CN113797221A - Beta-glucan composition and application thereof - Google Patents

Abstract

The invention provides a beta-glucan composition and application thereof. Specifically, the invention provides an application of a beta-glucan composition in preparing a composition for improving or treating immune-related diseases, wherein the beta-glucan is beta-glucan containing beta-1, 3 and beta-1, 6 glucosidic bonds in a molecule and has a molecular weight of 1-50 kDa. The beta-glucan has the advantages of novel structure, controllable quality, rich sources, simple preparation process, high product purity, strong biological activity, easy industrial production and the like, and the water-soluble beta-glucan composition obtained by the invention has better anti-tumor activity and is expected to be developed into a safe and effective novel class of drugs for improving or treating immune-related diseases and anti-tumor drugs.

Description

Beta-glucan composition and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a beta-glucan composition and application thereof.

Technical Field

Studies have shown that different sources of beta-1, 3-glucan have different biological activities, including anti-tumor, immunomodulatory, anti-aging, and anti-inflammatory properties. At present, beta-1, 3-glucan in the market is mostly from terrestrial organisms such as barley, oat, edible fungi (mushroom, grifola frondosa, schizophyllum commune), yeast and the like, and because of different raw material sources, the obtained beta-1, 3-glucan has great differences in molecular weight, connection mode, branching degree and the like, and the quality is difficult to control, for example, beta-glucan for injection is mainly from mushroom, is beta-1, 3-glucan (LNT) with beta-1, 6-branches, and because the molecular weight of the beta-1, 3-glucan is as high as 400-plus 800kDa, the water solubility is poor, the separation and purification is difficult, and the impurity content is high. With the continuous expansion of the application field of the beta-glucan and the continuous increase of the market demand, the existing mushroom beta-glucan cannot meet the market demand.

Therefore, the development of beta-glucan which has controllable quality, abundant sources, simple preparation process, high product purity, strong biological activity and easy industrial production is urgently needed in the field.

Disclosure of Invention

The invention aims to resolve a specific structure of beta-1, 3/1, 6-glucan and application of the composition in preparing a composition for treating immune-related diseases.

In a first aspect of the invention, there is provided a composition of beta-1, 3/1, 6-glucan, the composition comprising a beta-glucan having a structure represented by formula (I) and/or formula (II),

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).

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

Formula (III): glc β 1-;

formula (IV): glc β 1-3Glc β 1-or Glc β 1-6Glc β 1-;

formula (V): glc beta 1-3Glc beta 1-or Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc β 1-3Glc β 1-6Glc β 1-or Glc β 1-6Glc β 1-6Glc β 1-;

formula (VI):

glc beta 1-3Glc beta 1-or

Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-or

Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-6Glc beta 1-3Glc beta 1-6Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-or

Glcβ1-6Glcβ1-6Glcβ1-6Glcβ-。

Preferably, in the beta-glucan composition, the pentasaccharide-decasaccharide is 0-50.0% by weight, the decasaccharide-didesaccharide is 0-80.0% by weight, the icose-eicosapentaenoic saccharide is 0-25.0% by weight, the icose-tridecose is 0-45.0% by weight, the triacontose-tetraose is 0-30.0% by weight, the forty-pentasaccharide is 0-15.0% by weight, the fifty-hexasaccharide is 0.1-55.0% by weight, the sixty-heptasaccharide is 0.1-20.0% by weight, the heptadecasaccharide is 0.1-15.0% by weight, and the greater than eighty saccharide is 0-40.0% by weight.

Preferably, in the beta-glucan composition, the weight content of pentasaccharide-decasaccharide is 0-45.0%, the weight content of decasaccharide-ditridesaccharide is 0-75.0%, the weight content of icose-icose is 0-20.0%, the weight content of icose-tridecose is 0-40.0%, the weight content of beta-glucan with a degree of polymerization of 30-40 is 0-25.0%, the weight content of forty-fifty saccharide is 0-10.0%, the weight content of fifty-sixty saccharide is 0.1-50.0%, the weight content of sixty-heptasaccharide is 0.1-15.0%, the weight content of heptadecasaccharide is 0.1-10.0%, and the weight content of more than octasaccharide is 0-35.0%.

Preferably, the beta-glucan composition consists of beta-glucan having a degree of polymerization of 20-80, wherein the weight content of didodecan-icosanose is 13.2-19.8%, the weight content of icosanose-triacontose is 29.1-43.7%, the weight content of triacontose-forty-sugar is 18.2-27.4%, the weight content of forty-fifty-sugar is 7.3-10.9%, the weight content of fifty-sixty-sugar is 4.5-6.7%, the weight content of sixty-heptadecasugar is 3.5-5.3%, and the weight content of heptadecasugar-octadecasugar is 4.2-6.2%.

Preferably, the beta-glucan composition consists of beta-glucan having a degree of polymerization of 20 to 80, wherein the weight content of didodecan-icosanose is 16.5%, the weight content of icosanose-triacontose is 36.4%, the weight content of triacontose-forty-ose is 22.8%, the weight content of forty-fifty-ose is 9.1%, the weight content of fifty-sixty-sugar is 5.6%, the weight content of sixty-heptadecaose is 4.4%, and the weight content of heptadecaose-octadecaose is 5.2%.

Preferably, the beta-glucan composition consists of beta-glucan having a degree of polymerization of 10 to 80, wherein the weight content of decaose-icosanose is 40.6 to 60.8%, the weight content of icosanose-pentacose is 13.4 to 20.0%, the weight content of icosanose-triacontose is 7.7 to 11.5%, the weight content of triacontose-tetrantaose is 7.6 to 11.4%, the weight content of forty-pentaose is 4.2 to 6.2%, the weight content of fifty-sixtose is 2.3 to 3.5%, the weight content of sixty-heptadecaose is 1.6 to 2.4%, and the weight content of heptadecaose-octadecaose is 0.8 to 1.2%.

Preferably, the beta-glucan composition consists of beta-glucan having a degree of polymerization of 10 to 80, wherein the decaose-icosanose content is 50.7% by weight, the icosanose-icosanose content is 16.7% by weight, the icosanose-triacontose content is 9.6% by weight, the triacontose-forty-ose content is 9.5% by weight, the forty-fifty-ose content is 5.2% by weight, the fifty-sixty-sugar content is 2.9% by weight, the sixty-seventy-sugar content is 2.0% by weight, and the seventy-eighty-sugar content is 1.0% by weight.

Preferably, the beta-1, 3/1, 6-glucan has a molecular weight of 1-50 kDa; preferably, 2 to 30 kDa; more preferably, it is 2-10 kDa.

Preferably, the beta-1, 3/1, 6-glucan has a specific rotation of no less than-15.0 °; preferably-15 to-25; more preferably from-16 to 21.

Preferably, in the beta-1, 3/1, 6-glucan, the sulfate radical content is 0.01 wt% -2 wt%; preferably, 0.01 wt% to 0.5 wt%.

Preferably, the beta-1, 3/1, 6-glucan contains 0.01-2 wt% of chloride ions; preferably, 0.01 wt% to 0.5 wt%.

Preferably, the content of protein in the beta-1, 3/1, 6-glucan is 0.01 wt% -5 wt%; preferably, 0.01 wt% to 0.5 wt%.

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

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

Preferably, at least 20% of the side chains of said β -1,3/1, 6-glucan are 1 or 2 glucose residues in length.

