Yellow-rose yellow-meat boletus polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fungal polysaccharide application, and particularly relates to a yellow-rose yellow-meat boletus polysaccharide as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting crude polysaccharide from boletus rosea sporocarp through a water extraction and alcohol precipitation method, and then carrying out deproteinization, decoloration and DEAE-52 cellulose column chromatography separation and purification to obtain the purified polysaccharide BPP-1 of the boletus rosea water component. CCl4 is selected as an inducer of oxidative stress injury of the MIHA cells to construct a hepatic cell oxidative injury model, and the model is utilized to evaluate the protective effect of BPP-1 on CCl4-induced MIHA cell hepatic injury. The result shows that the BPP-1 reduces the release of ALT and AST, improves the GSH level and the SOD enzyme activity at the same time, inhibits the generation of a lipid peroxidation product MDA, and effectively reduces the damage of CCl4 to liver cells. The results show that BPP-1 has a certain protection effect on CCl4-induced hepatocyte injury, and BPP-1 may be a potential antioxidant and can alleviate oxidative injury of liver.

Description

Technical Field

[0001] This invention belongs to the field of fungal polysaccharide application technology, specifically relating to a polysaccharide of Boletus macrantha, its preparation method, and its application. Background Technology

[0002] Based on the exploration of Boletaceae fungal species resources, the *Boletus macrantha*, which is abundant in the wild and widely consumed in folk medicine, was selected. Butyriboletus roseoflavus As a research subject, the yellow-fleshed bolete is not only delicious and nutritious, but its protective effect against oxidative liver damage has not yet been reported. Summary of the Invention

[0003] Based on the above problems, the first technical solution of this application discloses a polysaccharide of Boletus macrantha, which uses a pyranose ring as the basic skeleton and is linked by β-glycosidic bonds. The monosaccharide composition and molar percentage are as follows: glucose (45.26%), galactose (30.45%), mannose (13.04%), fucose (8.97%), glucuronic acid (0.74%), ribose (0.49%), xylose (0.49%), arabinose (0.33%), rhamnose (0.16%) and galacturonic acid (0.07%).

[0004] The second technical solution of this application discloses a method for preparing the above-mentioned *Boletus edulis* polysaccharide, including the following steps: S1. Extraction of crude polysaccharide: The fruiting body powder of Boletus macrantha was extracted and concentrated by distilled water bath. The concentrated solution was dissolved in CaCl2, and the pH was adjusted to 8-9 and heated in a water bath to obtain the supernatant. The supernatant was decolorized and freeze-dried to obtain crude polysaccharide. S2. The crude polysaccharide was purified by column chromatography using distilled water as the eluent to obtain the polysaccharide of Boletus macrantha.

[0005] The third technical solution of this application discloses the application of the above-mentioned *Boletus macrantha* polysaccharide in the preparation of drugs to protect hepatocytes from oxidative damage.

[0006] Compared with the prior art, the present invention has the following beneficial effects: (1) In this invention, crude polysaccharide was extracted from the fruiting body of *Boletus macrantha* via water extraction and alcohol precipitation. The extracted polysaccharide was then purified by deproteinization, decolorization, and DEAE-52 cellulose column chromatography to obtain purified polysaccharide BPP-1 from the aqueous fraction of *Boletus macrantha*. Aqueous phase GPC analysis showed that the weight-average molecular weight (Mw) of BPP-1 was approximately 1.89 × 10⁻⁶. 4HPLC analysis showed that BPP-1 is mainly composed of glucose (Glc), galactose (Gal), and mannose (Man). UV spectroscopy revealed that BPP-1 contains almost no or only trace amounts of nucleic acids and proteins. Infrared spectroscopy analysis showed that BPP-1 contains characteristic peaks of OH stretching vibrations in sugar molecules, CH bond stretching vibrations specific to polysaccharides, and C=O bond stretching vibrations, exhibiting a pyranose ring structure and containing β-glycosidic bonds.

