Canthaxanthin and its preparation method and application

Abstract

The application discloses a Cantharellus cibarius polysaccharide, a preparation method and application thereof. The Cantharellus cibarius polysaccharide is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography and dialysis of Cantharellus cibarius fruiting bodies. The Cantharellus cibarius polysaccharide is a heteropolysaccharide composed of beta-D-glucose and alpha-D-galactose, and the molar ratio of residues of beta-D-glucose and alpha-D-galactose is 1:19. The Cantharellus cibarius polysaccharide prepared by the application has significant immunoregulation and anti-tumor activity, and can be applied in medicines, health products or food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fungal polysaccharide applications, and particularly relates to a Cantharellus cibarius polysaccharide as well as a preparation method and application thereof. BACKGROUND

[0002] Edible fungi fruiting bodies contain rich nutrients such as proteins, vitamins, mineral elements, amino acids and polysaccharides, and have low heat and fat. Edible fungus polysaccharides have good antitumor, hypoglycemic and hypolipidemic, immunocompetent and other biological activity characteristics, and are safe, non-toxic and without side effects, which makes them widely concerned in the fields of health food and biological medicine.

[0003] Cantharellus cibarius, also known as yellow silk fungus and apricot fungus, is an ectomycorrhizal fungus belonging to the fungal kingdom, Basidiomycota, Basidiomycetes, Cantharellales, Cantharellaceae and Cantharellus. The fruiting body of Cantharellus cibarius is trumpet-shaped with a smooth surface. It is mainly distributed in Yunnan, Guizhou, Sichuan, Hunan and Fujian provinces of China. Cantharellus cibarius has high nutritional value, and its fruiting body contains 8 kinds of amino acids, VA, VC, carotene and various mineral elements such as calcium, iron and phosphorus necessary for humans.

[0004] Yang et al. obtained neutral sugar homologous primary fraction WCCP-N-b and acidic sugar homologous primary fraction WCCP-A-b by water extraction, alcohol precipitation, ion exchange chromatography and gel chromatography purification of Cantharellus cibarius fruiting bodies, with molecular weights of 18 kDa and 10 kDa, respectively. WCCP-N-b is a linear galactan. WCCP-A-b is a linear glucan containing a small amount of glucan, mannose and gluconic acid side chains. Analysis of the activation effect of WCCP-N-b and WCCP-A-b on RAW264.7 cells showed that WCCP-N-b had a stronger activation effect, indicating that linear galactan had a stronger activation effect on RAW264.7 cells than linear glucan. RAW264.7 cells, HCT116 cells and HT29 cells were treated with different concentrations of WCCP-N-b (25-200 μg / ml). The results showed that 200 μg / ml WCCP-N-b could slightly inhibit the survival of RAW264.7 cells, and the other concentrations had no significant effect on the survival of RAW264.7 cells. For HCT116 cells and HT29 cells, WCCP-N-b had no significant effect on cell viability at different concentrations. This indicates that the Cantharellus cibarius polysaccharide does not have a direct killing effect on colon cancer cells, but when the concentration is high, the Cantharellus cibarius polysaccharide shows a slight killing effect on RAW264.7 cells (Yang G. Study on the effect of Cantharellus cibarius 3-O-Me-galactan on macrophage activation[D]. Northeast Normal University, 2019.).

[0005] Currently, the research on Cantharellus cibarius in the prior art mainly focuses on the fine structure of linear galactan isolated from Cantharellus cibarius and the role thereof in activating macrophages and inhibiting colon cancer cells. However, the research on the fine structure of other Cantharellus cibarius polysaccharides (such as linear glucan containing a small amount of galactose branches) and the antitumor (such as gastric cancer) and immunomodulatory activities thereof is relatively insufficient. SUMMARY

[0006] In order to overcome the defects existing in the prior art, the present application provides a Cantharellus cibarius polysaccharide and a preparation method and application thereof.

[0007] In a first aspect of the present application, a Cantharellus cibarius polysaccharide is provided, which is a heteropolysaccharide composed of galactose and glucose, and the molar ratio thereof is 1:19.

