Application of pleurotus eryngii purified polysaccharide in preparation of intestinal care medicine

Abstract

The invention discloses an application of pleurotus eryngii purified polysaccharide in preparation of an intestinal care medicine. The pleurotus eryngii purified polysaccharide component has high immunocompetence, high-activity and high-purity polysaccharide is obtained after ethanol precipitation and organic solvent purification, and the high biological activity of the polysaccharide is reserved; the composition has the biological activity of increasing the production of short-chain fatty acid, reducing the inflammation level, improving the intestinal flora structure, influencing the body metabolism condition and protecting the intestinal barrier. Compared with other components in the pleurotus eryngii polysaccharide extraction process, the polysaccharide component obtained by the preparation method has the highest purity and the strongest biological activity, and can be used for preparing intestinal care medicines.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology of Pleurotus eryngii, and particularly relates to the application of purified polysaccharides from Pleurotus eryngii in the preparation of intestinal care drugs. Background Technology

[0002] As the main active ingredient of Pleurotus eryngii, Pleurotus eryngii polysaccharides have been extensively studied and proven to possess various biological activities, primarily including immunomodulation, metabolic regulation, digestive enhancement, anti-tumor effects, and liver protection. They have broad health and medicinal value and a significant market presence. Current research mainly focuses on Pleurotus eryngii polysaccharides extracted by water extraction and alcohol precipitation, with limited research on the polysaccharide composition, structure, and activity of other components involved in the extraction process. Comparative studies of different components during the water extraction and alcohol precipitation of Pleurotus eryngii polysaccharides will be beneficial for the preparation of highly active Pleurotus eryngii polysaccharides, the exploration of their potential applications, improving the utilization efficiency of active Pleurotus eryngii polysaccharides, and reducing resource waste. Summary of the Invention

[0003] To overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide the application of purified polysaccharides from Pleurotus eryngii in the preparation of intestinal care drugs.

[0004] This invention is achieved through the application of purified polysaccharides from *Pleurotus eryngii* in the preparation of intestinal care drugs. The purified polysaccharides from *Pleurotus eryngii* include water-extracted polysaccharides and alcohol-soluble polysaccharides from *Pleurotus eryngii* prepared by the following methods:

[0005] (1) Slice king oyster mushrooms, freeze-dry them, grind them, and pass them through a 60-mesh sieve. Soak them in a freeze-dried powder: 85% ethanol solution at a mass ratio of 1:15 for 24 hours to decolorize and defatt them. Filter the solution to collect the precipitate. Extract the solution in a hot water bath at 60°C for 3-4 hours at a mass ratio of freeze-dried powder: water. Filter the solution through filter paper to remove the precipitate and obtain the extract. Rotary evaporate the extract to remove 2 / 3 of the water and concentrate it to obtain the concentrate.

[0006] (2) The concentrate and 95% to 100% ethanol were precipitated overnight in a volume ratio of 1:4. The mixture was then centrifuged at 10000g for 10 minutes to obtain the precipitate as component 1 and the supernatant as component 2.

[0007] (3) Component 1 was reconstituted with water at a mass ratio of precipitate:water = 1:1, and after removing the residual ethanol by rotary evaporation, it was freeze-dried to obtain water-extracted polysaccharide from king oyster mushroom; Component 2 was removed by rotary evaporation and then freeze-dried to obtain alcohol-soluble polysaccharide from king oyster mushroom.

[0008] Preferably, the purified polysaccharide from *Pleurotus eryngii* further includes high-purity *Pleurotus eryngii* polysaccharide prepared by the following method:

[0009] (4) The concentrated liquid: sevag = volume ratio 4:1 is stirred and mixed thoroughly for 20 minutes, centrifuged at 10000g for 15 minutes to remove denatured protein and organic solution layer, repeated 3 to 5 times until no protein is produced, and the organic solvent is removed by rotary evaporation to obtain supernatant;

[0010] (5) The supernatant obtained in step (4) was precipitated overnight with 95% to 100% ethanol in a volume ratio of 1:4. The precipitate was obtained by centrifugation at 10000g for 10 minutes, and then dissolved in water and freeze-dried to obtain high-purity Pleurotus ostreatus polysaccharide.

