Tremella aurantialba polysaccharide with immunoregulatory activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses tremella aurantialba polysaccharide with immunomodulatory activity and a preparation method and application thereof. The preparation method comprises the following steps: carrying out water extraction and alcohol precipitation on tremella aurantialba, and carrying out column chromatography separation to obtain tremella aurantialba polysaccharide TAP-2a; meanwhile, the structure of the TAP-2a is determined by adopting methods such as high performance gel permeation chromatography, precolumn derivatization, methylation combined with gas chromatography-mass spectrometry, nuclear magnetic resonance and the like, and the obtained TAP-2a is brand new tremella aurantialba polysaccharide. Meanwhile, the immunoregulation activity of the TAP-2a is further researched, the TAP-2a can promote proliferation of macrophages, up-regulate expression of iNOS, TNF-alpha, IL-6 and IL-1beta mRNA, promote secretion of NO, TNF-alpha, IL-6 and IL-1beta and enhance the phagocytic ability of the macrophages, and the result shows that the polysaccharide has the immunoregulation activity, can be used as a safe natural immunomodulator and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharides and biomedicine, specifically, it relates to a type of golden ear fungus with immunomodulatory activity. Tremella aurantialba Polysaccharides, their preparation methods, and applications. Background Technology

[0002] my country boasts abundant higher fungi resources, many of which have a long history of medicinal, edible, or dual-use applications. As food, they are delicious, rich in protein, carbohydrates, minerals, and low in fat, making them popular. As medicine, they are an important component of natural medicinal resources. Medical classics such as the *Shennong Bencao Jing* and *Compendium of Materia Medica* record and describe the medicinal effects of various higher fungi. Modern research confirms that polysaccharides from higher fungi are key components for their biological activity, exhibiting various biological activities including lowering blood lipids and blood sugar, immunomodulation, antioxidation, antiviral activity, and antitumor activity. They are particularly significant in regulating immunity, while also possessing high safety, making them a focus of attention and research in the biopharmaceutical and health product industries.

[0003] Golden Ear ( Tremella aurantialba *Auricularia auricula-judae*, belonging to the Basidiomycetes, is a higher fungus used both as food and medicine. The *Compendium of Materia Medica* records that "the golden variety (Auricularia auricula-judae) treats phlegm accumulation, abdominal pain, and sores." *Chinese Materia Medica of Medicinal Fungi* describes *Auricularia auricula-judae* as having expectorant, antitussive, and antiasthmatic effects, and can significantly improve the body's immune function. As a top-tier gelatinous fungus, *Auricularia auricula-judae* has high nutritional value, and studies have shown that it possesses immune-enhancing biological activities. Polysaccharides, especially glucuronic acid-xylomannan (GXM), which constitutes a significant proportion, may be the material basis for its activity. Other polysaccharides in *Auricularia auricula-judae* and their activities require further research. Therefore, it is necessary to improve the understanding of the composition and structural characteristics of other polysaccharides in *Auricularia auricula-judae* and explore their potential immunomodulatory activities to promote a comprehensive understanding and utilization of *Auricularia auricula-judae* polysaccharides. Summary of the Invention

[0004] The purpose of this invention is to provide an immunomodulatory polysaccharide (TAP-2a) from *Auricularia auricula-judae* and its preparation method, and to evaluate the immunomodulatory activity of the polysaccharide, so as to provide relevant data for the research and development of *Auricularia auricula-judae* polysaccharide TAP-2a.

[0005] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution: This invention first provides a polysaccharide called TAP-2a, which is composed of glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), xylose (Xyl), and glucuronic acid (GlcA) in a molar ratio of (9.90±1.00):(6.29±1.00):(5.57±1.00):(2.47±0.50):(1.31±0.50):(1.00±0.50). The weight-average molecular weight of TAP-2a is 14 k to 20 kDa, and the polydispersity index is between 1.0 and 1.8.

