A blue-purple laccaria polysaccharide, and a preparation method and application thereof

Abstract

The application discloses blue-purple Laccaria amethystina polysaccharide and a preparation method and application thereof. The blue-purple Laccaria amethystina polysaccharide is composed of mannose and glucose, and the molar ratio is 2:5; the chemical structure of the blue-purple Laccaria amethystina polysaccharide is that the mannose residue is (4→6)-alpha-D-Manp, the glucose residue contains (4→6)-alpha-D-Glcp, (1→4)-alpha-D-Glcp and (1→4, 6)-beta-D-Glcp, and the ratio is 2:3:1:1. The blue-purple Laccaria amethystina polysaccharide is obtained through hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography, concentration and dialysis of the blue-purple Laccaria amethystina fruiting body. The blue-purple Laccaria amethystina polysaccharide has significant immunomodulatory activity and antitumor activity, and can be applied in medicines, health products or food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of application of fungal polysaccharides, and particularly relates to a Laccaria moshuijun 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, 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 pathways without side effects on the body.

[0003] Laccaria moshuijun belongs to Basidiomycota, Agaricales, Cortinarius and Laccaria, is commonly known as skin fungus, and is mainly distributed in Sichuan, Yunnan, Guangxi and other places. The fruiting body of Laccaria moshuijun is purple, the cap is 2-5 cm wide, is initially flat and spherical, is then gradually flat and expanded, is sunken in the center and is navel-shaped, is blue-purple or lotus root powder color, is like wax when wet, is dark in color, is grayish white with purple color when dry, and the edge is wavy or petal-shaped with thick stripes. The flesh is the same color as the cap, and is thin. The gills are blue-purple, are straight or nearly curved, are wide, are sparse, and are unequal in length. The stem is 3-8 cm long, is 0.2-0.8 cm thick, has fluff, is fibrous, is solid, and the lower part is often curved. The spores are colorless, are spherical, and are 8.7-13.8 μm.

[0004] At present, the research on polysaccharides of Laccaria is only directed to Laccaria laccata polysaccharides. Zhang Shuahua et al. reported in "Ultrasonic-assisted extraction technology of Laccaria laccata polysaccharides and its in vitro antioxidant activity" (Northern Horticulture, 2018(17):133-138.) the influence of liquid-solid ratio, ultrasonic power, ultrasonic time, ultrasonic temperature and extraction times on the yield of Laccaria laccata polysaccharides. On the basis of single-factor experiment, the process conditions were optimized by orthogonal experiment, and the DPPH and ABTS+ scavenging of the extracted polysaccharides was studied. The results showed that the extraction times had the greatest influence on the polysaccharide extraction rate, followed by the liquid-solid ratio, and the extraction time had the least influence. The optimal extraction process conditions were as follows: extraction for 3 times, liquid-solid ratio 20:1 mL / g, ultrasonic power 300 W, ultrasonic temperature 70℃, and ultrasonic time 40 min, and the polysaccharide extraction rate reached 6.27% under the conditions. The antioxidant activity research showed that the polysaccharides had certain scavenging effect on DPPH and ABTS+, the DPPH scavenging rate could reach 87% when the polysaccharide concentration was 5.5 g / L, and the ABTS+ scavenging rate could reach more than 90% when the polysaccharide concentration was 5.0 g / L. However, there is no report on the research on Laccaria moshuijun polysaccharides.

[0005] It can be seen that the prior art lacks research on the fine structure of the Lentinula edodes polysaccharide and the application of the Lentinula edodes polysaccharide in immunoregulatory activity. SUMMARY

[0006] The present application overcomes the defects in the prior art and provides a Lentinula edodes polysaccharide, a preparation method and application thereof.

[0007] In a first aspect, the present application provides a Lentinula edodes polysaccharide, which is a heteropolysaccharide composed of mannose and glucose, wherein the molar ratio of the residues of the mannose and the glucose is 2:5.

[0008] Further, the chemical structure of the Lentinula edodes polysaccharide comprises (4→6)-mannose residues, (4→6)-glucose residues, (1→4, 6)-glucose residues and →1)-glucose residues, and the molar ratio is 2:3:1:1.

