods and biomedicine. Furthermore, edible fungal polysaccharides are non-specific immune enhancers; they can improve the body's immune function through multiple pathways without side effects.

[0003] *Russula adusta*, belonging to the order Agaricales, family Russulaceae, and genus *Russula*, is also known as black charcoal mushroom or fire charcoal mushroom. It mainly grows in parts of Fujian, Guangxi, Sichuan, and Jiangxi provinces. *Russula adusta* grows singly, in groups, or in clumps on the ground in broad-leaved and coniferous forests during summer and autumn. Its fruiting body is medium-sized, with a cap diameter of 9.5-11 cm, flattened hemispherical later concave, smooth, and a stipe 1.5-6.5 cm long and 1-2.8 cm thick. During its growth period, the flesh of *Russula adusta* is initially white, turning pale smoky, brownish-gray to dark brownish-gray after harvesting, and finally black. *Russula adusta* has a thick flesh and a delicious flavor, and is commonly used in traditional medicine to treat dysentery and other gastrointestinal problems; it is a dual-purpose edible and medicinal mushroom. Currently, most of the more than 60 edible Russula species in my country are rich in polysaccharides, triterpenes, amino acids, vitamins, and various minerals. They are also characterized by low fat, low calories, and no cholesterol, possessing high nutritional and edible value and certain health benefits, making them suitable as functional foods. In recent years, numerous studies have shown that polysaccharides from most Russula species exhibit strong biological activity, with Russula smokyis polysaccharides demonstrating certain antioxidant activity.

[0004] Li Yimeng et al. reported in "Optimization of Extraction Process and Study on Physicochemical Properties and Antioxidant Activity of Polysaccharides from Black Fungus" ([J]. Journal of Henan University of Technology (Natural Science Edition), 2020, 41(03):19-26.) that they used the fruiting bodies of *Russula smokyans* as raw material and adopted the polysaccharide yield as an indicator to optimize the extraction process of polysaccharides from *Russula smokyans* through single-factor and response surface experiments. They found that the optimal process conditions for RAP extraction of polysaccharides from *Russula smokyans* were a liquid-to-solid ratio of 26:1 (mL / g), a water temperature of 80℃, and an extraction time of 185 min. Under these conditions, the polysaccharide yield was 9.42%. Molecular structural analysis of RAP revealed a molecular weight of 5763 Da, primarily containing five monosaccharides: rhamnose, fucose, mannose, glucose, and galactose, in a ratio of 1:5.53:8.83:10.62:16.00. NMR analysis showed that RAP exists in both α- and β-glycosidic configurations, with the α-configuration being more prevalent than the β-configuration. Antioxidant assays demonstrated that RAP exhibited antioxidant activity against ·OH and O2 within the range of 0.25-8 mg / mL. 2- All exhibit a certain scavenging ability, and this ability is positively correlated with their concentration. When the RAP concentration is 8 mg / mL, the scavenging rate for ·OH is 78.32%, and for O2... 2－ The clearance rate reached 34.71%, indicating that RAP has certain in vitro antioxidant activity. However, it did not disclose the specific linkage mode of the five monosaccharides, rhamnose, fucose, mannose, glucose and galactose. In addition, there is a lack of research on the immunomodulatory activity and anti-tumor effects of Rhodotorula fuciformis polysaccharide.

[0005] Patent document CN1272024C discloses a composition derived from plants, foods, and their extracts. This composition, in a specific combination, can be used as a food additive to effectively combat malignant tumors and reduce immune function. The composition includes soybeans, mushrooms, and mung beans. The main active components of soybeans, mushrooms, and mung beans are genistein, protozoanin A, estradiol, inositol hexaphosphate, β-glucan, and exogenous lectins. The mushroom can be *Russula flavum*. However, it lacks research on the fine structure of *Russula flavum* polysaccharides, only mentioning glucan. Not all polysaccharides extracted from *Russula flavum* possess antitumor and immunomodulatory activities; these effects are closely related to the type, linkage, and ratio of monosaccharides.

[0006] It is evident that existing technologies lack research on the fine structure of Russula fuciformis polysaccharide, as well as its immunomodulatory activity and antitumor applications. Summary of the Invention

[0007] This invention overcomes the shortcomings of the prior art and provides a polysaccharide from *Russula smokyosa*, its preparation method, and its application.