[0008] Further, the galactose is D-galactose, L-galactose or D / L-galactose, and the glucose is D-glucose, L-glucose or D / L-glucose.

[0009] Further, the D-galactose is α-D-galactose, β-D-galactose or α / β-D-galactose, and the D-glucose is α-D-glucose, β-D-glucose or α / β-D-glucose.

[0010] Further, the L-galactose is α-L-galactose, β-L-galactose or α / β-L-galactose, and the L-glucose is α-L-glucose, β-L-glucose or α / β-L-glucose.

[0011] Preferably, the Cantharellus cibarius polysaccharide is a heteropolysaccharide composed of D-galactose and D-glucose.

[0012] More preferably, the Cantharellus cibarius polysaccharide is a heteropolysaccharide composed of α-D-galactose and β-D-glucose.

[0013] Further, the chemical structure of the Cantharellus cibarius polysaccharide comprises 1,6-linked glucose residues, 1,4-linked glucose residues, 1,4,6-linked glucose residues and 1-linked galactose residues, and the molar ratio thereof is 9:9:1:1. The above-mentioned Cantharellus cibarius polysaccharide comprises multiple connection modes.

[0014] Preferably, the chemical structure of the Cantharellus cibarius polysaccharide comprises 1,6-linked β-D-glucose residues, 1,4-linked β-D-glucose residues, 1,4,6-linked β-D-glucose residues and 1-linked α-D-galactose residues, and the molar ratio thereof is 9:9:1:1. The above-mentioned Cantharellus cibarius polysaccharide comprises multiple connection modes.

[0015] Further, in the chemical structure of the Cantharellus cibarius polysaccharide, the main chain is composed of 1, 6-linked glucose residues and 1, 4-linked glucose residues, and the side chain is composed of 1, 4, 6-linked glucose residues and 1-linked galactose residues.

[0016] Preferably, in the chemical structure of the Cantharellus cibarius polysaccharide, the main chain is composed of 1, 6-linked β-D-glucose residues and 1, 4-linked β-D-glucose residues, and the side chain is composed of 1, 4, 6-linked β-D-glucose residues and 1-linked α-D-galactose residues.

[0017] Further, the Cantharellus cibarius polysaccharide comprises the following structure:

[0018]

[0019] Wherein, n is an integer of 2-10 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0020] Further, the weight average molecular weight of the Cantharellus cibarius polysaccharide is 8000-20000 Da (such as 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000 Da).

[0021] In some embodiments of the present application, the weight average molecular weight of the Cantharellus cibarius polysaccharide is 8305 Da.

[0022] In a second aspect of the present application, a preparation method of Cantharellus cibarius polysaccharide is provided, which comprises the step of extracting Cantharellus cibarius fruiting bodies.

[0023] Further, the preparation method comprises the step of extracting crude polysaccharide by water extraction and alcohol precipitation.

[0024] Further, the preparation method further comprises the step of purifying the crude polysaccharide (for example, by ion exchange column chromatography).

[0025] In some embodiments of the present application, the preparation method comprises the following steps:

[0026] (1) Take Cantharellus cibarius fruiting body powder, extract with water (such as cold water or hot water), and obtain crude polysaccharide by concentrating, alcohol precipitation and removing protein in sequence from the obtained water extract;

[0027] (2) The crude polysaccharide obtained in step (1) is subjected to ion exchange column chromatography, eluted, and the eluate is collected;

[0028] (3) The eluate obtained in step (2) is subjected to dialysis using a dialysis bag.

[0029] Optionally, (4) The liquid in the dialysis bag after step (3) is completed is freeze-dried.

[0030] Further, in step (1), the ratio of the chicken oil fungus fruiting body powder to water (W / V, mg / mL) is 1:1-10 (such as 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.

[0031] Further, in step (1), the temperature of the extraction is 80-100°C (such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100°C). In an embodiment of the present application, the extraction temperature is 98°C.

[0032] Further, in step (1), the number of extractions is 1 or more (such as 2, 3, 4, 5 times). In an embodiment of the present application, the number of extractions is 3.

