A baishen mushroom polysaccharide, a preparation method and application thereof

Abstract

The application discloses a kind of white ginseng fungus polysaccharide (SC-P) and its preparation method and application.The composition of the SC-P includes alpha-D-glucose, the chemical structure of SC-P includes 1,4-connected alpha-D-glucose residue, 1-connected alpha-D-glucose residue, 1,4,6-connected alpha-D-glucose residue and 1,6-connected alpha-D-glucose residue, and the molar ratio of the residues of 1→4-Glu, 1→-Glu, 1→4,6-Glu and 1→6-Glu is 3:1:1:4.The SC-P is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography, dialysis and concentration.The SC-P of the application has significant immunomodulatory activity and antitumor activity, and can be applied in drugs, health products or food.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of application of fungal polysaccharides, in particular to a schizophyllum commune polysaccharide and a preparation method and application thereof. BACKGROUND

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

[0003] Schizophyllum commune, also known as schizophyllum commune, bazi chai, and xuelian mushroom, is a rare edible and medicinal mushroom belonging to the fungal kingdom, basidiomycota, agaricales, schizophyllaceae and schizophyllum. The fruiting body of schizophyllum commune is small, and is clustered or grouped, like a chrysanthemum. The cap is fan-shaped or kidney-shaped, with a diameter of 1-5 cm, and the fresh product of artificial cultivation weighs 50-100 g per single flower. The flesh is thin, tough, white to grayish white or brownish in color. Schizophyllum commune has multiple lobes, with the edges often longitudinally split and involuted, and the surface covered with fluff; the base is narrow, and the gills grow radially from the base; the stem is short or absent; the flesh is tough, white to grayish white, covered with fluff, fan-shaped or kidney-shaped, with multiple lobes; the gills are narrow, radiating from the base; the stem is short or absent.

[0004] In "Purification and characterization of high molecular weight schizophyllum commune polysaccharide" ([J], Food and Fermentation Industry, 2008, 34(12).), Zhou Lin et al. reported that the fermentation broth of schizophyllum commune was centrifuged, decolorized by activated carbon water bath, concentrated at 60℃ under vacuum, deproteinized by Sevag method and precipitated with 95% ethanol for 24h to obtain a crude polysaccharide product; then the product was purified by Sephacryl S-400HR filler, eluted with 0.05mol / L NaCl solution, concentrated, dialyzed, and freeze-dried to obtain refined schizophyllum commune polysaccharide SPG. Gel column chromatography and HPLC detection showed that SPG was a homogeneous component. The weight average molecular weight (Mw) and number average molecular weight (Mn) were 2.5×10 7 Da and 1.2×10 7 Da, respectively. HPLC, ultraviolet spectrum and infrared spectrum analysis showed that it was β-glucan. Through amylase and cellulase degradation test, GC-MS and 13CNMR data analysis confirmed that its structure is β-(1→3) main chain, every 3 glucose units produce 1 β-(1→6) branch. The surface morphology of the prepared high molecular weight schizophyllum polysaccharide and bacterial cellulose is very similar, which is observed by scanning electron microscope, it is inferred that the high molecular weight schizophyllum polysaccharide is expected to be applied in food, biological materials and other fields.

[0005] Yu et al. reported in Structure and bioactivity of polysaccharide from a subseafloor strain of Schizophyllum commune 20R-7-F01 ([J], International Journal of Biological Macromolecules, 2022, 610-619.) that the monosaccharide composition of the polysaccharide EPS obtained from the fruiting body of white ginseng fungus is glucose, and the molecular weight is 6.088 x 10 5 Da, and the methylation and nuclear magnetic resonance analysis show that the backbone of EPS is (1→3)-β-D-glucan, and every third residue has a side chain (1→6)-β-D-glucan, and the biological activity analysis results show that the EPS has strong antioxidant activity, and can enhance the cell viability and phagocytosis of RAW264.7 cells.

[0006] It can be seen that the prior art lacks research on the fine structure of white ginseng fungus polysaccharide and the application of white ginseng fungus polysaccharide in immunomodulatory activity. SUMMARY

[0007] The present application overcomes the defects in the prior art and provides a white ginseng fungus polysaccharide and a preparation method and application thereof.

[0008] The first aspect of the present application provides a white ginseng fungus polysaccharide (SC-P), which is a single polysaccharide composed of α-D-glucose.

