A polysaccharide of laetiporus curreus and a preparation method and application thereof

Abstract

The application discloses a kind of flower face fragrant mushroom polysaccharide and its preparation method and application.The composition of the flower face fragrant mushroom polysaccharide includes glucose, galactose, the chemical structure of flower face fragrant mushroom polysaccharide includes 1,4-connected D-glucose residue, 1,4-connected D-galactose residue, 1,6-connected galactose residue, 2-connected alpha-D-glucose residue, 1,4,6-connected glucose residue, 1,6-connected glucose residue, and the molar ratio of the residue of 1→4-Glc, 1→4-Gal, 1→6-Gal, 2→-Glc, 1→4,6-Glc and 1→6-Glc is about 3:2:3:2:2:1.The flower face fragrant mushroom polysaccharide is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography, dialysis and concentration.The flower face fragrant mushroom polysaccharide of the application has significant immunomodulatory activity and antitumor activity, and can be applied in medicine, health care products or food.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of application of fungal polysaccharides, and particularly relates to Lepistasordida polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] Edible fungi, commonly known as mushrooms, are a kind of large fungi, and the fruiting bodies thereof are rich in nutrients such as protein, vitamins, mineral elements, amino acids and polysaccharides. Edible fungus polysaccharides have biological activities such as antiviral, antioxidant, antitumor, hypolipidemic, promotion of proliferation and differentiation of immune cells and secretion of lymphokines, activation of complement and immunomodulation, and are safe and non-toxic, and are widely concerned in the fields of health food and biological medicine. Moreover, edible fungus polysaccharides are a kind of non-specific immune enhancer, which can improve the immune function of the body through various ways, and has no side effects on the body.

[0003] Lepistasordida belongs to Agaricales, Tricholomatacete and Lepista, and is mainly distributed in Yunnan, Heilongjiang, Liaoning, Sichuan and Guizhou of China. Lepistasordida has a strong fragrance, a crisp taste and a pleasing color. The cap of Lepistasordida is 4cm-8cm in diameter, purple in color, rough to have pitting, the stem is 4cm-6.5cm in length and solid, and the base is mostly curved; the flesh is light purple in color and thin, and is waterlogged; the gills are straight, sometimes slightly curved or slightly extended; the fruiting body is purple or light purple when young, and is easily faded to be milky white or powder purple after maturation. Even the same species will have different phenotypic characteristics at different development stages. It has been found that the extract of Lepistasordida has certain antioxidant and antitumor activities. In addition, two diterpenoid compounds (lepistal and lepistol) isolated from the fermentation broth of Lepistasordida have the biological activity of inducing differentiation of human anemic leukemia cells, and lepistal also has antibacterial and antifungal effects.

[0004] Hu Xinlei et al. in "Optimization of extraction conditions of polysaccharides from Lentinus rugatus mycelium and its in vitro immunological activity" ([J]. Food Science, 2017, 38(20): 185-190.) reported that Lentinus rugatus mycelium was used as raw material, and the extraction conditions of hot water extraction method were optimized by orthogonal test, and the optimal extraction conditions were obtained as follows: extraction temperature 65℃, extraction time 2.5h, liquid-solid ratio 40:1(mL / g), and the extraction rate of polysaccharide was 15.88% under the above conditions; and the extract was used to mouse macrophage Ana-1, and it was found that within the effective dose range of 0.05-1.6mg / mL, Lentinus rugatus mycelium polysaccharide could promote the proliferation of mouse macrophage Ana-1 to different degrees, increase the secretion of cytokines TNF-α and IL-6, and enhance the neutral red phagocytosis ability, membrane surface protein TLR2 and TLR4 expression capacity, and the effect of 1.6mg / mL was the best. It is shown that Lentinus rugatus mycelium polysaccharide has excellent immunological activity.