[0033] Further, in step (1), the extraction time is 1-10 hours (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours) each time. In an embodiment of the present application, the extraction time is 6 hours each time.

[0034] In an embodiment of the present application, the extraction step in step (1) comprises taking chicken oil fungus fruiting body powder, mixing it with water, and extracting by heating in a water bath.

[0035] Further, in step (1), in the alcohol precipitation step, the volume ratio of alcohol to concentrated water extract is 1-10:1 (such as 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 3:1.

[0036] In an embodiment of the present application, in the alcohol precipitation step, the alcohol is ethanol.

[0037] Further, in step (1), the Sevage method is used for protein removal.

[0038] In one embodiment of the present application, step (1) comprises: taking Cantharellus cibarius fruiting body powder, hot water extraction, collecting supernatant, concentration, adding anhydrous ethanol, collecting precipitate, drying, removing protein therein, and obtaining crude polysaccharide.

[0039] Further, in step (2), the ion exchange column is a cellulose column, and the filler of the cellulose column is DEAE-cellulose.

[0040] Further, in step (2), the eluent used for elution is a NaCl solution. Specifically, the concentration of the NaCl solution is 0.001-0.30 mol / L (such as 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30 mol / L).

[0041] Further, in step (2), the elution is gradient elution.

[0042] In some embodiments of the present application, the Cantharellus cibarius polysaccharide is from the eluate with a concentration of 0.1 mol / L NaCl solution in the gradient elution.

[0043] 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, and collecting the eluate.

[0044] Further, in step (3), the molecular weight cut-off of the dialysis bag is 5000-10000 Da (such as 5000, 6000, 7000, 8000, 9000, 10000 Da). In one embodiment of the present application, the molecular weight cut-off is 7000 Da.

[0045] In one embodiment of the present application, step (3) comprises: placing the eluate obtained in step (2) in a dialysis bag for dialysis for 2 days.

[0046] The preparation method of the present application can prepare the Cantharellus cibarius polysaccharide of the first aspect.

[0047] The present application also provides a crude polysaccharide obtained by the above preparation method.

[0048] In the third aspect of the present application, a composition comprising the Cantharellus cibarius polysaccharide is provided.

[0049] Further, the composition further comprises an auxiliary material, such as a pharmaceutically acceptable auxiliary material, a food or health product acceptable auxiliary material.

[0050] Further, the pharmaceutically acceptable adjuvant is selected from the group consisting of: solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, compression aid, flavoring agent, preservative, suspending agent, coating agent, fragrance, anti-adhesion agent, antioxidant, antioxidant synergist, chelating agent, skin penetration enhancer, air displacement agent, pH regulator, adsorbent, plasticizer, surfactant, antifoaming agent, thickening agent, inclusion agent, protective agent, humectant, softening agent, absorbent, diluent, flocculating agent, filter aid, ink, pressure-sensitive adhesive, lyophilization filler, lyophilization protective agent, empty capsule, colloidal stabilizer, vaccine adjuvant, traditional Chinese medicine processing adjuvant.

[0051] Further, the food or health product acceptable adjuvant is selected from the group consisting of: acidity regulator, anti-caking agent, antifoaming agent, antioxidant, bleaching agent, leavening agent, gum base, colorant, color protection agent, emulsifier, enzyme preparation, flavor enhancer, flour treatment agent, film forming agent, moisture retaining agent, nutritional fortifier, preservative, stabilizer and coagulant, sweetener, thickening agent, food flavor, food processing aid.

[0052] Further, the composition is a pharmaceutical composition, a food composition or a health product composition.

[0053] Further, the pharmaceutical composition is an antitumor drug, an immune enhancer, an immune adjuvant, a bacteriostatic drug, a hypoglycemic drug or a lipid-lowering drug, etc.

[0054] Further, the Cantharellus cibarius polysaccharide is used alone or in combination with other active ingredients.

[0055] Further, the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, preferably an oral dosage form.

[0056] Further, the dosage form of the food composition or the health product composition is an oral dosage form.

[0057] Further, the oral dosage form is selected from the group consisting of: tablet, capsule, granule, suspension, solution, mixture, syrup, powder, drop pill, pill, tincture, decoction, wine, emulsion.