[0009] Further, the chemical structure of the polysaccharide comprises 1,4-linked α-D-glucose residues, 1-linked α-D-glucose residues, 1,4,6-linked α-D-glucose residues and 1,6-linked α-D-glucose residues, and the molar ratio of the residues is about 3:1:1:4.

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

[0011] Further, the weight average molecular weight of the polysaccharide is 10000-100000 Da (e.g. 10000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, 45000 Da, 50000 Da, 55000 Da, 60000 Da, 65000 Da, 70000 Da, 75000 Da, 80000 Da, 85000 Da, 90000 Da, 100000 Da), preferably, 10000-80000 Da, further preferably, 20000-70000 Da.

[0012] In one embodiment of the present application, the weight average molecular weight of the polysaccharide is 62032 Da.

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

[0014]

[0015] wherein n is an integer of 20-50 (e.g. 20, 25, 30, 35, 40, 45, 50), preferably, an integer of 30-40.

[0016] wherein Glu is glucose.

[0017] The second aspect of the present application provides a composition comprising the polysaccharide of the first aspect.

[0018] The third aspect of the present application provides a preparation method of the polysaccharide of the first aspect, comprising the step of extracting the fruiting body of the white Ganoderma applanatum as raw material.

[0019] Further preferably, the preparation method comprises the step of extracting the crude polysaccharide by water extraction and alcohol precipitation.

[0020] Further preferably, the preparation method further comprises the step of purifying the crude polysaccharide (e.g. by ion exchange column chromatography).

[0021] In one embodiment of the present application, the preparation method comprises the following steps:

[0022] (1) taking the fruiting body powder of the white Ganoderma applanatum, extracting with hot water, and obtaining the water extract, which is sequentially concentrated, precipitated with alcohol, and dried to obtain the crude polysaccharide;

[0023] (2) subjecting the crude polysaccharide obtained in step (1) to ion exchange column chromatography, eluting, and collecting the eluate;

[0024] (3) subjecting the eluate obtained in step (2) to dialysis and concentration with a dialysis bag.

[0025] Further, the preparation method further comprises step (4), specifically freezing and drying the liquid in the dialysis bag after step (3) is completed.

[0026] 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.

[0027] Further, in step (1), the mass ratio (W / V, mg / mL) of the white ginseng mushroom fruiting body powder 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 mass ratio is 1:3.

[0028] 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.

[0029] 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.

[0030] In an embodiment of the present application, the extraction step in step (1) can include taking white ginseng mushroom fruiting body powder, mixing with water, and boiling in a water bath.

[0031] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to water extract concentrate 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.

[0032] In an embodiment of the present application, in the above alcohol precipitation step, the alcohol is ethanol.

[0033] In an embodiment of the present application, step (1) includes taking white ginseng mushroom fruiting body powder, hot water extraction, collecting the supernatant, concentrating, adding anhydrous ethanol, collecting the precipitate, drying, removing the protein therein, and obtaining crude polysaccharides.

[0034] Further, in step (2), the ion exchange column can be a cellulose column, and the filler of the cellulose column can be DEAE cellulose.

[0035] Further, in step (2), the eluent used for elution can be a NaCl solution; specifically, the concentration of the NaCl solution is 0-0.3 mol / L (such as 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3) mol / L.

[0036] Further, in step (2), the elution can be gradient elution, and the eluent concentration can be 0-0.3 mol / L (e.g. 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3) mol / L.

[0037] In one embodiment of the present application, step (2) comprises: passing the aqueous solution of the crude polysaccharide obtained in step (1) through a cellulose column, gradient elution, collecting the eluate, and concentrating.

[0038] 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 one embodiment of the present application, the molecular weight cut-off is 7000 Da.

[0039] In one embodiment of the present application, step (3) comprises: dialysis of the eluate obtained in step (2) in a dialysis bag, and dialysis for two days.

[0040] The fourth aspect of the present application provides an application of the crude polysaccharide prepared by the method of the first aspect, the composition of the second aspect, or the method of the third aspect, and the application comprises:

[0041] (1) an application in the preparation of a product capable of enhancing immunity;

[0042] (2) an application in the preparation of a product having anti-tumor activity.

[0043] Further, the product is a food, health product, or pharmaceutical product.

[0044] Further, in the application, the polysaccharide can be used alone or in combination with other active ingredients.

[0045] Further, in the product, the concentration of the polysaccharide 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.

