Preparation method of lentinan extract, product and application thereof

Abstract

The application discloses a preparation method of lentinan extract, a product and application thereof, and discloses that the beta-glucan content of eight lentinus edodes varieties is quite different, and the beta-glucan content of the fruiting body of Hu Xiang F2 is the highest. The lentinan extract prepared by the application has the activity of repairing liver injury, and can inhibit the proliferation of tumor cells by activating macrophages and enhancing the phagocytosis of the macrophages.

Description

Technical Field

[0001] This invention belongs to the field of lentinan technology, specifically relating to a method for preparing lentinan extract, its products, and applications. Background Technology

[0002] Functional sugars, as an important ingredient in functional foods, have been widely used in many health and functional foods. Especially with the rapid development of functional foods, beverages, and dairy products containing functional sugars, their market size is expanding dramatically. Although numerous functional sugar products have appeared on the market, our understanding of their mechanisms of action, the evaluation and identification of their activity levels, and the relationship between structure and activity is still very limited. In particular, the lack of clarity regarding the structure-activity relationship has brought functional sugar research to a bottleneck, preventing researchers from designing, constructing, and screening highly active functional sugar formulations based on their structure.

[0003] β-glucan, as a functional sugar with anti-tumor activity, has attracted attention for its advantages of being non-toxic and highly effective. Its main chain is linked by β-1,3-glucosidic bonds and usually also has β-1,6 branches of a certain proportion and size.

[0004] Shiitake mushrooms (Lentinus edodes (Berk.) Pegler), second only to button mushrooms, are the world's second most consumed edible fungus. Shiitake mushrooms are a traditional Chinese medicine and a common edible fungus. They are recorded in traditional Chinese medicine books such as *Daily Materia Medica* and *Benjing Fengyuan* as having "neutral medicinal properties, a slightly sweet taste, and invigorating qi and nourishing the stomach; they can treat loss of appetite and chickenpox." Modern medical research has found that shiitake mushrooms are rich in nutrients, including protein, fat, polysaccharides, dietary fiber, and various vitamins. As a natural edible resource, shiitake mushrooms are receiving increasing attention for their redevelopment and utilization in the pharmaceutical, cosmetic, and other industries. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a method for preparing lentinan extract, which comprises the following steps:

[0007] (1) Dry and crush the fruiting bodies of shiitake mushrooms, add ethanol solution for ultrasonic extraction, collect the residue, add sodium chloride aqueous solution, heat and extract, centrifuge and collect the precipitate, add water to the precipitate, heat and extract, centrifuge and collect the precipitate, add NaOH solution and NaBH solution to the precipitate and let it stand, centrifuge and collect the supernatant, adjust the pH to neutral, centrifuge and collect the supernatant, and dialyze to obtain crude extract of shiitake mushroom polysaccharide.

[0008] (2) The crude extract of lentinan was mixed with a 0.02 mol / L aqueous solution of trifluoroacetic acid and heated to hydrolyze, thereby obtaining the lentinan extract.

[0009] As a preferred embodiment of the preparation method of the shiitake polysaccharide extract of the present invention, the shiitake mushroom strain is the Hu Xiang F2 strain.

[0010] As a preferred embodiment of the preparation method of the shiitake polysaccharide extract of the present invention: in step (1), the addition of ethanol solution for ultrasonic extraction is to add 8-10 times the volume of 95% ethanol solution of shiitake fruiting body and ultrasonically extract for 1 hour.

[0011] As a preferred embodiment of the preparation method of the shiitake polysaccharide extract of the present invention: in step (1), the addition of sodium chloride aqueous solution and heating extraction is to add 0.9wt% sodium chloride aqueous solution, wherein the mass-volume ratio of the residue to the 0.9wt% sodium chloride aqueous solution is 1g:20mL, and heating is carried out at 60℃ for 12h.

[0012] As a preferred embodiment of the preparation method of the shiitake polysaccharide extract of the present invention: in step (1), water is added to the precipitate and heated for extraction, the mass-volume ratio of residue to water is 1g:20mL, the heating temperature is 100℃, and the heating time is 2h.

[0013] As a preferred embodiment of the preparation method of the lentinan extract of the present invention: the step of adding NaOH solution and NaBH solution to the precipitate and letting it stand is to add 5wt% NaOH aqueous solution and 0.05% NaBH aqueous solution to the precipitate and let it stand at 20-25°C for 4-6 hours.