[0058] Further, the injection dosage form is selected from the group consisting of: injection solution, sterile powder for injection (including lyophilized powder for injection).

[0059] In a fourth aspect of the present application, a use of Cantharellus cibarius polysaccharide in the preparation of an antitumor drug is provided.

[0060] Further, the tumor is selected from the group consisting of: gastric cancer, colon cancer, sarcoma, liver cancer, ovarian cancer.

[0061] In some embodiments of the present application, the tumor is gastric cancer.

[0062] Further, the anti-tumor drug further comprises a pharmaceutically acceptable excipient.

[0063] Further, the Cantharis polysaccharide is used alone or in combination with other active ingredients.

[0064] In a fifth aspect of the present application, a use of the Cantharis polysaccharide in the preparation of an immune enhancer or an immune adjuvant is provided.

[0065] Further, the Cantharis polysaccharide can enhance the immune function (such as non-specific or specific immune function) of the body by activating immune cells (such as lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, platelets, etc.) or immunologically active substances (such as antibodies, lysozyme, complement, immunoglobulin, interferon, interleukin, tumor necrosis factor, etc. cytokines), so as to restore the low immune function to normal; the Cantharis polysaccharide can also act as an adjuvant to enhance the immunogenicity of the combined antigen and accelerate the induction of immune response.

[0066] Further, the immune enhancer or the immune adjuvant further comprises a pharmaceutically acceptable excipient.

[0067] Further, the Cantharis polysaccharide is used alone or in combination with other active ingredients.

[0068] In a sixth aspect of the present application, a use of the Cantharis polysaccharide in the preparation of an immune-enhancing food or health product is provided.

[0069] Further, the Cantharis polysaccharide can enhance the immune function (such as non-specific or specific immune function) of the body by activating immune cells (such as lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, platelets, etc.) or immunologically active substances (such as antibodies, lysozyme, complement, immunoglobulin, interferon, interleukin, tumor necrosis factor, etc. cytokines), so as to achieve the purpose of enhancing immunity.

[0070] Further, the immune-enhancing food or health product further comprises a food or health product acceptable excipient.

[0071] Further, the Cantharis polysaccharide is used alone or in combination with other active ingredients.

[0072] The present application isolates and purifies Cantharellus cibarius polysaccharide from Cantharellus cibarius, and analyzes and identifies the molecular weight, monosaccharide composition, chemical structure and the like of the polysaccharide, and determines the weight average molecular weight and structural composition of the polysaccharide. Cell experiments show that the polysaccharide has significant immunomodulatory and anti-tumor activity, especially at a concentration of 20 μg / mL, the proliferation rate of RAW264.7 cells is the highest; especially at a concentration of 10 μg / mL, the inhibition rate of MFC cells is the highest. Based on this, the polysaccharide can be used for preparing medicines, and can also be used for preparing health care products and food for enhancing immunity, and has better application prospect and commercial value. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 The HPGPC spectrum of CC-P is shown.

[0074] Figure 2 The FT-IR spectrum of CC-P is shown.

[0075] Figure 3 The HPLC spectrum of the hydrolysate of CC-P is shown, in which peak 1 is glucose and peak 2 is galactose.

[0076] Figure 4 The H NMR spectrum of CC-P is shown. 1

[0077] Figure 5 The C NMR spectrum of CC-P is shown. 13

[0078] Figure 6 The H-H COSY spectrum of CC-P is shown. 1 1

[0079] Figure 7 The HMQC spectrum of CC-P is shown.

[0080] Figure 8 The HMBC spectrum of CC-P is shown.

[0081] Figure 9 The chemical structure of CC-P is shown.

[0082] Figure 10 The experimental results of the effect of CC-P on the proliferation of RAW264.7 cells are shown.

[0083] Figure 11 The experimental results of the effect of CC-P on the proliferation of MFC cells are shown. DETAILED DESCRIPTION

[0084] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as commonly understood by one of ordinary skill in the art to which the present application pertains.​​​​

[0085] In the present invention, "Cantharellus cibarius" refers to Basidiomycota, Basidiomycetes, Cantharellales, Cantharellaceae and Cantharellus, which comprises fruiting bodies and mycelium.