[0046] Further, when the effect of promoting the proliferation of RAW 264.7 cells is to be enhanced, the concentration of the polysaccharide is preferably 5-20 μg / mL (e.g. 5 μg / mL, 10 μg / mL, 20 μg / mL), more preferably 20 μg / mL.

[0047] Further, when the inhibitory effect of MFC cells is to be improved, the concentration of the white ginseng mushroom polysaccharide is 5-20 μg / mL (such as 5 μg / mL, 10 μg / mL, 20 μg / mL), and more preferably 10 μg / mL.

[0048] The beneficial effects of the present application are:

[0049] The present application separates and purifies polysaccharide SC-P from white ginseng mushrooms, and analyzes and identifies the molecular weight, monosaccharide composition, and chemical structure of the polysaccharide SC-P, determines the weight average molecular weight and structural composition of the polysaccharide SC-P. Cell experiments show that the polysaccharide SC-P has significant immunomodulatory activity, and in particular, the RAW264.7 cell proliferation rate is highest at a concentration of 20 μg / mL; the polysaccharide SC-P also has significant anti-tumor activity, and in particular, the MFC cell inhibition rate is highest at a concentration of 10 μg / mL. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 shown is the HPGPC spectrum of SC-P; Figure 1 shown is the HPGPC spectrum of SC-P;

[0051] Figure 2 shown is the infrared spectrum of SC-P;

[0052] Figure 3 shown is the HLPC spectrum of SC-P;

[0053] Figure 4 shown is the HMQC spectrum of SC-P; 1 H NMR spectrum of SC-P;

[0054] Figure 5 C NMR spectrum of SC-P; 13 C NMR spectrum of SC-P;

[0055] Figure 6 H- 1 H- 1 H-COSY spectrum of SC-P;

[0056] Figure 7 HMQC spectrum of SC-P;

[0057] Figure 8 HMQC spectrum of SC-P;

[0058] Figure 9 shown are the experimental results of the effect of SC-P on RAW264.7 cell proliferation (1: blank group; 2-4: SC-P with a final concentration of 5, 10, and 20 μg / mL; 5: LPS with a final concentration of 10 μg / mL);

[0059] Figure 10The experimental results of the effect of SC-P on MFC cell proliferation are shown (1: blank group; 2-4: SC-P with final concentrations of 5, 10, and 20 μg / mL; 5: MAN with a final concentration of 10 μg / mL). DETAILED DESCRIPTION

[0060] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0061] In the present application, the term "Schizophyllum commune" refers to a fungus belonging to the kingdom Fungi, subphylum Basidiomycotina, order Agaricales, family Schizophyllaceae, and genus Schizophyllum, which includes fruiting bodies and mycelia.

[0062] The term "LPS" refers to lipopolysaccharide, which is a major component of the cell wall of gram-negative bacteria. The LPS solution used in the present experiment was purchased from Biosharp Company, China.

[0063] The term "MAN" refers to mannan peptide. The MAN solution used in the present experiment was purchased from Sichuan Aobang Pharmaceutical Company.

[0064] The term "CK" refers to Control check, the blank control group.

[0065] The technical solutions of the present application will be described below in conjunction with the embodiments of the present application, and obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0066] Example 1: Isolation, extraction, and structural identification of Schizophyllum commune polysaccharide SC-P

[0067] 1. Isolation and extraction of Schizophyllum commune polysaccharide SC-P

[0068] 1.1. Extraction of Schizophyllum commune crude polysaccharide by water extraction and alcohol precipitation

[0069] Take 200 g of dried Schizophyllum commune fruiting bodies and crush them. Add the crushed Schizophyllum commune fruiting bodies 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 it, and repeat this process 3 times. Finally, concentrate all the supernatant to 200 mL. Add three times the volume of anhydrous ethanol to precipitate the proteins, collect the precipitate, and dry it to remove the proteins in the extraction solution, thereby obtaining the crude polysaccharide of Schizophyllum commune.

[0070] 1.2. Isolation and purification of Schizophyllum commune crude polysaccharide by DEAE-cellulose column chromatography

[0071] Accurately weigh 50 g of DEAE cellulose and dissolve it in 1 L of ultrapure water with thorough stirring. Stop stirring if no cellulose particles are visible to the naked eye. 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.

