A polysaccharide of Armillariella mellea and its preparation method and application

Abstract

The present invention specifically discloses a polysaccharide of Armillariella tabescens (AT-P), its preparation method and application. The fruiting bodies of Armillariella tabescens are subjected to hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography and dialysis, and finally concentrated to obtain purified polysaccharide of Armillariella tabescens (AT-P). The present invention also discloses the composition of the polysaccharide of Armillariella tabescens (AT-P): galactose, glucose; the molar ratio of galactose to glucose residues is 8:3, wherein the molar ratio of (1→6)-galactose residues, (1→3,6)-galactose residues and →1)-galactose residues is 3:3:2, and the molar ratio of (1→6)-glucose residues and →4)-galactose residues is 2:1. The polysaccharide of Armillariella tabescens (AT-P) prepared by the present invention has significant immunomodulatory activity and can be applied in medicines, health products or foods. This application provides a new way for the development of Armillariella tabescens.

Description

Technical Field

[0001] The present invention relates to the technical field of the application of fungal polysaccharides, and particularly relates to an armillariella tabescens polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Edible fungi, commonly known as mushrooms, are a type of large fungus, and their fruiting bodies are rich in nutrients such as proteins, vitamins, mineral elements, amino acids, and polysaccharides.

[0003] Edible-fungus polysaccharides have biological activities such as antiviral, antioxidant, antitumor, lipid-lowering, promoting the proliferation and differentiation of immune cells and the secretion of lymphokines, activating complement, etc. They are safe and non-toxic, which has attracted extensive attention in the fields of health foods and biomedicines. Moreover, edible-fungus polysaccharides are a non-specific immune enhancer, and they can improve the immune function of the body through various ways without side effects on the body.

[0004] Armillariella tabescens (Scop. ex Fr.) Sing, also known as Armillariella mellea var. tabescens, Shuqiu, Qinggang mushroom, etc., belongs to Basidiomycota, Agaricales, Tricholomataceae, Armillaria. It is mainly distributed in Sichuan, Yunnan, Guangxi and other places. It has physiological activities such as treating hepatitis, antitumor, and immune regulation.

[0005] The fruiting body of Armillariella tabescens is brown, growing in clusters or solitary on the dead stumps or roots on the forest floor. Its hyphae are septate, branched, colorless and transparent, without clamp connections.

[0006] At present, the research on Armillariella tabescens mainly focuses on the optimization of the mycelium fermentation process, and there is less research on Armillariella tabescens polysaccharides. Ran Liang et al. obtained a yield of up to 9.42% of Armillariella tabescens polysaccharides by microwave-assisted extraction, which was significantly higher than that of the hot water extraction method (5.93%), and found that Armillariella tabescens polysaccharides had certain antioxidant activities. Kishinaka Shigehisa in Japan extracted, isolated and purified two polysaccharides from Armillariella tabescens, a water-soluble polysaccharide AT-HW composed of D-glucopyranose and D-galactose linked by β-(1→6); and a water-insoluble AT-AL mainly composed of (1→3)-α-D-glucan, and found that AT-HW could affect macrophages and T cells and other participate in immunity, and AT-AL mainly activated macrophages to produce SOA and activated lysosomal enzymes to mediate the immune regulation mechanism to achieve the antitumor effect.

[0007] It can be seen that there is a lack of research on the fine structure of Armillariella tabescens polysaccharides and the application of Armillariella tabescens polysaccharides in immune regulation activities in the prior art. Summary of the Invention

[0008] The present invention overcomes the defects existing in the prior art and provides an Armillariella tabescens polysaccharide, a preparation method thereof, and an application thereof.

[0009] The first aspect of the present invention provides a polysaccharide of Armillariella tabescens (AT-P), which is a heteropolysaccharide composed of galactose and glucose, and the molar ratio of galactose and glucose residues is 8:3.

[0010] Furthermore, the chemical structure of the polysaccharide contains (1→6)-galactose residues, (1→3,6)-galactose residues, →1)-galactose residues, (1→6)-glucose residues and →4)-galactose residues.

[0011] In one embodiment of the present invention, the polysaccharide is composed of (1→6)-galactose residues, (1→3,6)-galactose residues, →1)-galactose residues, (1→6)-glucose residues and →4)-glucose residues.

