A polysaccharide of coriolus versicolor and a preparation method and application thereof

Abstract

The application discloses a Coriolus versicolor polysaccharide, a preparation method and application thereof. The Coriolus versicolor polysaccharide is obtained by hot water extraction, ethanol precipitation, protein removal, ion exchange column chromatography and dialysis of Coriolus versicolor fruiting bodies. The Coriolus versicolor polysaccharide is a heteropolysaccharide composed of arabinose, glucose and galactose, and the molar ratio is 1:6:6. The Coriolus versicolor 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 Coriolus versicolor polysaccharide as well as a preparation method and application thereof. BACKGROUND

[0002] Edible mushroom fruiting bodies contain rich nutrients such as proteins, vitamins, mineral elements, amino acids and polysaccharides, and have low heat and fat content, thus having important application potential in the fields of health foods and biological medicines. In particular, lentinan has been widely concerned due to its good anti-tumor, antioxidant and antibacterial properties, as well as immunocompetence, and is safe, non-toxic and free of side effects.

[0003] In the existing research on lentinan, Zhao Shiyu et al. found that lentinan from three different producing areas had significant differences in monosaccharide composition and content ratio, antioxidant and antitumor activity, which indicated that lentinan from different producing areas had different structures and biological activities (see Zhao Shiyu, Zhao Tingting, Liang Rui, et al. Physicochemical properties and biological activities of lentinan from three different producing areas [J]. Mycosystema, 2020, 39(08): 1530-1537.). Yan Huidan found a new lentinan L2 in lentinan picked in Suizhou, Hebei Province, which was different from lentinan reported in many literatures, and L2 did not have a triple helix structure, was easy to lose activity after high-temperature treatment, and lacked some types of immune receptors (see Yan Huidan. Purification, identification and immunocompetence of a new lentinan [D]. South China University of Technology, 2013.). In addition, Geng Anjing found that high-molecular-weight (348,850 Da) lentinan had an inhibitory effect on the proliferation of colon cancer cells, and low-molecular-weight (11,572 Da) lentinan had no inhibitory effect on the proliferation of colon cancer cells, which indicated that lentinan had selectivity in terms of anti-tumor (see Geng Anjing. Preparation, structural characteristics and biological activities of lentinan [D]. South China University of Technology, 2010.).

[0004] Lentinus edodes, also known as shiitake mushroom, belongs to Basidiomycota, Agaricales, Tricholomataceae and Lentinula. It is a special wild shiitake mushroom species that grows on the dead branches of Chinese hackberry trees. Compared with other shiitake mushroom species, Lentinus edodes exhibits differences in morphology, growth environment, biological characteristics, gene sequence and nutritional value. In particular, the content of crude polysaccharides in Lentinus edodes is significantly higher than that in other shiitake mushroom species (see, for example, Pan Gaochao, He Yunsong, Li Feng, et al. Composition and distribution of wild shiitake mushrooms at different altitudes in Guizhou [J]. Chinese edible fungi, 2011, 30(03): 13-14. Xiong Xue, Li Peng, Liao Xiaofeng, et al. Identification and biological characteristics of wild Lentinus edodes [J]. Northern Horticulture, 2021(04): 118-123. Wu Chenying. Transcriptome analysis of Lentinula edodes fruiting bodies at different developmental stages and prediction of opening-related genes [D]. Guizhou University, 2023.).

[0005] The above studies show that Lentinus edodes, as a special species of shiitake mushroom, has obvious differences with other shiitake mushroom species in many aspects, which may lead to differences in the fine structure and biological activity of Lentinus edodes polysaccharides from other shiitake mushroom species. However, there is currently no research report on the fine structure of Lentinus edodes polysaccharides and their immunomodulatory and anti-tumor activities. SUMMARY

[0006] To overcome the defects of the prior art, the present application provides a Lentinus edodes polysaccharide, a preparation method and application thereof.

[0007] In a first aspect of the present application, a Lentinus edodes polysaccharide is provided, which is a heteropolysaccharide composed of arabinose, glucose and galactose, and the molar ratio is 1:6:6.

