Immunologic adjuvant containing paris polyphylla glucomannan as well as preparation method and application of immunologic adjuvant

Abstract

The invention discloses an immunologic adjuvant containing paris polyphylla glucomannan for the first time, and belongs to the technical field of biological medicines. The immunologic adjuvant containing the paris polyphylla glucomannan can promote the phagocytic function of macrophages, promote the secretion of NO and TNF-alpha, promote the maturation of dendritic cells, up-regulate the expression quantity of CD40, CD86 and MHCII, and promote the secretion of TNF-alpha, IL-6 and IL-12. In-vivo experiments show that the immunologic adjuvant containing the paris polyphylla glucomannan can significantly improve the antigen-specific IgG, IgG1 and IgG2a antibody levels, induce Th1 and Th2 type immune responses at the same time, increase the proportion of CD3 + and CD3 + and CD4 + cells in splenic lymphocytes and enhance humoral immunity and cellular immunity levels at the same time. The paris polyphylla gulcomannan in the immunologic adjuvant is obtained by extracting and purifying paris polyphylla plants of alaginaceae, the weight-average molecular weight of the paris polyphylla gulcomannan is 50-800 kDa, monosaccharide composition of the paris polyphylla gulcomannan is mannose and glucose, the ratio of the mannose to the glucose is (10: 1)-(20: 1), and the second site or the third site of the mannose is substituted by acetyl. The invention further discloses a preparation method of the immunologic adjuvant containing the paris polyphylla glucomannan and application of the immunologic adjuvant containing the paris polyphylla glucomannan to preparation of a vaccine preparation or a vaccine composition.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to Paris polyphylla glucomannan, a composition thereof with aluminum salt, a preparation method thereof, and use thereof as an immune adjuvant. Background Art

[0002] Vaccination is an important means to protect humans from the threat of infectious diseases. However, in many cases, vaccines consisting only of antigens cannot stimulate the body to produce sufficient immune responses to prevent infection. Therefore, adjuvants need to be added during vaccine preparation to enhance the immune response. Adjuvants are substances that enhance antigen-specific immune responses by triggering and regulating innate immunity and adaptive immunity. The addition of adjuvants can reduce the amount of antigens used, stimulate more rapid and lasting immune responses, and improve the immune effect of vaccines in special populations. Although adjuvants play an important role in vaccines, only a few of the currently approved vaccines can meet the requirements of high efficiency and low toxicity. Aluminum salts are currently the most widely used vaccine adjuvants, and 80% of human vaccines on the market use aluminum adjuvants. However, aluminum adjuvants usually only stimulate Th2-type immune responses and humoral immunity, but cannot enhance Th1-type immune responses or cytotoxic T lymphocyte (CTL) responses, which limits their application in vaccines targeting cellular immunity.

[0003] Paris refers to the Paris genus of the family Melanophyllaceae. Paris plants are extremely valuable medicinal plants, and the knowledge of their medicinal value has a history of thousands of years. In ancient Chinese herbal medicine books, they are often called Paris, Paris, Paris gold thread, Paris one arrow, worm, three layers of grass, flea, Chi, sting, iron lamp, seven leaves one flower, grass river cart, grass gansui, white gansui, etc. The earliest description in the "Shennong's Herbal Classic" is: "Flea, bitter and slightly cold, mainly treats epilepsy, shaking the head and tongue, heat in the abdomen, epilepsy, carbuncle, yin erosion, three worms, remove snake venom, one is Chi, born in the valley." It has the effects of clearing away heat and detoxification, reducing swelling and relieving pain, cooling the liver and calming the nerves. It is used for sore throat, snake and insect bites, convulsions, functional uterine bleeding, neuroinflammation, surgical inflammation and other diseases with significant efficacy. It is the main ingredient of Chinese patent medicines such as Paris polyphylla detoxification tincture, Gongxuening capsule, Yunnan Baiyao, Sanjie Zhitong ointment, etc. Polysaccharide is an important active substance in Paris polyphylla. After systematic literature review, no application of Paris polyphylla glucomannan or its combination with aluminum salt in the preparation of immune adjuvants has been found. Summary of the invention

[0004] The purpose of the present invention is to provide an immune adjuvant containing Paris polyphylla glucomannan, a preparation method thereof, and its application in preparing a vaccine preparation or a vaccine composition. At the same time, Paris polyphylla glucomannan and its aluminum salt composition are provided, and their application in preparing an immune adjuvant, a vaccine preparation or a vaccine composition.

[0005] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0006] An immune adjuvant containing Paris polyphylla glucomannan and its aluminum salt composition, wherein the Paris polyphylla glucomannan has the following structural characteristics:

[0007] The Paris polyphylla glucomannan is extracted and purified from Paris polyphylla plants of the family Melanophoraceae, and has a weight average molecular weight of 50 to 800 kDa, and monosaccharides are composed of mannose and glucose, and the ratio of the two is 10:1 to 20:1, and mannose is substituted with acetyl at position 2 or 3. The Paris polyphylla plants include but are not limited to Paris yunnanensis, Paris moutan, Paris polyphylla chinensis, Paris delavayi, Paris polyphylla Smith var. latifolia, Paris polyphylla smith var. stenophylla, Paris fargesii, and Paris bashanensis.

[0008] The Paris polyphylla glucomannan can promote the phagocytic function of macrophages and the secretion of immune factors.

[0009] The Paris polyphylla glucomannan can promote the maturation of dendritic cells, upregulate the expression of CD40, CD86 and MHCII, and promote the secretion of TNF-α, IL-6 and IL-12.

[0010] The inventors further discovered through animal experiments that the Paris polyphylla glucomannan can significantly increase the levels of antigen-specific IgG, IgG1, and IgG2a antibodies, while enhancing the humoral immunity and cellular immunity of mice.

[0011] In the present invention, the antigens include, but are not limited to, hepatitis B virus surface antigen (HBsAg), varicella-zoster virus glycoprotein gE, human papillomavirus L1 protein and model antigen hen ovalbumins (OVA).

[0012] The creative research results of the present invention show that Paris polyphylla glucomannan and its aluminum salt composition have pharmacological activity as an immune adjuvant. Therefore, Paris polyphylla glucomannan has the use of being an immune adjuvant.

[0013] Further, the present invention discloses an immune adjuvant containing Paris polyphylla glucomannan or its aluminum salt composition, wherein the immune adjuvant contains an effective dose of Paris polyphylla glucomannan, or optionally contains or does not contain aluminum salt. Preferably, the immune adjuvant is a composition of Paris polyphylla glucomannan and aluminum salt. The aluminum salt is selected from one or both of aluminum hydroxide and aluminum phosphate.