[0007] (2) This application selected CCl4 as an inducer of oxidative stress injury in MIHA cells, successfully constructed a hepatocyte oxidative injury model, and used this model to evaluate the protective effect of BPP-1 against CCl4-induced liver injury in MIHA cells. The results showed that BPP-1 effectively reduced CCl4-induced hepatocyte damage by reducing the release of ALT and AST, while increasing GSH levels and SOD enzyme activity, and inhibiting the generation of lipid peroxidation product MDA. This indicates that BPP-1 has a certain protective effect against CCl4-induced hepatocyte injury, and this protective effect may be closely related to its antioxidant and free radical scavenging capabilities. Therefore, BPP-1 may be a potential antioxidant that can reduce oxidative damage to the liver. Attached Figure Description

[0008] Figure 1 Elution curve of crude polysaccharide from Boletus rosenbergii; Figure 2 This is the standard curve for glucose. Figure 3 The results are from the GPC assay of BPP-1. Figure 4 The HPLC chromatogram of a mixed standard of 12 monosaccharides; Figure 5 The HPLC chromatogram of monosaccharides in BPP-1 is shown. Figure 6 The image shows the ultraviolet spectrum of BPP-1. Figure 7 Infrared spectrum of BPP-1; Figure 8 Results of BPP-1 cell viability assay; Figure 9 Results of experiments on the activity of different concentrations of CCl4 on MIHA cells; Figure 10 The effect of BPP-1 on ALT levels in the supernatant of MIHA cells after oxidative damage; Figure 11 The effect of BPP-1 on AST levels in the supernatant of MIHA cells after oxidative damage; Figure 12The effect of BPP-1 on intracellular GSH levels in oxidatively damaged MIHA cells; Figure 13 The effect of BPP-1 on intracellular MDA levels in oxidatively damaged MIHA cells; Figure 14 The effect of BPP-1 on SOD activity in oxidatively damaged MIHA cells. Detailed Implementation

[0009] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0010] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0011] Unless otherwise specified, the technical terms in this specification have the same meaning as those generally understood by those skilled in the art; however, in case of any conflict, the definitions in this specification shall prevail.

[0012] Example 1: Extraction and Composition Analysis of Polysaccharides from Boletus rosaefolius Extraction, separation and purification of polysaccharides from Boletus rosaefolius (1) Extraction of crude polysaccharides Weigh 300 g of Boletus macrantha fruiting body powder, add 4 times the amount of 95% ethanol, sonicate at 30 °C for 30 min, then centrifuge the mixture (3500 rpm) for 20 min, stir appropriately, discard the supernatant, recover the ethanol, and retain the precipitate.

[0013] Add distilled water at a material-to-liquid ratio of 1:20, extract in a water bath at 80 °C for 80 min, filter and retain the filtrate, repeat the extraction 3 times, combine the filtrates, and concentrate by rotary evaporation at 65 °C.

[0014] Place the concentrate on a magnetic stirrer and add 5% by volume of CaCl2 while stirring. After it is completely dissolved, add NaOH solution to adjust the pH to 8-9. Then place the solution in a water bath and heat it to 80 °C. Stop heating and let it cool to room temperature.

[0015] The supernatant and SP825 macroporous resin were added to a stoppered Erlenmeyer flask at a ratio of 10:1 and placed in a constant temperature shaker (37°C, 150 r / min) for 6 h for decolorization.

[0016] After decolorization, the polysaccharide filtrate was obtained by filtering it through macroporous resin with gauze. Four times the volume of 95% ethanol was added to the filtrate to induce precipitation, and the precipitate was allowed to stand overnight at 4 °C. The supernatant was discarded, the ethanol was recovered, and the precipitate was obtained. The precipitate was then freeze-dried to obtain crude polysaccharide.

[0017] (2) Separation and purification DEAE-52 anion exchange chromatography: First, fix the chromatography column vertically and pretreat the DEAE-52 cellulose. Slowly pour the pretreated DEAE-52 cellulose into the chromatography column, add an appropriate amount of distilled water, and allow it to equilibrate with deionized water for 12 h. Weigh 1 g of crude polysaccharide from Boletus macrantha, dissolve it in distilled water, and then centrifuge it at 12000 rpm for 15 min. Collect the supernatant and load it onto the sample.

[0018] Elution was performed using distilled water and NaCl solutions of different concentrations (0.1, 0.2, 0.4, 0.8, 1.0 mol / L), with the elution rate carefully controlled. An automatic collection device was set up to collect samples every 9 minutes and 30 seconds, with 9 mL collected per tube. Seven small tubes were combined and concentrated into a large tube of 10–20 mL, and the absorbance was measured. The eluents of different components were finally concentrated to 50–100 mL and placed in dialysis bags (molecular weight cutoff MwCo = 3500 Da). The samples were then dialyzed with distilled water. Finally, the dialyzed solutions were freeze-dried to obtain the various eluent fractions of *Boletus macranthum* purified polysaccharide for later use.

