Polysaccharides from Agaricus bisporus with immunomodulatory activity and their uses

Abstract

This invention belongs to the field of pharmaceutical technology and relates to *Agaricus bisporus* polysaccharides with immunomodulatory activity and their uses. By optimizing process conditions such as alcohol concentration during alcohol precipitation, this invention uses a fractional precipitation method to prepare a polysaccharide mixture (*Agaricus bisporus* polysaccharide MP) and two high-purity homogeneous polysaccharides (*Agaricus bisporus* polysaccharide MP-1 and *Agaricus bisporus* polysaccharide MP-2). Experiments showed that these three *Agaricus bisporus* polysaccharides can restore the body weight of immunocompromised mice, increase spleen and thymus indices, improve splenic lesions, and enhance the release of IL-6, NO, and TNF-α from mouse serum. Compared to other unseparated *Agaricus bisporus* polysaccharide mixtures, these two polysaccharides exhibit significantly enhanced immunomodulatory activity. Therefore, they have potential applications in the preparation of health products or drugs that help enhance immunity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the polysaccharide of Agaricus bisporus with immunomodulatory activity and its uses. Background Technology

[0002] Button mushrooms (Agaricus bisporus), also known as white mushrooms or button mushrooms, belong to the genus Agaricus and are the most widely cultivated and consumed edible and medicinal fungus in the world. Besides being rich in nutrients beneficial to the human body such as protein, amino acids, vitamins, and minerals, button mushrooms also contain various bioactive components such as polyphenols, ergosterol, and polysaccharides. Pharmacological studies have shown that polysaccharides are the main active ingredient in button mushrooms, and existing research indicates that button mushroom polysaccharides possess antioxidant, immunomodulatory, and antitumor activities.

[0003] Immunity is a physiological function of the human body. The body uses immune organs, immune cells, and related immune factors to distinguish between "self" and "non-self" to maintain its health. Polysaccharides, especially fungal polysaccharides, play a particularly important role in regulating the immune system. They not only protect immune-related organs but also enhance the killing activity of natural killer cells, promote the secretion of TNF-α and IFN-γ by macrophages and lymphocytes, and fundamentally improve the body's immune function. They can also play a role by enhancing humoral immunity, such as activating the complement system, promoting the production of cytokines (interferons and interleukins), and inducing antibody production.

[0004] Polysaccharides are the main material basis for the immune activity of *Agaricus bisporus*. Currently, column chromatography, membrane separation technology, and fractionation precipitation are commonly used to separate polysaccharides to study the bioactivity of different polysaccharide components and promote further development and utilization of polysaccharides. However, existing methods for extracting *Agaricus bisporus* polysaccharides either involve numerous extraction steps and complex processes (requiring the addition of various organic reagents, enzymes, pH adjustment, or column chromatography purification), which are not conducive to industrial production; or the extracted *Agaricus bisporus* polysaccharides have low purity, consisting of a mixture of components with different molecular weights. For example, Chinese invention patent application "CN101891836A A method for ultrasonic-assisted extraction of *Agaricus bisporus* polysaccharides" requires ultrasonic treatment, Sevag method for protein removal (addition of chloroform and n-butanol), and macroporous resin decolorization, and the final polysaccharide purity is not high. Chinese invention patent application "CN105111322A A process for water bath oscillation-assisted enzymatic extraction of *Agaricus bisporus* polysaccharides" requires the addition of papain and pH adjustment, making the process complex and costly. The Chinese invention patent application "CN104877040B A method for extracting and purifying button mushroom polysaccharides from pre-cooked button mushroom liquid" requires pH adjustment and purification using ion exchange resin, making the process relatively complex.

[0005] Furthermore, literature reports that polysaccharides from different components exhibit varying bioactivities. Therefore, developing a rapid and precise method for separating different components of Agaricus bisporus polysaccharides is crucial for identifying the active polysaccharide components in mixed polysaccharides. However, there are currently no reports on the separation, preparation, and activity comparison of homogeneous polysaccharides with immunogenic activity from Agaricus bisporus. For example, Chinese invention patent application "CN105111322A A method for preparing and using Agaricus bisporus polysaccharides with immunogenic activity" uses ion exchange column chromatography and gel column chromatography to separate Agaricus bisporus polysaccharides with immunostimulatory effects. However, it does not analyze whether the separated Agaricus bisporus polysaccharides have superior immunostimulatory effects. Therefore, developing an extraction method for highly active and homogeneous Agaricus bisporus polysaccharides remains essential. However, on the one hand, there is a lack of research to confirm the most active components in Agaricus bisporus polysaccharides; on the other hand, adjustments to extraction parameters during the extraction process (e.g., the concentration of alcohol used in fractionation precipitation) significantly affect the composition of the extracted polysaccharides. Therefore, it is impossible to establish a method for extracting highly active Agaricus bisporus polysaccharides based on existing theories. This poses a significant obstacle to the efficient utilization of active substances in button mushrooms. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides Agaricus bisporus polysaccharide with immunomodulatory activity and its uses, with the aim of providing Agaricus bisporus polysaccharide with high activity in enhancing immunity, thereby making more efficient use of Agaricus bisporus resources.

[0007] A polysaccharide MP from Agaricus bisporus with immunomodulatory activity is prepared by a method comprising the following steps:

[0008] Step 1: Prepare an aqueous extract from button mushrooms;

[0009] Step 2: The water extract is separated using a membrane with a molecular weight cutoff of 500-3500 Da to obtain Agaricus bisporus polysaccharide MP.