[0009] Further, in the chemical structure of the Lentinula edodes polysaccharide, the main chain comprises (4→6)-mannose residues, (4→6)-glucose residues and (1→4, 6)-glucose residues, and the side chain comprises →1)-glucose residues.

[0010] Further, the Lentinula edodes polysaccharide comprises the following structure:

[0011]

[0012] wherein 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.

[0013] Further, the Lentinula edodes polysaccharide is a heteropolysaccharide composed of α-D-mannose, α-D-glucose, β-D-glucose and glucose, and the molar ratio is 2:3:1:1.

[0014] Preferably, the chemical structure of the Lentinula edodes polysaccharide comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→4, 6)-β-D-glucose residues and →1)-glucose residues, and the molar ratio is 2:3:1:1.

[0015] Preferably, in the chemical structure of the Lentinula edodes polysaccharide, the main chain comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues and (1→4, 6)-β-D-glucose residues, and the side chain comprises →1)-glucose residues.

[0016] Preferably, the Lentinula edodes polysaccharide comprises the following structure:

[0017]

[0018] wherein 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.

[0019] More preferably, the blue-purple Laccaria polysaccharide is a heteropolysaccharide composed of α-D-mannose, α-D-glucose and β-D-glucose, and the molar ratio is 2:4:1.

[0020] More preferably, the blue-purple Laccaria polysaccharide comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→4, 6)-β-D-glucose residues and →1)-α-D-glucose residues in the chemical structure, and the molar ratio is 2:3:1:1.

[0021] More preferably, the blue-purple Laccaria polysaccharide comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→4, 6)-β-D-glucose residues in the main chain, and →1)-α-D-glucose residues in the side chain.

[0022] More preferably, the blue-purple Laccaria polysaccharide comprises the following structure:

[0023]

[0024] wherein 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.

[0025] Further, the blue-purple Laccaria polysaccharide has a weight average molecular weight of 10000-20000 Da (such as 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000); in an embodiment of the present application, the blue-purple Laccaria polysaccharide has a weight average molecular weight of 17692 Da.

[0026] In a second aspect of the present application, a preparation method of the blue-purple Laccaria polysaccharide according to the first aspect is provided, which comprises the step of extracting the blue-purple Laccaria fruiting body.

[0027] Further, the preparation method comprises the step of extracting the crude polysaccharide by water extraction and alcohol precipitation.

[0028] Further, the preparation method further comprises the step of purifying the crude polysaccharide (e.g. by ion exchange column chromatography).

[0029] In an embodiment of the present application, the preparation method comprises the following steps:

[0030] (1) taking the powder of Laccaria amethystina fruiting bodies, extracting with water, and then sequentially concentrating, precipitating with alcohol, and removing protein to obtain the crude polysaccharide;

[0031] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate;

[0032] (3) subjecting the eluate obtained in step (2) to dialysis with a dialysis bag.

[0033] Optionally, (4) freeze-drying the liquid in the dialysis bag after step (3) is completed.

[0034] Further, in step (1), the ratio of the powder of Laccaria amethystina fruiting bodies to water (W / V, mg / mL) is 1:1-10 (e.g. 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10); in an embodiment of the present application, the ratio is 1:3.

[0035] Further, in step (1), the temperature of the extraction can be 80-100℃ (e.g. 80, 85, 90, 95, 100℃); in an embodiment of the present application, the extraction temperature is 100℃.

[0036] Further, in step (1), the number of extractions is 1-5 times (e.g. 1, 2, 3, 4, 5 times); in an embodiment of the present application, the number of extractions is 3 times.

[0037] Further, in step (1), the extraction time is 1-10 hours (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours) per time; in an embodiment of the present application, the extraction time is 6 hours per time.

[0038] In an embodiment of the present application, the extraction step in step (1) can comprise: taking the powder of Laccaria amethystina fruiting bodies, mixing with water, and boiling in a water bath.

[0039] 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 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1); in an embodiment of the present application, the volume ratio is 4:1.

[0040] In an embodiment of the present application, in step (1), in the alcohol precipitation step, the alcohol is ethanol.

[0041] Further, in step (1), the protein is removed by Sevage method.

