Beta-glucan composition and application thereof

Abstract

The invention relates to a beta-glucan composition and application thereof. Specifically, the invention provides an application of a beta-glucan composition in preparation of a composition for improving or treating immune-related diseases, the beta-glucan is beta-glucan of which the molecule contains beta-1, 3 and beta-1, 6 glucoside bonds, and the molecular weight of the beta-glucan is 1-50kDa. The beta-glucan has the advantages of being novel in structure, controllable in quality, rich in source, simple in preparation process, high in product purity, high in biological activity, easy to industrially produce and the like, and the obtained water-soluble beta-glucan composition has better anti-tumor activity and can be used for preparing medicines. The compound is expected to be developed into a novel safe and effective medicament for improving or treating immune-related diseases and resisting tumors.

Description

[0001] This application is a divisional application of the invention patent application with application date of June 15, 2020, application number 202080043546.9, and invention name “A β-glucan composition and its use”. Technical Field

[0002] The present invention relates to the field of medicines, and in particular to a beta-glucan composition and use thereof. Technical Background

[0003] Studies have shown that β-1,3-glucans from different sources have different biological activities, including anti-tumor, immunomodulatory, anti-aging and anti-inflammatory properties. At present, β-1,3-glucans on the market mostly come from terrestrial organisms such as barley, oats, edible fungi (shiitake mushrooms, maitake mushrooms, schizophyllum), yeast, etc. Due to the different sources of raw materials, the molecular weight, connection mode and branching degree of the obtained β-1,3-glucans vary greatly, and the quality is difficult to control. For example, the β-glucan for injection is mainly derived from shiitake mushrooms. It is a β-1,6-branched β-1,3-glucan (LNT). Due to its high molecular weight of 400-800kDa, it has poor water solubility, is difficult to separate and purify, and has a high impurity content. With the continuous expansion of the application field of β-glucan and the continuous growth of market demand, the existing shiitake mushroom β-glucan can no longer meet the needs of the market.

[0004] Therefore, there is an urgent need in this field to develop β-glucan with controllable quality, abundant sources, simple preparation process, high product purity, strong biological activity and easy industrial production. Summary of the invention

[0005] The purpose of the present invention is to analyze the specific structure of a β-1,3 / 1,6-glucan and the application of the composition in the preparation of a composition for treating immune-related diseases.

[0006] In a first aspect of the present invention, a β-1,3 / 1,6-glucan composition is provided, wherein the composition comprises β-glucan having a structure represented by formula (I) and / or formula (II),

[0007]

[0008]

[0009] wherein n is an integer selected from 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and R is H and / or no more than 4 glucose residues (e.g., 1, 2, 3 or 4 glucose residues).

[0010] Preferably, R in the structure of formula (I) or formula (II) is one or more of the structures of formula (III) or formula (IV) or formula (V) or formula (VI), wherein

[0011] Formula (III): Glcβ1-;

[0012] Formula (IV): Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-;

[0013] Formula (V): Glcβ1-3Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-3Glcβ1- or

[0014] Glcβ1-3Glcβ1-6Glcβ1- or Glcβ1-6Glcβ1-6Glcβ1-;

[0015] Formula (VI):

[0016] Glcβ1-3Glcβ1-3Glcβ1-3Glcβ1-or

[0017] Glcβ1-6Glcβ1-3Glcβ1-3Glcβ1-or

[0018] Glcβ1-3Glcβ1-6Glcβ1-3Glcβ1- or

[0019] Glcβ1-3Glcβ1-3Glcβ1-6Glcβ1- or

[0020] Glcβ1-6Glcβ1-6Glcβ1-3Glcβ1- or

[0021] Glcβ1-6Glcβ1-3Glcβ1-6Glcβ1-or

[0022] Glcβ1-3Glcβ1-6Glcβ1-6Glcβ1-or

[0023] Glcβ1-6Glcβ1-6Glcβ1-6Glcβ-.

[0024] Preferably, in the β-glucan composition, the weight content of pentasaccharide to decasaccharide is 0-50.0%, the weight content of decasaccharide to eicosaccharide is 0-80.0%, the weight content of eicosaccharide to pentacosaccharide is 0-25.0%, the weight content of pentacosaccharide to triaconosaccharide is 0-45.0%, the weight content of triaconosaccharide to tetraconosaccharide is 0-30.0%, the weight content of tetraconosaccharide to pentaconosaccharide is 0-15.0%, the weight content of pentaconosaccharide to hexasaccharide is 0.1%-55.0%, the weight content of hexasaccharide to septasaccharide is 0.1%-20.0%, the weight content of seventy-octaconosaccharide is 0.1%-15.0%, and the weight content of more than eighty-saccharide is 0-40.0%.

[0025] Preferably, in the β-glucan composition, the weight content of pentasaccharide to decasaccharide is 0-45.0%, the weight content of decasaccharide to eicosaccharide is 0-75.0%, the weight content of eicosaccharide to pentacosaccharide is 0-20.0%, the weight content of pentacosaccharide to triacontaosaccharide is 0-40.0%, the weight content of β-glucan with a degree of polymerization of 30-40 is 0-25.0%, the weight content of tetrasaccharide to pentacontaosaccharide is 0-10.0%, the weight content of pentacontaosaccharide to hexosacharide is 0.1%-50.0%, the weight content of hexosacharide to septosacharide is 0.1%-15.0%, the weight content of seventy-septosaccharide to octacontaosaccharide is 0.1%-10.0%, and the weight content of greater than eighty-saccharide is 0-35.0%.

[0026] Preferably, the β-glucan composition is composed of β-glucan with a degree of polymerization of 20-80, wherein the weight content of eicosaccharide-pentacosaccharide is 13.2%-19.8%, the weight content of pentacosaccharide-triaconose is 29.1-43.7%, the weight content of triaconose-tetraconose is 18.2-27.4%, the weight content of tetraconose-pentaconose is 7.3-10.9%, the weight content of pentaconose-sexuconose is 4.5%-6.7%, the weight content of hexaconose-septuconose is 3.5%-5.3%, and the weight content of seventy-septuconose is 4.2%-6.2%.

[0027] Preferably, the β-glucan composition is composed of β-glucan with a degree of polymerization of 20-80, wherein the weight content of eicosaccharide-pentacosaccharide is 16.5%, the weight content of pentacosaccharide-triaconose is 36.4%, the weight content of triaconose-tetraconose is 22.8%, the weight content of tetraconose-pentaconose is 9.1%, the weight content of pentaconose-sexuconose is 5.6%, the weight content of hexicosaccharide-septicosaccharide is 4.4%, and the weight content of seventy-septicosaccharide is 5.2%.

[0028] Preferably, the β-glucan composition is composed of β-glucan with a degree of polymerization of 10-80, wherein the weight content of decasaccharide to eicosaccharide is 40.6%-60.8%, the weight content of eicosaccharide to pentacosaccharide is 13.4%-20.0%, the weight content of pentacosaccharide to triaconosaccharide is 7.7%-11.5%, the weight content of triaconosaccharide to tetraconosaccharide is 7.6%-11.4%, the weight content of tetraconosaccharide to pentaconosaccharide is 4.2%-6.2%, the weight content of pentaconosaccharide to hexagonosaccharide is 2.3%-3.5%, the weight content of hexagonosaccharide to septagonosaccharide is 1.6%-2.4%, and the weight content of seventy-septagonosaccharide is 0.8%-1.2%.

[0029] Preferably, the β-glucan composition is composed of β-glucan with a degree of polymerization of 10-80, wherein the weight content of decasaccharide to eicosaccharide is 50.7%, the weight content of eicosaccharide to pentacosaccharide is 16.7%, the weight content of pentacosaccharide to triaconosaccharide is 9.6%, the weight content of triaconosaccharide to tetraconosaccharide is 9.5%, the weight content of tetraconosaccharide to fiscoconosaccharide is 5.2%, the weight content of pentosaccharide to hexicosaccharide is 2.9%, the weight content of hexicosaccharide to heptocosaccharide is 2.0%, and the weight content of seventy-septicosaccharide to eighty-saccharide is 1.0%.

[0030] Preferably, the molecular weight of the β-1,3 / 1,6-glucan is 1-50 kDa; more preferably, 2-30 kDa; more preferably, 2-10 kDa.

[0031] Preferably, the specific rotation of the β-1,3 / 1,6-glucan is not less than -15.0°; more preferably, -15° to -25°; more preferably, -16° to 21°.

[0032] Preferably, the sulfate content in the β-1,3 / 1,6-glucan is 0.01wt% to 2wt%; more preferably, 0.01wt% to 0.5wt%.