[0010] Preferably, the specific process of step 1 is as follows:

[0011] Step 1.1: Extract the button mushrooms with water at a material-to-liquid ratio of 1:10-60 (w / v) at an extraction temperature of 80-100℃. Take the supernatant and concentrate it to 10-40% of the volume to obtain the concentrate.

[0012] Step 1.2: Add 4 times the volume of ethanol to the concentrate obtained in Step 1.1 for alcohol precipitation, and the product is obtained.

[0013] In step 2, the separation method is as follows: dialysis with a dialysis membrane until the conductivity stabilizes below 3, or ultrafiltration with an ultrafiltration membrane until no sugar is detected in the filtrate.

[0014] This invention also provides a mushroom polysaccharide MP-1 with immunomodulatory activity, which is prepared by a method comprising the following steps:

[0015] Step A: Precipitate the above-mentioned Agaricus bisporus polysaccharide MP in an aqueous solution with a final ethanol concentration of 30-50 vt%.

[0016] Step B: Separate the precipitate and supernatant. The resulting precipitate is Agaricus bisporus polysaccharide MP-1.

[0017] Preferably, in step A, the alcohol precipitation process involves preparing a 2-20 mg / mL solution of the Agaricus bisporus polysaccharide MP with water, adding ethanol to achieve a final ethanol concentration of 30-50 vt%, and allowing it to stand.

[0018] Preferably, its molecular weight ranges from 8.0 to 10⁻⁶. 6 -9.0×10 6 Da, the monosaccharide composition includes glucose, and the infrared spectral signal includes a pyranose ring and an α-configuration, with the signal of the pyranose ring located at 1079 cm⁻¹. -1 The signal of the α configuration is located at 843 cm. -1 Or 925 cm -1 .

[0019] This invention also provides a mushroom polysaccharide MP-2 with immunomodulatory activity, which is prepared by a method comprising the following steps:

[0020] Step I: Collect the supernatant obtained in Step B above;

[0021] Step II: Add ethanol to the supernatant to make the final ethanol concentration reach 60-80 vt%, and let it stand.

[0022] Step III: Separate the precipitate, purify it, and obtain Agaricus bisporus polysaccharide MP-2.

[0023] Preferably, in step III, the specific purification process is as follows: after redissolving in water, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff greater than 100 kDa until no sugar is detected in the filtrate, and the filtrate is collected; or dialysis is performed using a dialysis membrane with a molecular weight cutoff greater than 100 kDa until the conductivity stabilizes below 3.

[0024] Preferably, its molecular weight ranges from 1.5 × 10⁻⁶. 4 -2.0×10 4 Da, the monosaccharide composition includes glucose and galactose, and the infrared spectral signal includes the pyranose ring and the α configuration, with the signal of the pyranose ring located at 1079 cm⁻¹. -1 The signal of the α configuration is located at 843 cm. -1 Or 925 cm -1 .

[0025] The present invention also provides the use of the above-mentioned Agaricus bisporus polysaccharide MP, Agaricus bisporus polysaccharide MP-1 or Agaricus bisporus polysaccharide MP-2 in the preparation of health products or medicines that help enhance immunity.

[0026] The present invention also provides a health product or medicine that helps to enhance immunity, which is obtained by adding pharmaceutically acceptable excipients or auxiliary ingredients to the above-mentioned Agaricus bisporus polysaccharide MP, Agaricus bisporus polysaccharide MP-1 or Agaricus bisporus polysaccharide MP-2 as active substances.

[0027] In this invention, the ratio of material to liquid (w / v) is expressed in units of 1g:1mL.

[0028] This invention, through optimized process conditions such as alcohol concentration during alcohol precipitation, utilizes a fractional precipitation method to prepare a polysaccharide mixture (Agaricus bisporus polysaccharide MP) and two high-purity homogeneous polysaccharides (Agaricus bisporus polysaccharide MP-1 and Agaricus bisporus polysaccharide MP-2). The method provided by this invention is simple, has low production costs, and is suitable for large-scale application. Experiments have shown that these three Agaricus bisporus polysaccharides exhibit significantly enhanced immune-boosting activity, demonstrating promising applications in the production of immune-enhancing health products or drugs.

[0029] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0030] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Attached Figure Description

[0031] Figure 1 High performance gel chromatography coupled with multi-angle laser scattering and differential refractometer (HPSEC-MALLS-RID) for polysaccharides and isolated components from Agaricus bisporus.

[0032] Figure 2 Chromatograms (HPLC) of monosaccharide composition of Agaricus bisporus polysaccharide and its isolated components;

[0033] Figure 3 The infrared spectral characteristics (FT-IR) of Agaricus bisporus polysaccharide and its isolated components are shown.

[0034] Figure 4 Polysaccharides from Agaricus bisporus and their isolated components 1 H NMR spectrum;

[0035] Figure 5 Polysaccharides from Agaricus bisporus and their isolated components 13 C NMR spectrum;

[0036] Figure 6 The effect of Agaricus bisporus polysaccharide and its isolated components on the proliferation of RAW264.7 mouse macrophages ( Figure 6 A) Cells release NO ( Figure 6 B), IL-6 Figure 6 C) and TNF-α Figure 6 The effect result diagram of D);

[0037] Figure 7 Figure showing the effect of Agaricus bisporus polysaccharide and its isolated components on mouse body weight;

[0038] Figure 8 The effect of Agaricus bisporus polysaccharide and its isolated components on the spleen index in mice ( Figure 8 A) and thymus index ( Figure 8 B) Impact Result Diagram;

[0039] Figure 9 Figure 1 shows the effects of Agaricus bisporus polysaccharide and its isolated components on the morphology of mouse spleen tissue.

