CN115651088A - Preparation method and application of ginseng total polysaccharide, ginseng total polysaccharide vaccine adjuvant and vaccine composition thereof - Google Patents

Abstract

The application provides a preparation method and application of ginseng total polysaccharide, a ginseng total polysaccharide vaccine adjuvant and a vaccine composition thereof. Compared with the prior art, the vaccine adjuvant can remarkably improve the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, and can effectively enhance the immune response activity of rabies vaccines, influenza vaccines, hepatitis B vaccines, hepatitis A vaccines, hepatitis C vaccines, hand-foot-and-mouth vaccines, HPV vaccines and novel coronavirus vaccines. The vaccine adjuvant has the advantages of good immune effect, convenience in use and the like, and provides a new adjuvant selection for vaccines.

Description

Preparation method and application of ginseng total polysaccharide, ginseng total polysaccharide vaccine adjuvant and vaccine composition thereof

Technical Field

The application belongs to the field of medicines, and particularly relates to a preparation method and application of ginseng total polysaccharide, a ginseng total polysaccharide vaccine adjuvant and a vaccine composition thereof.

Background

The immunoadjuvant is a substance capable of nonspecifically changing or enhancing the specific immune response of an organism to an antigen, and is required to have no toxicity, high purity, certain adsorption capacity and stable property. The action mechanism of the immunologic adjuvant is mainly that the surface area of an antigen can be increased, and the immunogenicity is improved; the slow-release effect on the antigen is realized, and the retention time of the antigen in tissues is prolonged; promoting inflammatory reaction and stimulating active immune response. Most of the currently marketed vaccine adjuvants are aluminum salt adjuvants, but aluminum salts can cause inflammation at injection sites and stimulate local erythema, granuloma and subcutaneous nodules, and meanwhile, the aluminum salt adjuvants can delay the generation of neutralizing antibodies of partial vaccines, so that the application is limited.

Ginseng, a plant of Araliaceae, mainly contains multiple active ingredients such as saponin, polysaccharide, flavone and the like, and the root, rhizome, leaf and other parts of the ginseng have a long medicinal history and have the effects of tonifying qi, promoting the production of body fluid and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the application provides a preparation method and application of ginseng total polysaccharide, a ginseng total polysaccharide vaccine adjuvant and a vaccine composition thereof. Through activity tracking and activity verification, the research finds that the ginseng total polysaccharide (GPS) has good immunologic adjuvant activity, and can obviously improve the immune response of experimental individuals to specific vaccines.

Specifically, the present application relates to the following aspects:

1. the preparation method of the ginseng total polysaccharide is characterized by comprising the following steps:

extracting Ginseng radix with water to obtain a first extract,

adding protease into the first extract, culturing, extracting to obtain a second extract,

extracting the second extract with ethanol and water to obtain crude Ginseng radix polysaccharide,

dissolving the crude polysaccharide of Ginseng radix, dialyzing, removing protein in trapped fluid,

extracting the trapped fluid after removing protein with ethanol to obtain Ginseng radix total polysaccharide.

2. The method according to claim 1, wherein the protease is a neutral protease.

3. The process according to item 1, wherein the protease is cultured at 30 to 40 ℃ for 1 to 3 hours.

4. The method according to item 1, wherein the molecular weight cutoff for dialysis is 800 to 2500kDa, preferably 1000 to 1500kDa.

5. The method according to item 1, wherein the protein in the retentate is removed using Sevag reagent.

6. A vaccine adjuvant is characterized by comprising ginseng total polysaccharide.

7. The vaccine adjuvant according to item 6, further comprising normal saline or water for vaccine injection, and more preferably the vaccine adjuvant comprises ginseng total polysaccharide and normal saline or water for vaccine injection.

8. The vaccine adjuvant according to the item 6, wherein the ginseng total polysaccharides are obtained by the preparation method according to any one of the items 1 to 5.

9. A vaccine composition comprising the vaccine adjuvant according to any one of claims 6 to 8 and a vaccine antigen or DNA encoding the antigen.

10. The vaccine composition of claim 8, further comprising a pharmaceutical excipient and a second vaccine adjuvant.

11. The vaccine composition according to item 8, wherein the vaccine is a rabies vaccine, an influenza vaccine, a hepatitis b vaccine, a hepatitis a vaccine, a hepatitis c vaccine, a hand-foot-and-mouth vaccine, an HPV vaccine, or a novel coronavirus vaccine.

12. The vaccine composition according to item 9, wherein the dosage ratio of the ginseng total polysaccharides and the vaccine antigen is (0.1-1000) to (0.10-200) μ g/IU or (0.1-1000): (0.1-500) mu g/mu g, preferably 10-500 mu 0-50 or 1-150 mu-1 IU.

13. The vaccine composition according to item 10, wherein the vaccine type is an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine, or a polypeptide vaccine.

14. Use of a vaccine adjuvant according to any one of claims 6 to 8 in the preparation of a vaccine formulation, a vaccine composition.

Compared with the prior art, the method has the following technical effects:

(1) The active ingredient GPS in the vaccine adjuvant and the vaccine composition is from natural plants, the source is clear, the resource is rich, and the ingredients have better safety and stability.

(2) The vaccine adjuvant can remarkably improve the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, and can effectively enhance the immune response activity of rabies vaccines, influenza vaccines, hepatitis B vaccines, hepatitis A vaccines, hepatitis C vaccines, hand-foot-and-mouth vaccines, HPV vaccines and novel coronavirus vaccines.

(3) The vaccine adjuvant has the advantages of good immune effect, convenience in use, low side effect, applicability to various vaccines (wide adjuvant activity) and the like, and provides a new adjuvant selection for the vaccines.

Drawings

FIG. 1 is a standard curve of absorbance versus glucose content;

FIG. 2 is a standard curve of absorbance versus protein content;

FIG. 3 is a graph showing the effect of GPS on IgG antibody levels in rabies vaccine immunized mice;

FIG. 4 is a graph of the effect of GPS on neutralizing antibody levels in rabies vaccine immunized mice;

FIG. 5 is a graph of the effect of GPS on neutralizing antibody levels in influenza vaccine immunized mice;

FIG. 6 is a graph of the effect of different doses of GPS on IgG antibody titer in influenza vaccine immunized mice;

FIG. 7 is a graph showing the effect of GPS on IgG antibody levels in mice immunized with hepatitis B vaccine;

FIG. 8 is a graph of the effect of GPS on neutralizing antibody levels in mice immunized with hepatitis A vaccine;

FIG. 9 is a graph of the effect of different doses of GPS on neutralizing antibody levels in mice immunized with hepatitis A vaccine;

FIG. 10 is a graph of the effect of GPS on IgG antibody levels in mice immunized with hepatitis C vaccine;

FIG. 11 is a graph of the effect of GPS on neutralizing antibody levels in mice immunized with the hand-foot-mouth vaccine;

FIG. 12 is a graph of the effect of GPS on IgG antibody levels in mice immunized with the hands-and-feet vaccine;

FIG. 13 is a graph of the effect of different doses of GPS on neutralizing antibody levels in hand-foot-and-mouth vaccine immunized mice;

FIG. 14 is the effect of GPS on HPV vaccine immunized mouse IgG antibody levels;

FIG. 15 is a graph of the effect of different doses of GPS on HPV vaccine immunized mouse IgG antibody levels;

FIG. 16 is a graph of the effect of GPS on IgG antibody levels in mice immunized with the novel coronavirus vaccine;

FIG. 17 is a graph of the effect of different doses of GPS on IgG antibody levels in mice immunized with the novel coronavirus vaccine;

wherein, denotes P <0.05 compared to positive control group; * Denotes P <0.01 compared to positive control group; * Denotes P <0.005 compared to positive control group; * Denotes P <0.001 compared to positive control group; # denotes that P <0.05 compared to the negative control group; # indicates P <0.01 compared to negative control group; # indicates P <0.005 compared to negative control group; indicates # P <0.001 compared to the negative control group; & indicates that P is less than 0.05 compared with the same dose of the commercial ginseng total polysaccharide; % indicates that P is less than 0.05 compared with the ginseng total polysaccharide prepared in the comparative example 1 with the same dosage.

