CN102942614B - Preparation method and application of high-purity polysaccharopeptide - Google Patents
Preparation method and application of high-purity polysaccharopeptide Download PDFInfo
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Abstract
The invention relates to a preparation method and application of high-purity polysaccharopeptide. The preparation method comprises extracting and purifying polysaccharopeptide coarse powder and obtaining the polysaccharopeptide with high polysaccharide content and high protein level. The polysaccharopeptide can improve antibody levels of chickens, accordingly improve immunity of the chickens, be used for preparing human medicines or beast medicines with immunity or/and physiological accommodation activity and particularly serve as vaccine immunopotentiator of beasts and birds.
Description
Technical field
The present invention relates to for animals or human drugs production technical field, particularly a kind of preparation method of high purity polysaccharide-peptide and pharmaceutical applications.
Background technology
Bacterium and viral infectious are the formidable enemies that harm livestock industry is produced, and along with livestock industry is increasingly intensive, the prevention of transmissible disease seems more important.For virus disease, the pharmacological agent that How to choose is suitable is the problem that makes culturist's headache always, and the pharmacological agent of virus disease is simultaneously difficult to again prove effective, and must carry out immunization and be prevented.But in process of production, the immuning failure phenomenon of animal often occurs again, as vaccine transport, preserve improper, immune programme for children is unreasonable, inoculation method is wrong, dosage is inaccurate as too much in dosage of inoculation or very few etc., causes animal immune unsuccessfully, breaks out communicable disease.At present; in livestock and poultry breeding industry, the epidemic disease of widespread use (bacterium) seedling adjuvant is as aluminium glue salt solution, oily adjuvant etc.; because these products exist certain defect at aspects such as activity, viscosity, stability, make some epidemic disease (bacterium) seedling exist the problems such as protection ratio is low, immunoprophylaxis poor effect.Therefore; for improving the immune protective efficiency of plague of livestock and fowl (bacterium) seedling; extend immune duration; adapt to the development of purified vaccine, subunit vaccine and synthetic peptide vaccine, the important directions that development can biological metabolism, toxicological harmless, active strong immunomodulator or toughener have become problem in the urgent need to address and the research of current adjuvant as epidemic disease (bacterium) seedling adjuvant.
Immunostimulant can improve animal autoimmunity, removes animal immune and suppresses, and strengthens the resistibility of animal to bacterium and viral infectious.Therefore the research of immunostimulant in animal produces causes the great attention of Chinese scholars day by day with application.Immunostimulant also claims immunological adjuvant, by the different modes of action, as strengthened macrophage activity, the immunogenicity of enhancement antigen material and stability, synthetic and the secretion of enhancing antibody etc., thus enhancing animal body is non-specific and the material of specific immunity.Along with the raising of scientific and technical progress and veterinary science technology, select a kind of feature desirable immunostimulant definite, efficient, that stable, toxic side effect is little to be main path and the means of resisting disease future.
At present, the immunostimulant kind of having studied is very many, according to its source, can be divided into product and the large class of synthetics two, further can be subdivided into physiological active products, microbe-derived material, synthetic chemistry thing, herbal medicine etc.Belong to having of microorganism and component thereof: tubercule bacillus, bifidus bacillus and zymosan, bacteria lipopolysaccharide, mycobacterium cell walls and subunit's Muramyl dipeptide thereof etc.; What belong to physiological active products has thymic factor, cytokine, a transfer factor etc.; Belong to the multiple ester class that has LEVAMISOLE HCL, polynucleotide, surface active molecules and synthetic of chemosynthesis; Belong to herbal medicine if the Radix Astragali, Radix Et Caulis Acanthopanacis Senticosi etc. and Chinese herbal medicine extract are as polysaccharide, alkaloid, organic acid and multiple glucoside material, as astragalus polysaccharides, Berberine, ginsenoside etc.
In current domestic and foreign literature, research about polysaccharide-peptide is a lot, extraction and application for polysaccharide-peptide also have many reports, but lack a kind of extraction and purification process of high purity polysaccharide-peptide, also do not set it as the report of veterinary vaccines immunostimulant.
Summary of the invention
In view of the deficiencies in the prior art, order of the present invention is to study by the extraction and purification process to polysaccharide-peptide and inside and outside activity, and a kind of preparation method and pharmaceutical applications of high purity polysaccharide-peptide is provided.
