Lepista sordida polysaccharide as well as preparation method and application thereof

Abstract

The invention particularly discloses lepista sordida polysaccharide as well as a preparation method and application thereof. The lepista sordida polysaccharide comprises glucose and galactose, and the chemical structure of the lepista sordida polysaccharide comprises a 1, 4-linked D-glucose residue, a 1, 4-linked D-galactose residue, a 1, 6-linked galactose residue, a 2-linked alpha-D-glucose residue, a 1, 4, 6-linked glucose residue and a 1, 6-linked glucose residue. And the molar ratio of the residues 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 lepista sordida polysaccharide is obtained through hot water extraction, ethanol precipitation, deproteinization, ion exchange column chromatography, dialysis and concentration. The lepista sordida polysaccharide disclosed by the invention has remarkable immunoregulatory activity and antitumor activity, and can be applied to medicines, health-care products or foods.

Description

Technical Field

[0001] The invention relates to the technical field of fungal polysaccharide application, and in particular to a polysaccharide of Lentinula glabra and a preparation method and application thereof. Background Art

[0002] Edible fungi, commonly known as mushrooms, are large fungi whose fruiting bodies are rich in nutrients such as protein, vitamins, mineral elements, amino acids, and polysaccharides. Edible fungi polysaccharides have biological activities such as antiviral, antioxidant, anti-tumor, lipid-lowering, promoting the proliferation and differentiation of immune cells and the secretion of lymphokines, activating complement and other immune regulation. They are safe and non-toxic, and have received extensive attention in the fields of health food and biomedicine. Moreover, edible fungi polysaccharides are a non-specific immunopotentiator that can improve the body's immune function through various pathways without side effects on the body.

[0003] Lepista sordida belongs to the order Agaricales, family Tricholomatacete, genus Lepista. It is mainly distributed in Yunnan, Heilongjiang, Liaoning, Sichuan, Guizhou and other places in China. It has a strong fragrance, crisp taste and pleasant color. The cap of Lepista sordida is 4cm-8cm in diameter, violet, rough to pitted, and the stipe is 4cm-6.5cm long, solid, and mostly curved at the base; the flesh is light violet, thin, and water-soaked; the gills are straight, sometimes slightly curved or slightly extended; the fruiting bodies are purple or lavender when young, and fade easily after maturity, mostly turning into milky white or pink purple. Even the same species will have different phenotypic characteristics at different developmental stages. Studies have found that extracts of Lepista sordida have certain antioxidant and anti-tumor activities. In addition, two diterpenoid compounds (lepistal and lepistol) separated from the fermentation broth of the bacteria have biological activity of inducing differentiation of human anemic leukemia cells, among which lepistal also has antibacterial and antifungal effects.

[0004] Hu Xinlei et al. reported in "Optimization of Extraction Conditions of Polysaccharides from Mycelium of Scutellaria odoratum and Its In Vitro Immune Activity" ([J]. Food Science, 2017, 38(20): 185-190.) that the polysaccharides from Scutellaria odoratum mycelium were optimized by hot water extraction using orthogonal experiments. The optimal extraction conditions were 65°C, 2.5h, and 40:1 (mL / g) liquid-to-solid ratio. Under these conditions, the polysaccharide extraction rate was 15.88%. The extract was applied to mouse macrophages Ana-1. The study found that within the effective dose range of 0.05-1.6 mg / mL, polysaccharides from Scutellaria odoratum mycelium could promote the proliferation of mouse macrophages Ana-1 to varying degrees, increase the secretion of cytokines TNF-α and IL-6, enhance the phagocytosis of neutral red, and the expression of membrane surface proteins TLR2 and TLR4. The best effect was achieved at 1.6 mg / mL. This indicates that polysaccharides from Scutellaria odoratum mycelium have excellent immune activity.

[0005] Luo et al. reported in Structural characterization of an immunoregulatory polysaccharide from the fruiting bodies of Lepistasordida. ([J]. Carbohydrate Polymers, 2012, 88: 820–824.) that the molecular weight (Mw) of the polysaccharide LSP from the mushroom was about 4×10 4 Da. According to Fourier transform infrared (FT-IR) spectroscopy, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and gas chromatography analysis, the results showed that LSP has (1→6)-α-D-glucose residues and (1→2,6)-α-D-glucose residues as the main chain and (1→)-α-D-galactose residues as the side chain, with a molar ratio of 2:1.1:0.9. In vitro cell experiments showed that LSP can significantly increase the secretion of NO and TNF-α by mouse macrophages (P<0.05), and has certain immune activity.