[0005] Luo et al. in Structural characterization of an immunoregulatory polysaccharide from the fruiting bodies of Lepista sordida. ([J]. Carbohydrate Polymers, 2012, 88: 820-824.) reported that Lentinus rugatus was used as experimental material, and the molecular weight (Mw) of polysaccharide LSP was about 4×10 4 Da was detected by high performance gel permeation chromatography (HPGPC). According to Fourier transform infrared (FT-IR) spectrum, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and gas chromatography analysis, the results showed that LSP mainly contained (1→6)-α-D-glucosyl residues and (1→2,6)-α-D-glucosyl residues as backbone, and (1→)-α-D-galactosyl residues as branch, and the molar ratio was 2:1.1:0.9. In vitro cell test showed that LSP could significantly increase the secretion of NO and TNF-α of mouse macrophages (P<0.05), and had certain immunological activity.

[0006] It can be seen that the fine structure of Lentinus rugatus polysaccharide and the application of Lentinus rugatus polysaccharide in immunoregulatory activity are lacking in the prior art. SUMMARY

[0007] The present application overcomes the defects in the prior art and provides a Lentinus rugatus polysaccharide, a preparation method and application thereof.

[0008] The first aspect of the present application provides a Laetiporus coccinus polysaccharide (LS-P), which is a heteropolysaccharide consisting of glucose and galactose, wherein the molar ratio of residues of the glucose and galactose is about 8:5.

[0009] Further, the chemical structure of the polysaccharide comprises 1,4-linked D-glucose residues, 1,4-linked D-galactose residues, 1,6-linked galactose residues, 2-linked α-D-glucose residues, 1,4,6-linked glucose residues, 1,6-linked glucose residues.

[0010] Further, the chemical structure of the polysaccharide comprises a main chain composed of 1,6-linked glucose residues, 1,4-linked D-galactose residues, 1,4-linked D-glucose residues, 1,6-linked galactose residues, and a side chain composed of 1,4,6-linked glucose residues, 2-linked α-D-glucose residues.

[0011] Further, the weight average molecular weight of the polysaccharide is 8000-20000 Da (such as 8000 Da, 8500 Da, 9000 Da, 9500 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, 15000 Da, 16000 Da, 17000 Da, 18000 Da, 19000 Da, 20000 Da), preferably 10000-15000 Da.

[0012] In an embodiment of the present application, the weight average molecular weight of the polysaccharide is 13135 Da.

[0013] Further, the polysaccharide comprises the following structural formula:

[0014]

[0015] wherein n is an integer of 1-15 (such as 5, 6, 7, 8, 9, 10), preferably n is an integer of 4-8, and wherein Glcp is glucose and Galp is galactose.

[0016] The second aspect of the present application provides a composition comprising the polysaccharide of the first aspect.

[0017] The third aspect of the present application provides a preparation method of the Laetiporus coccinus polysaccharide of the first aspect, which comprises the step of extracting the Laetiporus coccinus fruiting body as raw material.

[0018] Further preferably, the preparation method comprises the step of extracting the crude polysaccharide by water extraction and alcohol precipitation.

[0019] Further preferably, the preparation method further comprises a step of purifying the crude polysaccharide (e.g. by ion exchange column chromatography).

[0020] In one embodiment of the present application, the preparation method comprises the following steps:

[0021] (1) taking the powder of the fruiting body of Laetiporus cremeatus, and extracting the powder with hot water to obtain an aqueous extract, and then sequentially concentrating, alcohol precipitating, and drying the aqueous extract to obtain a crude polysaccharide;

[0022] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate;

[0023] (3) subjecting the eluate obtained in step (2) to dialysis and concentration using a dialysis bag.

[0024] Further, the preparation method further comprises step (4), which is freezing and drying the liquid in the dialysis bag after step (3) is completed.

[0025] Further, in step (1), the temperature of the extraction can be 80-100℃ (e.g. 80, 85, 90, 95, 100℃); in one embodiment of the present application, the extraction temperature is 98℃.