[0011] Preferably, in step (4), the sevag is chloroform: n-butanol in a volume ratio of 4:1.

[0012] This invention overcomes the shortcomings of existing technologies and provides an application of purified polysaccharides from Pleurotus eryngii in the preparation of intestinal care drugs, such as... Figure 1 As shown, this invention involves freeze-drying sliced ​​king oyster mushrooms, grinding them through a 60-mesh sieve, soaking them in a freeze-dried powder: 85% ethanol ratio of 1:15 for 24 hours to decolorize and defatt them, filtering to collect the precipitate, and then extracting it in a freeze-dried powder: water ratio of 1:25 at 60°C for 3-4 hours. The precipitate is removed by filter paper filtration to obtain an extract. The extract is then concentrated by rotary evaporation to remove 2 / 3 of the water, yielding a concentrated solution. This concentrated solution is then precipitated overnight in a 95%-100% ethanol ratio of 1:4 (v / v), centrifuged at 10000g for 10 minutes to obtain a precipitate (component 1) and a supernatant (component 2). Component 1 is reconstituted with water at a precipitate: water ratio of 1:1, and after rotary evaporation to remove residual ethanol, it is freeze-dried to obtain king oyster mushroom water-extracted polysaccharide. Component 2 is also obtained by rotary evaporation to remove ethanol and freeze-drying to obtain king oyster mushroom alcohol-soluble polysaccharide. Alternatively, the concentrated solution and sevag are thoroughly mixed at a volume ratio of 4:1 for 20 minutes, centrifuged at 10,000g for 15 minutes to remove denatured proteins and organic solvent layers, and repeated 3 to 5 times until no protein is produced. The organic solvent is removed by rotary evaporation to obtain the supernatant. The supernatant is then precipitated overnight with 95%–100% ethanol at a volume ratio of 1:4, centrifuged at 10,000g for 10 minutes to obtain the precipitate, reconstituted with water, and freeze-dried to obtain high-purity Pleurotus eryngii polysaccharide.

[0013] In this invention, to increase the polysaccharide extraction rate and better maintain polysaccharide activity, the water bath temperature is set at 60°C and the time is set at 3-4 hours during the preparation of the extract. To reduce waste, in the preparation of the concentrate, water is removed by rotary evaporation to reduce the amount of ethanol used, and excessive water removal is limited to prevent excessively high solution viscosity and the interaction between proteins and polysaccharides in the solution. To improve the purity of high-purity Pleurotus eryngii polysaccharides, Sevag is used to remove proteins 3-5 times. At least 3 times is to remove more proteins mixed in with the polysaccharides, and no more than 5 times is to reduce the loss of polysaccharides chelated with proteins. The choice of Sevag is also to maintain the polysaccharide activity as much as possible.

[0014] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects:

[0015] (1) Compared with other components in the extraction process of Pleurotus eryngii polysaccharide, the polysaccharide component obtained by the preparation method of the present invention has the highest purity and the strongest biological activity;

[0016] (2) The polysaccharide component extracted from Pleurotus ostreatus in this invention has high immunomodulatory activity. It is obtained by ethanol precipitation and purification with organic solvents, resulting in highly active and pure polysaccharides that retain the high biological activity of polysaccharides. It has biological activities such as increasing the production of short-chain fatty acids, reducing inflammation levels, improving the structure of intestinal flora, affecting the body's metabolism, and protecting the intestinal barrier. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation method of purified polysaccharide from king oyster mushroom according to the present invention;

[0018] Figure 2 It refers to the polysaccharide content of different purified polysaccharide components from king oyster mushrooms;

[0019] Figure 3 It shows the monosaccharide composition of different purified polysaccharide components from king oyster mushrooms before and after fermentation, including standard sample spectrum, polysaccharide spectrum before fermentation, and polysaccharide spectrum after fermentation;

[0020] Figure 4 These are the molecular weights of different purified polysaccharide components from king oyster mushrooms before and after fermentation, including polysaccharide spectra before fermentation, polysaccharide spectra after fermentation, and fermentation broth spectra.