Detailed Description

The present application is further described below in conjunction with the following examples, which are included merely to further illustrate and explain the present application and are not intended to limit the present application.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or experimental applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present application is further described with reference to the following specific examples, which should not be construed as limiting the scope of the present application.

As used herein, the term "vaccine" refers to any preparation of antigen or immunogenic substance suitable for stimulating active immunity in an animal or human.

As used herein, the term "adjuvant" refers to any substance or mixture of substances that enhances, increases, upwardly modulates, alters or otherwise facilitates an immune response (e.g., a humoral or cellular immune response) to an antigen in an animal.

As used herein, the term "antigen" refers to any substance that, when introduced into an immunologically active 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 substance (including cells, cell fragments or cell-derived fragments) or a live (usually attenuated) organism or virus. Examples of suitable antigens include (but are not limited to): proteins, glycoproteins, lipoproteins, skin, carbohydrates/polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi, and parasites. Other suitable antigens include the smallest components of an antigen, such as (but not limited to) an antigenic determinant, epitope, or skin. Still other suitable antigens include those described in U.S. Pat. No. 5,855,894. Antigens may be natural (expressed or made naturally), synthetic, or derived from recombinant DNA methodologies familiar to those skilled in the art.

As used herein, the term "pharmaceutical excipient" refers to a substance that is added to a pharmaceutical formulation in order to solve the problem of moldability, effectiveness, stability and safety of the formulation, and that is a general term for all pharmaceutical materials in the formulation other than the principal drug, and that has been reasonably evaluated in terms of safety and is included in the pharmaceutical formulation. The pharmaceutic adjuvant has important functions of solubilization, dissolution assistance, sustained and controlled release and the like besides shaping, serving as a carrier and improving stability, and is an important component which possibly influences the quality, safety and effectiveness of the medicine. The pharmaceutical excipients described herein may be suitable carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (e.g., aluminum hydroxide adjuvant, oil adjuvant, freund's complete adjuvant, and Freund's incomplete adjuvant), and the like.

As used herein, the term "ginseng polysaccharide" refers to the total polysaccharide extracted from ginseng, a plant of the family araliaceae. In the present application, the term "ginseng total polysaccharides" is used interchangeably with the same meaning. The ginseng total polysaccharides can be prepared by methods known in the art, can be purchased commercially, and can be prepared by the methods for preparing ginseng total polysaccharides provided herein.

The preparation method of the ginseng total polysaccharide provided by the application comprises the following steps:

extracting Ginseng radix with water to obtain a first extract,

adding protease into the first extract, culturing, extracting to obtain a second extract,

extracting the second extract with ethanol and water to obtain crude Ginseng radix polysaccharide,

dissolving the crude polysaccharide of Ginseng radix, dialyzing, removing protein in the trapped fluid,

extracting the retentate with ethanol to obtain Ginseng radix total polysaccharide.

In a specific embodiment, the step of obtaining the first extract comprises:

pulverizing dried Ginseng radix 1kg to 10-20 mesh, adding 8 times of water (g: mL), decocting in boiling water bath for 8 hr, filtering, and collecting filtrate; to the residue was added further 10 times of water (g: mL) and heated in a boiling water bath for 8h. The two filtrates were combined and centrifuged at 6000rpm for 10min. Collecting supernatant, and steaming to obtain first extract.

In a specific embodiment, in the step of obtaining the second extract, the protease is incubated at 30 ℃ to 40 ℃ for 1 to 3 hours.

In a specific embodiment, the step of obtaining the second extract comprises:

adding neutral protease into the first extract, culturing at 40 deg.C for 3 hr, inactivating at 100 deg.C for 30min, centrifuging the mixed solution at 10000rpm to obtain supernatant, and evaporating to obtain second extract.

In a specific embodiment, the step of obtaining the ginseng crude polysaccharide comprises:

soaking the second extract in 10 volume times of 95% ethanol at low temperature for 2.0h. Filtering, dissolving the residue with 2 times volume of distilled water, reflux-extracting with boiling water for 3 times (each time for 1.0 hr), filtering with 120 mesh nylon cloth, mixing extractive solutions, concentrating to 500mL, centrifuging for 20 min (5000 rpm), and discarding the precipitate. Adding 95% ethanol into the supernatant to ethanol final concentration of 90%, and standing for 1 hr. Centrifuging for 20 min (5000 r/min), collecting precipitate, adding 400mL distilled water to the precipitate, adding 95% ethanol to the supernatant to reach final concentration of 80%, standing for 1h, centrifuging for 20 min (5000 r/min), standing overnight at room temperature, and collecting precipitate. The next day, the precipitate was washed twice with absolute ethanol, 95% ethanol in that order. And freeze-drying to obtain crude polysaccharide of Ginseng radix,

in a particular embodiment, the dialysis employs a molecular cut-off of 800 to 2500kDa, preferably 1000 to 1500kDa.

In a specific embodiment, sevag reagent is used to remove proteins from the retentate.

In one embodiment, the step of dissolving the ginseng crude polysaccharide and dialyzing, and removing the protein in the retentate comprises:

preparing the ginseng crude polysaccharide into a 30% aqueous solution, adding a 1000kDa dialysis bag for dialysis, and standing overnight. Taking the liquid in the dialysis bag, adding Sevag reagent with the volume of 1/4, standing, centrifuging, and removing the gel precipitate. The above operation was repeated 5 times.

In a specific embodiment, the step of extracting the retentate after removing the proteins with ethanol to obtain ginseng total polysaccharides comprises:

and combining the supernatant of the protein-removed retentate, and concentrating under reduced pressure to remove the organic reagent. Adding 95% ethanol to a final concentration of 80%, standing overnight at 4 deg.C, centrifuging, and removing supernatant. The precipitate was washed twice with 95% ethanol and absolute ethanol in this order. Freeze drying to obtain Ginseng radix total polysaccharide.

The ginseng total polysaccharide prepared by the method has the polysaccharide content of over 95 percent and the protein content is lower (about 1 percent). Compared with the total polysaccharide sold in the market at present, the polysaccharide has the characteristics of high polysaccharide content, few protein impurities and stronger adjuvant activity.

The present application also provides a vaccine adjuvant comprising ginseng total polysaccharides.

In a specific embodiment, the vaccine adjuvant may further comprise physiological saline or water for vaccine injection. Wherein, the content of the ginseng total polysaccharide can be adjusted according to actual needs.

In a specific embodiment, the vaccine adjuvant consists of ginseng total polysaccharides.

In a specific embodiment, the vaccine adjuvant consists of ginseng total polysaccharides and physiological saline or water for vaccine injection.

In a specific embodiment, the ginseng total polysaccharide is obtained by the above-mentioned preparation method of ginseng total polysaccharide.

The present application also provides a vaccine composition comprising a vaccine adjuvant comprising any one of the ginseng total polysaccharides and an antigen or a DNA encoding the antigen as described herein.

The amount of vaccine adjuvant in the vaccine composition is an amount effective to produce a therapeutic effect, which is an amount that enhances, increases, upwardly modulates, alters or otherwise facilitates an immune response to the antigen. In particular, a therapeutically effective amount is an amount that induces immunity in an animal susceptible to a disease caused by a pathogen, cancer cell, or allergen. As will be appreciated by those skilled in the art, the therapeutically effective amount will vary and be determined on a case-by-case basis, and in the present application, the dose of the vaccine adjuvant and the vaccine antigen is not particularly limited and is appropriately selected depending on the administration method, the subject, the age of the subject, the dosage form, the administration route, and the like.

