CN112316133A - Preparation method and application of tremella polysaccharide immunologic adjuvant for injection - Google Patents
Preparation method and application of tremella polysaccharide immunologic adjuvant for injection Download PDFInfo
- Publication number
- CN112316133A CN112316133A CN202011281304.1A CN202011281304A CN112316133A CN 112316133 A CN112316133 A CN 112316133A CN 202011281304 A CN202011281304 A CN 202011281304A CN 112316133 A CN112316133 A CN 112316133A
- Authority
- CN
- China
- Prior art keywords
- tremella polysaccharide
- injection
- polysaccharide
- vaccine
- tremella
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Abstract
The invention relates to a tremella polysaccharide for injection and an extraction and purification method and application thereof. The tremella polysaccharide monosaccharide for injection comprises mannose in a component ratio: 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 the tremella polysaccharide in water, carrying out activated carbon column chromatography, carrying out alcohol precipitation, hydrolyzing with hydrochloric acid, neutralizing with sodium hydroxide, and carrying out alcohol precipitation again to obtain the tremella polysaccharide for injection. The prepared tremella polysaccharide for injection has the characteristics of small molecular weight, high purity and good water solubility. The injection tremella polysaccharide has good immunity enhancing activity, can be used as an adjuvant, obviously improves the immunity prototypes of various vaccines, and is applied to the preparation of various vaccines.
Description
Technical Field
The invention relates to application of tremella polysaccharide for injection as an immunologic adjuvant in a medical product. Belongs to the technical field of biological pharmacy.
Background
Immunoadjuvants are non-specific immunopotentiators that are effective in enhancing the intensity of or modifying the type of immune response when injected with an antigen or previously into the body. 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 (tuberculosis bacilli, bacille calmette-guerin), brevibacterium, bordetella pertussis, endotoxins, bacterial extracts (muramyl dipeptide), etc.; synthetic adjuvants such as synthetic double-stranded polynucleotides (double-stranded polyadenylic acid, uridylic acid) and the like; in recent years, polysaccharide components derived from plants or microorganisms have also become components for the development of novel adjuvants, such as laminarin. The biological effects of immunological adjuvants include: the physical properties of the antigen are changed, so that the antigen substance can be slowly released, and the action time of the antigen is prolonged; after the adjuvant adsorbs the antigen, the surface area of the antigen is increased, so that the antigen is easy to be phagocytized by macrophages; the adjuvant can stimulate phagocyte to treat antigen; the adjuvant can promote the contact between lymphocytes and enhance the effect of helper T cells; can stimulate the division of sensitized lymphocytes and the production of antibodies by plasma cells. Therefore, the immune adjuvant can make non-immunogenic substance become effective immunogen. The screening and development of new immunologic adjuvants are more and more attracting attention.
Tremella (Tremella fuciformis Berk) also called Tremella, is a dried fruiting body of Tremella fuciformis of Tremellaceae of Basidiomycetes, is a common health-care medicine, has the efficacy of nourishing yin and moistening lung, and has been in medicinal history for hundreds of years. The main chemical components of the composition are polysaccharide compounds. Also contains lipids, proteins (enzymes, proteins, amino acids), inorganic salts, vitamin B group, etc. The tremella polysaccharide is mainly acidic heteropolysaccharide, and is composed of a series of acidic heteropolysaccharides with molecular weights of tens of thousands to hundreds of thousands; the component sugar is 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 spores, reducing blood sugar, resisting ulcer, enhancing immunity, resisting tumors, resisting toxic and side effects of radiotherapy and chemotherapy and the like; acute toxicity and long-term toxicity research show that the polysaccharide has no obvious toxic or side effect when being taken orally or injected. The tremella polysaccharide is easy to dissolve in hot water, slightly soluble in low-concentration ethanol, insoluble in high-concentration ethanol and other organic solvents, and the water solution is transparent and viscous. The results of the in vivo absorption, distribution and metabolism research of the tremella polysaccharide show that the tremella polysaccharide has extremely low oral absorption utilization rate (more than 1 percent), and the tremella polysaccharide has obviously higher effects of improving the immunologic function, increasing the white blood and resisting the cancers when being injected intramuscularly and administrated intravenously than when being orally taken. Because of low oral bioavailability and unobvious effect, the injection route should be selected when the medicine is used as a new medicine for research and development. However, the tremella polysaccharide has a large molecular weight and a complex structure, and the aqueous solution of the tremella polysaccharide has certain viscosity, so that the application of the tremella polysaccharide as an injection is influenced, and the evaluation and control of the quality and the large-scale production of the tremella polysaccharide are also influenced. Therefore, the degradation is carried out, the fragments with relatively simple structure, small molecular weight and good water solubility are prepared, and active sites with stronger action are obtained by screening, thereby having important significance for development and application of the fragments.