[0014] As a preferred embodiment of the preparation method of the lentinan extract of the present invention: step (2) is to mix the crude lentinan extract with a 0.02mol / L trifluoroacetic acid aqueous solution at a ratio of 1mg:200μL, and hydrolyze at 100-110℃ for 2h to obtain the lentinan extract.

[0015] The main component of the lentinan extract obtained by the aforementioned preparation method is β-glucan, and the monosaccharide composition of the lentinan extract is (%): glucose: galactose: mannose = 81.45: 13.55: 5

[0016] As another aspect of the present invention, the present invention also provides the application of the lentinan extract obtained by the preparation method described above in the preparation of antioxidant, liver damage repair, and anti-tumor products.

[0017] The beneficial effects of this invention: Using β-glucan content as a reference indicator, this invention found significant differences in β-glucan content among the fruiting bodies of eight shiitake mushroom varieties, with the highest β-glucan content observed in the fruiting bodies of Huxiang F2. The shiitake polysaccharide extract prepared by this invention exhibits liver damage repair activity and can inhibit tumor cell proliferation by activating macrophages and enhancing their phagocytic capacity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0019] Figure 1 This is a flowchart of the extraction process for lentinan LF3 from shiitake mushrooms.

[0020] Figure 2 Gel column chromatography images of 0.02 mol / L TFA hydrolyzed at different times.

[0021] Figure 3 These are column chromatography images of TFA hydrolysis gels at different concentrations.

[0022] Figure 4 Gel column chromatography images of 2 mol / LTFA hydrolyzed at different times.

[0023] Figure 5 Gel column chromatography images of hydrolysis with 0.02 mol / L FA for 2 h (A), 0.1 mol / L FA for 2 h (B), and 2 mol / L FA for 2 h (C).

[0024] Figure 6 The image shows the high-performance liquid chromatography (HPLC) spectrum of LF301.

[0025] Figure 7 This demonstrates the antioxidant effect of lentinan LF301.

[0026] Figure 8 The liver repair effects of lentinan LF301 on liver injury (A) alcoholic liver injury repair effect; (B) H2O2 liver injury repair effect.

[0027] Figure 9 The phagocytic function of LF301, a polysaccharide extract of shiitake mushroom.

[0028] Figure 10 To activate the Dectin-1 receptor activity of lentinan extract. Detailed Implementation

[0029] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0030] Experimental materials: The fruiting bodies of Shiitake mushroom F2 were commercially available seed mushrooms, provided by Shanghai Pengshi Mushroom Industry Co., Ltd.

[0031] Reagents and Instruments: Scleroglucan standard (SG) was purchased from Shanghai Xibao Biotechnology Co., Ltd.; yeast and mushroom β-glucan detection kits were purchased from Megazyme, Ireland; D-galactose, D-glucose, D-arabinose, L-fucose, L-rhamnose, D-mannose, D-xylose, glucosamine and galactosamine standards and trifluoroacetic acid were purchased from Sigma-Aldrich, USA; YEK-Blue... TM hDectin-Ia cells and their detection kits (including HEK-Blue) TM CLR selection, HEK-BlueT M detection, Normocin TM (Multi-effect antibiotics, etc.) were purchased from InvivoGen, USA; AlamarBlue TM Reagents were purchased from AbD Serotec, UK; all other reagents were domestically produced analytical grade.

[0032] Water2695 liquid chromatograph (Waters Corporation, USA); 5804R centrifuge (Eppendorf GmbH, Germany); DJ-10A traditional Chinese medicine pulverizer (Shanghai Longtuo Instrument Equipment Co., Ltd.); Cytiva Pure 25 protein purification system (Danaher Instruments, USA); DHG-9240A forced-air drying oven (Shanghai Longtuo Instrument Equipment Co., Ltd.); R210 / V850 rotary evaporator (Switzerland). (Company); Biotek-Synergy HT microplate reader (Bio-Tek, USA); Agilent Technologies 6120 Quadrupole LC / MS (Agilent Technologies, USA).

[0033] Example 1:

[0034] Extraction of polysaccharides from the fruiting body of *Lentinula edodes* F2: The fruiting bodies of *Lentinula edodes* F2 were dried at 60℃ to constant weight, pulverized and passed through a 100-mesh (0.15mm pore size) sieve, and active lentinan extract LF3 was prepared through a series of processes. For details, please refer to [link to specific process]. Figure 1 .