In a specific embodiment, the dosage ratio of the ginseng total polysaccharide to the vaccine antigen is (0.1 μ g-1000 μ g) GAPS: (0.10 IU to 200 IU) antigen, for example, may be 0.1. Mu.g: 0.10IU, 0.1. Mu.g: 200IU, 1000. Mu.g: 0.10IU, 1000. Mu.g: 200IU; or

(0.1. Mu.g-1000. Mu.g) GAPS: (0.1. Mu.g to 500. Mu.g) of the antigen may be, for example, 0.1. Mu.g: 0.1. Mu.g, 0.1. Mu.g: 500. Mu.g, 1000. Mu.g: 0.1. Mu.g, 1000. Mu.g: 500 μ g.

In a specific embodiment, the dosage ratio of the ginseng total polysaccharide to the vaccine antigen is 10-500. Mu.g/μ g.

In a specific embodiment, the ratio of the total polysaccharides of ginseng to the vaccine antigen is 1 to 150. Mu. To/IU.

One skilled in the art will readily recognize that the therapeutic dose and length of treatment may vary depending on the type, weight and condition of the patient to be treated, their individual response to the vaccine composition, and the particular route of administration selected. In some instances, dosage levels below the lower limit of the aforesaid range may be therapeutically effective, while in other cases still larger doses may be employed without causing any harmful side effects, provided that such larger doses are first divided into several small doses for administration throughout the day. The priming dose is considered ideal whenever secondary stress or exposure may occur.

The vaccine adjuvant and the antigen or the DNA encoding the antigen in the vaccine combination of the present application may be contained together in one composition and may be formulated in separate compositions, and when formulated in separate compositions, the administration routes of the vaccine adjuvant and the antigen or the DNA encoding the antigen may be the same or different. In the present application, the vaccine adjuvant and the antigen or the DNA encoding the antigen may be administered simultaneously or with time difference, i.e., the vaccine adjuvant and the antigen or the DNA encoding the antigen may be administered simultaneously or separately (e.g., the vaccine adjuvant is administered before or after administration of the vaccine antigen). Vaccine adjuvants and antigens or DNA encoding the antigens may be provided as kits containing them. However, from the viewpoint of reducing the burden on the patient, it is preferable that the vaccine adjuvant and the antigen or the DNA encoding the antigen are contained in one composition so that they can be administered simultaneously at the time of administration. Whether co-administered or co-administered, the mode of administration of the vaccine composition may be any suitable route by which the vaccine composition is delivered to the host, whether co-administered or co-administered.

In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application further comprises a pharmaceutical adjuvant and a second vaccine adjuvant.

The pharmaceutic adjuvant is as described above.

The vaccine adjuvants of the present application may be administered as part of a vaccine formulation, optionally containing an additional second vaccine adjuvant. The second vaccine adjuvant is other adjuvant different from the ginseng total polysaccharide adjuvant of the present application, and may be one or more than two, and examples of suitable second vaccine adjuvants include those known in the art,

the vaccine adjuvant or vaccine composition of the present application may further comprise one or more antioxidants selected from the group consisting of: sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium methioninate, L-ascorbic acid, erythorbic acid, acetylcysteine, cysteine, monothioglycerol, thioglycolic acid, thiolactic acid, thiamine, dithiothreitol, dithioerythrol, glutathione, ascorbic acid melic acid, butylated shinyleaf balsamisole, butylated light toluene, nordihydroguaiaretic acid, propylgallate, alpha-tocopherol, and mixtures thereof.

The vaccine adjuvants or vaccine compositions of the present application may further comprise one or more preservatives, examples of suitable preservatives include (but are not limited to): the phytolachong has a structure selected from the group consisting of benzal chloride, grandrell chloride, benzoic acid, tyrol, methyl p-hydroxybenzoate, ethylsnore p-hydroxybenzoate, propylsnore p-hydroxybenzoate, butylsnore p-hydroxybenzoate, sodium benzoate, phenol, and mixtures thereof. As will be appreciated by those skilled in the art, the presence or absence of a preservative will depend on the antigen. For example, if the antigen is a live bacterial antigen, no preservative is added.

The vaccine adjuvant or vaccine composition of the present application can be used for preventing or treating a disease caused by a pathogen, a cancer cell or an allergen in a human or an animal by administering a therapeutically effective amount of the adjuvant composition or vaccine to a human or animal susceptible to the disease.

According to the present application, the pathogen may be any pathogen including (but not limited to): bacteria, protozoa, worms, viruses and fungi. Diseases in animals caused by the pathogens include (but are not limited to): bovine respiratory disease, porcine respiratory disease, pneumonia, pasteurellosis, coccidiosis, anaplasmosis, and infectious keratitis.

According to the present application, the cancer cell may be any type of cancer cell in the art. According to the present application, the allergen may be any allergen known in the art.

For example, the vaccine composition of the present application may be a rabies vaccine, an influenza vaccine, a hepatitis b vaccine, a hepatitis a vaccine, a hepatitis c vaccine, a hand-foot-mouth vaccine, an HPV vaccine, a novel coronavirus vaccine, or the like.

In a specific embodiment, for the influenza vaccine, the mass ratio of the ginseng total polysaccharide to the vaccine antigen is 83.3-333.3 mug/mug.

In a specific embodiment, for the hepatitis A vaccine, the mass ratio of the ginseng total polysaccharide to the vaccine antigen is 5 to 100 mug/IU.

In a specific embodiment, for the hand-foot-and-mouth vaccine, the mass ratio of the total ginseng polysaccharide to the vaccine antigen is 1.25-12.5 mug/IU.

In a specific embodiment, for the HPV vaccine, the mass ratio of the total ginseng polysaccharide to the vaccine antigen is 12.5-125 mug/IU.

In a specific embodiment, for the novel coronavirus vaccine, the mass ratio of the ginseng total polysaccharides to the vaccine antigen is 10-50 μ g/μ g.

The vaccine type of the vaccine composition of the present application may be inactivated virus vaccine, attenuated vaccine, inactivated vaccine, protein vaccine, DNA vaccine, polypeptide vaccine, or the like.

The vaccine adjuvants or vaccine compositions of the present application can be used to protect or treat humans and non-human animals such as livestock and livestock animals, including (but not limited to) cattle, horses, sheep, pigs, goats, rabbits, cats, dogs and other mammals in need of treatment. Preferably, the vaccine adjuvant or vaccine composition of the present application is for the protection or treatment of humans. The vaccine adjuvant or vaccine composition of the present application to be administered may be selected based on the patient to be protected or treated, as will be appreciated by those skilled in the art.

The compositions of the present application may be prepared by a general method, wherein one or more pharmaceutically acceptable diluents or carriers are added, for example, in the form of oral drugs such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions and the like, or parenteral drugs such as external drugs, suppositories, injections, eye drops, intranasal agents, transpulmonary agents and the like. Preferred examples of formulations include injectable or intranasal solutions, or lyophilized formulations prepared by lyophilizing the solutions.

Examples of injectable solutions include emulsions and liposomes comprising aqueous and oleaginous compositions, such as aqueous solution formulations or aqueous suspension formulations in which vaccine adjuvants and antigens or DNA encoding the antigens are dissolved or dispersed in water, or oleaginous solution formulations or oleaginous suspension formulations in which vaccine adjuvants and antigens or DNA encoding the antigens are dissolved or dispersed in oil.