[0019] The yield of crude polysaccharide from *Boletus macrantha* in this embodiment was determined to be 9.56%, and it was a brownish-yellow powder. The elution curves after elution with distilled water and different concentrations of NaCl solutions (0.1, 0.2, 0.4, 0.8, 1.0 mol / L) in step (2) are shown below. Figure 1As shown, after elution with distilled water and NaCl solutions of different concentrations, crude polysaccharides from *Boletus macrantha* exhibited four significant elution peaks with marked differences in content. Based on the elution conditions, these peaks were named BPP-1 (eluted with distilled water), BPP-2 (eluted with 0.1 mol·L⁻¹), and BPP-3 (eluted with distilled water). -1 NaCl elution), BPP-3 (0.2 mol·L⁻¹) -1 NaCl elution), BPP-4 (0.4 mol·L⁻¹) -1 (NaCl elution). The eluates of each component were combined, concentrated, dialyzed, and freeze-dried to obtain purified polysaccharides. The total sugar content was then determined using the following method: ① Preparation of glucose standard solution: Weigh 1 g of glucose standard, dry it to constant weight at 105 °C, cool it for 30 min, accurately weigh 0.5 g, add an appropriate amount of distilled water to dissolve it, and dilute to volume in a 50 mL volumetric flask to prepare a glucose standard solution with a concentration of 10 mg / mL. Then accurately measure 1 mL of this solution and dilute to volume in a 100 mL volumetric flask to prepare a glucose standard solution with a concentration of 0.1 mg / mL for later use.

[0020] ② Establishment of the glucose standard curve: Preparation of 5% phenol solution: Place the brown bottle containing phenol in a 60 °C water bath and heat until the phenol is completely melted. Accurately measure 5 mL of the melted phenol and transfer it to a 100 mL brown volumetric flask. Add distilled water to the mark, mix well, and store in a refrigerator protected from light.

[0021] Add 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of 0.1 mg / mL glucose standard solution to six graduated tubes, respectively. Make up the volume to 2 mL with distilled water, then add 1 mL of phenol reagent to each tube, followed by 5 mL of concentrated sulfuric acid. Shake to mix thoroughly. Heat the mixture in a 100 °C water bath for 15 min, then quickly cool to room temperature. Measure the absorbance (OD) at 490 nm using a UV-Vis spectrophotometer. Repeat the measurement three times for each group to reduce random errors. Plot a glucose standard curve with the mass of the standard solution (mg) on ​​the x-axis and the absorbance at 490 nm on the y-axis. The standard curve is shown below. Figure 2 As shown.

[0022] ③ Determination of purified polysaccharide content: The content of purified polysaccharides was determined using the phenol-sulfuric acid method. Accurately weigh 10 mg of the purified polysaccharide aqueous fraction sample and place it in a 10 mL volumetric flask. Add a small amount of distilled water, gently shake, and then place in an ultrasonic cleaner for 30 min to accelerate dissolution. After complete dissolution, add distilled water to the mark to prepare a sample test solution with a concentration of 1 mg / mL. Measure 0.1 mL of the sample test solution into a graduated test tube and add distilled water to a final volume of 2 mL. Subsequent procedures were the same as for establishing the glucose standard curve. OD values ​​were measured at a wavelength of 490 nm. Each experiment was repeated three times to reduce random errors. Experimental data were processed and a standard curve was plotted. The regression equation was obtained through data analysis, and the content of purified polysaccharides was calculated. According to the standard curve calculation results, BPP-1 had the highest polysaccharide content, reaching 83.64%. Therefore, BPP-1 was selected as the research object for further purification and collection, and used in subsequent experiments.

[0023] Component analysis of polysaccharides from Boletus rosifolia (1) Determination of average molecular weight The molecular weight of BPP-1 polysaccharide from Boletus macrantha was determined using gel permeation chromatography (GPC). The specific steps were as follows: approximately 10 mg of sample was dissolved in 1 mL of water and mixed thoroughly. The dissolved solution was then filtered through a 0.22 µm molecular membrane using a syringe. 100 μL of the filtrate was injected into the GPC chromatography array using a microsyringe to begin the experiment.

