Low molecular weight high purity macrocybe cubensis glycoprotein and preparation method and application thereof

Abstract

The application provides a low-molecular-weight high-purity Stropharia rugosannulata glycoprotein and a preparation method and application thereof, and belongs to the technical field of biochemistry. The Stropharia rugosannulata glycoprotein has a molecular weight of 10-20 kDa, the protein content of the Stropharia rugosannulata glycoprotein is 75%-82%, and the polysaccharide content is 10%-18%; the protein part of the Stropharia rugosannulata glycoprotein contains 17 kinds of amino acids, i.e., glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine and methionine; and the polysaccharide part of the Stropharia rugosannulata glycoprotein mainly includes fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose and mannose. The Stropharia rugosannulata glycoprotein prepared by the application has a purity of more than 95% and stable quality, has an immune enhancement effect, and can be used as an immune enhancer raw material for the development of functional food, health products and medicines.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a low molecular weight, high purity *Agaricus macrocarpa* glycoprotein, its preparation method, and its application. Background Technology

[0002] Currently, research on giant puffball mushrooms mainly focuses on strain selection, cultivation techniques, and processing methods, while research on the preparation techniques of its functional active ingredients and its bioactivity is relatively limited.

[0003] Glycoproteins are complex and diverse biological macromolecules composed of polysaccharide or oligosaccharide groups covalently linked to proteins. Their structures typically have multiple branches, and their composition is predominantly protein. The proportion of sugars varies significantly among different glycoproteins, ranging from 2% to 60% of the protein's weight. Glycoproteins are widely found in nature; natural glycoprotein components can be extracted from animals, plants, and microorganisms. They play crucial structural and functional roles in organisms, involved in various life activities such as signal transduction, immune responses, cell recognition, and adhesion. Therefore, glycoproteins have broad development and application prospects in food, medicine, and biomaterials. Due to various limitations in glycoprotein isolation and analysis methods and differences in purification techniques, there are currently no research reports on *Stropharia macrocarpa* glycoproteins, and their types, structures, and biological activities remain uncertain. Summary of the Invention

[0004] The purpose of this invention is to provide a low molecular weight, high purity *Stropharia macrocarpa* glycoprotein, its preparation method, and its application. This low molecular weight, high purity *Stropharia macrocarpa* glycoprotein has an immune-enhancing effect and can be applied to drug development.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a low molecular weight, high-purity *Stropharia macrocarpa* glycoprotein with a molecular weight of 10-20 kDa. The glycoprotein contains 75%-82% protein and 10%-18% polysaccharides. The protein in the glycoprotein contains 17 amino acids, including glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline. The polysaccharides in the glycoprotein are packaged... The product includes fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose, and mannose, wherein the molar ratio of fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose, and mannose is (0.9~1.1):(1~1.3):(0.6~0.9):(0.1~0.4):(0.6~0.9):(17~18):(1.8~2.2):(1.8~2.2).

[0006] Preferably, the secondary structure of the protein moiety of the *Pleurotus ostreatus* glycoprotein is α-helix 5.6%, β-sheet 29.8%, β-turn 16.9%, and random coil 47.7%; the content ratio of glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline is (12.9~13.1):(13.8~14.0):(38.2~3). 8.4): (20.2~20.5): (29.2~29.5): (41.4~41.8): (126.4~126.8): (17.6~18.10): (15.7~16.1): (51.2~51.6): (10.2~10.7): (28.8~29.2): (19.0~19.4): (21.2~21.7): (20.1~20.6): (49.3~49.8): (88.5~89.0).

[0007] The present invention also provides a method for preparing the low molecular weight, high purity *Agaricus macrocarpa* glycoprotein, including the extraction, separation and purification of *Agaricus macrocarpa* glycoprotein; The extraction of the glycoprotein from *Stropharia macrocarpa* includes: mixing choline chloride and urea at a molar ratio of 1:2 to obtain a mixture; then adding 8% to 15% of the mixture's mass of water to the mixture; heating to a molten state; cooling; and then mixing thoroughly with *Stropharia macrocarpa* powder at a mass ratio of 1:(15 to 25); extracting for 1.5 to 2.5 hours; performing a first centrifugation to obtain a supernatant; adding 5 to 10 times the volume of water to the supernatant; incubating overnight at 3 to 6°C; and performing a second centrifugation to obtain a precipitate, which is the glycoprotein extract from *Stropharia macrocarpa*. The separation of the *Stropharia macrocarpa* glycoprotein includes: dissolving the *Stropharia macrocarpa* glycoprotein extract in water, then dialyzing it with a dialysis bag of 3000-4000 Da for 40-60 hours, collecting the liquid in the dialysis bag to obtain the dialysate; removing the free protein in the dialysate to obtain the supernatant, freeze-drying it to obtain the crude *Stropharia macrocarpa* glycoprotein. The purification of the *Pleurotus ostreatus* glycoprotein includes: dissolving the crude *Pleurotus ostreatus* glycoprotein in water, centrifuging, and filtering through a microporous membrane to obtain a sample solution; purifying the sample solution by anion exchange resin column chromatography, using ammonium sulfate solution as the eluent, concentrating the collected eluent, and then dialyzing with a 3000-4000 Da dialysis bag for 70-80 hours; and freeze-drying the dialysate to obtain the *Pleurotus ostreatus* glycoprotein.

[0008] Preferably, the extraction of the *Stropharia macrocarpa* glycoprotein includes: heating to a molten state at a temperature of 100℃~120℃; passing the dried *Stropharia macrocarpa* powder through a 40~80 mesh sieve; the extraction includes stirring extraction at a speed of 300~500 r / min; and centrifugation at a speed of 5000~10000 rpm / min for a time of 10~15 min.

[0009] Preferably, the separation of the Pleurotus ostreatus glycoprotein includes removing the free protein 3 to 4 times.

[0010] Preferably, the purification of the *Agaricus macrocarpa* glycoprotein includes: the centrifugation speed is 8000~12000 rpm / min; the pore size of the microporous filter membrane is 0.22~0.45 μm; the anion exchange resin column is of the DEAEEFocurose Fast Flow type, and the specifications of the DEAEFocurose Fast Flow are Φ 1.6 cm × 100 cm; the elution flow rate is 1~3 mL / min; the elution is gradient elution, and the gradient elution procedure is: (1) elution with water for 1 column volume; (2) elution with 0.1M ammonium sulfate solution for 1 column volume; (3) elution with 0.3M ammonium sulfate solution for 1 column volume; (4) elution with 0.5M ammonium sulfate solution for 1 column volume; (5) elution with 0.7M ammonium sulfate solution for 1 column volume; (6) elution with 1M ammonium sulfate solution for 1 column volume; (7) elution with water for 2 column volumes.

[0011] Preferably, after gradient elution, an automatic collector is used to collect 12 mL from each tube, collecting the eluent from tubes 40 to 46. The concentration is carried out under reduced pressure to 90 to 120 mL, and the temperature is 32°C to 38°C.

[0012] Preferably, the freeze-drying temperature is -50℃ to -40℃, and the freeze-drying time is 48h to 72h.

[0013] This invention provides the application of the low molecular weight, high purity *Agrospalum discolor* glycoprotein or the *Agrospalum discolor* glycoprotein prepared by the aforementioned method in the preparation of products that enhance immune activity.

[0014] Preferably, the product includes pharmaceuticals.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a low molecular weight, high purity *Stropharia macrocarpa* glycoprotein, which has a molecular weight of 10-20 kDa, a purity of over 95%, and stable quality.

[0016] The method for preparing *Agaricus macrocarpa* glycoprotein provided by this invention is simple, quick, and easy to operate, facilitating practical application. Through steps such as deep eutectic solvent extraction, dialysis separation, and gel chromatography, the low molecular weight, high purity *Agaricus macrocarpa* glycoprotein described above is obtained from the fruiting body of *Agaricus macrocarpa*.