[0035] The steps for extracting polysaccharides from the fruiting bodies of *Houttuynia cordata* F2 are as follows:

[0036] After crushing and sieving, add 8 times the volume of 95% ethanol aqueous solution to the fruiting body of *Lentinula edodes* F2, and ultrasonically extract for 1 hour. Discard the supernatant and collect the residue. Add 0.9 wt% sodium chloride aqueous solution to the residue, with a mass-to-volume ratio of 1 g: 20 mL. Heat at 60℃ for 12 hours, centrifuge at 7000 rpm, collect the supernatant, and dialyze to obtain the LF1 fraction of lentinan. Add deionized water to the precipitate, heat at 100℃ for 2 hours, centrifuge, collect the supernatant, and dialyze to obtain the LF2 fraction of lentinan. Add 5 wt% NaOH aqueous solution and 0.05% NaBH aqueous solution to the precipitate, let stand at 25℃ for 4 hours, centrifuge to collect the supernatant, and adjust the pH to 7 with 1 mol / L acetic acid. Centrifuge to collect the supernatant, dialyze to obtain the LF3 fraction of lentinan.

[0037] Example 2:

[0038] Hydrolysis of lentinan:

[0039] Different hydrolysis times: Lentinan LF3 was mixed with 0.02 mol / L trifluoroacetic acid (TFA) aqueous solution at a ratio of 1 mg: 200 μL and hydrolyzed in an oil bath at 110 °C for 2, 4, 6 and 8 h respectively. After hydrolysis, the sample was removed and placed in a crushed ice bath to quickly cool and terminate the reaction. The trifluoroacetic acid was dried by nitrogen blowing and 2 ml of methanol was added to continue drying. This process was repeated 4 times until the trifluoroacetic acid was completely removed. Ultrapure water was added to make up to 2 ml, and the sample was transferred to a 2 ml centrifuge tube and centrifuged at 12000 r / min for 10 min at room temperature. The supernatant was filtered through a 0.22 μm filter membrane and then subjected to G-15 gel column chromatography (1.6 cm × 100 cm). The results were detected by UV and differential refractive index detectors. The mobile phase was deionized water and the flow rate was 0.75 mL / min.

[0040] Hydrolysis with different concentrations of acid: Lentinan LF3 was mixed with 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 mol / L trifluoroacetic acid (TFA) solutions at a ratio of 1 mg: 200 μL, and hydrolyzed at 110 °C for 1 and 2 h, respectively. After oil bath, the samples were removed and rapidly cooled in a crushed ice bath to terminate the reaction. The trifluoroacetic acid was dried by nitrogen evaporation, and 2 ml of methanol was added for further drying. This process was repeated 4 times until the trifluoroacetic acid was removed. Ultrapure water was added to a final volume of 2 ml, transferred to a 2 ml centrifuge tube, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter and then subjected to G-15 gel column chromatography.

[0041] Preparation of the low molecular weight lentinan target fragment LF301: 10 mg of lentinan extract LF3 was weighed and mixed with 0.02 mol / L TFA solution at a ratio of 1 mg: 200 μL. The mixture was hydrolyzed at 110 °C for 2 h. After drying the trifluoroacetic acid with nitrogen, 2 ml of methanol was added and the mixture was dried further. This process was repeated four times until the trifluoroacetic acid was removed. Ultrapure water was added to a final volume of 2 ml, which was then transferred to a 2 ml centrifuge tube. The tube was centrifuged at 12000 r / min for 10 min, and the supernatant was collected. After filtering through a 0.22 μm filter, the mixture was subjected to G-15 gel column chromatography, and the first peak was collected.

[0042] Large-scale preparation of small molecule polysaccharide LF301: Lentinan extract LF3 was mixed with 0.02 mol / L trifluoroacetic acid (TFA) solution at a ratio of 1 mg: 200 μL. After hydrolysis at 110 °C for 2 h, the mixture was placed in a crushed ice bath to rapidly cool and terminate the reaction. After drying the trifluoroacetic acid, the mixture was dissolved in 30 times the amount of distilled water. Small molecule substances were removed by ultrafiltration through a UFP-1-C-6 hollow fiber membrane (molecular weight cutoff 1000 Da) for 1 day. The mixture was then concentrated and dried to constant weight.

[0043] Determination of β-glucan content in lentinan extract: 10 mg of lentinan extract was accurately weighed, and the β-glucan content was determined by aniline blue fluorescence detection method (Nitschke et al. 2011). Each treatment was repeated 3 times.

[0044] Determination of polysaccharide content in extracts: The polysaccharide content in extracts was determined using the phenol-sulfuric acid method. Each treatment was repeated three times.