[0008] The first aspect of the present invention provides a polysaccharide (RA-P) from Russula smokyis, which is a heteropolysaccharide composed of glucose and galactose, wherein the molar ratio of glucose and galactose residues is approximately 3:2.

[0009] Furthermore, the chemical structure of the polysaccharide comprises 1,6-linked galactose residues, 1,4-linked D-glucose residues, 1,4-linked D-galactose residues, 1,4,6-linked glucose residues, and 1-linked galactose residues.

[0010] Further, the weight-average molecular weight of the polysaccharide is 10000Da-3000Da (e.g., 10000Da, 11000Da, 12000Da, 13000Da, 14000Da, 15000Da, 16000Da, 17000Da, 18000Da, 19000Da, 19500Da, 19600Da, 19700Da, 19800Da, 19900Da, 20000Da, 20100Da, 20200Da, 203...). 00Da, 20400Da, 20500Da, 20600Da, 20700Da, 20800Da, 20900Da, 21000Da, 22000Da, 23000Da, 24000Da, 25000Da, 26000Da, 27000Da, 28000Da, 29000Da, 30000Da), preferably 15000Da-25000Da, and more preferably 18000Da-22000Da.

[0011] In one embodiment of the present invention, the polysaccharide has a weight-average molecular weight of 20576 Da.

[0012] Furthermore, the polysaccharide comprises the following structural formula:

[0013]

[0014] Wherein, n is an integer from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably, n is an integer from 2 to 15, and more preferably, n is an integer from 8 to 14.

[0015] In this context, Glcp is glucose and Galp is galactose.

[0016] A second aspect of the invention provides a composition comprising the polysaccharide described in the first aspect.

[0017] In some embodiments of the present invention, the composition is a pharmaceutical composition that further comprises one or more pharmaceutically acceptable excipients.

[0018] In some embodiments of the present invention, the composition is a health product composition, which further comprises one or more food or health product-acceptable excipients.

[0019] A third aspect of the present invention provides a method for preparing polysaccharides from *Russula smokyans* as described in the first aspect, comprising an extraction step using *Russula smokyans* fruiting bodies as raw materials.

[0020] More preferably, the preparation method includes the step of extracting crude polysaccharide by water extraction and alcohol precipitation.

[0021] More preferably, the preparation method further includes a step of purifying the crude polysaccharide (e.g., by ion exchange column chromatography).

[0022] In one embodiment of the present invention, the preparation method includes the following steps:

[0023] (1) Take the fruiting body powder of smoky red mushroom, extract it with hot water, and then concentrate, precipitate with alcohol and dry the resulting water extract to obtain crude polysaccharide.

[0024] (2) The crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, eluted, and the eluent is collected;

[0025] (3) The eluent obtained in step (2) is concentrated by dialysis using a dialysis bag.

[0026] Furthermore, the preparation method also includes step (4), specifically freeze-drying the liquid inside the dialysis bag after step (3).

[0027] Further, in step (1), the extraction temperature can be 80-100℃ (e.g., 80, 85, 90, 95, 100℃); in one embodiment of the present invention, the extraction temperature is 98℃.

[0028] Further, in step (1), the mass ratio (W / V, mg / mL) of the fruiting body powder of *Pleurotus eryngii* to water is 1:1-10 (e.g., 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in one embodiment of the present invention, the mass ratio is 1:3.

[0029] Further, in step (1), the number of extractions is 1-5 times (e.g., 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0030] Further, in step (1), the extraction time for each extraction is 1-10 hours (e.g., 1, 3, 6, 8, 10 hours); in one embodiment of the present invention, the extraction time for each extraction is 6 hours.

[0031] In one embodiment of the present invention, the extraction step in step (1) may include: taking the fruiting body powder of *Pleurotus eryngii*, mixing it with water, and boiling it in a water bath.

[0032] Further, in step (1), during the alcohol precipitation step, the volume ratio of alcohol to water extract concentrate is 1-10:1 (e.g., 1:1, 3:1, 4:1, 5:1, 10:1); in one embodiment of the present invention, the volume ratio is 3:1.

[0033] In one embodiment of the present invention, the alcohol in the above-described alcohol precipitation step is ethanol.

[0034] In one embodiment of the present invention, step (1) includes: taking the fruiting body powder of *Pleurotus eryngii*, 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.