Preferably, in the beta-glucan having the structure shown in the formula (I) or the formula (II), at least 3-20R are respectively and independently 1 or 2 glucose residues.

Preferably, in the beta-glucan with the structure shown in the formula (I) or the formula (II), at least 3-10 Rs are respectively and independently shown as the structure in the formula (III) or the formula (IV); wherein the structures of formula (III) and formula (IV) are as defined above.

Preferably, among 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.

Preferably, where the side chain (R) is 1 or 2 glucose residues in length, the side chains (R) are each independently of the other of formula (III) and formula (IV), wherein the structures of formula (III) and formula (IV) are as previously defined.

Preferably, when the side chains (R) are 3 or 4 glucose residues in length, the side chains (R) are each independently of the other a structure of formula (V) or formula (VI), wherein the structures of formula (V) or formula (VI) are as previously defined.

In a second aspect of the invention there is provided the use of a β -glucan as described in the first aspect or a composition of β -1,3/1, 6-glucan as described in the first aspect in the manufacture of a composition for the amelioration or treatment of an immune-related disease.

Preferably, the immune-related disease is a tumor or inflammation.

Preferably, the tumor is selected from colorectal cancer, lung cancer, fibrosarcoma.

In a third aspect of the present invention, there is provided a composition for ameliorating or treating an immune-related disorder, the composition comprising:

(1) a composition of a beta-glucan as described in the first aspect or a beta-1, 3/1, 6-glucan as described in the first aspect, and

(2) a pharmaceutically acceptable carrier.

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

(1) degreasing: drying and crushing Antarctic brown algae, soaking in an organic solvent, and stirring to obtain defatted algae powder;

(2) water extraction: stirring and extracting the defatted algae powder with water at room temperature to obtain water extract;

(3) grading: centrifuging the water extract obtained in the step (2), and adding 1-3 mol/L calcium chloride aqueous solution into the supernatant obtained by centrifuging; stirring, centrifuging, collecting supernatant, dialyzing or ultrafiltering for desalting, concentrating under reduced pressure, and drying to obtain crude polysaccharide;

(4) and (3) purification: and (3) dissolving the crude polysaccharide obtained in the step (3) by using distilled water and a sodium chloride aqueous solution as mobile phases, separating and purifying by using anion exchange resin, collecting water elution components, concentrating under reduced pressure, and freeze-drying to obtain the beta-1, 3/1, 6-glucan.

In another preferred embodiment, the anion resin separation and purification is strong anion resin separation and purification.

In another preferred embodiment, the anion resin is separated and purified into: separating and purifying by strong anion resin and then separating and purifying by weak anion resin; or, the weak anion resin is separated and purified firstly, and then the strong anion resin is separated and purified. By combining a weak anion resin and a strong anion resin, it is unexpectedly possible to more effectively remove impurities and obtain beta-1, 3/1, 6-glucan having longer side chain glucose residues, higher purity and a triple helix structure.

Preferably, the strong anion resin is an anion resin containing quaternary ammonium groups.

Preferably, the weak anionic resin is a diethylaminoethyl group-containing anionic resin.

Preferably, the Antarctic brown algae are Lacca, Phyllostachys Pubescens and/or Leptospira sp.

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

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

Preferably, the immune checkpoint drug is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody.

Preferably, the anti-PD-1 antibody or PD-L1 antibody is selected from the group consisting of dolvacizumab (Durvalumab), aleuzumab (Atezolizumab), Nivolumab (Nivolumab), BMS202, sibradizumab (Spartalizumab), carprilizumab (Camrelizumab), or a combination thereof.

Preferably, the chemotherapeutic agent is selected from cytotoxic chemotherapeutic agents.

Preferably, the chemotherapeutic agent is selected from one of the anthracyclines, 5-Fu classes, alkaloids classes.

Preferably, the chemotherapeutic agent is selected from one or more of cisplatin, carboplatin.

Preferably, the drug or formulation is administered simultaneously, sequentially or separately in combination with an immune checkpoint drug and/or chemotherapeutic agent.

In a sixth aspect of the present invention, there is provided a pharmaceutical combination comprising:

(i) a first active ingredient: the first active ingredient is a composition of β -1,3/1, 6-glucan as described in the first aspect;

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

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

Preferably, the immune checkpoint drug is as described previously.

Preferably, the chemotherapeutic agent is as previously described.

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

Preferably, the white blood cells are lymphocytes.

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

Preferably, the drug or formulation is also used in combination with an immune checkpoint drug.

Preferably, the drug or formulation is administered simultaneously, sequentially or separately in combination with an immune checkpoint drug.

Preferably, the medicament or formulation is also used in combination with at least one chemotherapeutic agent.

Preferably, the drug or formulation is administered simultaneously, sequentially or separately in combination with the chemotherapeutic agent.

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

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

Preferably, the medicament or formulation further comprises a pharmaceutically acceptable carrier or excipient.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows ESI-CID-MS/MS plots for Glc β 1-6Glc β in formula (IV) as the side chain.

FIG. 2 shows ESI-CID-MS/MS plots for Glc β 1-3Glc β in formula (IV) as the side chain.

FIG. 3 shows a diagram of ESI-CID-MS/MS in which the side chain has the structure in formula (V).

FIG. 4 shows a diagram of ESI-CID-MS/MS in which the side chain has the structure in formula (VI).

FIG. 5 shows the weight average molecular weight measurement of example 2.

FIG. 6 shows the weight average molecular weight measurement of example 7.

FIG. 7 shows the polymerization degree distribution analysis of example 7.

Figure 8 shows that the β -glucan composition can shift the congo red maximum absorption wavelength.

FIG. 9 shows beta-glucan compositions having the structure of formula (II)¹³C-NMR chart.

FIG. 10 shows that β -1,3/1, 6-glucan has higher affinity for monocytes than for granulocytes. (A-C) lymphocytes (red dot CD11b-), monocytes (green dot CD11b + Ly6Chi), granulocytes (blue dot CD11b + Ly6G +) flow cells. (D) Blood cells were incubated with beta-1, 3/1, 6-dextran-FITC (200. mu.g/ml) for 2 hours at 37 ℃. Subsequently, the erythrocytes are lysed and the cells are stained with the corresponding flow antibodies. Flow cytometry examined the binding affinity of β -1,3/1, 6-glucan-FITC to monocytes (green, CD11b +) and granulocytes (blue, CD11b +).

FIG. 11 shows that β -1,3/1, 6-glucan of the invention increases phagocytic activity of BMDMs without cytotoxicity. (A and B) incubation of GM-BMDM (A) and M-BMDM (B) with beta-1, 3/1, 6-glucan or LNT for 24 hours. The phagocytic activity of both macrophages was examined by the neutral red method. Cell viability was measured using the MTT method (C, D).

FIG. 12 shows that β -1,3/1, 6-glucan of the invention reduced tumor burden and improved spleen index in a DLD1 xenograft mouse model. Mice implanted with DLD1 tumors were treated with vehicle or the β -1,3/1, 6-glucan of the invention. Tumor volume (a) and body weight (E) are shown. Tumors were excised, photographed (B), weighed (C), and spleen index calculated (D).