[0045] Determination of polysaccharide molecular weight distribution by high performance liquid chromatography: Accurately weigh 5 mg of lentinan extract and dissolve it in the mobile phase (0.05 mol·L⁻¹). -1 NaH2PO4·2H2O and 0.15mol·L -1 Prepare a solution with a mass concentration of 2 mg / mL in NaNO3 solution (0.02% sodium azide, pH 7). -1 The solution was centrifuged at 12000 rpm at room temperature for 10 min, and the supernatant was used for molecular weight distribution analysis using a GPC column. Chromatographic conditions: flow rate 0.3 mL / min. -1 Column temperature 30℃; Detected with a Waters differential refractive index detector; Molecular weight standard is Waters' PEG label.

[0046] Anion chromatography was used to determine the monosaccharide composition: Accurately weigh 0.1000 g of D-galactose, D-glucose, D-arabinose, L-fucose, L-rhamnose, D-mannose, D-xylose, glucosamine, and galactosamine standards, and dilute to 10 mL with 80% acetonitrile-water solution. Transfer 1 mL of each solution to a volumetric flask, mix well, and dilute to 10 mL to obtain the monosaccharide standard solution.

[0047] Accurately weigh 2 mg of lentinan extract and dissolve it thoroughly in 3 mL of 2 mol·L⁻¹ solution. -1 Trifluoroacetic acid (TFA) was heated in an oil bath at 110℃ for 5 hours, then dried using a nitrogen evaporator. Approximately 3 mL of methanol was added and the mixture was dried again. This process was repeated 6 times to remove TFA. 1.5 mL of ultrapure water was added and transferred to a 2 mL centrifuge tube. After centrifugation at 12000 rpm for 5 minutes at room temperature, the supernatant was transferred to a sample vial and diluted 30 times before loading. The monosaccharide composition was determined using an ICS-2500 high-performance anion chromatography system (PA-20 column) following the method of Liu Haiyan et al.

[0048] Determination of oligosaccharide molecular weight by liquid chromatography-mass spectrometry: The molecular weights of LF302 and LF303 were determined by Agilent Technologies 6120 Quadrupole LC / MS single quadrupole mass spectrometer.

[0049] The toxicity of lentinan to RAW264.7 macrophages: Macrophages were cultured according to the method of LIN et al., and cells in the logarithmic growth phase were used to prepare 5 × 10⁶ cells / mL of colorless RPMI 1640 medium (containing 10% fetal bovine serum and 1% antibiotics). 5 A suspension of cells is prepared for use.

[0050] RAW264.7 cells were seeded into 96-well plates, with 180 μL of cell suspension (9 × 10⁶ cells / well) per well. 4 (each sample) was incubated at 37°C under 5% CO2 for 24 h. 20 μL of the sample was added, and 20 μL of PBS was added to the blank control group. After 48 h, the supernatant was removed, and 180 μL of colorless RPMI 1640 medium (containing 10% fetal bovine serum and 1% antibiotics) was added, followed by 20 μL of Alamar Blue. TM The reagents were used to culture the cells until the color of the blank control group changed from blue to red. When the color stabilized, the absorbance of the cell culture medium was measured at wavelengths of 570 nm and 600 nm. The cell viability was calculated using the following formula. Each sample was repeated three times.

[0051] Survival rate = [117216 × D] 570 (Sample)-80586×D 600 (sample)] / [117216×D 570 (Blank)-80586×D 600 (blank)].

[0052] DPPH free radical scavenging ability: According to the method of Yang Yan et al., take 1 mL of 1 mg·mL⁻¹ -1 The extract solution was placed in a stoppered test tube, and 3 mL of 0.1 mmol·L⁻¹ extract was added. -1DPPH solution (using anhydrous ethanol as solvent) was mixed thoroughly and reacted at room temperature in the dark for 30 min. The absorbance of the reaction solution was measured at 517 nm. A vitamin C solution of the same concentration was used as a positive control. Each sample was tested in triplicate. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0053]

[0054] In the formula, A1 is the absorbance value of the sample solution; A2 is the absorbance value of the anhydrous ethanol solution replacing the DPPH solution; and A0 is the absorbance value of the anhydrous ethanol solution replacing the sample solution.