[0033] Preferably, the chloride ion content of the β-1,3 / 1,6-glucan is 0.01wt% to 2wt%; more preferably, 0.01wt% to 0.5wt%.

[0034] Preferably, the protein content of the β-1,3 / 1,6-glucan is 0.01wt% to 5wt%; more preferably, 0.01wt% to 0.5wt%.

[0035] Preferably, the ultraviolet full-wavelength scanning spectrum of the β-1,3 / 1,6-glucan has no obvious absorption in the wavelength range of 300 to 900 nm; more preferably, there is no obvious absorption in the wavelength range of 230 to 900 nm.

[0036] Preferably, the ultraviolet full-wavelength scanning spectrum of the β-1,3 / 1,6-glucan has no absorption peak in the wavelength range of 260 to 280 nm.

[0037] Preferably, among the side chains of the β-1,3 / 1,6-glucan, at least 20% of the side chains have a length of 1 or 2 glucose residues.

[0038] Preferably, in the β-glucan having the structure represented by formula (I) or formula (II), at least 3 to 20 Rs are independently 1 or 2 glucose residues.

[0039] Preferably, in the β-glucan of formula (I) or (II), at least 3 to 10 Rs are independently of each other represented by formula (III) or (IV); wherein the structures of formula (III) and (IV) are as defined above.

[0040] Preferably, in the side chains of the β-1,3 / 1,6-glucan (when R≠H), at least 5% of the side chains (R) are 3 or 4 glucose residues in length (preferably, 5-15%, more preferably 5-10% of the side chains are 3 or 4 glucose residues in length), and the remaining side chains (R) are 1 or 2 glucose residues in length.

[0041] Preferably, when the side chain (R) is 1 or 2 glucose residues in length, the side chain (R) is independently of each other a structure of formula (III) and formula (IV), wherein the structures of formula (III) and formula (IV) are as defined above.

[0042] Preferably, when the side chain (R) is 3 or 4 glucose residues in length, each side chain (R) is independently a structure of formula (V) or formula (VI), wherein the structure of formula (V) or formula (VI) is as defined above.

[0043] In the second aspect of the present invention, there is provided a use of the β-glucan as described in the first aspect or the β-1,3 / 1,6-glucan composition as described in the first aspect in the preparation of a composition for improving or treating immune-related diseases.

[0044] Preferably, the immune-related disease is tumor or inflammation.

[0045] Preferably, the tumor is selected from colorectal cancer, lung cancer, and fibrosarcoma.

[0046] In the third aspect of the present invention, a composition for improving or treating immune-related diseases is provided, the composition comprising:

[0047] (1) the β-glucan as described in the first aspect or the β-1,3 / 1,6-glucan combination as described in the first aspect, and

[0048] (2) A pharmaceutically acceptable carrier.

[0049] In a fourth aspect of the present invention, there is provided a method for preparing the β-1,3 / 1,6-glucan as described in the first aspect or the β-1,3 / 1,6-glucan composition as described in the first aspect, comprising the steps of:

[0050] (1) Defatting: drying and crushing the Antarctic brown algae, soaking and stirring with an organic solvent to obtain defatted algae powder;

[0051] (2) water extraction: the defatted algae powder is extracted with water at room temperature to obtain a water extract;

[0052] (3) Classification: centrifuge the aqueous extract obtained in step (2), add 1-3 mol / L calcium chloride aqueous solution to the supernatant obtained by centrifugation; stir and centrifuge, take the supernatant for dialysis or ultrafiltration desalination, and concentrate and dry under reduced pressure to obtain crude polysaccharide;

[0053] (4) Purification: The crude polysaccharide of step (3) is dissolved in distilled water, and separated and purified by anion exchange resin using distilled water and sodium chloride aqueous solution as mobile phases, and the water elution fraction is collected, concentrated under reduced pressure, and freeze-dried to obtain the β-1,3 / 1,6-glucan.

[0054] In another preferred embodiment, the anion resin separation and purification is a strong anion resin separation and purification.

[0055] In another preferred embodiment, the anion resin separation and purification is: first separation and purification by strong anion resin and then separation and purification by weak anion resin; or, first separation and purification by weak anion resin and then separation and purification by strong anion resin. By combining weak anion resin and strong anion resin, unexpectedly, impurities can be removed more effectively and β-1,3 / 1,6-glucan with longer side chain glucose residues, higher purity and triple helix structure can be obtained.

[0056] Preferably, the strong anion resin is an anion resin containing a quaternary ammonium group.

[0057] Preferably, the weak anion resin is an anion resin containing diethylaminoethyl.

[0058] Preferably, the Antarctic brown algae is Durvillaea Antarctica, Durvillaea Antarctica and / or Durvillaea Antarctica.

[0059] In a fifth aspect of the present invention, there is provided use of the β-1,3 / 1,6-glucan composition as described in the first aspect in combination with an immune checkpoint drug and / or a chemotherapeutic agent.

[0060] Preferably, the immune checkpoint drug is selected from: programmed death 1 protein (PD-1) antagonist, PD-L1 antagonist, cytotoxic T lymphocyte antigen (CTLA-4) antagonist, lymphocyte activation gene-3 (LAG-3) antagonist, T cell immunoglobulin-3 (TIM-3) antagonist, T cell immunoglobulin, ITIM domain protein (TIGIT) antagonist, or a combination thereof.

[0061] Preferably, the immune checkpoint drug is selected from anti-PD-1 antibody and anti-PD-L1 antibody.

[0062] Preferably, the anti-PD-1 antibody or PD-L1 antibody is selected from Durvalumab, Atezolizumab, Nivolumab, BMS202, Spartalizumab, Camrelizumab, or a combination thereof.

[0063] Preferably, the chemotherapeutic agent is selected from cytotoxic chemotherapeutic agents.

[0064] Preferably, the chemotherapeutic agent is selected from anthracyclines, 5-Fu and alkaloids.

[0065] Preferably, the chemotherapeutic agent is selected from one or more of cisplatin and carboplatin.

[0066] Preferably, the drug or preparation is administered simultaneously, sequentially or separately in combination with an immune checkpoint drug and / or a chemotherapeutic agent.

[0067] In a sixth aspect of the present invention, a drug combination is provided, comprising:

[0068] (i) First active ingredient: The first active ingredient is the β-1,3 / 1,6-glucan composition as described in the first aspect;

[0069] (ii) a second active ingredient; the second active ingredient comprises an immune checkpoint drug and / or a chemotherapeutic agent.

[0070] Preferably, the first active ingredient and the second active ingredient are in a single dosage form or in separate dosage forms.

[0071] Preferably, the immune checkpoint drug is as described above.

[0072] Preferably, the chemotherapeutic agent is as described above.

[0073] In the seventh aspect of the present invention, there is provided a use of the 1,3 / 1,6-glucan composition as described in the first aspect in the preparation of a medicament or preparation for treating leukopenia and / or thrombocytopenia.

[0074] Preferably, the white blood cells are lymphocytes.

[0075] Preferably, the lymphocytes are B cells and / or T cells.

[0076] Preferably, the drug or preparation can also be used in combination with an immune checkpoint drug.

[0077] Preferably, the drug or preparation is used in combination with the immune checkpoint drug for simultaneous, sequential or separate administration.

[0078] Preferably, the drug or preparation can also be used in combination with at least one chemotherapeutic agent.

[0079] Preferably, the drug or formulation is used in combination with a chemotherapeutic agent for simultaneous, sequential or separate administration.

[0080] Preferably, the medicament or formulation is for treating cancer in an individual.

[0081] Preferably, the cancer is one or more of melanoma, colorectal cancer, lung cancer, kidney cancer, liver cancer, and breast cancer.

[0082] Preferably, the medicine or preparation further comprises a pharmaceutically acceptable carrier or excipient.

[0083] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The ESI-CID-MS / MS spectrum is shown when the side chain is Glcβ1-6Glcβ in formula (IV).

[0085] Figure 2 The ESI-CID-MS / MS spectrum is shown when the side chain is Glcβ1-3Glcβ in formula (IV).

[0086] Figure 3 The ESI-CID-MS / MS spectrum is shown when the side chain is the structure in formula (V).

[0087] Figure 4 The ESI-CID-MS / MS spectrum is shown when the side chain is the structure in formula (VI).

[0088] Figure 5 The weight average molecular weight determination for Example 2 is shown.

[0089] Figure 6 The weight average molecular weight determination for Example 7 is shown.

[0090] Figure 7 The degree of polymerization distribution analysis of Example 7 is shown.

