Vaccine adjuvant and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a vaccine adjuvant and application thereof. And the adjuvant contains one or more of total saponins of Hylocereus undatus, a saponin component HA and a saponin component HB. The total saponin and the monomer saponin with immunologic adjuvant activity are separated from the Honeysuckle flower, and the total saponin and the monomer saponin can non-specifically change or enhance immune response reactions of various vaccines including influenza vaccines, so that not only can humoral immunity of influenza vaccine immunized mice be enhanced, but also cellular immunity of the influenza vaccine immunized mice can be enhanced.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a vaccine adjuvant and application thereof. Background Art

[0002] Vaccination is an important measure to effectively prevent infectious diseases. 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. Immune adjuvants refer to substances that can non-specifically change or enhance the body's specific immune response to antigens. They are required to be non-toxic, high-purity, have a certain adsorption capacity and stable properties. 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. Currently, most commercially available vaccine adjuvants are aluminum salt adjuvants, but aluminum salts can cause inflammation at the injection site and stimulate local erythema, granulomas and subcutaneous nodules. At the same time, aluminum salt adjuvants may delay the production of neutralizing antibodies in some vaccines, so they are subject to various restrictions when used.

[0003] Traditional Chinese medicine is a traditional Chinese medicine. Modern pharmacology shows that most traditional Chinese medicines have the effects of improving immunity, anti-tumor, anti-fatigue, etc. The discovery of new natural products with immune activity has become a research focus, especially in the search for a new generation of vaccine adjuvants. Summary of the invention

[0004] The present invention aims to provide a vaccine adjuvant and its application.

[0005] To achieve the above purpose, the present invention adopts the following technical solution:

[0006] A vaccine adjuvant, the adjuvant containing one or more of the total saponins of Helianthus annuus, saponin component HA, and saponin component HB;

[0007] Among them, the saponin component HA contains sapogenin, with β-D-galactopyranosyl-(1→2)-β-D-pyranoglucuronic acid linked to the 3rd position, and β-D-xylopyranosyl (1→2)-β-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl linked to the 28th position;

[0008] The saponin component HB contains sapogenin, with β-D-galactopyranosyl (1→2)-β-D-glucuronopyranosyl linked at the 3-position and β-D-xylopyranosyl (1→4)-[β-D-glucopyranosyl (1→3)]-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl linked at the 28-position.

[0009] The total saponins of Helianthus annuus are obtained by extracting Helianthus annuus from Helianthus annuus with an organic solvent; the saponin component HA and the saponin component HB are obtained by purifying the total saponins of Helianthus annuus.

[0010] The total saponin of Heqinghua is prepared by crushing the raw Heqinghua, repeatedly extracting it with ethanol and / or water as solvent, concentrating the extract to obtain an extract, extracting the extract with petroleum ether, dichloromethane and n-butanol in sequence, dissolving the extract with methanol and precipitating it with ethyl acetate, and repeatedly processing to collect saponin powder, which is the total saponin of Heqinghua.

[0011] The solvent is preferably an ethanol aqueous solution, wherein the ethanol concentration is 45-75%.

[0012] The material-liquid ratio of the raw material to the solvent is 1:3-1:15, the number of repetitions is 2-4 times, and each extraction is performed under reflux at 60-100° C. for 1-5 hours.

[0013] The volume ratio of the extract to petroleum ether, dichloromethane and n-butanol is 1:1-5 respectively; the n-butanol layer extract is dissolved in methanol, and then the saponin is precipitated by ethyl acetate, the volume ratio of the extract to methanol is 1:1-3, and the volume ratio of methanol to ethyl acetate is 1:1-20.

[0014] The total saponin of Helanthus odoratus is sequentially subjected to macroporous resin, silica gel column chromatography, and thin layer chromatography detection, and the fraction containing the saponin component HA is collected and concentrated; and then repeatedly subjected to ODS RP-C18 reverse phase column medium and high pressure chromatography for separation and purification to obtain the saponin component HA;

[0015] Alternatively, the product is separated repeatedly by ODS RP-C18 reverse phase column medium and high pressure chromatography and gel LH-20, detected by thin layer chromatography, and the fractions containing the saponin component HB are combined and purified by C18 reverse phase preparation column to obtain the saponin component HB.

[0016] The macroporous resin elution separation adopts methanol-water gradient elution, wherein the volume ratio of methanol to water is 0:100-90:10;

[0017] Silica gel column chromatography was eluted with a dichloromethane, methanol and water system, and the volume ratio of dichloromethane, methanol and water was 9:1:0.1-6:4:1;

[0018] ODS RP-C18 reverse phase column chromatography, using methanol-water gradient elution, wherein the volume ratio of methanol to water is 0:100-90:10;

[0019] For thin layer chromatography detection, the developing solvent system used was: dichloromethane:methanol:water (v / v / v) 7:3:0.5 or n-butanol:acetic acid:water (v / v / v) 4:1:5.

[0020] The C18 reverse preparative column uses a mobile phase of methanol / water (containing 1‰ formic acid) in a ratio of 50-85% and acetonitrile / water (containing 1‰ formic acid) in a ratio of 20-40%.

[0021] An application of the vaccine adjuvant, wherein the adjuvant is used as an influenza vaccine adjuvant.

[0022] An influenza vaccine contains the adjuvant.

[0023] 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.