[0006] It can be seen that the prior art lacks research on the fine structure of Lentinula edodes polysaccharide and the application of Lentinula edodes polysaccharide in immunomodulatory activity. Summary of the invention

[0007] The invention overcomes the defects in the prior art and provides a polysaccharide of Lentinula floridula and a preparation method and application thereof.

[0008] The first aspect of the present invention provides a Lentinula scaly-faced polysaccharide (LS-P), which is a heteropolysaccharide composed of glucose and galactose, wherein the molar ratio of the glucose to galactose residues is about 8:5.

[0009] Furthermore, 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, and 1,6-linked glucose residues.

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

[0011] Furthermore, 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 one embodiment of the present invention, the weight average molecular weight of the polysaccharide is 13135 Da.

[0013] Furthermore, 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, wherein Glcp is glucose and Galp is galactose.

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

[0017] The third aspect of the present invention provides a method for preparing the polysaccharide of the tricholoma eryngii as described in the first aspect, the preparation method comprising the step of extracting the tricholoma eryngii fruiting body as a raw material.

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

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

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

[0021] (1) extracting the powder of the fruiting body of the tricholoma scaly-leached mushroom with hot water, and concentrating, precipitating with alcohol, and drying the obtained water extract to obtain crude polysaccharide;

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

[0023] (3) The eluate obtained in step (2) is dialyzed and concentrated using a dialysis bag.

[0024] Furthermore, the preparation method further comprises step (4), specifically freeze-drying the liquid in the dialysis bag after completing step (3).

[0025] Furthermore, in step (1), the extraction temperature may 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.

[0026] Furthermore, in step (1), the mass ratio (W / V, mg / mL) of the powder of the fruiting body of the Oleander 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 mass ratio is 1:3.

[0027] Furthermore, in step (1), the number of extractions is 1-5 times (such as 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0028] Furthermore, in step (1), each extraction time is 1-10 hours (such as 1, 3, 6, 8, 10 hours); in one embodiment of the present invention, each extraction time is 6 hours.

[0029] In one embodiment of the present invention, the extraction step in step (1) may include: taking the fruiting body powder of the Oleander mushroom, mixing it with water, and boiling it in a water bath.

[0030] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to the water extract concentrate 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 3:1;

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

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

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

[0034] Furthermore, 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] Furthermore, in step (2), the elution can be gradient elution, and the eluent concentration 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 invention, step (2) comprises: passing the aqueous solution of crude polysaccharide obtained in step (1) through a cellulose column, gradient eluting, collecting the eluate, and concentrating.

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

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

[0039] The fourth aspect of the present invention provides an application of the polysaccharide of Lentinula ovata as described in the first aspect, or the composition as described in the second aspect, or the crude polysaccharide prepared by the preparation method as described in the third aspect, the application comprising:

[0040] (1) Application in the preparation of products capable of enhancing immunity;

[0041] (2) Application in the preparation of products with anti-tumor activity.

[0042] Furthermore, the product is food, health care product or medicine.

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

[0044] Furthermore, the final concentration of the polysaccharide of Lentinula edodes 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] Furthermore, when the purpose is to enhance the proliferation effect of B cells, the final concentration of Lentinula edodes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 10 μg / mL.

[0046] Furthermore, when the purpose is to enhance the proliferation effect of T cells, the final concentration of Lentinula edodes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 20 μg / mL.

[0047] Furthermore, when the proliferation effect of RAW 264.7 cells is to be enhanced, the final concentration of Lentinula edodes polysaccharide is preferably 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 20 μg / mL.

[0048] Furthermore, when the inhibitory effect on MFC cells is to be enhanced, the final concentration of the polysaccharide of Lentinula edodes is 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 10 μg / mL.

[0049] The beneficial effects of the present invention are:

[0050] The present invention separates and purifies the polysaccharide of the shimeji mushroom fruiting body, analyzes and identifies its molecular weight, monosaccharide composition, chemical structure, etc., and determines 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 proliferation rate of B cells is the highest, at a final concentration of 20 μg / mL, the proliferation rate of T cells is the highest, and at a final concentration of 20 μg / mL, the proliferation rate of RAW264.7 cells 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 THE DRAWINGS

[0051] Figure 1 Shown is the HPGPC spectrum of LS-P;

[0052] Figure 2 Shown is the infrared spectrum of LS-P;

[0053] Figure 3 Shown is the HLPC spectrum of LS-P;

[0054] Figure 4 Shown is LS-P 1H NMR spectrum;

[0055] Figure 5 Shown is LS-P 13 C NMR spectrum;

[0056] Figure 6 Shown is LS-P 1 H- 1 H-COSY spectrum;

[0057] Figure 7 Shown is the HMQC spectrum of LS-P;

[0058] Figure 8 Shown is the HMBC spectrum of LS-P;

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

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

[0061] Fig.11 Shown are the experimental results of the effect of LS-P on the proliferation of RAW264.7 cells (1: blank group; 2-4: LS-P with final concentrations of 5, 10, and 20 μg / mL; 5: LPS with a final concentration of 10 μg / mL);

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

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

[0064] In the present invention, the term "Lepistasordida" refers to the phylum Basidiomycetes, class Agaricomycetes, order Agaricales, family Tricholomataceae, genus Lepistasordida, which includes fruiting bodies and mycelium.