[0042] In an embodiment of the present application, step (1) comprises: taking the powder of the fruiting body of Laccaria amethystina, hot water extraction, collecting the supernatant, concentration, adding anhydrous ethanol, collecting the precipitate, drying, removing the protein therein, and obtaining the crude polysaccharide.

[0043] Further, in step (2), the ion exchange column can be a cellulose column, and the filler of the cellulose column can be DEAE cellulose.

[0044] Further, in step (2), the eluent used for elution can be a NaCl solution; specifically, the concentration of the NaCl solution is 0.01-1.0 mol / L (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mol / L).

[0045] Further, in step (2), the elution can be gradient elution, and the concentration of the eluent can be 0.01-1.0 mol / L (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mol / L).

[0046] In an 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, and collecting the eluate and concentrating.

[0047] Further, in step (3), the molecular weight cut-off of the dialysis bag is 5000-10000 Da (e.g., 5000, 6000, 7000, 8000, 9000, 10000 Da); in an embodiment of the present application, the molecular weight cut-off is 7000 Da.

[0048] In an embodiment of the present application, step (3) comprises: placing the eluate obtained in step (2) in a dialysis bag for dialysis, and dialysis for two days.

[0049] In a third aspect of the present application, a composition comprising the blue-purple Lentinula edodes polysaccharide of the first aspect or the blue-purple Lentinula edodes polysaccharide prepared by the method of the second aspect is provided.

[0050] Further, the composition can further comprise a pharmaceutically acceptable excipient.

[0051] In a fourth aspect of the present application, the blue-purple Lentinula edodes polysaccharide of the first aspect or the blue-purple Lentinula edodes polysaccharide prepared by the method of the second aspect or the composition of the third aspect is used for preparing a medicament for preventing and / or treating a tumor.

[0052] Further, the tumor is selected from lung cancer, colon cancer, gastric cancer, leukemia, lymphoma, multiple myeloma, liver cancer, hepatocellular carcinoma, intestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer, intestinal cancer, pancreatic cancer, uterine cancer, melanoma.

[0053] In some embodiments of the present application, the tumor is lung cancer, colon cancer, gastric cancer.

[0054] Further, in the use, the blue-purple Lentinula edodes polysaccharide can be used alone or in combination with other active ingredients.

[0055] In a fifth aspect of the present application, the blue-purple Lentinula edodes polysaccharide of the first aspect or the blue-purple Lentinula edodes polysaccharide prepared by the method of the second aspect or the composition of the third aspect is used for preparing a food or health product for enhancing immunity.

[0056] Further, the blue-purple Lentinula edodes polysaccharide can enhance immunity, for example, has a proliferative effect on immune cells (e.g., B cells, T cells, RAW 264.7 cells).

[0057] Further, in the use, the blue-purple Lentinula edodes polysaccharide can be used alone or in combination with other active ingredients.

[0058] In a sixth aspect of the present application, the blue-purple Lentinula edodes polysaccharide of the first aspect or the blue-purple Lentinula edodes polysaccharide prepared by the method of the second aspect or the composition of the third aspect is used for preparing a medicament for proliferating B cells, T cells and RAW264.7 cells and / or improving the phagocytic function of RAW 264.7 cells.

[0059] This invention isolates and purifies the polysaccharide LMP-2 from *Heliotropium indicum*, and analyzes and identifies its molecular weight, monosaccharide composition, and chemical structure, determining its weight-average molecular weight and structural composition. Cellular experiments show that this polysaccharide possesses significant immunomodulatory activity, particularly at a concentration of 2.5 μg / mL, exhibiting the highest proliferation rate of B cells; at 10 μg / mL, the highest proliferation rate of T cells; and at 20 μg / mL, the highest proliferation rate of RAW 264.7 cells. This polysaccharide also exhibits significant antitumor activity, particularly at a concentration of 2.5 μg / mL, showing the highest inhibition rate of LLC cells; at 10 μg / mL, the highest inhibition rate of CT26.WT cells; and at 20 μg / mL, the highest inhibition rate of MFC cells. Based on these findings, this polysaccharide can be used to prepare pharmaceuticals, as well as immune-enhancing health products and foods, demonstrating promising application prospects and commercial value, and also enhancing the utilization value of *Heliotropium indicum*. Attached Figure Description

[0060] Figure 1 The image shows the GPC spectrum of LMP-2.