[0006] The polysaccharide TAP-2a structure of this invention is mainly composed of glucose, galactose, and mannose in the pyranose ring, and the main glycosidic bonds of Glc include: Glc p -(1→ (17.53±2.0)%,→6)-Glc p -(1→ (13.83±2.0)%,→3)-Glc p -(1→ (3.78±1.0)%、→3,6)-Glc p -(1→ (2.34±0.5)%; Gal has →6)-Gal p -(1→(18.81±2.0)%,→4)-Gal p -(1→ (5.70±1.0)%、→3,6)-Gal p -(1→ (4.14±1.0)%,→3)-Gal p -(1→ (3.71±1.0)%、→2,6)-Gal p - (1→ (3.67±1.0)%, Gal p -(1→(1.96±0.5)%; the glycosidic bonds of Man include →3)-Man p -(1→ (10.11±2.0)%,→2)-Man p -(1→ (3.59±1.0)%、→2,3)-Man p -(1→ (2.77±0.5)%, Xyl has glycosidic bonds Xyl p -(1→(2.82±0.5)% and →4)-Xyl p -(1→(2.65±0.5)%); Fuc has only one type of glycosidic bond. p -(1→(2.60±0.5)%).

[0007] This invention also provides a method for preparing the above-mentioned *Auricularia auricula-judae* polysaccharide, comprising the following steps: (1) Preparation of crude polysaccharide: Take the powder of Auricularia auricula-judae and extract it in hot water at 90-100℃ for 2-4 hours at a ratio of 1:(30-50) (g / mL). Repeat the extraction 2-4 times, combine the supernatant, add 3-4 times or more of 95% ethanol or anhydrous ethanol to precipitate, and freeze dry to obtain crude polysaccharide of Auricularia auricula-judae. (2) Isolation and purification of polysaccharides: The crude polysaccharide was loaded onto an anion exchange column and eluted sequentially with pure water and 0.1M, 0.3M and 0.5M sodium chloride solutions. The elution peaks of 0M and 0.1M sodium chloride solutions were collected, dialyzed and lyophilized. The lyophilized components were dissolved to a suitable concentration and loaded onto a gel chromatography column. The elution was carried out with 0.2M sodium chloride solution, and the elution peaks were collected. After desalting, the polysaccharide TAP-2a was obtained.

[0008] Further explanation is that the TAP-2a polysaccharide from *Auricularia auricula-judae* is non-cytotoxic and can promote the proliferation of RAW264.7 macrophages, promote the release of nitric oxide from macrophages, and promote the secretion of cytokines, including tumor necrosis factor-α, interleukin-6, and interleukin-1β.

[0009] Further, it was found that treatment with *Auricularia auricula-judae* polysaccharide significantly upregulated the expression of iNOS, TNF-α, IL-6, and IL-1β mRNA in macrophages; treatment with *Auricularia auricula-judae* polysaccharide also promoted the phagocytosis of FITC-dextran by macrophages.

[0010] The present invention also provides an application of the above-mentioned auricularia polysaccharide as a natural immune enhancer in pharmaceuticals, health products and functional foods.

[0011] The beneficial effects of this invention are as follows: 1. This invention involves hot water extraction, ethanol precipitation, and purification using a DEAE Sepharose Fast Flow anion exchange chromatography column to collect the eluent. The eluent eluted with 0.1 M NaCl solution is further purified using a Sepharose CL-6B agarose gel, and the main eluent is collected. Finally, after desalting and concentration, the polysaccharide of *Auricularia auricula-judae* (TAP-2a) is obtained. The preparation process is simple, the conditions are mild, the operation is convenient, and it is easy to scale up for production.

[0012] 2. The polysaccharide (TAP-2a) obtained in this invention is derived from the abundant auricularia auricula. It has a low weight-average molecular weight (14 k to 20 kDa) and a narrow polydispersity index (1.0 to 1.8), indicating that the polysaccharide components are uniform. At the same time, its monosaccharide composition and glycosidic bond structure are clear and well-defined, mainly composed of glucose, galactose and mannose in pyranose rings. The structure is regular, which is conducive to quality control and standardization.

