CN112316133B - Preparation method and application of tremella polysaccharide immunoadjuvant for injection - Google Patents
Preparation method and application of tremella polysaccharide immunoadjuvant for injection Download PDFInfo
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- CN112316133B CN112316133B CN202011281304.1A CN202011281304A CN112316133B CN 112316133 B CN112316133 B CN 112316133B CN 202011281304 A CN202011281304 A CN 202011281304A CN 112316133 B CN112316133 B CN 112316133B
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- injection
- tremella
- polysaccharide
- tremella polysaccharide
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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Abstract
The invention relates to tremella polysaccharide for injection, and an extraction and purification method and application thereof. The tremella polysaccharide monosaccharide for injection comprises mannose: glucuronic acid: glucose: xylose: fucose = 66.2:10.75:1.12:19.20:2.82. the extraction and purification method of the tremella polysaccharide for injection comprises the steps of dissolving tremella polysaccharide with water, performing column chromatography through activated carbon, performing alcohol precipitation, hydrolyzing with hydrochloric acid, neutralizing with sodium hydroxide, and performing alcohol precipitation again to obtain tremella polysaccharide for injection. The tremella polysaccharide for injection has the characteristics of small molecular weight, high purity and good water solubility. The tremella polysaccharide for injection has good immunity enhancing activity, can be used as an adjuvant, can obviously improve the immunity prototype of various vaccines, and is applied to the preparation of various vaccines.
Description
Technical Field
The invention relates to an application of tremella polysaccharide for injection as an immune adjuvant in medical products. Belongs to the technical field of biological pharmacy.
Background
An immunoadjuvant is a non-specific immunopotentiator that is effective in enhancing the intensity of an immune response or altering the type of immune response when injected with an antigen together or into the body in advance. Commonly used adjuvants can be divided into 4 classes: inorganic adjuvants such as aluminum hydroxide, alum, etc.; an organic adjuvant; microorganisms and their products such as mycobacteria (tubercle bacillus, bacillus calmette guerin), bacillus pumilus, pertussis, endotoxin, bacterial extracts (muramyl dipeptide), etc.; synthetic adjuvants such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenylation, uridylic acid) and the like; in recent years, polysaccharide components derived from plants or microorganisms have also become components for developing novel adjuvants, such as laminarin. The biological effects of immunoadjuvants include: the physical properties of the antigen are changed, so that the antigen substances can be slowly released, and the action time of the antigen is prolonged; after the antigen is adsorbed by the adjuvant, the surface area of the antigen is increased, so that the antigen is easy to be phagocytized by macrophages; adjuvants can stimulate the treatment of antigens by phagocytes; the adjuvant can promote the contact between lymphocytes and enhance the effect of helper T cells; can stimulate division of sensitized lymphocytes and antibody production by plasma cells. Thus, the action of the immunoadjuvant can change non-immunogenic substances into effective immunogens. The screening and development of new immunoadjuvants is getting more and more attention.