Examples of the aqueous solution, aqueous solution formulation or aqueous suspension formulation include an aqueous solution or aqueous suspension and the like containing distilled water for injection and optionally containing a buffer, a pH adjuster, a stabilizer, an isotonic agent and/or an emulsifier.

The vaccine adjuvant or vaccine composition of the present application may be administered by oral, intramuscular, intravenous, subcutaneous, intraocular, parenteral, topical, intravaginal or rectal routes. For administration to cattle, swine or other livestock, the adjuvant composition or vaccine adjuvant may be administered in feed or orally as a drench composition. In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application is injected intramuscularly, intravenously or subcutaneously.

The application also provides application of any one of the vaccine adjuvants in preparation of vaccine preparations and vaccine compositions.

Examples

Experimental materials

Aluminum salt adjuvant: produced by Croda Corp

Example 1

Preparing ginseng total polysaccharide:

pulverizing dried Ginseng radix 1kg to 10-20 mesh, adding 8 times of water (g: mL), decocting in boiling water bath for 8 hr, filtering, and collecting filtrate; to the residue was added further 10 times of water (g: mL) and heated in a boiling water bath for 8h. The two filtrates were combined and centrifuged at 6000rpm for 10min. Evaporating supernatant, adding neutral protease (Neutrase, enzyme concentration 800U/g) into the extract _{Extract of plant} ) Culturing at 40 deg.C for 3 hr, inactivating at 100 deg.C for 30min, centrifuging the mixed solution at 10000rpm to obtain supernatant, and steamingAnd (5) drying.

Soaking in 10 times volume of 95% ethanol at low temperature for 2.0 hr. Filtering, taking filter residue, adding 2 times volume of distilled water for dissolving, refluxing and extracting with boiling water for 3 times, each time for 1h, filtering with 120 mesh nylon cloth, mixing extractive solutions, concentrating to 500mL, centrifuging for 20 min (5000 rpm), and discarding precipitate. Adding 95% ethanol into the supernatant to ethanol final concentration of 90%, and standing for 1 hr. Centrifuging for 20 min (5000 r/min), collecting precipitate, adding 400mL distilled water to the precipitate, adding 95% ethanol to the supernatant to reach final concentration of 80%, standing for 1h, centrifuging for 20 min (5000 r/min), standing overnight at room temperature, and collecting precipitate.

The next day, the precipitate was washed twice with absolute ethanol, 95% ethanol in that order. And freeze-drying to obtain ginseng crude polysaccharide, preparing the ginseng crude polysaccharide into a 30% aqueous solution, adding into a 1000kDa dialysis bag for dialysis, and standing overnight. The liquid in the dialysis bag was taken, and Sevag reagent was added thereto in an amount of 1/4 volume, and after standing and centrifugation, the gel-like precipitate was removed. Repeating the above operation for 5 times, mixing the supernatants, concentrating under reduced pressure to remove organic reagent, adding 95% ethanol to final concentration of 80%, standing at 4 deg.C overnight, centrifuging, and removing the supernatant. The precipitate was washed twice with 95% ethanol and absolute ethanol in this order. Lyophilizing to obtain total polysaccharides of Ginseng radix.

Comparative example 1

Preparation of "comparative Ginseng radix Total polysaccharide":

pulverizing dried Ginseng radix 1kg to 10-20 mesh, adding 8 times of water (g: mL), decocting in boiling water bath for 8 hr, filtering, and collecting filtrate; 10 times of water (g: mL) is added to the filter residue and heated in a boiling water bath for 8h. Mixing the two filtrates, centrifuging at 6000rpm for 10min, and evaporating the supernatant. Soaking in 8 times volume of 95% ethanol at low temperature for 2.0 hr. Filtering, taking filter residue, adding 2 times volume of distilled water for dissolving, refluxing and extracting with boiling water for 3 times, each time for 1h, filtering with 120 mesh nylon cloth, mixing extractive solutions, concentrating to 500mL, centrifuging for 20 min (5000 rpm), and discarding precipitate. Adding 95% ethanol into the supernatant to ethanol final concentration of 90%, and standing for 1 hr. Centrifuging for 20 min (5000 r/min), collecting precipitate, adding 400mL distilled water to the precipitate, adding 95% ethanol to the supernatant to reach final concentration of 80%, standing for 1h, centrifuging for 20 min (5000 r/min), standing overnight at room temperature, and collecting precipitate.

The next day, the precipitate was washed twice with 95% ethanol. And freeze-drying to obtain ginseng crude polysaccharide, preparing the ginseng crude polysaccharide into a 30% aqueous solution, adding into a 1000kDa dialysis bag for dialysis, and standing overnight. Taking the liquid in the dialysis bag, adding Sevag reagent with the volume of 1/4 into the liquid, standing and centrifuging, and removing gelatinous precipitate. Repeating the above operation for 5 times, mixing the supernatants, concentrating under reduced pressure to remove organic reagent, adding 95% ethanol to final concentration of 80%, standing at 4 deg.C overnight, centrifuging, and removing the supernatant. The precipitate was washed twice with 95% ethanol and absolute ethanol in this order. Freeze drying to obtain Ginseng radix total polysaccharide.

Determination of polysaccharide content

The content of ginseng total polysaccharide and acidic polysaccharide thereof is determined by using a phenol-sulfuric acid method. A standard curve is drawn by taking glucose with different concentrations as an abscissa and an absorbance value at OD 490nm as an ordinate, and the polysaccharide content is calculated according to the glucose standard curve and the absorbance of the sample as shown in FIG. 1. The polysaccharide content of the total polysaccharides of ginseng of example 1 was 95.8%. The content of "total polysaccharides of Ginseng radix for comparison" in comparative example 1 was determined in the same manner, and it was 91.3%. The content of commercially available Ginseng radix total polysaccharide is determined by the same method, and is 90%.

Protein content determination

The protein content of Ginseng radix total polysaccharide and its acidic polysaccharide is determined by Bradford method. And (3) drawing a standard curve by taking the bovine serum albumin as an abscissa and the OD 595nm as an ordinate, and calculating the protein content according to the bovine serum albumin standard curve and the absorbance of the sample as shown in figure 2. The protein content of the total polysaccharides of ginseng of example 1 was 1.1%. The protein content of "total polysaccharides of comparative ginseng" in comparative example 1 was measured in the same manner, and the content was 2.5%. The content of commercially available Ginseng radix total polysaccharide (JY 2021062A, osbeckia Biotech Co., ltd.) was determined by the same method, and was 3.9%.

In the following examples, GPS refers to the ginseng total polysaccharide prepared in example 1, unless otherwise specified.

Example 2: immunological adjuvant activity of ginseng total polysaccharide (GPS) on rabies vaccine

Rabies vaccine: manufactured by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech GmbH.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": appropriate amounts of the ginseng total polysaccharide (GPS) and the diploid inactivated rabies vaccine prepared in the above example 1 are respectively measured, dissolved in normal saline, and filtered by a 0.22 mu m microporous membrane, and then aseptically packaged, wherein each milliliter of the solution contains 2500 mu g of GPS and 2.5IU of rabies vaccine.

Low dose "adjuvant-vaccine composition": appropriate amounts of the GPS inactivated rabies vaccine and the diploid inactivated rabies vaccine prepared in the example 1 are respectively measured, dissolved by normal saline, and filtered by a 0.22 mu m microporous membrane, and then sterilized and subpackaged, wherein each milliliter of the solution contains 250 mu g of GPS and 2.5IU of rabies vaccine.

Positive control aluminium salt "adjuvant-vaccine composition": appropriate amounts of the GPS inactivated rabies vaccine and the diploid inactivated rabies vaccine prepared in the above example 1 are measured respectively, dissolved by normal saline, and each milliliter of the solution contains 250mg of aluminum salt and 2.5IU of rabies vaccine, filtered by a 0.22 mu m microporous membrane, and packaged in an aseptic manner.