FIG. 13 shows that β -1,3/1, 6-glucan of the invention upregulates macrophage phagocytic activity and proinflammatory cytokine secretion in a DLD1 xenograft model. (A) 9 colorectal cancer cell lines (HCT-116, LS174T, SW480, DLD1, HT-29, LS180, HCT-15, LOVO, T84) were examined for phagocytosis by peritoneal macrophages of vehicle or β -1,3/1, 6-glucan treated mice of the present invention. (B-G) the V-PLEX mouse inflammatory factor kit (LabEx) was used to detect the levels of proinflammatory cytokines and chemokines in plasma in mice from the drug-loaded group and the beta-1, 3/1, 6-glucan group of mice of the present invention.

FIG. 14 shows that β -1,3/1, 6-glucan of the invention increases pro-inflammatory macrophages and B cells in the tumor microenvironment. (A) Flow cytometry detected the proportion of CD11b +, CD335+, CD19+, CD11c + cells in blood. (B) measuring the ratio of the blood neutrophil (CD11B + LY6G +) to the proinflammatory monocyte (CD11B + LY6 Chi). (C-F) the proportion of tumour infiltrating CD11b + cells, monocyte derived infiltrating macrophages (CD11b + LY6Chi), TAMs (CD11b + CD80+ or CD11b + CD206+), CD19+ cells was examined. (G) The ratio of CD45+ leukocytes in vehicle and beta-1, 3/1, 6-glucan of the present invention (small tumor group and large tumor group) was determined in the treatment of mouse tumors.

Figure 15 shows that β -1,3/1, 6-glucan of the invention reduces the tumor burden in AOM-DSS-induced colorectal cancer model. C57 mice untreated or AOM/DSS induced, or β -1,3/1, 6-glucan of the invention (1,3 and 9mg/kg) treated after induction. (A) Body weights were recorded after treatment for each group. (B) Colon length was determined after sacrifice. (C-E) Colon longitudinal dissection, tumor number (C and E) and diameter (D) were collected.

FIG. 16 shows that β -1,3/1, 6-glucan of the invention reverses the changes in immune cell composition caused by AOM-DSS administration. (A-D) the ratio of CD11B +, CD335+, CD19+, CD4+, CD8+, CD11C + in blood (A), the ratio of CD19+ in spleen (B), and the ratio of CD4+ and CD8+ in lymph node (C) were examined. (E, F, G, H, I) measuring the levels of proinflammatory cytokines and chemokines in plasma of normal or AOM/DSS mice treated with or without β -1,3/1, 6-glucan of the invention, respectively.

FIGS. 17A and B show UV full-wavelength scan patterns of beta-1, 3/1, 6-glucan prepared according to the methods of preparation example 1 and preparation example 2, respectively.

Fig. 18A and B show the experimental results of example 21. (A) The mean tumor weight and the inhibition rate in the mice after administration are shown, and (B) the weight of the glands in the mice after administration is shown.

Fig. 19A and B show the experimental results of example 22. (A) Shows the amount of β -1,3/1, 6-glucan and its anti-tumor growth effect in combination with cisplatin, and (B) shows the weight inhibition of male Kunming mice.

FIGS. 20A-C show that intravenous injection of β -1,3/1, 6-glucan in combination with PD-1 antibody in example 25(i) was effective in inhibiting the growth of subcutaneous transplantable tumors in mouse colon carcinoma MC38 mice; in the figure, PD1-200(qw) represents the group of PD-1 antibody (200. mu.g/mouse) administered alone, and PD1-200(qw) + BG0.3(biw), PD1-200(qw) + BG1(biw) and PD1-200(qw) + BG3(biw) represent the groups of PD1 antibody (200. mu.g/mouse) in combination with β -1,3/1, 6-glucan (0.3, 1 or 1mg/kg), respectively.

FIGS. 21A-F show the effect of intravenous injection of β -1,3/1, 6-glucan in combination with PD-1 antibody on the expression of immune-related cytokines in murine colon carcinoma MC3 mice.

FIGS. 22A-C show the effect of oral administration of β -1,3/1, 6-glucan in combination with PD-1 antibody on the growth of mouse colon carcinoma MC38 mouse transplants.

Detailed Description

Other advantages and features of the present invention will become more apparent from the following detailed description of the embodiments of the present invention when read in conjunction with the accompanying drawings, which are set forth in the appended claims and the accompanying drawings, but the scope of the invention is not limited to the embodiments. Modifications and adaptations of the present invention may occur to those skilled in the art based on the teachings herein and fall within the scope of the present invention.

Term(s) for

Unless defined otherwise, the following terms used in the specification and claims have the meanings that are commonly understood by those skilled in the art. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety unless otherwise indicated.

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

2) Polysaccharide (Polysaccharide): the term "sugar chain" means a sugar chain formed by glycosidic bonding and is a polymeric carbohydrate formed by condensation and dehydration of a plurality of monosaccharide residues.

3) Glucose (Glucose): glc; c₆H₁₂O₆The most widely distributed and important monosaccharide in nature.

4) Oligosaccharide (Oligosaccharide): also called oligosaccharides, are carbohydrate compounds formed by connecting 2-20 identical or different monosaccharide residues through glycosidic bonds.

5) Sugar residue: refers to the hydrolyzed group obtained after hydrolysis of the saccharide.

6) D, gluco-oligosaccharide: oligosaccharides are composed of glucose residues linked by glycosidic linkages.

7) And (3) glucan: polysaccharides consisting of glucose residues linked by glycosidic bonds.

8) ESI: electrospray ionization (ionization) is a more common ionization method used in mass spectrometry.

9) CID: collision Induced Dissociation (collisioninduced Dissociation), a process in which energy is transferred to an ion by collisions with neutral molecules, the energy transfer being sufficient to cause cleavage and rearrangement of the bond.

10) MS: mass spectrometry (mass spectrometry), an analytical method for measuring the charge-to-mass ratio (charge-to-mass ratio) of ions.

In the present invention, "β -1,3/1, 6-glucan" and "β -glucan" are used interchangeably, and their conceptual expressions are the same.

In the present invention, "β -1,3/1, 6-glucan" includes a structure of formula I, a structure of formula II, or a derivative form thereof, or a combination thereof, e.g., 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 the structure of formula II; or the structure of formula II may be 0-100% with the balance being the structure of formula I. In the present invention, the structure of formula II can be regarded as open ring formula I. In addition, formula I and formula II may be interconverted by suitable conditions or reagents.

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

In another preferred embodiment, the beta-1, 3/1, 6-glucan has an optical rotation greater than-15 deg., such as between-15 deg. and-25 deg., preferably between-16 deg. and-21 deg..

As used herein, the terms "strong anion resin" and "strong anion exchange resin" are used interchangeably to refer to an anion resin that contains stronger reactive groups such as quaternary amine groups and the like. In general, strong anion resins can be used to remove impurities having sulfonic acid, carboxyl, and the like groups.

As used herein, the terms "weak anion resin" and "weak anion exchange resin" are used interchangeably and refer to an anion resin that contains weaker reactive groups such as diethylaminoethyl groups. In general, weak anionic resins can be used to remove impurities such as nucleic acids, proteins, pigments, and the like.