[0024] Disclosed is an application of a flos odoratus extract and a purified product thereof, and the application of the flos odoratus extract and the purified product thereof as a vaccine adjuvant, wherein the flos odoratus extract is obtained by extracting the flos odoratus with an organic solvent, and the purified product is a single substance obtained by further purifying the extract.

[0025] The advantages of the present invention are:

[0026] The present invention separates total saponins and monomeric saponins (A / B) with immune adjuvant activity from flos odoratissimii. Both the total saponins and the compounds A / B can non-specifically change or enhance the immune response of various vaccines including influenza vaccines, and can enhance not only the humoral immunity of mice immunized with influenza vaccines but also their cellular immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of separation and purification of total saponins from Helanthus odoratus;

[0028] Figure 2 This is a flow chart for the separation and purification of saponin component A (HA);

[0029] Figure 3 It is the structural diagram of HA;

[0030] Figure 4 It is HA 1 H-NMR analysis spectrum;

[0031] Figure 5 It is HA 13 C-NMR analysis spectrum;

[0032] Figure 6 This is a graph showing the effect of HA on mouse spleen lymphocyte toxicity;

[0033] Figure 7 .A is the effect of HA and lipopolysaccharide on the proliferation of mouse spleen lymphocytes;

[0034] Figure 7.B is the effect of HA and concanavalin on the proliferation of mouse spleen lymphocytes;

[0035] Figure 7 .C is a graph showing the effect of HA-synergistic influenza vaccine on the proliferation of mouse spleen lymphocytes;

[0036] Figure 8 .A is the effect of HA on the IgG titer of mice immunized with influenza vaccine;

[0037] Figure 8 .B is the effect of HA on the IgG1 titer of mice immunized with influenza vaccine;

[0038] Figure 8 .C is the effect of HA on the IgG2a titer of mice immunized with influenza vaccine;

[0039] Figure 8 .D is the effect of HA on the IgG2a / IgG1 titer of mice immunized with influenza vaccine;

[0040] Fig. 9 This is a flow chart for the separation and purification of saponin component B (HB);

[0041] Fig.10 It is the structural diagram of HB;

[0042] Fig.11 It's HB 1 H-NMR analysis spectrum;

[0043] Fig.12 It's HB 13 C-NMR analysis spectrum;

[0044] Fig.13 This is a graph showing the effect of HB on mouse spleen lymphocyte toxicity;

[0045] Fig.14 .A is the effect of HB and lipopolysaccharide on the proliferation of mouse spleen lymphocytes;

[0046] Fig.14 .B is the effect of HB and concanavalin on the proliferation of mouse spleen lymphocytes;

[0047] Fig.14 .C is a graph showing the effect of HB-synergistic influenza vaccine on the proliferation of mouse spleen lymphocytes;

[0048] Fig.15 : Effect of HB on IgG titer of mice immunized with influenza vaccine. DETAILED DESCRIPTION

[0049] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0050] As used herein, the term "vaccine" refers to any preparation of antigenic or immunogenic substance suitable for stimulating active immunity in animals or humans.

[0051] As used herein, the term "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.

[0052] As used herein, the term "antigen" refers to any substance that stimulates a humoral and / or cell-mediated immune response when introduced into an immunocompetent human or animal. 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. Examples of suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, peptides, carbohydrates / polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi and parasites. Other suitable antigens include the smallest component of an antigen, such as, but not limited to, an antigenic determinant, an epitope or a peptide. Still other suitable antigens include those described in U.S. Pat. No. 5,855,894. Antigens may be natural (naturally expressed or produced), synthetic, or derived from recombinant DNA methodology familiar to those skilled in the art.

[0053] As used herein, the term "pharmaceutical excipient" 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 medicines and preparing prescriptions. It has been reasonably evaluated in terms of safety and is contained in the drug preparation. In addition to excipients, serving 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.

[0054] Hylomecon japonicum (Thunb.) Prantl is a plant of the genus Hylomecon in the family Papaveraceae. It has the functions of dispelling rheumatism, relaxing muscles and tendons, relieving blood stasis and swelling, relieving pain and stopping bleeding, and treating rheumatoid arthritis, strain and trauma. Modern research has found that Hylomecon japonicum has multiple pharmacological effects such as anti-inflammatory, analgesic, antiviral, antibacterial, and anti-tumor.

[0055] As used herein, the term "total saponins of Heqinghua" refers to the sum of Heqinghua saponins extracted from the whole Heqinghua herb, including but not limited to saponin component A (HA) and saponin component B (HB). Total saponins of Heqinghua can be prepared by methods known in the art or purchased through commercial channels.

[0056] The invention also provides a vaccine adjuvant comprising total saponin of flos lycopersicum.

[0057] In a specific embodiment, the total saponins of Helianthus annuus are obtained by the above-mentioned preparation method of total saponins of Helianthus annuus.

[0058] In a specific embodiment, the aluminum salt is selected from one or both of aluminum phosphate and aluminum hydroxide. For example, the aluminum salt is aluminum phosphate, or aluminum hydroxide, or a mixture of aluminum phosphate and aluminum hydroxide.

[0059] In a specific embodiment, the vaccine adjuvant may also include physiological saline or water for vaccine injection. The content of the total saponin or aluminum salt of Flos Cynoglossi in the vaccine adjuvant may be adjusted according to actual needs.