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

[0066] The term "LPS" refers to lipopolysaccharide, which is the abbreviation of Lipopolysaccharides and 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 mannan peptide, the abbreviation of Mannatide. The MAN solution used in this experiment was purchased from Sichuan Aobang Pharmaceutical.

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

[0069] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0071] 1. Separation and extraction of LS-P polysaccharide from Lentinula odoratum

[0072] 1.1. Extraction of crude polysaccharides from Oleander mushroom by water extraction and alcohol precipitation

[0073] Weigh 200g of dried shimeji mushroom fruiting bodies and crush them, add the crushed shimeji mushroom fruiting bodies and distilled water in a beaker at a ratio of 1:3, place in a water bath at 98°C for 6 hours, collect the supernatant and concentrate, repeat 3 times, and finally concentrate all the supernatant to 200mL. Add three times the volume of anhydrous ethanol to precipitate, collect and dry the precipitate, remove the protein in the extract, and obtain shimeji mushroom crude polysaccharide.

[0074] 1.2. Separation and purification of crude polysaccharides from Oleander by DEAE-cellulose column chromatography

[0075] Accurately weigh 50g DEAE cellulose and dissolve it in 1L ultrapure water. Stir thoroughly. Stop stirring if no cellulose particles are visible to the naked eye. Let stand for 24 hours, discard the supernatant and set aside. Prepare 0.5mol / L NaOH, soak the cellulose for 6 hours, wash with ultrapure water until neutral, discard the supernatant, add 0.5mol / L HCl and soak for 6 hours, wash with distilled water until neutral, discard the supernatant; add 0.5mol / L NaOH and soak again for 6 hours, wash with distilled water until neutral, and let stand for use.

[0076] After the activated cellulose is loaded into the column, the crude polysaccharide can be separated and purified after balancing the column with distilled water for 24 hours. The supernatant (5mL) after the crude polysaccharide is diluted is added to the DEAE cellulose column, and different concentrations of NaCl (0mol / L, 0.05mol / L, 0.1mol / L, 0.2mol / L, 0.3mol / L) are added for elution. The polysaccharide is determined by the sulfuric acid-phenol method. The eluate is concentrated to 5mL and the sample is purified on the cellulose column. Dialysis is performed with a dialysis bag (Mw≥7kDa) for 48 hours, and freeze-dried to obtain the flower-faced mushroom polysaccharide, named LS-P.

[0077] 2. Structural identification of LS-P polysaccharide from Lentinula odoratum

[0078] The structure of Lentinula ovata polysaccharide (LS-P) was analyzed using acid hydrolysis, methylation analysis, high performance gel permeation chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance technology.

[0079] 2.1. Determination of molecular weight

[0080] 10 mg of Lentinan LS-P sample was washed with 1 mL of ddHO. 2 O dissolution, ultrasonication for 5 min, and HPGPC analysis.

[0081] 2.2. Infrared spectrum analysis of LS-P polysaccharide from Lentinula ovata

[0082] 2 mg LS-P was mixed with 200 mg KBr and pressed into a pellet. The pellet was scanned at 4000 cm by an infrared spectrophotometer. -1 -400cm -1 scope.

[0083] 2.3 Analysis of monosaccharide composition of LS-P polysaccharide from Lentinula ovata

[0084] The seven standards and the LS-P sample after TFA acid hydrolysis were dissolved in the mobile phase (80% acetonitrile) and then subjected to HLPC analysis.

[0085] 2.4. NMR analysis of LS-P polysaccharide from Lentinula ovata

[0086] Take 50 mg of LS-P sample and dissolve it in 0.6 mL of heavy water (D 2 O), put it into a nuclear magnetic resonance tube, and detect it on a nuclear magnetic resonance instrument.

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

[0088] Weigh 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 is just insoluble and place it in a shaker to shake at room temperature for 1 hour. After the shaking is completed, add 1.5 mL of iodomethane, react in the dark for 1 hour, and add water to terminate the reaction after the reaction. Extract the product with chloroform and dry it to obtain methylated polysaccharides. After the methylated polysaccharide is completely hydrolyzed with TFA, wash it with water three times to obtain a methylated completely acid hydrolyzed product.