[0012] Furthermore, the molar ratio of the (1→6)-galactose residues, (1→3,6)-galactose residues and →1)-galactose residues is 3:3:2.

[0013] Furthermore, the molar ratio of the (1→6)-glucose residues and →4)-galactose residues is 2:1.

[0014] Furthermore, the weight-average molecular weight of the polysaccharide is 5000-30000 Da (such as 5000 Da, 6000 Da, 7000 Da, 7500 Da, 7600 Da, 7700 Da, 7800 Da, 7900 Da, 8000 Da, 8100 Da, 8200 Da, 8300 Da, 8400 Da, 8500 Da, 9000 Da, 10000 Da, 15000 Da, 16000 Da, 16500 Da, 17000 Da, 17100 Da, 17200 Da, 17300 Da, 17400 Da, 17500 Da, 17600 Da, 17800 Da, 17900 Da, 18000 Da, 19000 Da, 20000 Da, 21000 Da, 22000 Da, 23000 Da, 24000 Da, 25000 Da, 30000 Da), preferably 7000-25000 Da, and more preferably 8000-20000 Da.

[0015] In one embodiment of the present invention, the weight-average molecular weight of the polysaccharide is 17600 Da.

[0016] Furthermore, the chemical structure of the polysaccharide contains a main chain composed of (1→6)-galactose residues and (1→3,6)-galactose residues and a side chain composed of (1→6)-glucose residues and →4)-glucose residues, and the terminal sugar is composed of the 6-O linkage of →1)-galactose residues and (1→6)-glucose residues.

[0017] Furthermore, the above polysaccharide has the following structural formula:

[0018]

[0019] Among them, n is an integer from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably an integer from 2 to 15, and more preferably an integer from 3 to 10.

[0020] Among them, Galp is galactose and Glcp is glucose.

[0021] The second aspect of the present invention provides a method for preparing the polysaccharide of Armillariella tabescens as described in the first aspect, and the preparation method includes the step of extracting the fruiting body of Armillariella tabescens.

[0022] Furthermore, the preparation method includes the step of obtaining crude polysaccharide by water extraction and alcohol precipitation.

[0023] Furthermore, the preparation method also includes the step of purifying the crude polysaccharide (such as by ion exchange column chromatography).

[0024] In one embodiment of the present invention, the preparation method includes the following steps:

[0025] (1) Take the powder of the fruiting body of Armillariella tabescens, extract it with hot water, and sequentially concentrate, precipitate with alcohol, and dry the obtained water extract to obtain crude polysaccharide;

[0026] (2) Subject the crude polysaccharide obtained in step (1) to ion exchange column chromatography, elute, and collect the eluate;

[0027] (3) Dialyze and concentrate the eluate obtained in step (2) using a dialysis bag.

[0028] Preferably, (4) lyophilize the liquid in the dialysis bag after step (3) is completed.

[0029] Furthermore, in step (1), the extraction temperature can be 80 - 100 °C (such as 80, 85, 90, 95, 100 °C); in one embodiment of the present invention, the extraction temperature is 98 °C.

[0030] Furthermore, in step (1), the material - liquid ratio (W / V, mg / mL) of the powder of the fruiting body of Armillariella tabescens to water is 1:1 - 10 (such as 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in one embodiment of the present invention, the material - liquid ratio is 1:3.

[0031] Further, in step (1), the number of leaching times is 1 or more (such as 2, 3, 4, 5 times); in one embodiment of the present invention, the number of leaching times is 3 times.

[0032] Further, in step (1), the leaching time each time is 1 - 10 hours (such as 1, 3, 6, 8, 10 hours); in one embodiment of the present invention, the leaching time each time is 6 hours.

[0033] In one embodiment of the present invention, the leaching step in step (1) may include: taking the powder of Armillariella tabescens fruit bodies, mixing it with water, and boiling it in a water bath.

[0034] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to the concentrated solution of the water extract is 1 - 10:1 (such as 1:1, 3:1, 4:1, 5:1, 10:1); in one embodiment of the present invention, the volume ratio is 4:1;

[0035] In one embodiment of the present invention, in the above - mentioned alcohol precipitation step, the alcohol is ethanol.