[0060] The present application also provides a vaccine adjuvant comprising saponin component A or B.

[0061] In a specific embodiment, the saponin component A or B is obtained by the above-mentioned preparation method.

[0062] In a specific embodiment, the vaccine adjuvant may also include physiological saline or water for vaccine injection. The content of saponin component A (HA), saponin component B (HB) or aluminum salt in the vaccine adjuvant may be adjusted according to actual needs.

[0063] In a specific embodiment, the vaccine adjuvant consists of saponin component A or B.

[0064] In a specific embodiment, the vaccine adjuvant consists of saponin component A (HA) or saponin component B (HB), physiological saline, and water for vaccine injection.

[0065] The present invention also provides a vaccine composition, comprising a vaccine adjuvant containing any one of the total saponins of Helianthus annuus, saponin component A and saponin component B (HB) in the present application and an antigen or a DNA encoding the antigen.

[0066] The amount of vaccine adjuvant used in the vaccine composition is an amount effective to achieve a therapeutic effect, which is an amount that improves, increases, upregulates, alters or otherwise promotes the immune response to the antigen. Specifically, 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, a therapeutically effective amount will vary and is determined on a case-by-case basis.

[0067] The therapeutically effective amount will vary and be determined on a case-by-case basis. In the present application, the dosage of vaccine adjuvant and vaccine antigen is not particularly limited and is appropriately selected according to the administration method, subject, age of the subject, dosage form, administration route, etc.

[0068] In a specific embodiment, the dosage ratio of the vaccine adjuvant to the vaccine antigen is (0.1μg~1000μg):(0.10IU~200IU) antigen, for example, it can be 0.1μg:0.10IU, 0.1μg:200IU, 1000μg:0.10IU, 1000μg:200IU; or (0.1μg~1000μg):(0.1μg~500μg) antigen, for example, it can be 0.1μg:0.1μg, 0.1μg:500μg, 1000μg:0.1μg, 1000μg:500μg.

[0069] In a specific embodiment, the dosage ratio of the vaccine adjuvant to the vaccine antigen is 33.3 to 133.3 μg / μg.

[0070] In a specific embodiment, the dosage ratio of the vaccine adjuvant to the vaccine antigen is 40-80 μg / IU.

[0071] Those skilled in the art will readily recognize that the therapeutic dosage 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 cases, dosage levels below the lower limit of the aforementioned range may be therapeutically effective, while in other cases, still larger doses may be used without causing any adverse side effects, provided that such larger doses are first divided into several smaller doses for administration throughout the day. Whenever secondary stress or exposure is likely to occur, it is considered ideal to have a provocative dose.

[0072] The vaccine adjuvant and antigen or the DNA encoding the antigen in the vaccine combination of the present invention can be contained in a composition together and can be prepared in a separate composition. When prepared in a separate composition, the administration route of the vaccine adjuvant and the antigen or the DNA encoding the antigen can be the same or different. In the present application, the vaccine adjuvant and the antigen or the DNA encoding the antigen can be given simultaneously or with time differences, that is, the vaccine adjuvant and the antigen or the DNA encoding the antigen can be given simultaneously or separately (for example, the vaccine adjuvant is given before or after the vaccine antigen is given). The vaccine adjuvant and the antigen or the DNA encoding the antigen can be provided as a kit comprising them. However, from the perspective of reducing the burden on the patient, it is preferred that the vaccine adjuvant and the antigen or the DNA encoding the antigen are contained in a composition so that they can be given simultaneously when administered. Whether it is co-administration or simultaneous administration, the administration mode of the vaccine composition can be any appropriate approach, and the approach, whether it is co-administration or simultaneous administration, delivers the vaccine composition to the host.

[0073] In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application further comprises a pharmaceutical excipient and a second vaccine adjuvant. The pharmaceutical excipient is as described above.

[0074] The vaccine adjuvant of the present invention can be administered as part of a vaccine formulation, which optionally contains an additional second vaccine adjuvant. The second vaccine adjuvant is another adjuvant different from the total saponin adjuvant of the present application, which can be one or more than two. Examples of suitable second vaccine adjuvants include those known in the art,

[0075] The vaccine adjuvant or vaccine composition of the present invention may further comprise one or more antioxidants, wherein the one or more antioxidants are selected from the group consisting of sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium methyloxysulfate, L-ascorbic acid, isoascorbic acid, acetylcysteine, cysteine, monothioglycerol, mercaptoacetic acid, thiolactic acid, thiothreitol, dithiothreitol, dithioerythrocyanate, glutathione, ascorbyl palmitate, butylated hydroxytoluene, nordihydroguaiaretic acid, propyl gallate, α-tocopherol, and mixtures thereof.

[0076] The vaccine adjuvant or vaccine composition of the present invention may further comprise one or more preservatives, examples of suitable preservatives include, but are not limited to, benzalkonium chloride, benzyl chloride, benzoic acid, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl 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 needs to be added.

[0077] The vaccine adjuvant or vaccine composition of the present invention can be used to prevent or treat diseases caused by pathogens, cancer cells or allergens in humans or animals by administering a therapeutically effective amount of the adjuvant composition or vaccine to humans or animals susceptible to the disease.