[0086] 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 the 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.

[0087] The term "cancer" refers to a malignant tumor (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).

[0088] The disclosures of various publications, patents and published patent specifications referred to herein are hereby incorporated by reference in their entirety.

[0089] 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, but not 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.

[0090] Example 1: Isolation and extraction of Cantharellus cibarius polysaccharide CC-P

[0091] 1. Isolation and extraction of Cantharellus cibarius polysaccharide CC-P

[0092] 1.1. Extraction of crude polysaccharide from Cantharellus cibarius by water extraction and alcohol precipitation

[0093] Take 200 g of dried Cantharellus cibarius fruiting body powder, add it to the beaker with distilled water at a ratio of 1:3, and crush the Cantharellus cibarius fruiting body. Place the beaker in a water bath at 98°C for 6 hours. Collect the supernatant and concentrate it, repeat 3 times, and finally concentrate all the supernatant to 200 mL. Add three times the volume of anhydrous ethanol to precipitate the protein, collect the precipitate and dry it to remove the protein in the extract, and obtain the crude polysaccharide of Cantharellus cibarius.

[0094] 1.2, DEAE-cellulose column chromatography for separation and purification of crude polysaccharide of Cantharellus cibarius

[0095] Accurately weigh 50 g of DEAE cellulose and dissolve it in 1 L of ultrapure water, stir well, and stop stirring if there are no visible cellulose particles. Let it stand for 24 hours, discard the supernatant, and use it as needed. Prepare 0.5 mol / L NaOH, soak the cellulose for 6 hours, wash with ultrapure water until neutral, discard the supernatant, add 0.5 mol / L HCl and soak for 6 hours, wash with distilled water until neutral, discard the supernatant, and add 0.5 mol / L NaOH again and soak for 6 hours, wash with distilled water until neutral, and let it stand for use.

[0096] After the activated cellulose is packed into the column, it is balanced with distilled water for 24 hours before the separation and purification of the crude polysaccharide. Add the diluted supernatant of the crude polysaccharide (5 mL) to the DEAE cellulose column, and elute with different concentrations of NaCl (0.01 mol / L, 0.05 mol / L, 0.1 mol / L). The polysaccharide is determined by the sulfuric acid-phenol method. Concentrate the eluate to 5 mL, purify the sample on the cellulose column. Dialysis bag (Mw≥7 kDa) for 48 hours, freeze-drying, get Cantharellus cibarius polysaccharide, named CC-P. Cantharellus cibarius polysaccharide CC-P mainly exists in the gradient eluate with a concentration of 0.1 mol / L NaCl solution.

[0097] 2, Structure identification of Cantharellus cibarius polysaccharide CC-P

[0098] 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 Cantharellus cibarius polysaccharide (CC-P).

[0099] 2.1, Determination of molecular weight

[0100] Dissolve 10 mg of Cantharellus cibarius polysaccharide CC-P sample in 1 mL of ddH2O, ultrasonic for 5 min, and perform HPGPC analysis.

[0101] 2.2, Infrared spectrum analysis of Cantharellus cibarius polysaccharide CC-P

[0102] 2mg CC-P was mixed with KBr and pressed into a tablet, which was scanned by infrared spectrophotometer in the range of 4000cm -1 -500cm -1 .

[0103] 2.3, Monosaccharide composition analysis of Cantharellus cibarius polysaccharide CC-P

[0104] Six standard samples (fructose, rhamnose, arabinose, mannose, glucose, galactose) purchased from Chengdu Manster Biotechnology Co., Ltd. and CC-P sample after TFA acid hydrolysis were dissolved with mobile phase (75% acetonitrile) and analyzed by HPLC.

[0105] 2.4, Nuclear magnetic resonance analysis of Cantharellus cibarius polysaccharide CC-P

[0106] 50mg CC-P sample was dissolved in 0.6mL heavy water (D2O) and loaded into a nuclear magnetic tube for detection on a nuclear magnetic resonance instrument.