[0035] Furthermore, in step (2), the ion exchange column can be a cellulose column, and the packing material of the cellulose column is such as DEAE cellulose.

[0036] Further, in step (2), the eluent used for elution can be a NaCl solution; specifically, the concentration of the NaCl solution is 0-0.3 mol / L (e.g., 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mol / L).

[0037] Furthermore, in step (2), the elution can be gradient elution, and the concentration of the eluent can be 0.01-1.0 mol / L (e.g. 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0) mol / L.

[0038] In one embodiment of the present invention, step (2) includes: passing the aqueous solution of the crude polysaccharide obtained in step (1) through a cellulose column, performing gradient elution, collecting the eluent, and concentrating it.

[0039] 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.

[0040] In one embodiment of the present invention, step (3) includes: placing the eluent obtained in step (2) into a dialysis bag for dialysis for two days.

[0041] A fourth aspect of the present invention provides an application of the polysaccharide of *Russula smokyans* as described in the first aspect, or the composition as described in the second aspect, or the polysaccharide of *Russula smokyans* prepared by the preparation method as described in the third aspect, the application comprising:

[0042] (1) Application in the preparation of products that can enhance immunity;

[0043] (2) Application in the preparation of products with antitumor activity.

[0044] Furthermore, the product is food, health supplement, or medicine.

[0045] In one embodiment of the present invention, the product capable of enhancing immunity is a pharmaceutical product.

[0046] In one embodiment of the present invention, the product that can enhance immunity is a food or health product.

[0047] In one embodiment of the present invention, the product having antitumor activity is a pharmaceutical product.

[0048] Furthermore, the pharmaceutical product may also include pharmaceutically acceptable excipients selected from sweeteners, diluents, emulsifiers, dispersants, preservatives (specifically, benzoic acid and its salts (e.g., sodium benzoate), sorbic acid and its salts (e.g., potassium sorbate), hydroxyphenyl esters (e.g., methylparaben, ethylparaben, butylparaben), etc.), immunostimulants, lubricants (specifically, stearic acid, magnesium stearate, sodium fumarate stearate, etc.), and wetting agents (specifically, polyoxyethylene...). The following are one or more of the following: sorbitan fatty acid esters, poloxamer, polyoxyethylene castor oil derivatives, etc.; suspending agents (specifically, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethyl cellulose, methylcellulose, etc.); antibacterial agents; stabilizers (specifically, citric acid, fumaric acid, succinic acid, etc.); fillers (specifically, starch, sucrose, lactose, microcrystalline cellulose, etc.); colorants; flavor enhancers; surfactants; disintegrants; suspending agents; isotonic agents; and solvents.

[0049] Furthermore, the medicines mentioned are in dosage forms such as tablets, capsules, pills, granules, powders, suppositories, injections, solutions, suspensions, ointments, and patches.

[0050] Furthermore, the routes of administration of the drug include, but are not limited to, oral, rectal, transmucosal, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intramuscular, subcutaneous, and intravenous administration.

[0051] Furthermore, in the application, the polysaccharide can be used alone or in combination with other active ingredients.

[0052] Furthermore, the final concentration of the polysaccharide in the product is 5-20 μg / mL (e.g., 5 μg / mL, 10 μg / mL, 20 μg / mL), preferably 10-20 μg / mL.

[0053] Furthermore, when it is necessary to enhance the proliferation effect of RAW264.7 cells, the final concentration of Russula smokyis polysaccharide is preferably 5-20 μg / mL (e.g., 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 10 μg / mL.

[0054] Furthermore, when it is necessary to enhance the inhibitory effect on MFC cells, the final concentration of Russula fulva polysaccharide is 5-20 μg / mL (e.g., 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 20 μg / mL.

[0055] The beneficial effects of this invention are:

[0056] This invention discloses the polysaccharide of *Russula flavomarginata* obtained from the fruiting body of *Russula flavomarginata*, and analyzes and identifies its molecular weight, monosaccharide composition, and chemical structure, determining its weight-average molecular weight and structural composition. The *Russula flavomarginata* polysaccharide is a heteropolysaccharide composed of glucose and galactose, containing 1,6-linked galactose residues, 1,4-linked D-glucose residues, 1,4-linked D-galactose residues, 1,4,6-linked glucose residues, and 1-linked galactose residues. The molar ratio of 1-Galp, 1,6-Galp, 1,4,6-Glcp, 1,4-D-Galp, and 1,4-D-Glcp residues is approximately 2:1:2:1:4. The *Russula flavomarginata* polysaccharide of this invention exhibits significant immunomodulatory and antitumor activities, and can be subsequently applied in the pharmaceutical field. Attached Figure Description

[0057] Figure 1 The image shows the GPC spectrum of RA-P.