Tremella (Tremella fueiformis Berk) is also called Tremella, is a dried fruiting body of tremella of Tremellaceae of Basidiomycetes, is a common health medicine, has effects of nourishing yin and moisturizing lung, and has medicinal history for hundreds of years. The main chemical components are polysaccharide compounds. Also contains lipid, proteins (enzyme, protein, amino acid), inorganic salt, vitamin B group, etc. The tremella polysaccharide is mainly acidic heteropolysaccharide, and is composed of series acidic heteropolysaccharide with molecular weight of tens of thousands to hundreds of thousands; the sugar comprises xylose, glucose, fucose, mannose and glucuronic acid; a large number of chemical and pharmacological experiments prove that the tremella polysaccharide has obvious activities of resisting radiation, increasing white fungus, reducing blood sugar, resisting ulcer, enhancing immunity, resisting tumor, resisting toxic and side effects of radiotherapy and chemotherapy and the like; acute toxicity and long-term toxicity studies show that the polysaccharide has no obvious toxic or side effect after oral administration or injection administration. The tremella polysaccharide is easy to dissolve in hot water, slightly dissolve in low-concentration ethanol, and is insoluble in high-concentration ethanol and other organic solvents, and the aqueous solution is transparent and viscous. The research results of the absorption, distribution and metabolism of tremella polysaccharide in vivo show that the oral absorption and utilization rate is extremely low (more than 1%), and the effects of tremella polysaccharide on improving immunity, increasing white and resisting cancer are obviously higher than those of oral administration during intramuscular injection and intravenous administration. Because of low oral bioavailability and insignificant effect, the route of injection administration should be selected when the drug is developed as a new drug. However, because tremella polysaccharide has large molecular weight and complex structure, the aqueous solution of tremella polysaccharide also has certain viscosity, which affects the application of tremella polysaccharide as an injection and also affects the evaluation, control and mass production of tremella polysaccharide. Therefore, the preparation method is used for degrading the active components to prepare fragments with relatively simple structure, small molecular weight and good water solubility, and active components with stronger effects are obtained by screening the fragments, so that the preparation method has important significance for development and application of the active components.
The application of tremella polysaccharide for injection as an immune adjuvant is not found before the completion of the invention.
Disclosure of Invention
The invention aims at providing an immune adjuvant which is prepared by matching natural polysaccharide with influenza vaccine, and can enhance the immune function of organisms.
The tremella polysaccharide for injection with smaller molecular weight, high purity and high yield is prepared after separation, purification and degradation of tremella polysaccharide. The tremella polysaccharide for injection has obvious effects in improving the immune function of organisms and the like; meanwhile, due to good water solubility, the injection can also meet the requirement of administration in an injection mode, and the adjuvant applied to the immunopotentiator and the vaccine for the first time has obvious technical innovation.
Preferably, the vaccine is an H5N1 influenza vaccine and a rabies vaccine.
The invention has the beneficial effects that: the natural tremella polysaccharide is used as an active ingredient, and is matched with the influenza vaccine to play a synergistic effect in effect, so that the activity of the influenza vaccine can be enhanced, and an ideal immune effect can be obtained.
In particular, the influenza vaccine using the immunoadjuvant of the present invention has significantly improved antibody titer of immune expression throughout the whole immune cycle compared to the influenza vaccine without the immunoadjuvant.
The invention further provides a preparation method of tremella polysaccharide for injection, which comprises the following steps:
a. taking tremella polysaccharide, adding purified water, stirring for 8 hours at 40 ℃, centrifuging, collecting centrifugate, passing through a granular activated carbon column, collecting eluent, concentrating to 0.3g tremella polysaccharide per ml, standing to room temperature, slowly adding 1.6 times of 95% ethanol under stirring, continuing stirring for 20 minutes, centrifuging, collecting centrifugate, continuing adding 10 times of 95% ethanol under stirring, continuing stirring for 20 minutes, centrifuging, collecting precipitate, and naturally drying to obtain crude polysaccharide;
b. taking coarse polysaccharide powder, adding 20 times of hydrochloric acid aqueous solution, hydrolyzing in 90 ℃ water bath, taking out after hydrolysis is finished, standing to room temperature, dripping sodium hydroxide aqueous solution to adjust pH to neutrality, adding 0.5 times of 95% ethanol under stirring, continuously stirring for 2 hours, centrifuging, collecting precipitate, adding 10 times of injection water for dissolving, filtering with microporous filter membrane, and freeze-drying to obtain tremella polysaccharide for injection.
The preparation method of the tremella polysaccharide for injection is characterized in that the feed liquid ratio of the tremella polysaccharide to distilled water in the step a is 1:20g/mL, and the rotation speed was 500rpm during stirring.
The preparation method of the tremella polysaccharide for injection is characterized in that the concentration of the hydrochloric acid aqueous solution in the step b is 0.1mol/L, and the hydrolysis time is 4-10 hours.