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

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

[0010] Further, the L-arabinose is a-L-arabinose, b-L-arabinose or a / b-L-arabinose, the L-glucose is a-L-glucose, b-L-glucose or a / b-L-glucose, and the L-galactose is a-L-galactose, b-L-galactose or a / b-L-galactose.

[0011] Preferably, the Morinda officinalis How polysaccharide is a heteropolysaccharide composed of D-arabinose, D-glucose and D-galactose.

[0012] More preferably, the Morinda officinalis How polysaccharide is a heteropolysaccharide composed of b-D-arabinose, a-D-glucose, b-D-glucose and a-D-galactose, and the molar ratio is 1:2:4:6.

[0013] Further, the Morinda officinalis How polysaccharide comprises 1,4-linked glucose residues, 1,6-linked glucose residues, 1,4,6-linked glucose residues, 1,4-linked galactose residues, 1,6-linked galactose residues, 1-linked galactose residues and 1,4-linked arabinose residues in the chemical structure, and the molar ratio is 2:2:2:2:2:2:1. The above-mentioned Morinda officinalis How polysaccharide comprises multiple linkage modes.

[0014] Preferably, the Morinda officinalis How polysaccharide comprises b-D-glucose residues linked by 1,4, a-D-glucose residues linked by 1,6, b-D-glucose residues linked by 1,4,6, a-D-galactose residues linked by 1,4, a-D-galactose residues linked by 1,6 and a-D-galactose residues linked by 1 in the chemical structure, and the molar ratio is 2:2:2:2:2:2:1. The above-mentioned Morinda officinalis How polysaccharide comprises multiple linkage modes.

[0015] Further, in the chemical structure of the Morinda officinalis How polysaccharide, the main chain is composed of glucose residues linked by 1,6, glucose residues linked by 1,4, galactose residues linked by 1,6, galactose residues linked by 1,4 and glucose residues linked by 1,4,6, and the side chain is composed of arabinose residues linked by 1,4 and galactose residues linked by 1.

[0016] Preferably, in the chemical structure of the Morinda officinalis How polysaccharide, the main chain is composed of a-D-glucose residues linked by 1,6, b-D-glucose residues linked by 1,4, a-D-galactose residues linked by 1,6, a-D-galactose residues linked by 1,4 and b-D-glucose residues linked by 1,4,6, and the side chain is composed of b-D-arabinose residues linked by 1,4 and a-D-galactose residues linked by 1.

[0017] Further, the Morinda officinalis polysaccharide comprises the following structure:

[0018]

[0019] wherein n is an integer from 3 to 10 (such as 3, 4, 5, 6, 7, 8, 9, 10).

[0020] Further, the Morinda officinalis polysaccharide has a weight average molecular weight of 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 Morinda officinalis polysaccharide has a weight average molecular weight of 13541 Da.

[0022] In a second aspect of the present application, a preparation method of Morinda officinalis polysaccharide is provided, which comprises the step of extracting Morinda officinalis 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) Morinda officinalis fruiting body powder is extracted with water (such as cold water or hot water), and the obtained water extract is sequentially concentrated, precipitated with alcohol, and deproteinated to obtain crude polysaccharide;

[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 temperature of the extraction is 80-100℃ (e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100℃). In one embodiment of the present application, the temperature of the extraction is 98℃.

[0031] Further, in step (1), the number of the extraction is 1 or more (e.g. 2, 3, 4, 5 times). In one embodiment of the present application, the number of the extraction is 3 times.

[0032] Further, in step (1), the time of each extraction is 1-10 hours (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours). In one embodiment of the present application, the time of each extraction is 6 hours.

[0033] Further, in step (1), the ratio of the powder of the fruiting body of Coriolus versicolor to water (W / V, mg / mL) is 1:1-10 (e.g. 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10). In one embodiment of the present application, the ratio is 1:3.

[0034] In one embodiment of the present application, the extraction step in step (1) comprises: taking the powder of the fruiting body of Coriolus versicolor, mixing with water, and extracting by heating in a water bath.

[0035] Further, in step (1), in the alcohol precipitation step, the volume ratio of the alcohol to the concentrated solution of the water extract is 1-10:1 (e.g. 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1). In one embodiment of the present application, the volume ratio is 3:1.

[0036] In one 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 removing the protein.