The main advantages and beneficial effects of the invention include:

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

In particular, the inventor further improves the preparation method of the beta-1, 3/1, 6-glucan, and obtains the beta-1, 3/1, 6-glucan which only has an absorption peak at the end in the ultraviolet full wavelength scanning spectrum and has no obvious absorption peak in the range of 230-900nm (especially in the range of 260-280 nm) and further increases the optical rotation (such as the range of-15 to-21 degrees) through the combined purification of strong anion chromatography purification and weak anion chromatography purification.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not indicated, are generally carried out according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Example 1 preparation of beta-1, 3/1, 6-glucan

Preparation example 1

(1) Degreasing: drying and crushing Antarctic brown algae, soaking in an organic solvent, and stirring to obtain defatted algae powder;

(2) water extraction: stirring and extracting the degreased algae powder with distilled water at room temperature to obtain water extract;

(3) grading: centrifuging the water extract obtained in the step (2), and adding 1-3 mol/L calcium chloride aqueous solution into the supernatant obtained by centrifuging; stirring, centrifuging, collecting supernatant, dialyzing with distilled water or ultrafiltering for desalting, concentrating under reduced pressure, and drying to obtain crude polysaccharide;

(4) and (3) purification: and (3) dissolving the crude polysaccharide obtained in the step (3) by using distilled water and a sodium chloride aqueous solution as mobile phases, separating and purifying by using strong anion exchange resin, collecting water elution components, concentrating under reduced pressure, and freeze-drying to obtain the beta-1, 3/1, 6-glucan.

Unless otherwise specified, the beta-1, 3/1, 6-glucan or validated beta-1, 3/1, 6-glucan used in examples 2-19 was prepared as described in this preparation.

Preparation example 2

(1) Degreasing: drying and crushing Antarctic brown algae, soaking in an organic solvent, and stirring to obtain defatted algae powder;

(2) water extraction: stirring and extracting the degreased algae powder with distilled water at room temperature to obtain water extract;

(3) grading: centrifuging the water extract obtained in the step (2), and adding 1-3 mol/L calcium chloride aqueous solution into the supernatant obtained by centrifuging; stirring, centrifuging, collecting supernatant, dialyzing with distilled water or ultrafiltering for desalting, concentrating under reduced pressure, and drying to obtain crude polysaccharide;

(4) and (3) purification: dissolving the crude polysaccharide obtained in the step (3) by using distilled water, taking distilled water and a sodium chloride aqueous solution as mobile phases, separating and purifying by using strong anion exchange resin, collecting water elution components, and concentrating under reduced pressure;

(5) and (3) second purification: separating and purifying the water elution component in the step (4) by using distilled water as a mobile phase through weak anion exchange resin, and collecting the water elution component;

(6) and (3) decoloring: and (5) separating and purifying the water elution component by using an active carbon column, taking distilled water as a mobile phase, and collecting the water elution component. Concentrating under reduced pressure, and lyophilizing to obtain the beta-1, 3/1, 6-glucan.

Unless otherwise specified, the beta-glucan or validated beta-glucan used in examples 20-25 was prepared as in this preparation.

Examples 2-7 analysis of beta-glucan compositions

The beta-glucan composition in this example was analyzed by High Performance Gel Permeation Chromatography (HPGPC) in combination with an eighteen angle laser light scatterometer (MALLS) and a differential detector (RI):

the determination method comprises the following steps: using 0.1mol/L Na₂SO4 the beta-glucan composition was formulated into solutions of varying concentrations and injected sequentially from low to high concentration into the DNDC meter to calculate the dn/dc of the beta-glucan composition. The analysis is carried out by using High Performance Gel Permeation Chromatography (HPGPC) in combination with an eighteen-angle laser scattering instrument (MALLS) and a differential detector (RI), and the chromatographic conditions are as follows: column TSK-Gel G3000PW (7.5X 300 mm); mobile phase 0.1mol/L Na₂SO 4; the column temperature is 35 ℃; the flow rate is 0.5 mL/min; and a differential detector and an eighteen-angle laser detector are used together. The weight average molecular mass and distribution analysis of the β -glucan composition were obtained by substituting the dn/dc measurements of the β -glucan composition (results are shown in table 1 and fig. 5-7).

The weight percentage of pentasaccharide-decasaccharide (Dp 5-10) β -glucan, decasaccharide-icosaccharide β -glucan, icosaccharide-pentacose β -glucan, icosaccharide-triacontose β -glucan, triacontose-forty saccharide β -glucan, forty saccharide-fifty saccharide β -glucan, fifty saccharide-sixty saccharide β -glucan, sixty saccharide-heptasaccharide β -glucan, seventy saccharide-eighty saccharide β -glucan, greater than eighty saccharide β -glucan was calculated.

The results obtained for examples 2-7 are shown in Table 1:

example 8 identification of the side chain length of beta-Glucan

1) Preparation of side chain oligosaccharides in beta-glucan composition: dissolving the beta-glucan composition to prepare about 5% of the beta-glucan composition, adding 10 mu L of endo-1, 3-beta-glucanase, and carrying out enzymolysis for 8h at 40 ℃. Boiling for 10min, centrifuging at 10000rpm for 10min, collecting supernatant, and freeze drying to obtain side chain oligosaccharide in the beta-glucan composition.

2) ESI-CID-MS/MS analysis of the structure of the side chain oligosaccharides in the beta-glucan composition of the invention. Mass spectrum conditions: an LTQ-Qrbitrap XL mass spectrometer, the capillary voltage is-3000V, the injection cone voltage is-50V, the ion source temperature is 80 ℃, the dissociation temperature is 150 ℃, the sheath flow rate is 8arb, and the sample flow rate is 3-5 mu L/min. The collision gas is helium, and the collision voltage is 15-30 eV.

3) The mass spectrum of the side chain oligosaccharide in the beta-glucan composition with each polymerization degree is shown in the attached figures 1-4. Attributing each signal peak in the mass spectrogram, and verifying the molecular structure of the side chain oligosaccharide in the beta-glucan composition, namely the structure shown in the general formula (III), the formula (IV), the formula (V) or the formula (VI). Wherein, FIG. 1 shows the mass spectrum of Glc beta 1-6Glc beta in the formula (IV) as the side chain, FIG. 2 shows the mass spectrum of Glc beta 1-3Glc beta in the formula (IV) as the side chain, FIG. 3 shows the mass spectrum of the formula (V) as the side chain, and FIG. 4 shows the mass spectrum of the formula (VI) as the side chain.

Example 9 advanced Structure information

The beta-glucan composition of the invention has a triple helix structure. Congo red can form a complex with a polysaccharide having a triple helical chain conformation, the maximum absorption wavelength of the complex being red-shifted compared to congo red. The beta-glucan composition of the invention can form a complex with congo red under alkaline conditions, so that the maximum absorption wavelength of the beta-glucan composition is red-shifted by more than 15 nm (shown in figure 8).

The reducing end of the beta-glucan composition of the invention has a sugar alcohol structure. 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 represented by the presence of a sugar alcohol at the reducing end¹³In C-NMR, the molecular weight distribution was characterized by 63.16ppm (FIG. 9).

Example 10: beta-1, 3/1, 6-glucan can enhance phagocytosis of bone marrow-derived macrophages

In this example, the beta-1, 3/1, 6-glucan saccharide of the present invention was examined for peripheral blood innate immune cellsTwo major populations, monocytes (CD11b + Ly 6C)^hiFig. 10A and 10B) and granulocytes (CD11B + Ly6G +, fig. 10A and 10C). As shown in fig. 10D, β -1,3/1, 6-glucan-FITC bound well to monocytes (12.8%) and to very few granulocytes (1.5%).

