A coriolus polysaccharide, and a preparation method and application thereof

Abstract

The application discloses a trametes versicolor polysaccharide (TV-P) and a preparation method and application thereof. The composition of the TV-P includes xylose, arabinose, glucose and galactose, and the molar ratio is 3:1:10:5. The TV-P is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography, dialysis and concentration of the trama of trames versicolor. The TV-P has significant immunoregulatory activity and antitumor activity, and can be applied in medicines, health products or food.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of application of fungal polysaccharides, and particularly relates to a polysaccharide of Trametes versicolor, 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.

[0003] Edible fungus polysaccharides have the characteristics of antiviral, antioxidant, antitumor, hypolipidemic, promoting the proliferation and differentiation of immune cells and the secretion of lymphokines, activating complement and other immunomodulatory biological activities, 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 non-specific immune enhancer, which can improve the immune function of the body through various pathways without side effects on the body.

[0004] Trametes versicolor, also known as Trametes versicolor, is a kind of fungus belonging to Basidiomycota, Hymenomycetes, Polyporales, Polyporaceae and Trametes. The fruiting body of Trametes versicolor is annual, and is made of leather and semi-fibrous, and is side-attached without handle, and is often covered with overlapping tiles, and is often connected to the left and right, and is often surrounded by a lotus seat. The fruiting body of Trametes versicolor is often surrounded by a lotus seat. Trametes versicolor is a kind of medicinal fungus, which can remove dampness, resolve phlegm, treat lung diseases, treat chronic bronchitis and chronic hepatitis, and is used as a drug for immunotherapy of liver cancer. Mycelium and polysaccharides extracted from fermentation broth have strong anticancer properties; the inhibition rates of sarcoma 180 and Ehrlich carcinoma of mice are 80% and 100%, respectively.

[0005] Ye Run et al. reported in "Purification Process of Trametes versicolor Polysaccharide in Xinyang Dabie Mountain Area and Its Antioxidant Activity" (Food Industry, 2020, 41(11): 70-73.) that the purity of Trametes versicolor polysaccharide purified by AB-8 macroporous resin increased from 46.81% to 81.24%, and had good scavenging effect on DPPH, ABTS and hydroxyl radicals, and the effect was enhanced with the increase of the mass concentration of the polysaccharide.

[0006] Jing et al. in Research Progress on the Extraction, Structure, and Bioactivities of Polysaccharides from Coriolus versicolor (Foods, 2022, 11(14): 2126.) reported that the main polysaccharide of C. versicolor is composed of glucose, with small amounts of mannose, rhamnose, galactose, and fucose. The main chain is composed of (1→4)-β- / (1→3)-β-d-glucopyranosyl and branches connected to the O-6 site. It has multiple biological activities such as antioxidant, antitumor, and immunomodulatory activities.

[0007] It can be seen that the prior art lacks research on the fine structure of C. versicolor polysaccharide and the application of C. versicolor polysaccharide in immunomodulatory activity. SUMMARY

[0008] The present application overcomes the defects in the prior art and provides a C. versicolor polysaccharide, a preparation method and application thereof.

[0009] The first aspect of the present application provides a C. versicolor polysaccharide (TV-P) which is a heteropolysaccharide composed of xylose, arabinose, glucose, and galactose, wherein the molar ratio of the residues of the xylose, arabinose, glucose, and galactose is 3:1:10:5.

[0010] Further, the chemical structure of the polysaccharide comprises 1,2,4-linked xylose residues, 1,4-linked xylose residues, 1,4-linked arabinose residues, 1-linked glucose residues, 6-linked glucose residues, 1,4-linked glucose residues, 1,4,6-linked glucose residues, and 1,4,6-linked galactose residues, and the molar ratio thereof is 1:2:1:6:1:2:1:5.

[0011] Further, the chemical structure of the polysaccharide comprises a main chain composed of 1,4,6-linked galactose, 1,4,6-linked glucose, 1,4,6-linked xylose, 1,4-linked glucose, 1,4-linked xylose, and 1,4-linked arabinose, and a side chain composed of 1-linked glucose residues and 6-linked glucose residues.