[0017] The *Stropharia macrocarpa* glycoprotein obtained by the above preparation method of the present invention has a molecular weight of 10-20 kDa, a purity of over 95%, and stable quality. In vitro bioactivity experiments show that the *Stropharia macrocarpa* glycoprotein has an immune-enhancing effect and can be used as a raw material for immune enhancers in drug development. Attached Figure Description

[0018] Figure 1 This is an SDS-PAGE gel electrophoresis image of the Pleurotus ostreatus glycoprotein in Example 2; where Marker represents the standard protein and SPGP-1 represents the Pleurotus ostreatus glycoprotein.

[0019] Figure 2 This is a high-performance anion chromatogram of SPGP-1, a glycoprotein from *Schefflera heptaphylla* in Example 2.

[0020] Figure 3 The infrared absorption spectrum of SPGP-1, a glycoprotein from *Schefflera heptaphylla*, in Example 2 is shown.

[0021] Figure 4 The image shows the UV absorption spectrum of SPGP-1, a glycoprotein from *Schefflera heptaphylla*, in Example 2.

[0022] Figure 5 The image shows a circular dichroism chromatogram of SPGP-1, a glycoprotein from *Schefflera heptaphylla* in Example 2.

[0023] Figure 6 This is the intrinsic fluorescence spectrum of SPGP-1, a glycoprotein from *Stropharia macrocarpa*, in Example 2.

[0024] Figure 7 The NMR spectrum of SPGP-1, a glycoprotein from *Pleurotus ostreatus*, in Example 2; where A is... 1H nuclear magnetic resonance spectrum; B is 13 C10 NMR spectrum; C10 represents DEPT NMR spectrum.

[0025] Figure 8 The images shown are scanning electron microscope (SEM) images of SPGP-1, a glycoprotein from *Agaricus macrocarpa*, in Example 2. Image A is a scanning electron microscope image at 20 μm, and image B is a scanning electron microscope image at 100 μm.

[0026] Figure 9 The results of Example 2 show the effect of Pleurotus ostreatus glycoprotein SPGP-1 on the cell survival rate of RAW 264.7 macrophages.

[0027] Figure 10 In Example 3, the *Stropharia macrocarpa* glycoprotein SPGP-1 was shown to promote the release of NO, TNF-α, IL-6, and IL-1β from RAW264.7 macrophages; compared with the control group, express p <0.05, express p <0.01, express p <0.001.

[0028] Figure 11 The results of the effect of Pleurotus ostreatus glycoprotein SPGP-1 on the expression of NF-κB signaling pathway-related proteins in RAW264.7 cells in Example 3 are shown; the differences between groups were analyzed by ANOVA and Tukey test (n=3); compared with the control group; express p <0.001, express p <0.0001).

[0029] Figure 12 The results of the effect of Pleurotus ostreatus glycoprotein SPGP-1 on the expression of MAPK signaling pathway-related proteins in RAW264.7 cells in Example 3 are shown; differences between groups were analyzed by ANOVA and Tukey test (n=3); compared with the control group express p <0.001, express p<0.0001; intergroup comparison ### indicates p <0.001.

[0030] Figure 13 Immunofluorescence images of RAW264.7 cell nuclear factor p-NF-κB p65 after treatment with different concentrations of SPGP-1 in Example 3. Detailed Implementation

[0031] This invention provides a low molecular weight, high-purity *Agaricus bisporus* glycoprotein. The molecular weight of the *Agaricus bisporus* glycoprotein is preferably 10-20 kDa, more preferably 11-18 kDa, even more preferably 12-15 kDa, and most preferably 12 kDa; the purity is preferably greater than 95%. The protein content of the *Agaricus bisporus* glycoprotein is preferably 75%-82%, more preferably 78%-81%, even more preferably 80%-81%, and most preferably 80.30%. The polysaccharide content of the *Agaricus bisporus* glycoprotein is preferably 10%-18%, more preferably 12%-17%, even more preferably 14%-17%, and most preferably 16.14%. The sugars and proteins in the *Agaricus bisporus* glycoprotein are covalently linked together.

[0032] In this invention, the glycoprotein of *Stropharia macrocarpa* contains 17 amino acids, namely glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline; glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine contain 17 amino acids. The preferred ratio of the content of acid, phenylalanine, histidine, lysine, arginine, and proline is (12.9~13.1): (13.8~14.0): (38.2~38.4): (20.2~20.5): (29.2~29.5): (41.4~41.8): (126.4~126.8): (17.6~18.10): (15.7~16.1): (51.2~51.6): (10.2~10) .7): (28.8~29.2): (19.0~19.4): (21.2~21.7): (20.1~20.6): (49.3~49.8): (88.5~89.0); Further preferred are (13.0~13.1): (13.9~14.0): (38.15~38.35): (20.3~20.4): (29.3~29.4): (41.5~41.6): (126). (5~126.7): (17.8~18.9): (15.8~16.0): (51.3~51.5): (10.4~10.6): (28.9~29.1): (19.1~19.3): (21.3~21.5): (20.2~20.4): (49.4~49.7): (88.6~88.9); As an optional embodiment, the *Pleurotus ostreatus* glycoprotein contains 13.03 glycine. The protein moiety of the *Agaricus bisporus* glycoprotein contains the following components: alanine 13.96 mg / g, aspartic acid 38.3 mg / g, threonine 20.33 mg / g, serine 29.38 mg / g, glutamic acid 41.59 mg / g, cysteine ​​126.59 mg / g, valine 17.86 mg / g, methionine 15.89 mg / g, isoleucine 51.42 mg / g, leucine 10.48 mg / g, tyrosine 29.00 mg / g, phenylalanine 19.19 mg / g, histidine 21.44 mg / g, lysine 20.33 mg / g, arginine 49.56 mg / g, and proline 88.74 mg / g. The secondary structure of the protein moiety of the *Agaricus bisporus* glycoprotein is α-helix 5.6%, β-sheet 29.8%, β-turn 16.9%, and random coil 47.7%.

[0033] In this invention, the polysaccharide of the *Stropharia macrocarpa* glycoprotein includes fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose, and mannose. The preferred molar ratio of fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose, and mannose is (0.9~1.1):(1~1.3):(0.6~0.9):(0.1~0.4):(0.6~0.9):(17~18):(1.8~2.2):(1.8~2.2); a more preferred ratio is (…). 0.95~1.05):(1.1~1.2):(0.7~0.8):(0.2~0.3):(0.7~0.8):(17.3~17.8):(1.9~2.1):(1.9~2.1); As an optional implementation method, the polysaccharide part is mainly composed of fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose and mannose, with a molar ratio of 1:1.15:0.75:0.23:0.77:17.61:2:2.07.

[0034] The present invention also provides a method for preparing the low molecular weight, high purity *Agaricus macrocarpa* glycoprotein, including the extraction, separation, and purification of the *Agaricus macrocarpa* glycoprotein.