[0107] 2.5, GC-MS analysis of Cantharellus cibarius polysaccharide CC-P after methylation and silylation derivatization

[0108] 20mg CC-P sample was weighed and the beaker was sealed. 2mL DMSO (dimethyl sulfoxide) was added to the sealed beaker, and the beaker was gently shaken to fully dissolve the CC-P. Then 200mg NaOH was added until the NaOH just did not dissolve, and it was placed in a shaker for 1h of room temperature shaking. After shaking, 1.5mL iodomethane was added, and the reaction was carried out in the dark for 1h. 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 methylation was obtained.

[0109] The above sample was fully reacted with 2mL hexamethyldisilazane, 1mL trimethylchlorosilane, and 2mL anhydrous pyridine, and was placed in a water bath at 50°C for 20min. A low-temperature high-speed centrifuge was used at a speed of 12000rpm / min, 4°C for 10min, the precipitate was discarded, and the upper layer solution was filtered with a 0.22μm filter for GC-MS analysis.

[0110] 3, Results

[0111] 3.1, Basic property results of Cantharellus cibarius polysaccharide CC-P

[0112] The HPGPC spectrum of CC-P is shown in Figure 1 , which shows that the weight average molecular weight of CC-P is 8305Da.

[0113] 3.2, FT-IR spectrum analysis of Cantharellus cibarius polysaccharide CC-P

[0114] The primary structure of CC-P was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 2 As shown, the wavenumber is at 3378 cm⁻¹ -1 2899cm -1 1682cm-1 and 1400-1200cm -1 The presence of typical polysaccharide absorption peaks and the absence of other impurity peaks indicates that the isolated and purified CC-P is a polysaccharide. The wavenumber is 3378 cm⁻¹. -1 The broad absorption peak is the stretching vibration peak of OH, at 2899 cm⁻¹. -1 The absorption peak within the range is the stretching vibration peak of CH, at 1682 cm⁻¹. -1 The peak is the C=O stretching vibration peak, 1402 cm⁻¹. -1 The peak represents the in-plane bending vibration of -CHO at 1200-1000 cm⁻¹. -1 The absorption peaks within the range are due to the absorption of pyranose ring lactones and hydroxyl groups, indicating that CC-P has a pyran ring. At 837 cm⁻¹ -1 The presence of an absorption peak indicates that CC-P contains β-pyranose, at 723 cm⁻¹. -1 The absorption peak at 1730 cm⁻¹ indicates that CC-P contains α-pyranose. Furthermore, the absorption peak at 1730 cm⁻¹... -1 The absence of absorption peaks nearby indicates that CC-P does not contain uronic acid.

[0115] 3.3 Monosaccharide composition analysis of Chanterelle Polysaccharide CCC-P

[0116] After complete hydrolysis of CC-P, its monosaccharide composition was analyzed by HPLC, and the results are as follows: Figure 3 As shown, peak 1 is glucose (Glc), with a retention time of 8.798 min; peak 2 is galactose (Gal), with a retention time of 9.417 min. The ratio of glucose to galactose is 19:1.

[0117] 3.4 NMR spectral analysis of Canarium praecox polysaccharide CCC-P

[0118] CC-P 1 The H NMR results are as follows Figure 4 As shown in the figure. The results show that CC-P has four anomeric hydrogen signals: δ5.32ppm, δ4.89ppm, δ4.45ppm, and δ4.43ppm, with an integrated area ratio of 1.0:0.78:8.97:9.58. The signals between δ3.0 and 4.2ppm are attributed to hydrogen signals from C2-C6 of the sugar residues.

[0119] CC-P 13 The C NMR results are as follows Figure 5As shown, CC-P has four anomeric carbon signals at δ 103.11 ppm, δ 103.02 ppm, δ 100.00 ppm and δ 97.87 ppm, which correspond to C, D, A, B four kinds of sugar residues respectively. The signals between δ 60-85 ppm are attributed to the carbon signals of C2-C6 in the sugar residues. The chemical shift of the end group carbon of the β-D configuration sugar residue is generally greater than δ 100 ppm, and the chemical shift of the end group carbon of the α-D configuration sugar residue is generally less than δ 100 ppm, so it can be obtained that C, D, A sugar residues are β-D configuration, and B sugar residue is β-D configuration.