The immunization scheme comprises the following steps:

the mice were randomly divided into 4 groups of 10 mice each. The composition is injected intramuscularly at a dose of 0.1 ml/body, and after one week of primary immunization, the second immunization is carried out. Serum of each group of mice is collected 14 days after the second immunization, and each serum is respectively detected for neutralizing antibody and IgG specific antibody titer by RFFIT and ELISA methods.

Experimental grouping and dosing:

GPS high dose adjuvant group: 0.25IU rabies vaccine +250 mug GPS/mouse;

GPS low dose adjuvant group: 0.25IU rabies vaccine +25 mug GPS per mouse;

aluminum salt positive control group: 0.25IU rabies vaccine +25 mug of aluminum salt adjuvant/vehicle;

negative control group: 0.25IU rabies vaccine/pig.

As a result:

as shown in fig. 3 and 4, aluminum salt and high and low doses of GPS both increased IgG antibody levels and neutralizing antibody levels in rabies-vaccinated mice compared to the negative control group. Compared with an aluminum salt positive control group, the high and low doses of GPS (250 mug and 25 mug) can obviously improve the IgG antibody level and the neutralizing antibody level of rabies vaccine mice (P < 0.1).

Example 3: immunological adjuvant activity of ginseng total polysaccharide (GPS) for influenza vaccine

Influenza vaccine: manufactured by Liaoning Daozhitong GmbH.

GPS: the GPS prepared in example 1 and comparative example 1, and the commercial ginseng total polysaccharide (JY 2021062A, kaye Biotech Co., ltd.) were selected.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech GmbH.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant (commercially available ginseng total polysaccharide) -vaccine composition": appropriate amount of commercially available GPS and influenza vaccine are measured respectively, dissolved with normal saline, and each milliliter of the solution contains 2500 mu g of GPS and 15 mu g of influenza vaccine, and is filtered by a 0.22 mu m microporous membrane, and then is packaged in an aseptic way.

High dose "adjuvant (preparation of total ginseng polysaccharides in comparative example 1) -vaccine composition": respectively measuring appropriate amount of GPS and influenza vaccine prepared in comparative example 1, dissolving with normal saline, allowing each milliliter of solution to contain 2500 μ g of GPS and 15 μ g of influenza vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

High dose "adjuvant-vaccine composition": appropriate amounts of the GPS and influenza vaccines prepared in example 1 were measured, dissolved in normal saline, and each ml of the solution containing 2500. Mu.g of GPS and 15. Mu.g of influenza vaccine was filtered through a 0.22 μm microporous membrane and aseptically dispensed.

Low dose "adjuvant (commercially available ginseng total polysaccharide) -vaccine composition": respectively measuring appropriate amount of commercially available GPS and influenza vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of GPS and 15 μ g of influenza vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and influenza vaccine prepared in the above example 1 was measured separately, dissolved in normal saline, such that each milliliter of the solution contained 250 μ g of GPS and 15 μ g of influenza vaccine, filtered through a 0.22 μm microporous membrane, and aseptically packaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and influenza vaccine, dissolving with normal saline, allowing each ml solution to contain 250 μ g of aluminum salt and 15 μ g of influenza vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging.

The immunization scheme comprises the following steps:

the mice were randomly divided into 7 groups of 10 mice each. The composition was injected intramuscularly at a dose of 0.1 ml/mouse, and after 1 week of the initial immunization, the 2 nd immunization was performed. On day 14 after the 2 nd immunization, blood was collected from each mouse, and the level of neutralizing antibodies of mouse serum influenza vaccine was detected by hemagglutination inhibition assay.

Experimental groups and doses:

the commercial GPS high dose adjuvant group comprises 1.5 mu g of influenza vaccine +250 mu g of commercial GPS per mouse;

comparative example 1 preparation of GPS high dose adjuvant group 1.5. Mu.g influenza vaccine + 250. Mu.g GPS per example 1;

the GPS high dose adjuvant group comprises 1.5 mug influenza vaccine +250 mug GPS per mouse;

the commercial GPS low dose adjuvant group comprises 1.5 mu g of influenza vaccine and 25 mu g of commercial GPS per mouse;

the GPS low-dose adjuvant group comprises 1.5 mug of influenza vaccine and 25 mug of GPS per mouse;

the aluminum salt positive control group comprises 1.5 mu g of influenza vaccine and 25 mu g of aluminum salt adjuvant per mouse;

negative control group 1.5. Mu.g influenza vaccine/mouse.

As a result:

as shown in fig. 5, high doses of GPS significantly increased antibody levels in influenza-vaccinated mice compared to the negative control group (P < 0.0001). Meanwhile, compared with a positive control group (aluminum salt adjuvant), the high-dose GPS (250 mu g/mouse) can obviously improve the level of neutralizing antibodies (P is less than 0.001) in the blood of influenza vaccine-inoculated mice. Under the same dosage, the adjuvant activity of the GPS prepared by the method is obviously higher than that of the high-dose commercial GPS (P < 0.05) and the GPS prepared in the comparative example 1 (P < 0.05).

Example 4: optimal dosage of Ginseng radix total polysaccharide (GPS) as influenza vaccine adjuvant

Preparation of "adjuvant-vaccine composition":

an "adjuvant-vaccine composition": respectively measuring appropriate amount of GPS and influenza vaccine, dissolving with normal saline, preparing into five parts of composition solution, making each ml solution respectively contain 250, 1250, 2500, 5000, 10000 μ g of GPS and 15 μ g of influenza vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging. In this case, the ratios of the GPS adjuvant to the influenza vaccine in the composition were 16.7, 83.3, 166.7, 333.3, 666.7 (μ g: μ g), respectively.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and influenza vaccine, dissolving with normal saline, allowing each ml solution to contain 250 μ g aluminum salt and 15 μ g influenza vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Experimental groups and doses:

GPS adjuvant group 1 + 1.5 μ g influenza vaccine +25 μ g GPS per mouse;

GPS adjuvant group 2 + 1.5 μ g influenza vaccine +125 μ g GPS per mouse;

GPS adjuvant group 3 + 1.5 μ g influenza vaccine +250 μ g GPS per mouse;

GPS adjuvant group 4;

GPS adjuvant group 5 + 1.5 μ g influenza vaccine +1000 μ g GPS per mouse;

the aluminum salt positive control group comprises 1.5 mu g influenza vaccine and 25 mu g aluminum salt adjuvant/mouse;

negative control group 1.5. Mu.g influenza vaccine/mouse.

As a result:

as shown in fig. 6, when different doses of GPS were administered as adjuvants, it was found that for influenza vaccine, the antibody levels in the mice in the GPS adjuvant groups 2, 3, and 4 were significantly higher than those in the negative control group and the positive control group, and the antibody levels in the mice in the GPS adjuvant group 5 were significantly lower than those in the adjuvant groups 3 and 4, although they were somewhat elevated, and the amount of polysaccharide was larger. As described above, when GPS is used as an adjuvant for influenza vaccine, the effect is most excellent when the ratio of the amount of GPS to the amount of vaccine used is 83.3 to 333.3 (μ g: μ g).