[0038] In one embodiment of the present application, step (1) comprises: taking the powder of the fruiting body of Coriolus versicolor, hot water extraction, collecting the supernatant, concentration, adding anhydrous ethanol, collecting the precipitate, drying, removing the protein therein, and obtaining the 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 (e.g. 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. Specifically, the concentration of the eluent in the gradient elution is optionally two or more selected from 0.001-0.30 mol / L (e.g. 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).

[0042] In an embodiment of the present application, step (2) comprises: subjecting the aqueous solution of the crude polysaccharide obtained in step (1) to cellulose column, gradient elution, and collecting the eluate and concentrating.

[0043] 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.

[0044] In an embodiment of the present application, step (3) comprises: subjecting the eluate obtained in step (2) to dialysis in a dialysis bag for 2 days.

[0045] The present application also provides a crude polysaccharide prepared by the above method.

[0046] The method of the present application can be used to prepare the polysaccharide of the first aspect.

[0047] In a third aspect of the present application, a composition comprising the polysaccharide of the first aspect is provided.

[0048] Further, the composition further comprises an excipient, such as a pharmaceutically acceptable excipient, a food or health product acceptable excipient.

[0049] Further, the pharmaceutically acceptable adjuvant is selected from the group consisting of suspending agents, coating agents, fragrances, anti-adhesion agents, antioxidants, stabilizers, glidants, compression aids, flavoring agents, preservatives, antioxidant synergists, chelating agents, solvents, propellants, solubilizers, co-solvents, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, adsorbents, plasticizers, surfactants, lyophilization fillers, lyophilization protectants, empty capsules, colloidal stabilizers, vaccine adjuvants, traditional Chinese medicine processing adjuvants, foaming agents, antifoaming agents, thickening agents, inclusion agents, protectants, humectants, softening agents, absorbents, diluents, flocculants, filter aids, inks, pressure-sensitive adhesives, skin penetration enhancers, air displacement agents, pH adjusting agents.

[0050] Further, the food or health product acceptable adjuvant is selected from the group consisting of flavor enhancers, food flavors, food industry processing aids, flour treatment agents, coloring agents, color protection agents, emulsifiers, enzyme preparations, film forming agents, moisture retention agents, nutritional fortifiers, preservatives, gum base, stabilizers and coagulants, sweeteners, thickening agents, acidity regulators, anti-caking agents, antifoaming agents, antioxidants, bleaching agents, leavening agents.

[0051] Further, the composition is a pharmaceutical composition, a food composition or a health product composition.

[0052] Further, the pharmaceutical composition is an antitumor drug, an immune enhancer, an immune adjuvant, an antiviral drug, an antibacterial drug, etc.

[0053] Further, the Coriolus versicolor polysaccharide is used alone or in combination with other active ingredients.

[0054] Further, the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, preferably an oral dosage form.

[0055] Further, the dosage form of the food composition or the health product composition is an oral dosage form.

[0056] Further, the oral dosage form is selected from the group consisting of syrup, tablet, capsule, powder, granule, drop pill, pill, tincture, decoction, liquor, emulsion, granule, suspension, solution, mixture.

[0057] Further, the injection dosage form is selected from the group consisting of injection solution, sterile powder for injection (including lyophilized powder for injection).

[0058] In a fourth aspect of the present application, a Coriolus versicolor polysaccharide is provided for use in the preparation of an antitumor drug.

[0059] Further, the tumor is selected from the group consisting of gastric cancer, breast cancer, lung cancer, lung adenocarcinoma, liver cancer, lymphoma, cardia cancer, digestive tract cancer, colon cancer, sarcoma, Abelson ascites tumor, skin cancer, leukemia, cervical cancer, bladder cancer, and ovarian cancer.

[0060] In some embodiments of the present application, the tumor is gastric cancer.

[0061] Further, the Morinda polysaccharide is used alone or in combination with other active ingredients.

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

[0063] In a fifth aspect of the present application, there is provided a use of Morinda polysaccharide in the preparation of an immune enhancer or an immunological adjuvant.

[0064] Further, the Morinda 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.), so as to restore the low immune function to normal; the Morinda polysaccharide can also act as an adjuvant to enhance the immunogenicity of the combined antigen and accelerate the induction of immune response.