[0012] Further, the weight average molecular weight of the polysaccharide is 8000-20000 Da (e.g. 10000 Da, 11000 Da, 12000 Da, 13000 Da, 13010 Da, 135020 Da, 13030 Da, 13040 Da, 13050 Da, 13060 Da, 13070 Da, 13080 Da, 13090 Da, 14000 Da, 15000 Da, 16000 Da, 17000 Da, 18000 Da, 19000 Da, 20000 Da), preferably 12000-17000 Da, further preferably 13000-15000 Da.

[0013] In one embodiment of the present application, the weight average molecular weight of the polysaccharide is 13069 Da.

[0014] Further, the polysaccharide comprises the following structural formula:

[0015]

[0016] wherein n is an integer of 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably an integer of 2-15, more preferably an integer of 3-10.

[0017] wherein Glcp is glucose, Arap is arabinose, Xylp is xylose, and Galp is galactose.

[0018] The second aspect of the present application provides a composition comprising the polysaccharide of the first aspect.

[0019] The third aspect of the present application provides a preparation method of the polysaccharide of the first aspect, comprising the step of extracting from the fruiting body of Grifola frondosa.

[0020] Further, the preparation method comprises the step of extracting the crude polysaccharide by water extraction and alcohol precipitation.

[0021] Further, the preparation method further comprises the step of purifying the crude polysaccharide (e.g. by ion exchange column chromatography).

[0022] In one embodiment of the present application, the preparation method comprises the following steps:

[0023] (1) taking the fruiting body powder of Grifola frondosa, extracting with hot water, and obtaining the water extract, which is then concentrated, precipitated with alcohol, and dried to obtain the crude polysaccharide;

[0024] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate;

[0025] (3) Concentrating the eluate obtained in step (2) by dialysis.

[0026] Further, the preparation method further comprises step (4), specifically, freeze-drying the liquid in the dialysis bag after step (3) is completed.

[0027] Further, in step (1), the temperature of the extraction can be 80-100°C (such as 80, 85, 90, 95, 100°C); in an embodiment of the present application, the extraction temperature is 98°C.

[0028] Further, in step (1), the mass ratio (W / V, mg / mL) of the powder of the fruiting body of Ganoderma applanatum to water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in an embodiment of the present application, the ratio of the solid to the liquid is 1:3.

[0029] Further, in step (1), the number of extractions is 1-5 times (such as 1, 2, 3, 4, 5 times); in an embodiment of the present application, the number of extractions is 3 times.

[0030] Further, in step (1), the extraction time of each extraction is 1-10 hours (such as 1, 3, 6, 8, 10 hours); in an embodiment of the present application, the extraction time of each extraction is 6 hours.

[0031] In an embodiment of the present application, the extraction step in step (1) can comprise: taking the powder of the fruiting body of Ganoderma applanatum, mixing it with water, and placing it in a water bath to boil.

[0032] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to the concentrated water extract is 1-10:1 (such as 1:1, 3:1, 4:1, 5:1, 10:1); in an embodiment of the present application, the volume ratio is 3:1.

[0033] In an embodiment of the present application, in the above alcohol precipitation step, the alcohol is ethanol.

[0034] In an embodiment of the present application, step (1) comprises: taking the powder of the fruiting body of Ganoderma applanatum, hot water extraction, collecting the supernatant, concentrating, adding anhydrous ethanol, collecting the precipitate, drying, removing the protein therein, and obtaining crude polysaccharides.

[0035] Further, in step (2), the ion exchange column can be a cellulose column, and the filler of the cellulose column can be DEAE cellulose.

[0036] 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. 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0) mol / L.

[0037] 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. 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0) mol / L.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The fourth aspect of the present application provides a use of the polysaccharide of the first aspect or the polysaccharide prepared by the method of the third aspect, and the use comprises:

[0042] (1) a use in the preparation of a product capable of enhancing immunity;

[0043] (2) a use in the preparation of a product having anti-tumor activity.

[0044] Further, the product is a food, a health product or a pharmaceutical product.