[0014] Preferably, in the immune adjuvant containing the composition of Paris polyphylla glucomannan and its aluminum salt, the mass ratio of Paris polyphylla glucomannan to aluminum salt is 0.0001 to 10:1, preferably 0.01 to 0.5:1.

[0015] The immune adjuvant containing Paris polyphylla glucomannan and its aluminum salt composition, which is a composition of Paris polyphylla glucomannan and aluminum salt, can significantly increase the levels of antigen-specific IgG, IgG1, and IgG2a antibodies, and simultaneously enhance the humoral immunity and cellular immunity levels of mice.

[0016] Preferably, the immunoadjuvant containing Paris polyphylla glucomannan and its aluminum salt composition can be used as an injection, and the amount of Paris polyphylla glucomannan in the injection is 0.1 mg to 50 mg per kilogram of body weight.

[0017] The present invention discloses a method for preparing an immune adjuvant containing Paris polyphylla glucomannan and its aluminum salt composition, comprising the following steps: (1) extracting dry powder of Paris polyphylla rhizome with hot water to obtain a water extract;

[0018] (2) enzymatically hydrolyzing the aqueous extract to obtain non-starch polysaccharides;

[0019] (3) subjecting the non-starch polysaccharide to graded alcohol precipitation and collecting the precipitate;

[0020] (4) Purifying by one or more of gel exclusion chromatography, dialysis and ultrafiltration to obtain Paris polyphylla glucomannan;

[0021] (5) Alternatively, Paris polyphylla glucomannan is mixed with aluminum salt to form a composition.

[0022] The present invention further discloses the use of the immune adjuvant in preparing a vaccine preparation or a vaccine composition.

[0023] In addition, the present invention discloses a Paris polyphylla glucomannan or an aluminum salt composition thereof, wherein the Paris polyphylla glucomannan is extracted and purified from a Paris polyphylla plant of the family Melanophoraceae, has a weight average molecular weight of 50 to 800 kDa, and has a monosaccharide composition of mannose and glucose in a ratio of 10:1 to 20:1. The 2nd or 3rd position of mannose is substituted with an acetyl group, and its structural formula is as follows:

[0024]

[0025] In the formula, n represents the number of sugar repeating units, and n is a natural number, wherein the degree of acetyl substitution of Polygonum multiflorum glucomannan PPM is 35%;

[0026] The polysaccharide is prepared by the following method: extracting the dried powder of the rhizome of the Paris genus plant with hot water to obtain a water extract; performing enzymatic hydrolysis on the water extract to obtain a non-starch polysaccharide; performing graded alcohol precipitation on the non-starch polysaccharide and collecting the precipitate; and purifying the polysaccharide by one or more of a gel exclusion chromatography column method, a dialysis method and an ultrafiltration method to obtain Paris glucomannan. After the Paris glucomannan is mixed with an aluminum salt, an aluminum salt composition is formed.

[0027] Paris polyphylla glucomannan or its aluminum salt composition, the Paris polyphylla glucomannan is extracted and purified from Paris polyphylla plants of the family Melanophoraceae, the extraction is as follows: weighing dry Paris polyphylla or Paris polyphylla rhizome, adding 95% ethanol to reflux and extract twice, each time for 2 hours, filtering while hot, collecting filter cakes, and evaporating the residual ethanol in the filter cakes; adding deionized water to heat and extract at 80°C for 2 hours, centrifuging at 4000rpm for 10 minutes after the extraction, taking the supernatant, precipitating and extracting once, combining the supernatants, adding 0.1% (v / v) α-amylase, enzymolysis at 70°C for 2 hours, no starch can be detected; adding 95% ethanol to the enzymolysis supernatant until the final alcohol concentration is 40%, centrifuging to obtain 40% alcohol precipitate and 40% alcohol precipitate supernatant; the above 40% alcohol precipitate In the supernatant, 95% ethanol is added to the final alcohol concentration of 60% under stirring, centrifuged to obtain a 60% alcohol precipitate and an alcohol precipitation supernatant, the 60% alcohol precipitate is washed 3 times with 95% ethanol, and then lyophilized after reconstitution with water; in the above 60% alcohol precipitation supernatant, 95% ethanol is added to the final alcohol concentration of 80% under stirring, centrifuged to obtain an 80% alcohol precipitate and an alcohol precipitation supernatant, the 80% alcohol precipitate is washed 3 times with 95% ethanol, and then lyophilized after reconstitution with water to obtain crude Paris polyphylla glucomannan or coarse Paris polyphylla glucomannan; the purification is as follows: weigh the above crude Paris polyphylla glucomannan or coarse Paris polyphylla glucomannan, dissolve it in deionized water, centrifuge it at 4000rpm for 15min, filter the supernatant through a 0.45μm filter membrane, and then slowly and evenly inject it into Sephadex The top of the G100 gel column was eluted with 0.1M NaCl solution, the flow rate was controlled to be 0.1mL / min, 2mL was collected in each tube, and the polysaccharide content was detected by the phenol-sulfuric acid method. The absorbance value was used as the ordinate and the number of tubes was used as the abscissa to draw the elution curve of the polysaccharide component, and the eluates belonging to the same elution peak were combined, desalted with a 500Da dialysis bag, concentrated under reduced pressure, and vacuum freeze-dried to obtain purified Paris polyphylla glucomannan (PPM) or Paris polyphylla glucomannan (PMM).

[0028] The use of the Paris polyphylla glucomannan or its aluminum salt composition in the preparation of immune adjuvants or in the preparation of vaccine preparations and vaccine compositions.

[0029] Compared with the prior art, the beneficial effects of the present invention are: the present invention discloses for the first time an immune adjuvant containing Paris polyphylla glucomannan, which can significantly increase the levels of antigen-specific IgG, IgG1, and IgG2a antibodies, while enhancing humoral immunity and cellular immunity levels, and the immune adjuvant effect is better than the aluminum adjuvant commonly used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figures are the effects of Paris polyphylla glucomannan or its combination with aluminum salt on the phagocytic function of macrophages, wherein (A) is a neutral red experiment to study the effects of different concentrations of Paris polyphylla glucomannan on the phagocytic function of macrophages (compared with the Control group, *P<0.05; **P<0.01; ***P<0.001); (B) and (C) are flow cytometry experiments to study the effects of Paris polyphylla glucomannan or its combination with aluminum salt on the phagocytosis of the antigen FITC-OVA by macrophages (compared with the OVA group, **P<0.01; ***P<0.001).

[0031] Figure 2 The effects of different concentrations of Paris polyphylla glucomannan on the secretion levels of macrophage immune factors, including (A) NO, (B) TNF-α, and (C) IL-10 (compared with the Control group, **P<0.01; ***P<0.001).