[0061] Figure 2 The image shown is the infrared spectrum of LMP-2.

[0062] Figure 3 The HPLC spectrum of LMP-2 is shown below.

[0063] Figure 4 The image shows LMP-2. 1 H NMR spectrum.

[0064] Figure 5 The image shows LMP-2. 13 C10 NMR spectrum.

[0065] Figure 6 The image shows LMP-2. 1 H- 1 H-COSY spectrum.

[0066] Figure 7 The image shows the HMQC spectrum of LMP-2.

[0067] Figure 8 The image shows the HMBC spectrum of LMP-2.

[0068] Figure 9 The chemical structure of LMP-2 is shown.

[0069] Figure 10 The results show the effects of LMP-2 on B cell proliferation.

[0070] Figure 11Results of experiments showing the effect of LMP-2 on T cell proliferation are shown.

[0071] Figure 12 Results of experiments showing the effect of LMP-2 on RAW264.7 cell proliferation are shown.

[0072] Figure 13 Results of experiments showing the effect of LMP-2 on MFC cell proliferation are shown.

[0073] Figure 14 Results of experiments showing the effect of LMP-2 on CT26.WT cell proliferation are shown.

[0074] Figure 15 Results of experiments showing the effect of LMP-2 on LLC cell proliferation are shown. DETAILED DESCRIPTION

[0075] 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 application pertains.

[0076] In the present application, “Laccaria moshuijun” refers to a fungus of the phylum Basidiomycota, order Agaricales, family Laccariaceae, genus Laccaria, which comprises a fruiting body and mycelium.

[0077] The term “tumor” refers to an abnormal mass of tissue in which the growth of the mass exceeds and is not coordinated with the normal growth of the tissues. Tumors can be “benign” or “malignant” depending on the following characteristics: degree of cellular differentiation (including morphology and function), rate of growth, local invasion, and metastasis. “Benign tumors” are generally well-differentiated, characterized by slower growth than malignant tumors, and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain “benign” tumors can later give rise to malignant tumors, possibly due to additional genetic changes in a subpopulation of neoplastic cells of the tumor, and these tumors are referred to as “pre-malignant tumors.” “Malignant tumors” are generally poorly differentiated (anaplastic), and have characteristic rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissues. In addition, malignant tumors generally have the ability to metastasize to distant sites.

[0078] The term “cancer” refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).

[0079] The disclosures of various publications, patents and published patent specifications, referred to by date in this text are hereby incorporated by reference in their entirety into the present document.

[0080] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some 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.

[0081] Example 1: Isolation and extraction of Lycoperdon purpurellum polysaccharide LMP-2

[0082] 1. Isolation and extraction of Lycoperdon purpurellum polysaccharide LMP-2

[0083] 1.1 Extraction of Lycoperdon purpurellum crude polysaccharide by water extraction and alcohol precipitation

[0084] Take 200g of completely dried Lycoperdon purpurellum (purchased from Xichang City, Sichuan Province, identified as Lycoperdon purpurellum by morphological observation and ITS sequencing) fruiting body, grind into powder with a high-speed grinder, add distilled water at a solid-liquid ratio of 1:3, 100℃ water bath for 6h, centrifuge at 8000rpm for 30min, collect the supernatant, repeat 3 times, concentrate to 200mL at 80℃. Add the above concentrated solution to anhydrous ethanol at a ratio of 1:4, precipitate with alcohol for 24h, discard the supernatant, collect the flocculent precipitate. Evaporate the remaining ethanol in a fume hood, and dry in a 60℃ oven after the ethanol is completely evaporated. Remove the protein, dry again and weigh to obtain Lycoperdon purpurellum crude polysaccharide, which is stored for use.

[0085] 1.2 Separation and purification of Lycoperdon purpurellum crude polysaccharide by DEAE cellulose column chromatography

[0086] 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 standing for 24h, and wait for use. Prepare 0.5mol / L NaOH, soak the cellulose for 6h, wash with ultrapure water until neutral, discard the supernatant, and then add 0.5mol / L HCl and soak for 6h. Wash with distilled water until neutral, discard the supernatant, and then add 0.5mol / L NaOH and soak for 6h. Wash with distilled water until neutral, and stand for use.