In order to realize the above invention, the following technical scheme is adopted:
1. determination of physicochemical properties of tremella polysaccharide for injection
1.1 neutral sugar content determination- -phenol-sulfuric acid method
Taking 100mg of tremella polysaccharide for injection, adding 50ml of water for dissolution, taking 1ml, and measuring according to an appendix IX S of 2000 edition of Chinese pharmacopoeia, wherein the tremella polysaccharide for injection is not less than 75% in terms of mannose.
1.2 determination of uronic acid content- -m-hydroxybiphenyl method
1.2.1 preparation of control solution
Precisely weighing 5mg of glucuronic acid reference substance dried to constant weight at 105 ℃, placing into a 100ml volumetric flask, adding water for dissolution, diluting to scale, and shaking uniformly to obtain the final product (each 1ml contains glucuronic acid 50 mug).
1.2.2 preparation of standard curve
Precisely sucking reference solution 0ml, 0.1ml, 0.2ml, 0.3ml and 0.4ml, respectively adding water to supplement the volume to 0.4ml, mixing, adding 2.4ml of 0.0125mol/L sodium tetraborate-sulfuric acid solution (taking sodium tetraborate to 0.475 g and adding 100ml of concentrated sulfuric acid for dissolution) into ice water bath, mixing, heating in boiling water bath for 5 minutes, taking out and rapidly cooling to room temperature, adding 0.15% m-hydroxybiphenyl to 40 μl, immediately mixing, taking 0 tube as blank, measuring absorbance at 525nm wavelength for 20 minutes according to spectrophotometry (one part of the appendix V A of the 2010 edition), and calculating regression equation according to absorbance corresponding to glucuronic acid μg.
1.2.3 assay
Accurately weighing 3350mg of the test sample, placing in a 100ml measuring flask, adding water to dilute to a scale, shaking uniformly to obtain a test sample solution, accurately sucking 0.2ml of the test sample solution, adding water to supplement the volume to 0.4ml, determining the absorbance according to the normal operation from 'adding in ice water bath' under the preparation item of a standard curve, and calculating the acid sugar content by a regression equation.
1.2.4 uronic acid content measurement results
TABLE 1 absorption values of standard uronic acid solutions
According to the above data, the regression equation was found to be y=0.0309x+0.0315, r=0.9985, with uronic acid content on the abscissa and absorption on the ordinate.
According to the experimental result, the tremella fuciformis sporin for injection contains acid sugar which is not less than 10% in terms of glucuronic acid.
1.3 determination of proteins
Taking 100mg of tremella polysaccharide for injection, adding 50ml of water for dissolution, taking 1ml, and measuring by an appendix IX S of the 2000 edition of Chinese pharmacopoeia, wherein the requirements are met.
2. Analysis of tremella polysaccharide composition sugar for injection
2.1 preparation of standard monosaccharide solutions
Fuc (fucose), rha (rhamnose), ara (arabinose), xyl (xylose), man (mannose), gal (galactose), glu (glucose), galA (galacturonic acid) GlcA (glucuronic acid) monosaccharide reference substances with proper amounts are taken, and distilled water is respectively added to prepare monosaccharide standard solutions with the concentration of 2 mmol/L.
2.2 preparation of hydrolysis solution of Tremella sporine for injection
Accurately weighing 20mg of tremella polysaccharide sample for injection in a test tube with a plug, adding 2ml of 2mol/L trifluoroacetic acid, sealing, hydrolyzing at 100 ℃ for 2 hours, and drying with nitrogen. Adding 1ml of methanol into the residue, drying, repeating for 4 times, removing trifluoroacetic acid, and derivatizing.
2.3 preparation of derivatives
Taking 3ml of each of the prepared 2mmol/L standard monosaccharide solution and the aqueous solution of the tremella polysaccharide sample for injection, respectively placing the solution into different test tubes with plugs, sequentially adding 6ml of 0.5mol/L PMP methanol solution and 0.3mol/L NaOH solution, uniformly mixing, carrying out water bath reaction at 70 ℃ for 30min, taking out, cooling for 5min, neutralizing with 5ml of 0.3mol/L HCl, adding equal volume chloroform for extraction, fully oscillating, taking a water layer, and repeating for 3 times. 300. Mu.L of each PMP-derivatized monosaccharide was mixed and passed through a 0.45 μm microporous filter.