[0024] Chromatographic conditions: Differential refractive index detection [Shimadzu RID-20A, Japan], chromatographic column (Shimadzu GPC-20A gel permeation chromatograph, Japan), column temperature 35 ℃, mobile phase 0.1N NaNO3 + 0.06% NaN3 aqueous solution, flow rate 0.6 mL / min, injection volume 100 μL.

[0025] like Figure 3 As shown in Table 1 (GPC determination results of BPP-1) and Table 1 (molecular weight determination results of BPP-1), a total of one elution peak was detected in the aqueous phase GPC of BPP-1. Figure 3 The weight-average relative molecular mass Mw is approximately 1.89 × 10⁻⁶. 4 Da (Table 1) indicates that the molecular weight distribution of polysaccharides is relatively narrow and the uniformity is relatively good.

[0026] Table 1. Molecular weight determination results of BPP-1 Peak Mn Mw Polydispersity 1 12622 18854 1.47 Note: Mn is the number-average molecular mass, Mw is the weight-average relative molecular mass, and Polydispersity is the molecular mass distribution coefficient.

[0027] (2) Determination of monosaccharide composition The monosaccharide composition of BPP-1 polysaccharide from Boletus roxburghii was determined and analyzed by high performance liquid chromatography (HPLC).

[0028] Control solution: Accurately weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, and fucose reference standards, dissolve and dilute with water to a mixed control solution containing 50 μg of each per mL.

[0029] Derivatization procedure: Accurately pipette 250 μL of the mixed control solution into a 5 mL EP tube, add 250 μL of 0.6 mol / L NaOH and 500 μL of 0.4 mol / L PMP-methanol, and react at 70 °C for 1 h. Cool in cold water for 10 min; neutralize with 500 μL of 0.3 mol / L HCl, then add 1 mL of chloroform, vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, and extract three times. Use the supernatant for HPLC analysis.

[0030] Hydrolysis of solid test samples: Accurately weigh an appropriate amount of sample into a 10 mL ampoule, add 3.0 mL of 2 mol / L TFA to the 10 mL ampoule, fill with nitrogen, seal the ampoule, and acid hydrolyze at 120 °C for 4 h. Take out 1.0 mL, add methanol and nitrogen to evaporate the TFA, and add 1 mL of water to reconstitute.

[0031] Hydrolysis of liquid test samples: Accurately transfer 0.2 mL of sample to a 10 mL ampoule, add 3.0 mL of 2 mol / L TFA to the 10 mL ampoule, fill with nitrogen, seal the ampoule, and acid hydrolyze at 120 °C for 4 h. Remove the above solution, add methanol and nitrogen to evaporate the TFA, and redissolve in 1 mL of water.

[0032] Sample solution derivatization: Accurately pipette 250 μL of sample solution into a 5 mL EP tube, add 250 μL of 0.6 mol / L NaOH and 500 μL of 0.4 mol / L PMP-methanol, and react at 70 ℃ for 1 h. Cool in cold water for 10 min; neutralize with 500 μL of 0.3 mol / L HCl, then add 1 mL of chloroform, vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, and extract three times. Collect the supernatant as the final product.

[0033] HPLC detection conditions: Instrument model: Shimadzu LC-20AD Chromatographic conditions: Xtimate C18 column (4.6 mm × 200 mm), particle size 5 μm; mobile phase: 0.05 M potassium dihydrogen phosphate solution (adjusted to pH 6.7 with sodium hydroxide solution) - acetonitrile = 83-17; detection wavelength: 250 nm; column temperature: 30 ℃; flow rate: 1 mL / min; injection volume: 20 μL.

[0034] Figure 4 The HPLC chromatogram is shown for a mixture of 12 monosaccharide standards. Figure 5 The figure shows the HPLC chromatogram of monosaccharides in BPP-1. By comparing the chromatograms with those of a mixed standard of 12 monosaccharides (including mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, and fucose), it can be determined that BPP-1 is composed of 10 monosaccharides, among which the main monosaccharide components are glucose (Glc), galactose (Gal), and mannose (Man).

[0035] Ultraviolet spectroscopy detection: The purified polysaccharide of Boletus rosaefolius was prepared into an aqueous solution of 1 mg / mL, and distilled water was used as a blank. The ultraviolet absorption spectrum was scanned in the range of 200~400 nm.