[0072] After the activated cellulose is packed into the column, it is equilibrated with distilled water for 24 h before crude polysaccharide separation and purification. The diluted supernatant (5 mL) of the crude polysaccharide is added to the DEAE cellulose column, and different concentrations of NaCl (0, 0.05, 0.1 mol / L) are added for elution. The polysaccharide is determined by the sulfuric acid-phenol method. The eluate from the distilled water section is concentrated to 5 mL, and the sample is purified on a cellulose column. Dialysis is performed in a dialysis bag (Mw≥7 kDa) for 48 h, and freeze-drying is performed to obtain white ginseng fungus polysaccharide, which is named SC-P.

[0073] 2. Structural identification of white ginseng fungus polysaccharide SC-P

[0074] Acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance techniques are used to analyze the structure of white ginseng fungus polysaccharide (SC-P).

[0075] 2.1. Determination of molecular weight

[0076] Dissolve 10 mg of white ginseng fungus polysaccharide SC-P sample in 1 mL of ddH2O, sonicate for 5 min, and perform HPGPC analysis.

[0077] 2.2. Infrared spectroscopy analysis of white ginseng fungus polysaccharide SC-P

[0078] Mix 2 mg of SC-P with KBr, press into a tablet, and scan the range of 4000 cm -1 -400 cm -1 by an infrared spectrophotometer.

[0079] 2.3. Analysis of monosaccharide composition of white ginseng fungus polysaccharide SC-P

[0080] Dissolve seven standard samples and SC-P sample after TFA acid hydrolysis in mobile phase (85% acetonitrile) and perform HLPC analysis.

[0081] 2.4. Nuclear magnetic resonance analysis of white ginseng fungus polysaccharide SC-P

[0082] Take 50 mg SC-P sample dissolved in 0.6 mL heavy water (D2O), loaded into a nuclear magnetic tube, detected on a nuclear magnetic resonance instrument.

[0083] 2.5, GC-MS analysis of SC-P after methylation and silylation derivation

[0084] Take 20 mg SC-P sample, seal the beaker, add 2 mL DMSO (dimethyl sulfoxide) to the sealed beaker, gently shake the beaker to fully dissolve SC-P. Then add 200 mg NaOH until NaOH just does not dissolve and place it in a shaker at room temperature for 1 h. After shaking, add 1.5 mL iodomethane, avoid light for 1 h, after reaction, add water to terminate the reaction. Extract the product with chloroform, dry to obtain 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.

[0085] The above sample is fully reacted with 2 mL hexamethyl disilazane, 1 mL trimethylsilyl chloride and 2 mL anhydrous pyridine, and is water bathed at 50°C for 20 min, centrifuged at 12000 rpm / min at 4°C for 10 min using a low-temperature high-speed centrifuge, the precipitate is discarded, filtered with a 0.22 μm filter, and the upper layer solution is used for GC-MS analysis.

[0086] 3, Results

[0087] 3.1, Basic property results of SC-P

[0088] The HPGPC spectrum of SC-P is shown in Figure 1 , which shows that the weight average molecular weight of SC-P is 62032 Da.

[0089] 3.2, FTIR spectrum analysis of SC-P

[0090] The primary structure of SC-P was characterized by Fourier infrared spectroscopy, and the results are shown in Figure 2 . The wave number at 3393.94 cm -1 , 2922.02 cm -1 and 1400-1200 cm -1 , etc. There are typical polysaccharide absorption peaks, and no other impurity peaks, indicating that the separated and purified SC-P is a polysaccharide substance. The wide absorption peak of O-H at 3393.94 cm -1 , the absorption peak in the range of 2922.02 cm -1 is the stretching vibration peak of -CH2, 1 646.80 cm -1 is the stretching vibration peak of C=O, 1365.26 cm -1The peak for the in-plane bending vibration of CH-CHO is 1043.05 cm⁻¹. -1 The peak represents the CO stretching vibration peak of carbohydrates. It is located in the 1200-1000 cm⁻¹ range. -1 The absorption peaks within the range are due to the absorption of pyranose ring lactones and hydroxyl groups, indicating that SC-P has a pyran ring. At 670.41 cm⁻¹ -1 The presence of an absorption peak indicates that SC-P contains an α-configuration. Furthermore, at 1730 cm⁻¹... -1 The absence of absorption peaks nearby indicates that SC-P does not contain uronic acid.

[0091] 3.3 Monosaccharide composition analysis of *Gynostemma pentaphyllum* polysaccharide SC-P

[0092] After complete hydrolysis of SC-P, its monosaccharide composition was analyzed by HPLC. The peak times of the standard and the target sample are shown in Table 4. The results for the target sample are as follows: Figure 3 As shown, the single symmetrical peak is glucose (Glu), with a retention time of 10.039 min.

