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WO2024169040A1 - 一种人参杂多糖及其分离方法和应用 - Google Patents

一种人参杂多糖及其分离方法和应用 Download PDF

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Publication number
WO2024169040A1
WO2024169040A1 PCT/CN2023/089771 CN2023089771W WO2024169040A1 WO 2024169040 A1 WO2024169040 A1 WO 2024169040A1 CN 2023089771 W CN2023089771 W CN 2023089771W WO 2024169040 A1 WO2024169040 A1 WO 2024169040A1
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Prior art keywords
heteropolysaccharide
vaccine
gaps
group
ginseng
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PCT/CN2023/089771
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English (en)
French (fr)
Inventor
殷军
周荔葆
翟健秀
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Shenyang Pharmaceutical University
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Shenyang Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of medical technology, and in particular to the separation, identification and application of ginseng polysaccharides.
  • Immune adjuvants refer to substances that can non-specifically change or enhance the body's specific immune response to antigens, and are an important component of vaccines.
  • the mechanism of action of immune adjuvants is mainly to increase the surface area of antigens and improve immunogenicity; to have a sustained release effect on antigens and prolong the retention time of antigens in tissues; to promote inflammatory responses and stimulate active immune responses.
  • Ginseng is the dried root and rhizome of Panax ginseng.A.Meyer, a plant of the Araliaceae family. It has the effects of replenishing vital energy, promoting the production of body fluids and nourishing blood, calming the nerves and improving intelligence. Modern pharmacology shows that ginseng has the effects of improving immunity, anti-tumor, and anti-fatigue. It is widely distributed in Asian countries such as China, Japan, and South Korea. Ginseng mainly contains saponins, polysaccharides, flavonoids and other active ingredients.
  • the present application separates a heteropolysaccharide from ginseng, and through activity tracking and in vivo experiments, it has been proved that the heteropolysaccharide has immune adjuvant activity for a variety of vaccines.
  • a heteropolysaccharide comprising galacturonic acid, arabinose, galactose, glucose, rhamnose, xylose and mannose.
  • heteropolysaccharide comprising 1, 1, 5, 1, 3, 5 linked arabinose residues; 1, 4 linked galacturonic acid residues; 1, 3, 4 linked rhamnose residues; 1, 1,4, 1,3,4 linked galactose residues; 1,1,4, 1,3,4 linked glucose residues; 1,2 linked mannose residues.
  • heteropolysaccharide according to item 1 or 2 wherein the main chain of the heteropolysaccharide is composed of 1,4-linked galacturonic acid alternately linked, and has different degrees of methylation and acetylation, and the side chains of the heteropolysaccharide are composed of arabinan, galactoarabinan and heteropolysaccharide.
  • heteropolysaccharide according to item 3 wherein the heteropolysaccharide is composed of galacturonic acid, arabinose, galactose, glucose, rhamnose, xylose and mannose.
  • heteropolysaccharide according to any one of items 1 to 6, equivalent to 100 moles of the heteropolysaccharide, wherein the molar amount of the following units is: Galacturonic acid: 20.00 ⁇ 65.00, Arabinose: 8.00 ⁇ 40.00, Galactose: 5.00 ⁇ 25.00, Glucose: 5.00 ⁇ 25.00, Rhamnose: 3.00-20.00, Xylose: 0.05 ⁇ 5.00, Mannose: 0.05 ⁇ 5.00.
  • the molar amounts of the following units are: Galacturonic acid: 40.00 ⁇ 50.00, Arabinose: 10.00 ⁇ 20.00, Galactose: 10.00 ⁇ 20.00, Glucose: 5.00 ⁇ 15.00, Rhamnose: 5.00-15.00, Xylose: 0.2 ⁇ 3.00, Mannose: 0.2 ⁇ 3.00.
  • a method for preparing the heteropolysaccharide described in any one of items 1 to 9, comprising:
  • ginseng After ginseng is defatted, it is extracted, precipitated with alcohol, concentrated and dried to obtain crude ginseng polysaccharide.
  • the crude ginseng polysaccharide is eluted by ion exchange column chromatography to obtain a heteropolysaccharide extract;
  • the heteropolysaccharide extract is purified by a gel column to obtain the heteropolysaccharide.
  • the ion exchange column is an anion exchange resin column, and preferably, the filler is DEAE Sepharose Fast Flow.
  • the solid-liquid ratio is 1:10-40, the number of times is 2-4 times, and the extraction time is 1-5 hours.
  • the exchange column is eluted with distilled water and sodium chloride aqueous solution in sequence, and the elution flow rate is 0.03 to 12 mL/min.
  • the gel column is eluted with 0.9-2.5 M sodium chloride, and the elution flow rate is 0.01-0.08 mL/min.
  • a composition comprising the heteropolysaccharide described in any one of items 1 to 9 or the heteropolysaccharide prepared according to the method described in any one of items 10 to 14 and a pharmaceutically acceptable carrier and/or excipient.
  • heteropolysaccharide described in any one of items 1 to 9, or the heteropolysaccharide prepared by the method described in any one of items 10 to 14, or the composition described in item 15 is used as a vaccine adjuvant.
  • the vaccine includes but is not limited to influenza vaccine, rabies vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine or new coronavirus vaccine vaccine.
  • heteropolysaccharide described in any one of items 1 to 9, or the heteropolysaccharide prepared by the method described in any one of items 10 to 14, or the composition described in item 15 in enhancing the expression of GATA-3, T-bet, IFN- ⁇ or IL-4 mRNA genes in splenic lymphocytes.
  • a method for preventing and/or treating a disease comprising administering the vaccine composition of item 15 to a subject.
  • the disease is selected from rabies, influenza, hepatitis B, hepatitis A, hepatitis C, hand, foot and mouth disease, HPV disease or new coronavirus disease.
  • GAPS-FL ginseng heteropolysaccharide with immune adjuvant activity
  • ginseng heteropolysaccharide GAPS-FL
  • immune adjuvant activity is a new polysaccharide substance isolated from ginseng roots and can non-specifically change or enhance the immune response of various vaccines including influenza vaccines, and can not only enhance the humoral immunity of mice immunized with influenza vaccines but also enhance their cellular immunity.
  • FIG1 is a flow chart of separation and purification of GAPS-FL
  • FIG2 is a DEAE elution curve of GAPS-FL
  • FIG3 is a gel column chromatography elution curve of GAPS-FL
  • FIG4A is a UV spectrum of GAPS-FL
  • FIG4B is an infrared spectrum of GAPS-FL
  • Fig. 5 is a standard curve made by glucose standard
  • FIG6 is a HPGPC spectrum of GAPS-FL
  • FIG7 is an ion chromatography analysis spectrum of GAPS-FL
  • FIG8 is a GC-MS analysis spectrum of GAPS-FL
  • FIG9A is a 1 H-NMR analysis spectrum of GAPS-FL
  • FIG9B is a 13 C-NMR analysis spectrum of GAPS-FL.
  • FIG9C is a DEPT-135 analysis spectrum of GAPS-FL.
  • FIG9D is a 1 H- 1 H COSY analysis spectrum of GAPS-FL;
  • FIG9E is a HSQC analysis spectrum of GAPS-FL
  • FIG9F is a HMBC analysis spectrum of GAPS-FL.
  • FIG9G is a NOESY analysis spectrum of GAPS-FL
  • Figure 9H is the HSQC-TOCSY NMR analysis spectrum of GAPS-FL;
  • FIG10 is the structural formula and structural repeating unit of GAPS-FL;
  • FIG11A is a scanning electron micrograph of GAPS-FL
  • FIG11B is an atomic force microscopy image of GAPS-FL
  • FIG12 is a diagram showing the effect of GAPS-FL on mouse spleen lymphocyte toxicity
  • FIG13 is a diagram showing the effect of GAPS-FL in combination with lipopolysaccharide on the proliferation of mouse spleen lymphocytes
  • A GAPS-FL (500 ⁇ g/mL) group
  • B GAPS-FL (50 ⁇ g/mL)
  • C aluminum salt adjuvant group
  • D influenza vaccine group
  • FIG14 is a diagram showing the effect of GAPS-FL in collaboration with concanavalin on the proliferation of mouse spleen lymphocytes
  • A GAPS-FL (500 ⁇ g/mL) group
  • B GAPS-FL (50 ⁇ g/mL)
  • C aluminum salt adjuvant group
  • D influenza vaccine group
  • FIG15 is a graph showing the effect of GAPS-FL synergistic influenza vaccine on the proliferation of mouse spleen lymphocytes
  • A GAPS-FL (500 ⁇ g/mL) group
  • B GAPS-FL (50 ⁇ g/mL)
  • C aluminum salt adjuvant group
  • D influenza vaccine group
  • FIG16A is a graph showing the effect of GAPS-FL on the IgG titer of mice immunized with influenza vaccine, in which groups A, B, and C are significantly different from group D (P ⁇ 0.05, P ⁇ 0.01);
  • FIG. 16B is a graph showing the effect of GAPS-FL on the IgG1 titer of mice immunized with influenza vaccine, in which groups A and C have significant differences compared with group D (P ⁇ 0.0001);
  • FIG. 16C is a graph showing the effect of GAPS-FL on the titer of IgG2a in mice immunized with influenza vaccine, in which group B showed a significant difference compared with group D (P ⁇ 0.0001);
  • FIG16D is a graph showing the effect of GAPS-FL on the titer of IgG2a/IgG1 in mice immunized with influenza vaccine, in which group B showed a significant difference compared with group D (P ⁇ 0.01);
  • FIG16E is a graph showing the effect of GAPS-FL on the neutralizing antibody titer of mice immunized with influenza vaccine, in which groups A, B, and C were significantly different from group D (P ⁇ 0.05, P ⁇ 0.001, P ⁇ 0.0001);
  • FIG17A is a graph showing the effect of GAPS-FL on the IgG titer of mice immunized with rabies vaccine, in which groups A, B, and C have significant differences compared with group D (P ⁇ 0.0001), and group A has significant difference compared with group C (P ⁇ 0.01);
  • FIG. 17B is a graph showing the effect of GAPS-FL on the IgG1 titer of mice immunized with rabies vaccine, in which groups A and C have significant differences compared with group D (P ⁇ 0.01, P ⁇ 0.0001), and group A has significant difference compared with group C (P ⁇ 0.01);
  • FIG. 17C is a graph showing the effect of GAPS-FL on the titer of IgG2a in mice immunized with rabies vaccine, in which groups A and C have significant differences compared with group D (P ⁇ 0.05);
  • FIG. 17D is a graph showing the effect of GAPS-FL on the titer of IgG2a/IgG1 in mice immunized with rabies vaccine, in which groups A and C have significant differences compared with group D (P ⁇ 0.05);
  • FIG18A is a graph showing the effect of GAPS-FL on the IgG titer of mice immunized with hand, foot and mouth disease vaccine, in which groups A, B, and C are significantly different from group D (P ⁇ 0.0001), and group A is significantly different from group C (P ⁇ 0.05);
  • FIG18B is a graph showing the effect of GAPS-FL on the IgG1 titer of mice immunized with hand, foot and mouth disease vaccine, in which groups A and B have significant differences compared with group D (P ⁇ 0.05), and group A has significant difference compared with group C (P ⁇ 0.05);
  • FIG18C is a graph showing the effect of GAPS-FL on the IgG2a titer of mice immunized with hand, foot and mouth disease vaccine, in which groups A, B, and C have significant differences compared with group D (P ⁇ 0.05, P ⁇ 0.0001), and group A has significant difference compared with group C (P ⁇ 0.05);
  • FIG. 18D is a graph showing the effect of GAPS-FL on the titer of IgG2a/IgG1 in mice immunized with hand, foot and mouth disease vaccine, in which groups A, B and C showed significant differences compared with group D (P ⁇ 0.0001);
  • FIG19A is a graph showing the effect of GAPS-FL on the IgG titer of mice immunized with hepatitis A vaccine, in which groups A, B, and C have significant differences compared with group D (P ⁇ 0.0001), and group A has significant difference compared with group C (P ⁇ 0.05);
  • FIG19B is a graph showing the effect of GAPS-FL on the IgG1 titer of mice immunized with hepatitis A vaccine, in which groups A and C have significant differences compared with group D (P ⁇ 0.05, P ⁇ 0.01), and group A has significant difference compared with group C (P ⁇ 0.05);
  • FIG. 19C is a graph showing the effect of GAPS-FL on the IgG2a titer of mice immunized with hepatitis A vaccine, in which groups A, B, and C were significantly different from group D (P ⁇ 0.0001);
  • FIG19D is a graph showing the effect of GAPS-FL on the titer of IgG2a/IgG1 in mice immunized with hepatitis A vaccine, in which groups A, B, and C have significant differences compared with group D (P ⁇ 0.01, P ⁇ 0.0001), and group A has significant difference compared with group C (P ⁇ 0.05);
  • FIG20 is an electrophoresis graph of total RNA of spleen lymphocytes in Example 15;
  • FIG21A is a diagram showing the effect of GAPS-FL on GATA-3 gene expression, in which groups A, B, and C are significantly different from group D (P ⁇ 0.01, P ⁇ 0.001);
  • FIG21B is a diagram showing the effect of GAPS-FL on T-bet gene expression, in which groups A, B, and C are significantly different from group D (P ⁇ 0.001, P ⁇ 0.0001);
  • FIG21C is a graph showing the effect of GAPS-FL on IFN- ⁇ gene expression, in which groups A and B have significant differences compared with group D (P ⁇ 0.05);
  • FIG21D is a graph showing the effect of GAPS-FL on IL-4 gene expression, in which groups A and C have significant differences compared with group D (P ⁇ 0.001, P ⁇ 0.0001);
  • FIG. 22A is a graph showing the effect of GAPS-FL on IFN- ⁇ spleen lymphocyte factor, in which group A has a significant difference compared with group D (P ⁇ 0.0001);
  • FIG22B is a graph showing the effect of GAPS-FL on IL-4 spleen lymphocyte factor, in which group A is significantly different from group D (P ⁇ 0.001);
  • FIG22C is a graph showing the effect of GAPS-FL on IFN- ⁇ spleen lymphocyte factor
  • FIG22D is a graph showing the effect of GAPS-FL on IL-4 spleen lymphocyte factor
  • FIG. 23A is a diagram showing the effect of GAPS-FL on CD3 + CD4 + T lymphocytes, in which group A is significantly different from group D (P ⁇ 0.05);
  • FIG23B is a diagram showing the effect of GAPS-FL on CD3 + CD8 + T lymphocytes, in which group B is significantly different from group D (P ⁇ 0.05);
  • FIG23C is a graph showing the effect of GAPS-FL on CD3 + CD4 + T lymphocytes
  • FIG23D is a graph showing the effect of GAPS-FL on CD3 + CD8 + T lymphocytes
  • Figures 24A-24D are HE staining results of mouse spleen tissue
  • FIG24A is a HE staining result of spleen tissue of mice in group A;
  • FIG24B is a diagram showing the HE staining results of spleen tissue of mice in group B;
  • Figure 24C is a diagram showing the HE staining results of spleen tissue of mice in group C;
  • Figure 24D is a diagram showing the HE staining results of spleen tissue of mice in group D.