[0036] In one embodiment of the present invention, step (1) includes: taking the powder of Armillariella tabescens fruit bodies, hot - water leaching, collecting the supernatant, concentrating, adding anhydrous ethanol, collecting the precipitate, drying, and removing the protein therein to obtain crude polysaccharide.

[0037] Further, in step (2), the ion - exchange column can be a cellulose column, and the packing of this cellulose column is such as DEAE - 52 cellulose.

[0038] Further, in step (2), the eluent used for elution can be an NaCl solution; specifically, the concentration of this NaCl solution is 0.01 - 1.0 mol / L (such as 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0) mol / L.

[0039] Further, in step (2), the elution can be gradient elution, and the concentration of the eluent can be 0.01 - 1.0 mol / L (such as 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0) mol / L.

[0040] In one embodiment of the present invention, step (2) includes: passing the aqueous solution of the crude polysaccharide obtained in step (1) through a cellulose column, gradient eluting, collecting the eluate, and concentrating.

[0041] Further, in step (3), the cut-off molecular weight of the dialysis bag is 5000-10000 Da (such as 5000, 6000, 7000, 8000, 9000, 10000 Da); in one embodiment of the present invention, the cut-off molecular weight is 7000 Da.

[0042] In one embodiment of the present invention, step (3) includes: placing the eluate obtained in step (2) in a dialysis bag for dialysis for two days.

[0043] The third aspect of the present invention provides a crude polysaccharide prepared by the method described in the second aspect.

[0044] The fourth aspect of the present invention provides an application of the Armillariella mellea polysaccharide as described in the first aspect in the preparation of health products and foods for enhancing immunity.

[0045] Further, in the above application, the polysaccharide can be used alone or in combination with other active ingredients.

[0046] The inventors of the present invention isolated and purified polysaccharide AT-P from Armillariella mellea, and analyzed and identified its molecular weight, monosaccharide composition, chemical structure, etc., and determined its weight average molecular weight and structural composition. Cell experiments show that this polysaccharide has significant immunomodulatory activity. Especially at a concentration of 20 μg / mL, the proliferation rate of B cells is the highest; especially at a concentration of 10 μg / mL, the promoting proliferation rate of T cells is the highest; at a concentration of 2.5 μg / mL, the proliferation rate of RAW264.7 cells is the highest.

[0047] Based on this, this polysaccharide can be used to prepare health products and foods for enhancing immunity, has good application prospects and commercial value, and can also improve the utilization value of Armillariella mellea. Description of the Drawings

[0048] Figure 1 Shown is the HPGPC spectrum of AT-P.

[0049] Figure 2 Shown is the infrared spectrum of AT-P.

[0050] Figure 3 Shown is the HLPC spectrum of AT-P (where (A-G): monosaccharide standards, (H): Armillariella mellea polysaccharide (AT-P)).

[0051] Figure 4 Shown is the 1H NMR spectrum of AT-P.

[0052] Figure 5 Shown is the 13 13C NMR spectrum of AT-P.

[0053] Figure 6Shown is that of AT-P 1 H- 1 H-COSY spectrogram.

[0054] Figure 7 Shown is the HMQC spectrogram of AT-P.

[0055] Figure 8 Shown is the HMQC spectrogram of AT-P.

[0056] Figure 9 Shown is the chemical structure of AT-P.

[0057] Figure 10 Shown are the experimental results of the effect of AT-P on B cell proliferation.

[0058] Figure 11 Shown are the experimental results of the effect of AT-P on T cell proliferation.

[0059] Figure 12 Shown are the experimental results of the effect of AT-P on the proliferation of RAW264.7 cells. Specific implementation manners

[0060] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains.

[0061] In the present invention, "Armillariella tabescens" refers to a fungus of the Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, Armillariella genus, which includes a fruiting body and mycelium.