[0058] Figure 2 The image shown is the FT-IR spectrum of RA-P.

[0059] Figure 3 The image shows the HLPC spectrum of RA-P.

[0060] Figure 4 The image shows RA-P. 1 H-NMR spectrum;

[0061] Figure 5 The image shows RA-P. 13 C-NMR spectrum;

[0062] Figure 6 The image shows RA-P. 1H- 1 H-COSY spectrum;

[0063] Figure 7 The image shows the HMQC spectrum of RA-P;

[0064] Figure 8 The image shows the HMBC spectrum of RA-P;

[0065] Figure 9 The results show the effects of each group on the proliferation of RAW264.7 cells (where A is the cell image corresponding to the CK group; B is the cell image corresponding to the RA-P group with a concentration of 5 μg / mL; C is the cell image corresponding to the RA-P group with a concentration of 10 μg / mL; D is the cell image corresponding to the RA-P group with a concentration of 20 μg / mL; E is the cell image corresponding to the LPS group; and F is the absorbance and inhibition rate of RAW264.7 cells under each group of reagents).

[0066] Figure 10 The experimental results of the effects of each group on the proliferation of MFC cells are shown (where A is the cell image corresponding to the CK group; B is the cell image corresponding to the RA-P group with a concentration of 5 μg / mL; C is the cell image corresponding to the RA-P group with a concentration of 10 μg / mL; D is the cell image corresponding to the RA-P group with a concentration of 20 μg / mL; E is the cell image corresponding to the MAN group; and F is the absorbance and inhibition rate of MFC cells under each group of reagents). Detailed Implementation

[0067] 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.

[0068] In this invention, the term "Russula adusta" refers to the phylum Basidiomycota, class Agaricales, order Agaricales, family Russulaceae, genus Russula, which includes the fruiting body and mycelium.

[0069] The term "RA-P" is an abbreviation for Russula adusta polysaccharide.

[0070] The term "LPS" is an abbreviation for lipopolysaccharides, a major component of the cell wall of Gram-negative bacteria. The LPS solution used in this experiment was purchased from Bioharp Pharmaceuticals in China.

[0071] The term "MAN" refers to mannan peptide, an abbreviation for mannatide. The MAN solution used in this experiment was purchased from Sichuan Aobang Pharmaceutical Co., Ltd.

[0072] The term "CK" stands for blank control group, an abbreviation for control check.

[0073] 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.

[0074] Example 1: Isolation, extraction and structural identification of polysaccharide RA-P from *Russula smokyosa*

[0075] 1. Isolation and extraction of polysaccharide RA-P from *Russula smoky*

[0076] 1.1 Extraction of crude polysaccharides from *Russula fumigatus* using water extraction and alcohol precipitation method

[0077] Weigh 200g of dried *Russula smokyans* fruiting bodies and crush them. Add the crushed 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 protein from the extract, thus obtaining crude polysaccharide from *Russula smokyans*.

[0078] 1.2. DEAE-cellulose column chromatography for the separation and purification of crude polysaccharides from *Russula fumigatus*.

[0079] 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.

[0080] 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, followed by elution with different concentrations of NaCl (0 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L). The polysaccharide in the eluent was determined using the sulfuric acid-phenol method. The eluent from the distilled water section was concentrated to 5 mL, dialyzed through a dialysis bag (Mw ≥ 7 kDa) for 48 hours, and then freeze-dried to obtain the polysaccharide from *Russula flavophila*, named RA-P.

[0081] 2. Structural identification of polysaccharide RA-P from *Russula smoky*

[0082] The structure of RA-P polysaccharide was determined using acid hydrolysis, methylation analysis, gel permeation chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance.

[0083] 2.1 Determination of molecular weight

[0084] Dissolve 10 mg of Rhododendron smokyans polysaccharide RA-P sample in 1 mL ddH2O, sonicate for 5 min, and perform GPC analysis.