  • the present application provides a heteropolysaccharide.
  • the heteropolysaccharide contains galacturonic acid, arabinose, galactose, glucose, rhamnose, xylose, and mannose.
  • connection mode of the above monosaccharide residues is 1, 1, 5, 1, 3, 5-linked arabinose residues; 1, 4-linked galacturonic acid residues, 1, 3, 4-linked rhamnose residues; 1, 1, 4, 1, 3, 4-linked galactose residues; 1, 1, 4, 1, 3, 4-linked glucose residues; 1, 2-linked mannose residues.
  • the heteropolysaccharide in the structure of the heteropolysaccharide, its main chain is composed of 1, 4-linked galacturonic acid alternately connected, and has different degrees of methylation and acetylation.
  • the main chain structure is as follows:
  • 1 ⁇ p ⁇ 80 preferably, 1 ⁇ p ⁇ 10, R1, R2, and R3 are branched structures;
  • R1 is arabinan
  • R2 is galactoarabinan
  • R3 is a heterosaccharide
  • the 1-position of arabinose in R1 is linked to the 3-position of galacturonic acid
  • the 3-position of galactose in R2 is linked to the 1-position of arabinose.
  • the 4-position of glucose in R3 is linked to the 1-position of mannose, forming a heterosaccharide-like structure of R3.
  • R1, R2, and R3 are, for example,
  • the present application provides a heteropolysaccharide having a structure as shown in formula (I),
  • the heteropolysaccharide of the present application is composed of galacturonic acid, arabinose, galactose, glucose, rhamnose, xylose and mannose.
  • the composition ratio of each monosaccharide equivalent to 100 moles of the heteropolysaccharide can be as follows, in terms of molar amount, wherein:
  • Galacturonic acid 20.00-65.00, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, 60.
  • the molar amount of galacturonic acid is 40.00-50.00
  • Arabinose 8.00-40.00, for example, 8, 9, 10, 11, 12, 13, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 36, 37, 38, 39.
  • the molar amount of arabinose is 10.00-20.00
  • Galactose 5.00-25.00, for example, 6, 7, 8, 9, 10, 11, 12, 13, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.
  • the molar amount of galactose is 10.00-20.00
  • Glucose 5.00-25.00, for example, 6, 7, 8, 9, 10, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.
  • the molar amount of glucose is 5.00-15.00
  • Rhamnose 3.00-20.00, for example, 4, 5, 6, 7, 8, 9, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 12, 13, 14, 15, 16, 17, 18, 19.
  • the molar amount of rhamnose is 5.00-15.00
  • Xylose 0.05-5.00, for example, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5.
  • the molar amount of xylose is 0.2-3.00,
  • Mannose 0.05-5.00, for example, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5.
  • the molar amount of mannose is 0.2-3.00.
  • the weight average molecular weight of the heteropolysaccharide of the present application is 4 ⁇ 10 3 Da to 7 ⁇ 10 6 Da, for example, it can be 5 ⁇ 10 3 Da, 6 ⁇ 10 3 Da, or 7 ⁇ 10 3 Da.
  • the source of the heteropolysaccharide of the present application is not limited, as long as it conforms to the structure provided in the present application, it is within the protection scope of the present application.
  • the heteropolysaccharide can be extracted from ginseng.
  • the present application further provides a method for extracting the heteropolysaccharide from ginseng, comprising:
  • ginseng After ginseng is defatted, it is subjected to water extraction, alcohol precipitation, concentration and drying to obtain crude ginseng polysaccharide.
  • the crude ginseng polysaccharide is eluted by ion exchange column chromatography to obtain a heteropolysaccharide extract;
  • the heteropolysaccharide extract is purified by a gel column to obtain the heteropolysaccharide.
  • ginseng refers to the Araliaceae plant, which has many types according to different classification methods. For example, according to the growth environment, it can be divided into three categories: wild ginseng, transplanted ginseng, and garden ginseng; according to the processing method, it can be divided into red ginseng, sugar ginseng, raw sun-dried ginseng, preserved ginseng, active ginseng, etc.; according to the origin, it can be divided into Jilin ginseng, Korean ginseng, American ginseng, Changbai Mountain ginseng, Liao ginseng, etc.; according to the species, it can be divided into the root of ginseng of the Araliaceae plant and the root of American ginseng of the Araliaceae plant.
  • the ginseng of the present application can be any of the above ginseng.
  • the ginseng used in the present application can be any part of the ginseng plant, such as the stems, leaves, ginseng flowers (ginseng fruit), fruits (ginseng seeds), roots, whiskers, etc.
  • the ginseng refers to ginseng roots.
  • Ginseng contains a high level of lipids, which can affect the polysaccharide extraction process and hinder the aqueous solution from penetrating into the interior of the plant material, thus reducing the polysaccharide extraction rate and content.
  • Defatting ginseng before processing can reduce the interference of lipids, allowing more polysaccharides to dissolve into the extract, thereby increasing the polysaccharide extraction rate.
  • Lipids are usually colored, and defatting can also help decolorize the extract.
  • the present application may use any of the methods commonly used in the art for the defatting of ginseng, such as using an organic solvent to soak and remove the lipids.
  • the organic solvent may be acidic, alkaline, neutral, or a mixed solvent.
  • a preferred embodiment uses ethanol for the defatting of ginseng. Degreasing, the concentration of the ethanol is preferably 70-95%, the soaking time is not limited, and the preferred embodiment is soaking for 8-36 hours.
  • the ginseng residue after soaking is collected and dried at a temperature of 45-60° C. It can be dried in an oven or by other methods. After drying to constant weight, it is crushed into powder for water extraction.
  • the extraction refers to the process of leaching soluble active ingredients from raw materials using appropriate solvents and methods.
  • the extraction method is also called liquid-solid extraction, which usually uses a volatile organic solvent to transfer certain components in the raw material to the solvent phase, and then recovers the organic solvent by evaporation, distillation and other means to obtain the required relatively pure extraction components.
  • the extraction method used in this application is not limited, and can be any method commonly used in the art, such as water extraction, alcohol extraction, etc.
  • the present application uses water extraction, preferably 2-6 times, for example, 3, 4, 5 times, each extraction 1 to 5 hours, for example, 2h, 3h, 4h, during the extraction process, the solid-liquid ratio is preferably 1:10 to 40, for example, 1:15, 1:20, 1:25, 1:30, 1:35.
  • the extract is subjected to alcohol precipitation, which refers to the use of the characteristics that the effective components are soluble in ethanol and the impurities are insoluble in ethanol.
  • alcohol precipitation refers to the use of the characteristics that the effective components are soluble in ethanol and the impurities are insoluble in ethanol.
  • ethanol is added to the mixed component solution, the effective components are transferred into the ethanol and the impurities are precipitated out.
  • the alcohol precipitation method of the present application can be any common method in the art without limitation.
  • the liquid after alcohol precipitation is concentrated and dried.
  • freeze-drying is used for drying, and then crude ginseng polysaccharides can be obtained.
  • the ginseng crude polysaccharide is chromatographed on an ion exchange column to obtain a heteropolysaccharide extract.
  • distilled water and sodium chloride aqueous solution are preferably used for elution in sequence, and the elution flow rate is preferably 0.03-12 mL/min, for example, 0.05 mL/min, 0.1 mL/min, 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min, 5 mL/min, 6 mL/min, 7 mL/min, 8 mL/min, 9 mL/min, 10 mL/min, 11 mL/min.
  • the heteropolysaccharide extract is purified by a gel column to obtain the heteropolysaccharide, wherein the elution process is preferably carried out with a 0.9-2.5M sodium chloride solution, for example, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2M, 2.2M, 2.3M, 2.4M sodium chloride solution can be used, and the elution flow rate is preferably 0.01-0.08mL/min, for example, 0.02mL/min, 0.03mL/min, 0.04mL/min, 0.05mL/min, 0.06mL/min, 0.07mL/min.
  • a 0.9-2.5M sodium chloride solution for example, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2M, 2.2M, 2.3M, 2.4M sodium chloride solution
  • the elution flow rate is preferably 0.01-0.08mL/min, for example, 0.02mL/min, 0.03mL/
  • the present application further provides a composition, which comprises any one of the heteropolysaccharides provided in the present application as described above and a pharmaceutically acceptable carrier and/or excipient.
  • the present application also provides any one of the heteropolysaccharides provided in the present application or the above-mentioned composition as Use as a vaccine adjuvant.
  • heteropolysaccharide of the present application can non-specifically change or enhance the immune response of the vaccine, and can not only enhance the humoral immunity of mice but also enhance their cellular immunity, for example,
  • heteropolysaccharide aqueous solutions provided by the present application at different concentrations had no toxic effect on mouse splenic lymphocytes, and some heteropolysaccharide aqueous solutions at certain concentrations could even enhance the proliferation of mouse splenic lymphocytes, proving that the heteropolysaccharide of the present application has immunostimulatory activity;
  • the heteropolysaccharide of the present application can cooperate with LPS (lipopolysaccharide), Con A (concanavalin) or influenza vaccine to enhance the proliferation of mouse spleen lymphocytes;
  • the present application has verified through mouse animal experiments that the heteropolysaccharide of the present application can be applied as a vaccine adjuvant to influenza vaccine, rabies vaccine, hand, foot and mouth vaccine, hepatitis A vaccine, etc., which can enhance the immune response of the vaccine, and can significantly increase the IgG, IgG2a, IgG2a/IgG1 and neutralizing antibody levels of vaccine mice, and can also significantly increase the IgG1 antibody level of vaccine mice, and the antibody level generally shows an upward trend;
  • the present application further studies the effects of heteropolysaccharides on the gene expression of GATA-3, T-bet, IFN- ⁇ , and IL-4, and finds that the heteropolysaccharide can significantly increase the expression of GATA-3, T-bet, IFN- ⁇ , and IL-4 mRNA genes in mouse splenic lymphocytes, further illustrating that the heteropolysaccharide of the present application can enhance the role of influenza vaccine adjuvants in cellular immunity; and through the study of heteropolysaccharides on IFN- ⁇ and IL-4 splenic lymphocyte factors, it is found that the heteropolysaccharide of the present application can significantly increase the proliferation of IFN- ⁇ and IL-4 cytokines in mouse splenic lymphocytes, indicating that the heteropolysaccharide of the present application can simultaneously promote the Th1 and Th2 immune responses of splenic lymphocytes; through the study of heteropolysaccharides on CD3+CD4+ and CD3+CD8+T
  • heteropolysaccharide or composition of the present application can be used as a vaccine adjuvant in vaccines.
  • the vaccines include but are not limited to influenza vaccine, rabies vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine or new coronavirus vaccine.
  • vaccine refers to any preparation of antigenic or immunogenic substance suitable for stimulating active immunity in animals or humans.