[0065] Further, the Morinda polysaccharide is used alone or in combination with other active ingredients.

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

[0067] In a sixth aspect of the present application, there is provided a use of Morinda polysaccharide in the preparation of an immune-enhancing food or health product.

[0068] Further, the Morinda 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.), so as to achieve the purpose of enhancing immunity.

[0069] Further, the Morinda polysaccharide is used alone or in combination with other active ingredients.

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

[0071] In a seventh aspect, the present invention provides the use of *Malus sarmentosus* polysaccharide in the preparation of a drug for inhibiting tumor cells (such as inhibiting the growth, proliferation, and differentiation of tumor cells).

[0072] Further, the concentration of *Amanita muscaria* polysaccharide in the drug is 0.01-20 μg / mL (e.g., 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20 μg / mL), preferably 10-20 μg / mL.

[0073] Furthermore, the tumor is selected from: gastric cancer, breast cancer, lung cancer, lung adenocarcinoma, liver cancer, lymphoma, cardia cancer, digestive tract cancer, colon cancer, sarcoma, Ehrlich ascites tumor, skin cancer, leukemia, cervical cancer, bladder cancer, and ovarian cancer.

[0074] In some embodiments of the present invention, the tumor is gastric cancer.

[0075] In some embodiments of the present invention, the tumor cells are gastric cancer cells (such as MFC cells).

[0076] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0077] Furthermore, the *Amanita muscaria* polysaccharide can be used alone or in combination with other active ingredients.

[0078] This invention isolates and purifies *Mallotus spp.* polysaccharide, and analyzes and identifies its molecular weight, monosaccharide composition, and chemical structure, determining its weight-average molecular weight and structural composition. Cell experiments show that this polysaccharide possesses significant immunomodulatory and antitumor activities, particularly at a concentration of 20 μg / mL, where it exhibits the highest proliferation rate in RAW264.7 cells; and at a concentration of 10 μg / mL, it shows the highest inhibition rate in MFC cells. Based on this, the polysaccharide can be used to prepare drugs, as well as health products and foods that enhance immunity, demonstrating promising application prospects and commercial value. Attached Figure Description

[0079] Figure 1 The image shown is the GPC spectrum of LE-P.

[0080] Figure 2 The image shown is the FT-IR spectrum of LE-P.

[0081] Figure 3The HPLC chromatogram of LE-P hydrolysate is shown, where peak 1 is arabinose, peak 2 is glucose, and peak 3 is galactose.

[0082] Figure 4 The image shown is of LE-P. 1 H NMR spectrum.

[0083] Figure 5 The image shown is of LE-P. 13 C10 NMR spectrum.

[0084] Figure 6 The image shown is of LE-P. 1 H- 1 H-COSY spectrum.

[0085] Figure 7 The image shows the HMQC spectrum of LE-P.

[0086] Figure 8 The image shown is the HMBC spectrum of LE-P.

[0087] Figure 9 The chemical structure of LE-P is shown.

[0088] Figure 10 The results show the effects of LE-P on MFC cell proliferation.

[0089] Figure 11 The results show the effects of LE-P on the MFC cytoskeleton.

[0090] Figure 12 The results show the effects of LE-P on the proliferation of RAW264.7 cells. Detailed Implementation

[0091] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0092] In this invention, "Lentinus edodes" belongs to the class Basidiomycetes, order Agaricales, family Stomataceae, and genus Lentinus.

[0093] The term "tumor" refers to an abnormal mass of tissue in which the growth of the mass exceeds and is not coordinated with the growth of normal tissue. Tumors can be "benign" or "malignant," depending on the following characteristics: degree of cellular differentiation (including morphology and function), rate of growth, local invasion, and metastasis. "Benign tumors" are generally well-differentiated, characterized by slower growth than malignant tumors, and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors can later give rise to malignant tumors, possibly due to additional genetic changes in a subpopulation of neoplastic cells of the tumor, and these tumors are referred to as "pre-malignant tumors." "Malignant tumors" are generally poorly differentiated (anaplastic), and are characterized by rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissue. In addition, malignant tumors generally have the ability to metastasize to distant sites.