[0035] The extraction of *Stropharia macrocarpa* glycoprotein in this invention includes: mixing choline chloride and urea at a molar ratio of 1:2 to obtain a mixture; then, preferably adding 8% to 15% (by mass) of water to the mixture; heating to a molten state; cooling; and then thoroughly mixing with *Stropharia macrocarpa* powder at a material-to-liquid ratio of 1:(15 to 25) (w / w); extracting for 1.5 to 2.5 hours; performing a first centrifugation to obtain a supernatant; adding 5 to 10 times the volume of water to the supernatant; incubating overnight at 3 to 6°C; and performing a second centrifugation to obtain a precipitate, which is the *Stropharia macrocarpa* glycoprotein extract. When adding water to the mixture for melting, preferably 10% (by mass) of water is added. The preferred temperature for heating and melting is 100°C to 120°C, more preferably 105°C to 110°C. The cooling temperature is to cool to room temperature, which is 20 to 25°C. After cooling, the mixture is preferably thoroughly mixed with *Stropharia macrocarpa* powder at a material-to-liquid ratio of 1:(15~25) (w / w), more preferably 1:(18~22) (w / w), and even more preferably 1:(19~21) (w / w). As a preferred embodiment, after cooling, the mixture is preferably thoroughly mixed with *Stropharia macrocarpa* powder at a material-to-liquid ratio of 1:20 (w / w), i.e., 1g of *Stropharia macrocarpa* powder is mixed with 20g of a molten mixture of choline chloride and urea. The *Stropharia macrocarpa* powder is preferably passed through a 40~80 mesh sieve, more preferably 50~70 mesh sieve, and even more preferably 60 mesh sieve. The extraction includes stirring extraction, and the stirring speed is preferably 300~500 r / min, more preferably 400 r / min. The centrifugation process for extracting glycoprotein from *Stropharia macrocarpa* of this invention consists of a first centrifugation and a second centrifugation. The preferred speed for the first centrifugation is 5000-10000 rpm / min, more preferably 5000-8000 rpm / min, and even more preferably 6000 rpm / min. The preferred centrifugation time is 10-15 min, more preferably 12-15 min, and even more preferably 15 min. The supernatant from the first centrifugation is preferably added with 5-8 times its volume of water, more preferably 5-6 times its volume, and most preferably 5 times its volume. After adding water, the supernatant from the first centrifugation is preferably incubated overnight at 4°C, meaning it stands for at least 12 hours, preferably 12-16 hours. The preferred speed for the second centrifugation is 5000-10000 rpm / min, more preferably 5000-8000 rpm / min, and even more preferably 6000 rpm / min. The preferred centrifugation time is 10-15 min, more preferably 10-12 min, and even more preferably 10 min.

[0036] The separation of *Stropharia macrocarpa* glycoprotein in this invention includes: dissolving *Stropharia macrocarpa* glycoprotein extract in water, then dialyzing with a dialysis bag containing 3000-4000 Da for 40-60 hours, collecting the liquid in the dialysis bag to obtain dialysate; removing free protein from the dialysate to obtain supernatant, lyophilizing to obtain crude *Stropharia macrocarpa* glycoprotein. During dissolution, the mass-to-volume ratio of the *Stropharia macrocarpa* glycoprotein extract to water is 1 g:(100-500) mL, more preferably 1 g:(200-300) mL, and even more preferably 1 g:200 mL. The molecular weight cutoff of the dialysis bag is preferably 3500 Da. The dialysis duration is preferably 42-50 hours, more preferably 46-50 hours, and even more preferably 48 hours. The dialysate is the solution of the *Stropharia macrocarpa* glycoprotein extract in the dialysis bag. The removal of free proteins from the dialysate is preferably performed using the Sevage method, which is described in the reference: Du Jingdong, Tian Fengjuan, Chen Peixiong, et al. Optimization of polysaccharide extraction method from Yunwu green tea and research on protein removal process using the Sevage method. Central South Pharmaceutical Journal, 2015, 12:1281-1283. The free proteins are removed 3 to 4 times, more preferably 3 times. The freeze-drying is preferably performed at a temperature of -45℃ for 60 hours.

[0037] The purification of *Agaricus spp.* glycoprotein in this invention includes: dissolving crude *Agaricus spp.* glycoprotein in water, centrifuging, and filtering through a microporous membrane to obtain a sample solution; purifying the sample solution by anion exchange resin column chromatography using ammonium sulfate solution as the eluent, collecting and concentrating the eluent, and then dialyzing with a 3000-4000 Da dialysis bag for 70-80 hours; freeze-drying the dialysate to obtain *Agaricus spp.* glycoprotein. The mass-to-volume ratio of crude *Agaricus spp.* glycoprotein to water is 1 g:(100-500) mL, more preferably 1 g:(200-300) mL, and even more preferably 1 g:200 mL. The centrifugation speed after dissolving the crude *Agaricus spp.* glycoprotein in water is preferably 8000-12000 rpm / min, more preferably 9000-11000 rpm / min, and even more preferably 10000 rpm / min; the centrifugation time is preferably 10-15 min, and even more preferably 10-12 min. The pore size of the microporous filter membrane is preferably 0.22 μm; the anion exchange resin column is preferably DEAE Focurose Fast Flow, and the DEAE Focurose Fast Flow column is preferably Φ 1.6 cm × 100 cm; the elution flow rate is preferably 1~3 mL / min, more preferably 2 mL / min; the elution is preferably gradient elution, and the gradient elution procedure is preferably: (1) elution with water for 1 column volume; (2) elution with 0.1M ammonium sulfate solution for 1 column volume; (3) elution with 0.3M ammonium sulfate solution for 1 column volume; (4) elution with 0.5M ammonium sulfate solution for 1 column volume; (5) elution with 0.7M ammonium sulfate solution for 1 column volume; (6) elution with 1M ammonium sulfate solution for 1 column volume; (7) elution with water for 2 column volumes. After gradient elution, an automatic collector is used to collect 12 mL from each tube, collecting the eluent from tubes 40 to 46. The concentration is performed under reduced pressure to 90-120 mL at a temperature of 32°C-38°C. Preferably, the concentration is made to 100 mL under reduced pressure; the concentration temperature is preferably 33°C-36°C, more preferably 35°C. The freeze-drying temperature is -50°C to -40°C, more preferably -45°C, and the freeze-drying time is 48-72 hours, more preferably 60 hours.

[0038] A low molecular weight, high-purity *Agaricus spp.* glycoprotein was prepared using the method described above in this invention. The molecular weight of the *Agaricus spp.* glycoprotein is 12 kDa, and its purity is greater than 95%. The preparation process is simple, quick, and convenient, facilitating practical application. In this invention, the *Agaricus spp.* glycoprotein exhibits excellent conformational stability within the physiologically relevant temperature range of 25–35°C, with a purity reaching 96.44%.

[0039] This invention provides the application of the low molecular weight, high purity *Pseudomonas aeruginosa* glycoprotein or the *Pseudomonas aeruginosa* glycoprotein prepared by the aforementioned method in the preparation of products that enhance immune activity. The products include pharmaceuticals. As an optional embodiment, the pharmaceutical can be an immune enhancer.

[0040] This invention provides a glycoprotein from *Stropharia macrocarpa* with a purity exceeding 95% and stable quality, prepared using the above-described method. This glycoprotein stimulates the release of NO from mouse macrophages (RAW264.7) in vitro, and also stimulates the release of cytokines such as TNF-α, IL-6, and IL-1β from RAW264.7 cells. Furthermore, the obtained *Stropharia macrocarpa* glycoprotein can, in a concentration-dependent manner, increase the expression levels of proteins such as p-IKKα / β, p-IκBα, and p-NF-κB p65 in the NF-κB signaling pathway of RAW264.7 cells, and also increase the phosphorylation levels of proteins such as p-p38, p-ERK, and p-JNK in the MAPK signaling pathway, thus exerting an immune-enhancing effect. It can be used as an immune enhancer or as a raw material for drug development.

[0041] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0043] Example 1 Preparation method of Pleurotus ostreatus glycoprotein 490 g of choline chloride and 420 g of urea (molar ratio 1:2) were mixed, and 90 g (10% by mass) of distilled water was added. The mixture was heated to 110°C on a magnetic stirrer until it reached a molten state, and then cooled to room temperature (20°C~25°C) to obtain a deep eutectic solvent. The fruiting bodies of *Stropharia macrocarpa* (purchased from Xixia, Henan) were pulverized and passed through a 60-mesh sieve. 50 g of *Stropharia macrocarpa* fruiting body powder was weighed and added to the above deep eutectic solvent (830~850 mL). Extraction was carried out at 25°C with a magnetic stirrer at 400 r / min for 2 h. The mixture was then centrifuged at 6000 rpm / min for 15 min to obtain the supernatant. Five times the volume of distilled water was added to the supernatant, and the mixture was incubated overnight at 4°C. The precipitate obtained was the *Stropharia macrocarpa* glycoprotein extract.