  • adjuvant refers to any substance or mixture of substances that enhances, increases, upregulates, alters or otherwise promotes an immune response (eg, a humoral or cellular immune response) to an antigen in an animal.
  • the term "antigen" refers to any substance that, when introduced into an immunocompetent human or animal, stimulates a humoral and/or cell-mediated immune response.
  • the antigen may be a pure substance, a mixture of substances, or particulate matter (including cells, cell fragments, or cell-derived fragments) or a live (usually attenuated) organism or virus.
  • suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, peptides, carbohydrates/polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi, and parasites.
  • Antigens may be natural (naturally expressed or produced), synthetic, or derived from recombinant DNA methodology familiar to those skilled in the art.
  • the term "pharmaceutical excipients” refers to a general term for all medicinal materials other than the main drug added to the prescription to solve the formability, effectiveness, stability and safety of the preparation when producing drugs and preparing prescriptions. It is a substance that has been reasonably evaluated in terms of safety and is contained in the drug preparation. In addition to excipients, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained release. They are important ingredients that may affect the quality, safety and effectiveness of drugs.
  • the pharmaceutical excipients described in this application can be appropriate carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (such as aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants and Freund's incomplete adjuvants), etc.
  • adjuvants such as aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants and Freund's incomplete adjuvants
  • an ultraviolet spectrophotometer is used to determine whether there are proteins and nucleic acids in the heteropolysaccharide
  • the molecular weight of the heteropolysaccharide was determined using an HPGPC analytical instrument, which included a high performance liquid chromatograph equipped with a differential detector;
  • Soak ginseng root (2.0 kg) in 70-95% ethanol for 24 hours to degrease. Collect the residue and dry it in a 50°C oven to constant weight, grind it into powder and pass it through a 150-mesh sieve for later use;
  • the ginseng polysaccharide freeze-dried powder solution obtained by DEAE separation was further purified by gel chromatography column, and eluted with distilled water and 0.9-2.5M sodium chloride solution in sequence.
  • the elution curve is shown in Figure 3.
  • the polysaccharide content was determined by phenol-sulfuric acid method and the main peak was collected, concentrated, dialyzed overnight at 4-15°C, and freeze-dried to obtain ginseng heteropolysaccharides (GAPS-FL).
  • the separation and purification flow chart is shown in Figure 1.
  • the polysaccharide content, uronic acid content and protein content of GAPS-FL were determined by phenol-sulfuric acid method, m-hydroxybiphenyl method and Bradford method respectively. The results are shown in Table 1.
  • the GAPS-FL sample prepared in Example 1 was prepared into a 0.04-1.0 mg/mL aqueous solution and scanned with a UV spectrophotometer in the range of 800-200 nm. As shown in FIG4A , there were no characteristic absorption peaks at 260 nm and 280 nm, indicating that GAPS-FL had no protein or nucleic acid.
  • the OH stretching vibration absorption peak at 3425 cm -1 is the characteristic peak of sugars.
  • the GAPS-FL sample prepared in Example 1 and standards of different molecular weights were accurately weighed, the sample was prepared into a 5 mg/mL solution, centrifuged at 12000 rpm for 10 min, the supernatant was filtered with a 0.22 ⁇ m microporous filter membrane, and then the sample was transferred to a 1.8 ml injection vial.
  • HPPC high performance gel permeation chromatography
  • the results are shown in FIG6 .
  • the acetylation product samples were analyzed by gas chromatography-mass spectrometry; the GC-MS program temperature conditions were: starting temperature 120°C, heating at 3°C/min to 250°C/min; maintaining for 5 minutes; injection port temperature 250°C, detector temperature 250°C/min, carrier gas helium, flow rate 1mL/min.
  • the analysis spectrum is shown in Figure 8, and the specific methylation analysis results are shown in Table 2.
  • the signals at 5.73, 5.06, 5.01, and 5.00 ppm correspond to the H-1 of T-Ara, 3,5-Ara, and 1,5-Ara, respectively.
  • the signals at 5.24 and 5.00 ppm correspond to the H-1 of 1,3,4-GalA and 1,4-GalA, respectively.
  • the signal at 5.23 ppm corresponds to the H-1 of 1,4,6-Glcp.
  • the signal near 4.88 ppm can be attributed to the H-1 of 1,3,4-Rhap, T-Gal, 1,4-Glc, 1,2-Man, and T-Xyl.
  • the 13 C-NMR end group peaks of GAPS-FL show that the signals at 107.6, 107.4, and 107.3 ppm correspond to the C-1 of T-Ara, 3,5-Ara, and 1,5-Ara.
  • the signals at 104.6, 102.8, and 100.2 ppm correspond to the C-1 of Galp with different substitutions.
  • the signal at 99.3 ppm corresponds to the C-1 of GalA. 100.9, 99.9, 98.7, and 97.6 ppm correspond to the H-1 of 1,2-Man, 1,4-Glc, T-Xyl, and 1,3,4-Rha, respectively.
  • the signals at 96.3 and 92.1 correspond to the C-1 of Glc with different substitutions.
  • the GAPS-FL prepared in Example 1 was subjected to scanning electron microscopy and atomic force microscopy analysis ( Figures 9A and 9B).
  • the microstructure of the heteropolysaccharide was analyzed using a scanning electron microscope under high vacuum conditions with an accelerating voltage of 5.0 kV.
  • GAPS-FL was prepared into a 10 ⁇ g/mL aqueous solution and ultrasonically treated for 15 min, the sample was transferred to a mica plate and then dried at 120°C for 30 s for atomic force microscopy analysis.
  • Example 9 Study on the toxicity of GAPS-FL to mouse spleen lymphocytes
  • Reagents and drugs red blood cell lysis solution (Beijing Solebow Technology Co., Ltd.), RPMI-1640 culture medium (Dalian Meilun Biotechnology Co., Ltd.), fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd.), CCK-8 (Dalian Meilun Biotechnology Co., Ltd.).
  • Example 10 Study on the effect of GAPS-FL on the proliferation of mouse spleen lymphocytes in combination with LPS (lipopolysaccharide), Con A (concanavalin A) or influenza vaccine
  • Reagents and drugs Lipopolysaccharide (LPS, Sigma), concanavalin A (Con A, Sigma), red blood cell lysis buffer (Beijing Solebow Technology Co., Ltd.), RPMI-1640 culture medium (Dalian Meilun Biotechnology Co., Ltd.), fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd.), CCK-8 (Dalian Meilun Biotechnology Co., Ltd.).
  • mice 50 female C57BL/6 mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.
  • Influenza vaccine preparations containing GAPS-FL adjuvant Weigh 2.5 mg, 0.5 mg of GAPS-FL and 15 ⁇ g of influenza vaccine respectively, dissolve in 10 ml of physiological saline, filter with a 0.22 ⁇ m microporous filter membrane, and aseptically package, 1 ml per tube. Each 1 ml contains 2.5 mg, 0.5 mg of GAPS-FL and 15 ⁇ g of influenza vaccine. Prepare four different preparations according to the following grouping and dosage.
  • mice were randomly divided into 4 groups, 10 mice in each group. 0.1 ml of the above-mentioned preparations of groups A, B, C, and D were injected intramuscularly.
  • the second immunization was performed. 14 days after the second immunization, spleen lymphocyte suspensions of mice in each group were prepared according to the method of Example 9, and the cell concentration was adjusted to (1-5) ⁇ 10 6 /mL with RPMI-1640 medium containing 10% fetal bovine serum, and evenly spread in three 96-well plates, plate 1, plate 2, and plate 3, respectively.
  • Plate 1 plate 2 and plate 3 respectively use different reagents to stimulate lymphocyte proliferation in an in vitro culture environment, and examine the proliferation activity of spleen lymphocytes in mice of different groups after administration.
  • Plate 1 uses lipopolysaccharide as a stimulator
  • plate 2 uses concanavalin as a stimulator
  • plate 3 uses influenza vaccine stock solution as a stimulator.
  • the incubation wells corresponding to the four groups A, B, C, and D contain 100 ⁇ L spleen lymphocyte suspension, 80 ⁇ L complete culture medium (RPMI 1640 + 10% fetal bovine serum) and the following components.
  • Plate 3 Each well contains 1 ⁇ g/mL of influenza vaccine stock solution.
  • the negative control wells correspond to the mice in the influenza vaccine group D, and the spleens were taken and ground to obtain the spleen cell group.
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • Influenza vaccine preparation containing GAPS-FL adjuvant Weigh 2.5 mg, 0.5 mg of GAPS-FL and 15 ⁇ g of influenza vaccine respectively, dissolve in 10 ml of normal saline, filter with 0.22 ⁇ m microporous filter membrane, and aseptically package, 1 ml per vial. Each 1 ml contains 2.5 mg, 0.5 mg of GAPS-FL and 15 ⁇ g of influenza vaccine.
  • Immunization scheme 40 mice were randomly divided into 4 groups, 10 mice in each group. 0.1 ml/mouse was injected intramuscularly, and the second immunization was performed one week after the first immunization. 14 days after the second immunization, the IgG, IgG1, IgG2a, and neutralizing antibody titers in the mouse serum were detected by ELISA and hemagglutination inhibition assay, and the IgG2a/IgG1 ratio was calculated.
  • high-dose GAPS-FL 500 ⁇ g
  • low-dose GAPS-FL 50 ⁇ g
  • IgG, IgG2a/IgG1 ratio and neutralizing antibody levels of mice immunized with influenza vaccine P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.0001
  • Other doses also showed an upward trend compared with the influenza vaccine group.
  • Example 12 Study on the adjuvant activity of GAPS-FL on rabies vaccine
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice 50 female C57BL/6 mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.
  • Rabies vaccine preparation containing GAPS-FL adjuvant Weigh 2.5mg, 0.5mg of GAPS-FL and 25IU of rabies vaccine respectively, dissolve in 10ml of physiological saline, filter with 0.22 ⁇ m microporous membrane, and aseptically package, 1ml per vial. Each 1ml contains 2.5mg, 0.5mg of GAPS-FL and 2.5IU of rabies vaccine.
  • Immunization scheme 40 mice were randomly divided into 4 groups, 10 mice in each group. 0.1 ml/mouse was injected intramuscularly, and the second immunization was performed one week after the first immunization. 14 days after the second immunization, the IgG, IgG1, and IgG2a antibody titers in the mouse serum were detected by ELISA experiment, and the IgG2a/IgG1 ratio was calculated.
  • High-dose GAPS-FL 500
  • high-dose GAPS-FL 500 ⁇ g
  • IgG and IgG1 antibody levels of mice immunized with rabies vaccine P ⁇ 0.01.
  • high-dose GAPS-FL 500 ⁇ g
  • Low-dose GAPS-FL 50 ⁇ g
  • Other doses also showed an upward trend compared with the rabies vaccine group.
  • Example 13 Study on the adjuvant activity of GAPS-FL on hand, foot and mouth disease vaccine
  • Hand, foot and mouth disease vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • Hand, foot and mouth disease vaccine preparation containing GAPS-FL adjuvant Weigh 2.5mg, 0.5mg of GAPS-FL and 50U of hand, foot and mouth disease vaccine respectively, dissolve in 10ml of normal saline, filter with 0.22 ⁇ m microporous membrane, and aseptically package, each containing 1ml. Each 1ml contains 2.5mg, 0.5mg of GAPS-FL and 50U of hand, foot and mouth disease vaccine.
  • Immunization scheme 40 mice were randomly divided into 4 groups, 10 mice in each group. 0.1 ml/mouse was injected intramuscularly, and the second immunization was performed one week after the first immunization. 14 days after the second immunization, the IgG, IgG1, and IgG2a antibody titers in the mouse serum were detected by ELISA experiment, and the IgG2a/IgG1 ratio was calculated.
  • the high-dose GAPS-FL 500 ⁇ g
  • the low-dose GAPS-FL (50 ⁇ g) and high-dose GAPS-FL (500 ⁇ g) could significantly increase the IgG, IgG1, IgG2a and IgG2a/IgG1 antibody levels of mice immunized with hand, foot and mouth disease vaccine (P ⁇ 0.05, P ⁇ 0.0001).
  • Other doses also showed an upward trend compared with the hand, foot and mouth disease vaccine group.
  • Example 14 Study on the adjuvant activity of GAPS-FL on hepatitis A vaccine
  • Hepatitis A vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • Hepatitis A vaccine preparation containing GAPS-FL adjuvant Weigh 2.5mg, 0.5mg of GAPS-FL and 4IU of hand, foot and mouth disease vaccine respectively, dissolve in 10ml of normal saline, filter with 0.22 ⁇ m microporous membrane, and aseptically package, 1ml per vial. Each 1ml contains 2.5mg, 0.5mg of GAPS-FL and 4IU of hepatitis A vaccine.
  • Immunization scheme 40 mice were randomly divided into 4 groups, 10 mice in each group. 0.1 ml/mouse was injected intramuscularly, and the second immunization was performed one week after the first immunization. 14 days after the second immunization, the IgG, IgG1, and IgG2a antibody titers in the mouse serum were detected by ELISA experiment, and the IgG2a/IgG1 ratio was calculated.