[0094] The term "cancer" refers to a malignant tumor (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).

[0095] The disclosures of various publications, patents and published patent specifications, referred to herein are hereby incorporated by reference in their entirety.

[0096] 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 are within the scope of protection of the present application.

[0097] Example 1: Isolation and extraction of polysaccharide LE-P from Coriolopsis floccosa

[0098] 1. Isolation and extraction of polysaccharide LE-P from Coriolopsis floccosa

[0099] 1.1. Extraction of crude polysaccharide from Coriolopsis floccosa by water extraction and alcohol precipitation

[0100] Take 200 g of dried Coriolopsis floccosa (collected from Kunming, Yunnan, identified as Coriolopsis floccosa by morphological observation and ITS sequencing) and crush it. Add the crushed Coriolopsis floccosa fruiting body to distilled water in a beaker at a ratio of 1:3 of solid to liquid, and heat 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 it, collect the precipitate and dry it, and remove the protein in the extraction solution to obtain the crude polysaccharide of Coriolopsis floccosa.

[0101] 1.2. Isolation and purification of crude polysaccharide from Coriaria sinica by DEAE-cellulose column chromatography

[0102] 50 g of DEAE-cellulose was accurately weighed and dissolved in 1 L of ultrapure water with thorough stirring. If no cellulose particles were visible to the naked eye, stirring was stopped. After standing for 24 h, the supernatant was discarded and prepared for use. 0.5 mol / L NaOH was prepared, and the cellulose was soaked for 6 h. After washing with ultrapure water until neutral, the supernatant was discarded. Then, 0.5 mol / L HCl was added and soaked for 6 h. After washing with distilled water until neutral, the supernatant was discarded. Then, 0.5 mol / L NaOH was added again and soaked for 6 h. After washing with distilled water until neutral, it was left standing for use.

[0103] After the activated cellulose was packed into a column, it was equilibrated with distilled water for 24 h before the isolation and purification of crude polysaccharide. The supernatant (5 mL) of the diluted crude polysaccharide was added to the DEAE-cellulose column, and different concentrations of NaCl (0.01 mol / L, 0.05 mol / L, 0.1 mol / L) were added for elution. The polysaccharide was determined by the sulfuric acid-phenol method. The eluate was concentrated to 5 mL, and the sample was purified on a cellulose column. Dialysis was performed in a dialysis bag (Mw≥7 kDa) for 48 h, and freeze-drying was performed to obtain Coriaria sinica polysaccharide, which was named LE-P.

[0104] 2. Structural identification of Coriaria sinica polysaccharide LE-P

[0105] Acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance techniques were used to analyze the structure of Coriaria sinica polysaccharide (LE-P).

[0106] 2.1. Determination of molecular weight

[0107] 10 mg of Coriaria sinica polysaccharide LE-P sample was dissolved in 1 mL of ddH2O and ultrasonicated for 5 min for GPC analysis.

[0108] 2.2. Infrared spectroscopy analysis of Coriaria sinica polysaccharide LE-P

[0109] 2 mg of LE-P was mixed with KBr and pressed into a tablet, which was scanned by an infrared spectrophotometer in the range of 4000 cm -1 - 500 cm -1 .

[0110] 2.3. Analysis of monosaccharide composition of Coriaria sinica polysaccharide LE-P

[0111] Six standard samples (fructose, rhamnose, arabinose, mannose, glucose, and galactose) (purchased from Chengdu Manster Biological Technology Co., Ltd.) and LE-P sample after TFA acid hydrolysis were dissolved with a mobile phase (80% acetonitrile) for HPLC analysis.

[0112] 2.4. NMR analysis of polysaccharide LE-P from Coriaria sinica

[0113] 50 mg of LE-P sample was dissolved in 0.6 mL of heavy water (D2O) and loaded into a NMR tube for detection on a NMR spectrometer.

[0114] 2.5. GC-MS analysis of polysaccharide LE-P from Coriaria sinica after methylation and silylation derivatization

[0115] 20 mg of LE-P sample was weighed and the beaker was sealed. 2 mL of DMSO (dimethyl sulfoxide) was added to the sealed beaker, and the beaker was gently shaken to fully dissolve the LE-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. 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 stop the reaction. The product was extracted with chloroform, and after drying, the methylated polysaccharide was obtained. After complete acid hydrolysis of the methylated polysaccharide by TFA, it was washed with water three times to obtain the completely acid hydrolyzed product of the methylated polysaccharide.