Example 5: immunological adjuvant activity of ginseng total polysaccharide (GPS) to hepatitis B vaccine

Hepatitis B vaccine: produced by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech, inc.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hepatitis B vaccine prepared in the above example 1 was measured separately, dissolved in normal saline, and each milliliter of the solution contained 2500 μ g of GPS and 25 μ g of hepatitis B vaccine, filtered through a 0.22 μm microporous membrane, and then aseptically packaged.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hepatitis B vaccines prepared in the above example 1 are measured respectively, dissolved by normal saline, each milliliter of the solution contains 250 mu g of GPS and 25 mu g of hepatitis B vaccine, filtered by a 0.22 mu m microporous membrane, and then sterilized and subpackaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively weighing appropriate amount of aluminum salt and hepatitis B vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of aluminum salt and 25 μ g of hepatitis B vaccine, filtering with 0.22 μm microporous membrane, and packaging under sterile condition.

Immunization protocol:

the mice were randomly divided into 4 groups of 10 mice each. The composition was injected intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the initial immunization, the 2 nd immunization was performed. On day 14 after the 2 nd immunization, blood was collected from the mice, and serum hepatitis B antibody levels of the mice were measured by ELISA assay.

Experimental grouping and dosing:

the GPS high-dose adjuvant group comprises 2.5 mu g hepatitis B vaccine +250 mu g GPS;

the GPS low-dose adjuvant group comprises 2.5 mu g hepatitis B vaccine +25 mu g GPS;

the aluminum salt positive control group comprises 2.5 mu g hepatitis B vaccine and 25 mu g aluminum salt adjuvant/mouse;

negative control group 2.5. Mu.g hepatitis B vaccine/mouse.

As a result:

as shown in FIG. 7, compared with the negative control group, aluminum salt and high and low doses of GPS can significantly improve the antibody level (P < 0.001) in the mice immunized with hepatitis B vaccine. Meanwhile, compared with a positive control group (aluminum salt adjuvant), the high-dose GPS (250 mu g/mouse) can obviously improve the antibody level (P is less than 0.0001) in a hepatitis B vaccine immune mouse, and the adjuvant activity of the low-dose GPS (25 mu g/mouse) on the hepatitis B vaccine is similar to the effect of aluminum salt.

Example 6: adjuvant activity of ginseng total polysaccharide (GPS) on hepatitis A vaccine

Diploid hepatitis A vaccine: manufactured by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech GmbH.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": respectively weighing appropriate amount of the total polysaccharides of Ginseng radix (GPS) and hepatitis A vaccine prepared in example 1, dissolving with normal saline, filtering with 0.22 μm microporous membrane to obtain solution containing 2500 μ g of GPS and 50IU of hepatitis A vaccine, and packaging under sterile condition.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hepatitis A vaccines prepared in the above example 1 are measured respectively, dissolved by normal saline, each milliliter of the solution contains 250 mug of GPS and 50IU of hepatitis A vaccine, filtered by a 0.22 mu m microporous membrane, and then sterilized and subpackaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively weighing appropriate amount of aluminum salt and hepatitis A vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of aluminum salt and 50IU of hepatitis A vaccine, filtering with 0.22 μm microporous membrane, and packaging under sterile condition.

Immunization protocol:

the mice were randomly divided into 4 groups of 10 mice each. The above composition was administered intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the primary immunization, a secondary immunization was performed, and blood of each mouse was collected on day 14 after the secondary immunization, and the level of neutralizing antibody in serum was measured.

Experimental grouping and dosing:

GPS high dose adjuvant group: 5IU hepatitis A vaccine +250 mug GPS/mouse;

GPS low dose adjuvant group: 5IU hepatitis A vaccine +25 mug GPS per mouse;

aluminum salt positive control group: 5IU hepatitis A vaccine +25 mug aluminum salt adjuvant/mouse;

negative control group: 5IU hepatitis A vaccine/mouse.

As a result:

as shown in FIG. 8, aluminum salt and high and low doses of GPS significantly increased the antibody level in mice immunized with hepatitis A vaccine (P < 0.01) compared to the negative control group. Meanwhile, compared with an aluminum salt control group, the high and low doses of GPS (250 mu g/mouse) can obviously improve the level of neutralizing antibodies of hepatitis A vaccine mice (P is less than 0.05).

Example 7: optimal dosage of Ginseng radix total polysaccharide (GPS) as hepatitis A vaccine adjuvant

Preparation of "adjuvant-vaccine composition":

an "adjuvant-vaccine composition": respectively measuring appropriate amount of GPS and hepatitis A vaccine, dissolving with normal saline, preparing into five parts of composition solution, making each ml solution respectively contain 125, 250, 2500, 5000, 10000 μ g of GPS and 50IU of hepatitis A vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging. In this case, the ratio of the GPS adjuvant to the hepatitis A vaccine in the composition was 2.5, 5, 50, 100, 200 (. Mu.g: IU), respectively.

Positive control aluminium salt "adjuvant-vaccine composition": respectively weighing appropriate amount of aluminum salt and hepatitis A vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of aluminum salt and 50IU of hepatitis A vaccine, filtering with 0.22 μm microporous membrane, and packaging under sterile condition.

Experimental grouping and dosing:

the GPS adjuvant group 1 is composed of 5IU hepatitis A vaccine +12.5 mug GPS;

the GPS adjuvant group 2 comprises 5IU hepatitis A vaccine +25 mug GPS;

the GPS adjuvant group is 3;

the GPS adjuvant group is 4;

the GPS adjuvant group is 5;

aluminum salt positive control group: 5IU hepatitis A vaccine +25 mug aluminum salt adjuvant/mouse;

negative control group: 5IU hepatitis A vaccine/mouse.

As a result:

as shown in fig. 9, when different doses of GPS were administered as an adjuvant, it could be found that the antibody levels in the mice of the GPS adjuvant groups 2, 3, and 4 were significantly higher than those of the negative control group and the positive control group for the hepatitis a vaccine. Compared with a negative control group, the antibody level in the mice of the GPS adjuvant group 5 is improved to a certain extent, but is obviously lower than that of the adjuvant groups 3 and 4, and the dosage of the polysaccharide is larger. As described above, when GPS is used as an adjuvant for hepatitis A vaccine, the effect is best when the ratio of the amount of GPS to the amount of vaccine is 5 to 100 (μ g: IU).

Example 8: adjuvant activity of ginseng total polysaccharide (GPS) on hepatitis C vaccine

Hepatitis C vaccine: manufactured by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech, inc.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hepatitis C vaccines prepared in the above example 1 are measured respectively, dissolved by normal saline, each milliliter of the solution contains 5000 mug of GPS and 50 mug of hepatitis C vaccine, filtered by a 0.22 μm microporous membrane, and then sterilized and subpackaged.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hepatitis C vaccines prepared in the above example 1 are measured respectively, dissolved by normal saline, each milliliter of the solution contains 500 mu g of GPS and 50 mu g of hepatitis C vaccine, filtered by a 0.22 mu m microporous membrane, and then sterilized and subpackaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and hepatitis C vaccine, dissolving with normal saline, making each ml solution contain 500 μ g aluminum salt and 50 μ g hepatitis C vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Immunization protocol:

the mice were randomly divided into 4 groups of 10 mice each. The composition was administered intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the primary immunization, the secondary immunization was performed, and on day 14 after the secondary immunization, blood of each mouse was collected and the level of specific antibody IgG in the serum was measured.

Experimental grouping:

GPS high dose adjuvant group: 5 mug hepatitis C vaccine +500 mug GPS per mouse;

GPS low dose adjuvant group: 5 mug hepatitis C vaccine +50 mug GPS per mouse;

aluminum salt positive control group: 5 mug hepatitis C vaccine +50 mug aluminium salt adjuvant/stick;

negative control group: 5 μ g hepatitis C vaccine/mouse.

As a result:

as shown in fig. 10, aluminum salt and high and low doses of GPS significantly increased the antibody level in mice immunized with hepatitis c vaccine (P < 0.001) compared to the negative control group. Meanwhile, compared with an aluminum salt positive control group, the IgG antibody level (P is less than 0.05) of hepatitis C vaccine mice can be remarkably improved by high-dose GAPS (500 mu g/mouse) and low-dose GAPS (50 mu g/mouse).