[0055] In vitro activation of Dectin-1 receptor activity assay: The in vitro activation activity of the Dectin-1 receptor in the samples was detected according to the instructions of the Dectin-1 assay kit. HEK-Blue... TM hDectin-1a cells were cultured in DMEM complete medium containing 10% fetal bovine serum at 5% CO2 and 37°C. Cells in the logarithmic growth phase were collected and prepared into a 3×10⁶ m³ / mL HEK-Blue Detection medium. 5 Cell suspensions were seeded into 96-well plates, with 180 μL of cell suspension per well. 20 μL of different concentrations of lentinan extract solution was added to each well, at a concentration of 0.01 mol·L⁻¹. -1 PBS (pH 7.2) and 10 μg / mL -1 Stibillin (SG) served as both a negative and positive control. After incubation at 5% CO2 and 37°C for 24 hours, the supernatant was collected, and the absorbance was measured at 630 nm. Three replicates were performed for each group.

[0056] Liver injury repair capacity: Normal hepatocytes L-O2 used in the experiment were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured at 37°C in a 5% CO2 incubator.

[0057] Alcoholic liver injury: When normal hepatocytes reach approximately 90% L-O2, discard the old culture medium, wash twice with PBS, add 0.5 mL of trypsin to digest until clear gaps are visible between cells, then stop digestion with 1 mL of complete culture medium, gently pipette to mix, and obtain a cell suspension. Adjust the cell concentration, add 100 μL to each well of a 96-well plate, 8 × 10⁸ cells / well. 3 Cells / well were cultured for 24 hours. The experimental group was then given the same volume of different concentrations of sample, while the control group was given the same volume of sample solvent. After 16 hours of culture, 1.2M anhydrous ethanol was added to each well of the experimental group, while the control group was given the same volume of PBS. The cells were then cultured for another 8 hours.

[0058] H2O2 Liver Injury: When normal hepatocytes reach approximately 90% L-O2 concentration, discard the old culture medium, wash twice with PBS, add 0.5 mL of trypsin to digest until clear gaps are visible between cells, then stop digestion with 1 mL of complete culture medium, gently pipette to mix, and obtain a cell suspension. Adjust the cell concentration, add 100 μL to each well of a 96-well plate, 8 × 10⁸ cells / well. 3 Cells per well were cultured for 24 hours. Then, the same volume of different concentrations of sample were added to the wells of the experimental group, while the same volume of sample solvent was added to the wells of the control group. Cultures were continued for another 18 hours. Afterward, 0.75 mM H2O2 was added to each well of the experimental group, while the same volume of PBS was added to the control group. Cultures were continued for another 6 hours.

[0059] MTT assay for cell viability: After the experiment, discard the culture medium and replace it with fresh serum-free medium containing 0.05 mg / mL diphenyltetrazolium bromide. Incubate in the dark for 4 hours. Then, discard the medium again, add 200 μL of dimethyl sulfoxide to each well, shake at room temperature for 15 minutes, and measure the absorbance at 490 nm using a microplate reader. Calculate the cell viability using the following formula:

[0060] Cell viability (%) = (OD1 - OD3) ÷ (OD2 - OD3) × 100%

[0061] OD1: Experimental group; OD2: Control group, no analyte added; OD3: Cell-free blank control group.

[0062] Macrophage phagocytic function: RAW264.7 macrophages in the logarithmic growth phase were selected, digested, and then prepared into 1×10⁶ cells / cells using colored DMEM medium containing 10% FBS. 6 Cells / mL suspension were seeded into 24-well plates, with 500 μL of cell suspension added to each well. After incubation at 37°C for 4 h in a 5% CO2 incubator, 500 μL of sample solution was added. PBS was used as the negative control, and 10 μg / mL LPS was used as the positive control. Each sample was tested in triplicate. After incubation at 37°C for 47 h in a 5% CO2 incubator, 1×10⁻⁶ cells / mL suspension was added. 8 Add 200 μL of fluorescent microspheres per 1 mL and continue culturing at 37°C and 5% CO2 for 1 h. Aspirate the culture medium, add 1 mL of PBS to wash away excess fluorescent microspheres, add 2.5% EDTA-trypsin, and digest in a 37°C, 5% CO2 incubator for 2 min. Add DMEM culture medium to terminate digestion. Transfer the cell suspension to centrifuge tubes and centrifuge at 1000 g for 3 min. Discard the supernatant, add 1 mL of PBS to wash 1-2 times, centrifuge at 1000 g for 3 min to discard the supernatant, and finally resuspend the cells in 500 μL of PBS. Analyze the sample using flow cytometry and calculate the phagocytosis rate using the formula.

[0063] Phagocytosis percentage (PP) = Macrophages phagocytosing fluorescent microspheres / Total number of macrophages × 100%.