The object of the present invention is achieved like this:
A preparation method for high purity polysaccharide-peptide, comprises the steps:
(1) get polysaccharide-peptide meal, by material-water ratio, 1:35-60 adds 5-8%(w/v) NaHCO
3solution, be heated to 70-72 DEG C, insulation lixiviate 1.5-2.5h, 5000-6000rpm is centrifugal, get supernatant liquor, 80 DEG C of following heating concentrate 8-10 doubly, then add the dehydrated alcohol of 5-7 times of volume, leave standstill after 18-36h under the environment of 1-5 DEG C, 4000-5000rpm is centrifugal, abandon supernatant liquor, precipitation is successively washed 1-3 time respectively with 95% ethanol, dehydrated alcohol, acetone, ether, dries to obtain polysaccharide-peptide work in-process;
(2) get polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.2-0.3 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 15-30min, 4000-5000rpm is centrifugal, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.20-0.30 μ m dialysis membrane, flowing water dialysis 36-60h, distill water dialysis 18-30h, after 60 DEG C of following heating are concentrated, add the dehydrated alcohol of 3-5 times of volume fully to vibrate leaves standstill after 18-36h under the environment of 1-5 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, washing with acetone, dry to obtain polysaccharide-peptide finished product.
Preferably, the preparation method of described high purity polysaccharide-peptide, wherein step (1) is: get polysaccharide-peptide meal, by material-water ratio, 1:45-50 adds 6%(w/v) NaHCO
3solution, be heated to 70-72 DEG C, insulation lixiviate 1.5-2.5h, 5000-6000rpm is centrifugal, get supernatant liquor, 80 DEG C of following heating concentrate 9 times, then add the dehydrated alcohol of 6 times of volumes, leave standstill after 18-36h under the environment of 1-3 DEG C, 4000-5000rpm is centrifugal, abandon supernatant liquor, precipitation is successively washed respectively 2 times with 95% ethanol, dehydrated alcohol, acetone, ether, dries to obtain polysaccharide-peptide work in-process.
Preferably, the preparation method of described high purity polysaccharide-peptide, wherein step (2) is: get polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.2-0.3 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 15-30min, 4000-5000rpm is centrifugal, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.22 μ m dialysis membrane, flowing water dialysis 40-48h, distill water dialysis 18-30h, after 60 DEG C of following heating are concentrated, add the dehydrated alcohol of 4 times of volumes fully to vibrate leaves standstill after 24-30h under the environment of 1-3 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, acetone washs respectively 2 times, dry to obtain polysaccharide-peptide finished product.
Study by vitro tests, polysaccharide-peptide prepared by the present invention can improve the antibody horizontal of chicken, thereby can improve chicken group's immunizing power.Therefore, second object of the present invention is to provide polysaccharide-peptide prepared by aforesaid method to have immunity or/and the application in the medicine of physiological regulation activity in preparation.
Preferably, described medicine is veterinary drug, human drugs.
Further preferably, described veterinary drug is veterinary vaccines immunostimulant.
Compared with prior art, the high purity polysaccharide-peptide preparation method tool the present invention relates to has the following advantages and marked improvement:
(1) polysaccharide and protein content are high.After testing, its polysaccharide content reaches more than 61% the polysaccharide-peptide that embodiment 1-3 prepares, and protein content reaches more than 9.7%.Compare polysaccharide-peptide meal, polysaccharide content and protein content significantly improve (in Table 1-3).
(2) polysaccharide-peptide can improve chicken group's immunizing power.From the table 4 of embodiment 4, for immune chicken, different treatment has certain improvement effect to the sickness rate of chicken, and control group chicken sickness rate is 27.2%, and the sickness rate of polysaccharide-peptide drinking-water treatment group chicken is 4.5%, has reduced by 22.7%.This explanation polysaccharide-peptide can improve the antibody horizontal of chicken, thereby improves the immune level of chicken, reduces sickness rate.And for the mortality ratio of chicken after morbidity, polysaccharide-peptide has reached comparatively significant improvement effect.
Embodiment
Be below concrete preparation example of the present invention, technical scheme of the present invention is done to further description, but protection scope of the present invention is not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
The extraction purifying of embodiment 1 polysaccharide-peptide
Get polysaccharide-peptide meal 1kg, by material-water ratio, 1:48 adds 6%(w/v) NaHCO
3the aqueous solution, is heated to 70-72 DEG C, insulation lixiviate 2h, 5500rpm is centrifugal, get supernatant liquor, be heated to concentrated 9 times of 75 DEG C of insulations, then add the dehydrated alcohol of 6 times of volumes, under the environment of 1-3 DEG C, leave standstill after 24h, 4500rpm is centrifugal, abandons supernatant liquor, and precipitation is successively washed respectively (4000rpm 2 times with 95% ethanol, dehydrated alcohol, acetone, ether, 5min), dry to obtain 492.5g polysaccharide-peptide work in-process.