[0021] Figure 5 The effects of different purified polysaccharide components from Pleurotus ostreatus on the production of short-chain fatty acids during in vitro fermentation are shown in the figure. The left figure shows the effect of different purified polysaccharide components from Pleurotus ostreatus on the production of acetic acid during in vitro fermentation, and the right figure shows the effect of different purified polysaccharide components from Pleurotus ostreatus on the production of butyric acid during in vitro fermentation.

[0022] Figure 6 The effect of different purified polysaccharide components from Pleurotus ostreatus on the amount of ROS produced in RAW264.7 cells before and after fermentation;

[0023] Figure 7 The effects of different purified polysaccharide components from Pleurotus ostreatus on alkaline phosphatase activity in Caco2-HT-29 cells before and after fermentation;

[0024] Figure 8 The effects of different purified polysaccharide components from Pleurotus ostreatus on the permeability of intestinal epithelial cells before and after fermentation;

[0025] Figure 9 The effects of different purified polysaccharide components from Pleurotus ostreatus on the integrity of intestinal epithelial cells before and after fermentation;

[0026] Figure 10 This is a flowchart of the experiment on mice;

[0027] Figure 11 The effect of high-purity Pleurotus ostreatus polysaccharide on intestinal barrier permeability in mice;

[0028] Figure 12 The effect of high-purity Pleurotus ostreatus polysaccharide on the proportion of immune cells in mice. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] Example 1

[0031] (1) Slice king oyster mushrooms, freeze-dry them, grind them, and pass them through a 60-mesh sieve. Soak them in a freeze-dried powder: 85% ethanol solution at a mass ratio of 1:15 for 24 hours to decolorize and defatt them. Filter the solution to collect the precipitate. Extract the solution in a hot water bath at 60°C for 3-4 hours at a mass ratio of freeze-dried powder: water. Filter the solution through filter paper to remove the precipitate and obtain the extract. Rotary evaporate the extract to remove 2 / 3 of the water and concentrate it to obtain the concentrate.

[0032] (2) The concentrate and 95% to 100% ethanol were precipitated overnight in a volume ratio of 1:4. The mixture was then centrifuged at 10000g for 10 minutes to obtain the precipitate as component 1 and the supernatant as component 2.

[0033] (3) Component 1 was reconstituted with water at a mass ratio of precipitate:water = 1:1, and after removing the residual ethanol by rotary evaporation, it was freeze-dried to obtain water-extracted polysaccharide from king oyster mushroom; Component 2 was removed by rotary evaporation and then freeze-dried to obtain alcohol-soluble polysaccharide from king oyster mushroom.

[0034] Example 2

[0035] (1) Slice king oyster mushrooms, freeze-dry them, grind them, and pass them through a 60-mesh sieve. Soak them in a freeze-dried powder: 85% ethanol solution at a mass ratio of 1:15 for 24 hours to decolorize and defatt them. Filter the solution to collect the precipitate. Extract the solution in a hot water bath at 60°C for 3-4 hours at a mass ratio of freeze-dried powder: water. Filter the solution through filter paper to remove the precipitate and obtain the extract. Rotary evaporate the extract to remove 2 / 3 of the water and concentrate it to obtain the concentrate.

[0036] (2) The concentrate: sevag = volume ratio 4:1 is stirred and mixed thoroughly for 20 minutes, centrifuged at 10000g for 15 minutes to remove denatured protein and organic solution layer, repeated 3 to 5 times until no protein is produced, and the organic solvent is removed by rotary evaporation to obtain supernatant;

[0037] (3) The supernatant obtained was precipitated overnight with 95% to 100% ethanol in a volume ratio of 1:4. The precipitate was obtained by centrifugation at 10000g for 10 minutes, and then dissolved in water and freeze-dried to obtain high-purity Pleurotus ostreatus polysaccharide.

[0038] Effect Example

[0039] The following experiments were conducted using purified polysaccharides from Pleurotus eryngii (including Pleurotus eryngii water-extracted polysaccharide and Pleurotus eryngii alcohol-soluble polysaccharide prepared in Example 1 and high-purity Pleurotus eryngii polysaccharide prepared in Example 2).