[0026] Further, in step (1), the mass ratio (W / V, mg / mL) of the powder of the fruiting body of Laetiporus cremeatus to water is 1:1-10 (e.g. 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in one embodiment of the present application, the mass ratio is 1:3.

[0027] Further, in step (1), the number of extractions is 1-5 times (e.g. 1, 2, 3, 4, 5 times); in one embodiment of the present application, the number of extractions is 3 times.

[0028] Further, in step (1), the extraction time of each extraction is 1-10 hours (e.g. 1, 3, 6, 8, 10 hours); in one embodiment of the present application, the extraction time of each extraction is 6 hours.

[0029] In one embodiment of the present application, the extraction step in step (1) can comprise: taking the powder of the fruiting body of Laetiporus cremeatus, mixing the powder with water, and boiling in a water bath.

[0030] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to the concentrated aqueous extract is 1-10:1 (e.g. 1:1, 3:1, 4:1, 5:1, 10:1); in one embodiment of the present application, the volume ratio is 3:1.

[0031] In one embodiment of the present application, in the above alcohol precipitation step, the alcohol is ethanol.

[0032] In one embodiment of the present application, step (1) comprises: taking the powder of the fruiting body of Armillaria mellea, hot water extraction, collecting the supernatant, concentrating, adding anhydrous ethanol, collecting the precipitate, drying, removing the protein therein, and obtaining the crude polysaccharide.

[0033] Further, in step (2), the ion exchange column can be a cellulose column, and the filler of the cellulose column can be DEAE cellulose.

[0034] Further, in step (2), the eluent used for elution can be a NaCl solution; specifically, the concentration of the NaCl solution is 0-0.3 mol / L (such as 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3) mol / L.

[0035] Further, in step (2), the elution can be gradient elution, and the concentration of the eluent can be 0-0.3 mol / L (such as 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3) mol / L.

[0036] In one embodiment of the present application, step (2) comprises: passing the aqueous solution of the crude polysaccharide obtained in step (1) through a cellulose column, gradient elution, collecting the eluate, and concentrating.

[0037] Further, in step (3), the molecular weight cut-off of the dialysis bag is 5000-10000 Da (such as 5000, 6000, 7000, 8000, 9000, 10000 Da); in one embodiment of the present application, the molecular weight cut-off is 7000 Da.

[0038] In one embodiment of the present application, step (3) comprises: placing the eluate obtained in step (2) in a dialysis bag for dialysis, and dialyzing for two days.

[0039] The fourth aspect of the present application provides an application of the polysaccharide of Armillaria mellea according to the first aspect or the crude polysaccharide prepared by the preparation method according to the third aspect, and the application comprises:

[0040] (1) an application in the preparation of a product capable of enhancing immunity;

[0041] (2) an application in the preparation of a product having anti-tumor activity.

[0042] Further, the product is a food, a health product, or a medicine.

[0043] Further, in the application, the polysaccharide can be used alone or in combination with other active ingredients.

[0044] Further, the final concentration of the L. lilaceipes polysaccharide in the product is 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), preferably 10-20 μg / mL.

[0045] Further, when the proliferation effect of B cells is to be improved, the final concentration of the L. lilaceipes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 10 μg / mL.

[0046] Further, when the proliferation effect of T cells is to be improved, the final concentration of the L. lilaceipes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 20 μg / mL.

[0047] Further, when the proliferation effect of RAW 264.7 cells is to be improved, the final concentration of the L. lilaceipes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 20 μg / mL.

[0048] Further, when the inhibition effect of MFC cells is to be improved, the final concentration of the L. lilaceipes polysaccharide is 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 10 μg / mL.