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

[0046] Further, in the product, the concentration of the polysaccharide of K. lycoperdon is 0.5-25 μg / mL (e.g. 0.5 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 25 μg / mL), preferably 0.6-20 μg / mL.

[0047] Furthermore, when enhancing the proliferation of RAW 264.7 and immune T cells, the concentration of Coriolus versicolor polysaccharide is preferably 20 μg / mL; when enhancing the proliferation of immune B cells, the concentration of Coriolus versicolor polysaccharide is preferably 2.5 μg / mL.

[0048] Furthermore, when enhancing the inhibitory effect on MFC and LLC cells, the concentration of Coriolus versicolor polysaccharide is preferably 20 μg / mL; when enhancing the inhibitory effect on CT26.WT cells, the concentration of Coriolus versicolor polysaccharide is preferably 1.25 μg / mL.

[0049] The beneficial effects of this invention are:

[0050] This invention isolates and purifies the polysaccharide TV-P from *Trametes versicolor*, 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, where the proliferation rate of B cells is highest; and at a concentration of 20 μg / mL, the proliferation rate of T cells and RAW264.7 cells is highest. This polysaccharide also exhibits significant antitumor activity, particularly at a concentration of 1.25 μg / mL, where the proliferation rate of CT26.WT cells is highest; and at a concentration of 20 μg / mL, the inhibition rate of MFC cells and LLC cells is highest. Attached Figure Description

[0051] Figure 1 The image shown is the GPC spectrum of TV-P.

[0052] Figure 2 The image shown is the infrared spectrum of TV-P.

[0053] Figure 3 The HPLC chromatogram of TV-P is shown below.

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

[0055] Figure 5 The image shown is of TV-P. 13 C NMR spectrum;

[0056] Figure 6 The image shown is of TV-P. 1 H- 1 H-COSY spectrum;

[0057] Figure 7 The image shows the HMQC spectrum of TV-P;

[0058] Figure 8 The image shows the HMQC spectrum of TV-P;

[0059] Figure 9 The experimental results of the effect of TV-P on B cell proliferation are shown.

[0060] Figure 10 The experimental results of the effect of TV-P on T cell proliferation are shown.

[0061] Figure 11 The experimental results of the effect of TV-P on RAW264.7 cell proliferation are shown.

[0062] Figure 12 The experimental results of the effect of TV-P on MFC cell proliferation are shown.

[0063] Figure 13 The experimental results of the effect of TV-P on CT26.WT cell proliferation are shown.

[0064] Figure 14 The experimental results of the effect of TV-P on LLC cell proliferation are shown. DETAILED DESCRIPTION

[0065] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as are commonly understood by one of ordinary skill in the art to which this application pertains.

[0066] In the present application, "Trametes versicolor" refers to a basidiomycota, hymenomycetes, polyporales, polyporaceae, trametes fungus, which includes fruiting body and mycelium.

[0067] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0068] Example 1 Isolation and extraction of Trametes versicolor polysaccharide TV-P

[0069] 1. Isolation and extraction of Trametes versicolor polysaccharide TV-P

[0070] 1.1 Extraction of Trametes versicolor crude polysaccharide by water extraction and alcohol precipitation

[0071] Take 200 g of dried Trametes versicolor fruiting body powder, add the crushed Trametes versicolor fruiting body and distilled water in a beaker at a ratio of 1:3, and heat in a water bath at 98℃ for 6 hours. Collect the supernatant and concentrate, repeat 3 times, and finally concentrate all the supernatant to 200 mL. Add three times the volume of anhydrous ethanol to precipitate it, collect the precipitate and dry it to remove the protein in the extraction solution, thereby obtaining Trametes versicolor crude polysaccharide.

[0072] 1.2DEAE-cellulose column chromatography for isolation and purification of crude polysaccharide from Trametes versicolor

[0073] Accurately weigh 50 g of DEAE-cellulose and dissolve it in 1 L of ultrapure water, stir thoroughly, and stop stirring if no cellulose particles are visible. Let stand for 24 h, discard the supernatant, and use as needed. Prepare 0.5 mol / L NaOH, soak the cellulose for 6 h, wash with ultrapure water until neutral, discard the supernatant, add 0.5 mol / L HCl again, soak for 6 h, wash with distilled water until neutral, discard the supernatant, and add 0.5 mol / L NaOH again for 6 h of soaking, wash with distilled water until neutral, and let stand for use.