[0085] 2.2 Infrared Spectroscopic Analysis of Polysaccharide RA-P from *Russula smokyosa*

[0086] 2 mg RA-P and 200 mg KBr were mixed and compressed into tablets, and the tablets were scanned at 4000-400 cm⁻¹ using an infrared spectrophotometer. -1 scope.

[0087] 2.3 Monosaccharide composition analysis of polysaccharide RA-P from *Russula smoky*

[0088] Seven standards and RA-P samples hydrolyzed with trifluoroacetic acid (TFA) were dissolved in the mobile phase (80% acetonitrile) and then analyzed by HLPC.

[0089] 2.4 Nuclear Magnetic Resonance Analysis of Polysaccharide RA-P from *Russula fumigatus*

[0090] Dissolve 50 mg of RA-P sample in 0.6 mL of heavy water (D2O), place it in an NMR tube, and detect it on an NMR spectrometer.

[0091] 2.5. GC-MS analysis of RA-P polysaccharide derived from *Russula fumigatus* after methylation and silanization.

[0092] Weigh 20 mg of RA-P sample, seal the beaker, add 2 mL of dimethyl sulfoxide (DMSO) to the sealed beaker, and gently shake the beaker to fully dissolve RA-P. Then add 200 mg of NaOH until the NaOH just stops dissolving, and place the beaker in a shaker at room temperature and shake for 1 h. After shaking, add 1.5 mL of iodomethane, and react in the dark for 1 h. After the reaction, add water to terminate the reaction. Extract the product with chloroform, and dry to obtain methylated polysaccharide. The methylated polysaccharide is completely acid-hydrolyzed by TFA, and washed three times with water to obtain the fully acid-hydrolyzed methylated product.

[0093] 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.

[0094] 3. Results

[0095] 3.1 Results of basic properties of polysaccharide RA-P from *Russula smokyosa*

[0096] The GPC spectrum of RA-P is as follows Figure 1 As shown, the weight-average molecular weight (Mw) of RA-P is 20576 Da, the number-average molecular weight (Mn) is 13185 Da, the polydispersity index is 1.561, and the peak molecular weight (MP) is 19921 Da.

[0097] 3.2 FT-IR Spectroscopic Analysis of Polysaccharide RA-P from *Russula smokyosa*

[0098] The primary structure of RA-P was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 2 As shown, the wavenumber is at 3395 cm⁻¹ -1 2919cm -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 RA-P is a polysaccharide. The wavenumber is 3395 cm⁻¹. -1 The broad absorption peak of OH stretching vibration peak at 2919 cm⁻¹ -1 The absorption peak within the range is the stretching vibration peak of -CH2, at 1677 cm⁻¹. -1 The peak is the C=O stretching vibration peak, 1350 cm⁻¹. -1 The in-plane bending vibration peak of -CHO is 1075 cm⁻¹. -1 The absorption peak corresponds to the CO stretching vibration peak of carbohydrates. It is located in the 1200-1000 cm⁻¹ range. -1 The absorption peaks within the range are due to the absorption of pyranose ring lactones and hydroxyl groups, indicating that RA-P has a pyran ring. At 683 cm⁻¹ -1 The absorption peak is the in-plane bending vibration peak of RA-P =CH. Additionally, at 1730 cm⁻¹... -1 The absence of absorption peaks nearby indicates that RA-P does not contain uronic acid.

[0099] 3.3 Monosaccharide composition analysis of polysaccharide RA-P from *Russula smoky*

[0100] After complete hydrolysis of RA-P, its monosaccharide composition was analyzed by HPLC, and the results are as follows: Figure 3As shown, peak 1 is glucose (Glc), with a retention time of 8.995 min; peak 2 is galactose (Gal), with a retention time of 9.675 min. The peak area ratio of glucose to galactose is approximately 3:2. Table 1 shows the elution retention times of the seven monosaccharide standards and the RA-P total hydrolysis products, as well as the proportion of the RA-P total hydrolysis products.

[0101] Table 1. Monosaccharide composition analysis of RA-P

[0102]

[0103]

[0104] 3.4 NMR spectral analysis of polysaccharide RA-P from *Russula smoky*

[0105] RA-P 1 H-NMR results are as follows Figure 4 As shown in the figure. The results show that RA-P has three anomeric hydrogen signals at δ5.01ppm, δ4.94ppm, and δ4.87ppm. The signals between δ3.0-4.3ppm are attributed to hydrogen signals from C2-C6 of the sugar residues.