[0049] The present application has the following beneficial effects:

[0050] The L. lilaceipes polysaccharide is separated and purified from L. lilaceipes fruiting bodies, and its molecular weight, monosaccharide composition, chemical structure, etc. are analyzed and identified to determine its weight average molecular weight and structural composition. Cell experiments show that the polysaccharide has significant immunomodulatory activity, especially at a final concentration of 10 μg / mL, the B cell proliferation rate is the highest, at a final concentration of 20 μg / mL, the T cell proliferation rate is the highest, at a final concentration of 20 μg / mL, the RAW 264.7 cell proliferation rate is the highest; the polysaccharide also has significant anti-tumor activity, especially at a final concentration of 10 μg / mL, the MFC cell inhibition rate is the highest. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The HPGPC spectrum of LS-P is shown;

[0052] Figure 2 The infrared spectrum of LS-P is shown;

[0053] Figure 3 The HLPC spectrum of LS-P is shown;

[0054] Figure 4 The of LS-P is shown 1H NMR spectrum;

[0055] Figure 5 HMQC spectrum of LS-P is shown 13 C NMR spectrum;

[0056] Figure 6 HMQC spectrum of LS-P is shown 1 H- 1 H-COSY spectrum;

[0057] Figure 7 HMBC spectrum of LS-P is shown

[0058] Figure 8 HMBC spectrum of LS-P is shown

[0059] Figure 9 Experimental results of the effect of LS-P on B cell proliferation are shown (1: blank group; 2-4: LS-P with final concentrations of 5, 10, 20 μg / mL; 5: LPS with final concentration of 10 μg / mL);

[0060] Figure 10 Experimental results of the effect of LS-P on T cell proliferation are shown (1: blank group; 2-4: LS-P with final concentrations of 5, 10, 20 μg / mL; 5: LPS with final concentration of 10 μg / mL);

[0061] Figure 11 Experimental results of the effect of LS-P on RAW264.7 cell proliferation are shown (1: blank group; 2-4: LS-P with final concentrations of 5, 10, 20 μg / mL; 5: LPS with final concentration of 10 μg / mL);

[0062] Figure 12 Experimental results of the effect of LS-P on MFC cell proliferation are shown (1: blank group; 2-4: LS-P with final concentrations of 5, 10, 20 μg / mL; 5: MAN with final concentration of 10 μg / mL). DETAILED DESCRIPTION

[0063] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0064] In the present application, the term "Lepista sordida" refers to Basidiomycota, Agaricomycetes, Agaricales, Tricholomataceae, Lepista, which includes fruiting bodies and mycelium.

[0065] The term "LS-P" refers to Lepista sordida polysaccharide, the abbreviation of Lepista sordida polysaccharide.

[0066] The term "LPS" refers to Lipopolysaccharides, the abbreviation of Lipopolysaccharides, which is the main component of the cell wall of Gram-negative bacteria. The LPS solution used in this experiment was purchased from Biosharp Company in China.

[0067] The term "MAN" refers to Mannatide, the abbreviation of Mannatide. The MAN solution used in this experiment was purchased from Sichuan Aobang Pharmaceutical Co., Ltd.

[0068] The term "CK" refers to Control check, the abbreviation of Control check.

[0069] The technical solutions of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0070] Example 1 Isolation, extraction and structural identification of Lycoperdon gemmatum polysaccharide LS-P

[0071] 1. Isolation and extraction of Lycoperdon gemmatum polysaccharide LS-P

[0072] 1.1 Extraction of Lycoperdon gemmatum polysaccharide LS-P by water extraction and alcohol precipitation

[0073] Take 200g of dried Lycoperdon gemmatum fruiting body powder, add it to a beaker with the crushed Lycoperdon gemmatum fruiting body and distilled water in a ratio of 1:3, and place it in a 98℃ water bath for 6 hours. Collect the supernatant and concentrate it, repeat 3 times, and finally concentrate all the supernatant to 200mL. Add three times the volume of anhydrous ethanol to precipitate it, collect the precipitate and dry it, and remove the protein in the extraction solution to obtain Lycoperdon gemmatum polysaccharide.