[0091] Figure 8 It was shown that the β-glucan composition can shift the maximum absorption wavelength of Congo red.

[0092] Fig. 9 It is shown that when the β-glucan composition has the structure of formula (II) 13 C-NMR spectrum.

[0093] Fig.10 It shows that β-1,3 / 1,6-glucan has a higher affinity for monocytes than for granulocytes. (AC) Flow cytometry of lymphocytes (red dots CD11b-), monocytes (green dots CD11b+Ly6Chi), and granulocytes (blue dots CD11b+Ly6G+). (D) Blood cells were incubated with β-1,3 / 1,6-glucan-FITC (200 μg / mL) at 37°C for 2 hours. Subsequently, red blood cells were lysed and the cells were stained with the corresponding flow antibodies. Flow cytometry was used to detect the binding affinity of β-1,3 / 1,6-glucan-FITC to monocytes (green, CD11b+) and granulocytes (blue, CD11b+).

[0094] Fig.11 It is shown that the β-1,3 / 1,6-glucan of the present invention increases the phagocytic activity of BMDMs without cytotoxicity. (A and B) GM-BMDM (A) and M-BMDM (B) were incubated with β-1,3 / 1,6-glucan or LNT for 24 hours. The phagocytic activity of the two macrophages was detected by the neutral red method. The cell viability was detected by the MTT method (C, D).

[0095] Fig.12 It is shown that the β-1,3 / 1,6-glucan of the present invention reduces tumor burden and improves spleen index in a DLD1 xenograft mouse model. Mice implanted with DLD1 tumors were treated with vehicle or β-1,3 / 1,6-glucan of the present invention. Tumor volume (A) and body weight (E) are shown. Tumors were excised, photographed (B), weighed (C), and spleen index calculated (D).

[0096] Fig.13It is shown that the β-1,3 / 1,6-glucan of the present invention upregulates macrophage phagocytic activity and pro-inflammatory cytokine secretion in the DLD1 xenograft model. (A) Detection of phagocytosis of 9 colorectal cancer cell lines (HCT-116, LS174T, SW480, DLD1, HT-29, LS180, HCT-15, LOVO, T84) by peritoneal macrophages of mice treated with vehicle or β-1,3 / 1,6-glucan of the present invention. (BG) The levels of plasma pro-inflammatory cytokines and chemokines in mice in the drug-loaded group and the β-1,3 / 1,6-glucan group of the present invention were detected using the V-PLEX mouse inflammatory factor kit (LabEx).

[0097] Fig.14 It is shown that the β-1,3 / 1,6-glucan of the present invention increases pro-inflammatory macrophages and B cells in the tumor microenvironment. (A) Flow cytometry was used to detect the ratio of CD11b+, CD335+, CD19+, and CD11c+ cells in the blood. (B) The ratio of blood neutrophils (CD11b+LY6G+) and pro-inflammatory monocytes (CD11b+LY6Chi) was detected. (CF) The ratio of tumor-infiltrating CD11b+ cells, monocyte-derived infiltrating macrophages (CD11b+LY6Chi), TAMs (CD11b+CD80+ or ​​CD11b+CD206+), and CD19+ cells was detected. (G) The ratio of CD45+ leukocytes in tumors of mice treated with vehicle and β-1,3 / 1,6-glucan of the present invention (small tumor group and large tumor group) was measured.

[0098] Fig.15 The β-1,3 / 1,6-glucan of the present invention is shown to reduce the tumor burden of the AOM-DSS-induced colorectal cancer model. C57 mice were treated with untreated or AOM / DSS-induced groups, or β-1,3 / 1,6-glucan (1, 3 and 9 mg / kg) of the present invention after induction. (A) The body weight of each group after treatment was recorded. (B) The colon length was measured after sacrifice. (CE) The colon was longitudinally cut and the number (C and E) and diameter (D) of tumors were collected.

[0099] Fig.16It is shown that the β-1,3 / 1,6-glucan of the present invention reverses the changes in immune cell composition caused by the administration of AOM-DSS. (AD) The ratios of CD11b+, CD335+, CD19+, CD4+, CD8+, and CD11c+ in the blood (A), the ratio of CD19+ in the spleen (B), and the ratio of CD4+ and CD8+ in the lymph nodes (C) were detected. (E, F, G, H, I) The levels of proinflammatory cytokines and chemokines in the plasma of normal or AOM / DSS mice treated with or without the β-1,3 / 1,6-glucan of the present invention were respectively detected.

[0100] Fig.17 A and B show the full-wavelength ultraviolet scanning spectra of β-1,3 / 1,6-glucan prepared according to the methods of Preparation Example 1 and Preparation Example 2, respectively.

[0101] Fig.18 A and B show the experimental results of Example 21. (A) shows the average tumor weight and inhibition rate in mice after administration, and (B) shows the gland weight in mice after administration.

[0102] Fig.19 A and B show the experimental results of Example 22. (A) shows the anti-tumor growth effect of β-1,3 / 1,6-glucan and its combination with cisplatin, and (B) shows the inhibition of male Kunming mouse weight.

[0103] Fig. 20 AC shows that intravenous injection of β-1,3 / 1,6-glucan combined with PD-1 antibody in Example 25(i) can effectively inhibit the growth of subcutaneous transplanted tumors in mouse colon cancer MC38 mice; in the figure, PD1-200(qw) represents the group administered with PD-1 antibody alone (200 μg / mouse), PD1-200(qw)+BG0.3(biw), PD1-200(qw)+BG1(biw) and PD1-200(qw)+BG3(biw) represent the groups of PD1 antibody (200 μg / mouse) combined with β-1,3 / 1,6-glucan (0.3, 1 or 1 mg / kg), respectively.

[0104] Fig.21 AF shows the effect of intravenous injection of β-1,3 / 1,6-glucan combined with PD-1 antibody on the expression of immune-related cytokines in mouse colon cancer MC3 mouse transplanted tumors.

[0105] Fig. 22 AC shows the effect of oral administration of β-1,3 / 1,6-glucan combined with PD-1 antibody on the growth of mouse colon cancer MC38 mouse xenografts. DETAILED DESCRIPTION

[0106] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. After reading the specific embodiments of the present invention in conjunction with the accompanying drawings, other advantages and features of the present invention will become clearer, but the scope of protection claimed in the present invention is not limited to the scope of the embodiments. Those skilled in the art can make some modifications and adjustments to the present invention based on the essence of the following disclosure, and these adjustments also belong to the scope of the present invention.

[0107] the term

[0108] Unless otherwise defined, the following terms used in the specification and claims have the meanings commonly understood by those skilled in the art. Unless otherwise indicated, all patents, patent applications, and public materials cited in this article are incorporated herein by reference in their entirety.

[0109] 1) Degree of Polymerization: dp refers to the number of repeating structural units in a polymer macromolecule. In carbohydrate compounds, the degree of polymerization generally refers to the number of monosaccharide residues in the compound.

[0110] 2) Polysaccharide: refers to a sugar chain formed by glycosidic bonds, which is composed of multiple monosaccharide residues condensed,

[0111] Polymeric carbohydrates formed by loss of water.

[0112] 3) Glucose: Glc; C6H 12 O6 is the most widely distributed and important monosaccharide in nature.

[0113] 4) Oligosaccharide: also known as oligosaccharide, is a carbohydrate compound formed by 2-20 identical or different monosaccharide residues connected by glycosidic bonds.

[0114] 5) Sugar residue: refers to the hydrolyzed group obtained after the hydrolysis of sugar substances.

[0115] 6) Gluco-oligosaccharides: oligosaccharides composed of glucose residues connected by glycosidic bonds.

[0116] 7) Glucan: A polysaccharide composed of glucose residues connected by glycosidic bonds.

[0117] 8) ESI: Electrospray ionization, a commonly used ionization method in mass spectrometry. 9) CID: Collision Induced Dissociation, a process in which energy is transferred to ions through collisions with neutral molecules. The energy transfer is sufficient to cause bond cleavage and rearrangement.

[0118] 10) MS: mass spectrometry, an analytical method that measures the charge-to-mass ratio of ions.

[0119] In the present invention, "β-1,3 / 1,6-glucan" and "β-glucan" can be used interchangeably, and their conceptual expressions have the same meaning.

[0120] In the present invention, "β-1,3 / 1,6-glucan" includes the structure of formula I, the structure of formula II, or a derivative form thereof, or a combination thereof. For example, in the β-1,3 / 1,6-glucan composition of the present invention, the structure of formula I may be 0-100%, and the balance is the structure of formula II; or the structure of formula II may be 0-100%, and the balance is the structure of formula I. In the present invention, the structure of formula II may be regarded as the ring-opened formula I. In addition, formula I and formula II may be mutually converted by appropriate conditions or reagents.