Get 500g polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.25 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 20min, the centrifugal 10min of 4500rpm, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.22 μ m dialysis membrane, flowing water dialysis 45h, distill water dialysis 24h, be heated to 58 DEG C concentrated after, add the dehydrated alcohol of 4 times of volumes fully to vibrate leaves standstill after 24h under the environment of 1-3 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, acetone washs respectively (4000rpm 2 times, 5min), dry to obtain 141.3g polysaccharide-peptide finished product.
Detected and found by the polysaccharide to sample and protein content, can make the polysaccharide content of polysaccharide-peptide improve 43.07% through purifying, protein content improves 6.10%(in table 1).
The content of polysaccharide and albumen in table 1 polysaccharide-peptide leaching process
The extraction purifying of embodiment 2 polysaccharide-peptides
Get polysaccharide-peptide meal 1kg, by material-water ratio, 1:40 adds 8%(w/v) NaHCO
3the aqueous solution, is heated to 70-72 DEG C, insulation lixiviate 1.5h, 6000rpm is centrifugal, get supernatant liquor, be heated to concentrated 8 times of 78 DEG C of insulations, then add the dehydrated alcohol of 5 times of volumes, under the environment of 1-3 DEG C, leave standstill after 20h, 5000rpm is centrifugal, abandons supernatant liquor, and precipitation is successively washed respectively (4000rpm 2 times with 95% ethanol, dehydrated alcohol, acetone, ether, 5min), dry to obtain 489.8g polysaccharide-peptide work in-process.
Get 500g polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.2 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 15min, the centrifugal 10min of 4500rpm, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.22 μ m dialysis membrane, flowing water dialysis 50h, distill water dialysis 30h, be heated to 58 DEG C concentrated after, add the dehydrated alcohol of 5 times of volumes fully to vibrate leaves standstill after 24h under the environment of 1-3 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, acetone washs respectively (4000rpm 2 times, 5min), dry to obtain 142.4g polysaccharide-peptide finished product.
Detected and found by the polysaccharide to sample and protein content, can make the polysaccharide content of polysaccharide-peptide improve 40.80% through purifying, protein content improves 5.64%(in table 2).
The content of polysaccharide and albumen in table 2 polysaccharide-peptide leaching process
The extraction purifying of embodiment 3 polysaccharide-peptides
Get polysaccharide-peptide meal 1kg, by material-water ratio, 1:55 adds 7%(w/v) NaHCO
3the aqueous solution, is heated to 70-72 DEG C, insulation lixiviate 2.5h, 5000rpm is centrifugal, get supernatant liquor, be heated to concentrated 10 times of 78 DEG C of insulations, then add the dehydrated alcohol of 7 times of volumes, under the environment of 1-3 DEG C, leave standstill after 30h, 4500rpm is centrifugal, abandons supernatant liquor, and precipitation is successively washed respectively (4000rpm 2 times with 95% ethanol, dehydrated alcohol, acetone, ether, 5min), dry to obtain 485.0g polysaccharide-peptide work in-process.
Get 500g polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.3 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 25min, the centrifugal 10min of 5000rpm, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.22 μ m dialysis membrane, flowing water dialysis 40h, distill water dialysis 20h, be heated to 58 DEG C concentrated after, add the dehydrated alcohol of 5 times of volumes fully to vibrate leaves standstill after 28h under the environment of 1-3 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, acetone washs respectively (4000rpm 2 times, 5min), dry to obtain 137.5g polysaccharide-peptide finished product.
Detected and found by the polysaccharide to sample and protein content, can make the polysaccharide content of polysaccharide-peptide improve 39.26% through purifying, protein content improves 5.75%(in table 3).
The content of polysaccharide and albumen in table 3 polysaccharide-peptide leaching process
Embodiment 4 polysaccharide-peptide Virulent Newcastle Disease Virus are attacked the protection test research of chicken
Take polysaccharide-peptide 1g prepared by embodiment 1, be dissolved in 100m1 physiological saline, 0.45 μ m membrane filtration degerming, obtains 1% polysaccharide-peptide injection liquid.Select 1 age in days SPF healthy chick, be divided at random 3 processing, 24 chickens of each processing, 3 repetitions of each processing.