[0032] Figure 3 The effect of the combination of Paris polyphylla glucomannan and aluminum salt as an adjuvant on the secretion level of macrophage immune factors, where (A) is NO, (B) is TNF-α, and (C) is IL-10 (compared with the OVA group, **P<0.01; ***P<0.001).

[0033] Figure 4 The figure shows the effect of Paris polyphylla glucomannan as an adjuvant on the expression of co-stimulatory molecules in BMDC cells, where A and B are CD40, C and D are CD86, E and F are MHCII, and G and H are MHCI (compared with the OVA group, **P<0.01; ***P<0.001).

[0034] Figure 5 The effect of the combination of Paris polyphylla glucomannan and aluminum salt as an adjuvant on the secretion level of BMDC cell immune factors, where (A) is TNF-α, (B) is IL-6, and (C) is IL-12 (compared with the OVA group, ***P<0.001).

[0035] Figure 6The figure shows the effect of Paris polyphylla glucomannan or its combination with aluminum salt as adjuvant on antigen-specific antibodies, wherein A is the IgG antibody level on the 14th day after the primary immunization; B is the IgG antibody level on the 28th day after the primary immunization; C is the IgG antibody level on the 42nd day after the primary immunization; D is the IgG1 antibody level on the 42nd day after the primary immunization; E is the IgG2a antibody level on the 42nd day after the primary immunization; F is the ratio of the IgG2a antibody level to the IgG1 antibody level on the 42nd day after the primary immunization (compared with the OVA group, *P<0.05; **P<0.01; ***P<0.001; compared with the Alum / OVA group, #P<0.05; ##P<0.01; ###P<0.001).

[0036] Figure 7 The effect of Paris polyphylla glucomannan or its combination with aluminum salt as adjuvant on the proportion of mouse spleen lymphocytes, wherein (A) is CD3 + , (B) is CD19 + , (C) is CD3 + / CD19 + , (D) is CD3 + CD4 + cells, (compared with the OVA group, *P<0.05; **P<0.01; ***P<0.001). DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in the present invention. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more related listed items.

[0039] Example 1

[0040] Preparation of Paris polyphylla glucomannan.

[0041] 1.1 Extraction of Paris polyphylla glucomannan.

[0042] Weigh 300 g of dry rhizome of Paris yunnanensis or Paris mairei, add 3.0 L 95% ethanol, reflux extract twice, each time for 2 hours, filter while hot, collect the filter cake, and evaporate the residual ethanol in the filter cake; add 3 L deionized water, heat and extract at 80°C for 2 hours, centrifuge at 4000 rpm for 10 minutes after the extraction, take the supernatant, precipitate and extract again once, combine the supernatants, add 0.1% (v / v) α-amylase, enzymolysis at 70°C for about 2 hours, no starch can be detected; add 95% ethanol to the supernatant of enzymolysis to a final alcohol concentration of 40%, centrifuge, and obtain 40% alcohol precipitate the 40% alcohol precipitation supernatant was added with stirring 95% ethanol to a final alcohol concentration of 60%, and the mixture was centrifuged to obtain a 60% alcohol precipitate and an alcohol precipitation supernatant, the 60% alcohol precipitate was washed 3 times with 95% ethanol, and the mixture was reconstituted with water and then freeze-dried; the 60% alcohol precipitate was added with stirring 95% ethanol to a final alcohol concentration of 80%, and the mixture was centrifuged to obtain an 80% alcohol precipitate and an alcohol precipitation supernatant, the 80% alcohol precipitate was washed 3 times with 95% ethanol, and the mixture was reconstituted with water and then freeze-dried to obtain crude Paris polyphylla glucomannan or crude Paris polyphylla glucomannan.

[0043] 1.2 Purification of Paris polyphylla glucomannan.

[0044] Weigh about 200 mg of the above-mentioned crude Polygonum multiflorum glucomannan or crude Polygonum multiflorum glucomannan, dissolve it in 10 mL of deionized water, centrifuge at 4000 rpm for 15 min, filter the supernatant through a 0.45 μm filter membrane, and slowly and evenly inject it into the top of the Sephadex G100 gel column. Elute with 0.1 M NaCl solution. Control the flow rate to 0.1 mL / min, collect 2 mL in each tube, and detect its polysaccharide content by phenol-sulfuric acid method. Draw the elution curve of the polysaccharide component with the absorbance value as the ordinate and the number of tubes as the abscissa. Combine the eluates belonging to the same elution peak, desalt with a 500 Da dialysis bag, concentrate under reduced pressure, and freeze-dry in vacuum to obtain purified Polygonum multiflorum glucomannan (PPM) or Polygonum multiflorum glucomannan (PMM).

[0045] 1.3 Chemical characterization of Paris polyphylla glucomannan.

[0046] (1) Molecular weight determination: The weight average molecular weights of PPM and PMM were 92 kDa and 724 kDa, respectively, as determined by high performance gel exclusion chromatography-differential detection.

[0047] (2) Monosaccharide composition analysis: A sample of Paris polyphylla glucomannan was hydrolyzed into monosaccharides with acid, derivatized with PMP, and then passed through a C18 chromatographic column. The monosaccharide composition and ratio of PPM and PMM were analyzed by HPLC and found to be 15:1 and 18.8:1, respectively.

[0048] (3) Methylation analysis: Take a sample of Paris polyphylla glucomannan and use a methyl halide reagent such as iodomethane under alkaline conditions to methylate the polysaccharide. After hydrolysis under alkaline conditions, reduce it with sodium borohydride, acetylate the methylated sugar alcohol with anhydride, extract and fix the volume, and then perform gas chromatography-mass spectrometry (GC-MS) analysis.

[0049] (4) Infrared spectroscopy analysis: 2 mg of sample was vacuum dried at 40 °C for 24 h, and the sample was analyzed by KBr pelleting method using Tensor27 Fourier transform mid-infrared spectrometer at 4000-400 cm -1 Scan and record the spectrum.

[0050] (5) Nuclear magnetic resonance analysis: A sample of Paris polyphylla glucomannan was dissolved in heavy water, vacuum freeze-dried, and repeated three times of heavy water exchange, then dissolved in heavy water and measured using a Bruker 800MHz nuclear magnetic resonance spectrometer. 1 H / 13 C NMR spectra and two-dimensional spectra ( 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 1 H ROESY, 1 H- 13 C HMBC, 1 H- 13 C HSQC-TOCSY and 1 H- 13 CHSQC) and analyzed using MestReNova and Topspin software.

[0051] The results of methylation analysis showed that the polyphylla glucomannan contained β(1→4) mannose glycosidic bonds, a small amount of β(1→4) glucose glycosidic bonds, β(1→) mannose glycosidic bonds and β(1→) glucose glycosidic bonds. The polyphylla glucomannan contained β(1→4) mannose glycosidic bonds, a small amount of β(1→4) glucose glycosidic bonds and β(1→) mannose glycosidic bonds.