  • high-dose GAPS-FL 500 ⁇ g
  • IgG, IgG1 and IgG2a/IgG1 antibody levels of mice immunized with hepatitis A vaccine P ⁇ 0.05
  • high-dose GAPS-FL 500 ⁇ g
  • IgG1, IgG2a and IgG2a/IgG1 antibody levels of mice immunized with hepatitis A vaccine P ⁇ 0.01, P ⁇ 0.0001).
  • Low-dose GAPS-FL (50 ⁇ g) can significantly increase the IgG, IgG2a and IgG2a/IgG1 antibody levels of mice immunized with hepatitis A vaccine (P ⁇ 0.0001). Other doses also showed an upward trend compared with the hepatitis A vaccine group.
  • Example 15 Study on the expression of GATA-3, T-bet, IFN- ⁇ and IL-4 genes by GAPS-FL
  • Example 10 Prepare the mouse spleen lymphocyte suspension in each group according to the method of Example 9, and adjust the cell concentration to (6-8) ⁇ 10 7 /mL.
  • the animals were grouped according to Example 10 and plated in 6-well culture plates. Add 1 mL of cell suspension to each well, then add 100 ⁇ L of influenza vaccine stock solution and culture for 48 hours. Centrifuge and collect the cells for later use.
  • the total RNA from spleen lymphocytes was extracted using the classic guanidine isothiocyanate method.
  • RNA Take 4 ⁇ L of total RNA, dilute to 1000 ⁇ L with DEPC water, and measure the OD 260 and OD 280 of total RNA with an ultra-micro spectrophotometer. Take another 0.5 g of agarose, add 30 mL of 0.1% DEPC water, heat in a microwave oven until melted, cool to 50°C, add 2 ⁇ L of EP solution, mix well, add the gel to the gel tank, take 5 ⁇ L of total RNA for sample loading, electrophoresis, observe under ultraviolet light and take pictures to identify the integrity of RNA. The results are shown in Figure 18.
  • mice cytokines GATA-3, T-bet, IFN- ⁇ , IL-4 and internal standard ⁇ -actin were obtained from Genebank and are shown in Table 4.
  • the PCR system was as follows: template cDNA 4 ⁇ L, Hieff qPCR SYBR Green Master Mix 10 ⁇ L, upstream and downstream primers 0.4 ⁇ L each, and sterile ultrapure water was added to 20 ⁇ L.
  • the amplification program was pre-denaturation at 95°C and then cycled 40 times according to the following program: 95°C for 10s, 60°C for 20s, and 72°C for 20s. Then, the extension was performed at 72°C for 10 minutes.
  • Example 16 Study of GAPS-FL on IFN- ⁇ and IL-4 Splenic Lymphocyte Factors
  • Example 9 Prepare the mouse spleen cell suspension of each group according to the method of Example 9, and adjust the cell concentration to (1-2) ⁇ 10 5 /mL, and evenly inoculate in a 96-well culture plate according to the operating instructions of the ELISPOT kit, add 100 ⁇ L of cell suspension to each well, add only 100 ⁇ L of culture medium (background control), 100 ⁇ L of cell suspension (negative control), 100 ⁇ L of cell suspension plus 10 ⁇ L of PMA stimulant (positive control) and each experimental well of the animal grouping according to Example 10. After all samples and stimulants are added, place them in an incubator and culture for 48 hours.
  • the spleen cell suspension of each group of mice was prepared, and the cell concentration was adjusted to 3-6 ⁇ 10 6 /mL, and inoculated into a 24-well culture plate, 1000 ⁇ L of cell suspension and 100 ⁇ L of influenza vaccine stock solution were added to each well, and the plate was placed in an incubator for 48 hours. Centrifugation was performed to collect the cells, and 100 ⁇ L of sterile PBS was added to resuspend them. 2 ⁇ g of APC-CD3, FITC-CD4, and PE-CD8 antibodies were added to each group for staining.
  • high-dose GAPS-FL 500 ⁇ g
  • low-dose GAPS-FL 50 ⁇ g
  • the proportion of CD3 + CD8 + T lymphocyte subsets was significantly different from that of the influenza vaccine group, indicating that GAPS-FL has strong cellular immune activity as an influenza vaccine adjuvant.
  • Example 18 HE staining of mouse spleen tissue
  • mice treated with the preparations in groups A, B, C, and D in Example 10 were randomly selected, killed by dislocation, and the spleen was removed under sterile conditions, and fixed in 4% paraformaldehyde. After dehydration in alcohol solutions of different concentrations, the spleen was embedded in paraffin and HE stained, and the pathological sections were observed under an optical microscope. The results are shown in Figure 24.
  • the high-dose GAPS-FL 500 ⁇ g
  • WP central zone
  • MZ marginal zone
  • spleen white pulp a large number of red blood cells appeared in the spleen red pulp (RP).
  • HE staining of mouse spleen tissue suggested that GAPS-FL can enhance the immune response of influenza vaccine.

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Abstract

本申请公开了一种杂多糖，包含半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖。具体来说是一种人参杂多糖(GAPS-FL)，为一种从人参根中分离得到的新的多糖物质，能够非特异性地改变或增强包括流感疫苗在内的多种疫苗的免疫应答反应，不仅可以增强流感疫苗免疫小鼠的体液免疫还可以增强其细胞免疫。

Description

一种人参杂多糖及其分离方法和应用 技术领域
本申请涉及医药技术领域，具体涉及一种人参杂多糖的分离、鉴定及应用。
背景技术
自2020年新冠疫情在全球爆发以来，严重影响人类的生命财产安全并对日常生活带来了很多不便，接种疫苗是有效预防传染病的重要措施。免疫佐剂是指能够非特异性改变或增强机体对抗原的特异性免疫应答的物质，并且是疫苗的重要组成部分。免疫佐剂的作用机理主要是可以增加抗原表面积，提高免疫原性；对抗原起缓释作用，延长抗原在组织中的滞留时间；促进炎症反应，刺激主动免疫应答。目前我国市售疫苗佐剂多为铝盐佐剂，但是铝盐可引起注射部位的炎症并刺激局部红斑、肉芽肿和皮下结节，同时，铝盐佐剂会延缓疫苗的中和抗体的产生，因此在应用时受到多种限制，所以开发新型疫苗佐剂是非常必要且有意义的。
人参为五加科人参属植物人参Panax ginseng.A.Meyer的干燥根及根茎，具有大补元气、生津养血、安神益智等功效。现代药理学表明人参具有提高免疫力、抗肿瘤、抗疲劳等功效，广泛分布于中国、日本、韩国等亚洲国家。人参中主要含有皂苷、多糖、黄酮等多种活性成分。
发明内容
本申请从人参中分离出一种杂多糖，经活性追踪，通过体内实验已经证明了所述杂多糖对多种疫苗具有免疫佐剂活性。
具体的，本申请采用如下技术方案，
1、一种杂多糖，包含半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖。
2、根据项1所述的杂多糖，所述杂多糖包含1、1，5、1，3，5连接的阿拉伯糖残基；1，4连接的半乳糖醛酸残基、1，3，4连接的鼠李糖残基；1、 1，4、1，3，4连接的半乳糖残基；1、1，4、1，3，4连接的葡萄糖残基；1，2连接的甘露糖残基。
3、根据项1或2所述的杂多糖，所述杂多糖的主链由1，4连接的半乳糖醛酸交替连接组成，并有不同程度的甲基化和乙酰化，所述杂多糖的支链由阿拉伯聚糖、半乳阿拉伯聚糖和杂聚糖组成。
4、根据项1-3中任一项所述的杂多糖，其重复单元结构式如式(I)所示，
其中， 1≤n≤30，1≤m≤50，1≤p≤80。
5、一种杂多糖，其重复单元结构式如式(I)所示，
其中， 1≤n≤30，1≤m≤50，1≤p≤80。
6、根据项3所述的杂多糖，所述杂多糖由半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖组成。
7、根据项1-6中任一项所述的杂多糖，相当于100摩尔的杂多糖，其中，下述单元的摩尔量为：
半乳糖醛酸：20.00～65.00，
阿拉伯糖：8.00～40.00，
半乳糖：5.00～25.00，
葡萄糖：5.00～25.00，
鼠李糖：3.00～20.00，
木糖：0.05～5.00，
甘露糖：0.05～5.00。
优选地，下述单元的摩尔量为：
半乳糖醛酸：40.00～50.00，
阿拉伯糖：10.00～20.00，
半乳糖：10.00～20.00，