[0040] I. In vitro fermentation of purified polysaccharides from king oyster mushrooms:

[0041] 1. Preparation of basic culture

[0042] Yeast extract 2.00 g / L, peptone 2.00 g / L, NaCl 0.10 g / L, KH2PO4 0.04 g / L, K2HPO4 0.04 g / L, CaCl2·6H2O 0.01 g / L, MgSO4·7H2O 0.01 g / L, NaHCO3 2.00 g / L, heme chloride 0.02 g / L, cysteine ​​salt 0.50 g / L, bile salt 0.50 g / L, resazurin 1.00 mg / L, Tween 80 2.00 mL / L, vitamin K 10 μL / L, pH adjusted to 7.0 with 0.1 mol / L HCl, sterilized at 121℃ for 20 min, and stored in an anaerobic, sealed environment for later use.

[0043] 2. Preparation of fecal microbial suspension

[0044] Six healthy adults (aged 20-30, 3 men and 3 women) participated in the study, and none of them had taken antibiotics or other medications in the past three months. 5g of fresh fecal matter from each volunteer was weighed and added to a 50mL sterile centrifuge tube. 45mL of sterile saline solution was added, and the mixture was vortexed until homogeneous. The supernatant was collected by filtering through multiple layers of gauze. Then, equal volumes of fecal matter from each volunteer were mixed thoroughly to obtain a fecal microbiota suspension.

[0045] 3. In vitro anaerobic fermentation

[0046] The experiment included a blank control group and an experimental group. The blank control group consisted of 0.5 mL of fecal bacterial suspension and 4.5 mL of basal culture mixed in a 10 mL Erlenmeyer flask; the experimental group consisted of 0.5 mL of fecal bacterial suspension and 4.5 mL of basal culture mixed in a 10 mL Erlenmeyer flask, containing 50 mg of polysaccharide.

[0047] After mixing all samples thoroughly, immediately place them into an anaerobic bag containing an anaerobic pack, seal the bag, and incubate at 37°C for 24 hours. Afterward, remove the samples and perform vacuum evaporation and desalination treatment, then filter them through a 0.22μm filter membrane for later use.

[0048] II. Polysaccharide Content Determination

[0049] The determination was performed using the phenol-sulfuric acid method, with glucose as the standard curve. The results are as follows: Figure 2 As shown, high-purity Pleurotus ostreatus polysaccharide has the highest polysaccharide content.

[0050] III. Determination of Monosaccharide Composition

[0051] Accurately weigh 5 mg of polysaccharide into an ampoule, add 2 mL of 2M trifluoroacetic acid, and hydrolyze at 120 °C for 2 h. Accurately pipette the hydrolyzed solution and blow it dry under nitrogen. Add 2 mL of methanol and evaporate to dryness, repeating this process three times to remove residual trifluoroacetic acid. Add 400 μL of deionized water to completely dissolve the polysaccharide, add 400 μL of 0.5 M PMP and 200 μL of 0.3 M NaOH, vortex to mix, heat in a 70 °C water bath for 30 min, cool to room temperature, and neutralize with 200 μL of 0.3 M HCl. Add 2 mL of deionized water and 4 mL of chloroform for vortex extraction, centrifuge at 2000 rpm for 5 min, discard the lower chloroform layer, repeat three times, and filter the supernatant through a 0.22 μm filter membrane for analysis. Analyze using an Agilent 1100 high-performance liquid chromatograph (HPLC) with an Agilent C100 micrometer. 18 (250mm×4.6mm, 5μm) chromatographic column, mobile phase: tetrahydrofuran:100mM ammonium acetate:acetonitrile = 81:17:2, flow rate 0.5mL / min, injection volume 20μL, column temperature 25℃.

[0052] The results of the monosaccharide composition determination are as follows: Figure 3 As shown, all three polysaccharide samples contained four monosaccharides: mannose, glucose, galactose, and xylose. The monosaccharide composition of the high-purity *Pleurotus eryngii* polysaccharide was similar to that of the water-extracted *Pleurotus eryngii* polysaccharide, with glucose being the main monosaccharide. In contrast, galactose had the highest proportion in the alcohol-soluble *Pleurotus eryngii* polysaccharide. After in vitro fermentation, almost no other polysaccharides besides mannose were detected in any of the three polysaccharides. During fermentation, the polysaccharides were largely converted into mannose by the gut microbiota.

