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WO2024169040A1 - Hétéropolysaccharide de panax ginseng, son procédé de séparation et son utilisation - Google Patents

Hétéropolysaccharide de panax ginseng, son procédé de séparation et son utilisation 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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English (en)
Chinese (zh)
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

La présente demande divulgue un hétéropolysaccharide, comprenant de l'acide galacturonique, de l'arabinose, du galactose, du glucose, du rhamnose, du xylose et du mannose. Plus particulièrement, la présente demande concerne un hétéropolysaccharide de Panax ginseng (GAPS-FL), qui est une nouvelle substance polysaccharidique séparée des racines de Panax ginseng, peut changer ou améliorer de manière non spécifique la réaction de réponse immunitaire de multiples types de vaccins comprenant des vaccins contre la grippe, peut améliorer l'immunité humorale des souris immunisées par le vaccin contre la grippe, et peut également améliorer l'immunité cellulaire associée.
PCT/CN2023/089771 2023-02-16 2023-04-21 Hétéropolysaccharide de panax ginseng, son procédé de séparation et son utilisation Ceased WO2024169040A1 (fr)

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