[0036] Figure 6 The image shows the ultraviolet spectrum of BPP-1. As can be seen from the image, the ultraviolet scanning curve of BPP-1 is relatively smooth, and no obvious absorption peaks appear at wavelengths of 260 nm and 280 nm, indicating that the polysaccharide does not contain or contains only trace amounts of impurities such as nucleic acids and proteins.

[0037] Infrared spectroscopy analysis: Weigh 2 mg of purified polysaccharide from Boletus roxburghii and mix it with an appropriate amount of KBr, compress it into tablets, and incubate at 4000~400 cm⁻¹. -1 Measure the infrared absorption spectrum.

[0038] Figure 7 The infrared spectrum of BPP-1 is shown in the figure, at 3397.96 cm⁻¹. -1 A broad and strong absorption peak appears at 2927.89 cm⁻¹, which is a characteristic peak of the stretching vibration of the OH group in the sugar molecule; while the mutual absorption peak at 2927.89 cm⁻¹ reflects the stretching vibration of the CH bond unique to polysaccharides; and the absorption peak at 1632.92 cm⁻¹... -1 The strong absorption peak is attributed to the stretching vibration of the C=O bond; 1418.87 cm⁻¹ -1 and 1373.07cm -1 The absorption peak at 1077.04 cm⁻¹ reflects the stretching and angle-shifting vibrations of the CH bonds in the polysaccharide. -1 The absorption peak at 1041.37 cm⁻¹ is due to the CO stretching vibration of the galactopyran ring. -11130.56 cm -1 The absorption peak at 886.61 cm⁻¹ is caused by vibrational absorption generated by the COH and COC structures. -1 The absorption peak at 806.58 cm⁻¹ corresponds to the absorption peak of the β-pyranoside bond. -1 The characteristic absorption peak of mannose is at 567 cm⁻¹. -1 The absorption peaks at the [specific location] are presumably due to the pyranose ring as the basic framework, consistent with the results of monosaccharide composition analysis. In summary, the infrared spectrum of BPP-1 exhibits typical characteristics of carbohydrate compounds, suggesting the presence of a pyranose ring structure and β-glycosidic bonds.

[0039] The above analysis shows that the molecular composition of the *Boletus macrantha* polysaccharide obtained in this application is as follows: it has a pyranose ring as its basic structure and is linked by β-glycosidic bonds to 45.26% glucose, 30.45% galactose, 13.04% mannose, 8.97% fucose, 0.74% glucuronic acid, 0.49% ribose, 0.49% xylose, 0.33% arabinose, 0.16% rhamnose, and 0.07% galacturonic acid.

[0040] Example 2: Protective effect of BPP-1 against CCl4-induced oxidative damage in MIHA cells Sample preparation Preparation of RPMI-1640 complete culture medium: Add 45 mL of RPMI-1640 cell basal culture medium to a 50 mL centrifuge tube, then add 5 mL of fetal bovine serum and 500 μL of penicillin-dextrose antibody, mix thoroughly, filter through a 0.22 μm microporous membrane for sterilization, and store at 4 ℃ for later use.

[0041] Preparation of saturated carbon tetrachloride solution: Add excess carbon tetrachloride solution to a centrifuge tube containing RPMI-1640 complete culture medium, seal the centrifuge tube, and shake for 10 min. Then, incubate the centrifuge tube at 37 ℃ for 24 h. Carbon tetrachloride crystals will precipitate at the bottom of the centrifuge tube, and the supernatant is the saturated carbon tetrachloride solution at 37 ℃. Aspirate the supernatant solution and store it at 37 ℃ for later use. Mix the saturated carbon tetrachloride solution thoroughly with normal RPMI-1640 low-serum culture medium at ratios of 1:3, 1:1, 3:1, and 1:0 to prepare 25%, 50%, 75%, and 100% carbon tetrachloride solutions, respectively.

[0042] Preparation of silymarin solution: silymarin was prepared to the required concentration using DMSO as a dissolving agent, and then filtered through a 0.22 μm microporous membrane for sterilization before use.

[0043] Polysaccharide sample preparation: Dissolve 8 mg of purified polysaccharide in 10 mL of RPMI-1640 complete medium, filter through a 0.22 μm microporous membrane for sterilization, and then serially dilute with complete medium to the required concentration. Prepare fresh before use.