[0106] RA-P 13 C-NMR results are as follows Figure 5 As shown, RA-P exhibits three anodic carbon signals at δ102.19 ppm, δ101.47 ppm, and δ97.88 ppm. The signals between δ60 and 82 ppm are attributed to C2-C6 carbon signals in sugar residues.

[0107] RA-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 signal in part A is δ5.01 / 3.75, the H1 / H2 signal in part B is δ5.01 / 3.80, the H1 / H2 signal in part C is δ4.94 / 3.71, the H1 / H2 signal in part D is δ4.94 / 3.95, and the H1 / H2 signal in part E is δ4.87 / 3.75.

[0108] All the chemical shifts of hydrogen are summarized in Table 2.

[0109] The HMQC spectrum of RA-P is as follows Figure 7 As shown, it can identify short-range related factors. 1 H and 13The coupling relationship between C and D. The signal of H1 / C1 in part A is δ5.01 / 97.88, the signal of H1 / C1 in part B is δ5.01 / 101.47, the signal of H1 / C1 in part C is δ4.94 / 97.88, the signal of H1 / C1 in part D is δ4.94 / 102.19, and the signal of H1 / C1 in part E is δ4.87 / 97.88. The integral areas of parts A, B, C, D, and E are 1.12, 0.55, 1.04, 0.50, and 1.99, respectively, with a ratio of approximately 2:1:2:1:4.

[0110] The HMBC spectrum of RA-P is as follows Figure 8 As shown, it can identify remote related... 1 H and 13 The coupling relationships between residues C and D are as follows: H2 / C4 signal of residue A is δ3.75 / 68.27, H5 / C3 signal of residue B is δ3.73 / 68.69, H1 / C3 signal of residue C is δ4.94 / 69.50, H2 / C4 signal of residue D is δ3.95 / 68.25, and H1 / C3 signal of residue E is δ4.87 / 68.62.

[0111] All the chemical shifts of carbon are summarized in Table 3.

[0112] Table 2RA-P 1 Chemical shift of H

[0113]

[0114] Table 3RA-P 13 Chemical shift of C

[0115]

[0116] 3.5 Gas Chromatography and Mass Spectrometry Analysis of Polysaccharide RA-P from *Russula smokyosa*

[0117] The methylation results are shown in Table 4, indicating that the main chain of RA-P consists of 1,6-linked galactose residues, 1,4-linked D-glucose residues, and 1,4-linked D-galactose residues, while the side chains consist of 1,4,6-linked glucose residues and 1-linked galactose residues.

[0118] Table 4 Analysis of RA-P methylation results

[0119]

[0120] Example 2: Study on the immunomodulatory and antitumor activities of polysaccharide RA-P from Russula fumigatus.

[0121] The immunomodulatory and antitumor activities of polysaccharide RA-P from *Russula flavum* were determined in vitro using the CCK-8 assay.

[0122] 1. Reagents

[0123] The CCK-8 kit, RPIM1640, FBS, DMSO, and bispecific antibodies are all commercially available products.

[0124] 2. Instruments

[0125] ELISA reader; cell culture incubator.

[0126] 3. Methods

[0127] Effects of RA-P on the proliferation of immune cells and tumor cells (RAW264.7 cells and MFC cells)

[0128] The effect of Rhododendron simsii polysaccharide (RA-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 per well, and the plates were incubated in a CO2 incubator for 24 h. After 24 h, the experimental groups (RA-P group) were treated with different concentrations of RA-P solution (final concentrations of 5, 10, and 20 μg / mL), the positive control groups (LPS group / MAN group) were treated with 100 μL of LPS solution (as a positive control for RAW264.7 cell proliferation activity) / MAN solution (as a positive control for MFC cell proliferation activity) (the final concentrations of LPS and MAN were both 10 μg / mL), and the blank group (CK group) was treated with 100 μL of cell culture medium. After 24 h of incubation in a CO2 incubator, images of each group of cells were taken under an inverted microscope. Then, 10 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 at 450 nm using a microplate reader.