[0074] After the activated cellulose is packed into a column, it is equilibrated with distilled water for 24 h before the isolation and purification of crude polysaccharide. Add the diluted supernatant (5 mL) of crude polysaccharide to the DEAE-cellulose column, and elute with different concentrations of NaCl (0.01 mol / L, 0.05 mol / L, and 0.1 mol / L). Determine the polysaccharide using the sulfuric acid-phenol method. Concentrate the eluate to 5 mL, purify the sample on a cellulose column, dialyze in a dialysis bag (Mw≥7 kDa) for 48 h, freeze-dry, and obtain Trametes versicolor polysaccharide, designated as TV-P.

[0075] 2. Structural identification of Trametes versicolor polysaccharide TV-P

[0076] Use acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance techniques to analyze the structure of Trametes versicolor polysaccharide (TV-P).

[0077] 2.1 Determination of molecular weight

[0078] Dissolve 10 mg of Trametes versicolor polysaccharide TV-P sample in 1 mL of ddH2O, sonicate for 5 min, and perform GPC analysis.

[0079] 2.2 Infrared spectroscopy analysis of Trametes versicolor polysaccharide TV-P

[0080] Mix 2 mg of TV-P with KBr, press into a tablet, and scan the range of 4000 cm -1 -400 cm -1 using an infrared spectrophotometer.

[0081] 2.3 Analysis of monosaccharide composition of Trametes versicolor polysaccharide TV-P

[0082] Dissolve the six standard samples and the TV-P sample after TFA acid hydrolysis in the mobile phase (85% acetonitrile) and perform HPLC analysis.

[0083] 2.4 NMR analysis of polysaccharide from Trametes versicolor TV-P

[0084] 50 mg of TV-P sample was dissolved in 0.6 mL of heavy water (D2O) and loaded into a NMR tube for detection on a NMR instrument.

[0085] 2.5 Methylation and silylation of polysaccharide from Trametes versicolor TV-P followed by GC-MS analysis

[0086] 20 mg of TV-P sample was weighed into a beaker, which was sealed, 2 mL of DMSO (dimethyl sulfoxide) was added to the sealed beaker, and the beaker was gently shaken to fully dissolve the TV-P. Then 200 mg of NaOH was added until the NaOH was just not dissolved, and it was placed in a shaker at room temperature for 1 h of shaking. After the shaking was completed, 1.5 mL of iodomethane was added, and the reaction was carried out in the dark for 1 h. After the reaction, water was added to terminate the reaction. The product was extracted with chloroform, and after drying, the methylated polysaccharide was obtained. The methylated polysaccharide was completely acid hydrolyzed by TFA, washed with water three times, and the completely acid hydrolyzed product of the methylated polysaccharide was obtained.

[0087] The above sample was fully reacted with 2 mL of hexamethyldisilazane, 1 mL of trimethylsilyl chloride, and 2 mL of anhydrous pyridine, and was placed in a water bath at 50°C for 20 min. A low-temperature high-speed centrifuge was used at a speed of 12000 rpm / min, 4°C, for 10 min, the precipitate was discarded, and a 0.22 μm filter was used to filter, and the upper layer solution was used for GC-MS analysis.

[0088] 3. Results

[0089] 3.1 Results of basic properties of polysaccharide from Trametes versicolor TV-P

[0090] The GPC spectrum of TV-P is shown in Figure 1 , which shows that the weight average molecular weight of TV-P is 14992 Da.