[0078] According to the present invention, the pathogen may be any pathogen, including but not limited to bacteria, protozoa, worms, viruses and fungi. Diseases in animals caused by the pathogen include but are not limited to bovine respiratory disease, porcine respiratory disease, pneumonia, pasteurella disease, coccidiosis, anaplasmosis, and infectious keratitis.

[0079] 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.

[0080] The vaccine type of the vaccine composition of the present invention can be an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine, etc.

[0081] The vaccine adjuvant or vaccine composition of the present invention can be used to protect or treat humans and non-human animals such as livestock and domestic 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 used to protect or treat humans. As is known to those skilled in the art, the vaccine adjuvant or vaccine composition of the present application to be administered can be selected based on the patient to be protected or treated.

[0082] The composition of the present invention can 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, etc., or parenteral drugs, such as external drugs, suppositories, injections, eye drops, intranasal agents, pulmonary agents, etc. Preferred examples of the preparation include injectable or intranasal solutions, or lyophilized preparations prepared by lyophilizing the solution.

[0083] Examples of injectable solutions include emulsions and liposomes containing aqueous solutions and oily compositions, such as aqueous solution preparations or aqueous suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in water, or oily solution preparations or oily suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in oil.

[0084] Examples of the aqueous solution, aqueous solution preparation or aqueous suspension preparation include an aqueous solution or aqueous suspension containing distilled water for injection and optionally containing a buffer, a pH adjuster, a stabilizer, an isotonic agent and / or an emulsifier, and the like.

[0085] The vaccine adjuvant or vaccine composition of the present application can be administered orally, intramuscularly, intravenously, subcutaneously, intraocularly, parenterally, topically, intravaginally or rectally. For administration to cattle, pigs or other livestock, the adjuvant composition or vaccine adjuvant can be administered orally as a feed or as a gavage composition. In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application is injected intramuscularly, intravenously or subcutaneously.

[0086] The experimental materials in the following examples: influenza vaccine (quadrivalent), aluminum hydroxide adjuvant, were purchased from Chengda Biotechnology Co., Ltd.

[0087] Example 1

[0088] Extraction and purification of total saponins from Helanthus odoratus, see the flow chart Figure 1

[0089] (1) Extraction of total saponins from Heqinghua: Grind the whole Heqinghua herb and pass it through a 40-mesh sieve. Add 10 times the volume of 50% ethanol aqueous solution, extract twice at 85°C, each time for 2.5 hours, filter, combine the filtrate, and concentrate under reduced pressure to recover ethanol. Then, extract the extract with 3 times the volume of petroleum ether and 3 times the volume of ethyl acetate until the upper liquid is almost colorless, and then extract with 5 times the volume of water-saturated n-butanol until the extract is almost colorless. Combine the n-butanol extracts, and recover n-butanol under reduced pressure to obtain n-butanol extracts.

[0090] (2) Purification of saponins from Helianthus annuus: The n-butanol layer extract was dissolved in methanol twice the mass of the solid, and ethyl acetate 10 times the mass of the methanol was added. With the addition of ethyl acetate, a large amount of powder was precipitated. The saponin powder was collected and dissolved in methanol. Excess ethyl acetate was added to precipitate the powder again. This step was repeated 3 times. After the powder was dried, the total saponins from Helianthus annuus were obtained, and the saponin content was determined to be 89%.

[0091] Example 2: Preparation of saponin component HA

[0092] like Figure 2 As shown, the obtained total saponin powder of Helanthus odoratus was dissolved in water, and loaded on a D101 macroporous resin column at a ratio of m (sample amount): m (resin amount) = 1:8, the diameter-to-height ratio of the resin column was 8:15, and the loading flow rate was 2BV / h. After the drug solution passed through the resin column, it was adsorbed for 5 hours, and eluted with water, 50% and 70% ethanol aqueous solutions in sequence, and the eluents of each fraction were collected, and the solvent was recovered under reduced pressure.

[0093] The above 50% eluted fractions were separated by silica gel column chromatography (silica gel particle size 300 mesh), the mass ratio of the sample silica gel to the separation silica gel was 1:6, and the dichloromethane: methanol: water system was used, 9:1:0.1, 8:2:0.2, 7:3:0.5, 6:4:1 gradient elution was performed, and according to the thin layer chromatography detection, the developing solvent was dichloromethane: methanol: water with a ratio of 7:3:0.5, and the fractions containing saponin component A with Rf of 0.4 were merged, and the fractions were separated by ODS reverse medium pressure column chromatography, the mass ratio of the sample ODS to the separation ODS was 1:10, and 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% methanol water gradient elution was performed, and according to the thin layer chromatography developing solvent was dichloromethane: methanol: water with a ratio of 7:3:0.5, the Rf 0.4 fraction was merged to obtain the purified saponin component A (HA) (see Figure 3-5 ).

[0094] Example 3: Study on the toxicity of saponin component A (HA) to mouse spleen lymphocytes

[0095] 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.).

[0096] Animals: SPF female ICR mice (Liaoning Chengda Biotechnology Co., Ltd.).