Before the invention is completed, the application of the tremella polysaccharide for injection as the immunologic adjuvant is not found.
Disclosure of Invention
The invention aims to provide an immunologic adjuvant which is used by matching natural polysaccharide with influenza vaccine and can enhance the immunologic function of organisms.
The invention prepares the tremella polysaccharide for injection with smaller molecular weight, high purity and high yield after separating, purifying and degrading the tremella polysaccharide. The tremella polysaccharide for injection has obvious effect on improving the immunity 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 immunopotentiators and vaccines 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 for use, and the natural tremella polysaccharide has a synergistic effect on the effect, can enhance the activity of the influenza vaccine, and obtains an ideal immune effect.
Specifically, the influenza vaccine adopting the immune adjuvant of the invention has obviously improved antibody titer of immune expression compared with the influenza vaccine without the immune adjuvant in the whole immune cycle.
The invention also provides a preparation method of the 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 until each ml contains 0.3g of tremella polysaccharide, standing to room temperature, slowly adding 1.6 times of 95% ethanol under stirring, continuing to stir for 20 minutes, centrifuging, collecting centrifugate, continuing to add 10 times of 95% ethanol under stirring, continuing to stir for 20 minutes, centrifuging, collecting precipitate, and naturally drying to obtain crude polysaccharide;
b. adding 20 times of hydrochloric acid aqueous solution into crude polysaccharide powder, hydrolyzing in a water bath at 90 ℃, taking out after hydrolysis, standing to room temperature, dropwise adding sodium hydroxide aqueous solution to adjust the pH to be neutral, adding 0.5 times of 95% ethanol under stirring, continuously stirring for 2 hours, centrifuging, collecting precipitate, adding 10 times of injection water to dissolve, filtering by using a microporous filter membrane, and freeze-drying to obtain the tremella polysaccharide for injection.
The preparation method of the tremella polysaccharide for injection is characterized in that the ratio of the tremella polysaccharide to the distilled water in the step a is 1: 20g/mL, and the rotation speed during stirring was 500 rpm.
The method for preparing the tremella polysaccharide for injection as described above, wherein the concentration of the aqueous hydrochloric acid solution in the step b is 0.1mol/L, and the hydrolysis time is 4 to 10 hours.
In order to realize the invention content, 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 dissolving, taking 1ml, and measuring according to appendix IX S of first part of Chinese pharmacopoeia 2000 edition, wherein the content of the tremella polysaccharide for injection is not less than 75% in terms of mannose.
1.2 uronic acid content determination- -m-hydroxybiphenyl method
1.2.1 preparation of control solutions
Precisely weighing 5mg of glucuronic acid reference substance dried at 105 deg.C to constant weight, placing in a 100ml volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain the final product (each 1ml contains 50 μ g of glucuronic acid).
1.2.2 preparation of Standard Curve
Precisely absorbing 0ml, 0.1ml, 0.2ml, 0.3ml and 0.4ml of reference substance solution, respectively adding water to supplement the volume to 0.4ml, uniformly mixing, adding 2.4ml of 0.0125mol/L sodium tetraborate-sulfuric acid solution (0.475 g of sodium tetraborate is taken and 100ml of concentrated sulfuric acid is added for dissolving) in an ice-water bath, uniformly mixing, heating for 5 minutes in a boiling water bath, taking out and rapidly cooling to room temperature, adding 40 mu L of 0.15% m-hydroxybiphenyl, immediately mixing uniformly, taking a 0 tube as a blank, measuring the absorbance within 20 minutes at the wavelength of 525nm according to the absorbance corresponding to the mu g of glucuronic acid by a spectrophotometric method (an appendix V A of 2010 edition in China), and calculating a regression equation.
1.2.3 assay
Precisely weighing 3350mg of a test sample, placing the test sample into a 100ml measuring flask, adding water to dilute the test sample to a scale, shaking the test sample to obtain a test sample solution, precisely sucking 0.2ml of the test sample solution, adding water to supplement the volume to 0.4ml, measuring the absorbance according to a method operation from the beginning of adding the test sample solution in an ice water bath under a standard curve preparation term, and calculating the content of the acidic sugar by using a regression equation.
1.2.4 measurement of uronic acid content
TABLE 1 absorption of standard uronic acid solution
From the above data, the regression equation was determined to be 0.0309X +0.0315 for Y and 0.9985 for R, using the uronic acid content as the abscissa and the absorption value as the ordinate.