[0013] 3. The applicant's experiments demonstrate that TAP-2a obtained in this invention, without cytotoxicity, significantly promotes the proliferation of RAW264.7 macrophages, enhances phagocytic function (such as phagocytosis of FITC-dextran), and induces the secretion of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Its mechanism of action involves upregulating the mRNA expression of iNOS, TNF-α, IL-6, and IL-1β, indicating that TAP-2a achieves its immune-enhancing effect by regulating immune-related genes. Within the experimental concentration range, TAP-2a did not exhibit cytotoxicity and showed good safety. TAP-2a can be used as a natural immune enhancer for the development of drugs, health products, or functional foods for treating immunodeficiency diseases. Its low molecular weight and structural stability are beneficial for formulation development and have broad application prospects. Attached Figure Description

[0014] Figure 1 This is a high-performance liquid chromatography gel permeation chromatogram of the molecular weight distribution of the auricularia polysaccharide TAP-2a of the present invention.

[0015] Figure 2 This is a high-performance liquid chromatogram of the monosaccharide composition analysis of the *Auricularia auricula-judae* polysaccharide TAP-2a of the present invention.

[0016] Figure 3 The infrared spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention is shown.

[0017] Figure 4 This is the total ion chromatogram of the TAP-2a methylated sugar alcohol acetyl derivative of the *Auricularia auricula-judae* polysaccharide of the present invention.

[0018] Figure 5 This is the hydrogen spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention.

[0019] Figure 6 This is the carbon spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention.

[0020] Figure 7 This is the COSY spectrum of the auricularia polysaccharide TAP-2a of the present invention.

[0021] Figure 8 This is the TOCSY spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention.

[0022] Figure 9 This is the HSQC spectrum of the auricularia polysaccharide TAP-2a of the present invention.

[0023] Figure 10 This is the HSQC-TOCSY spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention.

[0024] Figure 11This is the ROESY spectrum of the polysaccharide TAP-2a from *Auricularia auricula-judae* of this invention.

[0025] Figure 12 This is the HMBC spectrum of the auricularia polysaccharide TAP-2a of the present invention.

[0026] Figure 13 This invention relates to the effect of the auricularia polysaccharide TAP-2a on the proliferation ability of RAW264.7 cells.

[0027] Figure 14 This invention relates to the effect of the auricularia polysaccharide TAP-2a on the phagocytic ability of RAW264.7 cells.

[0028] Figure 15 The effects of the polysaccharide TAP-2a from *Auricularia auricula-judae* on NO (A) release and TNF-α (B), IL-6 (C), and IL-1β (D) secretion in RAW264.7 cells were investigated.

[0029] Figure 16 This invention investigates the effects of the auricularia polysaccharide TAP-2a on the mRNA expression levels of iNOS (A), TNF-α (B), IL-6 (C), and IL-1β (D) in RAW264.7 cells. Detailed Implementation

[0030] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0031] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example

[0032] This embodiment describes the preparation and structural analysis of the polysaccharide TAP-2a from *Auricularia auricula-judae*. The specific method is as follows: 1. Extraction and purification of polysaccharides from Auricularia auricula-judae Take 20g of dried *Auricularia auricula-judae* fruiting body powder and add pure water at a material-to-liquid ratio of 1:40 (w / v) for extraction. The extraction temperature is 95℃, the extraction time is 3 hours, and the extraction is repeated 3 times. Centrifuge the obtained extract at 4700 rpm for 15 minutes, combine the supernatants, and add anhydrous ethanol to precipitate the polysaccharide. Centrifuge the ethanol precipitation solution at 4700 rpm for 15 minutes, redissolve the precipitate in pure water, concentrate, and freeze-dry to obtain crude *Auricularia auricula-judae* polysaccharide.

[0033] 2. Isolation and purification of TAP-2a polysaccharide from Auricularia auricula-juda The crude polysaccharide from *Auricularia auricula-judae* was dissolved in an appropriate amount of pure water and then loaded onto a DEAE Sepharose Fast Flow anion exchange column. Gradient elution was performed sequentially with pure water, 0.1M sodium chloride solution, 0.3M sodium chloride solution, and 0.5M sodium chloride solution. The solutions were collected in separate tubes and detected using the phenol-sulfuric acid method. The elution peak obtained from the 0.1M sodium chloride elution was collected, dialyzed, and freeze-dried. The solution was then loaded onto a Sepharose CL-6B column with 0.2M sodium chloride solution as the eluent. The solutions were collected in separate tubes and detected using the phenol-sulfuric acid method. The collected solutions corresponding to the elution peaks were collected, dialyzed, and freeze-dried to obtain the *Auricularia auricula-judae* polysaccharide TAP-2a of this invention.