[0064] Data statistics: SPSS 13.0 software was used to perform statistical analysis on the obtained experimental data. The data are expressed as mean ± standard deviation, and ANOVA was used to test the significance of differences.

[0065] Experimental results:

[0066] Screening results of shiitake mushroom varieties with high β-glucan content:

[0067] Table 1. β-glucan content, total glucan content, and proportion of β-glucan to glucan in fruiting bodies of different shiitake mushroom varieties.

[0068]

[0069] Table 1 shows that the proportion of β-glucan in the fruiting bodies of shiitake mushrooms is generally high, but the β-glucan content varies greatly among different varieties of shiitake mushrooms, with Huxiang F2 having the highest β-glucan content.

[0070] Acid hydrolysis of polysaccharides:

[0071] Lentinan LF3 was hydrolyzed at 110℃ for 2, 4, 6, and 8 hours using 0.02 mol / L TFA. The hydrolysis products were centrifuged, filtered through a membrane, and then subjected to G15 gel column chromatography. The differential response values ​​are shown below. Figure 2 As shown, Figure 2 The images show gel column chromatography of 0.02 mol / L TFA hydrolysis at different times. The components hydrolyzed with 0.02 mol / L trifluoroacetic acid are mainly concentrated in the first peak with a higher degree of polymerization, with a small amount appearing in the second and third peaks with lower molecular weights. Due to the presence of 280 nm UV absorption, peak 2 is presumably composed of some protein. With increasing hydrolysis time, the peak height of the larger molecular weight peak 1 gradually decreases, while the peak height of the smaller molecular weight peak 2 gradually increases, and the peak height of the even smaller molecular weight peak 3 also slightly increases. This indicates that with prolonged hydrolysis time, larger molecular weight sugars are gradually hydrolyzed into smaller polysaccharide or oligosaccharide fragments. However, even when the hydrolysis time reaches 8 hours, the number of small molecular weight sugars increases significantly, but the absorption peaks are generally too low. Therefore, increasing the hydrolysis time with 0.02 mol / L TFA cannot effectively increase the content of small molecular weight sugars, but it can obtain a large amount of LF301 components.

[0072] The concentration of trifluoroacetic acid was further increased to 0.02, 0.05, 0.1, and 0.2 mol / L. Chromatographic results after 2 hours of hydrolysis of trifluoroacetic acid (TFA) were obtained. Figure 3It can be seen that peak 1 gradually decreases with increasing trifluoroacetic acid (TFA) concentration; peak 2 appears clearly at a concentration of 0.1 mol / L, and peak 3 appears when the concentration reaches 0.2 mol / L, indicating that with increasing acid concentration, high molecular weight polysaccharides are hydrolyzed into smaller polysaccharide fragments, but the peak height of peak 3 is still not high. Further increasing the acid concentration, the chromatographic results of hydrolysis for 1 h at concentrations of 0.5, 1.0, and 2.0 mol / L showed that peak 3 did not appear at concentrations of 0.5 and 1.0 mol / L, while peak 1 still existed; when the concentration reached 2.0 mol / L, peak 1 disappeared, and peak 3 was obtained as a relatively single, symmetrical, fine, sharp peak, indicating a high content. Further extending the hydrolysis time at a 2.0 mol / L acid concentration, the peak height of peak 3 gradually increased with increasing hydrolysis time, but the increase was not significant after 3 h. Therefore, hydrolysis with 2.0 mol / L trifluoroacetic acid for 2 h is sufficient to obtain a large quantity of low molecular weight peak 3.

[0073] Therefore, using 0.02 mol / L trifluoroacetic acid (TFA) for 2 hours of hydrolysis can yield a large amount of peak component LF301; using 0.1 mol / L trifluoroacetic acid (TFA) for 2 hours of hydrolysis can yield a large amount of peak component LF302; and using 2.0 mol / L trifluoroacetic acid (TFA) for 2 hours of hydrolysis can yield peak component LF303.

[0074] Analysis of lentinan components:

[0075] Table 2 shows the yield, polysaccharide content, and β-glucan content of lentinan extract.

[0076]

[0077]

[0078] The yields, polysaccharide contents, and β-glucan contents are shown in Table 2. The results showed significant differences in the extraction yield, polysaccharide content, and β-glucan content of lentinan extracts prepared using different processes. Extract LF3 had the highest extraction yield, reaching 3.59%, while the yields of other extracts were similar, around 1%. LF3 had the highest lentinan content, reaching 78.29%, and a β-glucan content of 68.26%. The yields of the hydrolyzed small molecule polysaccharide LF301 were 9.50%, the hydrolyzed oligosaccharide LF302 were 11.05%, and the LF303 was 1.44%, with polysaccharide contents of 71.39%, 17.86%, and 3.54%, respectively, and β-glucan contents of 72.69%, 4.00%, and 1.01%, respectively.