[0044] 2. Cell Culture Cell resuscitation: Thaw cryovials rapidly in a sterile water bath at 37 ℃. Once the cell suspension is completely thawed, transfer the cells to centrifuge tubes and centrifuge at 1000 r / min for 5 min. Discard the supernatant. Add 4 mL of complete culture medium and repeatedly pipette at least 120 times to fully resuspend the cells. Transfer the cell suspension to a T25 culture flask and gently agitate the flask using the cross-shading method to ensure even cell distribution. Finally, incubate the flasks at 37 ℃ in a 5% CO2 incubator.

[0045] Cell passage culture: When the cells reached 80% confluence, passage culture was performed. First, the old culture medium in the T25 culture flask was aspirated, then the cells were washed twice with PBS solution. Next, 2 mL of trypsin was added, and the mixture was gently shaken several times. The cells were then incubated at 37 ℃ in a 5% CO2 cell culture incubator for 2 minutes and 30 seconds for digestion. After digestion, 4 mL of complete culture medium was added to stop the digestion, and the cells were gently detached by pipetting. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged, and the supernatant was discarded. 1 mL of complete culture medium was added, and the cells were repeatedly pipetted. The cells were then passaged at a 1:3 ratio into new culture flasks and incubated at 37 ℃ in a 5% CO2 incubator for further culture.

[0046] Cell cryopreservation: After the cells have adhered and grown to confluence, digest them with 2 mL of trypsin, incubate them in a cell culture incubator (37 ℃, 5% CO2) for 2 minutes and 30 seconds, add 4 mL of complete culture medium to stop the digestion, centrifuge, discard the supernatant, add 1 mL of serum-free cell cryopreservation solution and mix well by pipetting, transfer the cell suspension into cryovials, label them, freeze them at -80 ℃ for 4–5 days, and finally place the cryovials in a liquid nitrogen tank for long-term storage.

[0047] Effects of BPP-1 on MIHA cell viability Once the cells had adhered to the culture medium and reached 80% confluence, they were digested with trypsin, and 4 mL of complete culture medium was added. The cell suspension was centrifuged, the supernatant was discarded, and an appropriate amount of complete culture medium was added again to adjust the cell density to 5 × 10⁶ cells / year. 4Cells were seeded at a concentration of 100 μL / mL into 96-well plates and cultured for 24 h. The culture medium was then replaced. After cell adhesion, the old medium was discarded, and 100 μL of purified polysaccharide solution at different concentrations (50, 100, 200, 400, and 800 μg / mL) was added to each well. A blank control group (without cells) and a normal control group (containing cells but without the drug) were also set up. After another 24 h of culture, the old medium was removed, and 110 μL of a mixture of CCK-8 and complete culture medium (CCK-8: complete culture medium = 1:10) was added to each well. The plates were incubated for another 1.5 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader.

[0048] Figure 8 The results of the BPP-1 cell viability assay are shown in the figure. Compared with the normal control group, the cell viability remained above 100% within the concentration range of 50–800 μg / mL of purified polysaccharide from *Boletus macrantha* (BPP-1). This result indicates that BPP-1 did not exhibit cytotoxicity within this concentration range and promoted MIHA cell proliferation. Based on the above cell viability assay results, 100, 400, and 800 μg / mL were selected as safe dosage concentrations for BPP-1 in subsequent experiments.

[0049] 4. Establishment of an in vitro carbon tetrachloride-induced injury model in MIHA cells MIHA cells were seeded into 96-well plates at a density of 100 μL per well, with a density of 5 × 10⁶ cells / well. 4 / mL. After culturing at 37 ℃ and 5% CO2 for 24 h, and after cell adhesion, the old culture medium was discarded, and 100 μL of saturated carbon tetrachloride solution of different concentrations (25%, 50%, 75%, and 100%) was added to each well. A normal control group (without saturated carbon tetrachloride solution) was also set up. After culturing for another 24 h, the old culture medium was removed, and 110 μL of a mixture of CCK-8 and complete culture medium (CCK-8:complete culture medium = 1:10) was added to each well. The cells were incubated for another 1.5 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader to screen for the optimal concentration of carbon tetrachloride solution that causes oxidative damage to cells.