According to the experimental result, the acid sugar content of the tremella polysaccharide for injection is not less than 10 percent calculated by glucuronic acid.
1.3 determination of proteins
100mg of tremella polysaccharide for injection is taken and dissolved in 50ml of water, 1ml is taken, and the measurement is carried out according to the appendix IX S of the first part of the Chinese pharmacopoeia 2000 edition, which meets the regulation.
2. Sugar analysis of composition of tremella polysaccharide for injection
2.1 Standard monosaccharide solution preparation
Taking a proper amount of Fuc (fucose), Rha (rhamnose), Ara (arabinose), Xyl (xylose), Man (mannose), Gal (galactose), Glu (glucose) and GalA (galacturonic acid) GlcA (glucuronic acid) monosaccharide reference substances, and respectively adding distilled water to prepare 2mmol/L monosaccharide standard solutions.
2.2 preparation of hydrolyzed solution of Tremella polysaccharide for injection
20mg of a tremella polysaccharide sample for injection is accurately weighed into a test tube with a stopper, 2ml of 2mol/L trifluoroacetic acid is added, the test tube is sealed, hydrolyzed at 100 ℃ for 2 hours, and dried by blowing with nitrogen. The residue was dried with 1ml of methanol and repeated 4 times to remove trifluoroacetic acid for derivatization.
2.3 preparation of the 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 solutions in 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 and cooling for 5min, neutralizing with 5mL0.3mol/L HCl, adding isometric chloroform for extraction, fully shaking, taking a water layer, and repeating for 3 times. Mixing 300 μ L of each PMP-derived monosaccharide, and filtering with 0.45 μm microporous membrane.
2.4HPLC analysis
Shimadzu LC-2010 Automation liquid chromatograph, ZORBAX eclipseXDB-C18 analytical column (Agilent) (phi 4.6 mm. times.150 mm); column temperature: 40 ℃; mobile phase: a: phosphate buffer (KH)2PO4-NaOH, PH 6.8): acetonitrile (85: 15, V/V); b: phosphate buffer (KH)2PO4-NaOH, PH 6.8): acetonitrile (60:40, V/V); flow rate: 0.9 mL/min; detection wavelength: 250 nm. And (3) gradient elution is started for 0-10 minutes, the proportion of the mobile phase B is increased from 0 to 8 percent for 10-30 minutes, the proportion of the mobile phase B is increased from 8 percent to 20 percent again, the mobile phase B is kept for 10 minutes, and the process is finished.
The analysis result of the PMP derivatization-HPLC method of the tremella polysaccharide for injection shows that the tremella polysaccharide for injection mainly comprises mannose, glucuronic acid, glucose, xylose and fucose; the mol ratio is as follows: 66.2: 10.75: 1.12: 19.20: 2.82.
2.4 determination of the molecular weight distribution
The tremella polysaccharide sample for injection is added with mobile phase to prepare 5mg/mL solution, and the determination is carried out according to Chinese pharmacopoeia. The molecular weight distribution range is 2000-50000Da, and the weight average molecular weight is 5900 Da.
3. Evaluation of immune effect of influenza vaccine by using injection tremella polysaccharide as adjuvant
Adjuvants are immunopotentiators in vaccines that, together with an antigen, are processed by the immune system to recognize and enhance or modulate the body's immune response to the antigen. The ideal adjuvant can efficiently stimulate the organism to generate immune response with less dosage, has no toxic or side effect on the organism, and is suitable for production and application.
3.1 design of the experiment
Periodically detecting the antibody titer generated in serum after the rat is injected with the influenza vaccine, and inspecting the immunogenicity of 25 mu g of influenza vaccine (H5N1) (HA) after the intraperitoneal inoculation; after the influenza vaccine (HA) is injected into a rat and different adjuvants are added, the antibody titer generated in serum is regularly detected, and the influence of different adjuvants and different doses on the generation of corresponding antibodies of the influenza vaccine is inspected. The influence of the intraperitoneal vaccination on the safety of the mice is examined by regularly detecting the weights of the mice of the experimental group and the control group after immunization. In addition, the tremella polysaccharide adjuvant for injection with different dosages of immunity is compared with the original aluminum adjuvant, and the influence of the novel adjuvant on the immunogenicity of the large influenza vaccine is investigated.