2.4HPLC analysis
Island liquid LC-2010 autosample high performance liquid chromatograph, ZORBAX EclipseXDB-C18 analytical column (Agilent) (phi 4.6 mm. Times.150 mm); column temperature: 40 ℃; mobile phase: a: phosphate buffer (KH) 2 PO 4 NaOH, ph=6.8): acetonitrile (85:15, V/V); b: phosphate buffer (KH) 2 PO 4 NaOH, ph=6.8): acetonitrile (60:40, V/V); flow rate: 0.9mL/min; detection wavelength: 250nm. Gradient elution is started for 0-10 minutes, the proportion of the mobile phase B is increased from 0 to 8 percent, the proportion of the mobile phase B is increased from 8 percent to 20 percent after 10-30 minutes, and the mobile phase B is maintained for 10 minutes and is ended.
The PMP derivatization-HPLC analysis result of the tremella fuciformis sporin for injection shows that the tremella fuciformis sporin for injection mainly comprises mannose, glucuronic acid, glucose, xylose and fucose; the molar ratio is as follows: 66.2:10.75:1.12:19.20:2.82.
2.4 determination of molecular weight distribution
The tremella polysaccharide sample for injection is added with a solution of 5mg/mL which is prepared by flowing and is measured according to Chinese pharmacopoeia. The molecular weight distribution range is 2000-50000Da, and the weight average molecular weight is 5900Da.
3. Evaluation of immune effect of tremella fuciformis sporin for injection as adjuvant on influenza vaccine
Adjuvants are immunopotentiators in vaccines, which, together with antigens, are recognized by the immune system and can enhance or mediate the immune response of the body to the antigen. The ideal adjuvant can efficiently stimulate the organism to generate immune response with a small dosage, has no toxic or side effect on the organism, and is suitable for production and application.
3.1 Experimental design
The immunogenicity of 25 μg influenza vaccine (H5N 1) (HA) after intraperitoneal inoculation was examined by periodically detecting the antibody titer generated in serum after influenza vaccine injection in rats; after different adjuvants are added into influenza vaccine (HA) injected into rats, the antibody titer generated in serum is periodically detected, and the influence of different adjuvants and different doses on the corresponding antibodies generated in the influenza vaccine is examined. The effect on the safety of mice after intraperitoneal vaccination was investigated by periodically detecting the body weights of mice in the experimental and control groups after immunization. In addition, the tremella polysaccharide adjuvant for simultaneous immunization of different doses is compared with the original aluminum adjuvant, and the immunogenicity influence of the novel adjuvant on the large influenza vaccine is examined.
3.2 Experimental methods
3.2.1 Experimental materials
Influenza vaccine (H5N 1) (HA); aluminum adjuvant: the Jilin province Asian Tai biological pharmacy is provided by the limited public pharmaceutical research center. Experimental animals: kunming mice (females): supplied by the animal room of the biological pharmaceutical company, yatai, jilin province. Tremella sporophore for injection: is provided by the research and development center of the university of vinca traditional Chinese medicine.
3.2.2 Experimental grouping
TABLE 2 grouping of experiments
| Group of | Grouping | Adjuvant dose | HA content | Number of immunizations |
| 1 | Tremella sporose+vaccine | 500 mug/dose | 25 μg/dose | 10 |
| 2 | Tremella sporose+vaccine | 250 mug/dose | 25 μg/dose | 10 |
| 3 | Tremella sporose+vaccine | 125 mug/dose | 25 μg/dose | 10 |
| 4 | Tremella sporose+half dose vaccine | 250 mug/dose | 12.5 μg/dose | 10 |
| 5 | Normal aluminium adjuvant vaccine immunization | 1000 mug/dose | 25 μg/dose | 10 |
| 6 | Adjuvant blank control | 25 μg/dose | 10 |
3.2.3 immunization programs
Female Kunming mice were immunized on day 0, 14, and each of the above groups was given 1ml by intraperitoneal injection after dilution with physiological saline. Venous blood was collected on day 35 after immunization, serum was isolated and stored at 2-8deg.C.