[0074] 1.2 Isolation and purification of Lycoperdon gemmatum polysaccharide by DEAE-cellulose column chromatography

[0075] Accurately weigh 50g of DEAE cellulose and dissolve it in 1L of ultrapure water, stir well, and stop stirring if no cellulose particles are visible. Discard the supernatant after 24h, and prepare 0.5mol / L NaOH. Soak the cellulose for 6h, wash it with ultrapure water until it is neutral, discard the supernatant, and add 0.5mol / L HCl again for 6h. Wash with distilled water until it is neutral, discard the supernatant, and add 0.5mol / L NaOH again for 6h. Wash with distilled water until it is neutral, and stand by.

[0076] After activated cellulose was loaded into the column, the column was equilibrated with distilled water for 24 h, and then the crude polysaccharide was separated and purified. The supernatant (5 mL) of the diluted crude polysaccharide was added to the DEAE cellulose column, and different concentrations of NaCl (0 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L) were added for elution. The polysaccharide was determined by the sulfuric acid-phenol method. The eluate was concentrated to 5 mL, and the sample was purified on a cellulose column. The sample was dialyzed in a dialysis bag (Mw≥7 kDa) for 48 h, and then freeze-dried to obtain the polysaccharide of Lycoperdon gemmatum, which was named LS-P.

[0077] 2. Structural identification of Lycoperdon gemmatum polysaccharide LS-P

[0078] The structure of Lycoperdon gemmatum polysaccharide (LS-P) was analyzed by acid hydrolysis, methylation analysis, high-performance gel permeation chromatography (HPGPC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy (IR), and nuclear magnetic resonance (NMR) techniques.

[0079] 2.1. Determination of molecular weight

[0080] A 10-mg sample of Lycoperdon gemmatum polysaccharide LS-P was dissolved in 1 mL of ddH2O and ultrasonicated for 5 min for HPGPC analysis.

[0081] 2.2. Infrared spectroscopy analysis of Lycoperdon gemmatum polysaccharide LS-P

[0082] A 2-mg sample of LS-P was mixed with 200 mg of KBr and pressed into a tablet, which was scanned by an infrared spectrophotometer in the range of 4000 cm -1 -400 cm -1 .

[0083] 2.3. Analysis of monosaccharide composition of Lycoperdon gemmatum polysaccharide LS-P

[0084] Seven standard samples and the LS-P sample after TFA acid hydrolysis were dissolved in a mobile phase (80% acetonitrile) for HPLC analysis.

[0085] 2.4. Nuclear magnetic resonance analysis of Lycoperdon gemmatum polysaccharide LS-P

[0086] A 50-mg sample of LS-P was dissolved in 0.6 mL of heavy water (D2O) and loaded into a nuclear magnetic tube for detection on a nuclear magnetic resonance instrument.

[0087] 2.5. GC-MS analysis of Lycoperdon gemmatum polysaccharide LS-P after methylation and silylation derivatization

[0088] Take 20 mg of LS-P sample, seal the beaker, add 2 mL of DMSO (dimethyl sulfoxide) to the sealed beaker, and gently shake the beaker to fully dissolve the LS-P. Then add 200 mg of NaOH until the NaOH just does not dissolve and place it in a shaker at room temperature for 1 h. After shaking is complete, add 1.5 mL of iodomethane, avoid light for 1 h, and after the reaction, add water to terminate the reaction. Extract the product with chloroform, dry to obtain the methylated polysaccharide. After complete acid hydrolysis of the methylated polysaccharide by TFA, wash with water three times to obtain the completely acid hydrolyzed product of the methylated polysaccharide.

[0089] The above sample is fully reacted with 2 mL of hexamethyl disilazane, 1 mL of trimethylchlorosilane, and 2 mL of anhydrous pyridine, and is placed in a water bath at 50°C for 20 min. A low-temperature high-speed centrifuge is used at a speed of 12000 rpm / min, 4°C, and centrifuged for 10 min. The precipitate is discarded, filtered with a 0.22 μm filter, and the upper layer solution is used for GC-MS analysis.