[0053] IV. Molecular weight determination

[0054] The molecular weight of polysaccharides was determined by high-performance liquid chromatography (HPLC). A TSK G4000 PWXL column (7.8 mm × 300 mm) equipped with an evaporative light scattering detector (ELSD) was used. Ultrapure water at a flow rate of 0.5 mL / min was used as the mobile phase, the column temperature was 30 °C, and the gas flow rate was 1.0 L / min. The sample concentration was 2 mg / mL, and the injection volume was 20 μL. A series of dextran standards (T-2000, T-70, T-40, T-10) were prepared into 1 mg / mL solutions using ultrapure water. A standard curve was plotted with retention time on the x-axis and the logarithm of molecular weight on the y-axis. The molecular weight of the polysaccharides was calculated using the standard curve.

[0055] The results are as follows Figure 4 As shown, high-purity Pleurotus eryngii polysaccharide has two components. Pleurotus eryngii alcohol-soluble polysaccharide has two components. Pleurotus eryngii water-extracted polysaccharide has three components. After fermentation, high-purity Pleurotus eryngii polysaccharide, alcohol-soluble polysaccharide, and water-extracted polysaccharide all have three components. The high molecular weight peak of the polysaccharide samples decreased significantly after fermentation, indicating that the polysaccharides were utilized by the microbial community.

[0056] V. Determination of Short-Chain Fatty Acids

[0057] Fermentation broth was collected at 0 and 48 hours. The pH of the supernatant was determined after centrifugation at 8000 rpm for 15 minutes. The concentration of SCFAs was determined by gas chromatography (GC) according to reported methods. Chromatographic separation was performed using an Agilent 8860GC system equipped with a DB-WAX (30 mm × 0.32 mm × 0.15 μm) and a flame ionization detector.

[0058] The conditions for gas chromatography analysis were as follows: nitrogen (carrier) flow rate was 1 mL / min, initial column temperature was 70℃, held for 1 min, then increased to 160℃ at a rate of 15℃ / min, held for 6 min, then increased to 210℃ at a rate of 30℃ / min, held for 3 min, injector and detector temperature was 250℃, and the flow rates of nitrogen, air and hydrogen in the detector were 25, 400 and 30 mL / min, respectively.

[0059] The results are as follows Figure 5As shown, all three polysaccharides can increase the production of short-chain fatty acids in the intestine, including acetic acid and butyric acid. Short-chain fatty acids have been shown to play a role in maintaining intestinal homeostasis, influencing intestinal epithelial cell function, promoting intestinal barrier integrity, and regulating immune cell differentiation and influencing inflammatory responses to maintain immune balance. Acetic acid is mainly related to regulating the growth of beneficial intestinal bacteria, while butyric acid is related to protecting the intestinal mucosal barrier. High-purity Pleurotus eryngii polysaccharides showed the most significant increase in short-chain fatty acids.

[0060] VI. ROS Measurement

[0061] RAW264.7 cells were cultured in a 37°C, 5% CO2 incubator using prepared complete medium (DMEM containing 10% FBS and 1% antibiotics), while HT-29 and Caco-2 cells were cultured in prepared complete medium (1640 medium containing 15% FBS and 1% antibiotics). Caco-2 and HT-29 cells were mixed at a 9:1 ratio and incubated at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / mL into the upper chamber of a Transwell cell culture plate and co-cultured in Caco-2-HT-29 for 21 days. RAW264.7 was then added to the lower chamber. The culture medium was changed every two days during the first week and daily thereafter.

[0062] Before the experiment, the complete culture medium was replaced with serum-free culture medium for synchronization treatment for 4 hours. Then, cultured in culture medium containing the corresponding polysaccharide for 12 hours, and 0.5 μg / mL LPS was added to the upper chamber of the Transwell cell culture plate for induction for 6 hours.