[0079] Polysaccharide molecular weight: As shown in Table 3, the weight-average molecular weight of LF3 is 6.91 × 10⁻⁶. 6 Da, M WThe ratio / Mn is an indicator of molecular homogeneity. The closer the ratio is to 1, the fewer the branches. A ratio with many branches can even reach 20-50. For LF3, the M... W The Mn ratio is close to 1, indicating that the polysaccharide LF3 extracted from shiitake mushrooms all have branches, but the branches are few in number. Meanwhile, the hydrolyzed small molecule polysaccharide LF301 has a weight-average molecular weight of 2445 Da. w / Mn is also close to 1, with fewer branches, while LF302 and LF303 are oligosaccharides.

[0080] Table 3. Molecular weight distribution range of polysaccharides in shiitake mushroom polysaccharides

[0081]

[0082] Monosaccharide composition of lentinan:

[0083] Table 4 Monosaccharide composition and molar percentage of lentinan

[0084]

[0085] Table 4 shows that the anion chromatography analysis of lentinan extract LF3 is mainly composed of glucose, accounting for 94.85%, with small amounts of galactose and mannose; the hydrolyzed small molecule polysaccharide LF301 contains 81.45% glucose, and also contains small amounts of galactose and mannose, while the hydrolyzed oligosaccharide LF302 contains 74.33% glucose, and also contains galactose and a small amount of mannose, and LF303 contains small amounts of galactose and glucuronic acid.

[0086] Antioxidant activity of lentinan LF301: such as Figure 7 As shown, the hydrolyzed small molecule polysaccharide LF301 of lentinan LF3 has good DPPH· scavenging activity, and this fragment may be the main antioxidant activity fragment of lentinan.

[0087] The liver repair function of lentinan LF301: Figure 8 The effects of lentinan on liver injury repair: (A) Repair of alcoholic liver injury; (B) Repair of H2O2-induced liver injury. In an ethanol-induced liver injury model, the small molecule dextran LF301 sample showed a certain alleviating effect on alcoholic liver injury, especially the high dose of 200 μg / mL (A); while in H2O2-induced liver injury, the LF301 sample also showed a certain alleviating effect. Lower concentrations seemed to be more effective (B). These results indicate that the small molecule dextran LF301 has a certain repair effect on both alcohol- and H2O2-induced liver injury.

[0088] Phagocytic function of lentinan LF301: Flow cytometry was used to detect the effect of different concentrations of LF301 on the phagocytic ability of Raw264.7 cells. Figure 9 The samples treated at concentrations of 100 μg / mL and 200 μg / mL showed significant differences compared to the negative control PBS group (p < 0.01), indicating that the LF301 sample can promote the phagocytosis of Raw264.7. Note: Data are mean ± standard deviation (n = 5). ＊ This indicates that p < 0.05; ＊＊ The statement indicates that p < 0.01; ＊＊＊ This means p < 0.001.

[0089] Cytotoxicity evaluation of lentinan against RAW264.7 cells: Experimental results showed that at concentrations of 50–1000 μg / mL... -1 Within the experimental concentration range, none of the lentinan polysaccharides inhibited the growth of RAW264.7 cells, indicating that they were non-toxic to RAW264.7 cells within the experimental concentration range.

[0090] Results of the assay for the activation of Dectin-1 receptor by lentinan extract: Figure 10 As shown, LF301 exhibits higher in vitro activated Dectin-1 receptor activity than LF3, and its β-glucan content is also higher than that of LF301, indicating a positive correlation between the β-glucan content and the in vitro activated Dectin-1 receptor activity of lentinan extract.

[0091] In summary, using β-glucan content as a reference indicator, this invention found significant differences in β-glucan content among the fruiting bodies of eight shiitake mushroom varieties, with Huxiang F2 exhibiting the highest β-glucan content. Huxiang F2 is a shiitake mushroom variety suitable for factory cultivation and can be produced on a large scale year-round.

[0092] The fruiting body of shiitake mushroom is rich in β-glucan. The yield results of shiitake polysaccharide extracts prepared by different processes show that the shiitake polysaccharide extract LF3, which is extracted with water at 80℃ and then with 5% NaOH, has the highest yield, reaching 3.59%, and also has the highest polysaccharide content, reaching 78.29%, with a β-glucan content of 68.26%.