[0090] 3. Results

[0091] 3.1. Basic property results of Armillaria luteoviridis polysaccharide LS-P

[0092] The HPGPC spectrum of LS-P is shown in Figure 1 , which shows that the weight average molecular weight of LS-P is 13135 Da.

[0093] 3.2. FTIR spectrum analysis of Armillaria luteoviridis polysaccharide LS-P

[0094] The primary structure of LS-P is characterized by Fourier infrared spectroscopy, and the results are shown in Figure 2 . There are typical polysaccharide absorption peaks at wave numbers of 3426.03 cm -1 , 2927.37 cm -1 , and 1400-1200 cm -1 , etc., and no other impurity peaks, indicating that the separated and purified LS-P is a polysaccharide substance. The wide absorption peak of O-H stretching vibration peak at 3426.03 cm -1 , the absorption peak in the range of 2927.37 cm -1 is the stretching vibration peak of -CH2, 1641.65 cm -1 is the C=O stretching vibration peak, 1415.80 cm -1 is the C-H in-plane bending vibration peak of -CHO, and 1079.67 cm -1 is the absorption peak of sugar C-O stretching vibration peak. The absorption peaks in the range of 1200-1000 cm -1 are absorption peaks produced by the absorption of pyranose ring lactone and hydroxyl groups, indicating that LS-P has a pyran ring. At 673.62 cm -1is the =C-H in-plane bending vibration peak of LS-P. In addition, there is no absorption peak near 1730 cm -1 , indicating that LS-P does not contain uronic acid.

[0095] 3.3, Monosaccharide composition analysis of Laetiporus cymbiformis polysaccharide LS-P

[0096] After complete hydrolysis of LS-P, HPLC was used to analyze the monosaccharide composition, and the results are shown in Figure 3 , wherein peak 1 is glucose (Glc) with a retention time of 9.154 min; peak 2 is galactose (Gal) with a retention time of 9.614 min. The peak area ratio of glucose and galactose is 59:41.

[0097] 3.4, NMR spectrum analysis of Laetiporus cymbiformis polysaccharide LS-P

[0098] LS-P 1 H NMR results are shown in Figure 4 . The results show that LS-P has five anomeric hydrogen signals at δ5.01 ppm, δ4.94 ppm, δ4.89 ppm, δ4.86 ppm and δ4.40 ppm. The signals between δ3.0-4.2 ppm are attributed to the hydrogen signals of C2-C6 in the sugar residue.

[0099] LS-P 13 C NMR results are shown in Figure 5 . LS-P has five anomeric carbon signals at δ102.98 ppm, δ101.67 ppm, δ98.26 ppm, δ98.19 ppm and δ98.01 ppm. The signals between δ60-80 ppm are attributed to the carbon signals of C2-C6 in the sugar residue.

[0100] LS-P 1 H- 1 H-COSY spectrum is shown in Figure 6 . According to it, the coupling relationship between adjacent hydrogen nuclei can be identified. The signals of H1 / H2 in part A are δ5.01 / 3.85, the signals of H1 / H2 in part B are δ4.94 / 3.62, the signals of H1 / H2 in part C are δ4.94 / 3.67, the signals of H1 / H2 in part D are δ4.89 / 3.73, the signals of H1 / H2 in part E are δ4.86 / 3.73, and the signals of H1 / H2 in part F are δ4.40 / 3.22.

[0101] The chemical shifts of all hydrogens are summarized in Table 1.

[0102] The HMQC spectrum of LS-P is shown in Figure 7 . According to it, the short-range related 1 H and C can be identified.13 The coupling relationship between C. The signal of H1 / C1 in part A is δ5.01 / 99.26, the signal of H1 / C1 in part B is δ4.94 / 98.26, the signal of H1 / C1 in part C is δ4.94 / 101.67, the signal of H1 / C1 in part D is δ4.89 / 98.19, the signal of H1 / C1 in part E is δ4.86 / 98.01, and the signal of H1 / C1 in part F is δ4.40 / 102.98. The ratio of the integral area of ​​the five parts A, B, C, D, E, and F is 1.28:0.80:1.24:0.79:0.84:0.43.