[0091] 3.2 FTIR spectrum analysis of polysaccharide from Trametes versicolor TV-P

[0092] The primary structure of TV-P was characterized by Fourier infrared spectroscopy, and the results are shown in Figure 2 . TV-P has obvious signal peaks in the range of 4000-500 cm -1 . In the characteristic peak region (4000-1250 cm -1 ), the strong wide peak at 3438.69 cm -1 is caused by the O-H stretching vibration of the sugar molecule, indicating that there are hydrogen bonds within or between the polysaccharide molecules; the peak at 2922.29 cm -1 is the C-H stretching vibration peak of methylene; the sharp peak at 1629.67 cm -1 is caused by the asymmetric stretching vibration of C=O; the peak at 1379.49 cm-1 The peak at this location is the in-plane bending vibration peak of CH. This is observed in the fingerprint spectrum region (1250–500 cm⁻¹). -1 ), 1085.20cm -1 The peak at this location corresponds to the CO stretching vibration of the pyranose ring. FT-IR data for TV-P indicate that it possesses characteristic absorption peaks of polysaccharides and contains a pyranose ring in its structure.

[0093] 3.3 Monosaccharide composition analysis of Turmeric polysaccharide TV-P

[0094] After complete hydrolysis of TV-P, its monosaccharide composition was analyzed by HPLC, and the results are as follows: Figure 3 As shown, Xyl is at 5.648 min, Ara at 6.009 min, Glc at 7.929 min, and Gal at 8.590 min. The peak area ratio of each hydrolyzed monosaccharide suggests that the content ratio of Xyl:Ara:Glc:Gal is 17.24:5.22:52.33:25.21, approximately 3:1:10:5.

[0095] 3.4 NMR Spectroscopic Analysis of Turmeric Polysaccharide TV-P

[0096] TV-P 1 The H NMR results are as follows Figure 4 As shown. The results show that TV-P has seven anodic hydrogen signals, but due to the solvent peak at δ4.70, the binding... 1 H- 1 For H COSY and HMQC, the signal at δ4.67 is still attributed to anomeric hydrogen signals. Therefore, TV-P has eight anomeric hydrogen signals: δ5.15, δ5.13, δ5.10, δ4.96, δ4.87, δ4.67, δ4.55, and δ4.42. The signals between δ3.0 and 4.2 are attributed to hydrogen signals from C2-C6 of the sugar residues.

[0097] TV-P 13 The C NMR results are as follows Figure 5 As shown, TV-P exhibits eight anodic carbon signals at δ101.11, δ87.81, δ91.76, δ101.45, δ97.81, δ102.34, δ95.69, and δ102.49. The signals between δ60 and 78 are attributed to carbon signals from C2 to C6 of the sugar residues.

[0098] TV-P 1 H- 1 H-COSY spectrum as shown Figure 6As shown, the coupling relationship between adjacent hydrogen nuclei can be identified. The H1 / H2 signal for part A is δ 5.15 / 3.86, for part B it is δ 5.13 / 3.29, for part C it is δ 5.10 / 3.45, for part D it is δ 4.96 / 3.67, for part E it is δ 4.87 / 3.72, for part F it is δ 4.67 / 3.27, for part G it is δ 4.55 / 3.16, and for part H it is δ 4.42 / 3.24.

[0099] All the chemical shifts of hydrogen are summarized in Table 1.

[0100] The HMQC spectrum of TV-P is as follows: Figure 7 As shown, it can identify short-range related factors. 1 H and 13 The coupling relationship between C. Due to 1 In the H NMR results, there was signal overlap with solvent peaks. Therefore, the integrated ratio of H1 / C1 signals in the HMQC plot was used to infer the proportion of each sugar residue. The H1 / C1 signal in part A is δ 5.15 / 101.11, in part B it is δ 5.13 / 87.81, in part C it is δ 5.10 / 97.76, in part D it is δ 4.96 / 101.45, in part E it is δ 4.87 / 97.81, in part F it is δ 4.67 / 102.34, in part G it is δ 4.55 / 95.69, and in part H it is δ 4.42 / 102.49. The signal ratio of each part is approximately 1:2:1:1:6:2:1:5.