[0093] Table 4

[0094]

[0095] 3.4 NMR spectral analysis of *Gynostemma pentaphyllum* polysaccharide SC-P

[0096] SC-P 1 The H NMR results are as follows Figure 4 As shown in the figure. The results show that SC-P has four anomeric hydrogen signals: δ5.27ppm, δ5.03ppm, δ4.89ppm, and δ4.42ppm, with an integrated area ratio of 0.74:0.19:0.23:1.03. The signals between δ3.1 and 4.2ppm are attributed to hydrogen signals from C2-C6 of the sugar residues.

[0097] SC-P 13 The C NMR results are as follows Figure 5 As shown, SC-P exhibits four anodic carbon signals at δ103.02ppm, δ99.39ppm, δ98.73ppm, and δ97.89ppm. The signals between δ60 and 82ppm are attributed to carbon signals from C2 to C6 of the sugar residues.

[0098] SC-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 in part A is δ5.27 / 3.59, the H1 / H2 signal in part B is δ5.03 / 3.92, the H1 / H2 signal in part C is δ4.89 / 3.79, and the H1 / H2 signal in part D is δ4.42 / 3.28.

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

[0100] The HMQC spectrum of SC-P is as follows: Figure 7 As shown, it can identify short-range related factors. 1 H and 13 The coupling relationship between C. The signal of H1 / C1 in part A is δ5.27 / 99.39, the signal of H1 / C1 in part B is δ5.03 / 98.73, the signal of H1 / C1 in part C is δ4.89 / 97.89, and the signal of H1 / C1 in part D is δ4.42 / 103.02.

[0101] The HMBC spectrum of SC-P is as follows: Figure 8 As shown, it can identify remote related... 1 H and 13 The coupling relationships between residues C and D are as follows: H2 / C4 signal is δ3.59 / 75.64, H6 / C4 signal is δ3.41 / 73.09, H6 / C4 signal is δ3.41 / 69.54, H1 / C3 signal is δ4.42 / 73.09 and H2 / C4 signal is 3.28 / 75.57.

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

[0103] Table 1 SC-P 1 Chemical shift of H

[0104]

[0105] Table 2SC-P 13 Chemical shift of C

[0106]

[0107] 3.5 Gas Chromatography and Mass Spectrometry Analysis of Gynostemma pentaphyllum Polysaccharide SC-P

[0108] The methylation results are shown in Table 3, indicating that the main repeating structural unit of SC-P consists of a backbone composed of 1,6-linked and 1,4-linked α-D-glucose residues, and a side chain composed of 1,4,6-linked and 1-linked α-D-glucose residues. Based on these findings, the structure of BA-T can be preliminarily deduced as follows:Figure 9 as shown.

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

[0110]

[0111] Example 2: Immune regulation and anti-tumor activity of white ginseng mushroom polysaccharide SC-P

[0112] In vitro, the immune regulation and anti-tumor activity of white ginseng mushroom polysaccharide SC-P were determined by CCK-8 method.

[0113] 1. Reagents

[0114] CCK-8 kit, RPIM1640, FBS, DMSO, double antibody, etc. are all commercially available products.

[0115] 2. Instruments

[0116] Microplate reader; cell incubator.

[0117] 3. Methods

[0118] 3.1 Effect of SC-P on the proliferation of RAW264.7 cells

[0119] The effect of white ginseng mushroom polysaccharide (SC-P) on the proliferation of RAW264.7 cells was determined by cell counting kit (CCK-8) method. 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 μL of the cell suspension was added to each well of a 96-well plate, and the 96-well plate was placed in a CO2 incubator for 24 h. After 24 h, different concentrations of SC-P solution (final concentration 5, 10, 20 μg / mL) were added to the experimental group (SC-P group), 100 μL of LPS solution (final concentration 10 μg / mL) was added to the positive control group (MAN group), and 100 μL of cell culture medium was added to the blank group (CK group). After incubation in a CO2 incubator for 24 h, cell photos were taken under an inverted microscope, and 10 μl of CCK-8 was added to each well, which was then placed in a CO2 incubator for 3 h. The absorbance value at 450 nm was detected on a microplate reader.