[0121] In another preferred embodiment, the β-1,3 / 1,6-glucan does not contain or substantially does not contain an absorption peak at 260-280 nm.

[0122] In another preferred embodiment, the optical rotation of the β-1,3 / 1,6-glucan is greater than -15°, such as between -15° and -25°, and preferably between -16° and -21°.

[0123] In this article, the terms "strong anion resin" and "strong anion exchange resin" are used interchangeably and refer to anion resins containing strong reactive groups such as quaternary amine groups. Generally, strong anion resins can be used to remove impurities with sulfonic acid groups, carboxyl groups, etc.

[0124] In this article, the terms "weak anion resin" and "weak anion exchange resin" can be used interchangeably to refer to anion resins containing weaker reactive groups such as diethylaminoethyl. Generally, weak anion resins can be used to remove impurities such as nucleic acids, proteins, pigments, etc.

[0125] The main advantages and beneficial effects of the present invention include:

[0126] The β-glucan composition of the present invention is a mixture of β-glucans with different polymerization degrees, different branches and side chains, the side chains are composed of β-1,3- and β-1,6-glucose, the side chain length does not exceed 4 sugar residues, the structure is novel, and the quality is controllable. The β-glucan composition obtained by the present invention has better anti-tumor activity and is expected to be developed into a new class of safe and effective anti-tumor drugs for improving or treating immune-related diseases.

[0127] In particular, the inventors further improved the preparation method of β-1,3 / 1,6-glucan. Through combined purification of strong anion chromatography purification and weak anion chromatography purification, β-1,3 / 1,6-glucan was obtained, which only had an absorption peak at the end in the ultraviolet full-wavelength scanning spectrum and had no obvious absorption peak in the range of 230-900nm (especially 260-280nm segment) and the optical rotation was further increased (such as the optical rotation in the range of -15° to -21°).

[0128] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0129] Example 1 Preparation of β-1,3 / 1,6-glucan

[0130] Preparation Example 1

[0131] (1) Defatting: drying and crushing the Antarctic brown algae, soaking and stirring with an organic solvent to obtain defatted algae powder;

[0132] (2) water extraction: the defatted algae powder is extracted with distilled water at room temperature to obtain a water extract;

[0133] (3) Classification: centrifuge the aqueous extract obtained in step (2), add 1-3 mol / L calcium chloride aqueous solution to the supernatant obtained by centrifugation; stir and centrifuge, take the supernatant, dialyze or ultrafiltrate with distilled water for desalination, and concentrate and dry under reduced pressure to obtain crude polysaccharide;

[0134] (4) Purification: The crude polysaccharide of step (3) is dissolved in distilled water, and separated and purified by a strong anion exchange resin using distilled water and sodium chloride aqueous solution as mobile phases, and the water elution fraction is collected, concentrated under reduced pressure, and freeze-dried to obtain the β-1,3 / 1,6-glucan.

[0135] Unless otherwise specified, the β-1,3 / 1,6-glucan or verified β-1,3 / 1,6-glucan used in Examples 2-19 was prepared according to the method of this Preparation Example.

[0136] Preparation Example 2

[0137] (1) Defatting: drying and crushing the Antarctic brown algae, soaking and stirring with an organic solvent to obtain defatted algae powder;

[0138] (2) water extraction: the defatted algae powder is extracted with distilled water at room temperature to obtain a water extract;

[0139] (3) Classification: centrifuge the aqueous extract obtained in step (2), add 1-3 mol / L calcium chloride aqueous solution to the supernatant obtained by centrifugation; stir and centrifuge, take the supernatant, dialyze or ultrafiltrate with distilled water for desalination, and concentrate and dry under reduced pressure to obtain crude polysaccharide;

[0140] (4) Purification: Dissolve the crude polysaccharide in step (3) in distilled water, use distilled water and sodium chloride aqueous solution as mobile phase, separate and purify through strong anion exchange resin, collect the water elution component, and concentrate under reduced pressure;

[0141] (5) Second purification: the water elution component of step (4) is separated and purified by a weak anion exchange resin using distilled water as the mobile phase, and the water elution component is collected;

[0142] (6) Decolorization: The water elution component of step (5) is separated and purified by an activated carbon column, and distilled water is used as the mobile phase, and the water elution component is collected, concentrated under reduced pressure, and freeze-dried to obtain the β-1,3 / 1,6-glucan.

[0143] Unless otherwise specified, the β-glucan or verified β-glucan used in Examples 20 to 25 was prepared according to the method of this Preparation Example.

[0144] Example 2-7: Analysis of β-glucan composition

[0145] The β-glucan composition in this example was analyzed by high performance gel permeation chromatography (HPGPC) in combination with 18-angle laser scattering (MALLS) and differential detector (RI):

[0146] Determination method: 0.1 mol / L Na2SO4 is used to prepare the β-glucan composition into solutions of different concentrations, which are injected into the DNDC instrument in order from low concentration to high concentration, and the dn / dc of the β-glucan composition is calculated. High performance gel permeation chromatography (HPGPC) is used in conjunction with an 18-angle laser scattering instrument (MALLS) and a differential detector (RI) for analysis. The chromatographic conditions are: chromatographic column TSK-Gel G3000PW (7.5×300mm); mobile phase 0.1 mol / L Na2SO4; column temperature 35°C; flow rate 0.5 mL / min; differential detector and 18-angle laser detector are used in conjunction. Substituting the dn / dc measurement value of the β-glucan composition, the weight-average molecular weight and distribution analysis of the β-glucan composition are obtained (the results are shown in Table 1 and the attached Figure 5-7 ).

[0147] The weight percentage of β-glucan of pentasaccharide-decasaccharide (Dp 5-10), β-glucan of decasaccharide-eicosaccharide, β-glucan of eicosaccharide-twentiesaccharide, β-glucan of pentasaccharide-twentiesaccharide, β-glucan of triasaccharide-tetraasaccharide, β-glucan of tetraasaccharide-pentaasaccharide, β-glucan of fisaccharide-sixtiesaccharide, β-glucan of sextuasaccharide-septuasaccharide, β-glucan of seventy-septuasaccharide-octosaccharide, and β-glucan of greater than eightiesaccharide was calculated.

[0148] The results obtained in Examples 2-7 are shown in Table 1:

[0149]

[0150] Example 8: Identification of β-glucan side chain length

[0151] 1) Preparation of side chain oligosaccharides in the β-glucan composition: The β-glucan composition was dissolved to a concentration of about 5%, 10 μL of endo-1,3-β-glucanase was added, and enzymatic hydrolysis was performed at 40° C. for 8 h. The mixture was boiled for 10 min, centrifuged at 10,000 rpm for 10 min, the supernatant was collected, and freeze-dried to obtain the side chain oligosaccharides in the β-glucan composition.

[0152] 2) ESI-CID-MS / MS analysis of the structure of the side chain oligosaccharides in the β-glucan composition of the present invention. Mass spectrometry conditions:

[0153] LTQ-Qrbitrap XL mass spectrometer, capillary voltage -3000 V, injection cone voltage -50 V, ion source temperature 80 °C, dissociation temperature 150 °C, sheath flow rate 8 arb, sample flow rate 3-5 μL / min. Collision gas helium, collision voltage 15-30 eV.

[0154] 3) The mass spectra of side chain oligosaccharides in the β-glucan compositions of different polymerization degrees are shown in the attached Figure 1-4 As shown. The signal peaks in the mass spectrum were assigned to verify the molecular structure of the side chain oligosaccharide in the β-glucan composition, that is, the structure shown by general formula (III) or formula (IV) or formula (V) or formula (VI). Figure 1 The side chain is shown as

[0155] Mass spectrum of Glcβ1-6Glcβ, Figure 2 The mass spectrum when the side chain is Glcβ1-3Glcβ in formula (IV) is shown. Figure 3 The mass spectrum of the side chain structure in formula (V) is shown. Figure 4 The mass spectrum is shown when the side chain is the structure of formula (VI).

[0156] Example 9: Advanced Structural Information

[0157] The β-glucan composition of the present invention has a triple helix structure. Congo red can form a complex with a polysaccharide having a triple helix chain conformation, and the maximum absorption wavelength of the complex is red-shifted compared with Congo red. The β-glucan composition of the present invention can form a complex with Congo red under alkaline conditions, causing its maximum absorption wavelength to be red-shifted by more than 15 nm (attached Figure 8 ).