[0062] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0063] Example 1 Isolation and extraction of Armillariella tabescens polysaccharide AT-P

[0064] 1. Isolation and extraction of Armillariella tabescens polysaccharide AT-P

[0065] 1.1. Extraction of crude Armillariella tabescens polysaccharide by water extraction and alcohol precipitation

[0066] Weigh 200 g of dried Armillariella tabescens fruit bodies, crush them, and add the crushed Armillariella tabescens fruit bodies and distilled water to a beaker at a ratio of 1:3 of material to liquid. Heat in a water bath at 98 °C for 6 hours, collect the supernatant and concentrate it, repeat 3 times, and finally concentrate all the supernatant to 200 mL. Add three times the volume of absolute ethanol to precipitate it, collect the precipitate and dry it to remove the protein in the extract, thereby obtaining crude Armillariella tabescens polysaccharide.

[0067] 1.2. Separation and purification of crude Armillariella tabescens polysaccharide by DEAE-52 cellulose column chromatography

[0068] Accurately weigh 50 g of DEAE cellulose, dissolve it in 1 L of ultrapure water, stir well, and stop stirring if there are no visible cellulose particles to the naked eye. Let it stand for 24 h, discard the supernatant for later use. Prepare 0.5 mol / L NaOH, soak the cellulose for 6 h, wash it with ultrapure water until neutral, then discard the supernatant. Add 0.5 mol / L HCl and soak for 6 h, wash with distilled water until neutral, and discard the supernatant; then add 0.5 mol / L NaOH again and soak for 6 h, wash with distilled water until neutral, and let it stand for later use.

[0069] After packing the activated cellulose into a column, perform column equilibration with distilled water for 24 h, and then the separation and purification of the crude polysaccharide can be carried out. Add the supernatant (5 mL) of the diluted crude polysaccharide to the DEAE cellulose column, and elute with different concentrations of NaCl (0.01 mol / L, 0.05 mol / L, 0.1 mol / L). The polysaccharide is determined by the sulfuric acid-phenol method. Concentrate the eluate to 5 mL, and purify the sample on the cellulose column. Dialyze with a dialysis bag (Mw ≥ 7 kDa) for 48 h, and freeze-dry to obtain Armillariella tabescens polysaccharide, named AT-P.

[0070] 2. Structural identification of Armillariella tabescens polysaccharide AT-P

[0071] Use acid hydrolysis, methylation analysis, high performance gel permeation chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance technology to analyze the structure of Armillariella tabescens polysaccharide (AT-P).

[0072] 2.1. Determination of molecular weight

[0073] Dissolve 10 mg of Armillariella tabescens polysaccharide AT-P sample in 1 mL of ddH2O, sonicate for 5 min, and perform HPGPC analysis.

[0074] 2.2. Infrared spectrum analysis of Armillariella tabescens polysaccharide AT-P

[0075] Mix 2 mg of AT-P with KBr and press into a tablet, and scan from 4000 cm -1 -400 cm-1 Range.

[0076] 2.3. Monosaccharide composition analysis of Armillaria mellea polysaccharide AT-P

[0077] Seven standard products and the AT-P sample after TFA acid hydrolysis were dissolved in the mobile phase (75% acetonitrile) and then analyzed by HLPC.

[0078] 2.4. Nuclear magnetic resonance analysis of Armillaria mellea polysaccharide AT-P

[0079] Take 50 mg of the AT-P sample, dissolve it in 0.6 mL of heavy water (D2O), put it into a nuclear magnetic resonance tube, and detect it on a nuclear magnetic resonance instrument.

[0080] 2.5. GC-MS analysis after methylation and silylation derivation of Armillaria mellea polysaccharide AT-P

[0081] Weigh 20 mg of the AT-P sample, seal the beaker, add 2 mL of DMSO (dimethyl sulfoxide) to the sealed beaker, gently shake the beaker to fully dissolve AT-P. Then add an excessive amount of NaOH until the NaOH just fails to dissolve and place it in a shaker for shaking at room temperature for 1 h. After the shaking is completed, add 1.5 mL of methyl iodide, react in the dark for 1 h, and add water to terminate the reaction after the reaction. Extract the product with chloroform, and obtain the methylated polysaccharide after drying. After the methylated polysaccharide is completely acid hydrolyzed by TFA, wash it three times with water to obtain the completely acid hydrolyzed product of methylation.