3.2.4 detection items
The blood coagulation inhibiting antibody titer was measured and the body weight of the mice was monitored periodically every week.
3.3 experimental results
3.3.1 hemagglutination inhibition antibody titres
TABLE 3 results of Tremella polysaccharide hemagglutination inhibition antibody titre experiments for injection
3.3.2 body weight
After all mice were randomly divided into 5 groups, the change in body weight of each group of mice throughout the immunization period was recorded. The body weight of each group of mice tended to increase over time. The normal growth state of mice immunized with influenza vaccine and influenza vaccine combined adjuvant should not be affected.
3.4 analysis of results
The test results showed that mice were immunized twice with a vaccine having an HA content of 25 μg, and serum was taken to detect HA antibody titer, the geometric mean of the antibody titer (GMT) was 1:40 25 μg HA vaccine HAs some immunogenicity. Mice were immunized twice with 1000 μg/dose of aluminum adjuvant, HA content 25 μg vaccine, serum was taken to detect HA antibody titer, the average antibody titer (GMT) was 1:171.48, the positive transfer rate is 100%, and 25 mug HA vaccine and aluminum adjuvant have good immunogenicity.
Different doses of tremella polysaccharide are used as an adjuvant to immunize mice, and antibodies are generated. The Geometric Mean (GMT) of antibody titers of the 500 μg tremella sporose+25 μg HA vaccine group, the 250 μg tremella sporose+25 μg HA vaccine group, the 50 μg tremella sporose+25 μg HA vaccine group, and the 250 μg tremella sporose+12.5 μg HA vaccine group were 1: 80. 1:172.81, 1:105.56, 1:105.56 all have the obvious effect of promoting antibody production. Wherein the promotion effect of the high-dose and low-dose tremella sporulation antibody generation is lower than that of the medium-dose tremella sporulation antibody generation.
The antibody titer of 250 mug tremella sporose plus 25 mug HA vaccine group is similar to the antibody titer of 1000 mug aluminum adjuvant vaccine. The 250 μg tremella sporosaccharide+25 μg HA vaccine group is significantly higher than the 12.5 μg HA vaccine group in the same dose of tremella sporosaccharide, and HAs HA dose dependence. The tremella polysaccharide adjuvant vaccine at each dose has immunogenicity, and 250 mug tremella polysaccharide+25 mug HA vaccine group has optimal immunogenicity. The weight of each group of mice has an increasing trend along with time, no obvious difference is generated from the normal control group, and the vaccine safety is good.
From the pharmacodynamics experiments, the tremella polysaccharide for injection has obvious effect of promoting antibody generation as an immune adjuvant.
Detailed Description
The present invention has been achieved (confirmed) by the following experimental examples.
Example 1 preparation method of Tremella Saccharum sinensis Roxb
1000 g of tremella polysaccharide is taken, purified water is added to prepare 5% (W/V) aqueous solution, and after stirring (500 rpm) is carried out for 8 hours at low temperature (40 ℃), the mixture is centrifuged (160 rpm,10 minutes). The centrifugate was collected and passed through a column of granular activated carbon (80 cmx10 cm) and the eluate was collected. Concentrating to 3L, standing to room temperature, stirring (500 rpm), slowly adding 95% ethanol for 5L, stirring for 20 min, centrifuging (1800 rpm,6 min), collecting centrifugate, stirring (500 rpm), adding 95% ethanol for 30L, stirring for 20 min, centrifuging (2000 rpm,10 min), collecting precipitate, and naturally drying to obtain crude polysaccharide about 100 g. Taking crude polysaccharide powder, adding 2.4 liters of 0.1mol/L HCL aqueous solution, stirring for 5 hours in a water bath at 90 ℃, taking out, standing to room temperature, and dripping 30% NaOH aqueous solution to adjust the pH of the polysaccharide acid hydrolysate to be neutral. Adding 12L of 95% ethanol under stirring (500 rpm), continuing stirring for 2 hours, centrifuging (2500 rpm,10 minutes), collecting precipitate, adding 1000ml of injectable water for dissolving, filtering (0.40 um filter membrane), and lyophilizing to obtain 50 g of injectable tremella polysaccharide.