葡萄糖：5.00～15.00，
鼠李糖：5.00～15.00，
木糖：0.2～3.00，
甘露糖：0.2～3.00。
8、根据项1-7中任一项所述的杂多糖，所述杂多糖的重均分子量为4×103Da～7×106Da。
9、根据项1-8中任一项所述的杂多糖，所述杂多糖是从人参中提取出来的。
10、一种制备项1-9中任一项所述杂多糖的方法，包括，
人参进行脱脂后，经过浸提、醇沉、浓缩、干燥，得到人参粗多糖；
所述人参粗多糖通过离子交换柱层析，洗脱，得到杂多糖提取液；
所述杂多糖提取液经过凝胶柱进行纯化，得到所述杂多糖。
11、根据项10所述的方法，所述离子交换柱为阴离子交换树脂柱，优选，填料为DEAE Sepharose Fast Flow。
12、根据项10或11所述的方法，所述水浸提过程中，料液比为1：10～40，次数为2～4次，浸提时间为1～5h。
13、根据项10-12中任一项所述的方法，所述通过离子交换柱层析过程中，以蒸馏水、氯化钠水溶液依次洗脱交换柱，洗脱流速为0.03～12mL/min。
14、根据项10-13中任一项所述的方法，所述经过凝胶柱进行纯化的过程中，以0.9～2.5M氯化钠洗脱凝胶柱，洗脱流速为0.01～0.08mL/min。
15、一种组合物，包括项1-9中任一项所述的杂多糖或根据项10-14中任一项所述的方法制备的杂多糖和药学上可接受的载体和/或辅料。
16、项1-9中任一项所述的杂多糖或项10-14中任一项所述的方法制备的杂多糖或项15所述的组合物作为疫苗佐剂用途。
17、根据项16所述的用途，其中，所述疫苗包括但不限于为流感疫苗、狂犬疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒 疫苗。
18、项1-9中任一项所述的杂多糖或项10-14中任一项所述的方法制备的杂多糖或项15所述的组合物在提升脾淋巴细胞中GATA-3、T-bet、IFN-γ或IL-4mRNA基因表达方面的用途。
19、项1-9中任一项所述的杂多糖或项10-14中任一项所述的方法制备的杂多糖或项15所述的组合物在提升脾淋巴细胞中IFN-γ和IL-4细胞因子的增殖方面的用途。
20、项1-9中任一项所述的杂多糖或项10-14中任一项所述的方法制备的杂多糖或项15所述的组合物在提高脾淋巴细胞中CD3+CD4+T或CD3+CD8+T淋巴细胞亚群的比例方面的用途。
21、一种预防和/或治疗疾病的方法，包括向受试者施用项15所述的疫苗组合物。
22、根据项21所述的方法，所述疾病选自狂犬病、流感病、乙肝病、甲肝病、丙肝病、手足口病、HPV病或新型冠状病毒病。
发明效果
提供一种具有免疫佐剂活性的人参杂多糖(GAPS-FL)，为一种从人参根中分离得到的新的多糖物质，能够非特异性地改变或增强包括流感疫苗在内的多种疫苗的免疫应答反应，不仅可以增强流感疫苗免疫小鼠的体液免疫还可以增强其细胞免疫。
附图说明
图1是GAPS-FL的分离纯化流程图；
图2是GAPS-FL的DEAE洗脱曲线；
图3是GAPS-FL的凝胶柱层析洗脱曲线；
图4A是GAPS-FL的紫外光谱图；
图4B是GAPS-FL的红外光谱图；
图5是葡萄糖标准品所做的标准曲线；
图6是GAPS-FL的HPGPC谱图；
图7是GAPS-FL的离子色谱分析谱图；
图8是GAPS-FL的GC-MS分析谱图；
图9A是GAPS-FL的1H-NMR分析谱图；
图9B是GAPS-FL的13C-NMR分析谱图；
图9C是GAPS-FL的DEPT-135分析谱图；
图9D是GAPS-FL的1H-1H COSY分析谱图；
图9E是GAPS-FL的HSQC分析谱图；
图9F是GAPS-FL的HMBC分析谱图；
图9G是GAPS-FL的NOESY分析谱图；
图9H是GAPS-FL的HSQC-TOCSY NMR分析谱图；
图10是GAPS-FL的结构式与结构重复单元；
图11A是GAPS-FL的扫描电镜图；
图11B是GAPS-FL的原子力显微镜图；
图12是GAPS-FL对小鼠脾淋巴细胞毒性的影响图；
与空白组相比，*P<0.05，**P<0.01，****P<0.0001；
图13是GAPS-FL协同脂多糖对小鼠脾淋巴细胞增殖的影响图；
A：GAPS-FL(500μg/mL)组；B：GAPS-FL(50μg/mL)；C：铝盐佐剂组；D：流感疫苗组；
A，B组与D组相比具有显著性差异(P＜0.05)；
图14是GAPS-FL协同刀豆蛋白对小鼠脾淋巴细胞增殖的影响图；
A：GAPS-FL(500μg/mL)组；B：GAPS-FL(50μg/mL)；C：铝盐佐剂组；D：流感疫苗组；
A，C组与D组相比具有显著性差异(P＜0.001)；
图15是GAPS-FL协同流感疫苗对小鼠脾淋巴细胞增殖的影响图；
A：GAPS-FL(500μg/mL)组；B：GAPS-FL(50μg/mL)；C：铝盐佐剂组；D：流感疫苗组；
A组与D组相比具有显著性差异(P＜0.05)；
图16A是GAPS-FL对流感疫苗免疫小鼠IgG效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.05，P＜0.01)；
图16B是GAPS-FL对流感疫苗免疫小鼠IgG1效价的影响图，图中A，C组与D组相比具有显著性差异(P＜0.0001)；
图16C是GAPS-FL对流感疫苗免疫小鼠IgG2a效价的影响图，图中B组与D组相比具有显著性差异(P＜0.0001)；
图16D是GAPS-FL对流感疫苗免疫小鼠IgG2a/IgG1效价的影响图，图中B组与D组相比具有显著性差异(P＜0.01)；
图16E是GAPS-FL对流感疫苗免疫小鼠中和抗体效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.05，P＜0.001，P＜0.0001)；
图17A是GAPS-FL对狂犬疫苗免疫小鼠IgG效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.0001)，A组与C组相比具有显著性差异(P＜0.01)；
图17B是GAPS-FL对狂犬疫苗免疫小鼠IgG1效价的影响图，图中A，C组与D组相比具有显著性差异(P＜0.01，P＜0.0001)，A组与C组相比具有显著性差异(P＜0.01)；
图17C是GAPS-FL对狂犬疫苗免疫小鼠IgG2a效价的影响图，图中A，C组与D组相比具有显著性差异(P＜0.05)；
图17D是GAPS-FL对狂犬疫苗免疫小鼠IgG2a/IgG1效价的影响图，图中A，C组与D组相比具有显著性差异(P＜0.05)；
图18A是GAPS-FL对手足口疫苗免疫小鼠IgG效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.0001)，A组与C组相比具有显著性差异(P＜0.05)；
图18B是GAPS-FL对手足口疫苗免疫小鼠IgG1效价的影响图，图中A，B组与D组相比具有显著性差异(P＜0.05)，A组与C组相比具有显著性差异(P＜0.05)；
图18C是GAPS-FL对手足口疫苗免疫小鼠IgG2a效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.05，P＜0.0001)，A组与C组相比具有显著性差异(P＜0.05)；
图18D是GAPS-FL对手足口疫苗免疫小鼠IgG2a/IgG1效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.0001)；
图19A是GAPS-FL对甲肝疫苗免疫小鼠IgG效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.0001)，A组与C组相比具有显著性差异(P＜0.05)；
图19B是GAPS-FL对甲肝疫苗免疫小鼠IgG1效价的影响图，图中A，C组与D组相比具有显著性差异(P＜0.05，P＜0.01)，A组与C组相比具有显著性差异(P＜0.05)；
图19C是GAPS-FL对甲肝疫苗免疫小鼠IgG2a效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.0001)；
图19D是GAPS-FL对甲肝疫苗免疫小鼠IgG2a/IgG1效价的影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.01，P＜0.0001)A组与C组相比具有显著性差异(P＜0.05)；图20是实施例15中脾淋巴细胞总RNA的电泳图；
图21A是GAPS-FL对GATA-3基因表达影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.01，P＜0.001)；
图21B是GAPS-FL对T-bet基因表达影响图，图中A，B，C组与D组相比具有显著性差异(P＜0.001，P＜0.0001)；
图21C是GAPS-FL对IFN-γ基因表达影响图，图中A，B组与D组相比具有显著性差异(P＜0.05)；
图21D是GAPS-FL对IL-4基因表达影响图，图中A，C组与D组相比具有显著性差异(P＜0.001，P＜0.0001)；
图22A是GAPS-FL对IFN-γ脾淋巴细胞因子的影响图，图中A组与D组相比具有显著性差异(P＜0.0001)；
图22B是GAPS-FL对IL-4脾淋巴细胞因子的影响图，图中A组与D组相比具有显著性差异(P＜0.001)；
图22C是GAPS-FL对IFN-γ脾淋巴细胞因子的影响图；
图22D是GAPS-FL对IL-4脾淋巴细胞因子的影响图；
图23A是GAPS-FL对CD3+CD4+T淋巴细胞的影响图，图中A组与D组相比具有显著性差异(P＜0.05)；
图23B是GAPS-FL对CD3+CD8+T淋巴细胞的影响图，图中B组与D组相比具有显著性差异(P＜0.05)；
图23C是GAPS-FL对CD3+CD4+T淋巴细胞的影响图；
图23D是GAPS-FL对CD3+CD8+T淋巴细胞的影响图；
图24A-24D是小鼠脾脏组织HE染色结果图
图24A是A组小鼠脾脏组织HE染色结果图；
图24B是B组小鼠脾脏组织HE染色结果图；
图24C是C组小鼠脾脏组织HE染色结果图；
图24D是D组小鼠脾脏组织HE染色结果图。
具体实施方式
以下对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解，应当将它们认为仅仅是示范性的。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围和精神。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
本申请提供了一种杂多糖，具体来说，所述杂多糖包含半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖。
一种具体的实施方式中，在所述杂多糖的结构中，上述单糖残基的连接方式为1、1，5、1，3，5连接的阿拉伯糖残基；1，4连接的半乳糖醛酸残基、1，3，4连接的鼠李糖残基；1、1，4、1，3，4连接的半乳糖残基；1、1，4、1，3，4连接的葡萄糖残基；1，2连接的甘露糖残基。
一种具体的实施方式中，在所述杂多糖的结构中，其主链由1，4连接的半乳糖醛酸交替连接组成，并有不同程度的甲基化和乙酰化，一种优选的实施方式中，主链结构如下所示，
其中，1≤p≤80，优选的，1≤p≤10，R1、R2、R3为支链结构；
一种优选的实施方式中，其中R1为阿拉伯聚糖，R2为半乳阿拉伯聚糖，R3为杂聚糖，其中R1阿拉伯糖的1位与半乳糖醛酸的3位相连，R2半乳糖的3位与阿拉伯糖的1位相连。R3葡萄糖的4位与甘露糖的1位相连，构成R3杂聚糖样的结构。具体的，R1、R2、R3例如为
其中，1≤n≤30，1≤m≤50。
本申请提供了一种如式(I)所示结构的杂多糖，
其中， 1≤n≤30，1≤m≤50，1≤p≤80。
一种优选的实施方式中，本申请的杂多糖由半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖组成。
一种优选的实施方式中，在如前所述的任一种本申请提供的杂多糖中，相当于100摩尔的杂多糖，各单糖的组成比例可以如下所示，以摩尔量计，其中，
半乳糖醛酸：20.00～65.00，例如可以为21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、41、42、43、44、45、46、47、48、49、50、52、54、56、58、60，一种优选的实施方式中，半乳糖醛酸的摩尔量为40.00～50.00，
阿拉伯糖：8.00～40.00，例如可以为8、9、10、11、12、13、14、14.1、14.2、14.3、14.4、14.5、14.6、14.7、14.8、14.9、15、16、17、18、19、20、21、22、23、24、25、30、35、36、37、38、39，一种优选的实施方式中，阿拉伯糖的摩尔量为10.00～20.00，
半乳糖：5.00～25.00，例如可以为6、7、8、9、10、11、12、13、13.6、13.7、13.8、13.9、14、14.1、14.2、14.3、14.4、15、16、17、18、19、20、21、22、23、24，一种优选的实施方式汇总，半乳糖的摩尔量为10.00～20.00，
葡萄糖：5.00～25.00，例如可以为6、7、8、9、10、11、11.1、11.2、11.3、11.4、11.5、11.6、11.7、11.8、11.9、12、13、14、15、16、17、18、19、20、21、22、23、24，一种优选的实施方式中，葡萄糖的摩尔量为5.00～15.00，
鼠李糖：3.00～20.00，例如可以4、5、6、7、8、9、9.6、9.7、9.8、9.9、10、10.1、10.2、10.3、10.4、10.5、10.6、10.7、10.8、10.9、11、12、13、14、15、16、17、18、19，一种优选的实施方式中，鼠李糖的摩尔量为5.00～15.00，