[0087] After activated cellulose was loaded into the column, it was balanced for 3h by distilled water before the separation and purification of crude polysaccharide. The supernatant (5mL) of diluted crude polysaccharide was added into the DEAE cellulose column, and eluted by adding different concentrations of NaCl (0, 0.05, 0.1, 0.2, 0.3mol / L). The polysaccharide was determined by sulfuric acid-phenol method. The eluent was concentrated to 10mL, dialyzed in a dialysis bag (Mw≥7kDa) for 48h, freeze-dried to obtain blue-purple Laccaria polysaccharide, named LMP-2.

[0088] 2. Structural identification of blue-purple Laccaria polysaccharide LMP-2

[0089] The structure of blue-purple Laccaria polysaccharide (LMP-2) was analyzed by acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance technology.

[0090] 2.1 Determination of molecular weight

[0091] 10mg of blue-purple Laccaria polysaccharide LMP-2 sample was dissolved in 1mL of ddH2O and ultrasonicated for 5min for GPC analysis.

[0092] 2.2 Infrared spectroscopy analysis of blue-purple Laccaria polysaccharide LMP-2

[0093] 2mg of LMP-2 was mixed with KBr and pressed into a tablet, which was scanned by infrared spectrophotometer in the range of 4000cm -1 -400cm -1 .

[0094] 2.3 Analysis of monosaccharide composition of blue-purple Laccaria polysaccharide LMP-2

[0095] Six standard samples and LMP-2 sample after TFA acid hydrolysis were dissolved in mobile phase (85% acetonitrile) for HPLC analysis.

[0096] 2.4 Nuclear magnetic resonance analysis of blue-purple Laccaria polysaccharide LMP-2

[0097] 50mg of LMP-2 was dissolved in 0.6mL of heavy water (D2O) and loaded into a nuclear magnetic tube for detection on a nuclear magnetic resonance instrument.

[0098] 2.5 GC-MS analysis of blue-purple Laccaria polysaccharide LMP-2 after methylation and silylation derivatization

[0099] Take 20 mg LMP-2 sample, seal the beaker, add 4 mL DMSO (dimethyl sulfoxide) to the sealed beaker, gently shake the beaker to fully dissolve LMP-2. Then add 200 mg of NaOH until the NaOH is just not dissolved and place it in a shaker at room temperature for 1 h. After shaking, add 3 mL of iodomethane, avoid light for 20-24 h, after reaction, add water to terminate the reaction. Extract the product with chloroform, dry, and wash with water three times 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.

[0100] 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 20 min. The precipitate is discarded, filtered with a 0.22 μm filter, and the upper layer solution is used for GC-MS analysis.

[0101] 3. Results

[0102] 3.1 Basic property results of blue-purple wax mushroom polysaccharide LMP-2

[0103] The GPC spectrum of LMP-2 is shown in Figure 1 , which shows that the weight average molecular weight of LMP-2 is 17692 Da.

[0104] 3.2 FTIR spectrum analysis of blue-purple wax mushroom polysaccharide LMP-2

[0105] The primary structure of LMP-2 was characterized by Fourier infrared spectroscopy, and the results are shown in Figure 2 . LMP-2 has obvious signal peaks in the range of 4000-500 cm -1 . The wide and strong absorption peak at 3436.51 cm -1 is assigned to the O-H stretching vibration peak; the absorption peaks at 2924.86 cm -1 , 1634.18 cm -1 and 1401.16 cm -1 are respectively -CH2 stretching vibration peak, C=O stretching vibration peak and C-H bending vibration peak. The C-O stretching vibration peak is located at 1079.50 cm -1 . The results of Fourier infrared spectrum show that LMP-2 has typical polysaccharide structure characteristics. In addition, absorption peaks appear at 1200-1000 cm -1 , indicating that the sugar rings in LMP-2 are all pyran type.

[0106] 3.3 Analysis of monosaccharide composition of blue-purple wax mushroom polysaccharide LMP-2

[0107] After complete hydrolysis of LMP-2, its monosaccharide composition was analyzed by HPLC, and the results are as follows: Figure 3 As shown, mannose was present at 7.078 min and glucose at 7.888 min. The ratio of mannose to glucose was 2:5.