[0052] Infrared spectrum of Polygonum multiflorum glucomannan (cm -1 ) Data: 3400~3430cm -1 Characteristic absorption of stretching vibration of hydroxyl group OH on sugar ring; 2920~2890cm -1 The CH stretching vibration of the methine on the sugar ring; 1730-1740cm -1 It is the characteristic absorption peak of carbonyl stretching vibration of acetyl; 1000~1060cm -1 COC stretching vibration on the sugar ring; 810~890cm-1 It is the characteristic absorption of the configuration of β-monosaccharide.

[0053] The chemical shifts of the terminal protons of Polygonum multiflorum glucomannan are 4.53, 4.73 / 4.76, 4.82, 4.91, 4.95 and 5.19 ppm, which are the non-reducing terminal β-D-Glc-(1→(abbreviated as D), →4)-β-D-Man-(1→(abbreviated as A / E), →4)-3-O-acetyl-β-D-Man-(1→(abbreviated as C), the reducing terminal β-D-Man (abbreviated as F), →4)-2-O-acetyl-β-D-Man-(1→(abbreviated as B) containing an acetyl group at the 2nd position and the terminal proton signal of α-D-Man (abbreviated as G) at the reducing end. The 4-position proton signal of 1,4-β-D-mannose is 3.82ppm and the corresponding C-4 chemical shift is 79.4 / 80.3ppm, which is significantly shifted to the downfield relative to the corresponding chemical shift of unsubstituted mannose, indicating that the 1st position of mannose A / E is The hydroxyl groups at the 2nd and 4th positions of mannose B are substituted; the 1,4-β-D-mannose containing acetyl groups at the 2nd and 4th positions has a proton signal of 5.52ppm and 3.82ppm, and the corresponding C-2 chemical shift is 74.5ppm and C-4 chemical shift is 79.5ppm, which is significantly shifted to the low field relative to the chemical shifts of carbon at the 2nd and 4th positions of unsubstituted mannose, indicating that the hydroxyl groups at the 2nd and 4th positions of mannosyl B are substituted; the 1,4-β-D-mannose containing acetyl groups at the 3rd and 4th positions has a proton signal of 5.52ppm and 3.82ppm, and the corresponding C-2 chemical shift is 74.5ppm and C-4 chemical shift is 79.5ppm, which is significantly shifted to the low field relative to the chemical shifts of carbon at the 2nd and 4th positions of unsubstituted mannose, indicating that the hydroxyl groups at the 2nd and 4th positions of mannose B are substituted; The 3-position proton signal of mannose is 5.11ppm, the 4-position proton signal is 4.04ppm, and the corresponding C-3 and C-4 chemical shifts are both 76.3ppm. The hydrogen-carbon chemical shifts at the 3rd and 4th positions of unsubstituted mannose are significantly shifted to the downfield, indicating that the hydroxyl groups at the 3rd and 4th positions of the mannose C are substituted. In addition, the hydrogen chemical shift at 2.19ppm and the carbon chemical shift at 176.2ppm are attributed to the methyl proton and carbonyl carbon signals of the acetyl group. 1 H and 13 C long-range correlation HMBC spectrum showed that C-1 (A1) of mannosyl A and H-4 (A4, B4, G4) of mannosyl A, B and G had correlation signals, C-1 (B1) of mannosyl B and H-4 (E4) of mannosyl E had correlation signals, C-1 (C1) of mannosyl C and H-4 (A4) of mannosyl A had correlation signals, and C-1 (E1) of mannosyl E and H-4 (A4) of mannosyl A and H-4 (C4) of mannosyl C had correlation signals. Therefore, the connection mode of mannosyl is as follows: mannosyl (A / E) forms the main chain with β(1→4) glycosidic bond, while the 2nd and 3rd acetyl-substituted mannosyl (B and C) are interspersed in the main chain and connected to mannosyl (A / E) with β(1→4) glycosidic bond.

[0054] The chemical structure of Polygonum multiflorum glucomannan is shown in the following general formula.

[0055]

[0056] In the formula, n represents the number of sugar repeating units, and n is a natural number. The acetyl substitution degree of the polyphylla glucomannan PPM is 35%.

[0057] Infrared spectrum of polyphylla glucomannan (cm -1 ) Data: 3400~3430cm -1 Characteristic absorption of stretching vibration of hydroxyl group OH on sugar ring; 2920~2890cm -1 The CH stretching vibration of the methine on the sugar ring; 1730-1740cm -1 It is the characteristic absorption peak of carbonyl stretching vibration of acetyl; 1000~1060cm -1 COC stretching vibration on the sugar ring; 810~890cm -1 It is the characteristic absorption of the configuration of β-monosaccharide.

[0058] The results of NMR analysis of glucomannan from Rhizoma Dioscoreae showed that the terminal proton chemical shifts at 4.52, 4.74 / 4.71, 4.84, 4.90, 4.94 and 5.17 ppm were respectively the non-reducing terminal β-D-Glc-(1→(abbreviated as D), →4)-β-D-Man-(1→(abbreviated as A / E), →4)-3-O-acetyl-β-D-Man-(1→(abbreviated as C), the reducing terminal The terminal proton signals of β-D-Man (abbreviated as F), 2-position acetyl-→4)-2-O-acetyl-β-D-Man-(1→(abbreviated as B) and reducing terminal α-D-Man (abbreviated as G). The 4-position proton signal of 1,4-β-D-mannose is 3.82ppm and the corresponding C-4 chemical shift is 79.4 / 80.3ppm, which is significantly shifted to the downfield relative to the corresponding chemical shift of unsubstituted mannose, indicating that mannose The hydroxyl groups at positions 1 and 4 of sugar moiety A / E are substituted; the proton signal at position 2 of 1,4-β-D-mannose containing an acetyl group is 5.50 ppm, and the proton signal at position 4 is 3.80 ppm, and the corresponding C-2 chemical shift is 74.5 ppm, and the C-4 chemical shift is 79.5 ppm, which is significantly shifted to the low field relative to the chemical shifts at positions 2 and 4 of unsubstituted mannose, indicating that the hydroxyl groups at positions 2 and 4 of mannosyl B are substituted; the 1,4- The 3-position proton signal of β-D-mannose is 5.10ppm, the 4-position proton signal is 4.02ppm, and the corresponding C-3 and C-4 chemical shifts are both 76.3ppm. The hydrogen-carbon chemical shifts at positions 3 and 4 of unsubstituted mannose are significantly shifted to the downfield, indicating that the hydroxyl groups at positions 3 and 4 of the mannosyl C are substituted. In addition, the hydrogen chemical shift at 2.19ppm and the carbon chemical shift at 176.2ppm are attributed to the methyl proton and carbonyl carbon signals of the acetyl group. 1 H and 13 C long-range correlation HMBC spectrum showed that C-1 (A1) of mannosyl A and H-4 (A4, B4, G4) of mannosyl A, B and G had correlation signals, C-1 (B1) of mannosyl B and H-4 (E4) of mannosyl E had correlation signals, C-1 (C1) of mannosyl C and H-4 (A4) of mannosyl A had correlation signals, and C-1 (E1) of mannosyl E and H-4 (A4) of mannosyl A and H-4 (C4) of mannosyl C had correlation signals. Therefore, the connection mode of mannosyl is as follows: mannosyl (A / E) forms the main chain with β(1→4) glycosidic bond, while the 2nd and 3rd acetyl-substituted mannosyl (B and C) are interspersed in the main chain and connected to mannosyl (A / E) with β(1→4) glycosidic bond.