3.2 Experimental methods
3.2.1 Experimental materials
Influenza vaccine (H5N1) (HA); aluminum adjuvant: provided by the drug research center of the Biopharmaceutical Co., Ltd, Adita, Jilin province. Experimental animals: kunming mouse (female): available from the animal house of the Biopharmaceutical Co., Ltd, Adita, Jilin province. Tremella polysaccharide for injection: provided by research and development center of Changchun Chinese medicine university.
3.2.2 Experimental groups
TABLE 2 Experimental groups
| Group of | Grouping | Adjuvant dose | HA content | Number of immunizations |
| 1 | Tremella polysaccharide vaccine | 500 mu g/dose | 25 mu g/dose | 10 |
| 2 | Tremella polysaccharide vaccine | 250 mu g/dose | 25 mu g/dose | 10 |
| 3 | Tremella polysaccharide vaccine | 125 mu g/dose | 25 mu g/dose | 10 |
| 4 | Tremella polysaccharide and half-dose vaccine | 250 mu g/dose | 12.5 μ g/dose | 10 |
| 5 | Normal aluminum adjuvant vaccine immunization | 1000 mug/dose | 25 mu g/dose | 10 |
| 6 | Adjuvant blank control | 25 mu g/dose | 10 |
3.2.3 immunization procedure
Female Kunming mice were immunized on days 0 and 14, and each group was diluted with physiological saline and injected with 1ml of the solution per abdominal cavity. Venous blood was collected on day 35 after immunization, serum was separated, and stored at 2-8 ℃.
3.2.4 test items
Hemagglutination inhibition antibody titers were measured and mouse body weights were monitored periodically weekly.
3.3 results of the experiment
3.3.1 hemagglutination inhibition antibody Titers
TABLE 3 result of hemagglutination inhibition antibody titer of Tremella polysaccharide for injection
3.3.2 body weight
After all mice were randomized into 5 groups, the weight change of each group was recorded throughout the immunization. The body weight of each group of mice increased with time. The normal growth state of the mice immunized by the influenza vaccine and the influenza vaccine combined adjuvant is not influenced.
3.4 analysis of results
Test results show that mice are immunized twice by using vaccine with the HA content of 25 mu g, serum is taken to detect the HA antibody titer, and the geometric mean value (GMT) of the antibody titer is 1: 40, 25 mug HA vaccine HAs certain immunogenicity. Mice were immunized twice with 1000 μ g/dose of aluminum adjuvant and with a HA content of 25 μ g vaccine, sera were taken to detect HA antibody titers, the mean antibody titer (GMT) was 1: 171.48, the positive conversion rate is 100%, and 25 mug HA vaccine plus aluminum adjuvant HAs good immunogenicity.
The mice immunized by using different doses of tremella polysaccharide as an adjuvant all produce antibodies. The geometric mean values (GMT) of antibody titers of the 500. mu.g of tremella polysaccharide + 25. mu.g of HA vaccine group, 250. mu.g of tremella polysaccharide + 25. mu.g of HA vaccine group, 50. mu.g of tremella polysaccharide + 25. mu.g of HA vaccine group, and 250. mu.g of tremella polysaccharide + 12.5. mu.g of HA vaccine group were 1: 80. 1: 172.81, 1: 105.56, 1: 105.56, all have the function of obviously promoting the generation of antibodies. Wherein the high-dose and low-dose tremellose group has a lower antibody production promoting effect than the medium-dose tremellose group.
The antibody titer of the 250 ug tremella polysaccharide +25 ug HA vaccine group was similar to the titer of 1000 ug aluminum adjuvant vaccine antibody. Compared with the 250 mu g of tremella polysaccharide and 25 mu g of HA vaccine group and the 250 mu g of tremella polysaccharide and 12.5 mu g of HA vaccine group, under the condition of the same dose of tremella polysaccharide, the 25 mu g of HA vaccine group is obviously higher than the 12.5 mu g of HA vaccine group, and HAs HA dose dependence. Each dose of the tremella polysaccharide adjuvant vaccine was immunogenic, and the 250 μ g of the tremella polysaccharide +25 μ g of the ha vaccine group was most immunogenic. The weight of each group of mice increases along with time, and the vaccine has no obvious difference with a normal control group and has good safety.
From the pharmacodynamic experiments, the tremella polysaccharide for injection has obvious effect of promoting antibody generation as an immunologic adjuvant.
Detailed Description
The present invention was achieved (confirmed) by the following experimental examples.