[0040] Figure 10 The effect of Agaricus bisporus polysaccharide and its isolated components on IL-6 in mouse serum ( Figure 10 A), NO ( Figure 10 B) and TNF-α Figure 10 C) Effect of content on the results. Detailed Implementation

[0041] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0042] Example 1: Agaricus bisporus polysaccharides MP, MP-1, and MP-2 and their preparation methods

[0043] The Agaricus bisporus polysaccharide in this embodiment was prepared by the following method:

[0044] (1) Dry and grind button mushrooms into powder. Extract them at 95°C for 1 h at a material-to-liquid ratio of 1:40 (w / v). Extract twice, combine the supernatants, and concentrate under reduced pressure to 20% volume.

[0045] (2) Add 4 times the volume of ethanol to the concentrate after vacuum concentration to precipitate, let stand for 12 h, centrifuge at 4000 g for 10 min, retain the precipitate, and redissolve it with water.

[0046] (3) After redissolving the precipitate, dialyze it using a 3500 Da dialysis bag for 4 days, changing the deionized water every 4 hours. Dialyze until the conductivity stabilizes (below 3). Finally, freeze-dry it to obtain the purified Agaricus bisporus polysaccharide, named Agaricus bisporus polysaccharide MP (hereinafter referred to as MP).

[0047] (4) The purified Agaricus bisporus polysaccharide was dissolved in deionized water to prepare a solution with a concentration of 10 mg / mL. 95% ethanol was added dropwise and stirred until the final ethanol concentration reached 38 vt%. After standing for 12 h, the solution was centrifuged at 6000 g for 15 min, and the precipitate was collected. The supernatant was collected. The precipitate was reconstituted with ultrapure water and freeze-dried to obtain the first isolated polysaccharide fraction, which was named Agaricus bisporus polysaccharide MP-1 (hereinafter referred to as MP-1).

[0048] (5) Add 95% ethanol to the clear liquid collected after centrifugation to make the final ethanol concentration reach 68 vt%. Let stand for 12 h, centrifuge at 6000 g for 15 min, retain the precipitate, add water to redissolve, transfer the redissolved liquid to an ultrafiltration centrifuge tube (molecular weight cutoff: >100 kDa, Millipore, Billerica, MA, USA), centrifuge at 4000 g for 1 h, centrifuge twice, collect the filtrate, add water and centrifuge multiple times, use phenol-sulfuric acid method to track and detect the sugar content in the filtrate until there is basically no sugar detected in the filtrate, freeze dry the filtrate to obtain the second separated component polysaccharide, named Agaricus bisporus polysaccharide MP-2 (hereinafter referred to as MP-2).

[0049] The technical solution of the present invention will be further illustrated by the following experiments. The samples MP, MP-1, and MP-2 tested in the following experimental examples were all prepared using the method of Example 1.

[0050] Experimental Example 1: Molecular Parameter Analysis

[0051] I. Experimental Methods

[0052] The molecular weight distribution of Agaricus bisporus polysaccharides before and after separation was determined by high-performance size exclusion chromatography (HPSEC-MALLS-RID) combining multi-angle laser scattering and differential refractive index detector. A Shodex OHpakSB-806M HQ (300 mm × 8.0 mm, id) gel column was used at 35 ℃. The mobile phase was 0.9% NaCl aqueous solution, the flow rate was 0.5 mL / min, the injection volume was 100 μL, and the dn / dc value of the mushroom polysaccharides was 0.138 mL / g. The sample concentration for MP and MP-1 detection was 0.6 mg / mL, while the sample concentration for MP-2 detection was 2 mg / mL.

[0053] II. Experimental Results

[0054] The results are as follows Figure 1 As shown, the original mushroom polysaccharide sample (MP) contained two polysaccharide components, while the polysaccharide MP-1 obtained through the first separation (fractional alcohol precipitation) and the mushroom polysaccharide MP-2 obtained through the second separation (fractional alcohol precipitation combined with ultrafiltration) each consisted of only one component. The results indicate that the first and second components of the mushroom polysaccharide were successfully separated and are homogeneous polysaccharides.

[0055] The average molecular weights of Agaricus bisporus polysaccharides before and after separation are shown in Table 1:

[0056] Table 1. Molecular weight and distribution of Agaricus bisporus polysaccharides and their isolated components

[0057]

[0058] Note: The molecular weight and dispersion coefficient of the two components of MP are different from those of MP-1 and MP-2. This is because MP is a crude extract of multiple components with lower purity, making the test results susceptible to the influence of impurities. In contrast, MP-1 and MP-2 are homogeneous polysaccharides with single components, resulting in more accurate test results.