[0059] The chemical structure of Paris polyphylla glucomannan is shown in the following general formula.

[0060]

[0061] In the formula, m and n represent the number of sugar repeating units, both of which are natural numbers, wherein m is independently 1, 2, 3, 4, 5, 6 or 7, and n is 150 to 300. The weight average molecular weight of polysaccharide glucomannan is acetyl substitution degree is 22%.

[0062] Example 2

[0063] Regulatory effect of Paris polyphylla glucomannan or its combination with aluminum salt on macrophages.

[0064] 2.1 Preparation of the composition formed by Paris polyphylla glucomannan and aluminum salt.

[0065] Preparation of the composition formed by Paris polyphylla glucomannan and aluminum salt (Alum): Paris polyphylla glucomannan was dissolved in physiological saline for injection to prepare a 10 mg / mL solution, and an equal volume of aluminum hydroxide adjuvant (Alum) or aluminum phosphate with a concentration of 40 mg / mL was added dropwise, and vortexed for 30 minutes to mix.

[0066] 2.2 Effect of Paris polyphylla glucomannan or its combination with aluminum salt on the phagocytic ability of macrophages.

[0067] 2.2.1 Method: The present invention firstly uses the neutral red experiment to study the effect of Paris polyphylla glucomannan on the ability of macrophages to phagocytize neutral red. RAW264.7 cells in the logarithmic growth phase were taken at 2×10 5 Each well was inoculated into a 24-well cell culture plate, 500 μL per well, and placed in an incubator for 24 hours. Polygonum erythrorhizon glucomannan PMM and Polygonum yunnanensis glucomannan PPM were added, respectively, and three concentration gradients of low (50 μg / mL), medium (100 μg / mL), and high (200 μg / mL) were set. The blank culture medium was used as the negative control group (Control), and 1 μg / mL of LPS was used as the positive control, and the culture treatment was continued for 24 hours. After washing lightly with PBS twice, 100 μL of 33.3% neutral red dye solution (PBS: 33.3% neutral red dye solution = 3:7) was added, and the cells were cultured in a cell culture incubator for 30 minutes, washed lightly with PBS 3 times, 100 μL of lysis solution / well (ethanol: glacial acetic acid = 1:1), lysed at room temperature for 2 hours, and the absorbance was measured at 540 nm. Three replicate wells were set for each group, and the experiment was conducted independently 3 times. The phagocytic rate was calculated according to the following formula: phagocytic rate = (A value of the experimental group - A value of the blank group) / (A value of the control group - A value of the blank group) × 100%.

[0068] Flow cytometry was further used to study the effect of Paris polyphylla glucomannan or its combination with Alum on the antigen phagocytosis of macrophages. RAW264.7 cells in the logarithmic growth phase were cultured at 2×10 5Each well was inoculated into a 24-well cell culture plate, 500 μL per well, and placed in an incubator for 24 hours. FITC-labeled model antigen chicken ovalbumin (FITC-OVA), Alum / FITC-OVA complex, PPM / FITC-OVA complex, PPM / Alum / FITC-OVA complex, PMM / FITC-OVA complex, PMM / Alum / FITC-OVA complex were added respectively, wherein the final concentration of OVA was 20 μg / mL, the final concentration of Alum was 400 μg / mL, and the final concentration of polysaccharide was 100 μg / mL. The cells were cultured for 12 hours at 37°C and 5% CO2, and the cells were collected. FITC-OVA that was not phagocytosed by macrophages was washed off with PBS, and the flow cytometer was used to detect at 488 nm.

[0069] 2.2.2 Results and analysis: Macrophages are professional phagocytes in the body. They eliminate pathogens and remove cell debris through phagocytosis, play an innate immune regulatory role, and further process the phagocytosed antigens and present them to lymphocytes to activate adaptive immune responses. Figure 1 As shown in A, compared with the blank control group, the glucomannan PPM from Paris polyphylla at a concentration of 100-200 μg / mL can significantly promote the phagocytosis of neutral red by macrophages (P<0.05), and the glucomannan PMM from Paris polyphylla can significantly promote the phagocytosis of neutral red by macrophages at a concentration range of 50-200 μg / mL (P<0.01). The results of flow cytometry experiments showed that ( Figure 1 In Figure 1B and Figure 1C), PPM alone did not significantly promote the phagocytosis of the model antigen FITC-OVA by macrophages, but PPM / Alum could promote the phagocytosis of FITC-OVA by macrophages (P<0.01). PMM and PMM / Alum could both promote the phagocytosis of FITC-OVA by macrophages (P<0.01).

[0070] 2.3 Effect of Paris polyphylla glucomannan or its combination with aluminum salt on the secretion of immune factors of macrophages.

[0071] 2.3.1 Methods: Griess method and ELISA method were used to detect the effect of Paris polyphylla glucomannan on the secretion level of immune factors of macrophages. RAW264.7 cells in logarithmic growth phase were cultured at 2×10 5 Each well was inoculated into a 24-well plate, 500 μL per well, and placed in an incubator for 24 h. 50, 100, and 200 μg / mL of PPM or PMM were added, respectively, with blank culture medium as the negative control group and 1 μg / mL of LPS as the positive control, and the culture treatment was continued for 24 h. The culture supernatant was collected, and the NO content in the supernatant was determined by the Griess method, and the TNF-α and IL-10 content in the supernatant was detected by ELISA.

[0072] The effects of Paris polyphylla glucomannan, aluminum salt, and OVA composition on the secretion of macrophage immune factors were further tested. The treatment conditions were: the final concentration of OVA was 8 μg / mL, the final concentration of Alum was 400 μg / mL, the final concentration of polysaccharide was 100 μg / mL, and 1 μg / mL of LPS was used as a positive control. Other treatment conditions and detection methods were the same as above.