[0082] React the above sample fully with 2 mL of hexamethyldisilazane, 1 mL of trimethylchlorosilane, and 2 mL of anhydrous pyridine, and carry out a water bath at 50 °C for 20 min. Use a low-temperature high-speed centrifuge to centrifuge at a speed of 12000 rpm / min at 4 °C for 10 min, discard the precipitate, filter it with a 0.22 μm filter, and take the upper layer solution for GC-MS analysis.

[0083] 3. Results

[0084] 3.1. Results of the basic properties of Armillaria mellea polysaccharide AT-P

[0085] The HPGPC chromatogram of PC-1 is as Figure 1 shown, which shows that the weight-average molecular weight of AT-P is 17600 Da.

[0086] 3.2. FTIR spectrum analysis of Armillaria mellea polysaccharide AT-P

[0087] The primary structure of AT-P was characterized by Fourier transform infrared spectroscopy, and the results are as Figure 2 shown. The wave numbers are at 3425 cm -1 , 2928 cm -1and 1400 - 1200 cm -1 etc. have typical polysaccharide absorption peaks and no other miscellaneous peaks, indicating that the isolated and purified AT-P is a polysaccharide substance. Table 4-1 is the functional group analysis table of AT-P. 4000 - 1250 cm -1 is the characteristic peak region. The broad and strong absorption peak at 3425.88 cm -1 is assigned to the O-H stretching vibration. The signal peak at 2928.49 cm -1 is assigned to the -CH2 stretching vibration peak. The signal peaks at 1632.92 cm -1 and 1404.47 cm -1 are respectively assigned to the C=O stretching vibration peak and the C-H bending vibration peak. In the fingerprint peak region of 1250 - 400 cm -1 , the signal peak at 1077.59 cm -1 is assigned to the C-O stretching vibration peak. The above indicates that AT-P contains pyranose. In addition, there is no absorption peak near 1730 cm -1 , indicating that AT-P does not contain uronic acid.

[0088] 3.3. Monosaccharide composition analysis of Armillariella mellea polysaccharide AT-P

[0089] After completely hydrolyzing AT-P, HPLC was used for its monosaccharide composition analysis. The results are as Figure 3 shown. The elution times of each monosaccharide standard are Rha (4.838 min), Xyl (5.638 min), Ara (6.363 min), Fru (7.120 min), Man (8.704 min), Glc (8.386 min), and Gal (8.949 min) respectively. There are two retention times of 7.934 and 8.486 min in the HPLC results after complete hydrolysis of AT-P. Compared with the elution times of the monosaccharide standards, they are respectively assigned to Glc and Gal. It shows that AT-P is composed of Glc and Gal, and the peak area ratio is about 3:8.

[0090] 3.4. NMR spectrum analysis of Armillariella mellea polysaccharide AT-P

[0091] The 1 1H NMR results of AT-P are as Figure 4 shown. The results show that AT-P has five anomeric hydrogen signals, namely: δ5.24, δ5.01, δ4.94, δ4.86, and δ4.38, and the integral area ratio is 2.52:2.03:3.52:2.65:0.61. The signals between δ3.0 - 4.2 are attributed to the hydrogen signals of C2 - C6 in the sugar residues.

[0092] The 13The \(^{13}\)C NMR results are as follows Figure 5 shown, AT-P has five anomeric carbon signals at δ102.64, δ101.61, δ99.27, δ98.21 and δ97.83. The signals between δ60 - 78 are assigned to the carbon signals of C2 - C6 in the sugar residue.

[0093] The 1 H- 1 H-COSY spectrum of AT-P is as Figure 6 shown, from which the coupling relationships between adjacent hydrogen nuclei can be identified. The signals of H1 / H2 in part A are δ5.24 / 3.53, those in part B are δ5.01 / 3.81, those in part C are δ4.94 / 3.66, those in part D are δ4.86 / 3.71, and those in part E are δ4.38 / 3.18.

[0094] The chemical shifts of all hydrogens are summarized in Table 1.

[0095] The HMQC spectrum of AT-P is as Figure 7 shown, from which the coupling relationships between the 1 H and 13 C in the short-range correlations can be identified. The signals of H1 / C1 in part A are δ5.24 / 99.27, those in part B are δ5.01 / 98.21, those in part C are δ4.94 / 101.61, those in part D are δ4.86 / 97.83, and those in part E are δ4.38 / 102.64.