[0097] 6-8 week old ICR mice were killed by cervical dislocation, and spleen lymphocyte suspension was prepared aseptically. The cell concentration was adjusted to (1-5)×10 in RPMI-1640 medium containing 10% fetal bovine serum. 6 / mL, evenly spread in a 96-well plate, 100 μL of cell suspension was added to each well, and divided into an experimental group and a blank control group. The blank control group was added with 100 μL of blank culture medium; the experimental groups were added with culture medium solutions containing HA at concentrations of 0.825 μm / mL, 1.75 μm / mL, 3.25 μm / mL, 7.5 μm / mL, 15 μm / mL, 30 μm / mL, and 50 μm / mL, respectively. After culturing in an incubator for 48 h, 20 μL of CCK-8 solution was added to each well, the absorbance of each well was measured on a microplate reader (450 nm), and statistical analysis was performed.

[0098] Results: See Figure 6 As shown, 0.825-30 μm / mL HA aqueous solution has no toxic effect on mouse spleen lymphocytes, and 6.25-15 μm / mL HA aqueous solution can enhance the proliferation of mouse spleen lymphocytes, proving that HA has immunostimulatory activity.

[0099] Example 4: Study on the effect of HA on the proliferation of mouse spleen lymphocytes in combination with LPS (lipopolysaccharide), Con A (concanavalin A) or influenza vaccine

[0100] Reagents and drugs: lipopolysaccharide (LPS, Sigma), concanavalin A (Con A, Sigma), erythrocyte lysate (Beijing Solebold Technology Co., Ltd.), RPMI-1640 medium (Dalian Meilun Biotechnology Co., Ltd.), fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd.), CCK8 (Dalian Meilun Biotechnology Co., Ltd.).

[0101] Animals: 50 female ICR mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.

[0102] Influenza vaccine preparation containing HA adjuvant: Dissolve 10.9 mg HA and 300 μg influenza vaccine in 10 ml of normal saline. After mixing, filter with a 0.22 μm microporous filter membrane and aseptically package, 1 ml per vial, to obtain a high-dose HA solution; take one of the high-dose HA solutions and dilute it 4 times with normal saline to obtain a low-dose HA solution.

[0103] At the same time, 10 mg of aluminum hydroxide adjuvant and 300 μg of influenza vaccine were dissolved in 10 ml of normal saline, mixed, filtered through a 0.22 μm microporous filter membrane, and aseptically packaged in 1 ml vials as a control.

[0104] Four groups of different preparations were prepared according to the following grouping and dosage.

[0105] Experimental groups and dosages:

[0106] A: Normal saline group

[0107] B: influenza vaccine group: 30 μg influenza vaccine / mL;

[0108] C: Aluminum salt adjuvant control group: 30 μg influenza vaccine + 1 mg aluminum salt adjuvant / mL;

[0109] D: HA low-dose group: 30 μg influenza vaccine + 0.27 mg HA / mL;

[0110] E: HA high-dose group: 30 μg influenza vaccine + 1.09 mg HA / mL;

[0111] Immunization scheme: 50 mice were randomly divided into 5 groups, 10 mice in each group. Each group was intramuscularly injected with 0.1 ml of the above AE preparations. One week after the first immunization, the second immunization was performed. 14 days after the second immunization, spleen lymphocyte suspensions were prepared from each group of mice according to the method of Example 3, and the cell concentration was adjusted to (1-5)×10 6 / mL, and evenly spread in three 96-well plates, plate 1, plate 2, and plate 3. Plate 1, plate 2, and plate 3 use different reagents to stimulate lymphocyte proliferation in an in vitro culture environment, and examine the proliferation activity of spleen lymphocytes in mice in different groups after administration. Plate 1 uses lipopolysaccharide as a stimulator, plate 2 uses concanavalin as a stimulator, and plate 3 uses influenza vaccine stock solution as a stimulator. After adding various reagents, the incubation wells corresponding to the 5 groups A, B, C, D, and E contain 100 μL of spleen lymphocyte suspension, 80 μL of complete culture medium (RPMI 1640 + 10% fetal bovine serum) and the following components.

[0112] Plate 1: Each incubation well contains 5 μg / mL LPS (lipopolysaccharide);

[0113] Plate 2: Each incubation well contains 10 μg / mL ConA;

[0114] Plate 3: Each well contains 1 μg / mL of influenza vaccine stock solution.

[0115] Plates 1, 2, and 3 were placed in an incubator for 48 hours. 4 hours before the end of the culture, 20 μL of CCK-8 solution was added to each well, and the absorbance of each well was measured on a microplate reader (450 nm), and the stimulation index SI (SI = OD value (drug group - blank group) / OD value (control group - blank group)) was calculated, where the control group corresponded to the mice in the influenza vaccine group B, and the spleen was ground to obtain the spleen cell group.

[0116] Results: In the experimental group, the effects of HA and LPS on the proliferation of mouse spleen lymphocytes were shown in Figure 7 As shown in Figure C, both high- and low-dose HA and 5 μg / mL LPS have a certain synergistic effect on the proliferation of mouse spleen lymphocytes, among which the high-dose HA group has the strongest synergistic ability on the proliferation of mouse spleen lymphocytes, with a significant difference.

[0117] In the experimental group, the effect of HA and Con A on the proliferation of mouse spleen lymphocytes was shown in Figure 7 As shown in .B, high and low doses of HA and 10 μg / mL of Con A had a certain proliferative effect on the proliferation of mouse spleen lymphocytes. There was no significant difference between the low and high dose groups, but there was an overall upward trend.