[0050] Figure 9 The results of the experiment on the activity of MIHA cells with different concentrations of CCl4 are shown in the figure. After treating MIHA cells with different concentrations (25%, 50%, 70% and 100%) of CCl4 for 24 h, the cell survival rate decreased in a dose-dependent manner with the increase of CCl4 concentration. When the CCl4 concentration was 100%, the cell survival rate dropped to about 50%, reaching the half-lethal concentration. Therefore, a 100% CCl4 concentration was selected to construct the cell oxidative damage model.

[0051] 5. Protective effect of BPP-1 against carbon tetrachloride-induced MIHA cell damage MIHA cells were seeded in T25 culture flasks at a density of 1.5 × 10⁶ cells / year. 6 / bottle, add 4 mL of complete culture medium to each bottle, incubate in a 37 ℃, 5% CO2 incubator for 24 h, then discard the old culture medium, wash the cell layer with PBS, and then replace with RPMI-1640 low serum culture medium for starvation treatment for 24 h. Discard the culture medium and wash twice with PBS buffer. Add three different concentrations of purified polysaccharide solution (100, 400 and 800 μg / mL, respectively) to each well, and continue to culture at 37 ℃, 5% CO2 for 24 h. Then discard the culture medium, and replace the other groups with 100% carbon tetrachloride saturated solution, except for the normal control group which is replaced with complete culture medium, and continue to culture at 37 ℃, 5% CO2 for 24 h. Collect the cell supernatant for subsequent assays; wash the cells with PBS, add cell lysis buffer for brief incubation, scrape off adherent cells, transfer cell debris and lysis buffer to centrifuge tubes, incubate on ice for 20 min, vortex 3 times during incubation, centrifuge, and transfer the cell pellet to a new centrifuge tube.

[0052] 6. Biochemical index determination Follow the instructions of the corresponding kit to detect the levels of ALT and AST in the cell supernatant, as well as the levels of GSH, MDA, and SOD in the cells using an ELISA reader.

[0053] Figure 10 The effect of BPP-1 on ALT levels in the supernatant of MIHA cells after oxidative damage is shown in the figure. Compared with the normal control group, the ALT level in the cell culture supernatant of the CCl4-damaged group was significantly increased. P < The result was 0.0001, confirming the successful establishment of the hepatocyte oxidative damage model. Compared with the model group, the addition of different concentrations of BPP-1 (100, 400 and 800 μg / mL) and the positive control drug silymarin (100 μg / mL) significantly reduced the ALT level in the cell supernatant. Among them, BPP-1 showed obvious dose dependence, that is, as the polysaccharide concentration increased, its effect on reducing ALT level gradually increased.

[0054] Figure 11 The effect of BPP-1 on AST levels in the supernatant of MIHA cells after oxidative damage is shown in the figure. Compared with the normal control group, the AST level in the cell culture supernatant of the CCl4-damaged group was significantly increased. P <The result was 0.01, indicating that the hepatocyte oxidative damage model was successfully established; compared with the model group, the positive control group silymarin (100 μg / mL) significantly reduced the alanine aminotransferase (AST) level in the cell supernatant. P < 0.01); Notably, compared to the model group, BPP-1 dose-dependently reduced AST levels in hepatocyte culture supernatant, and the differences were statistically significant at BPP-1 concentrations of 400 and 800 μg / mL ( ). P <0.05 or P < 0.01).

[0055] Figure 12 The effect of BPP-1 on intracellular GSH levels in oxidatively damaged MIHA cells is shown in the figure. Compared with the normal control group, the intracellular GSH level in the CCl4-damaged group was significantly reduced. P < The result was 0.0001, indicating that the hepatocyte oxidative damage model was successfully established; compared with the model group, the positive control group silymarin (100 μg / mL) could increase the intracellular GSH level ( P < 0.0001); Compared with the model group, different concentrations of BPP-1 (100, 400 and 800 μg / mL) significantly increased intracellular GSH levels, with the 100 μg / mL and 400 μg / mL groups showing highly significant differences ( ). P < 0.0001), the 800 μg / mL group showed a significant difference ( P <0.01).