[0096] The HMBC spectrum of AT-P is as Figure 8 shown, from which the coupling relationships between the 1 H and 13 C in the long-range correlations can be identified. The signals of H2 / C3 in residue A are δ3.53 / 77.05, those of H3 / C4 in residue B are δ3.96 / 68.33, those of H3 / C5 in residue C are δ3.91 / 72.84, those of H1 / C3 and H1 / C4 in residue D are δ4.86 / 66.55, those of H4 / C5 are δ3.54 / 71.77, those of H5 / C6 are δ3.75 / 68.19, and those of H2 / C1 in residue E are δ3.18 / 102.64.

[0097] The chemical shifts of all carbons are summarized in Table 2.

[0098] Table 1 Chemical Shifts of 1 H of AT-P

[0099]

[0100] In Table 2 of AT-P 13 The chemical shift of C

[0101]

[0102] 3.5. Gas Chromatography and Mass Spectrometry Analysis of Armillariella mellea Polysaccharide AT-P

[0103] The methylation results are shown in Table 3, indicating that the main repeating structural unit of AT-P consists of (1→6)-galactose residues and (1→3,6)-galactose residues as the main chain, (1→6)-glucose residues, →4)-glucose residues as the side chains, and →1)-galactose as the terminal sugar. In summary, the structure of AT-P can be preliminarily inferred as Figure 9 shown

[0104] Table 3 Analysis of Methylation Results of AT-P

[0105]

[0106]

[0107] Example 2: Study on the Immunomodulatory Activity of Armillariella mellea Polysaccharide AT-P

[0108] The immunomodulatory activity of Armillariella mellea polysaccharide AT-P was determined by two CCK-8 methods in vitro

[0109] 1. Reagents

[0110] CCK-8 kit, RPIM1640, FBS, DMSO, double antibodies, etc., are all commercially available products

[0111] 2. Instruments

[0112] Microplate reader; cell culture incubator

[0113] 3. Methods

[0114] The effect of AT-P on the proliferation of immune cells (B cells, T cells and RAW264.7 cells)

[0115] The effect of Armillariella mellea polysaccharide (AT-P) on the proliferation of T cells, B cells and RAW264.7 cells was determined by the Cell Counting Kit (CCK-8) method. T cells, B cells and RAW264.7 cells were cultured in vitro until the logarithmic growth phase. After counting with a cell counting plate, the cell suspension was diluted to 1×10 5cells / mL. The cell suspension was added to a 96-well plate, 100 μL per well, and the 96-well plate was placed in a CO2 incubator for 24 h. After 24 h, different concentrations of AT-P solution (final concentrations of 2.5, 5, 10, 20 μg / mL) were added to the experimental groups, 100 μL of LPS solution (final concentration 10 μg / mL) was added to the positive control group, and 100 μL of cell culture medium was added to the blank group. After culturing in a CO2 incubator for 24 h, 10 μL of CCK-8 was added to each well, and after incubation in a CO2 incubator for 2 h, the absorbance value was measured on an enzyme-linked immunosorbent assay (ELISA) reader (450 nm) and images were taken.

[0116] 4. Results

[0117] 4.1 Effect of AT-P on the proliferation of B cells

[0118] The results are as Figure 10 shown. Compared with the blank group, in the range of AT-P final concentrations of 1.25 - 20 μg / mL, AT-P could significantly (P < 0.05) stimulate the growth of B cells, and as the drug concentration increased, the proliferation rate also increased, with proliferation rates of 26.33%, 26.33%, 34.54%, 51.96%, and 70.16% respectively. Among them, when the concentration of AT-P was 20 μg / mL, the proliferation rate reached the highest value of 70.16%, which was much higher than the proliferation rate of 42.58% of the positive control group LPS (10 μg / mL).

[0119] 4.2 Effect of AT-P on the proliferation of T cells

[0120] The results are as Figure 11 shown. Compared with the blank group, AT-P could significantly promote cell proliferation in the range of final concentrations of 1.25 - 40 μg / mL, and the difference was statistically significant (P < 0.05). When the final concentration of AT-P was 10 μg / mL, the proliferation efficiency of T cells reached the maximum, with a proliferation rate of 38.28% (P < 0.01), which was close to the proliferation rate of 40.84% of the positive control group LPS (10 μg / mL).