木糖：0.05～5.00，例如可以为0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.2、0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5，一种优选的实施方式中，木糖的摩尔量为0.2～3.00，
甘露糖：0.05～5.00，例如可以0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.2、0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5，一种优选的实施方式中，甘露糖的摩尔量为0.2～3.00。
一种优选的实施方式中，本申请的杂多糖的重均分子量为4×103Da～7×106Da，例如可以为5×103Da、6×103Da、7×103Da、8×103Da、9×103Da、1×104Da、2×104Da、3×104Da、4×104Da、5×104Da、6×104Da、7×104Da、8×104Da、9×104Da、1×105Da、2×105Da、3×105Da、4×105Da、5×105Da、6×105Da、7×105Da、8×105Da、9×105Da、1×106Da、2×106Da、3×106Da、4×106Da、5×106Da、6×106Da。
本申请的杂多糖的来源不受限制，只要符合本申请所提供的结构均在本申请的保护范围之内，一种优选的实施方式中，所述杂多糖可以从人参中提取。
本申请进一步提供了一种从人参中提取所述杂多糖的方法，包括，
人参进行脱脂后，经过水浸提、醇沉、浓缩、干燥，得到人参粗多糖；
所述人参粗多糖通过离子交换柱层析，洗脱，得到杂多糖提取液；
所述杂多糖提取液经过凝胶柱进行纯化，得到所述杂多糖。
在上述方法中，人参是指五加科植物，按照不同的分类方法具有多种种类，例如按生长环境分类：可分为野山参、移山参、园参三类；按炮制方式分类：可分为红参、白糖参、生晒参、保鲜人参、活性人参等；按产地分类：可分为吉林人参、高丽参、西洋参、长白山参、辽参等；按物种分类：可分为五加科植物人参的根，以及五加科植物西洋参的根，本申请的人参可以是上述任何一种人参，同时，本申请使用的人参可以是人参的茎、叶、人参花(人参果)、果实(人参籽)、根、须等任何人参植物上的部位，一种优选的实施方式中，所述人参指人参根。
人参中含有较高的脂质，脂质会影响多糖提取的过程，阻碍水溶液渗透到植物原料的内部，使多糖的提取率和含量减少。在处理之前对人参进行脱脂处理可降低脂质的干扰，使多糖更多的溶入提取液中，提高多糖提取率，且脂质通常是有颜色的，脱脂还有助于提取液的脱色。本申请对人参的脱脂处理可以使用本领域内常用的任何一种方法，例如使用有机溶剂浸泡带走脂质，有机溶剂可以是酸性、碱性、中性，或混合溶剂，本申请中，一种优选的实施方式，使用乙醇进行 脱脂，所述乙醇的浓度优选为70～95％，浸泡时间不受限制，优选的实施方式为浸泡8-36小时。
收集浸泡后的人参残渣进行烘干，烘干温度为45-60℃，可以在烘箱内干燥也可以使用其他方法干燥，干燥至恒重后，粉碎成粉末用于水浸提。
所述浸提是指利用适当的溶媒和方法，从原料中将可溶性有效成分浸出的过程。浸提法也称液固萃取法，通常利用的是用挥发性有机溶剂将原料中的某些成分转移到溶剂相中，然后通过蒸发、蒸馏等手段回收有机溶剂，而得到所需的较为纯净的萃取组分。本申请使用的浸提方法不受限制，可以是本领域常用的任何方法，例如水浸提、醇浸提等，一种优选的实施方式，本申请使用水浸提，优选提取2-6次，例如可以为3、4、5次，每次提取1～5h，例如可以为2h、3h、4h，在浸提的过程中，料液比优选为1：10～40，例如可以为1：15、1：20、1：25、1：30、1：35。
浸提后对浸提液进行醇沉，所述醇沉即乙醇沉淀，是指利用有效组分溶于乙醇而杂质不溶于遗传的特性，在混合组分溶液中加入乙醇后，有效成分转入乙醇中而杂质则被沉淀出来。本申请的醇沉方法可以是本领域内常见的任何方法，不受限制。
醇沉后的液体进行浓缩、干燥，一种优选的实施方式，可以使用冻干进行干燥，然后即可得到人参粗多糖。
所述人参粗多糖经过离子交换柱进行层析后得到杂多糖提取液。其中，洗脱过程优选使用蒸馏水、氯化钠水溶液依次洗脱，洗脱流速优选为0.03～12mL/min，例如可以为0.05mL/min、0.1mL/min、1mL/min、2mL/min、3mL/min、4mL/min、5mL/min、6mL/min、7mL/min、8mL/min、9mL/min、10mL/min、11mL/min。
所述杂多糖提取液经过凝胶柱进行纯化后得到所述杂多糖，其中，洗脱过程优选以0.9～2.5M氯化钠溶液进行洗脱，例如可以使用1M、1.2M、1.4M、1.6M、1.8M、2M、2.2M、2.3M、2.4M的氯化钠溶液，洗脱流速优选为0.01～0.08mL/min，例如可以0.02mL/min、0.03mL/min、0.04mL/min、0.05mL/min、0.06mL/min、0.07mL/min。
本申请进一步提供了一种组合物，所述组合物包含如上所述任何一种本申请提供的杂多糖和药学上可接受的载体和/或辅料。
本申请还提供了如上所述任何一种本申请提供的杂多糖或者上述组合物作 为疫苗佐剂的用途。
通过本申请的实验研究发现，本申请的杂多糖能够非特异性地改变或增强疫苗的免疫应答反应，不仅可以增强小鼠的体液免疫还可以增强其细胞免疫，例如，
通过对小鼠脾淋巴细胞毒性的研究发现，不同浓度的本申请提供的杂多糖水溶液对小鼠脾淋巴细胞均无毒性作用，且有一部分浓度的杂多糖水溶液甚至可以增强小鼠脾淋巴细胞具有增殖作用，证明了本申请的杂多糖具有免疫刺激活性；
同时通过体内免疫后取小鼠脾脏进行体外的刺激实验，发现本申请的杂多糖可以协同LPS(脂多糖)、Con A(刀豆蛋白)或流感疫苗增强对小鼠脾淋巴细胞增殖作用；
以及，本申请通过小鼠动物实验，验证了本申请的杂多糖作为疫苗佐剂应用到流感疫苗、狂犬疫苗、手足口疫苗、甲肝疫苗等均具能够增强疫苗的免疫应答反应，能显著提升疫苗小鼠的IgG，IgG2a，IgG2a/IgG1和中和抗体水平，还能显著提升疫苗小鼠的IgG1抗体的水平，抗体水平总体呈现上升趋势；
本申请还进一步研究了杂多糖对GATA-3、T-bet、IFN-γ、IL-4基因表达的影响，发现所述杂多糖能够显著提升小鼠脾淋巴细胞中GATA-3、T-bet、IFN-γ、IL-4mRNA基因的表达，进一步说明本申请的杂多糖可以增强流感疫苗佐剂在细胞免疫中的作用；而通过杂多糖对IFN-γ和IL-4脾淋巴细胞因子的研究发现，本申请的杂多糖能显著提升小鼠脾淋巴细胞中IFN-γ和IL-4细胞因子的增殖，说明本申请的杂多糖可同时促进脾淋巴细胞Th1和Th2免疫反应；通过杂多糖对CD3+CD4+和CD3+CD8+T淋巴细胞亚群的研究发现，本申请的杂多糖能显著提升小鼠脾淋巴细胞中CD3+CD4+和CD3+CD8+T淋巴细胞亚群的比例，说明本申请的杂多糖作为流感疫苗佐剂具有强大的细胞免疫活性。
因此，本申请的杂多糖或组合物可以作为一种疫苗佐剂应用到疫苗中。
所述疫苗包括但不限于流感疫苗、狂犬疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒疫苗。
如本文所用，术语“疫苗“是指任何适于刺激动物或人类中的活性免疫性的抗原或致免疫性物质的制剂。
如本文所用，术语“佐剂＂，是指提高、增加、向上调节、改变或以其它方式促进动物中对抗原的免疫反应(例如，体液或细胞免疫反应)的任何物质或物质的混合物。
如本文所用，术语“抗原”是指当被引入至免疫活性的人或动物中时，刺激体液及/或细胞介导的免疫反应的任何物质。该抗原可为纯物质、物质的混合物或微粒物质(包括细胞、细胞片段或细胞衍生片段)或活的(通常经减毒)的生物或病毒。适当抗原的实例包括但不限于：蛋白质、糖蛋白、脂蛋白、肤、碳水化合物/多糖、脂多糖、毒素、病毒、细菌、真菌及寄生物。抗原可以为天然(自然表达或制得)的、合成的，或由那些本领域的技术人员熟悉的重组DNA方法学所衍生的。
如本文所用，术语“药用辅料”指生产药品和调配处方时，为解决制剂的成型性、有效性、稳定性、安全性加入处方中除主药以外的一切药用物料的统称，在安全性方面已进行了合理的评估，且包含在药物制剂中的物质。药用辅料除了赋形、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。本申请所述药用辅料可以是适当的载体或赋形剂、乳化剂、润湿剂、防腐剂、稳定剂、抗氧化剂、佐剂(例如氢氧化铝佐剂、油剂佐剂、弗氏完全佐剂和弗氏不完全佐剂)等。本申请提供了一种人参杂多糖(GAPS-FL)的一级结构鉴定方法：
首先采用紫外分光光度计确定所述杂多糖中有无蛋白质和核酸；
用红外光谱扫描仪确定特征峰，初步判定含有的官能团；
用HPGPC分析仪器，其中包括高效液相色谱仪配备示差检测器测定杂多糖的分子量；
然后用GC-MS杂多糖甲基化分析确定其糖苷键的种类，用离子色谱法测定单糖组成分析确定其单糖组成；
最后通过核磁分析推断糖苷键连接及其结构单元。
实施例
实施例1：GAPS-FL分离及纯化
1、人参根(2.0kg)经70～95％乙醇浸泡24小时进行脱脂。收集残渣于50℃烘箱内干燥至恒重后，粉碎成粉末后过150目筛网备用；
2、按料液比1：15～1：30加入蒸馏水，搅拌均匀后于90℃恒温水浴锅中提取4次，每次提取1～5h；
3、冷却至室温后离心取上清，浓缩至1/10体积，Sevag法除蛋白，取上清液 浓缩，使用终浓度为65～95％乙醇进行沉淀，4～15℃过夜，收集沉淀，进行冻干，即获得人参粗多糖(200g)；
4、取100g人参粗多糖用蒸馏水充分溶解，离心取上清，通过蒸馏水，氯化钠溶液依次快流速洗脱DEAE法(氯化钠浓度为0.05～1mol/L，流速为0.03～0.12mL/min)，洗脱曲线如图2所示，分别收集溶液，苯酚-硫酸法测定多糖含量并收集主峰，浓缩，4～15℃透析过夜，进行冻干；
5、将通过DEAE分离获得的人参多糖冻干粉溶液蒸馏水，以凝胶色谱柱进一步纯化，以蒸馏水，0.9～2.5M氯化钠溶液依次洗脱，洗脱曲线如图3所示，分管收集并使用苯酚-硫酸法测定多糖含量并收集主峰，浓缩，4～15℃透析过夜，进行冻干即获得人参杂多糖(GAPS-FL)，分离纯化流程图见图1。
6、通过苯酚-硫酸法、间羟基联苯法、Bradford法分别测定GAPS-FL的多糖含量、糖醛酸含量、蛋白质含量，结果见表1。
表1 GAPS-FL的多糖、糖醛酸、蛋白质含量结果
实施例2：GAPS-FL紫外光谱分析
将实施例1所制备的GAPS-FL样品配置成0.04～1.0mg/mL水溶液，用紫外分光光度计在800～200nm的范围内进行扫描。见图4A，其在260nm和280nm处均无特征吸收峰，表明GAPS-FL均无蛋白质和核酸。
实施例3：GAPS-FL红外色谱分析
取干燥的实施例1所制备的GAPS-FL样品1.0mg，以KBr压片，在4000-400cm-1的范围内进行红外光谱扫描。见图4B，吸收带在3600-3200cm-1是-OH的伸缩振动吸收峰，这个区域的吸收峰是糖类的特征峰。具体如下：
在3425cm-1是O-H的伸缩振动吸收峰，是糖类的特征峰。
在2935cm-1处有一个吸收峰，可能归属于C-H伸缩振动。
在1743cm-1处有一个吸收峰，可能归属于C＝O伸缩振动。
在1608cm-1处有一个吸收峰，可能归属于C＝O非对称伸缩振动。
在1373cm-1处、1330cm-1处有一个吸收峰，可能归属于C＝O对称伸缩振动。
在1419cm-1处、1247cm-1处、1143cm-1处、1099cm-1处有吸收峰，可能归属于C-O伸缩振动。
在1022cm-1处有吸收峰，可能归属于O-H变角振动。
在960cm-1处有吸收峰，可能归属于吡喃环的非对称环伸缩振动。
实施例4：GAPS-FL纯度和相对分子量测定
精密称取实施例1所制备的GAPS-FL样品和不同分子量的标准品，样品配制成5mg/mL溶液，12000rpm离心10min，上清液用0.22μm的微孔滤膜过滤，然后将样品转置于1.8ml进样小瓶中。
使用高效凝胶渗透色谱法(HPGPC)进行测定，色谱方法为色谱柱：BRT105-104-102串联凝胶柱(8×300mm)；流动相：0.05M NaCl溶液；流速：0.6ml/min，柱温：40℃；进样量：20μl；检测器：示差检测器RI-10A。
以以下不同相对分子质量的葡聚糖(Mw 5000，11600，23800，48600，80900，148000，273000，409800，667800)作为标准品，做出标准曲线(图5)，测定GAPS-FL的纯度及相对分子质量，经过计算，最终得到其分子量为63350Da，同时，从HPGPC测得的谱图(图6)中可以看出，HPGPC谱图中具有单个对称的峰，表明GAPS-FL是纯的多糖。
实施例5：GAPS-FL单糖组成分析
取16种单糖标准品(岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸、氨基半乳糖盐酸盐、盐酸氨基葡萄糖、N-乙酰-D氨基葡萄糖、古罗糖醛酸、甘露糖醛酸)配成标准母液溶液。
取各单糖标准溶液精密配置浓度标准品作为混标。根据绝对定量方法，测定不同单糖质量，根据单糖摩尔质量计算出摩尔比。取干燥的GAPS-FL样品5.0mg置于安瓿瓶中，加入3mL 2.0mol/L TFA，120℃水解3h。准确吸取酸水解溶液转移至管中氮气吹干，加入5mL水涡旋混匀，吸取50μL加入950μL去离子水，12000rpm离心5min。取上清进离子色谱(IC)分析。
结果见图6，GAPS-FL的单糖组成为鼠李糖：阿拉伯糖：半乳糖：葡萄糖：木糖：甘露糖：半乳糖醛酸(摩尔比)＝0.094：0.148：0.139：0.117：0.005：0.009： 0.455。
实施例6：GAPS-FL甲基化分析