[0063] Collect lower ventricular cells at a rate of 1×10 6 Cells / mL were resuspended in DCFH-DA dissolved in 2 μmol / L PBS and incubated at 37°C with 5% CO2 for 20 min. Unfilled DCFH-DA was removed by washing three times with PBS. Measurements were taken on black 96-well plates using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0064] The results are as follows Figure 6 As shown, LPS significantly increased ROS production in RAW264.7 cells, indicating that LPS significantly activated the inflammatory response in RAW264.7 cells, putting the cells under oxidative stress. The addition of Pleurotus eryngii polysaccharides significantly reduced cellular ROS production and alleviated cellular oxidative stress.

[0065] VII. Alkaline phosphatase activity assay

[0066] The culture medium from the upper chamber was collected, and the activity of alkaline phosphatase (AKP / ALP) in tissues and blood was measured according to the instructions of the kit. In an alkaline environment, AKP / ALP catalyzes the formation of free phenol from disodium phenyl phosphate; the phenol reacts with 4-aminoantipyrine and potassium ferricyanide to form a red quinone derivative, which exhibits characteristic light absorption at 510 nm to detect enzyme activity.

[0067] The results are as follows Figure 7 As shown, alkaline phosphatase plays a crucial role in maintaining the stability of the intestinal mucosal barrier and intestinal function. Alkaline phosphatase activity is closely related to the absorptive capacity of small intestinal epithelial cells, reducing the toxicity of intestinal lipopolysaccharides, preventing and reducing intestinal inflammation, regulating intestinal flora distribution, and inhibiting bacterial translocation. LPS significantly reduced alkaline phosphatase activity in the Caco-2-HT-29 intestinal epithelial cell model, while the addition of Pleurotus eryngii polysaccharide significantly increased alkaline phosphatase activity in the Caco-2-HT-29 intestinal epithelial cell model under the influence of LPS. Among these, high-dose fermented high-purity Pleurotus eryngii polysaccharide showed the most significant increase in alkaline phosphatase activity.

[0068] 8. Intestinal permeability

[0069] After rinsing the cell surface in the upper chamber twice with PBS, sodium fluorescein dissolved in PBS was added to the upper chamber. The chamber was then placed in a cell culture plate containing 2 mL of PBS. After 30 minutes, the PBS in the lower chamber was collected, and measurements were taken on a black 96-well plate using an excitation wavelength of 490 nm and an emission wavelength of 513 nm.

[0070] The results are as follows Figure 8 As shown, the experiment assessed the integrity and permeability of intestinal epithelial cells by monitoring the permeation of sodium fluorescein in an intestinal epithelial cell model. The results showed that LPS significantly increased sodium fluorescein permeation, indicating that LPS disrupted the intestinal epithelial cell model. The addition of polysaccharides significantly reduced the permeation of sodium fluorescein through the intestinal epithelial cell monolayer, meaning that polysaccharides mitigated the damage caused by LPS to the intestinal epithelial cell model. Specifically, high-dose fermented high-purity Pleurotus eryngii polysaccharide and high-dose fermented Pleurotus eryngii water-extracted polysaccharide significantly reduced the LPS-induced disruption of the intestinal epithelial cell model.

[0071] IX. Transmembrane resistance (intestinal epithelial integrity)

[0072] The resistance was measured using a resistance meter (Millicell ERS-2, Merck KgaA, Darmstadt, Germany).

[0073] The results are as follows Figure 9As shown, transmembrane resistance is a simple method for measuring the integrity of tight junction dynamics in epithelial monolayer cell culture models. Transmembrane resistance quantitatively reflects the integrity of the intestinal epithelial cell model. Results showed that LPS significantly reduced the transmembrane resistance of the intestinal epithelial cell model, indicating that LPS disrupted the integrity of intestinal epithelial cells. The addition of *Pleurotus eryngii* polysaccharide increased the transmembrane resistance of the intestinal epithelial cell model, indicating that *Pleurotus eryngii* polysaccharide mitigated the damage caused by LPS to the intestinal epithelial cell model. The effect was most significant with high-dose fermented, high-purity *Pleurotus eryngii* polysaccharide, with its measured transmembrane resistance value approaching that of the blank control group.