[0093] The small molecule polysaccharide LF301 can be obtained in large quantities by hydrolyzing LF3 at 110℃ for 2 hours with 0.02 mol / L trifluoroacetic acid (TFA); the monosaccharide component LF303 can be obtained by hydrolyzing LF3 at 2.0 mol / L trifluoroacetic acid (TFA) for 2 hours.

[0094] The antioxidant activity of different samples was evaluated using an antioxidant activity model. The results showed that all lentinan from shiitake mushrooms possessed antioxidant activity. LF3 could be hydrolyzed under different conditions to obtain the low-molecular-weight polysaccharide LF301 and oligosaccharide components LF302 and LF303. The hydrolyzed low-molecular-weight polysaccharide LF301 exhibited good DPPH scavenging activity and certain liver damage repair activity. Furthermore, it could inhibit tumor cell proliferation by activating macrophages and enhancing their phagocytic capacity.

[0095] Dectin-1 is a pattern recognition receptor (PRR) for β-glucan, recognizing glucans containing β-(1,3) and β-(1,6) glycosidic bonds, but not α-glucan, such as Ganoderma lucidum α-(1,3)-D-glucan. LF3 exhibits strong in vitro activation activity against the Dectin-1 receptor, with a β-glucan content reaching 68.26%. The low molecular weight polysaccharide LF301 has a β-glucan content of 72.69%, higher than LF3, and its Dectin-1 receptor activation activity is also higher than that of LF3.

[0096] LF3 was mixed with 0.02 mol / L trifluoroacetic acid (TFA) solution at a ratio of 1 mg: 200 μL, hydrolyzed at 100-110℃ for 2 h, and then rapidly cooled in a crushed ice bath to terminate the reaction. After drying the trifluoroacetic acid, it was dissolved in 30 times the amount of distilled water, and ultrafiltered with a hollow fiber membrane with a molecular weight cutoff of 1000 Da for 1 day. The solution was then concentrated and dried to constant weight to prepare LF301 on a large scale.

[0097] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing lentinan extract, characterized by: It is composed of the following steps, (1) drying and crushing the Lentinus edodes fruiting body, adding ethanol solution for ultrasonic extraction, collecting the residue, adding sodium chloride aqueous solution for heating extraction, centrifuging to collect the precipitate, adding water to the precipitate for heating extraction, centrifuging to collect the precipitate, adding NaOH solution and NaBH solution to the precipitate for standing, centrifuging to collect the supernatant, adjusting the pH to neutral, centrifuging to collect the supernatant, and obtaining the Lentinus edodes polysaccharide crude extract after dialysis; (2) mixing the Lentinus edodes polysaccharide crude extract with 0.02 mol / L trifluoroacetic acid aqueous solution for heating hydrolysis, removing small molecular substances by hollow fiber filter membrane ultrafiltration with a molecular weight cutoff of 1000 Da for 1 day, and concentrating and drying to constant weight to obtain the Lentinus edodes polysaccharide extract; The strain of the Lentinus edodes fruiting body is Shanghai F2 strain; In step (1), the ultrasonic extraction with ethanol solution is adding 8-10 times the volume of 95% ethanol solution to the Lentinus edodes fruiting body for ultrasonic extraction for 1 h; The heating extraction with sodium chloride aqueous solution is adding 0.9wt% sodium chloride aqueous solution, wherein the mass-volume ratio of the residue to 0.9wt% sodium chloride aqueous solution is 1g:20mL, and heating at 60℃ for 12h; The heating extraction with water in the precipitate is adding water to the precipitate, wherein the mass-volume ratio of the residue to water is 1g:20mL, the heating temperature is 100℃, and the heating time is 2h; The standing of the precipitate after adding NaOH solution and NaBH solution is adding 5wt% NaOH aqueous solution and 0.05% NaBH aqueous solution to the precipitate, and standing at 20-25℃ for 4-6h; In step (2), the Lentinus edodes polysaccharide crude extract is mixed with 0.02 mol / L trifluoroacetic acid aqueous solution at a ratio of 1 mg:200 μL, hydrolyzed at 100-110℃ for 2h to obtain the Lentinus edodes polysaccharide extract; The main component of the Lentinus edodes polysaccharide extract is β-glucan, and the monosaccharide composition of the Lentinus edodes polysaccharide extract is: glucose:galactose:mannose=81.45:13.55:5.

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