称量GAPS-FL样品(2-3mg)置于玻璃反应瓶中，加入1mL无水DMSO，快速加入甲基化试剂A液，封闭，在超声作用下溶解，再加入甲基化试剂B液。在磁力搅拌水浴30℃反应60min反应。最后将2mL超纯水加入到上述混合物中终止甲基化反应。取甲基化后的多糖，加入1mL的2M三氟乙酸(TFA)水解90min，旋转蒸发仪蒸干。残基加入2mL双蒸水，60mg硼氢化钠还原8小时，加入冰醋酸中和，旋蒸，101℃烘箱烘干，然后加入1mL乙酸酐乙酰化100℃反应1h，冷却。然后加入3mL甲苯，减压浓缩蒸干，重复4-5次，以除去多余的醋酐。将乙酰化后的产物用3mL CH2Cl2溶解后转移至分液漏斗，加入少量蒸馏水充分震荡后，除去上层水溶液，如此重复4次。CH2Cl2层以适量的无水硫酸钠干燥，定容10mL，放入液相小瓶。
分析采用气相色谱-质谱联用仪测定乙酰化产物样品；GC-MS程序升温条件为：起始温度120℃，以3℃/min升温至250℃/min；保持5min；进样口温度为250℃，检测器温度为250℃/min，载气为氦气，流速为1mL/min。分析谱图如图8所示，具体甲基化分析结果见表2。
表2 GAPS-FL的甲基化分析结果

实施例7：GAPS-FL NMR光谱测定
取冷冻干燥后的GAPS-FL样品50mg，溶于0.5mL D2O中，氢氘交换3次后，在AVANCE600核磁共振仪上测定其1H-NMR，13C-NMR，DEPT-135，H-HCOSY，HSQC，HMBC，NOESY，HSQC-TOCSY谱图，见图9A-9H。
GAPS-FL的1H-NMR和13C-NMR化学位移数据见表3。
据图9A的1H-NMR谱图显示，在5.73、5.06、5.01、5.00ppm处的信号分别对应T-Ara、3,5-Ara、1,5-Ara的H-1。5.24、5.00ppm处的信号分别对应1,3,4-GalA和1,4-GalA的H-1。5.23ppm处的信号对应1,4,6-Glcp的H-1。4.88ppm附近的信号可以归属为1,3,4-Rhap，T-Gal，1,4-Glc，1,2-Man，T-Xyl的H-1。
据图9B的13C-NMR谱图显示，GAPS-FL的13C-NMR端基峰显示在107.6、107.4、107.3ppm处的信号对应于T-Ara、3,5-Ara、1,5-Ara的C-1。104.6、102.8、100.2ppm处的信号对应于不同取代Galp的C-1。99.3ppm处的信号对应于GalA的C-1。100.9、99.9、98.7、97.6ppm分别对应1,2-Man，1,4-Glc，T-Xyl，1,3,4-Rha的H-1。96.3、92.1处的信号对应于不同取代的Glc的C-1。在160-180ppm处含有羰基碳信号，归属为GalA的C-6，由于化学位移值向高场移动，说明C-6由甲氧基的取代形成甲基化。
通过图9F的HMBC谱图将各个糖残基进行连接，残基A分别与残基B、C、D、H、I相连形成阿拉伯半乳糖聚糖侧链，残基B和C相连形成阿拉伯聚糖侧链，残基K、L、M相连形成葡聚糖侧链，残基D、E、F相连形成半乳糖醛酸主链，并通过二维核磁将各侧链与主链相连。根据GAPS-FL的相对分子质量确定重复片段个数并确定GAPS-FL的重复片段见图10。
综上可以推断GAPS-FL的重复单元结构式如图10所示。
实施例8：GAPS-FL三维结构测定
为了观察多糖的微观结构和形态特征，对实施例1制备的GAPS-FL进行扫描电镜和原子力显微镜分析(图9A和图9B)。GAPS-FL样品在镀金后，使用扫描电镜在高真空条件下以5.0kV的加速电压分析该杂多糖的微观结构。将GAPS-FL配置成10μg/mL的水溶液并超声处理15min后，将样品转移至云母板上，随后在120℃下干燥30s进行原子力显微镜分析。
从图11A和图11B可以看出，在聚集状态下，观察到具有不均匀表面的不规则分支片状结构，表明多糖无定形结构。多糖表面厚薄不均，出现波峰和凹陷，可能具有高度分支结构。
实施例9：GAPS-FL对小鼠脾淋巴细胞毒性的研究
试剂与药品：红细胞裂解液(北京索莱宝科技有限公司)，RPMI-1640培养基(大连美伦生物科技有限公司)，胎牛血清(大连美伦生物科技有限公司)，CCK-8(大连美伦生物科技有限公司)。
动物：SPF级雌性C57BL/6小鼠(辽宁成大生物科技股份有限公司)。
取6-8周龄C57BL/6小鼠，颈椎脱臼处死，无菌制备脾淋巴细胞悬液，用含10％胎牛血清的RPMI-1640培养基调整细胞浓度为(1～5)×106/mL，均匀铺于96孔板中，每孔加入100μL细胞悬液，分为实验组和空白对照组，空白对照组再加100μL空白培养基；实验组分别加入浓度为25ug/ml、50ug/ml、100ug/ml、200ug/ml、400ug/ml、800ug/ml、1600ug/ml、3200ug/ml、6400ug/ml的GAPS-FL溶液，于培养箱中培养44h后每孔加入20μL的CCK-8溶液，在酶标仪上测各孔的吸光值(450nm)，并进行统计学分析。
结果：见图12所示，25-6400μg/mL的GAPS-FL水溶液对小鼠脾淋巴细胞均无毒性作用，且1600、3200、6400μg/mL的GAPS-FL水溶液可以增强小鼠脾淋巴细胞具有增殖作用，证明GAPS-FL具有免疫刺激活性。
实施例10：GAPS-FL协同LPS(脂多糖)、Con A(刀豆蛋白)或流感疫苗对小鼠脾淋巴细胞增殖的研究
试剂与药品：脂多糖(LPS，Sigma公司)，刀豆蛋白(Con A，Sigma公司)，红细胞裂解液(北京索莱宝科技有限公司)，RPMI-1640培养基(大连美伦生物科技有限公司)，胎牛血清(大连美伦生物科技有限公司)，CCK-8(大连美伦生物科技有限公司)。
动物：雌性C57BL/6小鼠50只，6-8周龄，购自辽宁成大生物科技股份有限公司。
含GAPS-FL佐剂的流感疫苗制剂：分别称取GAPS-FL为2.5mg、0.5mg和流感疫苗15μg，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每1ml含GAPS-FL为2.5mg、0.5mg和流感疫苗15μg。按照以下分组与剂量，配制四组不同的制剂。
实验分组与剂量：
免疫方案：50只小鼠随机分成4组，每组10只。分别肌肉注射上述A、B、C、D四组制剂0.1ml/只，初次免疫1周后，然后进行第2次免疫。第2次免疫后14天将各组小鼠按实施例9方法制备脾淋巴细胞悬液，用含10％胎牛血清的RPMI-1640培养基调整细胞浓度为(1～5)×106/mL，分别均匀铺于板1、板2、板3三个96孔板中，
板1、板2和板3分别用不同的试剂在体外培养环境下刺激淋巴细胞增殖，并考察不同分组小鼠给药后脾淋巴细胞的增殖活性。其中板1使用脂多糖作为刺激物，板2使用刀豆蛋白作为刺激物，板3使用流感疫苗原液作为刺激物加入各项试剂后使得A、B、C、D四组所对应孵育孔分别含有100μL脾淋巴细胞悬液、80μL完全培养基(RPMI 1640+10％胎牛血清)及如下成分。
板1：每孵育孔内含有5μg/mL的LPS(脂多糖)；
板2：每孵育孔内含有10μg/mL的ConA；
板3:每孵育孔内含有1μg/mL的流感疫苗原液。
置培养箱培养48h。培养结束前4h，于每孔加入20μL的CCK-8溶液，在酶标仪上测各孔的吸光值(450nm)，计算刺激指数SI其中阴性对照孔对应的是D组流感疫苗组的小鼠，取其脾脏研磨得脾细胞组。
结果：实验组中，GAPS-FL协同LPS对小鼠脾淋巴细胞增殖的影响见图11所示，高、低剂量GAPS-FL与5μg/mL的LPS对小鼠脾淋巴细胞增殖均具有一定的协同作用，其中高剂量GAPS-FL(500μg)与Con A对小鼠脾淋巴细胞增殖的协同能力最强，低剂量GAPS-FL(50μg)次之，均具有显著性差异。
实验组中，GAPS-FL协同Con A对小鼠脾淋巴细胞增殖的影响见图12所示，高、低剂量GAPS-FL与10μg/mL的Con A对小鼠脾淋巴细胞增殖均具有一定的协同作用，其中高剂量GAPS-FL(500μg)与Con A对小鼠脾淋巴细胞增殖的协同能力最强，并具有显著性差异，低剂量GAPS-FL(50μg)Con A对小鼠脾淋巴细胞增殖的协同能力虽无显著性差异，但总体呈上升趋势。
实验组中，GAPS-FL协同流感疫苗对小鼠脾淋巴细胞增殖的影响见图13所示，高剂量GAPS-FL(500μg)与流感疫苗原液对小鼠脾淋巴细胞增殖均具有一定的协同作用，并具有显著性差异。
实施例11：GAPS-FL对流感疫苗的佐剂活性研究
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠40只，6-8周龄，购自辽宁成大生物科技股份有限公司。
含GAPS-FL佐剂的流感疫苗制剂：分别称取GAPS-FL为2.5mg、0.5mg和流感疫苗15μg，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每1ml含GAPS-FL为2.5mg、0.5mg和流感疫苗15μg。
实验分组与剂量：

免疫方案：40只小鼠随机分成4组，每组10只。肌肉注射0.1ml/只，初次免疫1周后，然后进行第2次免疫。第2次免疫后14天通过ELISA和血凝抑制实验检测小鼠血清中IgG、IgG1、IgG2a、中和抗体效价水平，并计算IgG2a/IgG1比率。
结果：见图16A-16E所示，与空白组相比，高剂量GAPS-FL(500μg)能显著提升流感疫苗免疫小鼠的IgG，IgG1，和中和抗体水平(P＜0.01，P＜0.0001)，低剂量GAPS-FL(50μg)能显著提升流感疫苗免疫小鼠的IgG，IgG2a/IgG1比例和中和抗体水平(P＜0.05，P＜0.01，P＜0.0001)，其他剂量与流感疫苗组相比也呈现上升趋势。
实施例12：GAPS-FL对狂犬疫苗佐剂活性研究
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠50只，6-8周龄，购自辽宁成大生物科技股份有限公司。
含GAPS-FL佐剂的狂犬疫苗制剂：分别称取GAPS-FL为2.5mg、0.5mg和狂犬疫苗25IU，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每1ml含GAPS-FL为2.5mg、0.5mg和狂犬疫苗2.5IU。
实验分组与剂量：
免疫方案：40只小鼠随机分成4组，每组10只。肌肉注射0.1ml/只，初次免疫1周后，然后进行第2次免疫。第2次免疫后14天通过ELISA实验检测小鼠血清中IgG、IgG1、IgG2a抗体效价水平，并计算IgG2a/IgG1比率。
结果：见图17A-17D所示，与铝盐佐剂组相比，高剂量GAPS-FL(500 μg)能显著提升狂犬疫苗免疫小鼠的IgG和IgG1抗体水平(P＜0.01)，与空白组相比，高剂量GAPS-FL(500μg)能显著提升狂犬疫苗免疫小鼠的IgG，IgG1，IgG2a，IgG2a/IgG1抗体水平(P＜0.05，P＜0.0001)，低剂量GAPS-FL(50μg)能显著提升流感疫苗免疫小鼠的IgG抗体的水平(P＜0.0001)，其他剂量与狂犬疫苗组相比也呈现上升趋势。
实施例13：GAPS-FL对手足口疫苗佐剂活性研究
手足口疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠40只，6-8周龄，购自辽宁成大生物科技股份有限公司。
含GAPS-FL佐剂的手足口疫苗制剂：分别称取GAPS-FL为2.5mg、0.5mg和手足口疫苗50U，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每1ml含GAPS-FL为2.5mg、0.5mg和手足口疫苗50U。
实验分组与剂量：
免疫方案：40只小鼠随机分成4组，每组10只。肌肉注射0.1ml/只，初次免疫1周后，然后进行第2次免疫。第2次免疫后14天通过ELISA实验检测小鼠血清中IgG、IgG1、IgG2a抗体效价水平，并计算IgG2a/IgG1比率。
结果：见图18A-18D所示，与铝盐佐剂组相比，高剂量GAPS-FL(500μg)能显著提升手足口疫苗免疫小鼠的IgG，IgG1和IgG2a抗体水平(P＜0.05)，与空白组相比，低剂量GAPS-FL(50μg)，高剂量GAPS-FL(500μg)能显著提升手足口疫苗免疫小鼠的IgG，IgG1，IgG2a和IgG2a/IgG1抗体水平(P＜0.05，P＜0.0001)，其他剂量与手足口疫苗组相比也呈现上升趋势。
实施例14：GAPS-FL对甲肝疫苗佐剂活性研究
甲肝疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠40只，6-8周龄，购自辽宁成大生物科技股份有限公司。
含GAPS-FL佐剂的甲肝疫苗制剂：分别称取GAPS-FL为2.5mg、0.5mg和手足口疫苗4IU，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每1ml含GAPS-FL为2.5mg、0.5mg和甲肝疫苗4IU。
实验分组与剂量：
免疫方案：40只小鼠随机分成4组，每组10只。肌肉注射0.1ml/只，初次免疫1周后，然后进行第2次免疫。第2次免疫后14天通过ELISA实验检测小鼠血清中IgG、IgG1、IgG2a抗体效价水平，并计算IgG2a/IgG1比率。
结果：见图19A-19D所示，与铝盐佐剂组相比，高剂量GAPS-FL(500μg)能显著提升甲肝疫苗免疫小鼠的IgG，IgG1和IgG2a/IgG1抗体水平(P＜0.05)，与空白组相比，高剂量GAPS-FL(500μg)能显著提升甲肝疫苗免疫小鼠的IgG，IgG1，IgG2a和IgG2a/IgG1抗体水平(P＜0.01，P＜0.0001)，低剂量GAPS-FL(50μg)能显著提升甲肝疫苗免疫小鼠的IgG，IgG2a和IgG2a/IgG1抗体水平(P＜0.0001)，其他剂量与甲肝疫苗组相比也呈现上升趋势。
实施例15：GAPS-FL对GATA-3、T-bet、IFN-γ、IL-4基因表达的研究
1、制备各组小鼠脾淋巴细胞悬液