[0158] The β-glucan composition of the present invention has a sugar alcohol structure at the reducing end. The difference from the structure of formula (I) is that the structure of formula (II) has a sugar alcohol structure at the reducing end, which is manifested as 13 In C-NMR, there is a characteristic signal at 63.16 ppm (attached Fig. 9 ).

[0159] Example 10: β-1,3 / 1,6-glucan can enhance the phagocytic activity of bone marrow-derived macrophages

[0160] In this example, the effects of the β-1,3 / 1,6-glucan of the present invention on two major populations of peripheral blood innate immune cells, monocytes (CD11b+Ly6C hi , Fig.10 A and 10B) and granulocytes (CD11b+Ly6G+, Fig.10 A and 10C) binding affinity and selectivity. Fig.10 As shown in D, β-1,3 / 1,6-glucan-FITC binds well to monocytes (12.8%) and only rarely to granulocytes (1.5%).

[0161] Next, the effect of the β-1,3 / 1,6-glucan of the present invention on the phagocytic activity of differentially polarized BMDMs was evaluated. β-1,3 / 1,6-glucan slightly upregulated the phagocytic activity of macrophages GM-BMDMs and M-BMDMs Fig.11 A and B. When the dose is higher than 10 μg / ml, the phagocytic effect of the structurally similar β-glucan LNT is higher than that of the β-1,3 / 1,6-glucan of the present invention ( Fig.11 B) However, if Fig.11 As shown in C and 11D, LNT significantly reduced the cell viability of BMDMs in a dose-dependent manner. No cytotoxicity was observed for the β-1,3 / 1,6-glucan of the present invention even at a concentration of 100 μg / ml.

[0162] Example 11: The β-1,3 / 1,6-glucan of the present invention reduces tumor burden and improves spleen index in a tumor transplantation mouse model

[0163] Blood monocytes are recruited into the tumor microenvironment and differentiate into macrophages, creating an immunosuppressive and tumor-promoting microenvironment. Remodeling immunosuppressive macrophages into a pro-inflammatory state will manipulate the tumor microenvironment and hinder tumor growth in vivo. The anti-tumor effect of the β-1,3 / 1,6-glucan of the present invention was detected in a human colorectal cancer cell DLD1 xenograft mouse model.

[0164] like Fig.12 As shown in AC, the β-1,3 / 1,6-glucan of the present invention inhibits tumor volume ( Fig.12 A. Fig.12 B) and tumor weight ( Fig.12 C). It is noteworthy that, unlike classical chemotherapeutic compounds, the β-1,3 / 1,6-glucan of the present invention inhibits tumor growth independently of dose. The low dose of 2 mg / kg of β-1,3 / 1,6-glucan has a stronger inhibitory effect on tumor growth than the high doses (4 mg / kg and 8 mg / kg) ( Fig.12 A). The β-1,3 / 1,6-glucan of the present invention, as an immunostimulatory factor, significantly up-regulated the spleen index of tumor-bearing mice, indicating that the activation and infiltration of immune cells increased ( Fig.12 D). All treatments had no significant effect on the body weight of mice ( Fig.12 E).

[0165] Example 12: β-1,3 / 1,6-glucan of the present invention upregulates macrophage phagocytic activity and secretion of pro-inflammatory cytokines / chemokines in vitro

[0166] In this example, peritoneal macrophages of mice in the control group and the β-1,3 / 1,6-glucan-treated group of the present invention were induced and co-cultured with 9 different colorectal cancer cell lines to detect the phagocytic function of the macrophages.

[0167] like Fig.13 As shown in A, the macrophages induced by the mice in the β-1,3 / 1,6-glucan group showed strong phagocytic activity against all the cancer cell lines we tested. The secretion of cytokines and chemokines is an important indicator of the activation of the body's immune function. The β-1,3 / 1,6-glucan of the present invention strongly increased the secretion of proinflammatory cytokines (IL-1β and TNFα, which are mainly secreted by monocytes / macrophages). β-1,3 / 1,6-glucan treatment can also upregulate the expression levels of proinflammatory cytokines such as IL-2, IL12p70 and chemokine CXCL1 produced by immune cells such as macrophages. The IFNγ levels of mice in the 2mg / kg and 4mg / kg groups of β-1,3 / 1,6-glucan were increased. These data suggest that the β-1,3 / 1,6-glucan of the present invention not only triggers the phagocytic activity of macrophages in vivo, but also promotes the secretion of proinflammatory cytokines / chemokines, exerting an anti-tumor effect.

[0168] Example 13: β-1,3 / 1,6-glucan of the present invention increases the infiltration of pro-inflammatory macrophages and B cells in the tumor microenvironment

[0169] The composition of immune cells in the circulating blood system was detected. It was found that the proportion of B cells (CD19+) and dendritic cells (CD11c+) increased, while the proportion of myeloid cells (CD11b+) decreased slightly ( Fig.14 A). β-1,3 / 1,6-glucan treatment induces the formation of myeloid subsets, pro-inflammatory monocyte-derived macrophages (CD11b+Ly6Chi) ( Fig.14 B).

[0170] The tumor was dissected and the percentage of tumor-infiltrating immune cells was calculated. In the tumor, the percentage of myeloid cells (CD11b+) was slightly increased after treatment with the β-1,3 / 1,6-glucan of the present invention ( Fig.14 C). In addition, β-1,3 / 1,6-glucan promoted the infiltration of pro-inflammatory monocyte-derived macrophage subsets (CD11b+Ly6Chi) ( Fig.14 D). Compared with the control group, in the β-1,3 / 1,6-glucan mice of the present invention, anti-tumor pro-inflammatory tumor-associated macrophages (TAMs) (CD11b+CD80+) were up-regulated, and pro-tumor immunosuppressive TAMs (CD11b+CD206+) were down-regulated ( Fig.14 E). These data suggest that the β-1,3 / 1,6-glucan treatment of the present invention can modulate the proportion of anti-tumor myeloid cells in tumors. Consistent with the induction of B cells in the blood system, the proportion of infiltrating B cells was also increased after β-1,3 / 1,6-glucan treatment of the present invention ( Fig.14 F). Therefore, the β-1,3 / 1,6-glucan of the present invention can regulate the composition of systemic and intratumoral immune cells, placing them in a pro-inflammatory and anti-tumor state, thereby hindering the growth of tumors in the body.

[0171] The β-1,3 / 1,6-glucan treated tumors were grouped according to their volume to detect immune cell (CD45+) infiltration. Large tumors refer to tumors with a volume higher than the average in the β-1,3 / 1,6-glucan treated group of the present invention, and vice versa. Fig.14 As shown in Figure 5, all tumors treated with the β-1,3 / 1,6-glucan of the present invention contained more immune cells (CD45+). In addition, large tumors had more immune cell infiltration than small tumors. In summary, these data suggest that the β-1,3 / 1,6-glucan of the present invention may trigger tumor infiltration of immune cells such as pro-inflammatory macrophages in vivo and inhibit cancer cell growth.

[0172] Example 14: β-1,3 / 1,6-glucan of the present invention reduces tumor burden in AOM-DSS-induced colorectal cancer model

[0173] In order to verify the anti-tumor effect of the β-1,3 / 1,6-glucan of the present invention in immune-competent mice, the anti-tumor effect of β-1,3 / 1,6-glucan was tested in the AOM / DSS-induced C57BL / 6J mouse colorectal cancer model. In the late stage of tumor progression, the body weight of the β-1,3 / 1,6-glucan group at 1 and 3 mg / kg doses was higher than that of the vehicle group, indicating that these treated mice were in better physical condition ( Fig.15 A). In addition, 3 mg / kg β-1,3 / 1,6-glucan treatment slightly increased colon length ( Fig.15 No significant differences were found except for colon length. Even after treatment with a low dose of 1 mg / kg β-1,3 / 1,6-glucan, the number of induced colorectal tumors per mouse was reduced ( Fig.15 C) Fig.15 As shown in D, after treatment with β-1,3 / 1,6-glucan, at a dose of 3 mg / kg, the number of tumors with a diameter of 2 mm to 4 mm and larger than 4 mm was reduced. After using AOM / DSS, 1 mg / kg of the β-1,3 / 1,6-glucan of the present invention induced a smaller number of tumors with a diameter larger than 4 mm than when using vehicle ( Fig.15 E).