[0118] In the experimental group, the effect of HA combined with influenza vaccine on the proliferation of mouse spleen lymphocytes was shown in Figure 7 As shown in .A, the high-dose HA group and the influenza vaccine concentrate both had a certain proliferative effect on the proliferation of mouse spleen lymphocytes, and there were significant differences.

[0119] Example 5: Study on the adjuvant activity of HA on influenza vaccine

[0120] Influenza vaccine: produced by Liaoning Chengda Biological Co., Ltd.

[0121] Animals: Forty female ICR mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.

[0122] Influenza vaccine preparation containing HA adjuvant: Dissolve 10.9 mg HA and 300 μg influenza vaccine in 10 ml of normal saline. After mixing, filter with a 0.22 μm microporous filter membrane and aseptically package, 1 ml per vial, to obtain a high-dose HA solution; take one of the high-dose HA solutions and dilute it 4 times with normal saline to obtain a low-dose HA solution.

[0123] At the same time, 10 mg of aluminum hydroxide adjuvant and 300 μg of influenza vaccine were dissolved in 10 ml of normal saline, mixed, filtered through a 0.22 μm microporous filter membrane, and aseptically packaged in 1 ml vials as a control.

[0124] Four groups of different preparations were prepared according to the following grouping and dosage.

[0125] Experimental groups and dosages:

[0126] A: Normal saline group

[0127] B: influenza vaccine group: 30 μg influenza vaccine / mL;

[0128] C: Aluminum salt adjuvant control group: 30 μg influenza vaccine + 1 mg aluminum salt adjuvant / mL;

[0129] D: HA low-dose group: 30 μg influenza vaccine + 0.27 mg HA / mL;

[0130] E: HA high dose group: 30μg influenza vaccine + 1.09mg HA / mL

[0131] Immunization scheme: 50 mice were randomly divided into 5 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, IgG, IgG1, and IgG2a in the mouse serum were detected by ELISA and hemagglutination inhibition test, and the IgG2a / IgG1 ratio was calculated.

[0132] Results: See Figure 8 As shown in .A-8D, compared with the blank group, the high-dose HA group can enhance the IgG, IgG1, and IgG2a of mice immunized with influenza vaccine, and reduce the IgG2a / IgG1 ratio to that of the blank group, indicating that saponin has a good adjuvant effect.

[0133] Example 6: Preparation of saponin component B (HB)

[0134] like Fig. 9As shown, the total saponin powder of Heqinghua obtained in the above example is dissolved in water, and loaded on a D101 macroporous resin column at a ratio of m (sample amount): m (resin amount) = 1:8, and the resin column diameter-to-height ratio is 8:15, and the loading flow rate is 2BV / h. After the drug solution passes through the resin column, it is adsorbed for 5 hours, and eluted with water, 50% and 70% ethanol aqueous solutions in sequence, and the eluents of each fraction are collected, and the solvent is recovered under reduced pressure.

[0135] 50% of the eluted fractions were separated by silica gel column chromatography (silica gel particle size 300 mesh), the mass ratio of sample silica gel to separation silica gel was 1:6, and dichloromethane: methanol: water system, 9:1:0.1, 8:2:0.2, 7:3:0.5, 6:4:1 gradient elution was adopted. According to the thin layer chromatography detection, the developing solvent was dichloromethane: methanol: water with a ratio of 7:3:0.5. The fractions containing B with Rf of 0.5 were combined, and the fractions were separated by ODS reverse medium pressure column chromatography, the mass ratio of sample ODS to separation ODS was 1:10, and 30%, 35%, 40%, 45% and 50% were used. 5%, 50%, 55%, 60%, 65%, 70% methanol water gradient elution, under the guidance of thin layer chromatography developing solvent of dichloromethane: methanol: water 7:3:0.5, the fraction containing saponin component B with Rf of 0.5 was combined, and the fraction was eluted with 70% methanol water using a gel LH-20 column, and according to thin layer chromatography detection, the developing solvent was dichloromethane: methanol: water 7:3:0.5, and the fraction containing B with Rf of 0.5 was combined, and the fraction was eluted with 70% methanol water (containing 1‰ formic acid) using a semi-preparative column to obtain saponin component B (HB) (see Figure 10-12 ).

[0136] Example 7: Study on the toxicity of HB to mouse spleen lymphocytes

[0137] 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.).

[0138] Animals: SPF female ICR mice (Liaoning Chengda Biotechnology Co., Ltd.).

[0139] 6-8 week old ICR mice were killed by cervical dislocation, and spleen lymphocyte suspension was prepared aseptically. The cell concentration was adjusted to (1-5)×10 in RPMI-1640 medium containing 10% fetal bovine serum. 6 / mL, evenly spread in a 96-well plate, 100 μL of cell suspension was added to each well, and divided into an experimental group and a blank control group. The blank control group was added with 100 μL of blank culture medium; the experimental group was added with HB culture medium solutions with concentrations of 0.825 μm / mL, 1.75 μm / mL, 3.25 μm / mL, 7.5 μm / mL, 15 μm / mL, 30 μm / mL, and 50 μm / mL, respectively. After culturing in an incubator for 48 h, 20 μL of CCK-8 solution was added to each well, and the absorbance of each well was measured on a microplate reader (450 nm), and statistical analysis was performed.

[0140] Results: See Fig.13 As shown, 0.825-30 μm / mL HB aqueous solution has no toxic effect on mouse spleen lymphocytes, and 6.25-15 μm / mL HB aqueous solution can enhance the proliferation of mouse spleen lymphocytes, proving that HB has immunostimulatory activity.