[0059] Experiment Example 2: Basic Chemical Composition Analysis

[0060] I. Experimental Methods

[0061] The total arsenic and inorganic arsenic contents of *Agaricus bisporus* polysaccharides and their purified fractions were determined according to the methods specified in GB 5009.11-2014. The total arsenic content in *Agaricus bisporus* polysaccharides was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to Part I, Method I of GB 5009.11; the inorganic arsenic content was determined by liquid chromatography-atomic fluorescence spectrometry (LC-AFS) according to Part II, Method I of GB 5009.11. In addition, the total polysaccharide, total protein, and total uronic acid contents of the mushroom polysaccharides before and after separation were determined by the phenol-sulfuric acid method, the Coomassie brilliant blue method, and the m-hydroxybiphenyl method, respectively. The sample concentration was 0.1 mg / mL for total sugar content, 2 mg / mL for protein content, and 3 mg / mL for uronic acid content.

[0062] II. Experimental Results

[0063] The results are shown in Table 2. The yield of the first component (MP-1) after separation and purification was 3.26% ± 0.35% (relative to the raw material of *Agaricus bisporus* powder), and the yield of the second component (MP-2) was only 0.188% ± 0.02% (relative to the raw material of *Agaricus bisporus* powder). This indicates that the first component is the main component in *Agaricus bisporus* polysaccharide, a result similar to that of HPSEC-MALLS-RID analysis. After separation using ethanol fractionation precipitation (38% ethanol final concentration), the separated first component (MP-1) accounted for 74.09% of the polysaccharide component (MP) before separation. Furthermore, the total arsenic and inorganic arsenic contents in *Agaricus bisporus* polysaccharide and its purified components were further determined. The results showed that no inorganic arsenic was detected in the mushroom polysaccharides before and after separation, and the total arsenic content was below 0.05 mg / kg, indicating that the total arsenic content in fresh *Agaricus bisporus* was below 0.26 mg / kg, meeting food safety standards. The total sugar contents of MP, MP-1, and MP-2, determined by colorimetric method, were 77.99%, 91.32%, and 88.16%, respectively. Except for the first polysaccharide fraction (MP-1), which was not detected, both MP and MP-2 contained small amounts of protein, with contents of 4.99% and 5.04%, respectively. Furthermore, the mushroom polysaccharides before separation contained a small amount of uronic acid (1.79%), while uronic acid was not detected in the separated mushroom polysaccharides. These results indicate that polysaccharides are the main components of the separated first (MP-1) and second (MP-2) polysaccharide fractions, and the total sugar content is significantly increased compared to MP, indicating that MP-1 and MP-2 have high purity.

[0064] Table 2. Basic chemical composition of Agaricus bisporus polysaccharides and their isolated components.

[0065]

[0066] Note: MP, dried mushroom slices polysaccharide; MP-1 and MP-2, the first and second polysaccharide samples isolated from MP, respectively; the superscript ac indicates a significant difference in the chemical composition and content of mushroom polysaccharides before and after isolation. p <0.05); nd indicates that the component was not detected in the sample.

[0067] Experimental Example 3: Sugar Composition Analysis

[0068] I. Experimental Methods

[0069] The monosaccharide composition of Agaricus bisporus polysaccharides before and after separation was determined by high performance liquid chromatography combined with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone. 6.0 mg of Agaricus bisporus polysaccharide was weighed and hydrolyzed with trifluoroacetic acid (2.0 mol / L) at 95 °C for 10 h. Subsequently, the trifluoroacetic acid was evaporated, and the hydrolysate was dissolved in 1.0 mL of deionized water. 50.0 µL of the hydrolysate was mixed with 50.0 µL of NaOH (0.6 mol / L) and 100 µL of PMP (0.5 mol / L), and reacted at 70 °C for 100 min. The mixture was then neutralized with hydrochloric acid and brought to a final volume of 1.0 mL with ultrapure water. The mixture was then extracted four times with chloroform, and after separation, the organic layer was removed. Finally, the mixture was passed through a 0.22 µm organic filter membrane to determine the monosaccharide composition. The mixed standard was prepared by mixing mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose in a specific ratio. At an operating temperature of 30 °C, 20 µL of the derivative was injected into a high-performance liquid chromatography (HPLC) system equipped with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, id 5 µm, Agilent Technologies Inc., CA, USA), using a mixture of phosphate buffer and acetonitrile (82:18, v / v) as the eluent at a flow rate of 1.0 mL / min. The DAD wavelength was set to 245 nm.

[0070] II. Experimental Results

[0071] The results are as follows Figure 2 As shown: the mushroom polysaccharide before separation consists of galactose, glucose, mannose, glucuronic acid and xylose, while the mushroom polysaccharide after separation consists mainly of galactose and glucose, as well as a small amount of mannose.

[0072] The molar composition of monosaccharides in Agaricus bisporus polysaccharides before and after separation is shown in Table 3:

[0073] Table 3. Molar ratio of monosaccharide composition of Agaricus bisporus polysaccharide and its isolated components

[0074]

[0075] Note: MP, polysaccharide from dried slices of Agaricus bisporus; MP-1 and MP-2, polysaccharides from the first and second components isolated from MP, respectively.

[0076] Experiment Example 4 Infrared Spectroscopy Analysis

[0077] I. Experimental Methods

[0078] Fourier transform infrared spectroscopy was used to determine the infrared spectra of Agaricus bisporus polysaccharides before and after separation. 1.0 mg of sample was accurately weighed and mixed with 100.0 mg of dry potassium bromide. After careful grinding, the mixture was compressed into tablets, and the absorbance of the sample in the wavenumber range of 4000-500 cm-1 was detected by infrared spectroscopy.