[0034] 3. Determination of the purity and molecular weight of TAP-2a polysaccharide from Auricularia auricula-judae by high performance gel permeation chromatography. The purity and molecular weight of the polysaccharide were determined by high-performance gel permeation chromatography (HPGPC). TAP-2a polysaccharide solution was prepared and analyzed using a Shimadzu LC-2030C 3D HPLC system. A Shodex OHpak SB-804 HQ column (8 × 300 mm) was used, and the column temperature was maintained at 35℃. 0.1 M NaCl solution was used as the mobile phase at a flow rate of 0.5 mL / min. A differential refractive index detector was used. Regression equations were established using the logarithmic molecular weight and retention time of different molecular weight standards. The relative molecular weight of the polysaccharide was calculated by substituting the peak time of TAP-2a into the regression equation.

[0035] 4. Determination of the monosaccharide composition of TAP-2a polysaccharide from Auricularia auricula-judae using pre-column derivatization method The TAP-2a solution prepared in this invention was hydrolyzed with trifluoroacetic acid. The hydrolysate was then concentrated under reduced pressure multiple times with methanol to remove trifluoroacetic acid. The hydrolyzed monosaccharide was added to a 1-phenyl-3-methyl-5-pyrazolone-methanol solution under alkaline conditions and derivatized at 70°C for 100 min. After derivatization, the solution was neutralized with hydrochloric acid, extracted three times with chloroform, and the aqueous layer was filtered through a membrane and analyzed by high-performance liquid chromatography (HPLC) using an EclipsePlus C18 column (4.6 × 250 mm, 5 µm) and a photodiode array detector (PDA). The mobile phase consisted of phosphate buffer (0.1 M, pH 6.7) and acetonitrile in a ratio of 83:17 (v / v). The mobile phase flow rate was 1.0 mL / min, the injection volume was 20 µL, the detection wavelength was 245 nm, and the column temperature was maintained at 30°C. Take 5 mg each of mannose, ribose, rhamnose, galacturonic acid, glucuronic acid, glucose, galactose, arabinose, xylose, and fucose, and prepare mixed standard solutions of different concentrations. Perform derivatization and injection analysis according to the above method.

[0036] 5. Infrared spectral analysis of TAP-2a polysaccharide from Auricularia auricula-judae Take 1 mg of thoroughly dried polysaccharide sample of this invention and mix it with approximately 100 mg of thoroughly dried potassium bromide. Grind the mixture thoroughly into powder in quartz agate and press it into thin tablets using a tablet press. Perform infrared spectral scanning on the tablets using a Nicolet iS50 infrared spectrometer, with the scanning range being 400-4000 cm⁻¹. -1 .

[0037] 6. Methylation analysis of TAP-2a polysaccharide from Auricularia auricula-judae Take 5 mg of thoroughly dried polysaccharide sample of this invention into a reaction flask, add 2 mL of anhydrous DMSO, and stir magnetically until the polysaccharide sample is completely dissolved. Quickly add approximately 50 mg of dry NaOH and stir until dissolved. Add 1.5 mL of CH3I and carry out the methylation reaction in a light-protected environment for 2 hours. Finally, add 2 mL of pure water to the mixture to terminate the methylation reaction. Extract the methylated polysaccharide three times with chloroform and dry. Repeat the above operation to 3500 cm⁻¹ infrared spectroscopy. –1 The disappearance of the nearby absorption peaks indicates that the polysaccharide hydroxyl groups have been completely methylated.

[0038] The methylated polysaccharide was hydrolyzed using 2M TFA solution at 120°C for 2 hours. The hydrolysate was evaporated to dryness using a rotary evaporator, and then 3 mL of methanol was added and evaporated to dryness under reduced pressure. This process was repeated three times. The residue was dissolved in 2 mL of pure water and reduced with 30 mg of NaBH4 for 3 hours. After the reaction, excess NaBH4 could be destroyed by adding glacial acetic acid. The reaction solution was repeatedly concentrated to dryness under reduced pressure by adding methanol. Then, 1.5 mL of acetic anhydride and 1.5 mL of pyridine were added, and an acetylation reaction was carried out at 100°C for 1 hour. The acetylation product was extracted with dichloromethane, and the organic phase was washed with water. This process was repeated three times to obtain the methylated sugar alcohol acetylated derivative. This derivative was analyzed using an Agilent 7890B-5977B gas chromatograph-mass spectrometer.