Example 9: adjuvant activity of ginseng total polysaccharide (GPS) hand-foot-mouth vaccine

Hand-foot-mouth vaccine: manufactured by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech, inc.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hand-foot-and-mouth vaccine prepared in the above example 1 was measured, dissolved in normal saline, and each milliliter of the solution contained 2500 μ g of GPS and 200IU of hand-foot-and-mouth vaccine, filtered through a 0.22 μm microporous membrane, and aseptically packaged.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and hand-foot-and-mouth vaccine prepared in the above example 1 is measured respectively, dissolved by normal saline, so that each milliliter of the solution contains 250 mug of GPS and 200IU of the hand-foot-and-mouth vaccine, filtered by a 0.22 mu m microporous membrane, and then sterilized and subpackaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and hand-foot-and-mouth vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of aluminum salt and 200IU of hand-foot-and-mouth vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Immunization protocol: the mice were randomly divided into 4 groups of 10 mice each. The composition was administered intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the primary immunization, a secondary immunization was performed, and on day 14 after the secondary immunization, blood of each mouse was collected and the levels of neutralizing antibodies and specific antibody IgG in the serum were measured.

Grouping experiments:

the GPS high-dose adjuvant group comprises 20IU hand-foot-mouth vaccine +250 mug GPS;

the GPS low-dose adjuvant group comprises 20IU hand-foot-mouth vaccine and 25 mug GPS;

the aluminum salt positive control group comprises 20IU hand-foot-mouth vaccine and 25 mug of aluminum salt adjuvant per mouse;

negative control group 20IU hand-foot-mouth vaccine/mouse.

As a result:

as shown in fig. 11 and fig. 12, aluminum salt and high and low doses of GPS both significantly increased the antibody level in mice immunized with the hand-foot-and-mouth vaccine (P < 0.05) compared to the negative control group. Meanwhile, compared with an aluminum salt positive control group, the high-dose GPS (250 mu g/mouse) and the low-dose GPS (25 mu g/mouse) can obviously improve the levels of neutralizing antibodies and IgG antibodies (P is less than 0.01) in the body of the hand-foot-and-mouth vaccination mouse.

Example 10: optimal dosage of Ginseng radix total polysaccharide (GPS) as hand-foot-and-mouth vaccine adjuvant

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": respectively measuring appropriate amount of GPS and hand-foot-and-mouth vaccine, dissolving with normal saline, preparing into five parts of composition solution, making each milliliter solution respectively contain 125, 250, 1250, 2500, 5000 μ g of GPS and 200IU of hand-foot-and-mouth vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging. In this case, the ratio of the GPS adjuvant to the hand-foot-and-mouth vaccine in the composition was 0.625, 1.25, 6.25, 12.5, 25 (μ g: IU), respectively.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and hand-foot-and-mouth vaccine, dissolving with normal saline to make each ml solution contain 250 μ g of aluminum salt and 200IU of hand-foot-and-mouth vaccine, filtering with 0.22 μm microporous membrane, and packaging under sterile condition.

Experimental grouping and dosing:

the GPS adjuvant group 1 is 20IU hand-foot-mouth vaccine +12.5 mu g GPS;

the GPS adjuvant group 2 is 20IU hand-foot-mouth vaccine +25 mu g GPS;

the GPS adjuvant group 3 is 20IU hand-foot-mouth vaccine +125 mu g GPS;

the GPS adjuvant group 4 comprises 20IU hand, foot and mouth vaccine +250 microgram GPS;

the GPS adjuvant group 5 is 20IU hand-foot-mouth vaccine +500 mu g GPS;

aluminum salt positive control group: 20IU hand-foot-mouth vaccine +25 mug aluminum salt adjuvant/mouse;

negative control group: 20IU vaccine for hand, foot and mouth.

As a result:

as shown in fig. 13, when different doses of GPS were administered as adjuvants, it was found that the antibody levels in the mice of the GPS adjuvant groups 2, 3, and 4 were significantly higher than those of the negative control group and the positive control group for the hand-foot-and-mouth vaccine. Compared with a negative control group, the antibody levels in the mice of the GPS adjuvant groups 1 and 5 are improved to a certain extent, but are obviously lower than those of the adjuvant groups 2 to 4. In conclusion, when the GPS is used as an adjuvant of the hand-foot-and-mouth vaccine, the effect is best when the ratio of the dosage of the GPS to the dosage of the vaccine is 1.25-12.5 (mug: IU).

Example 11 adjuvant Activity of Total polysaccharides of Panax Ginseng (GPS) against HPV vaccine

HPV vaccine: produced by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech GmbH.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": the appropriate amount of the GPS and HPV vaccines prepared in example 1 above were measured separately, dissolved in normal saline to make each ml of solution contain 2500. Mu.g of GPS and 20. Mu.g of HPV vaccine, filtered through a 0.22 μm microporous membrane, and aseptically dispensed.

Low dose "adjuvant-vaccine composition": the appropriate amount of the GPS and HPV vaccines prepared in the above example 1 are measured separately, dissolved in normal saline, so that each milliliter of the solution contains 250 microgram of GPS and 20 microgram of HPV vaccine, filtered by a 0.22 micrometer microporous membrane, and then aseptically packaged.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and HPV vaccine, dissolving with normal saline, allowing each ml solution to contain 250 μ g of aluminum salt and 20 μ g of HPV vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging.

The immunization scheme comprises the following steps: the mice were randomly divided into 4 groups of 10 mice each. The composition was administered intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the primary immunization, the secondary immunization was performed, and on day 14 after the secondary immunization, blood of each mouse was collected and the level of specific antibody IgG in the serum was measured.

Experimental grouping and dosing:

the GPS high-dose adjuvant group comprises 2 mug HPV vaccine +250 mug GPS;

the GPS high-dose adjuvant group comprises 2 mug HPV vaccine and 25 mug GPS per mouse;

the aluminum salt positive control group comprises 2 mu g of HPV vaccine and 25 mu g of aluminum salt adjuvant/mouse;

negative control group 2. Mu.g HPV vaccine/mouse.

As a result:

as shown in fig. 14, aluminum salt and high and low doses of GPS both significantly increased antibody levels in HPV vaccine immunized mice (P < 0.001) compared to the negative control group. Meanwhile, compared with an aluminum salt positive control group, the high-dose GPS (250 mu g/mouse) and the low-dose GPS (25 mu g/mouse) can obviously improve the IgG antibody level (P is less than 0.01) in the HPV vaccinated mice.

Example 12: optimal dosage of Ginseng radix total polysaccharide (GPS) as HPV vaccine adjuvant

Preparation of "adjuvant-vaccine composition":

an "adjuvant-vaccine composition": respectively measuring appropriate amount of GPS and HPV vaccine, dissolving with normal saline, preparing into five parts of composition solution, making each milliliter solution respectively contain 125 μ g, 250 μ g, 1250 μ g, 2500 μ g, 5000 μ g GPS and 20 μ g HPV vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging. In this case, the ratio of GPS adjuvant to HPV vaccine in the composition was 6.25, 12.5, 62.5, 125, 250 (μ g: μ g), respectively.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and HPV vaccine, dissolving with normal saline, allowing each ml solution to contain 250 μ g of aluminum salt and 20 μ g of HPV vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Experimental grouping and dosing:

GPS adjuvant group 1;

GPS adjuvant group 2 +2 μ g HPV vaccine +25 μ g GPS/mouse;

GPS adjuvant group 3;

GPS adjuvant group 4;

GPS adjuvant group 5;

aluminum salt positive control group: 2 μ g HPV vaccine +25 μ g aluminium salt adjuvant/vehicle;

negative control group: 2 μ g HPV vaccine/mouse.