[0079] II. Experimental Results

[0080] The results are as follows Figure 3 As shown: the infrared spectra of Agaricus bisporus polysaccharides before and after isolation are similar, indicating that the structures of the mushroom polysaccharides before and after isolation are quite similar. At 3400 cm⁻¹ -1 There is a strong absorption band at this point, which represents the stretching vibration of the hydroxyl group. 2928 cm⁻¹ -1 The absorption peak is caused by the asymmetric stretching vibration of CH, including the stretching vibrations of CH, CH2, and CH3. The 1152 cm⁻¹ peak... -1 The vibration at this point is an asymmetric COC vibration, indicating the presence of a methoxy group. 1079 cm⁻¹ -1 The vibration at this location indicates the presence of a pyranose ring in the mushroom polysaccharide. Furthermore, at 843 cm⁻¹... -1 Or 925 cm -1 The absorption peaks indicate the presence of the α configuration.

[0081] Experimental Example 5: Nuclear Magnetic Resonance Spectroscopy Analysis

[0082] I. Experimental Methods

[0083] 40 mg of Agaricus bisporus polysaccharide samples, both before and after separation, were weighed and dissolved in 1.0 mL of D2O. The solutions were allowed to stand overnight at room temperature, then freeze-dried, and 1.0 mL of D2O was added again. This process was repeated three times. Finally, the solutions were dissolved in 1.0 mL of D2O and placed in NMR tubes. One-dimensional NMR analysis of the polysaccharide samples was performed using an NMR spectrometer. 1 H and 13 C) Nuclear magnetic resonance spectrum.

[0084] II. Experimental Results

[0085] One-dimensional ( 1 H and 13 C) Nuclear magnetic resonance spectrum, such as Figure 4 and 5 As shown: The polysaccharide (MP) of Agaricus bisporus. 1 The H-NMR spectrum showed resonance peaks at δ5.41, δ5.15, δ5.08, and δ4.53. 13C-NMR spectra showed resonance peaks at δ102.9, δ99.8, and δ97.8, indicating that both α- and β-pyranoses exist in MP, but the proportion of α-pyranoses is much higher than that of β-pyranoses. Furthermore, based on NMR analysis results from literature on Agaricus bisporus polysaccharides, combined with the results of cellulose complex enzyme hydrolysis, monosaccharide composition, and infrared spectroscopy analysis in this experiment, the following residues are mainly present in Agaricus bisporus polysaccharides (MP): (1→4)-α-D-Glc, (1→6)-α-D-Glc, (1→6)-α-D-Gal, α-D-Gal-(1→), (1→4)-β-D-Glc, (1→6)-β-D-Glc, β-Man-(1→), and (1→2)-β-D-Man.

[0086] The specific analysis is as follows: The signal peak at δ 5.41 / 99.8 ppm is attributed to the H-1 / C-1 of (1→4)-α-Glc. Additionally, *Agaricus bisporus* polysaccharides also contain a high proportion of (1→6)-α-D-Glc, and the signal peak at δ 5.00 / 97.8 ppm can be attributed to the H-1 / C-1 of (1→6)-α-D-Glc. Furthermore, β-glucan has also been found in *Agaricus bisporus* polysaccharides, and the signal peak at δ 4.53 / 102.9 ppm is attributed to the H-1 / C-1 of (1→6)-β-D-Glc. Since cellulase (which hydrolyzes 1,4-β-D-glucan and converts it to glucose) can hydrolyze mushroom polysaccharides, producing a large amount of glucose hydrolysis products, this indicates that *Agaricus bisporus* polysaccharides contain β-1,4-glucan. Literature comparison further confirmed the presence of (1→4)-β-D-Glc residues in *Agaricus bisporus*. The chemical shift at δ 4.53 / 102.9 ppm was also attributed to the H-1 / C-1 signal in (1→4)-β-D-Glc. Monosaccharide composition results indicated that *Agaricus bisporus* polysaccharides contain a high proportion of glucose, as well as small amounts of galactose and mannose. NMR analysis also verified these results. The signal peaks at δ 5.08 / 97.8 ppm can be attributed to H-1 / C-1 of α-D-Gal-(1→). (1→6)-α-D-Gal residues are also present in mushroom polysaccharides, with the signal peak at δ 5.00 / 97.8 ppm corresponding to H-1 / C-1 in (1→6)-α-D-Gal. Additionally, the signal peak at δ 102.9 ppm is C-1 in β-Man-(1→). The chemical shift at δ 5.15 ppm is the H-1 signal in →2)-β-Man-(1→; the signal peak at 75.5 ppm corresponds to C-2 in →2)-β-Man-(1→).

[0087] The NMR spectrum shows that the first Agaricus bisporus polysaccharide fraction MP-1 after ethanol fractionation and precipitation has fewer signal peaks. Its anomeric hydrogen / anomeric carbon signals mainly occur at δ5.41 / δ99.8 ppm, indicating that the main chemical residues in MP-1 are (1→4)-α-D-Glc. The slight signal peak at δ5.00 ppm represents the anomeric hydrogen signals of (1→6)-α-D-Glc and (1→6)-α-D-Gal residues. The chemical shift at δ3.79 corresponds to the H-6 signal of β-Man-(1→ and →2)-β-Man-(1→). These results indicate that MP-1 is mainly composed of α-glucan, and may have a main chain consisting of repeating units of (1→4)-α-D-Glc, with (1→6)-α-D-Glc, (1→6)-α-D-Gal, α-D-Gal-(1→), and β-Man-(1→ and →2)-β-Man-(1→) residues as branches.