[0101] The HMBC spectrum of TV-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: H4 / C6 signal of residue A is δ3.79 / 68.20, H2 / C4 signal of residue B is δ3.29 / 75.06, H4 / C2 signal of residue C is δ3.40 / 73.07, H6 / C4 signal of residue D is δ3.52 / 78.46, H5 / C3 signal of residue E is δ3.90 / 68.28, H2 / C4 signal of residue F is δ3.27 / 75.07, H4 / C2 signal of residue G is δ3.28 / 75.07, and H2 / C4 signal of residue H is δ3.24 / 75.34.

[0102] All the chemical shifts of carbon are summarized in Table 2.

[0103] Table 1 Chemical shifts of TV-P 1 Chemical shifts of H

[0104]

[0105] Table 2 Chemical shifts of TV-P 13 Chemical shifts of C

[0106]

[0107] 3.5 Gas chromatography and mass spectrometry analysis of the trichloroacetic acid- precipitated polysaccharide TV-P

[0108] The methylation results are shown in Table 3, which indicates that the main repeating structural unit of TV-P consists of a backbone of 1,4,6-linked galactose, 1,4,6-linked glucose, 1,4,6-linked xylose, 1,4-linked glucose, 1,4-linked xylose and 1,4-linked arabinose, and a branch of 1-linked glucose residue and 6-linked glucose residue.

[0109] Table 3 Analysis of methylation results of TV-P

[0110]

[0111] Example 2 Determination of the immunomodulatory effect of trichloroacetic acid- precipitated polysaccharide TV-P (in vitro determination of the immunomodulatory activity of trichloroacetic acid-precipitated polysaccharide TV-P by two CCK-8 methods)

[0112] 1. Reagents

[0113] The CCK-8 kit, RPIM1640, FBS, DMSO, and double antibodies are all commercially available products.

[0114] 2. Instruments

[0115] Enzyme label instrument; cell incubator.

[0116] 3. Methods

[0117] Effect of TV-P on the proliferation of immune cells (B cells, T cells and RAW264.7 cells)

[0118] The effect of trichloroacetic acid-precipitated polysaccharide (TV-P) on the proliferation of T cells, B cells and RAW264.7 cells was determined by a 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 on a cell counting plate, the cell suspension was diluted to 1 x 10 5The cell suspension was added to a 96-well plate at 100 μL per well, and the 96-well plate was placed in a CO2incubator for 24 h. After 24 h, different mass concentrations of TV-P solution (final mass concentration: 0.625, 1.25, 2.5, 5, 10, and 20 μg / mL) were added to the experimental groups, 100 μL of LPS solution (final mass concentration: 5 μg / mL) was added to the positive control group, and 100 μL of cell culture solution was added to the blank group. After incubation in a CO2incubator for 24 h, 5 μL of CCK-8 was added to each well, and the plate was incubated in a CO2incubator for 3 h. The absorbance value was detected (450 nm) on an enzyme label instrument, and an image was taken.

[0119] 4. Results

[0120] 4.1 Effect of TV-P on B cell proliferation

[0121] The results are shown in Table 1. Figure 9 As shown in Table 1, compared with the blank group, the LPS group significantly (P < 0.05) promoted B cell proliferation, with a proliferation rate of 24.77%; when the final concentration of TV-P was in the range of 0.625-20 μg / mL, it significantly (P < 0.05) promoted B cell proliferation; and when the final concentration of TV-P was 2.5 μg / mL, TV-P had the most obvious effect on B cell proliferation, with a maximum proliferation rate of 30.73%.

[0122] 4.2 Effect of TV-P on T cell proliferation

[0123] The results are shown in Table 2. Figure 10 As shown in Table 2, compared with the blank group, the LPS group significantly (P < 0.05) promoted T cell proliferation, with a proliferation rate of 175.63%; when the final concentration of TV-P was in the range of 1.25-20 μg / mL, it significantly (P < 0.05) promoted T cell proliferation; and when the final concentration of TV-P was 20 μg / mL, TV-P had the most obvious effect on T cell proliferation, with a maximum proliferation rate of 96.88%.