[0044] The extract of *Stropharia macrocarpa* glycoprotein was dissolved in distilled water at a mass-to-volume ratio of 1 g: 200 mL, and then dialyzed through a 3500 Da dialysis bag for 24 h to obtain the dialysate. The dialysate was then subjected to the Sevage method to remove free protein, yielding a supernatant. This process was repeated 3-4 times. The supernatant was then freeze-dried using a Labconco FreeZone 12L freeze dryer at -45°C for 60 h to obtain crude *Stropharia macrocarpa* glycoprotein. 50 mg of the crude *Stropharia macrocarpa* glycoprotein was dissolved thoroughly in 5 mL of ultrapure water, centrifuged at 10,000 rpm, and filtered through a 0.22 μm microporous membrane to obtain the sample solution.

[0045] The sample solution was purified by chromatography using a DEAE Focurose Fast Flow anion exchange resin column (Φ 1.6 cm × 100 cm), with ammonium sulfate solution as the eluent, a flow rate of 2 mL / min, and the following elution program: (1) Elute with water for 1 column volume (200 mL); (2) Elute one column volume with 0.1M ammonium sulfate solution; (3) Elute one column volume with 0.3M ammonium sulfate solution; (4) Elute one column volume with 0.5M ammonium sulfate solution; (5) Elute one column volume with 0.7M ammonium sulfate solution; (6) Elute 1 column volume with 1M ammonium sulfate solution; (7) Wash with water for 2 column volumes.

[0046] An automated collector was used to collect 12 mL from each tube. The eluent from tubes 40 to 46 was collected based on the UV signal. The eluent was concentrated to 100 mL under reduced pressure at 35°C. Then, the tube was dialyzed with distilled water in a 3500 Da dialysis bag for 72 hours. The dialysate (the liquid inside the dialysis bag) was freeze-dried using a Labconco FreeZone 12L freeze dryer at -45°C for 60 hours to obtain high-purity Pleurotus ostreatus glycoprotein (28 mg), denoted as SPGP-1.

[0047] Referring to the BCA method for determining protein content (Cao Mingxiang, Xu Hua, Liu Biyun, et al. Comparison of the performance of three protein content determination kits for detecting trace proteins. Laboratory Detection, 2024, 2(07): 126-130), the protein content of *Stropharia stropharia* glycoprotein was determined to be 80.30%. Referring to the phenol-sulfuric acid method for determining polysaccharide content (Wen Wenjuan, Liu Shan, Huang Yuanli. Comparison of phenol-sulfuric acid method and anthrone-sulfuric acid method for determining polysaccharide content in shiitake mushrooms. Modern Food, 2020, 21:177-179), the polysaccharide content of *Stropharia stropharia* glycoprotein was determined to be 16.14%. The purity of *Stropharia stropharia* glycoprotein was 96.44%.

[0048] Example 2 Structural characterization of Pleurotus ostreatus glycoprotein 1. SDS-PAGE gel electrophoresis analysis of glycoproteins (1) Coomassie brilliant blue staining One mg of *Pleurotus ostreatus* glycoprotein sample was dissolved in 20 mL of distilled water and subjected to SDS-PAGE gel electrophoresis according to the method in the literature (Zhang Xiaohui, Wang Shiwei, Xi Xiaohu, et al. Study on protein analysis of donkey skin glue and common counterfeit glue by SDS-PAGE gel electrophoresis. Journal of Traditional Chinese Medicine, 2023, 29(2): 47-49). The protein in *Pleurotus ostreatus* glycoprotein was then analyzed by staining with Coomassie Brilliant Blue G-250.

[0049] 2) PAS staining One mg of *Agaricus bisporus* glycoprotein sample was dissolved in 20 mL of distilled water. Following the method described in the literature (Zhang Xiaohui, Wang Shiwei, Xi Xiaohu, et al. Study on protein analysis of donkey skin glue and common counterfeit glues by SDS-PAGE gel electrophoresis. Journal of Traditional Chinese Medicine, 2023, 29(2): 47-49), SDS-PAGE gel electrophoresis was performed. Then, following the method described in the literature (Wang Yuqi, Wu Guanghong, Lin Xianfeng, et al. Improvement of polyacrylamide gel electrophoresis staining method for polysaccharides and glycoproteins. Plant Physiology Communications, 2009, 45(2): 169-172), PAS (periodic acid-Schiff reaction) staining was performed to analyze the polysaccharides in *Agaricus bisporus* glycoprotein.

[0050] (3) Results Analysis like Figure 1 As shown, the *Stropharia macrocarpa* glycoprotein SPGP-1 was subjected to SDS-PAGE gel electrophoresis and then stained with Coomassie Brilliant Blue and PAS, respectively. Coomassie Brilliant Blue staining identified the protein in the *Stropharia macrocarpa* glycoprotein, while PAS staining identified the polysaccharide. The results showed that a single protein band and a polysaccharide band appeared at the same location, i.e., 12 kDa, which proves that this component is a glycoprotein. The sugar and protein are linked together by covalent bonds, rather than being a mixture of sugar and protein. The bands are relatively clear, the background is clear, the sample purity is high, and its molecular weight is approximately 12 kDa.

[0051] 2. Amino acid composition analysis Accurately weigh 50 mg of *Stropharia stolonifera* glycoprotein sample and determine the amino acid composition of the protein fraction of *Stropharia stolonifera* glycoprotein according to the method in the literature (Sun Xiaojie, Huang Xuexue, Zhao Yuqiang, et al. Determination and analysis of 12 sugar components and 20 amino acids in 5 kinds of honey in Yunnan. Food Science, 2025, 46(2): 148-155).

[0052] According to the results in Table 1, the amino acid composition of the protein portion of *Stropharia macrocarpa* glycoprotein includes (mg / g): glycine 13.03, alanine 13.96, aspartic acid 38.3, threonine 20.33, serine 29.38, glutamic acid 41.59, cysteine ​​126.59, valine 17.86, methionine 15.89, isoleucine 51.42, leucine 10.48, tyrosine 29.00, phenylalanine 19.19, histidine 21.44, lysine 20.33, arginine 49.56, and proline 88.74.

[0053] Table 1. Amino acid composition of Pleurotus ostreatus glycoprotein SPGP-1

[0054] 3. Monosaccharide component analysis Accurately weigh 10 mg of *Stropharia macrocarpa* glycoprotein sample and add it to a 100 mL volumetric flask. Add distilled water to bring the volume to 100 mL, shake until completely dissolved, and take 1 mL. Filter through a 0.22 μm filter membrane and dilute 10 times to obtain the test solution. The monosaccharide components of the *Stropharia macrocarpa* glycoprotein polysaccharide fraction were determined using a high-performance anion chromatography (Dionex, ICS5000+). The separation column was a Dionex CarboPac PA20 detection column (4 mm x 250 mm), the mobile phase was 480 mmol / L NaOH solution, the flow rate was 0.40 mL / min, the sample loading volume was 25 μL, and the column temperature was 30℃.

[0055] like Figure 2 As shown, the results indicate that the polysaccharide portion of the *Stropharia macrocarpa* glycoprotein is mainly composed of fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose, and mannose, with a molar ratio of 1:1.15:0.75:0.23:0.77:17.61:2:2.07.

[0056] 4. Infrared absorption spectroscopy analysis The infrared absorption spectrum of SPGP-1, a glycoprotein from *Agaricus bisporus*, was determined using a Nicolet iS5 infrared spectrometer. The infrared spectral acquisition range was 500–4000 cm⁻¹. -1 The resolution was set to 4 cm. -1 .