[0120] 3.2 Effect of SC-P on the proliferation of MFC cells

[0121] The effect of white ginseng mushroom polysaccharide (SC-P) on the proliferation of MFC cells was determined by cell counting kit (CCK-8) method. MFC cells were cultured in vitro to the logarithmic growth phase, and after counting with a cell counting plate, the cell suspension was diluted to 1 × 10 5The cell suspension was added to a 96-well plate at 100 μL per well, and the 96-well plate was placed in a CO2 incubator for 24 h. After 24 h, different concentrations of SC-P solution (5, 10 and 20 μg / mL) were added to the experimental group (SC-P group), 100 μL of MAN solution (10 μg / mL) was added to the positive control group (MAN group), and 100 μL of cell culture solution was added to the blank group (CK group). After 24 h of incubation in a CO2 incubator, cell photos were taken under an inverted microscope, and 10 μl of CCK-8 was added to each well. After 3 h of incubation in a CO2 incubator, the absorbance value at 450 nm was detected on a microplate reader.

[0122] 4. Results

[0123] 4.1 Effect of SC-P on the proliferation of RAW264.7 cells

[0124] The results are shown in Table 1. Figure 9 As shown in Table 1, compared with the blank group, the LPS group can significantly (P < 0.01) promote the proliferation of RAW264.7 cells, with a proliferation rate of 107.43%; when the final concentration of SC-P is 5, 10 and 20 μg / mL, it can significantly (P < 0.05) promote the proliferation of RAW264.7 cells; when the final concentration of SC-P is 20 μg / mL, the effect of SC-P on the proliferation of RAW264.7 cells is the most obvious, with a maximum proliferation rate of 90.98%. Therefore, the white ginseng fungus polysaccharide can promote the proliferation of immune cells and has an immunomodulatory effect.

[0125] 4.2 Effect of SC-P on the proliferation of MFC cells

[0126] The results are shown in Table 2. Figure 10 As shown in Table 2, compared with the blank group, the positive control group (MAN group) can significantly (P < 0.01) inhibit the proliferation of MFC cells, with an inhibition rate of 32.02%; when the final concentration of SC-P is 5, 10 and 20 μg / mL, it can significantly (P < 0.01) inhibit the proliferation of MFC cells; when the final concentration of SC-P is 10 μg / mL, the effect of SC-P on the inhibition of MFC cells is the most obvious, with a maximum inhibition rate of 48.11%. Therefore, the white ginseng fungus polysaccharide can inhibit the proliferation of cancer cells and has an antitumor activity.

[0127] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. 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.

[0128] 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.

[0129] The mere fact that method steps are recited in a certain order in the present disclosure does not necessarily constitute any limitation on the order in which such steps are performed.

Claims

1. A white ginseng mushroom polysaccharide, characterized in that, The polysaccharide is a single polysaccharide consisting of α-D- 1,4-linked glucose residues, 1 -linked α-D- - glucose residues, 1,4,6-linked α-D glucose residues and 1,6-linked α-D- - glucose residues, the molar ratio of the 1,4-linked α-D glucose residues, 1 -linked α-D- - glucose residues, 1,4,6-linked α-D glucose residues and 1,6-linked α-D- - glucose residues, the molar ratio of the 1,4-linked α-D glucose residues being 3 : 1 : 1 :

4. The polysaccharide comprises the following structure: Wherein, n is an integer of 20-50; The weight average molecular weight of the polysaccharide is 20000-70000 Da.

2. A composition characterized in that, The composition comprises the polysaccharide of claim 1.

3. The process for the preparation of polysaccharides as claimed in claim 1, wherein, The method comprises the step of extracting white mushroom fruiting body as raw material: (1) taking white mushroom fruiting body powder, extracting with hot water, and obtaining the water extract, then concentrating, alcohol precipitation, and drying to obtain crude polysaccharide; (2) the crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, elution, and collection of eluate; (3) the eluate obtained in step (2) is subjected to dialysis bag dialysis and concentration; In step (1), the extraction temperature is 80-100℃; The mass ratio of white mushroom fruiting body powder to water is 1:1-10; The volume ratio of alcohol to water extract concentrate in alcohol precipitation is 1-10:1; In step (2), the ion exchange column is a cellulose column, and the filler is DEAE cellulose; The eluent used for elution is NaCl solution; The elution is gradient elution.

4. Use of a polysaccharide according to claim 1, characterized in that, The application comprises: (1) application in preparing products capable of enhancing immunity; (2) application in preparing products with anti-tumor activity.

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