[0039] GC-MS conditions: capillary column: HP-5MS (30m×0.25mm×0.25μm); temperature program: 50℃ for 1 min, increase to 130℃ at 50℃ / min, increase to 230℃ at 3℃ / min, hold for 2 min; carrier gas: high-purity helium, flow rate: 1.0 mL / min.

[0040] 7. Nuclear magnetic resonance analysis of TAP-2a polysaccharide from Auricularia auricula-judae The chemical structure of TAP-2a was further characterized using nuclear magnetic resonance spectroscopy. 30 mg of TAP-2a was dissolved in 0.5 mL of heavy water and freeze-dried. The freeze-dried powder was then redissolved in heavy water and freeze-dried again; this process was repeated three times. One-dimensional and two-dimensional nuclear magnetic resonance spectra of TAP-2a were recorded using a Bruker 500 spectrometer at 500 MHz.

[0041] 8. Experimental Results (1) Purity and molecular weight The high-performance liquid chromatography gel permeation chromatogram of TAP-2a polysaccharide from Auricularia auricula-judae is shown below. Figure 1 As shown, the polysaccharide exhibits a single symmetrical peak, indicating that the polysaccharide of this invention is a homogeneous polysaccharide. A regression equation was established using the logarithmic molecular weight and retention time of different molecular weight standards. Substituting the elution time of the *Auricularia auricula-judae* polysaccharide TAP-2a into the regression equation, the relative molecular weight of the polysaccharide of this invention was calculated to be 16.94 kDa.

[0042] (2) Monosaccharide composition The monosaccharide composition of *Auricularia auricula-judae* polysaccharide TAP-2a was determined by PMP-pre-column derivatization, and the results are as follows: Figure 2 As shown, compared with the peak time of the monosaccharide standard, the TAP-2a polysaccharide in the present invention is composed of glucose, galactose, mannose, xylose, fucose and glucuronic acid, with a molar ratio of 9.90: 6.29: 5.57: 2.47: 1.31: 1.00.

[0043] (3) Infrared spectrum Infrared spectroscopy analysis of the characteristic absorption peaks of TAP-2a polysaccharide from Auricularia auricula-judae, results are attached. Figure 3 As shown in the figure. The infrared spectrum of TAP-2a, a polysaccharide from *Auricularia auricula-judae*, shows the characteristic absorption peaks of the polysaccharide, located at approximately 3400 cm⁻¹. -1 and 2930 cm -1 These correspond to the stretching vibrations of OH and CH, respectively. 1650 cm⁻¹ -1 The absorption peak at 1135 cm⁻¹ is attributed to water absorption. -1 1076 cm -1 and 1041cm -1 The absorption peak at approximately 890 cm⁻¹ is attributed to the CO bending vibration of the pyranose ring. -1 An absorption peak of β-glycosidic bond was clearly observed.

[0044] (4) Methylation Gas chromatography-mass spectrometry (GC-MS) analysis of TAP-2a polysaccharide from *Auricularia auricula-judae* after methylation treatment yielded the following results: Figure 4 As shown. The total ion chromatogram of methylation analysis indicates that the polysaccharide TAP-2a from *Auricularia auricula-judae* contains multiple glycosidic bonds, with the main glycosidic bonds of Glc including: Glcp-(1→, →6)-Glc. p -(1→、→3)-Glc p -(1→、→3,6)-Glc p -(1→;Gal has a glycosidic bond Gal p -(1→;→6)-Gal p -(1→、→4)-Gal p -(1→、→3)-Galp -(1→、→3,6)-Gal p -(1→、→2,6)-Gal p -(1→, Man's glycosidic bonds include →3)-Man p -(1→、→2)-Man p -(1→、→2,3)-Man p -(1→,Xyl glycosidic bond has Xyl p (1→and→4)-Xyl p -(1→;Fuc has only one type of glycosidic bond, Fuc p -(1→。The molar ratios of these glycosidic bond peaks can be obtained by integrating the area and calculating the molecular weight, as shown in Table 1.