[0073] 2.3.2 Results and analysis: Figure 2 As shown in the results, PPM at 100-200 μg / mL can significantly promote the secretion of NO by macrophages (P<0.01), at a concentration range of 50-200 μg / mL can promote the secretion of TNF-α by macrophages (P<0.01), and at a high concentration of 200 μg / mL can significantly promote the secretion of IL-10 by macrophages (P<0.01); PMM at a range of 50-200 μg / mL can significantly promote the secretion of cytokines NO, TNF-α and IL-10 by macrophages. Figure 3 As shown in the data, PPM / OVA can increase the secretion levels of NO and TNF-α of macrophages to a certain extent, PMM / OVA and PMM / Alum / OVA can increase the secretion levels of NO and TNF-α of macrophages (P<0.01), PMM / OVA can increase the secretion level of IL-10 of macrophages (P<0.01), while Alum / OVA cannot increase the secretion levels of NO, TNF-α and IL-10 of macrophages.

[0074] Example 3

[0075] Regulatory effect of Paris polyphylla glucomannan or its combination with aluminum salt on dendritic cells.

[0076] 3.1 Extraction of mouse bone marrow-derived dendritic cells (BMDC).

[0077] Balb / c mice were killed by cervical dislocation and soaked in 75% (v / v) alcohol for 3 minutes. The femurs and tibias of the mice were separated under sterile conditions. The bone marrow cavity was flushed with RPMI 1640 culture medium until the bone turned completely white. The bone marrow suspension was collected and centrifuged at 1200rpm for 5 minutes. The supernatant was discarded, and 2ml of ammonium chloride red blood cell lysis buffer was added to resuspend the cells. Incubate at room temperature for 3 minutes, add 10mL PBS to neutralize the lysis buffer, centrifuge at 1200rpm for 5 minutes, and discard the supernatant. Wash the cells once with 5ml of pre-cooled PBS, and then resuspend the cells with RPMI 1640 culture medium containing 10% FBS and 1% double antibody. After counting, the cell concentration was adjusted to 1×10 with RPMI 1640 complete culture medium containing 10% FBS. 6 / mL; inoculated into 24-well culture plates, 1mL cells per well, and recombinant mouse GM-CSF (20ng / mL) and IL-4 (10ng / mL) were added at the same time, and cultured in a 37°C, 5% CO2 incubator. The culture plates were gently shaken on the 2nd and 4th days, and then 3 / 4 of the volume was replaced with fresh culture medium and supplemented with cytokines. On the 6th day of culture, the culture medium was gently blown to collect the suspended cells and the cells that grew on the wall of thiocyanate.

[0078] 3.2 Flow cytometry was used to detect the effect of Paris polyphylla glucomannan on BMDC cell surface molecules.

[0079] 3.2.1 Method: BMDC cells extracted in 3.1 were cultured at 1×10 6 / mL was inoculated into the remaining 12-well plate, 1mL per well, and Paris polyphylla glucomannan (PPM or PMM), OVA, and the complex of Paris polyphylla glucomannan and OVA were added respectively, wherein the final concentration of OVA was 8μg / mL, and the concentration of Paris polyphylla glucomannan was 100μg / mL, 1μg / mL of LPS was used as a positive control, and the cells were cultured in a cell culture incubator for 24h. The cells were collected and washed twice with PBS, and the cells were resuspended with 100μL PBS at the last time. Antibodies PE anti-mouse CD11c, FITC anti-mouse CD86, PerCP Cy5.5anti-mouse CD40, and APCanti-mouse MHCII were added according to the flow cytometry antibody instructions, incubated at room temperature and away from light for 30min, 1mL PBS was added to each tube to wash twice, and 400μL PBS was added to resuspend the cells, and detected by flow cytometry.

[0080] 3.2.2 Results and analysis: Figure 4 As shown, compared with the OVA group, PMM and PMM / OVA could significantly promote the expression of co-stimulatory molecules CD40, CD86 and MHCII on the surface of BMDC cells (P<0.05), stimulate BMDC maturation, but could not stimulate the expression of MHCI.

[0081] 3.3 ELISA method to detect the effect of Paris polyphylla glucomannan or its combination with aluminum salt on the secretion of immune factors of BMDC cells

[0082] 3.3.1 Method: BMDC cells extracted in 3.1 were cultured at 1×10 6 / mL was inoculated into the remaining 12-well plate, 1mL per well, OVA, Paris polyphylla glucomannan (PPM or PMM), Paris polyphylla glucomannan and OVA complex, Paris polyphylla glucomannan / aluminum salt / OVA complex were added respectively, wherein the final concentration of OVA was 8μg / mL, the concentration of Paris polyphylla glucomannan was 100μg / mL, and the final concentration of Alum was 400μg / mL, 1μg / mL LPS was used as a positive control, and the cell culture was continued for 24h in the cell culture incubator. The culture supernatant was collected, and the ELISA method was used to detect the content of TNF-α, IL-6, and IL-12 in the culture supernatant.

[0083] 3.3.2 Results and analysis: Figure 5 As shown in the figure, compared with the OVA group, PMM, PMM / OVA, and PMM / Alum / OVA could significantly promote BMDC cells to secrete immune factors TNF-α, IL-6, and IL-12 (P<0.01) and enhance the immune response.

[0084] Example 4

[0085] In vivo immune adjuvant activity of Paris polyphylla glucomannan or its aluminum salt composition.

[0086] 4.1 Experimental methods

[0087] 4.1.1 Animal grouping and immunization procedure

[0088] In vivo immune adjuvant activity of Paris polyphylla glucomannan was studied using ovalbumin (OVA) as a model antigen. Fifty-six 6-8-week-old BALB / C mice were randomly divided into seven groups: saline group, OVA group, Alum / OVA group, PPM / OVA group, PPM / Alum / OVA group, PMM / OVA group, and PMM / Alum / OVA group. Each mouse was injected intramuscularly with 50 μL of the sample solution to be tested. The injection dose of OVA was 20 μg / mouse, the injection dose of Paris polyphylla glucomannan was 250 μg / mouse, and the injection dose of Alum was 1 mg / mouse. The day of injection was taken as day 0, and booster immunization was performed once on the 14th and 28th days, respectively. Blood was collected from the orbital venous plexus of mice on the 14th, 28th, and 42nd days, respectively. The mice were killed on the 42nd day, and the spleen was aseptically separated for subsequent extraction of spleen lymphocytes.

[0089] 4.1.2 Determination of OVA-specific antibody titers in serum samples by ELISA

[0090] 1) ELISA plate coating: OVA was diluted to 5 μg / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), added to a 96-well plate, 100 μL / well, and coated at 4°C for 16 h.

[0091] 2) Washing: discard the coating solution, add 200uL PBST washing solution to each well, let it stand for 5 minutes, discard the washing solution, repeat 3 times, and finally turn the reaction plate upside down on absorbent paper and pat dry.