Example 2 preparation method of Tremella Saccharum injection
Under aseptic condition, adding double distilled water into tremella polysaccharide for injection to prepare 5% aqueous solution, sterilizing with 0.24 μm microporous membrane, and standing for use.
Example 3 preparation method of influenza vaccine injection containing Tremella Saccharum sinensis Roxb as adjuvant
Under the aseptic condition, the tremella polysaccharide and the influenza vaccine are mixed with concentrated stock solution of the influenza vaccine to prepare tremella polysaccharide for injection, wherein the tremella polysaccharide and the influenza vaccine are 10:1, and subpackaging the solution into different doses according to the requirement to prepare the influenza vaccine injection containing tremella polysaccharide for injection as an adjuvant.
Example 4 preparation method of rabies vaccine lyophilized powder containing Tremella sportsman for injection as adjuvant
Mixing with concentrated stock solution of rabies vaccine under aseptic condition to obtain a mixture of 10:1, subpackaging into different doses according to the requirement, and freeze-drying to prepare the rabies vaccine freeze-dried powder injection taking tremella polysaccharide as an adjuvant.
Claims (4)
1. The application of tremella polysaccharide for injection in preparing vaccine adjuvant, wherein the vaccine is H5N1 influenza vaccine, and the preparation method of tremella polysaccharide for injection comprises the following steps:
a. taking tremella polysaccharide, adding purified water, stirring for 8 hours at 40 ℃, centrifuging, collecting centrifugate, passing through a granular activated carbon column, collecting eluent, concentrating to 0.3g tremella polysaccharide per ml, standing to room temperature, slowly adding 1.6 times of 95% ethanol under stirring, continuing stirring for 20 minutes, centrifuging, collecting centrifugate, continuing adding 10 times of 95% ethanol under stirring, continuing stirring for 20 minutes, centrifuging, collecting precipitate, and naturally drying to obtain crude polysaccharide;
b. taking coarse polysaccharide powder, adding 20 times of hydrochloric acid aqueous solution, hydrolyzing in 90 ℃ water bath, taking out after hydrolysis is finished, standing to room temperature, dripping sodium hydroxide aqueous solution to adjust pH to neutrality, adding 0.5 times of 95% ethanol under stirring, continuously stirring for 2 hours, centrifuging, collecting precipitate, adding 10 times of injection water for dissolving, filtering with microporous filter membrane, and freeze-drying to obtain tremella polysaccharide for injection.
2. The use of tremella fuciformis sporin injection according to claim 1 for preparing vaccine adjuvants, which is characterized in that the tremella fuciformis sporum polysaccharide for injection is not less than 75% calculated by mannose; the content of acidic sugar is not less than 10% based on glucuronic acid.
3. The application of the tremella polysaccharide for injection in preparing vaccine adjuvants according to claim 1, which is characterized in that the tremella polysaccharide for injection is prepared by the preparation method of step a, wherein the ratio of tremella polysaccharide to purified water is 1:20g/mL, and the rotation speed was 500rpm during stirring.
4. The use of tremella fuciformis spore sugar for injection according to claim 1 for preparing vaccine adjuvants, which is characterized in that the concentration of hydrochloric acid aqueous solution in the step b of the preparation method of tremella fuciformis spore sugar for injection is 0.1mol/L, and the hydrolysis time is 4-10 hours.
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