按照实施例9的方法制备各组小鼠脾淋巴细胞悬液，并调整细胞浓度为(6～8)×107/mL。按照实施例10的动物分组铺于6孔培养板中，每孔加入1mL细胞悬液，再加入100μL流感疫苗原液培养48h，离心并收集细胞备用。
2、总RNA的提取
采用经典的异硫氰酸胍方法提取脾淋巴细胞总RNA。
3、总RNA浓度测定及电泳鉴定
取4μL总RNA，用DEPC水稀释至1000μL，用超微量分光光度计测定总RNA的OD260及OD280。另取0.5g琼脂糖，加入30mL 0.1％DEPC水，在微波炉中加热至熔化，冷却至50℃，加入2μL EP溶液，混匀后将胶加到制胶槽内，取5μL总RNA上样，电泳，紫外灯下观察并拍照鉴定RNA的完整性，结果见图18，可以发现28S和18S两条明显的条带，并且28S的亮度是18S的两倍左右，且260/280紫外吸收比值在1.7-2.0之间，表明RNA没有发生明显的降解可以用于后续实验。
4、RP-PCR
由Genebank获取小鼠细胞因子GATA-3、T-bet、IFN-γ、IL-4及内标β-actin基因全序列见表4。
PCR系统如下：模板cDNA 4μL，Hieff qPCR SYBR Green Master Mix 10μL，上下游引物各0.4μL，无菌超纯水补足至20μL。扩增程序为预变性95℃后按下列程序循环40次，95℃10s，60℃20s，72℃20s。然后72℃延伸10min。
用Bio-Rad RT-PCR测定各组Ct值，并计算2-ΔΔCt。结果见图21A-21D，低剂量GAPS-FL(50μg)，高剂量GAPS-FL(500μg)能显著提升小鼠脾淋巴细胞中GATA-3、T-bet、IFN-γ、IL-4mRNA基因的表达，说明GAPS-FL可以增强流感疫苗佐剂在细胞免疫中的作用。
表4 GATA-3、T-bet、IFN-γ、IL-4及内标β-actin基因全序列

实施例16：GAPS-FL对IFN-γ和IL-4脾淋巴细胞因子的研究
按照实施例9的方法制备各组小鼠脾细胞悬液，并调整细胞浓度为(1～2)×105/mL，根据ELISPOT试剂盒的操作说明均匀接种于96孔培养板中，每孔加入100μL细胞悬液，仅加入100μL培养基(背景对照)，100μL细胞悬液(阴性对照)，100μL细胞悬液加10μL PMA刺激物(阳性对照)以及按照实施例10的动物分组的各实验孔。所有样品和刺激物加完后，放入培养箱中培养48h。经过细胞裂解、洗板、IFN-γ和IL-4检测抗体孵育、酶联亲和素孵育、显色等步骤，最终在读板机下读取各个孔的斑点数。各组斑点图片和统计结果见图22A-D，从图中可以看出，高剂量GAPS-FL(500μg)能显著提升小鼠脾淋巴细胞中IFN-γ和IL-4细胞因子的增殖，与流感疫苗组相比具有显著性差异，低剂量GAPS-FL(50μg)虽然无显著性差异，但总体呈升高趋势，说明GAPS-FL可同时促进脾淋巴细胞Th1和Th2免疫反应。
实施例17：GAPS-FL对CD3+CD4+和CD3+CD8+T淋巴细胞亚群的研究
按照实施例9的方法制备各组小鼠脾细胞悬液，并调整细胞浓度为3-6×106/mL，接种于24孔培养板中，每孔加入1000μL细胞悬液和100μL流感疫苗原液，置于培养箱中培养48h。离心，收集细胞，并加入100μL无菌PBS重悬，每组加入2μg APC-CD3、FITC-CD4、PE-CD8抗体进行染色，4℃孵育30min后，离心，收集细胞，并加入100μL无菌PBS重悬细胞，上机检测。各组染色图片和统计结果见图23A-23D，低剂量GAPS-FL(50μg)与高剂量GAPS-FL(500μg)能显著提升小鼠脾淋巴细胞中CD3+CD4+和CD3+CD8+T淋巴细胞亚群的比例，其中高剂量GAPS-FL(500μg)可以显著提高小鼠脾淋巴细胞CD3+CD4+T淋巴细胞亚群的比例，与流感疫苗组相比具有显著性差异，低剂量GAPS-FL(50μg)可以显著提高小鼠脾淋巴 细胞CD3+CD8+T淋巴细胞亚群的比例，与流感疫苗组相比具有显著性差异，说明GAPS-FL作为流感疫苗佐剂具有强大的细胞免疫活性。
实施例18：小鼠脾脏组织HE染色
随机取实施例10中A，B，C，D组制剂处理过的小鼠各3只，脱臼处死并在无菌条件下取脾脏，并置于4％多聚甲醛中固定，在不同浓度的酒精溶液中脱水后，将脾脏包埋于石蜡中，并进行HE染色，使用光学显微镜观察病理切片。结果见图24，高剂量GAPS-FL(500μg)脾白髓中心区(WP)和边缘区(MZ)淋巴细胞比例最高且脾红髓区(RP)出现大量红细胞，小鼠脾脏组织HE染色提示GAPS-FL可以增强流感疫苗的免疫反应。
尽管以上结合对本申请的实施方案进行了描述，但本申请并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本申请权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本申请保护之列。

Claims (22)

  1. 一种杂多糖，其特征在于，包含半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖。
  2. 根据权利要求1所述的杂多糖，其特征在于，所述杂多糖包含1、1，5、1，3，5连接的阿拉伯糖残基；1，4连接的半乳糖醛酸残基、1，3，4连接的鼠李糖残基；1、1，4、1，3，4连接的半乳糖残基；1、1，4、1，3，4连接的葡萄糖残基；1，2连接的甘露糖残基。
  3. 根据权利要求1或2所述的杂多糖，其特征在于，所述杂多糖的主链由1，4连接的半乳糖醛酸交替连接组成，并有不同程度的甲基化和乙酰化，所述杂多糖的支链由阿拉伯聚糖、半乳阿拉伯聚糖和杂聚糖组成。
  4. 根据权利要求1-3中任一项所述的杂多糖，其重复单元结构式如式(I)所示，
    其中， 1≤n≤30，1≤m≤50，1≤p≤80。
  5. 一种杂多糖，其特征在于，其重复单元结构式如式(I)所示，
    其中， 1≤n≤30，1≤m≤50，1≤p≤80。
  6. 根据权利要求3所述的杂多糖，其特征在于，所述杂多糖由半乳糖醛酸、阿拉伯糖、半乳糖、葡萄糖、鼠李糖、木糖、甘露糖组成。
  7. 根据权利要求1-6中任一项所述的杂多糖，其特征在于，相当于100摩尔 的杂多糖，其中，下述单元的摩尔量为：
    半乳糖醛酸：20.00～65.00，
    阿拉伯糖：8.00～40.00，
    半乳糖：5.00～25.00，
    葡萄糖：5.00～25.00，
    鼠李糖：3.00～20.00，
    木糖：0.05～5.00，
    甘露糖：0.05～5.00。
    优选地，下述单元的摩尔量为：
    半乳糖醛酸：40.00～50.00，
    阿拉伯糖：10.00～20.00，
    半乳糖：10.00～20.00，
    葡萄糖：5.00～15.00，
    鼠李糖：5.00～15.00，
    木糖：0.2～3.00，
    甘露糖：0.2～3.00。
  8. 根据权利要求1-7中任一项所述的杂多糖，其特征在于，所述杂多糖的重均分子量为4×103Da～7×106Da。
  9. 根据权利要求1-8中任一项所述的杂多糖，其特征在于，所述杂多糖是从人参中提取出来的。
  10. 一种制备权利要求1-9中任一项所述杂多糖的方法，其特征在于，包括，
    人参进行脱脂后，经过浸提、醇沉、浓缩、干燥，得到人参粗多糖；
    所述人参粗多糖通过离子交换柱层析，洗脱，得到杂多糖提取液；
    所述杂多糖提取液经过凝胶柱进行纯化，得到所述杂多糖。
  11. 根据权利要求10所述的方法，其特征在于，所述离子交换柱为阴离子交换树脂柱，优选，填料为DEAE Sepharose Fast Flow。
  12. 根据权利要求10或11所述的方法，其特征在于，所述水浸提过程中，料液比为1：10～40，次数为2～4次，浸提时间为1～5h。
  13. 根据权利要求10-12中任一项所述的方法，其特征在于，所述通过离子交换柱层析过程中，以蒸馏水、氯化钠水溶液依次洗脱交换柱，洗脱流速为 0.03～12mL/min。
  14. 根据权利要求10-13中任一项所述的方法，其特征在于，所述经过凝胶柱进行纯化的过程中，以0.9～2.5M氯化钠洗脱凝胶柱，洗脱流速为0.01～0.08mL/min。
  15. 一种组合物，其特征在于，包括权利要求1-9中任一项所述的杂多糖或根据权利要求10-14中任一项所述的方法制备的杂多糖和药学上可接受的载体和/或辅料。
  16. 权利要求1-9中任一项所述的杂多糖或权利要求10-14中任一项所述的方法制备的杂多糖或权利要求15所述的组合物作为疫苗佐剂用途。
  17. 根据权利要求16所述的用途，其中，所述疫苗包括但不限于为流感疫苗、狂犬疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒疫苗。
  18. 权利要求1-9中任一项所述的杂多糖或权利要求10-14中任一项所述的方法制备的杂多糖或权利要求15所述的组合物在提升脾淋巴细胞中GATA-3、T-bet、IFN-γ或IL-4 mRNA基因表达方面的用途。
  19. 权利要求1-9中任一项所述的杂多糖或权利要求10-14中任一项所述的方法制备的杂多糖或权利要求15所述的组合物在提升脾淋巴细胞中IFN-γ和IL-4细胞因子的增殖方面的用途。
  20. 权利要求1-9中任一项所述的杂多糖或权利要求10-14中任一项所述的方法制备的杂多糖或权利要求15所述的组合物在提高脾淋巴细胞中CD3+CD4+T或CD3+CD8+T淋巴细胞亚群的比例方面的用途。
  21. 一种预防和/或治疗疾病的方法，包括向受试者施用权利要求15所述的疫苗组合物。
  22. 根据权利要求21所述的方法，所述疾病选自狂犬病、流感病、乙肝病、甲肝病、丙肝病、手足口病、HPV病或新型冠状病毒病。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119060209A (zh) * 2024-08-28 2024-12-03 南京中医药大学 一种抗疲劳的黄精寡聚糖的制备方法和应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116082532A (zh) * 2023-02-16 2023-05-09 沈阳药科大学 一种人参杂多糖及其分离方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100221281A1 (en) * 2009-01-27 2010-09-02 World Force Technologies, Llc High molecular weight polysaccharide that binds and inhibits virus
CN102417544A (zh) * 2011-09-28 2012-04-18 陕西师范大学 一种紫阳富硒绿茶含硒多糖及其制备方法和用途
CN109678982A (zh) * 2018-12-26 2019-04-26 王秋红 一种麻黄均一多糖esp-b4及其制备方法和用途
CN113616670A (zh) * 2020-05-06 2021-11-09 长春中医药大学 一种红参果胶样多糖在制备降脂药物或保健品中的应用
CN114057907A (zh) * 2021-12-21 2022-02-18 中国药科大学 一种红参多糖的提取和分离纯化方法
CN115651088A (zh) * 2022-08-23 2023-01-31 辽宁成大生物股份有限公司 人参总多糖的制备方法和应用、人参总多糖疫苗佐剂及其疫苗组合物
CN116789865A (zh) * 2023-02-16 2023-09-22 沈阳药科大学 一种人参杂多糖及其分离方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101323873B (zh) * 2008-07-16 2011-08-10 珠海市星远科技有限公司 一种人参多糖提取工艺
CN112830978B (zh) * 2021-03-04 2022-07-22 牡丹江医学院 红参多糖、提纯方法及联合顺铂在抗癌药物中的应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100221281A1 (en) * 2009-01-27 2010-09-02 World Force Technologies, Llc High molecular weight polysaccharide that binds and inhibits virus
CN102417544A (zh) * 2011-09-28 2012-04-18 陕西师范大学 一种紫阳富硒绿茶含硒多糖及其制备方法和用途
CN109678982A (zh) * 2018-12-26 2019-04-26 王秋红 一种麻黄均一多糖esp-b4及其制备方法和用途
CN113616670A (zh) * 2020-05-06 2021-11-09 长春中医药大学 一种红参果胶样多糖在制备降脂药物或保健品中的应用
CN114057907A (zh) * 2021-12-21 2022-02-18 中国药科大学 一种红参多糖的提取和分离纯化方法
CN115651088A (zh) * 2022-08-23 2023-01-31 辽宁成大生物股份有限公司 人参总多糖的制备方法和应用、人参总多糖疫苗佐剂及其疫苗组合物
CN116789865A (zh) * 2023-02-16 2023-09-22 沈阳药科大学 一种人参杂多糖及其分离方法和应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119060209A (zh) * 2024-08-28 2024-12-03 南京中医药大学 一种抗疲劳的黄精寡聚糖的制备方法和应用

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