[0174] Example 15: β-1,3 / 1,6-glucan of the present invention reverses the changes in immune cell composition induced by AOM-DSS

[0175] The changes in various immune cells including myeloid cells (CD11b+) were observed after AOM / DSS induction and β-1,3 / 1,6-glucan treatment of the present invention. AOM / DSS increased the proportion of myeloid cells (CD11b+) in the circulating blood system, and β-1,3 / 1,6-glucan treatment of the present invention reversed this trend ( Fig.16 A). At the same time, β-1,3 / 1,6-glucan increased the blood ( Fig.16 A) and spleen ( Fig.16 B) and reversed the expression of CD4 T cells in the blood ( Fig.16 A) and lymph nodes ( Fig.16 C). Therefore, β-1,3 / 1,6-glucan treatment reversed the changes in immune cell composition caused by AOM / DSS administration. In addition, the secretion of inflammatory factor IFNγ ( Fig.16 D), TNF-α and chemokine CXCL1 upregulation ( Fig.16 E. Fig.16 F. Fig.16 G. Fig.16 H. and Fig.16 I).

[0176] These data indicate that the anti-tumor effect of the β-1,3 / 1,6-glucan of the present invention on immunocompetent mice is associated with the regulation of immune cell composition and the secretion of pro-inflammatory cytokines / chemokines as immunostimulatory factors.

[0177] Example 16: Inhibitory effect of the β-1,3 / 1,6-glucan of the present invention on nude mouse inoculation of sporadic human colon cancer cell lines

[0178] Mouse colon cancer cell line HCT-116 was resuscitated by liquid nitrogen and cultured in 5A medium containing 10% fetal bovine serum; cells were expanded and cultured in culture bottles until the required cell volume was reached, and then digested and collected; 5A medium containing 10% fetal bovine serum was used to dilute the suspension into 250 million / ml, and after routine disinfection, 0.2 ml per mouse was inoculated subcutaneously in the axilla of the right forelimb of the mouse. When the tumor grew to a tissue block of about 1g, the animal was sacrificed to peel off the tumor tissue, cut into tumor blocks of about 2-3 cubic millimeters, and implanted into the subcutaneous part of the nude mice by catheter method, and the cells were subcutaneously subcultured twice. The HT-29, SW-480, DLD-1 and RKO cell lines listed in the table were modeled and experimented in the same way. After the tumor of the nude mice bearing tumors grew to about 1g, the above method was used to transplant the tumor blocks for modeling, and the mice were randomly divided into groups and began to be administered the next day after transplantation.

[0179] The animals were randomly divided into groups, with 8 animals in each group. The dosage of each group of animals is as follows:

[0180]

[0181] The administration route was tail vein injection. The dosage for each animal was determined based on the most recent body weight of each animal before administration. Each group was administered twice a week, and the administration continued until the end of the experiment. At the end of the experiment, the tumor tissues of the animals that died unexpectedly and the animals that survived were euthanized, and the tumor weights were weighed to calculate the differences in tumor weights of each group and further calculate the tumor inhibition rate IR. TW As a reference indicator, the calculation formula is as follows:

[0182] IR TW (%)=(W 模型组 -W 给药组 ) / W 模型组 ×100%

[0183] It can be seen from the results in the table that the β-1,3 / 1,6-glucan of the present invention exhibits a relatively significant inhibitory effect on the growth of HCT-116, HT-29, SW-480, DLD-1 and RKO cell human colon cancer xenogeneic BALB / c nude mouse transplanted tumors under the experimental conditions of 1 mg / kg, 3 mg / kg and 9 mg / kg administration and twice a week.

[0184]

[0185]

[0186] Example 17: Inhibitory effect of the β-1,3 / 1,6-glucan of the present invention on the mouse S180 sarcoma residual cancer model

[0187] S180 mouse fibrosarcoma cells were cultured and expanded in DMEM high-glucose medium containing 10% FBS. The next step of the experiment can be carried out when the cells enter the logarithmic growth phase. The cells in the logarithmic growth phase were collected and centrifuged and counted. The cell concentration was adjusted and 0.2 ml / mouse was inoculated into the abdominal cavity of Kunming mice as the first generation of breeding mice. After one week of feeding, the ascites in the abdominal cavity of the first generation of breeding mice was centrifuged and counted, and the cell concentration was adjusted. 0.2 ml / mouse was inoculated into the abdominal cavity of Kunming mice as the second generation of breeding mice. After another week of feeding, the ascites in the abdominal cavity of the second generation of breeding mice was centrifuged and counted, and the cell concentration was adjusted. 0.2 ml / mouse was inoculated subcutaneously in the back of Kunming mice. The entire inoculation process was performed aseptically in the clean bench. After inoculation, the growth of mice was observed, and the mice were screened according to their growth status. After screening, they were randomly divided and administered. The experimental results are shown in the following table. β-1,3 / 1,6-glucan can significantly inhibit the regrowth of tumors after surgery at 3 mg / kg.

[0188]

[0189] Example 18: Inhibitory effect of the β-1,3 / 1,6-glucan of the present invention on the mouse Lewis lung cancer residual cancer model

[0190] Lewis cells of mouse lung cancer were cultured and expanded in DMEM high-glucose medium containing 10% FBS. The next step of the experiment can be carried out when the cells enter the logarithmic growth phase. The cells in the logarithmic growth phase were collected and centrifuged and counted. The cell concentration was adjusted and 0.2 ml / mouse was inoculated subcutaneously in the right armpit of C57BL / 6 mice as seed mice. When the tumor grew to more than 1000 mm 3 After that, the tumor was removed aseptically, weighed, diluted and ground with sodium chloride injection at a mass-to-volume ratio of 1:4, and inoculated subcutaneously in the axilla of the right forelimb of each mouse at a rate of 0.2 ml after routine disinfection for passage. 3After that, the second generation tumor was taken out aseptically, weighed, diluted and ground with sodium chloride injection at a mass volume ratio of 1:4, and inoculated subcutaneously on the back of mice at 0.2 ml per mouse after routine disinfection. The growth of mice was observed after inoculation, and the mice were screened according to their growth status. After screening, they were randomly divided and administered. The experimental results are shown in the following table. The β-1,3 / 1,6-glucan of the present invention can significantly inhibit the regrowth of tumors after surgery at 1 mg / kg, 3 mg / kg, and 9 mg / kg.

[0191]

[0192] Example 19: Inhibitory effect of β-1,3 / 1,6-glucan on AOM / DSS-induced inflammation-related colorectal cancer in mice

[0193] Cancer is one of the leading causes of death in humans, and the etiology and pathogenesis of about 1 / 4 of cancer cases are related to chronic inflammation. Inflammatory bowel disease (IBD) is a type of intestinal inflammatory disease with unknown etiology and pathogenesis. According to its pathological characteristics, it can be divided into ulcerative colitis (UC) and Crohn's disease (CD), which is increasing year by year and becoming younger. IBD patients have an increased risk of colorectal cancer (CRC), which increases by 0.5%-1% per year after 8-10 years. After 30 years, up to 18% of IBD patients may develop CRC. Although IBD-related CRC only accounts for 1%-2% of all colorectal cancers, it is a common cause of death in IBD patients. The azoxymethane (AOM) / dextransodium sulfate (DSS)-induced colitis associated cancer (CAC) animal model has been widely used in the study of the efficacy and mechanism of new drugs because it successfully simulates the entire process of IBD-induced CRC. Elucidating its pathological changes and carcinogenesis principles will help discover new candidate targets for the treatment of colorectal cancer.

[0194] In this experiment, a mouse colorectal cancer model was established by intraperitoneal injection of AOM and periodic administration of DSS drinking solution. At the beginning of the experiment, 70 male C57BL / 6 mice aged 6-8 weeks were randomly divided into 7 groups with 10 mice in each group. Except for the first group as a normal control, the other groups were modeled with AOM / DSS. On the first day of the modeling group mouse experiment, a single intraperitoneal injection of 10 mg / kg azoxymethane (AOM) was given to the mice. Seven days later, the mice were given drinking water containing 1% sodium dextran (DSS). After seven days, fourteen days of normal drinking water were given, 7 days of 1% DSS drinking water and 14 days of normal drinking water as a cycle, and a total of three cycles were repeated, and finally CAC was induced in the mice.

[0195] At the end of the experiment, the mice were dissected, the entire colorectum was taken out, the mesentery and attached adipose tissue were carefully removed, and their lengths were measured. Then, the entire colorectal cavity was flushed clean with physiological saline and longitudinally cut open, and the macroscopic tumors were counted and measured with a vernier caliper under a dissecting microscope. Finally, the effect of the β-1,3 / 1,6-glucan of the present invention was evaluated from the aspects of colon length, tumor formation rate, and total number of tumors.