[0124] 4.3 Effect of TV-P on RAW264.7 cell proliferation

[0125] The results are shown in Table 3. Figure 11 As shown in Table 3, compared with the blank group, the LPS group significantly (P < 0.05) promoted RAW264.7 cell proliferation, with a proliferation rate of 102.72%; when the final concentration of TV-P was in the range of 0.625-20 μg / mL, it significantly (P < 0.05) promoted RAW264.7 cell proliferation; and when the final concentration of TV-P was 20 μg / mL, TV-P had the most obvious effect on RAW264.7 cell proliferation, with a maximum proliferation rate of 106.02%.

[0126] 4.4 Effect of TV-P on the proliferation of MFC cells

[0127] As shown in the results Figure 12 compared with the blank group, the MAN group can significantly (P<0.05) inhibit the proliferation of MFC cells, with an inhibition rate of 68.71%; when the final concentration of TV-P is in the range of 1.25-20 μg / mL, it can significantly (P<0.05) inhibit the proliferation of MFC cells; and when the final concentration of TV-P is 20 μg / mL, the inhibitory effect of TV-P on MFC cells is the most obvious, with a maximum inhibition rate of 61.89%.

[0128] 4.5 Effect of TV-P on the proliferation of CT26.WT cells

[0129] As shown in the results Figure 13 compared with the blank group, the MAN group can significantly (P<0.05) inhibit the proliferation of CT26.WT cells, with an inhibition rate of 34.06%; when the final concentration of TV-P is in the range of 1.25-20 μg / mL, it can significantly (P<0.05) inhibit the proliferation of CT26.WT cells; and when the final concentration of TV-P is 1.25 μg / mL, the inhibitory effect of TV-P on CT26.WT cells is the most obvious, with a maximum inhibition rate of 34.98%.

[0130] 4.6 Effect of TV-P on the proliferation of LLC cells

[0131] As shown in the results Figure 14 compared with the blank group, the MAN group can significantly (P<0.05) inhibit the proliferation of LLC cells, with an inhibition rate of 49.66%; when the final concentration of TV-P is in the range of 5-20 μg / mL, it can significantly (P<0.05) inhibit the proliferation of LLC cells; and when the final concentration of TV-P is 20 μg / mL, the inhibitory effect of TV-P on LLC cells is the most obvious, with a maximum inhibition rate of 54.19%.

[0132] The above only describes preferred embodiments of the present application and is not intended 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.

[0133] The foregoing embodiments and methods described in the present application can be different based on the ability, experience and preference of a person skilled in the art.

[0134] The steps of the method described in the present application do not constitute any limitation on the order of the steps of the method.

Claims

1. Use of a polysaccharide of Coriolus versicolor in the preparation of a medicine for enhancing immunity or in the preparation of an anti-tumor medicine, characterized in that, the polysaccharide of Coriolus versicolor is used in the preparation of a medicine for enhancing immunity or in the preparation of an anti-tumor medicine. The polysaccharide is a heteropolysaccharide composed of xylose, arabinose, glucose and galactose, and the molar ratio of residues of xylose, arabinose, glucose and galactose is 3:1:10:5; The weight average molecular weight of the polysaccharide is 13069 Da; The preparation method of the polysaccharide comprises the following steps: (1) taking the powder of the fruiting body of Trametes versicolor, extracting with hot water, and then concentrating, alcohol precipitating and drying to obtain the crude polysaccharide; (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting and collecting the eluate; (3) subjecting the eluate obtained in step (2) to dialysis and concentration with a dialysis bag; In step (1), in the alcohol precipitation step, the volume ratio of alcohol to the concentrated water extract is 3:1; In step (2), the ion exchange column is a DEAE cellulose column, and gradient elution is performed by sequentially adding 0.01 mol / L, 0.05 mol / L and 0.1 mol / L NaCl solutions; In step (3), the molecular weight cut-off of the dialysis bag is ≥7000 Da.

2. Use according to claim 1, wherein In step (1), the extraction temperature is 80-100℃, The mass ratio of the powder of the fruiting body of Trametes versicolor to water is 1:1-10; The alcohol is ethanol.

3. The use according to claim 1, wherein The concentration of the polysaccharide in the medicine is 0.5-25 μg / mL.

Images