Process 1: control group, in the time of chicken 7 age in days, the weak malicious seedling of immune newcastle disease clone-30, by the dosage collunarium of 0.05ml/ plumage, eye droppings immunity.Without polysaccharide-peptide.
Process 2: polysaccharide-peptide group, by 100mg/kg body weight inject, every 3d intramuscular injection once, to off-test.In the time of chicken 7 age in days, the weak malicious seedling of immune newcastle disease clone-30, by the dosage collunarium of 0.05ml/ plumage, eye droppings immunity.
Process 3: polysaccharide-peptide group, dosage and usage are same processes 2, but does not carry out immunity.
Immunity: test 7d, the weak malicious seedling of ND is pressed 0.05ml/ plumage collunarium, eye droppings immunity, immunity start injection polysaccharide on the same day, every 3 days are once.
Blood sampling: respectively at 7d, 14d wing venous blood collection after immunity, prepare serum.With Microhemagglutination inhibition method mensuration newcastle disease HI antibody horizontal.
Attack poison: when test chicken 22 age in days, proceed to strong malicious chamber, attacking Virulent Newcastle Disease Virus dosage is 105ELD
50, occur that with chicken the newcastle disease classical symptoms such as listless, expiratory dyspnea, salivation, diarrhea, neck distortion are judged as morbidity.Every day entry morbidity number of elements, dead number of elements, calculates sickness rate, mortality ratio.The results are shown in Table 4.
Table 4 polysaccharide-peptide is attacked the impact of malicious protection ratio on chicken
From table 4; for not immune chicken, when Virulent Newcastle Disease Virus is attacked, the sickness rate of chicken is up to 84.5%, and death rate of the onset is 100%; polysaccharide-peptide does not have effective any provide protection and therapeutic action to chicken; this proving again newcastle disease is a kind of deadly infectious disease, and its clinical therapeutic efficacy is very poor, in fact; in the market; also do not have a kind of effective medicine can treat the newcastle disease of chicken, for newcastle disease, can only carry out preventive vaccination.
For immune chicken, different treatment has certain improvement effect to the sickness rate of chicken, and control group chicken sickness rate is 27.2%, and the sickness rate of polysaccharide-peptide drinking-water treatment group chicken is 4.5%, has reduced by 22.7%.This explanation polysaccharide-peptide can improve the antibody horizontal of chicken, thereby improves the immune level of chicken, reduces sickness rate.And for the mortality ratio of chicken after morbidity, polysaccharide-peptide has reached comparatively significant improvement effect.
Claims (1)
1. a preparation method for high purity polysaccharide-peptide, comprises the steps:
(1) get polysaccharide-peptide meal, by material-water ratio, 1:45-50 adds 6%(w/v) NaHCO
3solution, be heated to 70-72 DEG C, insulation lixiviate 1.5-2.5h, 5000-6000rpm is centrifugal, get supernatant liquor, 80 DEG C of following heating concentrate 9 times, then add the dehydrated alcohol of 6 times of volumes, leave standstill after 18-36h under the environment of 1-3 DEG C, 4000-5000rpm is centrifugal, abandon supernatant liquor, precipitation is successively washed respectively 2 times with 95% ethanol, dehydrated alcohol, acetone, ether, dries to obtain polysaccharide-peptide work in-process;
(2) get polysaccharide-peptide work in-process, adding distilled water dissolves, add again the organic solvent of 0.2-0.3 times of volume, described organic solvent is chloroform and the propyl carbinol of volume ratio=5:1, thermal agitation mixes 15-30min, 4000-5000rpm is centrifugal, get supernatant liquor, repeat 2 times, merging supernatant liquor packs in 0.22 μ m dialysis membrane, flowing water dialysis 40-48h, distill water dialysis 18-30h, after 60 DEG C of following heating are concentrated, add the dehydrated alcohol of 4 times of volumes fully to vibrate leaves standstill after 24-30h under the environment of 1-3 DEG C, abandon supernatant, precipitation is successively with dehydrated alcohol, acetone washs respectively 2 times, dry to obtain polysaccharide-peptide finished product.
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| CN103408645B (en) * | 2013-08-27 | 2017-10-13 | 中科医药行业生产力促进中心有限公司 | A kind of method prepared by polysaccharide-peptide |
| CN104450826B (en) * | 2014-12-31 | 2017-11-17 | 华南师范大学 | A kind of coriolan extract and preparation method and application |
| CN110713513B (en) * | 2019-10-16 | 2021-04-06 | 江苏神华药业有限公司 | Method for extracting polysaccharide peptide from corious versicolor cells |
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