[0196] The experimental results are shown in the figure below. Compared with the blank group, the colon length of the model group was significantly shortened (P<0.01), and the tumor formation rate reached 77.78%, indicating that the AOM / DSS-induced mouse colorectal cancer model was successful. The tumor formation rate of the β-1,3 / 1,6-glucan (3mg / kg) group was 16.67%, which was lower than that of the model group, indicating that β-1,3 / 1,6-glucan (3mg / kg) had an inhibitory effect on AOM / DSS-induced mouse colorectal cancer.

[0197]

[0198] *p<0.05 vs. blank; **p<0.01 vs. blank; #p<0.05 vs. model; ##p<0.01 vs. model;

[0199] Example 20 Characterization of β-1,3 / 1,6-glucan obtained in Preparation Example 1 and Preparation Example 2

[0200] The test method is as follows:

[0201] (i) Ultraviolet full wavelength scanning spectrum: The β-glucan composition was dissolved to a concentration of 5 wt %, a baseline was established with pure water, the scanning wavelength was set to 190-900 nm, the scanning accuracy was 1 nm, and ultraviolet full wavelength scanning was performed.

[0202] (ii) The side chain length was determined by the method of Example 8

[0203]

[0204]

[0205] Example 21. Tail vein injection of β-1,3 / 1,6-glucan anti-mouse S-180 experiment

[0206] Animal experiment on mice with normal immune function (administration in the coccygeal vein)

[0207] 1. Experimental plan:

[0208] Tumor cells: S-180; Inoculation site: shoulder blade; Mouse species: KM (male); Number of mice: 13 per group; Administration started the day after tumor cell inoculation; Administration frequency: daily; Administration cycle: 20 days; Administration method: coccygeal intravenous injection;

[0209] Blank control group: model group (CN group)

[0210] Positive control group: cisplatin (2 mg / kg) (PC or CP group); Lentinan LNT (1.5 mg / kg)

[0211] Experimental groups: BL: low dose of β-1,3 / 1,6-glucan (0.3 mg / kg); BM: medium dose of β-1,3 / 1,6-glucan (1.5 mg / kg); BH: high dose of β-1,3 / 1,6-glucan (7.5 mg / kg).

[0212] 2. Experimental results

[0213] The results are as follows Fig.18 As shown in AB. The physiological status of mice in the positive control group and the experimental group was still very good 20 days after inoculation, and the animals were very active and agile before being sacrificed.

[0214] Compared with the model group, β-1,3 / 1,6-glucan can significantly inhibit the growth of S-180 tumors at low, medium and high doses, with a tumor inhibition rate of up to 60% to 70%. The thymus weight of mice in the cisplatin group was significantly lower than that in the model group, and the thymus weight of mice in the β-1,3 / 1,6-glucan administration group (0.3, 1.5, 7.5 mg / kg) was significantly higher than that in the cisplatin group. The thymus weight of mice in the high-dose BG136 administration group was equivalent to that of the model group. This indicates that β-1,3 / 1,6-glucan is likely to exert its anti-tumor effect by activating the immune system of mice.

[0215] Example 22 Oral β-1,3 / 1,6-glucan anti-tumor test

[0216] 1. Experimental plan:

[0217] Tumor cells: S-180; Inoculation site: axilla; Mouse species: Kunming mouse; Number of mice: 13 mice in each group (7 female mice and 6 male mice); Start time of administration: the day after inoculation; Frequency of administration: daily; Dosing cycle: 10 days; Mode of administration: oral; Dosage (mg / kg): ① Model (CN); ② Cisplatin (CP or PC): 1.5 mg; ③ β-1,3 / 1,6-glucan: 1 mg (B1); ④ β-1,3 / 1,6-glucan: 5 mg (B5); ⑤ β-1,3 / 1,6-glucan: 25 mg (B25); ⑥ Cisplatin 1.5 mg + BG 1 mg (CPB1); ⑦ Cisplatin 1.5 mg + BG 5 mg (CPB5); ⑧ Cisplatin 1.5 mg + BG 25 mg (CPB25).

[0218] 2. Experimental results:

[0219] The experimental results are as follows Fig.19 A and B. The experimental results show that oral administration of β-1,3 / 1,6-glucan has a significant inhibitory effect on S-180 tumors. When the oral dose of β-1,3 / 1,6-glucan is 1 mg / kg, the overall inhibition rate is more than 58%, and the tumor inhibition rate for male mice is as high as 70.4%. When BG136 is used in combination with cisplatin (1.5 mg / kg) at 25 mg / kg, the inhibition rate is further improved, reaching 77.3%.

[0220] Example 23 β-1,3 / 1,6-glucan combined with chemotherapy for mouse tumor model

[0221] Effects of β-1,3 / 1,6-glucan combined with chemotherapy on leukocytes and platelets

[0222] 3x10 5 The cell suspension of mouse melanoma cell line B16 (gift from PerkinElmer) was subcutaneously injected into C57BL / 6J mice (female, 6-8 weeks old, purchased from Jinan Pengyue Experimental Animal Company) (Overwijk &

[0223] Restifo, 2001). About 2 days after tumor implantation, carboplatin (30 mg / kg, twice a week) and BG136 (4 mg / kg) or Lentinan LNT (2 mg / kg) were injected intraperitoneally and the tumor volume was measured during the administration. On the 15th day, the animals were sacrificed and the tumor mass was measured. After the animals were sacrificed, blood was collected from the heart and placed in an EDTA-anticoagulant tube. After mixing, 50 μl of whole blood was collected and the immune cell concentration was measured by a blood analyzer.

[0224] The test results show that the β-1,3 / 1,6-glucan prepared in Preparation Example 2 can enhance the tumor inhibition effect of carboplatin, stimulate the immune response, and reverse the immunosuppression and platelet decrease after the administration of carboplatin.

[0225] Embodiment 24

[0226] Since immune cells play an important role in various tumor cell types, the anti-tumor and leukocyte and platelet-raising effects of β-1,3 / 1,6-glucan will play a role in various tumor cells (for example, lung cancer, kidney cancer, liver cancer, breast cancer, etc.) and immune cells also play a regulatory role in tumor metastasis and occurrence. Therefore, β-1,3 / 1,6-glucan may inhibit the metastasis and occurrence of tumor cells.

[0227] Example 25 Effect of the combination of β-1,3 / 1,6-glucan and PD-1 antibody on mouse tumor model

[0228] PD-1 antibody used: Name: in vivo MAb anti-mouse PD-1 (CD279); purchased from Bioxcell

[0229] (i) Effects of intravenous injection of β-1,3 / 1,6-glucan combined with PD-1 antibody on subcutaneous transplanted tumors in MC38 mice

[0230] In this example, mouse colon cancer cell line MC38 was selected to detect the effect of β-1,3 / 1,6-glucan combined with PD-1 antibody on the growth of mouse transplanted tumors. The test results are as follows: Fig. 20 As shown in AC.

[0231] Fig. 20 The results of A and B showed that tail vein injection of β-1,3 / 1,6-glucan combined with PD-1 antibody can effectively inhibit the growth of MC38 transplanted tumors. The inhibitory effect is significantly better than that of the PD-1 antibody group alone, with significant synergistic effects and no obvious drug toxicity ( Fig. 20 C) has good drug safety.

[0232] (ii) Effects of intravenous injection of β-1,3 / 1,6-glucan combined with PD-1 antibody on the expression of tumor immunity-related cytokines in mice

[0233] In this example, the expression of multiple cytokines in mouse prostate cancer cell MC38 transplanted tumors was quantified by RT-PCR. Fig.21 As shown in AF.

[0234] Depend on Fig.21Results A, B, and C show that after β-1,3 / 1,6-glucan was combined with PD-1 antibody, the expression of tumor necrosis factor α (TNFα), interleukin 1β (IL1-β), and nitric oxide synthase (iNOS) produced by macrophages in mouse tumors increased significantly, indicating that β-1,3 / 1,6-glucan can cooperate with PD-1 antibody to stimulate immune activation inside tumors;

[0235] Depend on Fig.21 The results of D, E, and F show that after β-1,3 / 1,6-glucan was combined with PD-1 antibody, the expression levels of pro-inflammatory Th1 polarized cytokines (interferon γ, IFN-γ), interleukin 2 (IL-2), and granzyme B (GZMB) increased, further demonstrating that β-1,3 / 1,6-glucan combined with PD-1 antibody can bridge the body's innate immunity and adaptive immunity to exert the best anti-tumor immunity.

[0236] (iii) Effects of oral administration of β-1,3 / 1,6-glucan combined with PD-1 antibody on mouse tumor models

[0237] The mouse colon cancer cell line MC38 was selected to detect the effect of β-1,3 / 1,6-glucan combined with PD-1 antibody on the growth of mouse transplanted tumors. Fig. 22 As shown in AC.