[0045]

[0046] (5) Nuclear magnetic resonance analysis The nuclear magnetic resonance spectroscopy analysis results of TAP-2a are as follows: Figure 5-12 As shown, where, Figure 5 The proton spectrum of TAP-2a Figure 6 The carbon spectrum of TAP-2a Figure 7 For COSY spectrum, Figure 8 For the TOCSY spectrum, Figure 9 For HSQC spectrum, Figure 10 This is the HSQC-TOCSY spectrum. Figure 11 For the ROESY spectrum, Figure 12 The results are HMBC spectra. The chemical shifts of each sugar residue in TAP-2a and the correlations between different sugar residues are shown in Table 2.

[0047]

[0048] Example 2: This example studies the immunomodulatory activity of the *Auricularia auricula-judae* polysaccharide TAP-2a prepared in this application. 1. Effect of Auricularia auricula polysaccharide TAP-2a on the proliferation ability of RAW264.7 cells RAW264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. Culture conditions were 5% CO2 and 37°C. Cells were seeded into plates at the logarithmic growth phase and cultured overnight. The medium was then aspirated, and different concentrations (15, 30, 60, 120, 240 μg / mL) of TAP-2a polysaccharide solution were added. The blank control group received the same volume of complete medium, and the positive control group received 1 μg / mL LPS solution. Incubation continued for 24 h. The supernatant was discarded, and 100 μL of 0.5 mg / mL LMT solution was added to each well. The plates were returned to the incubator and incubated for another 3–4 h. The solution in the wells was then aspirated. 150 μL of analytical grade DMSO was added to each well, and the plates were shaken to dissolve any crystals. The absorbance of each well was measured at 490 nm to calculate cell viability.

[0049] 2. Effect of Auricularia auricula polysaccharide TAP-2a on the phagocytic ability of RAW264.7 cells RAW264.7 cells in logarithmic growth phase were seeded in 48-well plates (2 × 10⁻⁶ cells per well). 5 Cells were cultured at different concentrations of TAP-2a in each well after cell adhesion (cells / mL). A blank control group and a positive control group were also included. Twenty-four hours after administration, FITC-dextran stock solution (40,000 Da) was added to each well to bring the final concentration of FITC-dextran to 1 mg / mL. Cells were then cultured at 37°C in the dark for another 1 hour. The culture medium was removed, and the cells were washed with PBS and collected. The fluorescence intensity of the collected cells was analyzed by flow cytometry to assess the effect of the samples on phagocytic capacity.

[0050] 3. Effects of Auricularia auricula-judae polysaccharide TAP-2a on NO and cytokine release in RAW264.7 cells Standard curve plotting: Dilute 1 mmol / L NaNO2 standard solution to a series of gradient concentrations (1, 2, 5, 10, 20, 40, 60, 100 μmol / L) with DMEM complete medium. Pipette 50 μL of each concentration of NaNO2 standard solution into a 96-well plate, then add equal volumes of Griess A and Griess B solutions sequentially, and incubate in the dark for 10 min. Measure the absorbance at 540 nm using a microplate reader to plot the standard curve.

[0051] After digesting RAW264.7 cells in the logarithmic growth phase, the cell density was adjusted to obtain a cell concentration of 4 × 10⁻⁶. 5Cell suspensions of 100 cells / mL were seeded into well plates and cultured overnight for adhesion. The liquid in the wells was discarded. Complete culture medium was added to the blank group, different concentrations of TAP-2a were added to the sample groups, and LPS was added to the positive control group. The cells were incubated for another 24 hours. The supernatant was collected for analysis. The amount of NO released from the cell supernatant was determined using the Griess method, and the release of TNF-α, IL-1β, and IL-6 from the cell supernatant was detected using an ELISA kit.