As a result:

as shown in fig. 15, when different doses of GPS were administered as an adjuvant, it was found that the antibody levels in the mice of the GPS adjuvant groups 2, 3, and 4 were significantly higher than those of the negative control group and the positive control group for HPV vaccine. Compared with a negative control group, the antibody levels in the mice of the GPS adjuvant groups 1 and 5 are improved to a certain extent, but are obviously lower than those of the adjuvant groups 2 to 4. In summary, when GPS is used as an adjuvant for HPV vaccine, the effect is best when the ratio of the amount of GPS to the amount of vaccine is 12.5-125 (μ g: μ g).

Example 13 adjuvant Activity of Total polysaccharides of Panax Ginseng (GPS) against New coronavirus vaccines (New coronavirus vaccines)

The new crown vaccine comprises: manufactured by Liaoning Daozhitong GmbH.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotech, inc.

Preparation of "adjuvant-vaccine composition":

high dose "adjuvant-vaccine composition": the ginseng total polysaccharide (GPS) prepared in the example 1 and a proper amount of the novel coronavirus vaccine are respectively measured and dissolved by normal saline, each milliliter of the solution contains 5000 microgram of GPS and 50 microgram of the novel coronavirus vaccine, and the solution is filtered by a 0.22 mu m microporous membrane and is packaged in a sterile way.

Low dose "adjuvant-vaccine composition": respectively measuring appropriate amount of the GPS prepared in the embodiment 1 and the new corona vaccine, dissolving with normal saline, filtering with 0.22 μm microporous membrane to obtain solution containing 500 μ g of GPS and 50 μ g of new corona vaccine, and packaging under aseptic condition.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and new corona vaccine, dissolving with normal saline to make each ml solution contain 500 μ g of aluminum salt and 50 μ g of new corona vaccine, filtering with 0.22 μm microporous membrane, and aseptically packaging.

Immunization protocol: the mice were randomly divided into 4 groups of 10 mice each. The composition was administered intramuscularly at a dose of 0.1 ml/mouse, and 2 weeks after the primary immunization, the second immunization was performed, and the blood of each mouse was collected on the 14 th day after the second immunization, and the level of specific antibody IgG in the serum was measured.

Experimental grouping and dosing:

the GPS high-dose adjuvant group comprises 5 mu g of new corona vaccine and 500 mu g of GPS;

the GPS low-dose adjuvant group comprises 5 mu g of new corona vaccine and 50 mu g of GPS;

the aluminum salt positive control group comprises 5 mu g of new corona vaccine and 50 mu g of aluminum salt adjuvant per mouse;

negative control group 5. Mu.g new corona vaccine/mouse.

As a result:

as shown in fig. 15, aluminum salt and high and low doses of GPS significantly increased the antibody levels in mice immunized with the new corona vaccine (P < 0.01) compared to the negative control group. Meanwhile, compared with an aluminum salt positive control group, the high-dose GPS (500 mu g/mouse) and the low-dose GPS (50 mu g/mouse) can obviously improve the IgG antibody level in a new corona vaccination mouse (P is less than 0.01, and P is less than 0.05).

Example 14: optimal dosage of Ginseng radix total polysaccharide (GPS) as adjuvant of novel coronavirus vaccine (neocoronavirus)

Preparation of "adjuvant-vaccine composition":

"adjuvant-vaccine composition": respectively measuring appropriate amount of GPS and new corona vaccine, dissolving with normal saline, preparing into five parts of composition solution, respectively containing 250 μ g, 500 μ g, 2500 μ g, 5000 μ g, 10000 μ g GPS and 50 μ g new corona vaccine per ml solution, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition. In this case, the ratio of the GPS adjuvant to the new corona vaccine in the composition was 6.25, 12.5, 62.5, 125, 250 (μ g: μ g), respectively.

Positive control aluminium salt "adjuvant-vaccine composition": respectively measuring appropriate amount of aluminum salt and new crown vaccine, dissolving with normal saline to make each ml solution contain 250 μ g aluminum salt and 50 μ g new crown vaccine, filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Experimental grouping and dosing:

the GPS adjuvant group 1 comprises 5 mug of new corona vaccine +25 mug of GPS;

GPS adjuvant group 2;

GPS adjuvant group 3 +5 μ g neo-corona vaccine +250 μ g GPS/mouse;

GPS adjuvant group 4;

GPS adjuvant group 5 μ g neo-corona vaccine +1000 μ g GPS/mouse;

aluminum salt positive control group: 5 μ g of neocorona vaccine +25 μ g of aluminium salt adjuvant/stick;

negative control group: 5 μ g of neocorona vaccine/mouse.

As a result:

as shown in fig. 15, with different doses of GPS administered as adjuvant, it was found that the antibody levels in the mice of the GPS adjuvant groups 2 and 3 were significantly higher than those of the negative and positive control groups for the new corona vaccine. Compared with a negative control group, the antibody level in the mice of the GPS adjuvant groups 1, 4 and 5 is improved to a certain extent, but is obviously lower than that of the adjuvant groups 2 to 4. In summary, when GPS is used as an adjuvant of a new corona vaccine, the effect is best when the ratio of the amount of GPS to the amount of the vaccine is 10 to 50 (μ g: μ g).

Claims (14)

1. The preparation method of the ginseng total polysaccharide is characterized by comprising the following steps:

extracting Ginseng radix with water to obtain a first extract,

adding protease into the first extract, culturing, extracting to obtain a second extract,

extracting the second extract with ethanol and water to obtain crude Ginseng radix polysaccharide,

dissolving the crude polysaccharide of Ginseng radix, dialyzing, removing protein in the trapped fluid,

extracting the retentate with ethanol to obtain Ginseng radix total polysaccharide.

2. The method according to claim 1, wherein the protease is a neutral protease.

3. The process according to claim 1, wherein the protease is cultured at 30 to 40 ℃ for 1 to 3 hours.

4. The method of claim 1, wherein the dialysis uses a molecular cut-off of 800 to 2500kDa, preferably 1000 to 1500kDa.

5. The method of claim 1, wherein the protein in the retentate is removed using Sevag reagent.

6. A vaccine adjuvant is characterized by comprising ginseng total polysaccharide.

7. The vaccine adjuvant according to claim 6, further comprising physiological saline or water for vaccine injection.

8. The vaccine adjuvant according to claim 6, wherein the ginseng total polysaccharides are obtained by the preparation method according to any one of claims 1 to 5.

9. A vaccine composition comprising the vaccine adjuvant of any one of claims 6 to 8 and a vaccine antigen or DNA encoding said antigen.

10. The vaccine composition of claim 8, further comprising a pharmaceutical adjuvant and a second vaccine adjuvant.

11. The vaccine composition according to claim 8, wherein the vaccine is a rabies vaccine, an influenza vaccine, a hepatitis b vaccine, a hepatitis a vaccine, a hepatitis c vaccine, a hand-foot-and-mouth vaccine, an HPV vaccine or a novel coronavirus vaccine.

12. The vaccine composition of claim 9, wherein the ratio of the total ginseng polysaccharide to the vaccine antigen is (0.1-1000) to (0.10-200) μ g/IU or (0.1-1000): (0.1-500) mu g/mu g, preferably 10-500 mu g/mu g or 1-150 mu g/IU.

13. The vaccine composition according to claim 10, wherein the vaccine type is an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine, or a polypeptide vaccine.

14. Use of a vaccine adjuvant according to any one of claims 6-8 in the preparation of a vaccine formulation, a vaccine composition.

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