[0092] 3) Blocking: Add 300 μL of 1% BSA to each well and block at 37°C for 1.5 h.

[0093] 4) Washing: Add 200uL of PBST to each well, let stand for 5 minutes, discard the washing solution, repeat 3 times, and finally turn the reaction plate upside down on absorbent paper and pat dry.

[0094] 5) Add serum samples: dilute the serum of the animal to be tested with 1% BSA and add it to a 96-well plate, 100 μL / well, and incubate at 37° C. for 2 h.

[0095] 6) Washing: Add 200uL of PBST to each well, let stand for 5 minutes, discard the washing solution, repeat 3 times, and finally turn the reaction plate upside down on absorbent paper and pat dry.

[0096] 7) Detection antibody incubation: add 100 μL Goat Anti-Mouse IgG (H+L)-HRP, Goat Anti-Mouse IgG1-HRP, Goat Anti-Mouse IgG2a-HRP (diluted 1:5000 with 1% BSA) to each well and incubate at 37°C for 1 h.

[0097] 8) Washing: Add 200uL of PBST to each well, let stand for 5 minutes, discard the washing solution, repeat 5 times, and finally turn the reaction plate upside down on absorbent paper and pat dry.

[0098] 9) Color development: Add TMB substrate color development solution, 100 μL / well, and incubate at room temperature in the dark for 15 minutes.

[0099] 10) Stop the reaction: Add color stop solution to stop color development, and read the sample absorbance at 450nm using an ELISA reader.

[0100] 4.1.3 Extraction of mouse spleen lymphocytes

[0101] Place the mouse spleen described in 4.1.1 on a 200-mesh cell sieve to which 3 mL of RPMI 1640 medium has been added in advance, and grind the spleen into a single-cell suspension using a sterile syringe needle, and centrifuge (1500 rpm, 5 min). Pour off the supernatant, retain the cell pellet, add 1 mL of sterile red blood cell lysis buffer, resuspend the cells with a pipette, and then make up to 3 mL, and let stand for 4 min to fully lyse the red blood cells. Add 10 mL of culture medium and mix well to terminate the lysis, and centrifuge (1500 rpm, 5 min). Pour off the supernatant, retain the cell pellet, add 1 mL of culture medium and resuspend the cells with a pipette, and then make up to 10 mL and mix well, and centrifuge (1500 rpm, 5 min) to wash away any residual red blood cell lysis buffer. Pour off the supernatant, add 1 mL of RPMI 1640 complete culture medium (10% FBS and 1% double antibody), and resuspend the cells with a pipette. Adjust the cell concentration to 5×10 6 / mL.

[0102] 4.1.4 Detection of lymphocytes and their subtypes in spleen lymphocytes by flow cytometry

[0103] The spleen lymphocytes obtained in 4.1.3 were counted at 5×10 6 Cells were inoculated into 6-well plates at 1 mL per well, and 1 mL of blank medium was added. OVA diluted with complete medium was added for in vitro stimulation (final concentration was 10 μg / mL). Incubate in a 5% CO2 incubator at 37°C for 48 h, pipette the cells into a centrifuge tube, and collect the cells by centrifugation (2000 rpm, 5 min). Wash the cells once with 1 mL of PBS and centrifuge (2000 rpm, 5 min). 100 μL of PBS solution containing APC anti-mouse CD3e, PerCP / Cy5.5anti-mouse CD4, and FITC anti-mouse CD8a flow cytometry antibodies was added to each tube, mixed thoroughly, and incubated at 25°C in the dark for 30 min. Wash twice with 1 mL of PBS in each tube, resuspend the cells in 400 uL of PBS, and detect by flow cytometry.

[0104] 4.2 Results and Analysis

[0105] like Figure 6As shown, Paris polyphylla glucomannan (PPM, PMM) or its aluminum salt composition can significantly increase the specific IgG level in mouse serum on the 14th, 28th and 42nd day after the initial immunization. At the immunization endpoint, the specific IgG and IgG1 levels of the PPM / Alum / OVA and PMM / OVA groups were comparable to those of the Alum / OVA group. The specific IgG and IgG1 levels of the PPM / OVA group were slightly lower than those of the positive control Alum / OVA, while the specific IgG and IgG1 levels of the PMM / Alum / OVA group were significantly higher than those of the positive control Alum / OVA. It is worth noting that PPM / Alum / OVA and PMM / Alum / OVA can also significantly enhance the specific IgG2a level in mouse serum, indicating that they can mediate both Th1 and Th2 immune responses, while traditional aluminum adjuvants can only mediate Th2 immune responses. Figure 7 As shown in Figure 2, PPM / OVA, PMM / OVA, PPM / Alum / OVA and PMM / Alum / OVA could significantly increase the expression of CD3 + 、CD3 + CD4 + cell ratio (P<0.05), further indicating that the combination of Paris polyphylla glucomannan and aluminum salt can simultaneously enhance the humoral immunity and cellular immunity of mice.

[0106] Similarly, the present invention found that Paris polyphylla glucomannan or its combination with aluminum salt can also increase the specific IgG, IgG1 and IgG2a antibody levels of hepatitis B virus surface antigen (HBsAg), varicella-zoster virus glycoprotein gE and / or human papillomavirus L1 protein antigen in the serum of immunized mice, and significantly increase the CD3 + 、CD3 + CD4 + cell ratio (P<0.05), further indicating that Paris polyphylla glucomannan or its combination with aluminum salt can simultaneously enhance the humoral immunity and cellular immunity of mice.

[0107] In summary, the present invention discloses for the first time a Paris polyphylla glucomannan or a combination thereof with aluminum salts having immune adjuvant function. The Paris polyphylla glucomannan can promote the phagocytic function of macrophages and the secretion of NO and TNF-α; at the same time, the Paris polyphylla glucomannan can also promote the maturation of dendritic cells, upregulate the expression of CD40, CD86, and MHCII, and promote the secretion of TNF-α, IL-6, and IL-12. In vivo experiments have shown that the Paris polyphylla glucomannan or a combination thereof with aluminum salts can significantly increase the levels of antigen-specific IgG, IgG1, and IgG2a antibodies, while inducing Th1 and Th2 immune responses, and can also increase the expression of CD3 in splenic lymphocytes. + 、CD3 + CD4+ cell ratio, and simultaneously enhanced the humoral immunity and cellular immunity levels of mice.