[0088] The HMR spectrum of *Agaricus bisporus* polysaccharide (MP-2) after a second fractional alcohol precipitation and ultrafiltration purification was generally similar to that of the pre-purification *Agaricus bisporus* polysaccharide. The signal peak at δ 5.41 / 99.8 ppm can be attributed to the H-1 / C-1 signal of the (1→4)-α-D-Glc residue. The weaker signal peak at δ 5.15 ppm is the anomeric hydrogen signal of (1→2)-β-D-Man. The signal peak at δ 5.08 ppm is the H-1 signal of α-D-Galp-(1→). The signal peak at δ 4.53 ppm is attributed to the anomeric hydrogens of (1→6)-β-D-Glc and (1→4)-β-D-Glc. It is noteworthy that... 1 In the H-NMR spectrum, the chemical peak signal at δ 5.01 ppm is stronger, representing the anomeric hydrogen signal of the (1→6)-α-D-Glc and (1→6)-α-D-Gal residues. 13The anomer region in the C-NMR spectrum is mainly represented by signals at δ102.9 ppm and δ97.8 ppm. The chemical shift at δ 102.9 ppm can be attributed to the anodic carbon signals of (1→4)-β-D-Glc, (1→6)-β-D-Glc, and β-Man-(1→) residues; the chemical shift at δ 97.8 ppm can be attributed to the anodic carbon signals of (1→6)-α-D-Glc, α-D-Galp-(1→), and (1→6)-α-D-Gal residues. The signal peaks at 75.5 and 73.0 ppm correspond to the C-2 signals of (1→2)-β-Man-(1→) residues and the C-3 signals of (1→4)-α-D-Glc residues, respectively. In addition, the monosaccharide composition results show that the molar ratio of Gal to Glc in MP-2 is close to 1:1, and the Gal content is significantly higher than that in MP and MP-2, and it also contains trace amounts of Man. Therefore, it can be inferred that MP-2 contains (1→6)-α-D-Gal, (1→6)-α-D-Glc, The main chain consists of (1→4)-β-D-Glc and (1→6)-β-D-Glc residues, and the side chains may contain (1→4)-α-D-Glc, α-D-Gal-(1→), β-Man-(1→ and →2)-β-Man-(1→) residues.

[0089] Experiment Example 6: Immunomodulatory Effects on Macrophages

[0090] I. Experimental Methods

[0091] (1) Culture of mouse macrophages RAW264.7

[0092] RAW264.7 cells were seeded in DMEM complete culture medium and cultured at 37°C with 5% CO2. When the cells reached about 80% confluence, they were passaged, and cells in the logarithmic growth phase were used for subsequent experiments.

[0093] (2) Assay of cell proliferation activity

[0094] MTT assay was used to assess the effect on RAW264.7 cell viability. RAW264.7 cell suspension (5 × 10⁴ cells / mL) was seeded into 96-well plates (100 μL / well) and cultured for 24 h at 37°C in a 5% CO₂ humidified incubator. After discarding the supernatant, MP, MP-1, and MP-2 at concentrations of 5, 20, 80, and 320 μg / mL were added. A blank control group and a positive control group of 1 μg / mL LPS were included, with three replicates per group. After 48 h of culture, 20 μL of MTT (5 μg / mL) solution was added, and the cells were cultured for another 4 h. The solution in the wells was removed, and 150 μL of DMSO was added to each well. The cells were shaken for 10 min to fully dissolve the crystals, and the OD value at 490 nm was measured using a microplate reader.

[0095] (3) Measurement of NO, TNF-α and IL-6 levels in RAW264.7 cells

[0096] The cell suspension concentration was adjusted to 2 × 10⁴ cells / mL, and 100 μL was seeded into 96-well plates and cultured at 37℃ and 5% CO₂ for 24 h. MP, MP-1, and MP-2 at concentrations of 5, 20, 80, and 320 μg / mL were added, with blank and positive control groups (LPS, 1 μg / mL) included, and each group was replicated in triplicate. The final volume of each well was adjusted to 200 μL, and the cells were cultured at 37℃ and 5% CO₂ for 48 h. The cell supernatant was then used for analysis. The levels of TNF-α and IL-6 cytokines were detected using an ELISA kit. Separately, another 96-well cell plate was filled with 100 μL of a 1:1 mixture of Griess A and Griess B reagent, and the plate was incubated in the dark for 20 min. The OD value was measured at 540 nm, and the NO content was calculated using NaNO₂ as the standard.

[0097] II. Experimental Results

[0098] The results of the cell proliferation activity experiment are as follows: Figure 6 As shown in Figure A, Agaricus bisporus polysaccharides at concentrations ranging from 5.0 to 320.0 μg / mL showed no toxicity to RAW264.7 macrophages after 48 h of treatment. Furthermore, the promoting effect on macrophages gradually increased with increasing Agaricus bisporus polysaccharide concentration. At a polysaccharide concentration of 80.0 μg / mL, the MP and MP-1 groups showed significantly stronger proliferative effects on RAW264.7 than the control group. Moreover, at a concentration of 320.0 μg / mL, the Agaricus bisporus polysaccharide groups (MP, MP-1, and MP-2) all showed significantly stronger proliferative effects on RAW264.7 than the control group.