[0238] Fig. 22 The results of A and B showed that oral administration of β-1,3 / 1,6-glucan combined with PD-1 antibody can effectively inhibit the growth of MC38 transplanted tumors, and the inhibitory effect is significantly better than that of the PD-1 antibody group alone, with significant synergistic effects and no obvious drug toxicity ( Fig. 22 C) has good drug safety.

[0239] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a β-glucan composition, characterized in that: The composition comprises β-glucan having a structure represented by formula (I) or formula (II): wherein n is an integer selected from 1 to 20, R is H and / or no more than 4 glucose residues; in the side chains of the β-glucan, when R≠H, 5 to 10% of the side chains R are 3 or 4 glucose residues in length; The β-glucan has a protein content of 0.01 wt% to 0.5 wt%, and the optical rotation of the β-glucan is between -16° and -21°; Includes steps: (1) Defatting: drying and crushing the Antarctic brown algae, soaking and stirring with an organic solvent to obtain defatted algae powder; (2) water extraction: the defatted algae powder is extracted with water at room temperature to obtain a water extract; (3) Classification: centrifuge the aqueous extract obtained in step (2), add 1-3 mol / L calcium chloride aqueous solution to the supernatant obtained by centrifugation; stir and centrifuge, take the supernatant for ultrafiltration and desalination, and concentrate and dry under reduced pressure to obtain crude polysaccharide; (4) Purification: Dissolve the crude polysaccharide in step (3) in distilled water, use distilled water and sodium chloride aqueous solution as mobile phase, separate and purify through anion exchange resin, collect the water elution component, concentrate under reduced pressure, and freeze-dry to obtain the β-glucan; The anion resin separation and purification is: first separation and purification by a strong anion resin and then separation and purification by a weak anion resin; or first separation and purification by a weak anion resin and then separation and purification by a strong anion resin. Furthermore, the strong anion resin is an anion resin containing a quaternary ammonium group, and the weak anion resin is an anion resin containing a diethylaminoethyl group.

2. The preparation method according to claim 1, characterized in that R in the structure of formula (I) or formula (II) is one or more of the structures of formula (III) or formula (IV) or formula (V) or formula (VI), wherein Formula (III): Glcβ1-; Formula (IV): Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-; Formula (V): Glcβ1-3Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-3Glcβ1- or Glcβ1-3Glcβ1-6Glcβ1- or Glcβ1-6Glcβ1-6-Glcβ1-; Formula (VI): Glcβ1-3Glcβ1-3Glcβ1-3Glcβ1-or Glcβ1-6Glcβ1-3Glcβ1-3Glcβ1-or Glcβ1-3Glcβ1-6Glcβ1-3Glcβ1- or Glcβ1-3Glcβ1-3Glcβ1-6Glcβ1- or Glcβ1-6Glcβ1-6Glcβ1-3Glcβ1- or Glcβ1-6-Glcβ1-3-Glcβ1-6Glcβ1-or Glcβ1-3Glcβ1-6Glcβ1-6Glcβ1-or Glcβ1-6Glcβ1-6Glcβ1-6Glcβ-.

3. The preparation method according to claim 1, characterized in that The preparation method has one or more of the following characteristics: (a) The anion resin separation and purification is as follows: first separation and purification by a strong anion resin and then separation and purification by a weak anion resin; (b) the β-glucan has a molecular weight of 1-50 kDa; (c) The ultraviolet full wavelength scanning spectrum of the β-glucan has no obvious absorption in the wavelength range of 300 to 900 nm, and the ultraviolet full wavelength scanning spectrum of the β-glucan has no absorption peak in the wavelength range of 260 to 280 nm.

4. The preparation method according to claim 1, characterized in that: In the β-glucan composition, the weight content of pentasaccharide-decasaccharide is 0-50.0%, the weight content of decasaccharide-eicosaccharide is 0-80.0%, the weight content of eicosaccharide-pentacosaccharide is 0-25.0%, the weight content of pentacosaccharide-triacosaccharide is 0-45.0%, the weight content of triasaccharide-tetraasaccharide is 0-30.0%, the weight content of tetrasaccharide-pentaosaccharide is 0-15.0%, the weight content of pentasaccharide-sexucasaccharide is 0.1%-55.0%, the weight content of hexosaccharide-septosaccharide is 0.1%-20.0%, the weight content of seventy-octasaccharide-octosaccharide is 0.1%-15.0%, and the weight content of more than eighty-saccharide is 0-40.0%; or, In the β-glucan composition, the weight content of pentasaccharide-decasaccharide is 0-45.0%, the weight content of decasaccharide-eicosaccharide is 0-75.0%, the weight content of eicosaccharide-pentacosaccharide is 0-20.0%, the weight content of pentacosaccharide-triacosaccharide is 0-40.0%, the weight content of β-glucan with a polymerization degree of 30-40 is 0-25.0%, the weight content of tetrasaccharide-pentaosaccharide is 0-10.0%, the weight content of pentasaccharide-sexicosaccharide is 0.1%-50.0%, the weight content of hexosaccharide-septosaccharide is 0.1%-15.0%, the weight content of seventy-octaosaccharide is 0.1%-10.0%, and the weight content of more than eighty-saccharide is 0-35.0%; or, The β-glucan composition is composed of β-glucan with a degree of polymerization of 20-80, wherein the weight content of eicosaccharide-pentacosaccharide is 13.2%-19.8%, the weight content of pentacosaccharide-triaconose is 29.1%-43.7%, the weight content of triaconose-tetraconose is 18.2%-27.4%, the weight content of tetraconose-pentaconose is 7.3%-10.9%, the weight content of pentaconose-sexuconose is 4.5%-6.7%, the weight content of hexosaconose-septuaconose is 3.5%-5.3%, the weight content of septuaconose-octaconose is 4.2%-6.2%, or, The β-glucan composition is composed of β-glucan with a degree of polymerization of 20-80, wherein the weight content of eicosaccharide-pentacosaccharide is 16.5%, the weight content of pentacosaccharide-triaconose is 36.4%, the weight content of triaconose-tetraconose is 22.8%, the weight content of tetraconose-pentaconose is 9.1%, the weight content of pentaconose-sexuconose is 5.6%, the weight content of hexicosaccharide-septuconose is 4.4%, and the weight content of septuconose-octaconose is 5.2%; or, The β-glucan composition is composed of β-glucan with a degree of polymerization of 10-80, wherein the weight content of decasaccharide-eicosaccharide is 40.6%-60.8%, the weight content of eicosaccharide-pentacosaccharide is 13.4%-20.0%, the weight content of pentacosaccharide-triacosaccharide is 7.7%-11.5%, the weight content of triacontaccharide-tetracontaccharide is 7.6%-11.4%, the weight content of tetracontaccharide-pentacontaccharide is 4.2%-6.2%, the weight content of pentacontaccharide-sexucontaccharide is 2.3%-3.5%, the weight content of hexaccharide-septuctaccharide is 1.6%-2.4%, and the weight content of septuctaccharide-octacontaccharide is 0.8%-1.2%; or, The β-glucan composition is composed of β-glucan with a degree of polymerization of 10-80, wherein the weight content of decasaccharide-eicosaccharide is 50.7%, the weight content of eicosaccharide-pentacosaccharide is 16.7%, the weight content of pentacosaccharide-triacosaccharide is 9.6%, the weight content of triaconta ...

5. The preparation method according to claim 1, characterized in that: Have one or more of the following characteristics: (a) The β-glucan has a sulfate content of 0.01 wt% to 2 wt%; (b) the β-glucan has a chloride ion content of 0.01 wt% to 2 wt%; (c) The ultraviolet full-wavelength scanning spectrum of the β-glucan has no obvious absorption in the wavelength range of 230 to 900 nm.

6. A β-glucan composition, characterized in that The β-glucan composition is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the β-glucan composition according to claim 6 in preparing a composition for improving or treating immune-related diseases.

8. Use of the β-glucan according to claim 6 in preparing a composition for improving or treating immune-related diseases, characterized in that: The immune-related disease is tumor or inflammation.

9. Use of the β-glucan according to claim 6 in preparing a composition for improving or treating immune-related diseases, characterized in that: The tumor is selected from colorectal cancer, lung cancer, fibrosarcoma, melanoma, kidney cancer, liver cancer, breast cancer, or a combination thereof.

10. A composition for improving or treating immune-related diseases, characterized in that: The composition comprises: (1) the β-glucan according to any one of claims 6 to 9, and (2) A pharmaceutically acceptable carrier.

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