[0074] 10. Measurement of intestinal barrier permeability in mice

[0075] The experimental procedure is as follows Figure 10 As shown, mice were fasted for 4 hours before the experiment and then administered 20 mL / kg of 25 mg / mL FITC-Dextran solution dissolved in PBS by gavage. Plasma was collected three hours later, and the plasma was diluted with PBS at a ratio of 1:3. A standard curve of FITC-Dextran was prepared using 25% normal mouse plasma diluted in PBS as the standard diluent. Measurements were taken on black 96-well plates using an excitation wavelength of 490 nm and an emission wavelength of 513 nm.

[0076] The results are as follows Figure 11 As shown, the effects of Pleurotus eryngii polysaccharide on in vivo were investigated using mouse experiments. High-purity Pleurotus eryngii polysaccharide significantly reduced the abnormally high intestinal permeability caused by LPS-induced intestinal barrier dysfunction in mice, and this effect was dose-dependent.

[0077] XI. Mouse Immune Cell Assay

[0078] After the experiment, mouse colon cells were collected and prepared into a single-cell suspension using PBS. 2 × 10⁻⁶ cells were then collected. 6 Cells were specifically stained with CD3, CD4, CD8, and CD45, and 10,000 cells were collected by FACSCalibur flow cytometry before FlowJo analysis.

[0079] The results are as follows Figure 12 As shown, LPS significantly reduced the number of helper T cells and cytotoxic T cells in the mouse colon, and CD4 + Cells significantly decreased to the point of disappearance, indicating immunosuppression and disruption of immune function in mice. Under high-dose, high-purity *Pleurotus eryngii* polysaccharide feeding, the number of helper T cells and cytotoxic T cells in mouse colonic immune cells significantly increased, as did CD4+. + An increase in cells indicates that the immune function is relatively intact.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of purified polysaccharides from Pleurotus eryngii in the preparation of intestinal care drugs, characterized in that, The purified polysaccharides from Pleurotus eryngii include Pleurotus eryngii water-extracted polysaccharides and Pleurotus eryngii alcohol-soluble polysaccharides prepared by the following methods: (1) Slice king oyster mushrooms, freeze-dry them, grind them, and pass them through a 60-mesh sieve. Soak them in a freeze-dried powder: 85% ethanol solution at a mass ratio of 1:15 for 24 hours to decolorize and defatt them. Filter the solution to collect the precipitate. Extract the solution in a hot water bath at 60°C for 3-4 hours at a mass ratio of freeze-dried powder: water. Filter the solution through filter paper to remove the precipitate and obtain the extract. Rotary evaporate the extract to remove 2 / 3 of the water and concentrate it to obtain the concentrate. (2) The concentrate and 95% to 100% ethanol were precipitated overnight in a volume ratio of 1:

4. The mixture was then centrifuged at 10000g for 10 minutes to obtain the precipitate as component 1 and the supernatant as component 2. (3) Component 1 was reconstituted with water at a mass ratio of precipitate:water = 1:1, and after removing the residual ethanol by rotary evaporation, it was freeze-dried to obtain water-extracted polysaccharide from king oyster mushroom; Component 2 was removed by rotary evaporation and then freeze-dried to obtain alcohol-soluble polysaccharide from king oyster mushroom.

2. The application as described in claim 1, characterized in that, The purified polysaccharide from Pleurotus ostreatus also includes high-purity Pleurotus ostreatus polysaccharide prepared by the following methods: (4) The concentrated liquid: sevag = volume ratio 4:1 is stirred and mixed thoroughly for 20 minutes, centrifuged at 10000g for 15 minutes to remove denatured protein and organic solution layer, repeated 3 to 5 times until no protein is produced, and the organic solvent is removed by rotary evaporation to obtain supernatant; (5) The supernatant obtained in step (4) was precipitated overnight with 95% to 100% ethanol in a volume ratio of 1:

4. The precipitate was obtained by centrifugation at 10000g for 10 minutes, and then dissolved in water and freeze-dried to obtain high-purity Pleurotus ostreatus polysaccharide.

3. The application as described in claim 2, characterized in that, In step (4), the sevag is chloroform: n-butanol in a volume ratio of 4:1.

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