[0052] 4. Effects of Auricularia auricula-judae polysaccharide TAP-2a on the expression of iNOS, TNF-α, IL-6 and IL-1β mRNA in RAW264.7 cells After digesting RAW264.7 cells in the logarithmic growth phase, the cell density was adjusted to obtain a cell concentration of 4 × 10⁻⁶. 5 Cell suspensions of 100 cells / mL were seeded into 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The liquid in the wells was discarded. Complete culture medium was added to the blank control group, different concentrations of TAP-2a were added to the sample groups, and LPS was added to the positive control group. The plates were incubated for another 24 hours. The culture medium in the wells was discarded, and the cells were washed with PBS to remove residual culture medium and cell debris. Total RNA was extracted from each sample using TRIzol reagent, followed by reverse transcription to cDNA. Primers targeting the detection indicators and internal reference genes were designed, and qPCR was performed using SYBR Green fluorescent dye. The instrument automatically calculated the Ct value for each sample, using a 2-1... -△△Ct The relative expression level of the target mRNA can be calculated using this method.

[0053]

[0054] 5. Results (1) Effect of TAP-2a polysaccharide from Auricularia auricula-judae on the proliferation ability of RAW264.7 cells The effect of TAP-2a on the proliferation of RAW264.7 macrophages was detected by MTT assay, and the results are as follows: Figure 13 As shown, TAP-2a significantly promoted cell proliferation within the concentration range used (15 μg / mL-240 μg / mL), indicating that the selected sample concentration was non-cytotoxic and could be used for subsequent experiments.

[0055] (2) Effect of TAP-2a polysaccharide on the phagocytic ability of RAW264.7 cells The FITC-dextran phagocytosis assay was used to detect the effect of the sample on the phagocytic ability of RAW264.7 cells. The results are as follows: Figure 14 As shown, compared with the blank control group, treatment with low concentration (15 μg / mL) of TAP-2a did not significantly enhance the phagocytic capacity of macrophages. P>0.05), with increasing concentration, the fluorescence intensity of RAW264.7 cells gradually increased. Within the concentration range of 30 μg / mL to 240 μg / mL, the mean fluorescence intensity (MFI) of RAW264.7 cells was significantly stronger than that of the blank control group. P <0.05), and exhibits a dose-dependent effect.

[0056] (3) Effects of TAP-2a polysaccharide on NO release and cytokine secretion in RAW264.7 cells The effect of TAP-2a on NO release from RAW264.7 macrophages was detected using the Griess method. The results are as follows: Figure 15 As shown, TAP-2a significantly promoted NO release within the range of concentrations used (15 μg / mL-240 μg / mL) in a dose-dependent manner. The effect of TAP-2a on cytokine release from RAW264.7 macrophages was detected using an ELISA kit, and the results are as follows: Figure 15 As shown, the release of TNF-α increased significantly after the addition of TAP-2a, exhibiting a clear dose-dependent effect. TAP-2a also significantly promoted the secretion of IL-6 and IL-1β within a certain concentration range (30 μg / mL-240 μg / mL), also showing a concentration-dependent effect.

[0057] (4) Effects of TAP-2a polysaccharide on the expression of iNOS, IL-6, IL-1β and TNF-α mRNA in RAW264.7 cells The effects of TAP-2a on the expression of iNOS, IL-6, IL-1β, and TNF-α genes in RAW264.7 macrophages are as follows: Figure 16 As shown in the figure, TAP-2a significantly promoted the relative expression level of iNOS mRNA in a dose-dependent manner within a certain concentration range, with the best effect observed at a concentration of 120 μg / mL. Compared with the blank control group, TAP-2a significantly increased the expression level of TNF-α mRNA in RAW264.7 cells within the concentration range used (15 μg / mL-240 μg / mL) in a dose-dependent manner. At low concentrations of TAP-2a (15 μg / mL, 30 μg / mL), the expression levels of IL-6 and IL-1β mRNA were not significantly upregulated compared with the blank group, but at high concentrations (60 μg / mL-240 μg / mL), it dose-dependently increased the relative expression levels of IL-6 and IL-1β mRNA.

[0058] In summary, this invention is the first to obtain a novel polysaccharide (TAP-2a) from *Auricularia auricula-judae* with a novel structure. Cellular experiments revealed that this polysaccharide possesses significant immunomodulatory activity and holds promise for development as a novel immunomodulatory drug, health product, or functional food. This invention provides a scientific basis for the further development and utilization of *Auricularia auricula-judae* polysaccharide (TAP-2a).