[0120] The HMQC spectrum of CC-P is shown in Figure 6. 1 H- 1 The COSY spectrum of CC-P is shown in Figure 5. Figure 6 As shown, the coupling relationship between the adjacent hydrogen nuclei can be identified according to the coupling constant, and the α / β configuration of the sugar residue can be judged. The sugar residue with a coupling constant of 2-4 Hz is generally in α configuration, and the sugar residue with a coupling constant of 4-8 Hz is generally in β configuration. The signals of H1 / H2 of A part are δ 5.32 / 3.85, the signals of H1 / H2 of B part are δ 4.89 / 3.75, the signals of H1 / H2 of C part are δ 4.45 / 3.41, and the signals of H1 / H2 of D part are δ 4.43 / 3.22. The coupling constant of H1 / H2 of A part is 8.0 Hz, the coupling constant of H1 / H2 of B part is 4.0 Hz, the coupling constant of H1 / H2 of C part is 8.0 Hz, and the coupling constant of H1 / H2 of D part is 7.2 Hz. A, C, D sugar residues are β configuration sugar residues, and B sugar residue is α configuration sugar residue.

[0121] The chemical shifts of all hydrogens are summarized in Table 1.

[0122] The HMQC spectrum of CC-P is shown in Figure 6. Figure 7 As shown, the coupling relationship between the near-range related 1 H and 13 C can be identified. The signals of H1 / C1 of A part are δ 5.32 / 100.00, the signals of H1 / C1 of B part are δ 4.89 / 97.88, the signals of H1 / C1 of C part are δ 4.45 / 103.11, and the signals of H1 / C1 of D part are δ 4.43 / 103.02.

[0123] The HMBC spectrum of CC-P is shown in Figure 7. Figure 8 As shown, the coupling relationship between the far-range related 1 H and 13The coupling relationship between C. The signal of H6 / C4 of A residue is δ3.39 / 75.56, the signal of H5 / C6 of B residue is δ3.84 / 69.57, the signal of H4 / C2 of C residue is δ3.63 / 68.32, and the signal of H2 / C1 of D residue is δ3.22 / 103.02.

[0124] The chemical shifts of all carbons are summarized in Table 2.

[0125] Table 1 CC-P of 1 The chemical shifts of H

[0126]

[0127] Table 2 CC-P in 13 The chemical shifts of C

[0128]

[0129] 3.5, Gas chromatography and mass spectrometry analysis of Cantharellus cibarius polysaccharide CC-P

[0130] The methylation results are shown in Table 3, which shows that the main repeating structural unit of CC-P is composed of 1,6-linked β-D-glucose residues and 1,4-linked β-D-glucose residues, and the branched chain is composed of 1,4,6-linked β-D-glucose residues and 1-linked α-D-galactose residues. Based on the above, it can be preliminarily inferred that the structure of CC-P is as shown in Figure 9 .

[0131] Table 3 Analysis of CC-P methylation results

[0132]

[0133]

[0134] Example 2: Anti-tumor and immunomodulatory activity of Cantharellus cibarius polysaccharide CC-P

[0135] In vitro, two CCK-8 methods were used to determine the anti-tumor and immunomodulatory activity of Cantharellus cibarius polysaccharide CC-P.

[0136] 1. Reagents

[0137] CCK-8 kit, RPIM1640, FBS, DMSO, double antibody, etc. are all commercially available products. Man (mannan peptide) is purchased from Chengdu Li'er Pharmaceutical Co., Ltd.

[0138] 2. Instruments

[0139] Microplate reader; cell incubator.