[0141] Example 8: Study on the effect of HB on the proliferation of mouse spleen lymphocytes in combination with LPS (lipopolysaccharide), Con A (concanavalin A) or influenza vaccine

[0142] Reagents and drugs: lipopolysaccharide (LPS, Sigma), concanavalin A (Con A, Sigma), erythrocyte lysate (Beijing Solebold Technology Co., Ltd.), RPMI-1640 medium (Dalian Meilun Biotechnology Co., Ltd.), fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd.), CCK8 (Dalian Meilun Biotechnology Co., Ltd.).

[0143] Animals: 50 female ICR mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.

[0144] Influenza vaccine preparation containing HB adjuvant: Dissolve 11.2mg HB and 300μg influenza vaccine in 10ml normal saline. After mixing, filter with a 0.22μm microporous filter membrane and aseptically package, 1ml per vial to obtain HB high-dose solution; take one vial of HB high-dose solution and dilute it 4 times with normal saline to obtain HB low-dose solution.

[0145] At the same time, 10 mg of aluminum hydroxide adjuvant and 300 μg of influenza vaccine were dissolved in 10 ml of normal saline, mixed, filtered through a 0.22 μm microporous filter membrane, and aseptically packaged in 1 ml vials as a control.

[0146] Four groups of different preparations were prepared according to the following grouping and dosage.

[0147] Experimental groups and dosages:

[0148] A: Normal saline group

[0149] B: influenza vaccine group: 30 μg influenza vaccine / mL;

[0150] C: Aluminum salt adjuvant control group: 30 μg influenza vaccine + 1 mg aluminum salt adjuvant / mL;

[0151] D: HA low-dose group: 30 μg influenza vaccine + 0.28 mg HB / mL;

[0152] E: HA high-dose group: 30 μg influenza vaccine + 1.12 mg HB / mL;

[0153] Immunization scheme: 50 mice were randomly divided into 5 groups, 10 mice in each group. Each group was intramuscularly injected with 0.1 ml of the above AE preparations. One week after the first immunization, the second immunization was performed. 14 days after the second immunization, spleen lymphocyte suspensions were prepared from each group of mice according to the method of Example 9, and the cell concentration was adjusted to (1-5)×10 6 / mL, and were evenly spread in three 96-well plates, plate 1, plate 2, and plate 3, respectively. Different reagents were used to stimulate lymphocyte proliferation in an in vitro culture environment in plate 1, plate 2, and plate 3, and the proliferation activity of spleen lymphocytes in mice in different groups after administration was examined.

[0154] Among them, plate 1 uses lipopolysaccharide as a stimulator, plate 2 uses concanavalin as a stimulator, and plate 3 uses influenza vaccine concentrate as a stimulator. After adding various reagents, the incubation wells corresponding to the four groups A, B, C, and D respectively contain 100 μL of spleen lymphocyte suspension, 80 μL of complete culture medium (RPMI 1640+10% fetal bovine serum) and the following components.

[0155] Plate 1: Each incubation well contains 5 μg / mL LPS (lipopolysaccharide);

[0156] Plate 2: Each incubation well contains 10 μg / mL ConA;

[0157] Plate 3: Each well contains 1 μg / mL of influenza vaccine stock solution.

[0158] Plates 1, 2, and 3 were placed in an incubator for 48 hours. 4 hours before the end of the culture, 20 μL of CCK-8 solution was added to each well, and the absorbance of each well was measured on a microplate reader (450 nm), and the stimulation index SI (SI = OD value (drug group - blank group) / OD value (control group - blank group)) was calculated, where the control group corresponded to the mice in the influenza vaccine group B, and the spleen was ground to obtain the spleen cell group.

[0159] Results: In the experimental group, the effects of HB and LPS on the proliferation of mouse spleen lymphocytes were shown in Fig.14As shown in Figure C, both high and low doses of HB and 5 μg / mL of LPS have a certain synergistic effect on the proliferation of mouse spleen lymphocytes, among which high doses of HB and LPS have the strongest synergistic ability on the proliferation of mouse spleen lymphocytes, with significant differences.

[0160] In the experimental group, the effect of HB and Con A on the proliferation of mouse spleen lymphocytes was shown in Fig.14 As shown in .B, high and low doses of HB and 10 μg / mL of Con A had a certain proliferative effect on the proliferation of mouse spleen lymphocytes. There was no significant difference between the low and high dose groups, but there was an overall upward trend.

[0161] In the experimental group, the effect of HB combined with influenza vaccine on the proliferation of mouse spleen lymphocytes was shown in Fig.14 As shown in .C, high-dose HB and influenza vaccine concentrate both have a certain proliferative effect on the proliferation of mouse spleen lymphocytes, and there are significant differences.

[0162] Example 9: Study on the adjuvant activity of HB on influenza vaccine

[0163] Influenza vaccine: produced by Liaoning Chengda Biological Co., Ltd.

[0164] Animals: Forty female ICR mice, 6-8 weeks old, were purchased from Liaoning Chengda Biotechnology Co., Ltd.