[0121] 4.3 Effect of AT-P on the proliferation of RAW264.7 cells

[0122] The results are as Figure 12 shown. Compared with the blank group, when the final concentration of AT-P was 1.25 - 20 μg / mL, it could significantly increase the cell proliferation rate, and the difference was significant (P < 0.05). When the concentration of AT-P was 2.5 μg / mL, it could extremely significantly promote the proliferation of RAW 264.7 cells (P < 0.0001), and the proliferation rate reached the highest of 49.65%.

[0123] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0124] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0125] Listing the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. An Armillaria mellea fruit body polysaccharide, characterized in that, The polysaccharide from the fruiting body of Armillariella mellea contains (1→6)-galactose residues, (1→3,6)-galactose residues, and →1)-galactose residues in a molar ratio of 3:3:2, and (1→6)-glucose residues and →4)-glucose residues in a molar ratio of 2:

1. The polysaccharide from the fruiting body of Armillariella mellea has the following structure: The weight-average molecular weight of the polysaccharide from the fruiting body of Armillariella mellea is 5000-30000 Da.

2. The polysaccharide of the fruiting body of Armillariella mellea as described in claim 1, characterized in that, The weight-average molecular weight of the polysaccharide from the fruiting body of Armillariella mellea is 7000-25000 Da.

3. The polysaccharide of Armillariella tabescens fruit body according to claim 2, wherein, The weight-average molecular weight of the polysaccharide from the fruiting body of Armillariella mellea is 8000-20000 Da.

4. The polysaccharide from the fruiting body of Armillariella mellea as claimed in claim 3, wherein, The weight-average molecular weight of the polysaccharide from the fruiting body of Armillariella mellea is 17600 Da.

5. The method for preparing the polysaccharide from the fruiting body of Armillariella mellea according to any one of claims 1-4, the preparation method comprising the following steps: (1) Take the powder of the fruiting body of Armillariella mellea, extract with water, and precipitate the obtained aqueous extract with alcohol to obtain a crude polysaccharide; (2) Subject the crude polysaccharide obtained in step (1) to ion exchange column chromatography, elute, and collect the eluate; (3) Dialyze and concentrate the eluate obtained in step (2) using a dialysis bag.

6. The preparation method according to claim 5, characterized in that, In step (1), the extraction temperature is 80-100 °C.

7. The preparation method according to claim 6, characterized in that, The extraction temperature is 98 °C.

8. The preparation method according to claim 5, characterized in that, In step (1), the material-liquid ratio of the powder of the fruiting body of Armillariella mellea to water is 1:1-10.

9. The preparation method according to claim 5, characterized in that, In step (1), the number of extraction times is 1-5 times, and the extraction time for each time is 1-10 hours.

10. The preparation method according to claim 5, characterized in that, In step (1), the volume ratio of the alcohol to the concentrated solution of the aqueous extract is 1-10:

1.

11. The preparation method according to claim 5, characterized in that, In step (1), the alcohol is ethanol.

12. The preparation method according to claim 5, characterized in that, In step (2), the ion exchange column is a cellulose column, and its packing is DEAE-52 cellulose.

13. The preparation method according to claim 5, wherein, In step (2), the eluent used for elution is an NaCl solution.

14. The preparation method according to claim 5, characterized in that, In step (2), the elution is gradient elution, and the concentration of the eluent used for elution is 0.01-1.0 mol / L.

15. The preparation method according to claim 5, characterized in that, In step (3), the cut-off molecular weight of the dialysis bag is 5000-10000 Da.

16. The preparation method according to claim 15, characterized in that, The cut-off molecular weight of the dialysis bag is 7000 Da.

17. The application of the polysaccharide from the fruiting body of Armillariella mellea according to any one of claims 1-4 or the polysaccharide from the fruiting body of Armillariella mellea prepared by the preparation method according to any one of claims 5-16 in the preparation of immune-enhancing health products.

18. The application of the polysaccharide from the fruiting body of Armillariella mellea according to any one of claims 1-4 or the polysaccharide from the fruiting body of Armillariella mellea prepared by the preparation method according to any one of claims 5-16 in the preparation of immune-enhancing foods.

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