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

[0090] 3. Results

[0091] 3.1 Basic properties of Lentinula edulis polysaccharide LS-P

[0092] The HPGPC spectrum of LS-P is as follows: Figure 1 As shown, it shows that the weight average molecular weight of LS-P is 13135 Da.

[0093] 3.2 FTIR spectrum analysis of Lentinula ovata polysaccharide LS-P

[0094] Fourier transform infrared spectroscopy was used to characterize the primary structure of LS-P. Figure 2 As shown, the wave number is 3426.03cm -1 , 2927.37cm -1 and 1400~1200cm -1 There are typical polysaccharide absorption peaks in the above places, and no other impurity peaks, indicating that the separated and purified LS-P is a polysaccharide substance. The wave number is 3426.03cm -1 The broad absorption peak of OH stretching vibration peak, 2927.37cm -1 The absorption peak in the range is -CH 2 Stretching vibration peak, 1641.65cm -1 C=O stretching vibration peak, 1415.80cm -1 The in-plane bending vibration peak of CH of -CHO is 1079.67 cm -1 The absorption peak is the CO stretching vibration peak of sugars. -1The absorption peaks in the range are the absorption peaks generated by the pyranose ring lactone and the hydroxyl group, indicating that LS-P has a pyranose ring. -1 The absorption peak is the in-plane bending vibration peak of =CH of LS-P. In addition, at 1730cm -1 There is no absorption peak nearby, indicating that LS-P does not contain uronic acid.

[0095] 3.3 Analysis of monosaccharide composition of LS-P polysaccharide from Lentinula ovata

[0096] After LS-P was completely hydrolyzed, its monosaccharide composition was analyzed by HPLC. Figure 3 As shown, 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 to galactose is 59:41.

[0097] 3.4 NMR spectrum analysis of LS-P polysaccharide from Lentinula odoratum

[0098] LS-P 1 H NMR results are as follows Figure 4 The results showed that LS-P had five anomeric hydrogen signals at δ5.01ppm, δ4.94ppm, δ4.89ppm, δ4.86ppm and δ4.40ppm. The signals between δ3.0 and 4.2ppm were assigned to the hydrogen signals of C2-C6 in the sugar residues.

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

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

[0101] All hydrogen chemical shifts are summarized in Table 1.

[0102] The HMQC spectrum of LS-P is shown in Figure 7 As shown, it can be used to identify the close related 1 H and 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, the signal of H1 / C1 in part F is δ4.40 / 102.98, and the integrated area ratios of parts A, B, C, D, E, and F are 1.28:0.80:1.24:0.79:0.84:0.43.

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

[0104] The chemical shifts of all carbons 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 LS-P polysaccharide from Lentinula odoratum

[0110] The methylation results are shown in Table 3, indicating that the main repeating structural unit of LS-P is composed of a main chain consisting 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 consisting of 1,4,6-linked glucose residues and 2-linked α-D-glucose residues. Table 4 shows the peak time of monosaccharide standards and monosaccharide components of LS-P after hydrolysis.

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

[0112]

[0113]

[0114] Table 4

[0115]

[0116] Example 2: Study on the immunomodulatory and anti-tumor activity of Lentinula scaly-leaf polysaccharide LS-P

[0117] The CCK-8 method was used to determine the immunomodulatory and antitumor activities of Lentinula ovata polysaccharide LS-P in vitro.

[0118] 1. Reagents

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

[0120] 2. Instruments

[0121] Microplate reader; cell culture incubator.

[0122] 3. Methods

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

[0124] The effect of Lentinula edodes polysaccharide (LS-P) on the proliferation of B cells, T cells, RAW264.7 cells and MFC cells was determined by the 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. After counting with a cell counting plate, the cell suspension was diluted to 1×10 5 The cell suspension was added to a 96-well plate, 100 μL per well, and the 96-well plate was placed in a CO 2 The cells were cultured in an incubator for 24 h. After 24 h, the experimental group (LS-P group) was added with LS-P solutions of different mass concentrations (final concentrations of 5, 10, and 20 μg / mL), the positive control group (LPS group / MAN group) was added with 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 mass concentrations were 10 μg / mL), and the blank group (CK group) was added with 100 μL of cell culture medium. 2After culturing in the incubator for 24 h, images of each group of cells were taken under an inverted microscope, and 10 μl of CCK-8 was added to each well. 2 After incubation in the incubator for 3 h, the absorbance value was measured on an enzyme-labeled instrument (450 nm).