[0108] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0109] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0110] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An immune adjuvant containing Paris polyphylla glucomannan or its aluminum salt composition, characterized in that: The Paris polyphylla glucomannan has the following characteristics: The Paris polyphylla glucomannan is extracted and purified from Paris polyphylla plants of the family Melanophoraceae, and has a weight average molecular weight of 50 to 800 kDa. The monosaccharide composition is mannose and glucose, and the ratio of the two is 10:1 to 20:

1. The 2nd or 3rd position of mannose is substituted with an acetyl group, and its structural formula is as follows: In the formula, n represents the number of sugar repeating units, and n is a natural number. The acetyl substitution degree of Paris polyphylla glucomannan PPM is 35%.

2. The immunoadjuvant containing Paris polyphylla glucomannan or its aluminum salt composition according to claim 1, characterized in that: The Paris polyphylla glucomannan can promote the maturation of dendritic cells, upregulate the expression of CD40, CD86, and MHCII, and promote the secretion of TNF-α, IL-6, and IL-12; it can significantly increase the serum specific IgG, IgG1, and IgG2a antibody levels of antigens, and simultaneously enhance the humoral immunity and cellular immunity levels.

3. The immunoadjuvant containing Paris polyphylla glucomannan or its aluminum salt composition according to claim 1, characterized in that: The aluminum salt composition is a composition formed by mixing Paris polyphylla glucomannan with aluminum salt.

4. The immunoadjuvant containing Paris polyphylla glucomannan or its aluminum salt composition according to claim 3, characterized in that: The aluminum salt is selected from one or both of aluminum hydroxide and aluminum phosphate; the mass ratio of Paris polyphylla glucomannan to the aluminum salt is 0.0001 to 10:1, preferably 0.01 to 0.5:

1.

5. The immunoadjuvant containing Paris polyphylla glucomannan or its aluminum salt composition according to claim 4, characterized in that: The aluminum salt composition of Paris polyphylla glucomannan can significantly increase the serum specific IgG, IgG1, and IgG2a antibody levels of antigens, and simultaneously enhance the humoral immunity and cellular immunity levels.

6. The method for preparing an immune adjuvant containing Paris polyphylla glucomannan or its aluminum salt composition according to claim 1, characterized in that: The method comprises the following steps: extracting dry powder of rhizomes of Paris genus plants with hot water to obtain a water extract; performing enzymatic hydrolysis on the water extract to obtain non-starch polysaccharides; performing graded alcohol precipitation on the non-starch polysaccharides and collecting the precipitates; purifying by using one or more of gel exclusion chromatography, dialysis and ultrafiltration to obtain Paris glucomannan; and further mixing the Paris glucomannan with aluminum salt to obtain an aluminum salt composition of Paris glucomannan.

7. Paris polyphylla glucomannan or its aluminum salt composition, characterized in that: The Paris polyphylla glucomannan is extracted and purified from Paris polyphylla plants of the family Melanophoraceae, and has a weight average molecular weight of 50 to 800 kDa. The monosaccharide composition is mannose and glucose, and the ratio of the two is 10:1 to 20:

1. The 2nd or 3rd position of mannose is substituted with an acetyl group, and its structural formula is as follows: In the formula, n represents the number of sugar repeating units, and n is a natural number, wherein the degree of acetyl substitution of Polygonum multiflorum glucomannan PPM is 35%; The polysaccharide is prepared by the following method: extracting dry powder of the rhizome of a Paris polyphylla plant with hot water to obtain a water extract; performing enzymatic hydrolysis on the water extract to obtain non-starch polysaccharides; performing graded alcohol precipitation on the non-starch polysaccharides and collecting the precipitate; purifying the polysaccharide by one or more of a gel exclusion chromatography column method, a dialysis method and an ultrafiltration method to obtain Paris polyphylla glucomannan; and further mixing the polysaccharide with an aluminum salt to obtain an aluminum salt composition of Paris polyphylla glucomannan.

8. Paris polyphylla glucomannan or its aluminum salt composition, characterized in that: The Paris polyphylla glucomannan is extracted and purified from Paris polyphylla plants of the family Melanophoraceae. The extraction is as follows: weighing dried Paris polyphylla or Paris polyphylla rhizome, adding 95% ethanol to reflux and extract twice, each time for 2 hours, filtering while hot, collecting filter cakes, and evaporating the residual ethanol in the filter cakes; adding deionized water to heat at 80°C for extraction for 2 hours, centrifuging at 4000rpm for 10 minutes after the extraction, taking the supernatant, precipitating and extracting once, combining the supernatants, adding 0.1% (v / v) α-amylase, and performing enzymolysis at 70°C for 2 hours, until no starch can be detected; adding 95% ethanol to the enzymolysis supernatant until the final alcohol concentration is 40%, centrifuging, and obtaining a 40% alcohol precipitate and a 40% alcohol precipitate supernatant; To the above 40% alcohol precipitation supernatant, add 95% ethanol while stirring until the final alcohol concentration is 60%, centrifuge to obtain 60% alcohol precipitation and alcohol precipitation supernatant, wash the 60% alcohol precipitation 3 times with 95% ethanol, add water to reconstitute and then freeze-dry; To the above 60% alcohol precipitation supernatant, 95% ethanol is added under stirring until the final alcohol concentration is 80%, and the mixture is centrifuged to obtain an 80% alcohol precipitate and an alcohol precipitation supernatant. The 80% alcohol precipitate is washed 3 times with 95% ethanol, and then re-dissolved with water and freeze-dried to obtain crude Paris polyphylla glucomannan or Paris polyphylla glucomannan. The purification step is as follows: the above crude Paris polyphylla glucomannan or Paris polyphylla glucomannan is weighed, dissolved in deionized water, centrifuged at 4000 rpm for 15 minutes, the supernatant is filtered through a 0.45 μm filter membrane, and then slowly and evenly injected into the top of a Sephadex G100 gel column, and filtered with a 0.1 M The product was eluted with NaCl solution at a flow rate of 0.1 mL / min, 2 mL was collected in each tube, and the polysaccharide content was detected by a phenol-sulfuric acid method. The elution curve of the polysaccharide component was plotted with the absorbance as the ordinate and the number of tubes as the abscissa. The eluates belonging to the same elution peak were combined, desalted with a 500 Da dialysis bag, concentrated under reduced pressure, and vacuum-frozen to obtain purified Paris polyphylla glucomannan (PPM) or Paris polyphylla glucomannan (PMM); the purified Paris polyphylla glucomannan (PPM) or Paris polyphylla glucomannan (PMM) was further mixed with aluminum salt to obtain an aluminum salt composition of Paris polyphylla glucomannan (PPM) or Paris polyphylla glucomannan (PMM).

9. Use of the Paris polyphylla glucomannan or its aluminum salt composition according to claim 7 or 8 in the preparation of immune adjuvants or in the preparation of vaccine preparations or vaccine compositions.

10. Use of the immune adjuvant according to claim 1 in the preparation of vaccine preparations and vaccine compositions.

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