[0116] 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 and a temperature of 4°C for 10 min, and the precipitate was discarded. A 0.22 μm filter was used to filter, and the upper layer solution was used for GC-MS analysis.

[0117] 3. Results

[0118] 3.1. Basic property results of polysaccharide LE-P from Coriaria sinica

[0119] The GPC spectrum of LE-P is shown in Figure 1 , which shows that the weight average molecular weight of LE-P is 13541 Da.

[0120] 3.2. FT-IR spectrum analysis of polysaccharide LE-P from Coriaria sinica

[0121] The primary structure of LE-P was characterized by Fourier infrared spectroscopy, and the results are shown in Figure 2 . There are typical polysaccharide absorption peaks at wave numbers of 3403 cm -1 , 2938 cm -1 , 1682 cm -1 , and 1400-1200 cm -1 , etc., and no other impurity peaks, indicating that the purified LE-P is a polysaccharide substance. The wide absorption peak of O-H at a wave number of 3403 cm -1 , 2938 cm -1stretching vibration peak of C-H, 1682 cm -1 is C=0 stretching vibration peak, 1405 cm -1 is C-H in-plane bending vibration peak of -CHO. In 1200-1000 cm -1 range, the absorption peak is the absorption peak produced by the absorption of pyranose ring lactone and hydroxyl, indicating that LE-P has a pyran ring. In 800 cm -1 There is an absorption peak to prove that LE-P has a pyranose ring structure. In addition, there is no absorption peak near 1730 cm -1 , indicating that LE-P does not contain uronic acid.

[0122] 3.3, monosaccharide composition analysis of Coriaria polysaccharide LE-P

[0123] After complete hydrolysis of LE-P, its monosaccharide composition was analyzed by HPLC, and the results are shown in Figure 3 , wherein peak 1 is arabinose (Ara) with a retention time of 8.191 min; wherein peak 2 is glucose (Glc) with a retention time of 8.794 min; peak 3 is galactose (Gal) with a retention time of 9.322 min. And the ratio of arabinose, glucose and galactose is 1:6:6.

[0124] 3.4, NMR spectrum analysis of Coriaria polysaccharide LE-P

[0125] The 1 H NMR results of LE-P are shown in Figure 4 . The results show that LE-P has seven anomeric hydrogen signals, which are δ5.22ppm, δ4.99ppm, δ4.93ppm, δ4.92ppm, δ4.87ppm, δ4.84ppm and δ4.65ppm, and the integral area ratio is 0.58:1.04:1.00:0.92:0.99:1.20:1.07. The signals between δ3.0-4.2ppm are attributed to the hydrogen signals of C2-C6 in the sugar residue.

[0126] The 13 C NMR results of LE-P are shown in Figure 5 . LE-P has seven anomeric carbon signals at δ99.33ppm, δ98.04ppm, δ101.49ppm, δ97.90ppm, δ97.91ppm, δ97.87ppm and δ101.65ppm. The signals between δ60-85ppm are attributed to the carbon signals of C2-C6 in the sugar residue.

[0127] The 1 H- 1 H-COSY spectrum of LE-P is shown in Figure 6As shown, the coupling relationship between adjacent hydrogen nuclei can be identified. The H1 / H2 signal for part A is δ 5.22 / 3.55, for part B it is δ 4.99 / 3.81, for part C it is δ 4.93 / 101.49, for part D it is δ 4.92 / 3.65, for part E it is δ 4.87 / 3.71, for part F it is δ 4.84 / 3.72, and for part G it is δ 4.65 / 3.95.

[0128] All the chemical shifts of hydrogen are summarized in Table 1.

[0129] The HMQC spectrum of LE-P is as follows: Figure 7 As shown, the coupling relationship between the short-range correlated 1H and 13C can be identified. The H1 / C1 signal is δ5.22 / 99.33 in part A, δ4.99 / 98.04 in part B, δ4.93 / 101.49 in part C, δ4.92 / 97.90 in part D, δ4.87 / 97.91 in part E, δ4.84 / 97.87 in part F, and δ4.65 / 101.65 in part G.