[0125] 4. Results

[0126] 4.1 Effect of LS-P on B cell proliferation

[0127] The results are as follows Fig. 9 As shown, compared with the blank group (CK group), the LPS group could significantly (P<0.01) promote the proliferation of B cells, with a proliferation rate of 83.63%; when the final concentration of LS-P was 5, 10 and 20 μg / mL, it could significantly (P<0.01) promote the proliferation of B cells; and when the final concentration of LS-P was 10 μg / mL, the effect of LS-P on the proliferation of B cells was the most obvious, with the maximum proliferation rate reaching 59.92%.

[0128] 4.2 Effect of LS-P on T cell proliferation

[0129] The results are as follows Fig.10 As shown, compared with the blank group (CK group), the LPS group could significantly (P<0.01) promote the proliferation of T cells, with a proliferation rate of 25.78%; when the final concentration of LS-P was 5, 10 and 20 μg / mL, it could significantly (P<0.01) promote the proliferation of T cells; when the final concentration of LS-P was 20 μg / mL, the effect of LS-P on the proliferation of T cells was the most obvious, with the maximum proliferation rate reaching 18.94%.

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

[0131] The results are as follows Fig.11 As shown, compared with the blank group (CK group), the LPS group could significantly (P<0.01) promote the proliferation of RAW264.7 cells, with a proliferation rate of 68.08%; when the final concentration of LS-P was 5, 10 and 20 μg / mL, it could significantly (P<0.05) promote the proliferation of RAW264.7 cells; when the final concentration of LS-P was 20 μg / mL, the proliferation effect of LS-P on RAW264.7 cells was most obvious, with the maximum proliferation rate reaching 51.16%.

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

[0133] The results are as follows Fig.12As shown, compared with the blank group (CK group), the MAN group could significantly (P<0.01) inhibit the proliferation of MFC cells, with an inhibition rate of 20.71%; when the final concentration of LS-P was 5, 10 and 20 μg / mL, it could significantly (P<0.01) inhibit the proliferation of MFC cells; when the final concentration of LS-P was 10 μg / mL, the inhibitory effect of LS-P on MFC cells was the most obvious, with the maximum inhibition rate reaching 27.36%.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0135] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0136] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A polysaccharide of Lentinula edulis, characterized in that: The polysaccharide is a heteropolysaccharide composed of glucose and galactose, wherein the molar ratio of the residues of glucose and galactose is about 8:5; The polysaccharide comprises the following structure: Wherein, n is an integer of 1-15, Glcp is glucose, and Galp is galactose.

2. The polysaccharide according to claim 1, characterized in that The weight average molecular weight of the polysaccharide is 8000-20000 Da, preferably 10000-15000 Da, particularly preferably 13135 Da.

3. The polysaccharide according to claim 1, characterized in that Said n is an integer of 4-8.

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

5. The method for preparing a polysaccharide according to claim 1, characterized in that: The steps of extracting the fruiting body of the tricholoma scaly-leaved mushroom as raw material include: Preferably, the preparation method comprises the steps of extracting crude polysaccharides by water extraction and alcohol precipitation; Further preferably, the preparation method further comprises the step of purifying the crude polysaccharide.

6. The preparation method according to claim 5, characterized in that: The preparation method comprises the following steps: (1) extracting the powder of the fruiting body of the tricholoma scaly-leached mushroom with hot water, and concentrating, precipitating with alcohol, and drying the obtained water extract to obtain crude polysaccharide; (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate; (3) The eluate obtained in step (2) is dialyzed and concentrated using a dialysis bag.

7. The preparation method according to claim 6, characterized in that: In step (1), the extraction temperature is 80-100° C.; The mass ratio of the powder of the fruiting body of the tricholoma scaly is 1:1-10 to the water. The volume ratio of alcohol to water extract concentrate in the alcohol precipitation is 1-10:1; Preferably, the alcohol in the alcohol precipitation is ethanol.

8. The preparation method according to claim 6, characterized in that: In step (2), the ion exchange column is a cellulose column, and its filler is DEAE cellulose; The eluent used for the elution is NaCl solution; The elution was gradient elution.

9. Use of the polysaccharide according to any one of claims 1 to 3, the composition according to claim 4, or the polysaccharide prepared by the preparation method according to any one of claims 5 to 9, characterized in that: The applications include: (1) Application in the preparation of products capable of enhancing immunity; (2) Application in the preparation of products with anti-tumor activity.

10. The use according to claim 9, characterized in that: The final concentration of Lentinula edodes polysaccharide in the product is 5-20 μg / mL.

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