Next, the effect of the β -1,3/1, 6-glucan of the invention on the phagocytic activity of differentially polarized BMDMs was evaluated. Phagocytic activity of the macrophages GM-BMDMs and M-BMDMs, which are slightly upregulated by β -1,3/1, 6-glucan FIGS. 11A and B. At doses above 10 μ g/ml, the phagocytosis of the structurally similar β -glucan LNT was higher than that of the β -1,3/1, 6-glucan of the invention (fig. 11B). However, as shown in fig. 11C and 11D, LNT significantly reduced cell viability of BMDMs in a dose-dependent manner. No cytotoxicity was observed with the beta-1, 3/1, 6-glucan of the present invention even at a concentration of 100. mu.g/ml.

Example 11: the beta-1, 3/1, 6-glucan of the invention reduces tumor burden and improves spleen index in a tumor transplantation mouse model

Blood monocytes are recruited into the tumor microenvironment to differentiate into macrophages, creating an immunosuppressive and tumor promoting microenvironment. Remodeling immunosuppressive macrophages to a pro-inflammatory state will manipulate the tumor microenvironment and prevent tumor growth in vivo. The anti-tumor effect of the beta-1, 3/1, 6-glucan of the invention in a human colorectal cancer cell DLD1 xenograft mouse model is tested.

As shown in fig. 12A-C, the β -1,3/1, 6-glucan of the present invention inhibited tumor volume (fig. 12A, fig. 12B) and tumor weight (fig. 12C). Notably, unlike classical chemotherapeutic compounds, the β -1,3/1, 6-glucan of the present invention inhibits tumor growth independent of dose. Low doses of 2mg/kg beta-1, 3/1, 6-glucan were more potent in inhibiting tumor growth than high doses (4mg/kg and 8mg/kg) (FIG. 12A). The beta-1, 3/1, 6-glucan of the invention, as an immunostimulant, significantly up-regulated the spleen index of tumor-bearing mice, suggesting increased activation and infiltration of immune cells (fig. 12D). Each treatment had no significant effect on mouse body weight average (fig. 12E).

Example 12: the beta-1, 3/1, 6-glucan of the invention up-regulates the phagocytic activity of macrophages in vitro and the secretion of proinflammatory cytokines/chemokines

In this example, macrophages were detected by inducing macrophages in mice from control and β -1,3/1, 6-glucan treated groups of mice and co-culturing with 9 different colorectal cancer cell lines.

As shown in fig. 13A, macrophages induced by β -1,3/1, 6-glucan group mice showed strong phagocytic activity against all cancer cell strains we detected. The secretion of cytokines and chemokines is an important indicator of the activation of immune functions in the body. The beta-1, 3/1, 6-glucan of the invention potently increases the secretion of pro-inflammatory cytokines (IL-1 beta and TNF alpha, which are primarily secreted by monocytes/macrophages). The beta-1, 3/1, 6-glucan treatment can also up-regulate the expression level of proinflammatory cytokines such as IL-2 and IL12p70 and the chemokine CXCL1 produced by immune cells such as macrophage cells. Beta-1, 3/1, 6-glucan 2mg/kg and 4mg/kg groups of mice IFN gamma levels were increased. These data suggest that the β -1,3/1, 6-glucan of the present invention not only triggers phagocytic activity of macrophages in vivo, but also promotes secretion of pro-inflammatory cytokines/chemokines, exerting an anti-tumor effect.

Example 13: the beta-1, 3/1, 6-glucan of the invention increases infiltration of pro-inflammatory macrophages and B cells in a tumor microenvironment

Detecting immune cell composition in the circulating blood system. The proportion of B cells (CD19+) and dendritic cells (CD11c +) was found to increase, while the proportion of myeloid cells (CD11B +) was slightly decreased (fig. 14A). β -1,3/1, 6-glucan treatment induced the formation of a myeloid subset, a pro-inflammatory monocyte-derived macrophage (CD11B + Ly6Chi) (fig. 14B).

Tumors were dissected and the percentage of tumor infiltrating immune cells was calculated. Within the tumor, the percentage of myeloid cells (CD11b +) was slightly up-regulated after β -1,3/1, 6-glucan treatment of the invention (fig. 14C). In addition, β -1,3/1, 6-glucan promoted infiltration of a proinflammatory monocyte-derived macrophage subpopulation (CD11b + Ly6Chi) (fig. 14D). Compared with the control group, in the beta-1, 3/1, 6-glucan mice of the invention, anti-tumor pro-inflammatory tumor associated macrophage cells (TAMs) (CD11b + CD80+) were up-regulated, and the prototumor immunosuppression TAMs (CD11b + CD206+) were down-regulated (FIG. 14E). These data indicate 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 treatment with β -1,3/1, 6-glucan of the invention (fig. 14F). Thus, the beta-1, 3/1, 6-glucan of the present invention can regulate the composition of systemic and intratumoral immune cells, placing them in pro-inflammatory and anti-tumor states, thereby hindering tumor growth in vivo.

Tumors were grouped according to the volume of β -1,3/1, 6-glucan treated tumors to detect immune cell (CD45+) infiltration. Large tumors refer to tumors that are higher than average in volume in the beta-1, 3/1, 6-glucan treated group of the invention, and vice versa. As shown in FIG. 14F, all tumors treated with β -1,3/1, 6-glucan of the invention contained more immune cells (CD45 +). In addition, large tumors have more immune cell infiltration than small tumors. In conclusion, these data suggest that the beta-1, 3/1, 6-glucan of the present invention may trigger tumor infiltration of immune cells such as pro-inflammatory macrophages and inhibit cancer cell growth in vivo.

Example 14: the beta-1, 3/1, 6-glucan of the invention reduces tumor burden in AOM-DSS-induced colorectal cancer models

To verify the anti-tumor effect of the beta-1, 3/1, 6-glucan of the present invention in immunocompromised mice, the anti-tumor effect of beta-1, 3/1, 6-glucan was examined in AOM/DSS-induced C57BL/6J mouse colorectal cancer model. In the later stages of tumor progression, the body weight average at 1 and 3mg/kg doses was higher in the β -1,3/1, 6-glucan group than in the vehicle group, indicating that these treated mice were in a better physical condition (FIG. 15A). No significant difference in colon length was found, except that 3mg/kg of beta-1, 3/1, 6-glucan treatment slightly increased the colon length (FIG. 15B). The number of colorectal tumors induced per mouse was reduced even after low dose 1mg/kg β -1,3/1, 6-glucan treatment (fig. 15C). As shown in FIG. 15D, the number of tumors with tumor diameters ranging from 2mm to 4mm and above 4mm was reduced at the dose of 3mg/kg after treatment with β -1,3/1, 6-glucan. After AOM/DSS, 1mg/kg of β -1,3/1, 6-glucan of the invention induced a smaller number of tumors greater than 4mm in diameter than vehicle (FIG. 15E).

Example 15: the beta-1, 3/1, 6-glucan of the invention reverses AOM-DSS-induced changes in immune cell composition

Changes in various immune cells including myeloid cells (CD11b +) were observed following AOM/DSS induction and treatment with β -1,3/1, 6-glucan according to the invention. AOM/DSS increased the proportion of myeloid cells (CD11b +) in the circulating blood system, and the β -1,3/1, 6-glucan treatment of the present invention reversed this trend (FIG. 16A). At the same time, β -1,3/1, 6-glucan increased down-regulated B cells in blood (fig. 16A) and spleen (fig. 16B) caused by AOM/DSS administration in vivo, and reversed the reduction of CD 4T cells in blood (fig. 16A) and lymph nodes (fig. 16C). Thus, β -1,3/1, 6-glucan treatment reversed the changes in immune cell composition caused by AOM/DSS administration. Furthermore, BETA-1, 3/1, 6-glucan of the present invention induced the up-regulation of the secretion of the inflammatory factor IFN γ (FIG. 16D), TNF- α, etc., and the chemokine CXCL1 (FIG. 16E, FIG. 16F, FIG. 16G, FIG. 16H, and FIG. 16I).