[0057] The infrared absorption spectrum of Pleurotus ostreatus glycoprotein is shown below. Figure 3 As shown, at 3370.7 cm -1 The broad and strong absorption peak at 1651.7 cm⁻¹ is attributed to the stretching vibrations of OH and NH in polysaccharides and proteins, indicating the presence of hydroxyl and amino groups; -1The absorption peak at 1414.0 cm⁻¹ corresponds to the amide I band (C=O stretching vibration), a characteristic signal of protein secondary structure; -1 and 1253.0 cm -1 The absorption peaks at 1132.4 cm⁻¹ may be related to the symmetric stretching vibration of the carboxyl group and the amide III band, respectively; -1 998.4 cm -1 The nearby absorption peaks are attributed to the COC stretching vibration of the pyranose ring, while the peak at 875.5 cm⁻¹ is... -1 This may be attributed to the contraction vibration of glycosidic bonds, confirming the presence of a polysaccharide structure. Based on the above analysis, this spectrum indicates that SPGP-1 is a typical glycoprotein, exhibiting infrared characteristic absorption peaks of both proteins and polysaccharides.

[0058] 5. Ultraviolet absorption spectroscopy analysis Following the method described in the literature (Fan Hanqiu, Chen Dongli, Liu Xiaojing, et al. Determination of protein content by ultraviolet-visible absorption spectroscopy and the effect of protein state. China Pharmaceutical Industry Journal, 2025, 56(5): 584-592), the ultraviolet-visible absorption spectrum of the glycoprotein solution was determined at a glycoprotein concentration of 0.4 mg / mL.

[0059] The glycoprotein SPGP-1 from *Stropharia macrocarpa* exhibits typical glycoprotein characteristics in its ultraviolet absorption spectrum within the 200–600 nm range, such as… Figure 4 The strong absorption peak at 200–220 nm is attributed to the π→π electronic transition of peptide bonds, a characteristic terminal absorption of protein macromolecules in the far-ultraviolet region, directly confirming the presence of protein components in glycoproteins. A gentle absorption peak is observed in the 250–280 nm range, corresponding to the π→π and n→π transitions in the benzene ring conjugated system of aromatic amino acid residues. The transition indicates that there are aromatic amino acids in the protein portion of SPGP-1. When the wavelength exceeds 300 nm, the absorbance decreases rapidly and approaches the baseline without any absorption peak. This is because polysaccharide molecules do not have conjugated double bonds and do not absorb in the ultraviolet region, indicating that the ultraviolet absorption signal of glycoproteins in this invention is mainly contributed by protein components.

[0060] 6. Circular dichroism analysis The circular dichroism (CD) spectrum of *Agaricus bisporus* glycoprotein (0.5 mg / mL in distilled water) was measured in the 190-240 nm range using a J-1500CD spectrophotometer (Jasco Co., Japan). The spectral resolution was 0.1 nm, the scan rate was 500 nm / min, and the bandwidth was 1 nm. The content of α-helices, β-sheets, β-turns, and random coils in the secondary structure of the glycoprotein was analyzed using DichroWeb (https: / / dichroweb.cryst.bbk.ac.uk).

[0061] The circular dichroism chromatogram of Pleurotus ostreatus glycoprotein is shown below. Figure 5 As shown, the secondary structure information of the protein portion of the glycoprotein of *Stropharia macrocarpa* is obtained according to calculation: α-helix 5.6%, β-sheet 29.8%, β-turn 16.9%, and random coil 47.7%.

[0062] 7. Intrinsic fluorescence spectroscopy analysis Following the method described in the literature (Hu Yaqi, Gao Yuhan, Li Mei, et al. Study on the interaction between a novel antihypertensive drug and bovine serum albumin by fluorescence spectroscopy. Chemical and Biological Engineering, 2026, 43(2): 50-55), the fluorescence spectrum of SPGP-1 glycoprotein from *Schefflera heptaphylla* was detected. The fluorescence spectrum of SPGP-1 was measured at three temperatures: 298 K, 303 K, and 308 K, with an excitation wavelength of 280 nm. The environment of tyrosine and tryptophan in the protein was analyzed.

[0063] like Figure 6 As shown, the fluorescence spectrum of SPGP-1 in the temperature range of 298-308 K exhibits a regular change: the fluorescence intensity gradually decreases with increasing temperature. This is because the increased temperature intensifies the thermal motion of molecules, increasing the probability of nonradiative transitions in excited-state molecules, which is a normal thermal quenching effect. The maximum emission wavelength remains stable at about 330 nm without significant redshift, indicating that the hydrophobic core structure of SPGP-1 is intact and the tryptophan residues are not exposed to the polar water environment due to the increase in temperature. At the same time, the high consistency of the peak shape also proves that its tertiary structure has not undergone significant conformational rearrangement, indicating that SPGP-1 has excellent conformational stability in the physiologically relevant temperature range of 25~35℃.

[0064] 8. Nuclear Magnetic Resonance Spectroscopy Analysis 30 mg of *Stropharia macrocarpa* glycoprotein SPGP-1 sample was dissolved in 0.5 mL of heavy water (D2O). One-dimensional NMR spectra were recorded at 25 °C using a Bruker VNMRS 600 instrument. Chemical shifts are expressed in ppm, with Me4Si as an internal standard.

[0065] Nuclear magnetic resonance1 H spectrum Figure 7 As shown in Figure A, the signal peaks at δ 6.5–8.6 ppm belong to aromatic amino acid residues in the protein. Specifically, the signal peak at δ 7.2–7.4 ppm corresponds to phenylalanine aromatic ring protons, the signal peak at δ 7.3–7.6 ppm corresponds to tyrosine aromatic ring protons, and the signal peak at δ 7.6–8.6 ppm corresponds to tryptophan indole ring and histidine imidazole ring protons. The presence of these signals proves that the sample contains a complete protein structure and that the microenvironment of aromatic amino acid residues has not undergone significant denaturation. The signal peaks at δ 0.5–5.0 ppm correspond to protons on aliphatic and peptide backbones. The strong signal peaks at δ 0.8–1.5 ppm correspond to alkyl protons on the protein side chains, possibly due to proton signals from the methyl and methylene groups of leucine, isoleucine, and valine, exhibiting typical characteristics of a hydrophobic protein core. The signals at δ 1.5-2.5 ppm correspond to the methylene groups of the aspartic acid and glutamate side chains, as well as the proline ring protons, indicating the presence of polar protein chains in the glycoprotein. The dense signal peaks at δ 3.0-4.5 ppm correspond to the α-carbon protons and some side chain protons of the protein, representing the core characteristic signals of the polypeptide backbone and proving the presence of a complete peptide chain structure in the sample. SPGP-1 has a low polysaccharide content, and the proton signals on the sugar rings should be lower than those in the protein portion. The proton signals on the sugar rings may be distributed between δ 3.0-5.5 ppm, with the weak signal peak at δ 1.9-2.1 ppm corresponding to the methyl protons of the N-acetyl group, a characteristic signal of acetylglucosamine in the N-linked sugar chain, indicating that this glycoprotein may contain N-glycosidic bonds. 13 C spectrum as shown Figure 7 As shown in B, the signal distribution is in the δ 10-210 ppm range, where δ 160-210 ppm may represent the carbonyl carbon region of the protein. The strong signal peak at δ 170-180 ppm corresponds to the carbonyl carbon of the protein peptide bond, providing direct evidence of the presence of the polypeptide chain and reflecting the intact primary structure of the protein in the sample. The weak signal peaks in the δ 120-140 ppm region may correspond to the aromatic ring carbons of aromatic amino acid residues: phenylalanine, tyrosine, and tryptophan indole. 1The 1H NMR aromatic signals corroborate each other, demonstrating that the microenvironment of aromatic residues in the protein's secondary structure has not undergone significant denaturation. The strong signal peaks in the δ 10-45 ppm range likely correspond to the alkyl carbons of the protein's side chains, such as the methyl and methylene carbons of leucine, isoleucine, and valine, as well as the methyl carbon of alanine, confirming the hydrophobic core of SPGP-1. The dense signal peaks in the δ 45-80 ppm range correspond to the α-carbon and polar side chain carbons of the protein, representing the core characteristic signals of the polypeptide backbone, further confirming the presence of a complete peptide chain structure. The weak signal peaks in the δ 20-25 ppm range represent the methyl carbon of the N-acetyl group, consistent with the 1H NMR analysis, indicating that this glycoprotein may contain N-linked glycosidic bonds. DEPT spectrum ( Figure 7 As shown in C), a carbonyl quaternary carbon signal appears at δ 181.05 ppm, proving the presence of tryptophan. An anterior carbon signal of the sugar ring is present at δ 107.85 ppm, and the sugar ring hydroxymethyl and backbone methylene signals are at δ 67.60 ppm and δ 59.16 ppm, respectively. A methyl signal is present near δ 19 ppm. The overall carbon signal distribution and chemical shift characteristics perfectly match the glycoprotein structure, clearly confirming that the sample is a glycoprotein with glycosyl modification.