[0130] The HMBC spectrum of LE-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: H1 / C5 signal of residue A is δ5.22 / 79.90, H1 / C6 signal of residue B is δ4.99 / 68.46, H5 / C3 signal of residue C is δ3.84 / 68.51, H2 / C5 signal of residue D is δ3.65 / 69.84, H5 / C3 signal of residue E is δ3.89 / 68.52, H1 / C5 signal of residue F is δ4.84 / 69.37, and H1 / C5 signal of residue G is δ4.65 / 76.86.

[0131] All the chemical shifts of carbon are summarized in Table 2.

[0132] Table 1 LE-P 1 Chemical shift of H

[0133]

[0134] Table 2 LE-P 13 Chemical shift of C

[0135]

[0136] 3.5. Gas chromatography and mass spectrometry analysis of polysaccharide LE-P of Coriaria sinica

[0137] The methylation results are shown in Table 3, which indicates that the main repeating structural unit of LE-P is composed of a main chain of 1,6-linked α-D-glucose residues, 1,4-linked β-D-glucose residues, 1,6-linked α-D-galactose residues, 1,4-linked α-D-galactose residues and 1,4,6-linked β-D-glucose residues, and a side chain of 1,4-linked β-D-arabinose residues and 1-linked α-D-galactose residues. Based on the above, it can be preliminarily inferred that the structure of LE-P is as shown in Figure 9 .

[0138] Table 3 Analysis of LE-P methylation results

[0139]

[0140]

[0141] Example 2: Anti-tumor and immunomodulatory activity of polysaccharide LE-P of Coriaria sinica

[0142] The anti-tumor and immunomodulatory activity of polysaccharide LE-P of Coriaria sinica was determined in vitro using two CCK-8 methods.

[0143] 1. Reagents

[0144] The CCK-8 kit, RPIM1640, FBS, DMSO, and double antibodies were all commercially available products.

[0145] Man (mannan peptide) was purchased from Chengdu Li'er Pharmaceutical Co., Ltd.

[0146] The cytoskeleton (actin microfilament, F-actin) green fluorescent staining kit was purchased from Shanghai Jimei Gene Pharmaceutical Technology Co., Ltd.

[0147] 2. Instruments

[0148] Enzyme label instrument; cell incubator; SP8 laser scanning confocal microscope.

[0149] 3. Method for detecting the effect of LE-P on the proliferation of RAW264.7 cells and MFC cells

[0150] The effect of polysaccharide LE-P on the proliferation of RAW264.7 cells and MFC cells was determined by the cell counting kit (CCK-8) method. RAW264.7 cells and 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 CO2incubator for 24 h. After 24 h, different mass concentrations of LE-P solution (5, 10, and 20 μg / mL) were added to the experimental groups, respectively. The positive control group (LPS group) of the RAW264.7 cell proliferation experiment was added with 100 μL of LPS (10 μg / mL in mass concentration), the positive control group (Man group) of the MFC cell proliferation experiment was added with 100 μL of Man (10 μg / mL in mass concentration), and the blank group was added with 100 μL of cell culture solution. After 24 h of incubation in a CO2incubator, 5 μL of CCK-8 was added to each well, and the absorbance value was detected (450 nm) on an enzyme label instrument after 3 h of incubation in a CO2incubator, and an image was taken. Finally, the cell proliferation rate or inhibition rate was calculated by the equation as follows:

[0151]

[0152] In the formula, 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.

[0153] 4. Method for detecting the effect of LE-P on the MFC cell skeleton

[0154] The mouse gastric cancer cells MFC were cultured with equal amounts of culture solution, LE-P solution (10 μg / mL), and MAN solution (10 μg / mL), respectively, and the blank control group, LE-P experimental group (LE-P 10 μg / mL), and positive control group (MAN 10 μg / mL, MAN group) were set. The cell skeleton experiment was performed by using a cell skeleton green fluorescent staining kit. The staining working solution was prepared and placed in an ice tank in a dark room for standby. The cells to be tested were inoculated and cultured until the plating rate reached 70% per well. According to the operation steps provided by the kit, the cells were treated by adding reagents to allow sufficient staining. After mounting, observation was performed immediately under a laser scanning confocal microscope under the conditions of excitation wavelength 488 nm and emission wavelength 530 nm, F-actin showed green fluorescence, and the fluorescence intensity was counted by using Fiji software.