These data indicate that the anti-tumor effect of BETA-1, 3/1, 6-glucan of the present invention on immunocompromised mice is associated with modulation of immune cell composition and proinflammatory cytokine/chemokine secretion as an immunostimulant.

Example 16: beta-1, 3/1, 6-glucan of the invention has effect of inhibiting inoculation of a naked mouse with a scattered human colon cancer cell line

Recovering the mouse colon cancer cell strain HCT-116 by liquid nitrogen, and culturing by using a 5A culture medium containing 10% fetal bovine serum; expanding the cultured cells in a culture bottle, digesting and collecting the cells after the required cell amount is reached; diluting with 5A culture medium containing 10% fetal calf serum to 25000 ten thousand/ml cell suspension, inoculating 0.2ml of the cell suspension into right forelimb axillary fossa subcutaneous part of mouse after conventional sterilization, sacrificing animal to remove tumor tissue when about 1g of tissue block grows, cutting into tumor blocks of about 2-3 cubic millimeters, implanting into subcultured nude mouse subcutaneous part by catheter method, and continuously subculturing for 2 times. HT-29, SW-480, DLD-1 and RKO cell lines listed in the tables were modeled and tested in the same manner. After the tumor of the subcultured tumor-bearing nude mice grows about 1g, the tumor mass is transplanted and modeled by adopting the method, and the tumor mass is randomly grouped and the drug administration is started the next day after the transplantation.

Animals were divided into groups of 8 animals each using a random grouping method. The following table is given for each group of animals:

the administration route is tail vein injection, and the dosage of each animal is determined according to the weight of the animal which is the most recent time before administration. Each group was dosed 2 times per week, continuously until the end of the experiment. After the experiment is finished, tumor tissues are required to be stripped after the animals which die accidentally and survive are euthanized, the weights of the tumors are weighed, the difference of the weights of the tumors in each group is calculated, and the tumor inhibition rate IR is further calculated_TWAs a reference index, the calculation formula is as follows:

IR_TW(％)＝(W_{model set}-W_{Administration set})/W_{Model set}×100％

As can be seen from the results in the table, the beta-1, 3/1, 6-glucan of the invention shows more remarkable inhibition effect on the growth of transplanted tumor of nude mouse of human colon cancer heterogeneous BALB/c nude of HCT-116, HT-29, SW-480, DLD-1 and RKO cells under the experimental conditions of 1mg/kg, 3mg/kg and 9mg/kg administration doses for 2 times per week.

Example 17 inhibitory Effect of beta-1, 3/1, 6-glucan of the present invention on mouse S180 sarcoma residual tumor model

S180 mouse fibrosarcoma cells were used, and culture expansion was performed using DMEM high-glucose medium containing 10% FBS. The cells enter logarithmic growth phase and are ready for further testing. Cells in the logarithmic growth phase were collected, centrifuged and counted. The cell concentration was adjusted and 0.2 ml/mouse was inoculated into the abdominal cavity of Kunming mouse as the first generation mouse. After raising for one week, taking ascites in the abdominal cavity of the first generation mouse, centrifuging and counting, adjusting the cell concentration, inoculating 0.2 ml/abdominal cavity of the Kunming mouse as a second generation mouse, after raising for one week, taking ascites in the abdominal cavity of the second generation mouse, centrifuging and counting, adjusting the cell concentration, and inoculating 0.2 ml/abdominal cavity of the Kunming mouse under the back subcutaneous surface. The whole inoculation process is carried out in an ultra-clean workbench in an aseptic operation mode. And observing the growth condition of the mice after inoculation, screening according to the growth state of the mice, and randomly grouping and administering the mice after screening. The experimental results are shown in the table below, and the beta-1, 3/1, 6-glucan can obviously inhibit the regrowth of the tumor after the operation at 3 mg/kg.

Example 18 inhibition of beta-1, 3/1, 6-glucan of the invention against the mouse Lewis lung carcinoma residual model

Mouse lung cancer Lewis cells are used, and a DMEM high-glucose medium containing 10% FBS is used for culture and amplification. The cells enter logarithmic growth phase and are ready for further testing. Cells in the logarithmic growth phase were collected, centrifuged and counted. The cell concentration was adjusted to 0.2 ml/mouse and inoculated subcutaneously in the right armpit of C57BL/6 mouse as a seed mouse. When the tumor grows to be larger than 1000mm³And then taking out aseptically, weighing, adding sodium chloride injection with the mass-volume ratio of 1:4, diluting and grinding, inoculating 0.2ml of the mixture into each mouse right forelimb axilla for subculture after conventional disinfection. When the tumor grows to be larger than 1000mm³And then, taking the second-generation tumor, taking out the second-generation tumor aseptically, weighing, adding sodium chloride injection with the mass-volume ratio of 1:4, diluting and grinding, inoculating 0.2ml of the second-generation tumor into the subcutaneous back of the mouse after conventional disinfection, observing the growth condition of the mouse after inoculation, screening according to the growth state of the mouse, and randomly grouping and administering after screening. The results of the experiments are shown in the following table,the beta-1, 3/1, 6-glucan of the invention can obviously inhibit the regrowth of the tumor after the operation at 1mg/kg, 3mg/kg and 9 mg/kg.

Example 19 inhibition of inflammation-associated colorectal cancer in mice induced by AOM/DSS by beta-1, 3/1, 6-glucan

Cancer is one of the leading causes of death in humans, and the etiology and pathogenesis of about 1/4 cancer cases are associated with chronic inflammation. Inflammatory Bowel Disease (IBD) is an intestinal inflammatory disease with unknown etiology and pathogenesis, which is classified into Ulcerative Colitis (UC) and Crohn's Disease (CD) according to its pathological features, and has a tendency to increase and become younger year by year. IBD patients have an increased risk of developing colorectal cancer (CRC), which increases by 0.5-1% per year after 8-10 years, and up to 18% of IBD patients may develop CRC after 30 years. Although IBD-associated CRC accounts for only 1% -2% of all colorectal cancers, it is a common cause of death in IBD patients. Because the complete process of IBD induced CRC is successfully simulated, the animal model of the Colitis Associated Cancer (CAC) induced by Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) is widely used for researching the drug effect and mechanism of a new medicine, and the pathological change and canceration principle of the model are clarified to help to find a new candidate target for treating colorectal cancer.

In the experiment, a colorectal cancer model of mice is established by adopting a method of intraperitoneal injection of AOM (argon oxygen decarburization) and periodic administration of DSS (DSS) drinking solution, and 70 male C57BL/6 mice aged 6-8 weeks are randomly grouped into 10 mice each group and 7 groups in total at the beginning of the experiment. Except for the first group, which was a normal control, the other groups were molded with AOM/DSS. Mice were treated with a single intraperitoneal injection of 10mg/kg Azoxymethane (AOM) on the first day of the start of the experimental study, and seven days later, drinking water containing 1% sodium dextran sulfate (DSS) was administered to the mice. Seven days later, fourteen more days of regular drinking water, 7 days of 1% DSS drinking water and 14 days of regular drinking water were given as one cycle, and thus three cycles were repeated in total, finally inducing the mice to develop CAC.