[0056] Figure 13 The effect of BPP-1 on intracellular MDA levels in oxidatively damaged MIHA cells is shown in the figure. Compared with the normal control group, the intracellular MDA level in the CCl4-damaged group was significantly increased. P < The result was 0.0001, indicating that the hepatocyte oxidative damage model was successfully established; compared with the model group, the positive control group silymarin (100 μg / mL) significantly reduced the intracellular MDA level ( P < 0.0001); Compared with the model group, the addition of different concentrations of BPP-1 (100, 400 and 800 μg / mL) all reduced intracellular MDA levels and showed different degrees of significant differences. P < 0.0001 P < 0.0001 or P < 0.001), among which the MDA reduction effect was most significant when the BPP-1 concentration was 100 μg / mL ( P < 0.0001).

[0057] Figure 14The effect of BPP-1 on SOD activity in oxidatively damaged MIHA cells is shown in the figure. Compared with the normal control group, the intracellular SOD activity in the CCl4-damaged group was significantly reduced. P < The result was 0.0001, indicating that the hepatocyte oxidative damage model was successfully established; compared with the model group, the positive control group silymarin (100 μg / mL) significantly increased intracellular SOD activity ( P < 0.01); Compared with the model group, different concentrations of BPP-1 (100, 400 and 800 μg / mL) significantly increased intracellular SOD activity, with the 100 μg / mL and 800 μg / mL groups showing significant differences. P < 0.001), while the 400 μg / mL group showed the most significant increase in SOD activity ( P The result was < 0.0001, which is speculated to be due to the cells developing drug resistance at high drug concentrations.

[0058] In summary, this invention extracts crude polysaccharide from the fruiting body of *Boletus macrantha* via water extraction and alcohol precipitation, followed by deproteinization, decolorization, and purification by DEAE-52 cellulose column chromatography to obtain purified polysaccharide BPP-1 from the aqueous fraction of *Boletus macrantha*. Aqueous phase GPC analysis results show that the weight-average molecular weight (Mw) of BPP-1 is approximately 1.89 × 10⁻⁶. 4 HPLC analysis showed that BPP-1 is mainly composed of glucose (Glc), galactose (Gal), and mannose (Man). UV spectroscopy revealed that BPP-1 contains almost no or only trace amounts of nucleic acids and proteins. Infrared spectroscopy analysis showed that BPP-1 contains characteristic peaks of OH stretching vibrations in sugar molecules, CH bond stretching vibrations specific to polysaccharides, and C=O bond stretching vibrations, exhibiting a pyranose ring structure and containing β-glycosidic bonds.

[0059] This application selected CCl4 as an inducer of oxidative stress injury in MIHA cells, successfully constructed a hepatocyte oxidative injury model, and used this model to evaluate the protective effect of BPP-1 against CCl4-induced liver injury in MIHA cells. The results showed that BPP-1 effectively alleviated CCl4-induced hepatocyte damage by reducing the release of ALT and AST, while simultaneously increasing GSH levels and SOD enzyme activity, and inhibiting the generation of lipid peroxidation product MDA. This indicates that BPP-1 has a certain protective effect against CCl4-induced hepatocyte injury, and this protective effect may be closely related to its antioxidant and free radical scavenging capabilities. Therefore, BPP-1 may be a potential antioxidant capable of alleviating oxidative damage to the liver.

[0060] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A polysaccharide from *Boletus luridus*, characterized in that, The basic backbone is a pyranose ring linked by β-glycosidic bonds. The monosaccharide composition and molar percentage are as follows: glucose: 45.26%, galactose: 30.45%, mannose: 13.04%, fucose: 8.97%, glucuronic acid: 0.74%, ribose: 0.49%, xylose: 0.49%, arabinose: 0.33%, rhamnose: 0.16%, and galacturonic acid: 0.07%.

2. The method for preparing the polysaccharide of *Boletus edulis* according to claim 1, characterized in that, Includes the following steps: S1. Extraction of crude polysaccharides: The fruiting body powder of Boletus macrantha was extracted and concentrated by distilled water bath, and the concentrated solution was dissolved in CaCl2. The pH was then adjusted to 8-9 and heated in a water bath to obtain the supernatant. The supernatant was decolorized and freeze-dried to obtain crude polysaccharide; S2. The crude polysaccharide was purified by column chromatography to obtain the polysaccharide of Boletus macrantha.

3. The *Boletus edulis* polysaccharide prepared by the method according to claim 2.

4. The use of the *Boletus macrantha* polysaccharide according to claim 1 or 3 in the preparation of a drug to protect against oxidative damage to hepatocytes.

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