[0108] 3.4 NMR spectral analysis of LMP-2 polysaccharide from *Tetrapanax papyriferus*

[0109] LMP-2 1 The H NMR results are as follows Figure 4 As shown. The results indicate that LMP-2 has three anomeric hydrogen signals: δ5.00, δ4.86, and δ4.40. The signals between δ3.0 and 4.2 are attributed to hydrogen signals from C2-C6 of the sugar residues. Because... 1 In the H-NMR results, the anomeric hydrogen signal at δ4.66 overlaps with the solvent peak (δ4.70). Therefore, the integral ratio of the H1 / C1 signal in the HMQC plot is used to infer the proportion of each sugar residue. The results suggest that the integral ratio of the A to D signals is approximately 1:2:1:3.

[0110] LMP-2 13 The C NMR results are as follows Figure 5 As shown, LMP-2 has four anodic carbon signals at δ102.98, δ101.66, δ97.99, and δ97.67. The signals between δ60 and 78 are attributed to carbon signals from C2 to C6 of the sugar residues.

[0111] LMP-2 1 H- 1 H-COSY spectrum as shown Figure 6 As shown, the coupling relationship between adjacent hydrogen nuclei can be identified. Signals A, B, C, and D are chemical signals between the anomeric hydrogen and the hydrogen on the adjacent carbon in LMP-2. Taking signal A (δ5.00 / δ3.79) as an example: its anomeric hydrogen chemical shift is δ5.00, and the corresponding adjacent hydrogen chemical shift is δ3.79. Therefore, the signal peak at δ5.00 / δ3.79 is attributed to the resonance coupling signal between H1 and H2 on the (4→6)-α-D-glucose residue. Signal B (δ4.86 / δ3.70) is attributed to the resonance coupling signal between H1 and H2 on the (4→6)-α-D-mannose residue. Signal C (δ4.66 / δ3.40) is attributed to the resonance coupling signal between H1 and H2 on the (1→4,6)-β-D-glucose residue. Signal D (δ4.40 / δ3.18) is attributed to the resonance coupling signal of H1 and H2 on (4→6)-α-D-glucose residues.

[0112] All the chemical shifts of hydrogen are summarized in Table 1.

[0113] The HMQC spectrum of LMP-2 is as follows:Figure 7 As shown, it can identify short-range related factors. 1 H and 13 The coupling relationship between C and C. In LMP-2, signals A (H1 / C1:δ5.00 / δ97.99), B (H1 / C1:δ4.86 / δ97.69), C (H1 / C1:δ4.66 / δ101.66), and D (H1 / C1:δ4.40 / δ103.00) are respectively assigned to the H1 and C1 coupling signals of (1→1)-α-D-glucose residues, (4→6)-α-D-mannose residues, (1→4,6)-β-D-glucose residues, and (4→6)-α-D-glucose residues, and are related to... 1 H- 1 In the H COSY spectrum, signals A, B, C, and D correspond to each other.

[0114] The HMBC spectrum of LMP-2 is as follows: Figure 8 As shown, it can identify remote related... 1 H and 13 The coupling relationships between C residues are as follows: Signal A (H6 / C4:δ3.99 / δ66.34) is assigned to the resonance signal of H6 and C4 of (→1)-α-D-glucose residues; Signal B (H6 / C4:δ3.53 / δ66.64) is assigned to the coupling signal of H6 and C4 of (4→6)-α-D-mannose residues; Signal C (H6 / C4:δ3.24 / δ75.28) is assigned to the coupling signal of H6 and C4 of (1→4,6)-β-D-glucose residues; Signal D (H4 / C2:δ3.28 / δ78.52) is assigned to the coupling signal of H4 and C2 of (4→6)-α-D-glucose residues.

[0115] All the chemical shifts of carbon are summarized in Table 2.

[0116] Table 1 Chemical shifts of 1H in LMP-2

[0117]

[0118] Table 2 Chemical shifts of 13C in LMP-2

[0119]

[0120] 3.5 Gas Chromatography and Mass Spectrometry Analysis of LMP-2 Polysaccharide from *Tetrapanax papyriferus*

[0121] The methylation results are shown in Table 3, indicating that the major repeating structural units of LMP-2 are composed of alternating links of (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→1)-α-D-glucose residues, and (1→4,6)-β-D-glucose residues.