After the experiment is finished, the mouse is dissected, the whole colorectal is taken out, mesentery and attached adipose tissues are carefully removed, the length of the mesentery and the attached adipose tissues is measured, then the intestinal cavity of the whole colorectal is washed clean by normal saline and longitudinally dissected, macroscopic tumors are counted and measured by a vernier caliper under a dissecting microscope, and finally the effect of the beta-1, 3/1, 6-glucan is evaluated from the aspects of colon length, tumor formation rate, total number of tumors and the like.

The experimental results are shown in the table below, compared with the blank group, the colon length is obviously shortened (P <0.01), the tumor formation rate reaches 77.78%, and the AOM/DSS induces the mouse colorectal cancer model successfully. The tumor formation rate of the beta-1, 3/1, 6-glucan (3mg/kg) is 16.67%, and is reduced compared with that of a model group, which shows that the beta-1, 3/1, 6-glucan (3mg/kg) has an inhibiting effect on mouse colorectal cancer induced by AOM/DSS.

P <0.05vs. blank; p <0.01vs. blank; model # p <0.05 vs; model # p <0.01 vs;

EXAMPLE 20 characterisation of beta-1, 3/1, 6-glucan obtained in preparation 1 and preparation 2

The test method is as follows:

(i) and (3) ultraviolet full-wavelength scanning atlas, dissolving the beta-glucan composition to prepare a concentration of 5 wt%, establishing a base line by using pure water, setting the scanning wavelength to be 190-900 nm, and the scanning precision to be 1nm, and performing ultraviolet full-wavelength scanning.

(ii) Side chain length was determined as in example 8

Example 21 Tail intravenous beta-1, 3/1, 6-glucan anti-mouse S-180 experiment

Animal experiment for mice with normal immune function (caudal vertebra vein administration)

1. The experimental scheme is as follows:

tumor cells: s-180; inoculation part: at the scapula; mouse species: KM (male mouse); number of mice: each group comprises 13; inoculating the tumor cells, and starting administration the next day; the administration frequency is as follows: daily administration; the administration period is as follows: 20 days; the administration mode comprises the following steps: intravenous injection into the caudal vertebra;

blank control group: model group (CN group)

Positive control group: cisplatin (2mg/kg) (PC or CP group); lentinan LNT (1.5mg/kg)

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

2. Results of the experiment

The results are shown in FIGS. 18A-B. And the positive control group and the experimental group of mice still have good physiological state after being inoculated for 20 days, and the animals are very active and quick to move before sacrificing.

Compared with a model group, the beta-1, 3/1, 6-glucan can obviously inhibit the growth of S-180 tumor at low, medium and high doses, and the tumor inhibition rate is up to 60-70%. The weight of the thymus of the mice in the cis-platinum group is obviously lower than that of the mice in the model group, the weight of the thymus of the mice in the beta-1, 3/1, 6-glucan administration group (0.3, 1.5 and 7.5mg/kg) is obviously higher than that of the thymus of the mice in the cis-platinum group, and the weight of the thymus of the mice in the high-dosage BG136 administration group is equivalent to that of the thymus of the model group. It is suggested that beta-1, 3/1, 6-glucan has a high potential to exert an anti-tumor effect by activating the immune system of mice.

Example 22 oral beta-1, 3/1, 6-glucan antitumor assay

1. The experimental scheme is as follows:

tumor cells: s-180; inoculation part: axilla region; mouse species: kunming mice (Kunming); number of mice: 13 mice per group (7 female mice 6 male mice); administration start time: administration is carried out the next day after inoculation; the administration frequency is as follows: daily administration; the administration period is as follows: 10 days; the administration mode comprises the following steps: orally taking; dose administered (mg/kg): model (CN); ② cisplatin (CP or PC): 1.5 mg; ③ beta-1, 3/1, 6-glucan: 1mg (B1); beta-1, 3/1, 6-glucan: 5mg (B5); beta-1, 3/1, 6-glucan: 25mg (B25); sixthly, 1.5mg of cisplatin and BG1mg (CPB 1); (vii) cisplatin 1.5mg + BG5mg (CPB 5); (viii) 1.5mg of cisplatin + BG 25mg (CPB 25).

2. The experimental results are as follows:

the results of the experiment are shown in FIGS. 19A and B. The experimental result shows that the oral beta-1, 3/1, 6-glucan has obvious inhibition effect on S-180 tumor, the total inhibition rate of the oral beta-1, 3/1, 6-glucan is more than 58% when the oral dosage of the beta-1, 3/1, 6-glucan is 1mg/kg, and the tumor inhibition rate of male mice is as high as 70.4%. When BG136 is combined with cisplatin (1.5mg/kg) medicines at 25mg/kg, the inhibition rate is further improved, and the tumor inhibition rate reaches 77.3%.

Example 23 beta-1, 3/1, 6-Glucan combination chemotherapy for use in mouse tumor models

Effect of beta-1, 3/1, 6-glucan combination chemotherapy on leukocytes and platelets

Mix 3x10⁵Cell suspension of mouse melanoma cell line B16(Perkinelmer gift) was injected subcutaneously (Overwijk) into C57BL/6J mice (female, 6-8 weeks old, purchased from England-Hay laboratory animals Co., Ltd.)&Restifo, 2001). Approximately 2 days after tumor implantation, carboplatin (30mg/kg, twice weekly) and BG136(4mg/kg) or lentinan LNT (2mg/kg) were intraperitoneally injected, tumor volume was measured during dosing, and tumor mass was measured on day 15 after animal sacrifice. Blood is taken from the heart after the animal is sacrificed and is put into an EDTA-anticoagulation tube, and after the blood is uniformly mixed, 50 mu l of whole blood is taken, and the concentration of the immune cells is detected by a blood analyzer.

The test results showed that β -1,3/1, 6-glucan prepared in preparation example 2 can enhance the tumor suppressive effect of carboplatin and can stimulate the immune response, reverse the immunosuppression after administration of carboplatin, and decrease platelets.

Example 24

Since immune cells play an important role in a variety of tumor cell types, the anti-tumor and leukocyte and platelet increasing effects of β -1,3/1, 6-glucan are exerted in a variety of tumor cells (e.g., lung cancer, kidney cancer, liver cancer, breast cancer, etc.) and immune cells also play a regulatory role in tumor metastasis and occurrence, and thus β -1,3/1, 6-glucan may inhibit metastasis and occurrence of tumor cells.

Example 25 Effect of β -1,3/1, 6-glucan in combination with PD-1 antibody on a mouse tumor model

PD-1 antibody used: name: in vivo MAb anti-mouse PD-1(CD 279); purchased from Bioxcell

(i) Effect of intravenous injection of beta-1, 3/1, 6-glucan in combination with PD-1 antibody on subcutaneous transplantation tumor of colon cancer MC38 mice

In this example, a mouse colon cancer cell line MC38 was selected and tested for the effect of β -1,3/1, 6-glucan in combination with PD-1 antibody on the growth of mouse transplantable tumors, as shown in FIGS. 20A-C.

The results of fig. 20A and B show that the beta-1, 3/1, 6-glucan combined with PD-1 antibody injected into tail vein can effectively inhibit the growth of MC38 transplanted tumor, the inhibition effect is obviously better than that of the PD-1 antibody group administered alone, and the compound has significant synergistic effect, no obvious drug toxicity (fig. 20C), and good medication safety.