[0099] The experimental results for NO, TNF-α, and IL-6 levels are as follows: Figure 6 As shown in B, C, and D: Agaricus bisporus polysaccharides and their isolated components significantly promoted the release of NO, the immunomodulatory factor IL-6, and TNF-α from mouse macrophages. As the sample concentration increased from 5.0 µg / mL to 320.0 µg / mL, the concentrations of NO, IL-6, and TNF-α released by mouse macrophages continuously increased, demonstrating that the ability of Agaricus bisporus polysaccharides to promote the release of NO, IL-6, and TNF-α from mouse macrophages was dose-dependent. Compared with the unisolated mushroom polysaccharide (MP), the isolated mushroom polysaccharides (MP-1 and MP-2) significantly promoted the release of NO, IL-6, and TNF-α from mouse macrophages under concentration gradients of 5.0, 20.0, 80.0, and 320.0 µg / mL.

[0100] The above in vitro experimental results show that the isolated Agaricus bisporus polysaccharides (MP-1 and MP-2) are homogeneous immunomodulatory components, and their activity is superior to that of the unisolated polysaccharide (MP).

[0101] The regulatory effect of Experiment 7 on immunocompromised mice

[0102] I. Experimental Methods

[0103] (1) Construction of a mouse immunosuppression model

[0104] Eighty SPF-grade female BALB / c mice (6 weeks old, weighing 19–21 g) were randomly divided into 8 groups (n = 10) and fed with free access to water and food. Except for a normal control group of 10 mice that received no treatment, the other 7 groups were intraperitoneally injected with 60 mg / kg cyclophosphamide (CTX) for 5 consecutive days to establish a CTX-induced immunosuppressive mouse model. Except for the CTX group, the other 6 groups were administered MP, MP-1, and MP-2 by gavage at doses of 100 mg / kg, 200 mg / kg, and 14 mg / kg respectively, starting from day 6, for 14 consecutive days. Blood was collected from the eyeballs of the animals the morning after the last administration. Mice were then sacrificed by cervical dislocation, and the blood was centrifuged to obtain serum, which was stored at 4°C. The thymus and spleen of the mice were collected.

[0105] (2) Effects on mouse body weight

[0106] During the experiment, the mice were observed daily for signs such as consciousness, mental state, appetite, and activity after the intervention. The weight of 8 groups of mice was weighed and recorded every day, and a curve was plotted based on the weight changes.

[0107] (3) Calculation of organ index

[0108] The thymus and spleen were dissected and their masses were precisely weighed using an electronic analytical balance. The thymus index and spleen index were calculated using the following formulas.

[0109] Thymus index = average thymus mass (mg) / average mouse body weight (g);

[0110] Spleen index = average spleen mass (mg) / average mouse body weight (g).

[0111] (4) Organizational morphology analysis

[0112] The spleen was fixed in 10% formalin solution, routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin, dehydrated and mounted, and the pathological morphology of the tissue was observed under an optical microscope.

[0113] (5) Determination of serum cytokine levels

[0114] Blood was collected from the eyeballs of animals the morning after the last administration of medication. Serum was obtained after centrifugation at 3000 r / min for 15 min and stored at 4℃. The NO content in the mouse serum was then measured using Griess reagent. The TNF-α and IL-6 levels in the serum were measured according to the ELISA kit instructions.

[0115] II. Experimental Results

[0116] (1) Effect on mouse body weight

[0117] The result is as follows Figure 7 As shown, mice in the Control group gained weight steadily over 20 days, while mice in the Model group (CTX group) experienced a sharp decline in weight starting from day 2, indicating successful model establishment. During treatment (starting on day 6), mice in the high-dose Agaricus bisporus polysaccharide group showed a stepwise increase in weight, eventually recovering to levels similar to the Control group, indicating that Agaricus bisporus polysaccharide can reverse CTX-induced weight loss in mice and possesses a certain immunomodulatory capacity. Furthermore, the isolated component MP-2 of Agaricus bisporus polysaccharide showed even better weight recovery in immunocompromised mice.

[0118] (2) Calculation of organ index

[0119] The result is as follows Figure 8 As shown, compared with the control group, the spleen and thymus indices of the model group mice (CTX group) were significantly reduced, indicating that CTX has a severe damaging effect on the spleen and thymus of mice, signifying successful model establishment. After administration of Agaricus bisporus polysaccharide, the immune organ indices of this group of mice increased linearly with increasing polysaccharide dosage. Compared with the model group, the spleen and thymus indices of the MP-1 and MP-2 groups at a concentration of 200 mg / kg were significantly increased. In addition, after administration of Agaricus bisporus polysaccharide, the immune organ indices of all groups of mice recovered, indicating that Agaricus bisporus polysaccharide and its isolated components have the effect of improving spleen and thymus damage in immunocompromised mice.

[0120] (3) Organizational morphology analysis

[0121] The result is as follows Figure 9 As shown, compared with the control group, the model group exhibited extensive congestion of spleen cells and infiltration of inflammatory factors, indicating that CTX had a severe damaging effect on the spleen of mice, and the model was successfully established. Compared with the model group, the spleen tissue structure damage in mice was partially restored after administration of Agaricus bisporus polysaccharide, and the improvement in spleen damage increased with increasing dosage. In addition, high-dose groups of Agaricus bisporus polysaccharide and its isolated components significantly improved the spleen lesions in immunocompromised mice, and the improved condition was close to that in the control group.