[0066] The above analysis shows that the aromatic and fatty regions of the SPGP-1 protein have intact NMR signals, proving that the sample maintains a stable protein secondary structure, and the distribution of the hydrophobic core and polar side chains is consistent with the conformational characteristics of natural glycoproteins. 1 The N-acetyl protons corresponding to δ 1.9-2.1 ppm in the H spectrum, 13 The signals of N-acetyl methyl carbon at δ 20-25 ppm and N-acetyl carbonyl carbon at δ 170-180 ppm in the C spectrum correspond, confirming the presence of sugar chains and N-glycosidic bonds.

[0067] 9. Scanning electron microscopy analysis like Figure 8 As shown, SPGP-1 exhibits an irregular blocky morphology with a relatively dense surface and rough or slightly curled edges, displaying typical amorphous aggregation characteristics. This morphology is usually due to the strong hydrophilicity of the polysaccharide chains and the intermolecular hydrogen bonding interactions during the freeze-drying process, leading to the self-assembly of glycoprotein molecules into a relatively compact aggregate structure. The absence of a clear crystal structure or fibrous network indicates that this glycoprotein component exists in an amorphous solid form, a microscopic characteristic consistent with its macromolecular composition and natural origin.

[0068] Example 3 Evaluation of the in vitro immunomodulatory activity of Pleurotus ostreatus glycoprotein 1. Cytotoxicity of *Stropharia macrocarpa* glycoprotein against mouse RAW264.7 macrophages (1) Experimental methods The cytotoxic effect of *Stropharia macrocarpa* glycoprotein SPGP-1 on mouse macrophage RAW264.7 cells was determined using the Alamar blue assay. RAW264.7 macrophage suspensions were inoculated into 96-well plates at a cell count of 5 × 10⁶ cells / mL. 5 200 μL of culture medium was added to each well and incubated at 37 °C for 24 h in an incubator containing 5% CO2. The cell culture medium in the wells was then removed and replaced with fresh culture medium (200 μL) containing different concentrations of Pleurotus ostreatus glycoprotein (final concentrations: 12.5, 25, 50, 100 μg / mL). After incubation at 37 °C for 48 h, 20 μL of Alamar Blue reagent was added to each well for further incubation. The effect of SPGP-1 on the viability of RAW264.7 cells within the selected concentration range was evaluated using the Alamar Blue method. Each sample was tested in triplicate.

[0069] Cell viability = (2) Results Analysis The effect of Pleurotus ostreatus glycoprotein SPGP-1 on the viability of RAW264.7 cells as follows: Figure 9 As shown, the results indicated that 12.5 μg / mL of SGP-1 had no significant effect on cell viability; however, when the SPGP-1 concentration increased to 100 μg / mL, cell viability decreased significantly. The results also showed that SPGP-1 was non-toxic to RAW 264.7 cells at doses of 12.5–50 μg / mL, but exhibited significant cytotoxicity at a dose of 100 μg / mL. Therefore, subsequent experiments used SPGP-1 at concentrations of 12.5, 25, and 50 μg / mL.

[0070] 2. Effects of Pleurotus ostreatus glycoprotein SPGP-1 on NO and cytokine (IL-1β, IL-6, TNF-α) release from mouse macrophage RAW264.7 cells. (1) Experimental methods RAW264.7 cells were diluted with colorless RPMI 1640 medium to a concentration of 5 × 10⁶ cells / mL. 5A suspension of cells was added to each well of a 96-well plate (200 μL). Cells were cultured at 37 °C until fully adherent. The culture medium was then removed from the wells and replaced with 200 μL of fresh culture medium containing different concentrations of *Amanita muscaria* glycoprotein (final concentrations: 12.5, 25, and 50 μg / mL). PBS was used as a blank control, and 2 μg / mL LPS was used as a positive control. After culturing at 37 °C for 48 h, 100 μL of the supernatant was added to 50 μL of Griess reagent, and the reaction was allowed to proceed for 10 min. The absorbance at 543 nm was measured to determine the NO release. Cells were cultured using the same method, with the addition of LPS and *Amanita muscaria* glycoprotein samples. After culturing at 37 °C for 48 h, the supernatant was collected, centrifuged at 10000 g for 10 min, and the levels of inflammatory factors IL-1β, IL-6, and TNF-α were measured strictly according to the ELISA kit requirements.

[0071] (2) Results Analysis like Figure 10 As shown, using LPS as a positive control, the effects of different concentrations of SPGP-1 on the release of related immune mediators in RAW264.7 cells were examined. The results showed that, compared with the control group, LPS significantly induced the production of key inflammatory mediators such as NO, TNF-α, IL-6, and IL-1β. P <0.001), TNF-α, IL-6, and IL-1β are involved in macrophage phagocytosis and immune regulation, and play important roles in inflammation and cellular activities. After SPGP-1 treatment, NO production and the secretion of TNF-α, IL-6, and IL-1β in RAW264.7 cells showed a concentration-dependent increase. At a concentration of 12.5 μg / mL, only NO and TNF-α showed a slight increase; when the concentration was increased to 25 μg / mL, all four inflammatory mediators were significantly higher than those in the control group ( P <0.01 or P <0.001); at a concentration of 50 μg / mL, the secretion levels of NO, IL-6, and IL-1β were close to those in the LPS group. The results indicate that SPGP-1 can exert an immune-activating effect by activating the release of immune cytokines from RAW264.7 cells, and the intensity of this effect increases with increasing concentration, suggesting that SPGP-1 may participate in immune regulation by modulating the immune signaling pathways of macrophages.

[0072] 3. Effects of Pleurotus ostreatus glycoprotein SPGP-1 on the expression of key proteins in the classical immune signaling pathways of NF-κB and MAPK in macrophages. (1) Western Blot experimental method The expression levels of NF-κB and MAPK pathway-related proteins and their phosphorylated forms in RAW264.7 macrophages were determined, and GAPDH was used as a reference for protein quantification.

[0073] Cell culture: RAW264.7 cells were diluted with colorless RPMI 1640 medium to a concentration of 5 × 10⁶ cells / mL. 5 A suspension of cells was added to each well of a 96-well plate at a density of 200 μL. Cells were cultured at 37 °C until complete adhesion. The culture medium was then removed from the wells and replaced with 200 μL of fresh culture medium containing different concentrations of *Agaricus bisporus* glycoprotein (final concentrations: 12.5, 25, and 50 μg / mL). PBS was used as a blank control group, and 2 μg / mL LPS was used as a positive control group. After culturing at 37 °C for 48 h, the supernatant was discarded, and cells were collected for protein extraction.