(ii) Effect of intravenous injection of beta-1, 3/1, 6-glucan in combination with PD-1 antibody on expression of mouse tumor immune-related cytokines

In this example, the expression of various cytokines in mouse prostate cancer cell MC38 transplants was quantified by RT-PCR. The results are shown in FIGS. 21A-F.

As can be seen from the results of FIG. 21A, B and C, the expression levels of tumor necrosis factor alpha (TNF α), interleukin 1 β (IL1- β) and nitric oxide synthase (iNOS) produced by macrophage cells in the tumors of mice were significantly increased by combining β -1,3/1, 6-glucan with PD-1 antibody, indicating that β -1,3/1, 6-glucan can stimulate immune activation in the tumors in cooperation with PD-1 antibody;

from the results of fig. 21D, E and F, it can be seen that the expression levels of pro-inflammatory Th1 polarized cytokines (interferon γ, IFN- γ), interleukin 2(IL-2) and granzyme b (gzmb) are increased after the combination of β -1,3/1, 6-glucan and PD-1 antibody, further indicating that the combination of β -1,3/1, 6-glucan and PD-1 antibody can bridge the innate immunity and adaptive immunity of the body and exert the optimal anti-tumor immunity.

(iii) Effect of oral administration of beta-1, 3/1, 6-Glucan in combination with PD-1 antibody on mouse tumor models

A mouse colon cancer cell line MC38 is selected to detect the influence of beta-1, 3/1, 6-glucan and PD-1 antibody on the growth of mouse transplantable tumor. The results are shown in FIGS. 22A-C.

The results of fig. 22A and B show that oral administration of β -1,3/1, 6-glucan in combination with PD-1 antibody can effectively inhibit the growth of MC38 transplanted tumors, and the inhibition effect is significantly better than that of the PD-1 antibody group administered alone, and the oral administration has significant synergistic effect, no significant drug toxicity (fig. 22C), and good drug safety.

All documents referred to herein are incorporated by reference into this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A β -glucan composition comprising a 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 not more than 4 glucose residues.

2. The beta-glucan composition according to claim 1, wherein R in the structure of formula (I) or formula (II) is one or more of the structures of formula (III), formula (IV), formula (V) or formula (VI), wherein

Formula (III): glc β 1-;

formula (IV): glc β 1-3Glc β 1-or Glc β 1-6Glc β 1-;

formula (V): glc beta 1-3Glc beta 1-or Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc β 1-3Glc β 1-6Glc β 1-or Glc β 1-6Glc β 1-;

formula (VI): glc beta 1-3Glc beta 1-or

Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-or

Glc beta 1-6Glc beta 1-3Glc beta 1-or

Glc beta 1-6-Glc beta 1-3-Glc beta 1-6Glc beta 1-or

Glc beta 1-3Glc beta 1-6Glc beta 1-or

Glcβ1-6Glcβ1-6Glcβ1-6Glcβ-。

3. The beta-glucan composition according to claim 1, wherein in the beta-glucan composition, the weight content of pentasaccharide-decasaccharide is 0-50.0%, the weight content of decasaccharide-icose is 0-80.0%, the weight content of icose-icose is 0-25.0%, the weight content of icose-triacontose is 0-45.0%, the weight content of triacontose-forty-sugar is 0-30.0%, the weight content of forty-fifty-sugar is 0-15.0%, the weight content of fifty-sixty-sugar is 0.1-55.0%, the weight content of sixty-heptasaccharide is 0.1-20.0%, the weight content of heptadecasaccharide-octadecasaccharide is 0.1-15.0%, and the weight content of more than octadecasaccharide is 0-40.0%.

4. The beta-glucan composition according to claim 3, wherein in the beta-glucan composition, the weight content of pentasaccharide-decasaccharide is 0-45.0%, the weight content of decasaccharide-icose is 0-75.0%, the weight content of icose-icose is 0-20.0%, the weight content of icose-triacontose is 0-40.0%, the weight content of beta-glucan with the degree of polymerization of 30-40 is 0-25.0%, the weight content of forty-fifty-saccharide is 0-10.0%, the weight content of fifty-sixty-saccharide is 0.1-50.0%, the weight content of sixty-heptasaccharide is 0.1-15.0%, the weight content of heptadecasaccharide-octaose is 0.1-10.0%, and the weight content of more than octaose is 0-35.0%.

5. The beta-glucan composition according to claim 4, consisting of beta-glucan having a degree of polymerization of 20-80, wherein the weight content of icosanose-icosanose is 13.2-19.8%, the weight content of icosanose-triacontose is 29.1-43.7%, the weight content of triacontose-tetrantaose is 18.2-27.4%, the weight content of forty-fifty-ose is 7.3-10.9%, the weight content of fifty-sixty-sugar is 4.5-6.7%, the weight content of sixty-heptadecaose is 3.5-5.3%, and the weight content of heptadecaose-octadecaose is 4.2-6.2%.

6. The beta-glucan composition according to claim 4,

the beta-glucan composition consists of beta-glucan having a degree of polymerization of 20 to 80, wherein the weight content of icosyl-icosyl is 16.5%, the weight content of icosyl-triacontose is 36.4%, the weight content of triacontose-forty-ose is 22.8%, the weight content of forty-fifty-ose is 9.1%, the weight content of fifty-sixty-sugar is 5.6%, the weight content of sixty-heptadecaose is 4.4%, and the weight content of heptadecaose-octadecaose is 5.2%; or

The beta-glucan composition is composed of beta-glucan with a polymerization degree of 10-80, wherein the weight content of decaose-icosanose is 40.6-60.8%, the weight content of icosanose-eicosapentaenoic sugar is 13.4-20.0%, the weight content of icosanose-triacontose is 7.7-11.5%, the weight content of triacontose-tetradotose is 7.6-11.4%, the weight content of forty-fifty sugar is 4.2-6.2%, the weight content of fifty-sixty sugar is 2.3-3.5%, the weight content of sixty-heptadecaose is 1.6-2.4%, and the weight content of heptadecaose-octadecaose is 0.8-1.2%; or

The beta-glucan composition is composed of beta-glucan having a degree of polymerization of 10 to 80, wherein the weight content of decaose-icosanose is 50.7%, the weight content of icosanose-eicosapentaenoic sugar is 16.7%, the weight content of icosanose-triacontose is 9.6%, the weight content of triacontose-forty sugar is 9.5%, the weight content of forty-fifty sugar is 5.2%, the weight content of fifty-sixty sugar is 2.9%, the weight content of sixty-seventy sugar is 2.0%, and the weight content of heptadecaose-eighty sugar is 1.0%.

7. Use of the β -glucan according to any one of claims 1-6 in the preparation of a composition for ameliorating or treating an immune-related disorder.

8. Use of beta-glucan according to claim 7 for the preparation of a composition for ameliorating or treating an immune-related disorder, wherein said immune-related disorder is a tumor or inflammation.

9. Use of beta-glucan according to claim 8 for the preparation of a composition for ameliorating or treating an immune related disorder, wherein said tumor is selected from the group consisting of colorectal cancer, lung cancer, fibrosarcoma, melanoma, renal cancer, liver cancer, breast cancer, or a combination thereof.

10. A composition for ameliorating or treating an immune-related disorder, said composition comprising:

(1) the beta-glucan as claimed in the preceding claim, and

(2) a pharmaceutically acceptable carrier.

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