[0103] The HMBC spectrum of LS-P is as follows Figure 8 As shown, it can identify remote related... 1 H and 13 The coupling relationships between residues C and C are as follows: H1 / C3 signal of residue A is δ5.01 / 68.79, H3 / C5 signal of residue B is δ3.83 / 68.55, H2 / C4 signal of residue C is δ3.67 / 69.49, H1 / C3 signal of residue D is δ4.89 / 69.49, H5 / C3 signal of residue E is δ3.64 / 68.26, and H5 / C3 signal of residue F is δ3.53 / 67.19.

[0104] All the chemical shifts of carbon are summarized in Table 2.

[0105] Table 1 LS-P 1 Chemical shift of H

[0106]

[0107] Table 2 LS-P 13 Chemical shift of C

[0108]

[0109] 3.5 Gas Chromatography and Mass Spectrometry Analysis of Polysaccharide LS-P from Mushroom Flourula 'Hua Lian Xiang'

[0110] The methylation results are shown in Table 3, indicating that the main repeating structural unit of LS-P consists of a backbone composed of 1,6-linked glucose residues, 1,4-linked D-galactose residues, 1,4-linked D-glucose residues, and 1,6-linked galactose residues, and a side chain composed of 1,4,6-linked glucose residues and 2-linked α-D-glucose residues. Table 4 shows the peak times of the monosaccharide standard and the hydrolyzed LS-P monosaccharide components.

[0111] Table 3 Analysis of LS-P methylation results

[0112]

[0113]

[0114] Table 4

[0115]

[0116] Example 2: Immunomodulatory and Anti-tumor Activity of Ls-Polysaccharide from Laccaria papyracea

[0117] The immunomodulatory and anti-tumor activity of LS-Polysaccharide from Laccaria papyracea was determined in vitro using CCK-8 method.

[0118] 1. Reagents

[0119] CCK-8 kit, RPIM1640, FBS, DMSO, double antibody, etc. are all commercially available products.

[0120] 2. Instruments

[0121] Enzyme marker; cell incubator.

[0122] 3. Methods

[0123] Effect of LS-P on the proliferation of immune cells (B cells, T cells, RAW264.7 cells and MFC cells)

[0124] The effect of LS-Polysaccharide from Laccaria papyracea (LS-P) on the proliferation of B cells, T cells, RAW264.7 cells and MFC cells was determined by cell counting kit (CCK-8) method. B cells, T cells, RAW264.7 cells and MFC cells were cultured in vitro to the logarithmic growth phase, and after counting with a cell counting plate, the cell suspension was diluted to 1×10 5 μg / mL, 100 μL of the cell suspension was added to each well of a 96-well plate, and the 96-well plate was placed in a CO2 incubator for 24 h. After 24 h, different concentrations of LS-P solution (final concentration 5, 10, 20 μg / mL) were added to the experimental group (LS-P group), 100 μL of LPS solution (as a positive control group for the proliferation activity of B cells, T cells and RAW264.7 cells) / MAN solution (as a positive control group for the proliferation activity of MFC cells) (final concentration 10 μg / mL) were added to the positive control group (LPS group / MAN group), and 100 μL of cell culture medium was added to the blank group (CK group). After 24 h of incubation in a CO2 incubator, the cell images of each group were taken under an inverted microscope, and then 10 μl of CCK-8 was added to each well, incubated in a CO2 incubator for 3 h, and then the absorbance value was detected (450 nm) on an enzyme marker.

[0125] 4. Results

[0126] 4.1 LS-P on the proliferation of B cells

[0127] The results are shown in Figure 9 Compared with the blank group (CK group), the LPS group can significantly promote the proliferation of B cells (P <0.01), and the proliferation rate is 83.63%; when the final concentration of LS-P is 5, 10 and 20 μg / mL, it can significantly promote the proliferation of B cells (P <0.01); and when the final concentration of LS-P is 10 μg / mL, the effect of LS-P on the proliferation of B cells is the most obvious, and the maximum proliferation rate reaches 59.92%.