[0122] Based on the above inference, the structural formula of LMP-2 is as shown in Figure 9 .

[0123] Table 3 Analysis of LMP-2 methylation results

[0124]

[0125] Example 2: Determination of the immunomodulatory activity of LMP-2 (determination of the immunomodulatory and anti-tumor activity of LMP-2 by two CCK-8 methods in vitro)

[0126] 1. Reagents

[0127] CCK-8 kit, RPIM1640, FBS, DMSO, double antibody, etc. are all commercially available products.

[0128] 2. Instruments

[0129] Microplate reader; cell incubator.

[0130] 3. Methods

[0131] 3.1 Effect of LMP-2 on the proliferation of immune cells (B cells, T cells and RAW264.7 cells)

[0132] The effect of LMP-2 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 to the logarithmic growth phase, and after counting with a cell counting plate, the cell suspension was diluted to 1 × 10 5 cells / mL with fresh culture medium, and the cell suspension was added to a 96-well plate at 100 μL per well. The 96-well plate was placed in a CO2 incubator for 24 h. After 24 h, different concentrations of LMP-2 solution were added to the experimental group (the final concentration was 0.625, 1.25, 2.5, 5, 10, 20 μg / mL), 100 μL of LPS solution was added to the positive control group (the final concentration was 5 μg / mL), and 100 μL of cell culture medium was added to the blank group. After 24 h of incubation in a CO2 incubator, 5 μl of CCK-8 was added to each well, and the plate was incubated in a CO2 incubator for 3 h. The absorbance value was detected on a microplate reader (450 nm) and the image was taken.

[0133] 3.2 Effect of LMP-2 on the proliferation of tumor cells (MFC cells, CT26.WT cells and LLC cells)

[0134] CCK-8 method was used to detect the inhibitory effect of polysaccharides on MFC, CT26.WT and LLC cells. Except for the positive control group (MAN group) which added the same volume of mannose peptide solution (MAN) with a final concentration of 5 μg / mL, the rest was the same as 3.1.

[0135] 4. Results

[0136] 4.1 Effect of LMP-2 on the proliferation of B cells

[0137] The results are shown in Table 4.2. Compared with the blank group, the LPS group could significantly (P<0.05) promote the proliferation of T cells, with a proliferation rate of 62.68%; when the final concentration of LMP-2 was in the range of 2.5-20 μg / mL, it could significantly (P<0.05) promote the proliferation of T cells; when the final concentration of LMP-2 was 10 μg / mL, the effect of LMP-2 on the proliferation of T cells was the most obvious, with a maximum proliferation rate of 49.27%. Figure 10 4.2 Effect of LMP-2 on the proliferation of T cells

[0138] The results are shown in Table 4.2. Compared with the blank group, the LPS group could significantly (P<0.05) promote the proliferation of T cells, with a proliferation rate of 62.68%; when the final concentration of LMP-2 was in the range of 2.5-20 μg / mL, it could significantly (P<0.05) promote the proliferation of T cells; when the final concentration of LMP-2 was 10 μg / mL, the effect of LMP-2 on the proliferation of T cells was the most obvious, with a maximum proliferation rate of 49.27%.

[0139] Figure 11 4.3 Effect of LMP-2 on the proliferation of RAW264.7 cells

[0140] The results are shown in Table 4.3. Compared with the blank group, the LPS group could significantly (P<0.05) promote the proliferation of RAW264.7 cells, with a proliferation rate of 80.52%; when the final concentration of LMP-2 was in the range of 0.625-20 μg / mL, it could significantly (P<0.05) promote the proliferation of RAW264.7 cells; when the final concentration of LMP-2 was 20 μg / mL, the effect of LMP-2 on the proliferation of RAW264.7 cells was the most obvious, with a maximum proliferation rate of 84.90%.