[0155] 5. Results

[0156] 5.1. Effect of LE-P on the proliferation of MFC cells

[0157] The results are as follows: Figure 10As shown, compared with the blank group, the Man group significantly (P<0.05) inhibited the proliferation of MFC cells, and the inhibition was 37.16%; when the mass concentration of LE-P was 10 and 20 μg / mL, LE-P could extremely significantly (P<0.01) inhibit the proliferation of MFC cells; when the mass concentration of LE-P was 10 μg / mL, LE-P had the most obvious inhibitory effect on MFC cells, and the maximum inhibition rate reached 42.24%.

[0158] 5.2, Effect of LE-P on the skeleton of MFC cells

[0159] As shown in the results, Figure 11 compared with the blank group, the fluorescence intensity of the MAN group (10 μg / mL) and the LE-P group (10 μg / mL) was extremely significantly lower than that of the blank control group (P<0.01); among them, the fluorescence intensity of the MAN group was the lowest, which was 849.4 a.u., and the fluorescence intensity of the LE-P group was 2015.0 a.u. By comparing the cell map of the blank group, it can be seen that the uniform network structure of actin in the MFC cells of the MAN group and the LE-P group was destroyed, and the actin in the cells was unevenly distributed in the form of dots, indicating that LE-P and MAN can promote the breaking of F-actin filaments in MFC cells, thereby inhibiting the normal growth of MFC.

[0160] 5.3, Effect of LE-P on the proliferation of RAW264.7 cells

[0161] As shown in the results, Figure 12 compared with the blank group, the LPS group could extremely significantly (P<0.01) promote the proliferation of RAW264.7 cells, and the proliferation rate was 84.71%; when the mass concentration of LE-P was 5, 10 and 20 μg / mL, there was a trend that the higher the concentration, the stronger the effect of promoting the proliferation of RAW264.7 cells; when the mass concentration of LE-P was 10 and 20 μg / mL, LE-P extremely significantly (P<0.01) promoted the proliferation of RAW264.7 cells, and when the mass concentration of LE-P was 20 μg / mL, LE-P had the most obvious effect on the proliferation of RAW264.7 cells, and the maximum proliferation rate reached 35.14%.

[0162] The above only describes the 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 should be included in the protection scope of the present application.

[0163] 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.

[0164] In the present application, the steps of the method are only listed in a certain order, which does not constitute any limitation on the order of the steps of the method.

Claims

1. A Coriolus versicolor polysaccharide, said Coriolus versicolor polysaccharide being a heteropolysaccharide consisting of β - D - arabinose, α - D - glucose, β - D - glucose, α - D - galactose in a molar ratio of 1 :2:4:6, comprising the following structure: wherein n is an integer from 3 to 10; The weight average molecular weight of the polysaccharide of Coriolus versicolor is 8000-20000 Da.

2. A preparation method of the polysaccharide of Coriolus versicolor according to claim 1, comprising the following steps: (1) taking the powder of the fruiting body of Coriolus versicolor, extracting with water, and obtaining the 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 temperature of the extraction is 80-100℃; In step (1), the extraction is performed for 1-5 times, and each extraction is performed for 1-10 hours; In step (1), the solid-liquid ratio of the powder of the fruiting body of Coriolus versicolor to water is 1:1-10; In step (1), in the alcohol precipitation step, the volume ratio of alcohol to the concentrated solution of the water extract is 1-10:1; In step (2), the ion exchange column is a cellulose column, and the filler of the cellulose column is DEAE-cellulose; In step (2), the eluent used for the elution is a NaCl solution.

3. A composition comprising the polysaccharide of Coriolus versicolor according to claim 1.

4. Use of the polysaccharide of Coriolus versicolor according to claim 1 in the preparation of an antitumor drug, an immune enhancer, an immune adjuvant, and a health care product for enhancing immunity.

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