[0074] Cell protein extraction and quantification: The culture medium was aspirated, and cells were thoroughly pipetted into the buffer using cold PBS buffer. Cells from each experimental group were collected in 2 mL centrifuge tubes, centrifuged at 12000 rpm, and the supernatant was discarded; the precipitate was the collected cells. 300 μL of cell lysis buffer containing a phosphatase inhibitor (RIPA) and 2 μL of stop solution (PMSF) were added to the cells. The cells were incubated on ice for 1 h for lysis. After complete lysis, the cells were centrifuged at 14000 rpm for 5 min, and the supernatant was collected as the cell protein sample. The extracted proteins were analyzed according to the BCA kit instructions. The protein samples from each group were diluted to the same concentration, and an appropriate amount of protein loading buffer was added. The samples were incubated in a boiling water bath at 100 ℃ for 5 min and immediately placed on ice. The proteins were then stored at -40 ℃ for later use.

[0075] Preparation of electrophoresis buffer: Dilute 5×Tris-glycine electrophoresis buffer with distilled water to make 1× electrophoresis buffer. Mount the precast gel into the electrophoresis tank, add an appropriate amount of electrophoresis buffer, and allow it to stand to check for leakage. If no leakage is observed, add the remaining electrophoresis buffer. The sample loading volume per well is approximately 40 µg of protein. Electrophoresis conditions are 100 V; the time is determined by the position of the bromophenol blue.

[0076] Transfer: The transfer solution was prepared by diluting 10× electrotransfer solution, methanol, and distilled water at a ratio of 1:2:7 to obtain 1× transfer solution. The strip was cut and transferred onto a polyvinylidene fluoride (PVDF) membrane under the following conditions: 350 mA, 120 min ice bath.

[0077] Blocking: Preparation of TBST washing solution: Dilute 10×TBST with distilled water to make 1×TBST. Preparation of blocking solution: Weigh an appropriate amount of skim milk powder and prepare a 5% skim milk powder solution with TBST. Place the PVDF membrane in the blocking solution and shake at room temperature for 1 hour.

[0078] Antibody incubation: The PVDF membrane was incubated with the primary antibody overnight at 4 °C, followed by washing three times with TBST for 15 min each time. The PVDF membrane was then incubated with HRP-conjugated secondary antibody at room temperature for 1 h, followed by washing three times with TBST for 15 min each time. Protein bands were stained using an enhanced chemiluminescence detection kit and scanned using ImageJ software. GAPDH was used as a reference for protein quantification.

[0079] (2) Results Analysis Western blot analysis was used to detect the expression of key proteins in two classic immune-related signaling pathways, NF-κB and MAPK, in macrophages. The mechanism of action of the *Stropharia macrocarpa* glycoprotein SPGP-1 in enhancing immunity at the cellular level was investigated, confirming its in vitro immune-enhancing activity. Nuclear factor κB (NF-κB), as a key transcription factor, coordinates various biological processes, such as cell proliferation, differentiation, immune responses, and inflammation. Aberrant activation of the NF-κB pathway is considered to be associated with the development of various diseases, such as inflammatory diseases, cancer, and autoimmune diseases. Furthermore, the NF-κB signaling pathway is downstream of the MyD88-dependent pathway and can be triggered by the MAPK signaling pathway. NF-κB can bind to inhibitory proteins such as IκB and exists in the cytoplasm as an inactive complex. Once cells are subjected to various stimuli (such as inflammatory intervertebral arthritis, viral infection, oxidative stress), IκB is phosphorylated and degraded, subsequently releasing NF-κB into the nucleus, regulating the expression of target genes, such as cytokines, chemokines, and adhesion molecules.

[0080] like Figure 11 As shown, compared with the control group, the expression of phosphorylated IKKα / β, IκB-α, phosphorylated IκB-α and phosphorylated NF-κBp65 in RAW264.7 cells was significantly higher after exposure to different concentrations of SPGP-1 (…). P <0.05 or P The expression of phosphorylated NF-κB (p65) was significantly upregulated when the concentration was <0.01, showing a dose-dependent effect. The highest dose of SPGP-1 (50 μg / mL) significantly promoted the expression of phosphorylated NF-κB p65, with its expression level even exceeding that of the LPS group. Conversely, different concentrations of SPGP-1 did not promote the expression of IKKα, IKKβ, and NF-κB (p65) proteins, and there was no significant difference in the expression levels of the three proteins. P >0.05). The results indicate that SPGP-1 may activate the immune response of macrophages by activating the expression of phosphorylated IKKα / β, IκB-α, phosphorylated IκB-α and phosphorylated NF-κBp65 in the NF-κB signaling pathway.

[0081] MAPK is a key intracellular signaling pathway that plays a central role in regulating cellular responses to various extracellular signals, such as stress, cytokines, hormones, and growth factors. The MAPK signaling pathway includes several family members, such as extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (JNK), and p38 MAPK, all of which are involved in cell proliferation, differentiation, immunity, and inflammation. Figure 12 As shown, compared with the control group, the LPS group significantly induced increased phosphorylation levels of p38, ERK, and JNK proteins, while having no significant effect on the expression of p38, ERK, and JNK proteins. This is consistent with the classic mechanism by which LPS activates the MAPK signaling pathway through the TLR4 receptor and promotes the synthesis of downstream pro-inflammatory factors. Different concentrations of SPGP-1 were applied to RAW264.7 cells, and the expression of phosphorylated p38, ERK, and JNK proteins was significantly enhanced compared with the control group, showing a dose-dependent effect. In particular, 50 μg / mL SPGP-1 significantly promoted the expression of phosphorylated p38 protein, with a better promoting effect than the LPS group. Conversely, after treatment with different concentrations of SPGP-1, the expression of ERK, p38, and JNK proteins did not differ significantly from the control group. P >0.05). The results indicate that SPGP-1 may activate the MAPK signaling pathway by promoting the expression of phosphorylated p38, ERK, and JNK proteins, thereby enhancing immunity.

[0082] In summary, the *Stropharia macrocarpa* glycoprotein SPGP-1 can activate the NF-κB signaling pathway by binding to TLR receptors on the cell membrane and promoting the recruitment of related proteins and phosphorylation of IκBα. This pathway is also activated via an IKK-dependent phosphorylation mechanism through extracellular receptor signaling via MAPKs. Simultaneously, the expression levels of phosphorylated p38, JNK, and ERK proteins in the MAPK signaling pathway are upregulated in a dose-dependent manner. Therefore, *Stropharia macrocarpa* glycoprotein SPGP-1 mediates the immune enhancement effect of RAW264.7 macrophages by activating proteins such as p-IKKα / β, p-IκBα, and p-NF-κB p65 in the NF-κB signaling pathway and upregulating the expression of proteins such as p-p38, p-ERK, and p-JNK in the MAPK signaling pathway.

[0083] 4. Immunofluorescence analysis (1) Experimental methods Cell culture: RAW264.7 macrophages were cultured at 5 × 10⁻⁶ cells / year. 4 The concentration of cells / mL was plated into 1 mL in a laser confocal cell culture dish (30 mm × 10 mm), and incubated at 37 °C for 12 h. Then, the cells were loaded at the previous sample concentration, and after loading, they were incubated at 37 °C for another 24 h before being processed.