[0165] Influenza vaccine preparation containing HB adjuvant: Dissolve 11.2mg HB and 300μg influenza vaccine in 10ml normal saline. After mixing, filter with a 0.22μm microporous filter membrane and aseptically package, 1ml per vial to obtain HB high-dose solution; take one vial of HB high-dose solution and dilute it 4 times with normal saline to obtain HB low-dose solution.

[0166] At the same time, 10 mg of aluminum hydroxide adjuvant and 300 μg of influenza vaccine were dissolved in 10 ml of normal saline, mixed, filtered through a 0.22 μm microporous filter membrane, and aseptically packaged in 1 ml vials as a control.

[0167] Four groups of different preparations were prepared according to the following grouping and dosage.

[0168] Experimental groups and dosages:

[0169] A: Normal saline group

[0170] B: influenza vaccine group: 30 μg influenza vaccine / mL;

[0171] C: Aluminum salt adjuvant control group: 30 μg influenza vaccine + 1 mg aluminum salt adjuvant / mL;

[0172] D: HA low-dose group: 30 μg influenza vaccine + 0.28 mg HB / mL;

[0173] E: HA high-dose group: 30 μg influenza vaccine + 1.12 mg HB / mL

[0174] Immunization scheme: 40 mice were randomly divided into 5 groups, 8 mice in each group. 0.1 ml / mouse was injected intramuscularly, and the second immunization was performed 1 week after the first immunization. IgG in mouse serum was detected by ELISA and hemagglutination inhibition test 14 days after the second immunization.

[0175] Results: See Fig.15 As shown, compared with the blank group, high-dose HB can enhance the IgG of mice immunized with influenza vaccine, indicating that saponin has a good adjuvant effect.

Claims

1. A vaccine adjuvant, characterized in that The adjuvant contains one or more of the total saponins of Helanthus odoratus, saponin component HA, and saponin component HB; Among them, the saponin component HA contains sapogenin, with β-D-galactopyranosyl-(1→2)-β-D-pyranoglucuronic acid linked to the 3rd position, and β-D-xylopyranosyl (1→2)-β-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl linked to the 28th position; The saponin component HB contains sapogenin, with β-D-galactopyranosyl (1→2)-β-D-glucuronopyranosyl linked at the 3-position and β-D-xylopyranosyl (1→4)-[β-D-glucopyranosyl (1→3)]-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl linked at the 28-position.

2. The vaccine adjuvant according to claim 1, characterized in that The total saponins of Helianthus annuus are obtained by extracting Helianthus annuus from Helianthus annuus with an organic solvent; the saponin component HA and the saponin component HB are obtained by purifying the total saponins of Helianthus annuus.

3. The vaccine adjuvant according to claim 2, characterized in that The total saponin of Heqinghua is prepared by crushing the raw Heqinghua, repeatedly extracting it with ethanol and / or water as solvent, concentrating the extract to obtain an extract, extracting the extract with petroleum ether, dichloromethane and n-butanol in sequence, dissolving the extract with methanol and precipitating it with ethyl acetate, and repeatedly processing to collect saponin powder, which is the total saponin of Heqinghua.

4. The vaccine adjuvant according to claim 3, characterized in that The material-liquid ratio of the raw material to the solvent is 1:3-1:15, the number of repetitions is 2-4 times, and each extraction is performed under reflux at 60-100° C. for 1-5 hours.

5. The vaccine adjuvant according to claim 3, characterized in that The volume ratio of the extract to petroleum ether, dichloromethane and n-butanol is 1:1-5 respectively; the n-butanol layer extract is dissolved in methanol, and then the saponin is precipitated by ethyl acetate, the volume ratio of the extract to methanol is 1:1-3, and the volume ratio of methanol to ethyl acetate is 1:1-20.

6. The vaccine adjuvant according to claim 2 or 3, characterized in that The total saponin of Helanthus odoratus is sequentially subjected to macroporous resin, silica gel column chromatography, and thin layer chromatography detection, and the fraction containing the saponin component HA is collected and concentrated; and then repeatedly subjected to ODS RP-C18 reverse phase column medium and high pressure chromatography for separation and purification to obtain the saponin component HA; Alternatively, the product is separated repeatedly by ODS RP-C18 reverse phase column medium and high pressure chromatography and gel LH-20, detected by thin layer chromatography, and the fractions containing the saponin component HB are combined and purified by C18 reverse phase preparation column to obtain the saponin component HB.

7. The vaccine adjuvant according to claim 6, characterized in that The macroporous resin elution separation adopts methanol-water gradient elution, wherein the volume ratio of methanol to water is 0:100-90:10; Silica gel column chromatography was eluted with a dichloromethane, methanol and water system, and the volume ratio of dichloromethane, methanol and water was 9:1:0.1-6:4:1; ODS RP-C18 reverse phase column chromatography was performed using a methanol-water gradient elution, wherein the volume ratio of methanol to water was 0:100-90:

10.

8. Use of the vaccine adjuvant according to claim 1, characterized in that: The adjuvant is used as an influenza vaccine adjuvant.

9. An influenza vaccine, characterized in that: The vaccine contains the adjuvant according to claim 1.

10. An application of a Helanthus odoratus extract and a purified product thereof, characterized in that: The invention relates to the application of flos cerasifera extract and its purified product as vaccine adjuvants, wherein the flos cerasifera extract is obtained by extracting flos cerasifera with an organic solvent, and the purified product is a single substance obtained by further purifying the extract.

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