[0129] 4. Results

[0130] 4.1 Effect of RA-P on the proliferation of RAW264.7 cells

[0131] The results are as follows Figure 9As shown, compared with the CK group, the LPS group significantly promoted the proliferation of RAW264.7 cells (P<0.01). When the final concentration of RA-P was 5, 10 and 20 μg / mL, it significantly promoted the proliferation of RAW264.7 cells (P<0.01). When the final concentration of RA-P was 10 μg / mL, the proliferation effect of RA-P on RAW264.7 cells was the most obvious, with a maximum proliferation rate of 61.04%.

[0132] The polysaccharide RA-P from *Russula smokyris* of this invention exhibits a significant proliferation effect on RAW264.7 cells, reaching approximately 61.04%. In contrast, other plant polysaccharides in the prior art do not show significant proliferation effects on RAW264.7 cells. For example, the proliferation rate of peanut leaf polysaccharide on RAW264.7 cells is only 32.25% (Lu Ruijia, Xia Youlin, You Yu, et al. Structural analysis and in vitro antitumor and immunomodulatory activity of Tianfu peanut leaf polysaccharide AHL-P [J]. Food Science, 2023, 44(23):45-54.); the proliferation rate of *Termitomyces albuminosus* polysaccharide on RAW264.7 cells is only 34.64% (Comparative Studies on the Structure and Biological Activities of Two New Polysaccharides from *Tricholomasinoportentosum* (TS-P) and *Termitomyces albuminosus* (TA-P) Polymers (IF 5) PubDate:2023-05-08,DOI:10.3390 / polym15092227); The proliferation rate of new polysaccharides from Armillariella tabescens (AT-P) and Tricholoma portentosum (TP-P) was only 49.65% in RAW264.7 cells (The structure characterization and biological activities of new polysaccharides from Armillariella tabescens (AT-P) and Tricholoma portentosum (TP-P)); The proliferation rate of Agaricus blazei from Agaricus blazei on RAW264.7 cells was only 29.59% (Journal of Food Measurement and Characterization PubDate:2024-04-10,DOI:10.1007 / s11694-024-02519-5Liqian Zhou,Xiang Ding,Yiling Hou).

[0133] 4.2 Effect of RA-P on the proliferation of MFC cells

[0134] The results are as follows Figure 10 As shown, compared with the CK group, the MAN group significantly inhibited MFC cell proliferation (P<0.01), with an inhibition rate of 66.53%. When the final concentration of RA-P was 5, 10, and 20 μg / mL, it significantly inhibited MFC cell proliferation (P<0.01). When the final concentration of RA-P was 20 μg / mL, the inhibitory effect of RA-P on MFC cells was the most obvious, with a maximum inhibition rate of 64.35%.

[0135] 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.

[0136] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0137] 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 Russula nigricans polysaccharide, characterized in that, The polysaccharide is a heteropolysaccharide composed of glucose and galactose, wherein the molar ratio of the residues of the glucose and the galactose is 3:2; comprising 1,6-linked galactose residues, 1,4-linked D glucose residues, 1,4-linked D galactose residues, 1,4,6-linked glucose residues and 1 -linked galactose residues; The polysaccharide comprises the following structure: Wherein, n is an integer of 1-20; The weight average molecular weight of the polysaccharide is 10000 Da-30000 Da.

2. The polysaccharide of claim 1, wherein, The weight average molecular weight of the polysaccharide is 15000 Da-25000 Da.

3. A composition characterized in that, The composition comprises the polysaccharide of claim 1 or 2.

4. The process for the preparation of polysaccharides as claimed in claim 1, wherein, The method comprises the step of extracting from the Russula ochroleucum fruiting body: (1) taking the Russula ochroleucum fruiting body powder, extracting with hot water, and obtaining the water extract, and then sequentially concentrating, alcohol precipitating, and drying to obtain the crude polysaccharide; (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate; (3) subjecting the eluate obtained in step (2) to dialysis and concentration in a dialysis bag; in step (1), the extraction temperature is 80-100 ℃; The mass ratio of the Russula ochroleucum fruiting body powder to water is 1:1-10; The volume ratio of the alcohol to the water extract concentrate is 1-10:1; The alcohol is ethanol; In step (2), the ion exchange column is a cellulose column, and the filler thereof is DEAE cellulose; The eluent used for elution is a NaCl solution; The elution is gradient elution.

5. Use of a polysaccharide according to claim 1 or 2, characterised in that, The application comprises: (1) application in the preparation of a product capable of enhancing immunity; or (2) application in the preparation of a product having anti-tumor activity.

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