[0122] (4) Determination of serum cytokine levels

[0123] The result is as follows Figure 10 As shown, compared with the Control group, the levels of IL-6 and NO in the CTX group were significantly decreased, proving the successful establishment of the model. After administration of Agaricus bisporus polysaccharide, the levels of IL-6, NO, and TNF-α in mouse serum increased to normal levels, and the levels of each factor in the serum of mice in the polysaccharide group gradually increased with increasing polysaccharide dosage. This indicates that Agaricus bisporus polysaccharide can effectively regulate the abnormal cytokine secretion induced by CTX. In addition, in the low-dose groups of the three polysaccharide groups, MP-2 significantly enhanced the secretion of IL-6 and TNF-α factors in mouse serum compared with MP and MP-1. In the high-dose polysaccharide groups, MP-1 and MP-2 were more effective in promoting the release of IL-6, NO, and TNF-α cytokines.

[0124] The above in vivo experimental results further demonstrate that the isolated Agaricus bisporus polysaccharides (MP-1 and MP-2) are homogeneous immunomodulatory components.

[0125] As can be seen from the above embodiments and experimental examples, the present invention has extracted two novel Agaricus bisporus polysaccharides (MP-1 and MP-2), which can restore the body weight of immunocompromised mice, increase the spleen index and thymus index, improve spleen lesions, and enhance the release of IL-6, NO, and TNF-α in mouse serum, with better effects than the unextracted polysaccharide (MP). This indicates that these two novel polysaccharides have great application prospects in the preparation of health products or drugs that help enhance immunity.

Claims

1. A polysaccharide MP-1 from Agaricus bisporus with immunomodulatory activity, characterized in that: The Agaricus bisporus polysaccharide MP-1 is prepared by a method comprising the following steps: Step 1: Prepare an aqueous extract from button mushrooms; Step 2: The water extract is separated using a membrane with a molecular weight cutoff of 500-3500 Da to obtain Agaricus bisporus polysaccharide MP; Step 3: Precipitate the Agaricus bisporus polysaccharide MP in an aqueous solution with a final ethanol concentration of 30-50 vt%. Step 4: Separate the precipitate and supernatant. The precipitate obtained is Agaricus bisporus polysaccharide MP-1. The specific process of step 1 is as follows: Step 1.1: Extract the button mushrooms with water at a material-to-liquid ratio of 1:10-60 (w / v) at an extraction temperature of 80-100℃. Take the supernatant and concentrate it to 10-40% of the volume to obtain the concentrate. Step 1.2: Add 4 times the volume of ethanol to the concentrate obtained in Step 1.1 for alcohol precipitation, and the product is obtained. In step 2, the separation method is as follows: dialysis with a dialysis membrane until the conductivity stabilizes below 3, or ultrafiltration with an ultrafiltration membrane until no sugar is detected in the filtrate.

2. The Agaricus bisporus polysaccharide MP-1 according to claim 1, characterized in that: In step 3, the alcohol precipitation process involves preparing a solution of Agaricus bisporus polysaccharide MP with water at a concentration of 2-20 mg / mL, adding ethanol to achieve a final ethanol concentration of 30-50 wt%, and allowing it to stand.

3. The Agaricus bisporus polysaccharide MP-1 according to claim 1, characterized in that: The molecular weight range of the Agaricus bisporus polysaccharide MP-1 is 8.0 × 10⁻⁶. 6 -9.0×10 6 Da, the monosaccharide composition includes glucose, and the infrared spectral signal includes a pyranose ring and an α-configuration, with the signal of the pyranose ring located at 1079 cm⁻¹. -1 The signal of the α configuration is located at 843 cm. -1 Or 925 cm -1 .

4. A polysaccharide MP-2 from Agaricus bisporus with immunomodulatory activity, characterized in that: The Agaricus bisporus polysaccharide MP-2 is prepared by a method comprising the following steps: Step I: Collect the supernatant obtained in step 4 of the method for preparing the immunomodulatory polysaccharide MP-1 from Agaricus bisporus according to claim 1; Step II: Add ethanol to the supernatant to make the final ethanol concentration reach 60-80 vt%, and let it stand. Step III: Separate the precipitate, purify it, and obtain Agaricus bisporus polysaccharide MP-2.

5. The Agaricus bisporus polysaccharide MP-2 according to claim 4, characterized in that: In step III, the specific purification process is as follows: after redissolving in water, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff greater than 100 kDa until no sugar is detected in the filtrate, and the filtrate is collected; or dialysis is performed using a dialysis membrane with a molecular weight cutoff greater than 100 kDa until the conductivity stabilizes below 3.

6. The Agaricus bisporus polysaccharide MP-2 according to claim 5, characterized in that: The molecular weight range of the Agaricus bisporus polysaccharide MP-2 is 1.5 × 10⁻⁶. 4 -2.0×10 4 Da, the monosaccharide composition includes glucose and galactose, and the infrared spectral signal includes the pyranose ring and the α configuration, with the signal of the pyranose ring located at 1079 cm⁻¹. -1 The signal of the α configuration is located at 843 cm. -1 Or 925 cm -1 .

7. Use of the Agaricus bisporus polysaccharide MP-1 according to any one of claims 1-3 or the Agaricus bisporus polysaccharide MP-2 according to any one of claims 4-6 in the preparation of health products or medicines that help enhance immunity.

8. A health product or medicine that helps boost immunity, characterized in that: The health product or medicine is obtained by adding pharmaceutically acceptable excipients to Agaricus bisporus polysaccharide MP-1 as described in any one of claims 1-3 or Agaricus bisporus polysaccharide MP-2 as described in any one of claims 4-6 as the active substance.

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