[0128] 4.2 LS-P on the proliferation of T cells

[0129] The results are shown in Figure 10 Compared with the blank group (CK group), the LPS group can significantly promote the proliferation of T cells (P <0.01), and the proliferation rate is 25.78%; when the final concentration of LS-P is 5, 10 and 20 μg / mL, it can significantly promote the proliferation of T cells (P <0.01); and when the final concentration of LS-P is 20 μg / mL, the effect of LS-P on the proliferation of T cells is the most obvious, and the maximum proliferation rate reaches 18.94%.

[0130] 4.3 LS-P on the proliferation of RAW264.7 cells

[0131] The results are shown in Figure 11 Compared with the blank group (CK group), the LPS group can significantly promote the proliferation of RAW264.7 cells (P <0.01), and the proliferation rate is 68.08%; when the final concentration of LS-P is 5, 10 and 20 μg / mL, it can significantly promote the proliferation of RAW264.7 cells (P <0.05); and when the final concentration of LS-P is 20 μg / mL, the effect of LS-P on the proliferation of RAW264.7 cells is the most obvious, and the maximum proliferation rate reaches 51.16%.

[0132] 4.4 LS-P on the proliferation of MFC cells

[0133] The results are shown in Figure 12 Compared with the blank group (CK group), the MAN group can significantly inhibit the proliferation of MFC cells (P <0.01), and the inhibition rate is 20.71%; when the final concentration of LS-P is 5, 10 and 20 μg / mL, it can significantly inhibit the proliferation of MFC cells (P <0.01); and when the final concentration of LS-P is 10 μg / mL, the effect of LS-P on the inhibition of MFC cells is the most obvious, and the maximum inhibition rate reaches 27.36%.

[0134] The above merely describes preferred embodiments of the present application, but is not used to limit the present application, and any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0135] The foregoing embodiments and methods described in the present application can vary based on the ability, experience and preference of the person skilled in the art.

[0136] The fact that the steps of the method are listed in a certain order in the present application does not constitute any limitation on the order of the steps of the method.

Claims

1. A polysaccharide from the spotted mushroom, characterized in that, The polysaccharide is a heteropolysaccharide composed of glucose and galactose, wherein the molar ratio of glucose and galactose residues is 8:5; The polysaccharide comprises the following structure: ， Wherein, n is an integer from 1 to 15, and the weight-average molecular weight of the polysaccharide is 8000-20000 Da.

2. The polysaccharide as described in claim 1, characterized in that, The weight-average molecular weight of the polysaccharide is 10,000-15,000 Da.

3. The polysaccharide as described in claim 1, characterized in that, The n is an integer between 4 and 8.

4. A composition, characterized in that, The composition comprises the polysaccharide according to any one of claims 1-3.

5. The method for preparing polysaccharides according to claim 1, characterized in that, This includes the extraction steps using the fruiting bodies of *Mushroom floribunda* as raw material: (1) Take the fruiting body powder of Mushroom floridae, extract it with hot water, and then concentrate, precipitate with alcohol and dry the resulting water extract to obtain crude polysaccharide; (2) The crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, eluted, and the eluent is collected; (3) The eluent obtained in step (2) is concentrated by dialysis using a dialysis bag; In step (1), the extraction temperature is 80-100℃; The mass ratio of the mushroom fruiting body powder to water is 1:1-10; The volume ratio of alcohol in the alcohol precipitation to the concentrated water extract is 1-10:1; In step (2), the ion exchange column is a cellulose column and its packing material is DEAE cellulose; The eluent used for elution is a NaCl solution; The elution is gradient elution.

6. The application of the polysaccharide according to any one of claims 1-3, characterized in that, The applications include: (1) Application in the preparation of products that can enhance immunity; or, (2) Application in the preparation of products with antitumor activity.

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