[0140] 3. Method for detecting the influence of CC-P on the proliferation of RAW264.7 cells and MFC cells

[0141] The influence of Cantharellus cibarius polysaccharide (CC-P) on the proliferation of RAW264.7 cells and MFC cells was determined by a cell counting kit (CCK-8) method. RAW264.7 cells and MFC cells were cultured in vitro to the logarithmic growth phase, and after counting in a cell counting plate, the cell suspension was diluted to 1 x 10 5 μg / mL with fresh culture solution, 100 μL of the cell suspension was added to each well of a 96-well plate, and the 96-well plate was placed in a CO2incubator for 24 h. After 24 h, different concentrations of CC-P solution (5, 10, and 20 μg / mL) were added to the experimental groups, 100 μL of LPS (10 μg / mL) was added to the positive control group (LPS group) of the RAW264.7 cell proliferation experiment, 100 μL of Man (10 μg / mL) was added to the positive control group (Man group) of the MFC cell proliferation experiment, and 100 μL of 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 determined at 450 nm on an enzyme label meter, and an image was taken. Finally, the cell proliferation rate or inhibition rate was calculated by the following equation:

[0142]

[0143] where p is the cell proliferation rate (or inhibition rate), A0is the average absorbance value of the culture solution, A2is the average absorbance value of the blank group, and A1is the average absorbance value of the drug group or the positive group.

[0144] 4. Results

[0145] 4.1. Influence of CC-P on the proliferation of RAW264.7 cells

[0146] The results are shown in Table 1. Figure 10 Compared with the blank group, the LPS group could significantly promote the proliferation of RAW264.7 cells (P < 0.01), with a proliferation rate of 55.65%. When the concentration of CC-P was 5, 10, and 20 μg / mL, it could significantly promote the proliferation of RAW264.7 cells (P < 0.01). When the concentration of CC-P was 20 μg / mL, the effect of CC-P on the proliferation of RAW264.7 cells was the most obvious, with a maximum proliferation rate of 39.97%.

[0147] 4.2. Influence of CC-P on the proliferation of MFC cells

[0148] The results are shown in Table 2. Figure 11As shown, compared with the blank group, the Man group can significantly (P<0.05) inhibit the proliferation of MFC cells, and the inhibition is 62.29%; when the mass concentration of CC-P is 5, 10 and 20 μg / mL, CC-P can extremely significantly (P<0.01) inhibit the proliferation of MFC cells; when the mass concentration of CC-P is 10 μg / mL, the inhibition effect of CC-P on MFC cells is the most obvious, and the maximum inhibition rate reaches 61.84%.

[0149] 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.

[0150] The foregoing embodiments and methods described in the present application can be different based on the ability, experience and preference of the person skilled in the art.

[0151] The steps of the method described in the present application are only listed in a certain order, and do not constitute any limitation on the order of the steps of the method.

Claims

1. A Cantharellus polysaccharide comprising 1,6-linked β - D glucose residues, 1,4-linked β - D glucose residues, 1,4,6-linked β - D glucose residues, and 1-linked α - D galactose residues in a molar ratio of 9:9:1:1, comprising the structure: wherein, n is an integer from 2 to 10; The chicken oil fungus polysaccharide has a weight average molecular weight of 8000-20000 Da.

2. A method for preparing the chicken oil fungus polysaccharide according to claim 1, comprising the step of extracting chicken oil fungus fruiting bodies. The preparation method comprises the following steps: (1) taking chicken oil fungus 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) subjecting the eluate obtained in step (2) to dialysis with a dialysis bag; In step (1), the ratio of chicken oil fungus fruiting body powder to water is 1:1-10; the temperature of the extraction is 80-100℃; the extraction is performed 1-5 times, and each extraction is performed for 1-10 hours; in the alcohol precipitation step, the volume ratio of alcohol to the concentrated water extract is 1-10:1; the alcohol is ethanol; In step (2), the ion exchange column is a cellulose column, and the filler of the cellulose column is DEAE-cellulose; The eluent used in the elution is a NaCl solution.

3. A composition comprising the chicken oil fungus polysaccharide according to claim 1.

4. The composition of claim 3, wherein The composition further comprises an auxiliary material selected from the group consisting of a pharmaceutically acceptable auxiliary material, a food or health product acceptable auxiliary material.

5. Use of the chicken oil fungus polysaccharide according to claim 1 in the preparation of an antitumor drug, an immune enhancer, an immune adjuvant, and an immune-enhancing health product.

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