[0141] 4.4 Effect of LMP-2 on the proliferation of MFC cells Figure 12 The results are shown in Table 4.4. Compared with the blank group, the LPS group could significantly (P<0.05) promote the proliferation of MFC cells, with a proliferation rate of 80.52%; when the final concentration of LMP-2 was in the range of 0.625-20 μg / mL, it could significantly (P<0.05) promote the proliferation of MFC cells; when the final concentration of LMP-2 was 20 μg / mL, the effect of LMP-2 on the proliferation of MFC cells was the most obvious, with a maximum proliferation rate of 84.90%.

[0142] 4.4 Effect of LMP-2 on the proliferation of MFC cells

[0143] Figure 13 ​​As shown, compared with the control group, the MAN group significantly (P<0.05) inhibited the proliferation of MFC cells, with an inhibition rate of 83.39%. When the final concentration of LMP-2 was in the range of 1.25-20 μg / mL, it significantly (P<0.05) inhibited the proliferation of MFC cells. Moreover, when the final concentration of LMP-2 was 20 μg / mL, the inhibitory effect of LMP-2 on MFC cells was the most obvious, with a maximum inhibition rate of 83.26%.

[0144] Effect of 4.5 LMP-2 on the proliferation of CT26.WT cells

[0145] The results are as follows Figure 14 As shown, compared with the blank group, the MAN group significantly (P<0.05) inhibited the proliferation of CT26.WT cells, with an inhibition rate of 35.05%. When the final concentration of LMP-2 was in the range of 0.625-20 μg / mL, it significantly (P<0.05) inhibited the proliferation of CT26.WT cells. Moreover, when the final concentration of LMP-2 was 10 μg / mL, the inhibitory effect of LMP-2 on CT26.WT cells was the most obvious, with a maximum inhibition rate of 36.07%.

[0146] 4.6 Effects of LMP-2 on LLC cell proliferation

[0147] The results are as follows Figure 15 As shown, compared with the blank group, the MAN group significantly (P<0.05) inhibited LLC cell proliferation, with an inhibition rate of 25.45%. When the final concentration of LMP-2 was in the range of 0.625-20 μg / mL, it significantly (P<0.05) inhibited LLC cell proliferation. Moreover, when the final concentration of LMP-2 was 2.5 μg / mL, the inhibitory effect of LMP-2 on LLC cells was the most obvious, with a maximum inhibition rate of 34.06%.

[0148] 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 principles of the present invention should be included within the protection scope of the present invention.

[0149] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0150] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. Laricifomes violeus polysaccharide, which is a heteropolysaccharide composed of α-D-mannose, α-D-glucose and β-D-glucose with a molar ratio of 2:4:

1. The chemical structure of the Laricifomes violeus polysaccharide comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→4, 6)-β-D-glucose residues and →1)-α-D-glucose residues with a molar ratio of 2:3:1:

1. The chemical structure of the Laricifomes violeus polysaccharide comprises (4→6)-α-D-mannose residues, (4→6)-α-D-glucose residues, (1→4, 6)-β-D-glucose residues in the main chain and →1)-α-D-glucose residues in the side chain. The Laricifomes violeus polysaccharide comprises the following structure: ， wherein n is an integer from 1 to 20, and the weight average molecular weight of the Laricifomes violeus polysaccharide is 10,000-20,000 Da. 2.A method for preparing the Laricifomes violeus polysaccharide according to claim 1, which comprises the step of extracting Laricifomes violeus fruiting bodies. The method comprises the following steps: (1) taking Laricifomes violeus fruiting body powder, extracting with water, and obtaining crude polysaccharide by concentrating, alcohol precipitation and removing protein in sequence from the obtained water extract; (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting and collecting the eluate; (3) dialyzing the eluate obtained in step (2) with a dialysis bag; In step (1), the ratio of Laricifomes violeus fruiting body powder to water is 1:1-10; The extraction temperature is 80-100℃; In the alcohol precipitation step, the volume ratio of alcohol to water extract concentrate is 1-10:1; The alcohol is ethanol; In step (2), the ion exchange column is a cellulose column, and the filler is DEAE cellulose; The eluent used in the elution is a NaCl solution. 3.A composition comprising the Laricifomes violeus polysaccharide according to claim 1. 4.Use of the Laricifomes violeus polysaccharide according to claim 1 in the preparation of a medicine for preventing and / or treating tumors or a health care product for enhancing immunity.

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