[0059] The above embodiments are only used to describe the specific implementation of the present invention in detail and should not be construed as limiting the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., that can be made by those skilled in the art without inventive effort within the scope of the claims of the present invention are still within the scope of protection of this patent.

Claims

1. A polysaccharide from Auricularia auricula, characterized in that: The gold ear polysaccharide is simply referred to as TAP-2a, the weight average molecular weight ranges from 14 k to 20 kDa, the polydispersity index is between 1.0 and 1.8; the monosaccharide composition includes glucose, galactose, mannose, fucose, xylose and glucuronic acid, and the corresponding molar ratio is (9.90±1.00):(6.29±1.00):(5.57±1.00):(2.47±0.50):(1.31±0.50):(1.00±0.50).

2. The Auricularia auricula polysaccharide of claim 1, characterized in that: The main glycosidic linkages of Glc in the polysaccharide from Auricularia auricula-judae include: Glc p - (1→ (17.53 ± 2.0) %,→ 6)-Glc p - (1→ (13.83 ± 2.0) %,→ 3)-Glc p - (1→ (3.78 ± 1.0) %,→ 3, 6)-Glc p - (1→ (2.34 ± 0.5) %; the glycosidic linkages of Gal have→ 6)-Gal p - (1→ (18.81 ± 2.0) %,→ 4)-Gal p - (1→ (5.70 ± 1.0) %,→ 3, 6)-Gal p - (1→ (4.14 ± 1.0) %,→ 3)-Gal p - (1→ (3.71 ± 1.0) %,→ 2, 6)-Gal p - (1→ (3.67 ± 1.0) %, Gal p - (1→ (1.96 ± 0.5) %; the glycosidic linkages of Man include→ 3)-Man p - (1→ (10.11 ± 2.0) %,→ 2)-Man p - (1→ (3.59 ± 1.0) %,→ 2, 3)-Man p - (1→ (2.77 ± 0.5) %, the glycosidic linkages of Xyl have Xyl p - (1→ (2.82 ± 0.5) % and→ 4)-Xyl p - (1→ (2.65 ± 0.5) %; there is only one glycosidic linkage of Fuc, Fuc p - (1→ (2.60 ± 0.5) %.

3. The method for preparing Auricularia auricula polysaccharide according to any one of claims 1-2, characterized in that, The method comprises the following steps: (1) Preparation of crude polysaccharide: take the gold ear powder, extract in 90-100℃ hot water at a ratio of 1:(30-50) (g / mL) for 2-4 hours, repeat 2-4 times, combine the supernatant, add 3-4 times volume of 95% ethanol or anhydrous ethanol for precipitation, freeze-dry to obtain the crude polysaccharide of gold ear; (2) Isolation and purification of polysaccharide: load the crude polysaccharide on an anion exchange column, sequentially use pure water and 0.1M, 0.3M and 0.5M sodium chloride solution for gradient elution, collect the 0M and 0.1M sodium chloride solution elution peak, dialysis, freeze-drying; Dissolve the freeze-dried component to a suitable concentration and continue to load on a gel chromatography column, elute with 0.2M sodium chloride solution, collect the elution peak, desalt and freeze-dry to obtain the gold ear polysaccharide TAP-2a.

4. The Auricularia auricula polysaccharide according to any one of claims 1-2, characterized in that: The gold ear polysaccharide TAP-2a has no cytotoxicity, and can promote the proliferation of macrophage RAW264.7 cells, promote the release of nitric oxide by macrophages, and also promote the secretion of cytokines, including tumor necrosis factor-α, interleukin-6 and interleukin-1β.

5. The Auricularia auricula polysaccharide of claim 4, characterized in that: After treatment with the gold ear polysaccharide, the iNOS, TNF-α, IL-6 and IL-1β mRNA expression in macrophages is significantly up-regulated; after treatment with the gold ear polysaccharide, the phagocytosis of macrophages to FITC-dextran is promoted.

6. The gold ear polysaccharide as claimed in any one of claims 1-2 as a natural immune enhancer for application in pharmaceuticals, health products and functional foods.

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