EXAMPLE 1 preparation of injectable Tremella saccharide
1000 g of tremella polysaccharide was added with purified water to prepare a 5% (W/V) aqueous solution, stirred at low temperature (40 ℃) for 8 hours (500rpm), and centrifuged (1600rpm, 10 minutes). The centrate was collected and passed through a granular activated carbon column (80cmx10cm) to collect the eluate. Concentrating to 3L, standing at room temperature, slowly adding 95% ethanol 5L under stirring (500rpm), stirring for 20 min, centrifuging (1800rpm, 6 min), collecting centrifugate, adding 95% ethanol 30L under stirring (500rpm), stirring for 20 min, centrifuging (2000rpm, 10 min), collecting precipitate, and naturally drying to obtain crude polysaccharide 100 g. Adding 0.1mol/L HCL aqueous solution 2.4L into crude polysaccharide powder, stirring in 90 deg.C water bath for 5 hr, taking out, standing to room temperature, and adding 30% NaOH aqueous solution dropwise to adjust pH of polysaccharide acid hydrolysis solution to neutrality. Adding 95% ethanol 12L under stirring (500rpm), stirring for 2 hr, centrifuging (2500rpm, 10 min), collecting precipitate, adding 1000ml water for injection to dissolve, filtering (0.40um filter membrane), and freeze drying to obtain 50 g Tremella polysaccharide for injection.
EXAMPLE 2 preparation of an injectable Tremella polysaccharide injection
Adding double distilled water into Tremella polysaccharide for injection under aseptic condition to obtain 5% water solution, sterilizing with 0.24 μm microporous membrane, and standing.
EXAMPLE 3 preparation of influenza vaccine injection containing Tremella saccharide for injection as adjuvant
Under the aseptic condition, the mixture is mixed with concentrated preparation liquid of influenza vaccine to prepare the injection tremella polysaccharide and influenza vaccine which are 10: the solution of 1 is subpackaged into different dosages according to the requirement to prepare the influenza vaccine injection with the tremella polysaccharide for injection as the adjuvant.
EXAMPLE 4 preparation of rabies vaccine lyophilized powder containing Tremella saccharide for injection as adjuvant
Under aseptic conditions, the mixture with the concentrated solution of the rabies vaccine is 10: the solution of 1 is subpackaged into different dosages according to the requirement, and is frozen and dried to prepare the rabies vaccine freeze-dried powder injection taking the tremella polysaccharide as the adjuvant.
Claims (9)
1. The tremella polysaccharide for injection is characterized in that monosaccharide composition and molar ratio are mannose: glucuronic acid: glucose: xylose: fucose ═ 66.2: 10.75: 1.12: 19.20: 2.82; the molecular weight distribution range is 2000-50000Da, and the weight average molecular weight is 5900 Da.
2. The tremella polysaccharide for injection according to claim 1, wherein the polysaccharide content is not less than 75% in terms of mannose; contains acidic sugar not less than 10% calculated by glucuronic acid.
3. The process for producing a tremella polysaccharide for injection according to claim 1, comprising the steps of:
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 until each ml contains 0.3g of tremella polysaccharide, standing to room temperature, slowly adding 1.6 times of 95% ethanol under stirring, continuing to stir for 20 minutes, centrifuging, collecting centrifugate, continuing to add 10 times of 95% ethanol under stirring, continuing to stir for 20 minutes, centrifuging, collecting precipitate, and naturally drying to obtain crude polysaccharide;
b. adding 20 times of hydrochloric acid aqueous solution into crude polysaccharide powder, hydrolyzing in a water bath at 90 ℃, taking out after hydrolysis, standing to room temperature, dropwise adding sodium hydroxide aqueous solution to adjust the pH to be neutral, adding 0.5 times of 95% ethanol under stirring, continuously stirring for 2 hours, centrifuging, collecting precipitate, adding 10 times of injection water to dissolve, filtering by using a microporous filter membrane, and freeze-drying to obtain the tremella polysaccharide for injection.
4. The method for preparing tremella polysaccharide for injection according to claim 3, wherein the ratio of the tremella polysaccharide to the distilled water in the step a is 1: 20g/mL, and the rotation speed during stirring was 500 rpm.
5. The method of claim 3, wherein the aqueous hydrochloric acid solution of step b has a concentration of 0.1mol/L and the hydrolysis time is 4 to 10 hours.
6. Use of a tremella polysaccharide for injection according to claim 1, in the preparation of an immunopotentiator.
7. Use of a tremella polysaccharide for injection according to claim 1, in the preparation of a vaccine adjuvant.
8. The vaccine adjuvant according to claim 7, characterized in that: the vaccine is an H5N1 influenza vaccine.
9. The vaccine adjuvant according to claim 7, characterized in that: the vaccine is a rabies vaccine.
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