[0084] Cell treatment: Remove the culture medium, wash twice with cold PBS, then fix cells with 1 mL of 4% cold paraformaldehyde at room temperature for 15 minutes. Remove the paraformaldehyde, wash three times with cold PBS, and then permeate the cells with 1 mL of 0.25% Triton X-100 at 37°C for 30 minutes. Remove the permeabilization solution, wash twice with cold PBS, and block with 2 mL of 5% BSA in PBS solution for 1 hour while gently shaking. Then add 5% BSA in PBS solution and p-NFκB p65 specific primary antibody, and incubate overnight at 4°C (12-16 h). The next day, gently shake for 30 min at room temperature, then wash twice with PBS for 5-10 minutes each time; add 2 mL of 5% BSA in PBS solution, and incubate with Cy3 (1:500) labeled secondary antibody at room temperature in the dark for 1 hour. Then wash three times with PBS solution for 5 minutes each time; add about 200 μL of DAPI solution, use a fluorescence inverted microscope to check whether the secondary antibody (Cy3 red fluorescence) has successfully bound, and then use a laser confocal microscope to obtain images.

[0085] (2) Results Analysis The nuclear translocation of phosphorylated NF-κB p65 was detected by immunofluorescence double staining. Figure 13 As shown, in the control group RAW264.7 cells, phosphorylated NF-κB p65 was mainly located in the cytoplasm, with extremely weak fluorescence signal in the nucleus. After LPS stimulation, the red fluorescence signal of phosphorylated NF-κB p65 was significantly enhanced, and it translocated extensively into the nucleus, forming obvious co-localization with the blue nuclei labeled with DAPI, verifying the classic effect of LPS as an NF-κB pathway activator. Compared with the control group, SPGP-1 treatment could induce the expression and nuclear translocation of phosphorylated NF-κB p65 in RAW264.7 cells in a concentration-dependent manner. 12.5 μg / mL of SPGP-1 could slightly enhance the fluorescence intensity and induce some nuclear translocation. As the concentration increased to 25 and 50 μg / mL, the red fluorescence intensity increased significantly, and the nuclear co-localization signal showed a gradient enhancement. The degree of nuclear translocation in the 50 μg / mL group was close to that in the LPS-positive control group. Previous studies have confirmed that phosphorylation and nuclear translocation of NF-κB p65 are core transcriptional regulatory links in macrophage activation, release of NO and cytokines (TNF-α, IL-6, IL-1β). Combined with the above findings, this indicates that SPGP-1 can activate the NF-κB signaling pathway, induce NF-κB p65 phosphorylation and nuclear translocation, thereby initiating the immune activation program of RAW264.7 cells and exerting an immune-enhancing effect.

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A low molecular weight, high-purity glycoprotein from *Agaricus macrocarpa*, characterized in that, The molecular weight is 10-20 kDa. The *Stropharia macrocarpa* glycoprotein contains 75%-82% protein and 10%-18% polysaccharides. The protein in the *Stropharia macrocarpa* glycoprotein contains 17 amino acids, including glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline. The polysaccharide portion of the *Stropharia macrocarpa* glycoprotein... It includes fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose and mannose, wherein the molar ratio of fucose, rhamnose, arabinose, galactose, glucosamine, glucose, xylose and mannose is (0.9~1.1):(1~1.3):(0.6~0.9):(0.1~0.4):(0.6~0.9):(17~18):(1.8~2.2):(1.8~2.2).

2. The low molecular weight, high purity *Agaricus macrocarpa* glycoprotein according to claim 1, characterized in that, The secondary structure of the protein portion of the *Pleurotus ostreatus* glycoprotein is characterized by 5.6% α-helix, 29.8% β-sheet, 16.9% β-turn, and 47.7% random coil. The content ratio of glycine, alanine, aspartic acid, threonine, serine, glutamic acid, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline is (12.9~13.1):(13.8~14.0):(38.2~38). 4): (20.2~20.5): (29.2~29.5): (41.4~41.8): (126.4~126.8): (17.6~18.10): (15.7~16.1): (51.2~51.6): (10.2~10.7): (28.8~29.2): (19.0~19.4): (21.2~21.7): (20.1~20.6): (49.3~49.8): (88.5~89.0).

3. The method for preparing low molecular weight, high purity *Agaricus macrocarpa* glycoprotein according to claim 1 or 2, characterized in that, This includes the extraction, separation, and purification of glycoproteins from *Stropharia macrocarpa*. The extraction of the glycoprotein from *Stropharia macrocarpa* includes: mixing choline chloride and urea at a molar ratio of 1:2 to obtain a mixture; then adding 8% to 15% of the mixture's mass of water to the mixture; heating to a molten state; cooling; and then mixing thoroughly with *Stropharia macrocarpa* powder at a mass ratio of 1:(15 to 25); extracting for 1.5 to 2.5 hours; performing a first centrifugation to obtain a supernatant; adding 5 to 10 times the volume of water to the supernatant; incubating overnight at 3 to 6°C; and performing a second centrifugation to obtain a precipitate, which is the glycoprotein extract from *Stropharia macrocarpa*. The separation of the *Stropharia macrocarpa* glycoprotein includes: dissolving the *Stropharia macrocarpa* glycoprotein extract in water, then dialyzing it with a dialysis bag of 3000-4000 Da for 40-60 hours, collecting the liquid in the dialysis bag to obtain the dialysate; removing the free protein in the dialysate to obtain the supernatant, freeze-drying it to obtain the crude *Stropharia macrocarpa* glycoprotein. The purification of the *Pleurotus ostreatus* glycoprotein includes: dissolving the crude *Pleurotus ostreatus* glycoprotein in water, centrifuging, and filtering through a microporous membrane to obtain a sample solution; purifying the sample solution by anion exchange resin column chromatography, using ammonium sulfate solution as the eluent, concentrating the collected eluent, and then dialyzing with a 3000-4000 Da dialysis bag for 70-80 hours; and freeze-drying the dialysate to obtain the *Pleurotus ostreatus* glycoprotein.

4. The preparation method according to claim 3, characterized in that, The extraction of the glycoprotein from *Stropharia macrocarpa* includes: heating to a molten state at a temperature of 100℃~120℃; passing *Stropharia macrocarpa* powder through a 40~80 mesh sieve; the extraction includes stirring extraction at a speed of 300~500 r / min; and centrifugation at a speed of 5000~10000 rpm / min for a time of 10~15 min.

5. The preparation method according to claim 3, characterized in that, The separation of the Pleurotus ostreatus glycoprotein includes the removal of the free protein 3 to 4 times.

6. The preparation method according to claim 3, characterized in that, The purification of the *Agaricus bisporus* glycoprotein includes: centrifugation at a speed of 8000-12000 rpm / min; microporous membrane with a pore size of 0.22-0.45 μm; anion exchange resin column of type DEAE Focurose Fast Flow with a specification of Φ1.6 cm × 100 cm; elution flow rate of 1-3 mL / min; gradient elution, the gradient elution procedure being: (1) elution with water for 1 column volume; (2) elution with 0.1 M ammonium sulfate solution for 1 column volume; (3) elution with 0.3 M ammonium sulfate solution for 1 column volume; (4) elution with 0.5 M ammonium sulfate solution for 1 column volume; (5) elution with 0.7 M ammonium sulfate solution for 1 column volume; (6) elution with 1 M ammonium sulfate solution for 1 column volume; (7) elution with water for 2 column volumes.

7. The preparation method according to claim 6, characterized in that, After gradient elution, an automatic collector is used to collect 12 mL from each tube, collecting the eluent from tubes 40 to 46. The concentration is carried out under reduced pressure to 90 to 120 mL at a temperature of 32°C to 38°C.

8. The preparation method according to claim 3, characterized in that, The freeze-drying temperature is -50℃ to -40℃, and the freeze-drying time is 48h to 72h.

9. The use of the low molecular weight, high purity *Agaricus macrocarpa* glycoprotein as described in claim 1 or 2, or the *Agaricus macrocarpa* glycoprotein prepared by any of the preparation methods described in claims 3 to 8, in the preparation of products that enhance immune activity.

10. The application according to claim 9, characterized in that, The products include pharmaceuticals.

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