CN103599071B - A kind of preparation method of polyinosinic-polycytacidic acidic dry powder - Google Patents
A kind of preparation method of polyinosinic-polycytacidic acidic dry powder Download PDFInfo
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- CN103599071B CN103599071B CN201310552020.5A CN201310552020A CN103599071B CN 103599071 B CN103599071 B CN 103599071B CN 201310552020 A CN201310552020 A CN 201310552020A CN 103599071 B CN103599071 B CN 103599071B
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- polyinosinic
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- polycytacidic
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- 239000000843 powder Substances 0.000 title claims abstract description 68
- 230000002378 acidificating effect Effects 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000000203 mixture Substances 0.000 claims abstract description 28
- 238000002156 mixing Methods 0.000 claims abstract description 26
- 239000002253 acid Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 16
- 239000003381 stabilizer Substances 0.000 claims abstract description 13
- 238000001816 cooling Methods 0.000 claims abstract description 11
- 238000001035 drying Methods 0.000 claims abstract description 11
- 239000002244 precipitate Substances 0.000 claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 239000000376 reactant Substances 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 239000003795 chemical substances by application Substances 0.000 claims description 17
- 239000002671 adjuvant Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 206010013786 Dry skin Diseases 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 9
- 229920002472 Starch Polymers 0.000 claims description 8
- 229930027917 kanamycin Natural products 0.000 claims description 8
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 8
- 229960000318 kanamycin Drugs 0.000 claims description 8
- 229930182823 kanamycin A Natural products 0.000 claims description 8
- 239000008107 starch Substances 0.000 claims description 8
- 235000019698 starch Nutrition 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 5
- 229920001661 Chitosan Polymers 0.000 claims description 4
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 229920001353 Dextrin Polymers 0.000 claims description 2
- 239000004375 Dextrin Substances 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- 235000019425 dextrin Nutrition 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 7
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
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- 238000000746 purification Methods 0.000 abstract description 2
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 27
- 238000009413 insulation Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000007873 sieving Methods 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229920001345 ε-poly-D-lysine Polymers 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- XQRLCLUYWUNEEH-UHFFFAOYSA-L diphosphonate(2-) Chemical compound [O-]P(=O)OP([O-])=O XQRLCLUYWUNEEH-UHFFFAOYSA-L 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N isopropyl alcohol Natural products CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- IBUIVNCCBFLEJL-UHFFFAOYSA-M sodium;phosphoric acid;chloride Chemical compound [Na+].[Cl-].OP(O)(O)=O IBUIVNCCBFLEJL-UHFFFAOYSA-M 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical group CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 241000934136 Verruca Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000000260 Warts Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
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- 238000003808 methanol extraction Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
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- 201000010153 skin papilloma Diseases 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a kind of preparation method of polyinosinic-polycytacidic acidic dry powder, described method comprises: polyinosinic acid and each personal phosphate buffer of poly dissolve by (1), add stabilizing agent, mix homogeneously, be incubated at 40 ~ 100 DEG C after mixing; (2) mix with organic solvent after step (1) reactant liquor natural cooling, staticly settle, drying precipitate, obtain described polyinosinic-polycytacidic acidic dry powder; Beneficial effect of the present invention is mainly reflected in: the preparation method that the invention provides a kind of polyinosinic-polycytacidic acidic dry powder, the polyinosini dry powder made has complete double-spiral structure characteristic sum physiologically active, quality and stability will well much than the polyinosini under solution state, enter polyinosini more exposed in organism and have stronger resistance to enzymolysis performance, thus enhance curative effect.And to make in the process of dry powder at double-strand polyinosini solution purification has been carried out to it, eliminate a lot of small-molecule substance and impurity, reduce toxic and side effects, improve the quality of double-strand polyinosini.
Description
Technical field
The present invention relates to a kind of preparation method of polyinosinic-polycytacidic acidic dry powder.
Background technology
Double-strand polyinosini is a kind of double stranded RNA of synthetic, can induce generation interferon, has broad-spectrum disease resistance toxic action; In addition also have conditioner body immunity function, strengthen human body non-specific immunity and some specific immune function, reach anti-hepatic necrosis and antitumor action etc.
Double-strand polyinosini is applied in cosmetics can eliminate the skin vegetations such as small pox, verruca plana.
Double-strand polyinosini is mainly used in prevention and therapy poultry, virosis of livestock in veterinary drug, easy to use, extensive, can use with many medicines simultaneously, has collaborative or summation action.This product, without species specificity, can extensively use on the animals such as poultry, pig, cattle, and it also has antiviral spectrum extensively simultaneously, the advantage that toxicity is little, safety range is large.
Double-strand polyinosini is a kind of animal immunity potentiator, can be used as disease treatment adjuvant drug, strengthens animal organism immunity, improves the curative effect of medicine.Meanwhile, double-strand polyinosini is also a kind of immunological adjuvant, uses while with vaccine immunity, can significantly improve immune effect, and have epidemic prevention stress and the generation of other viral disease.
On the one hand, current Powdered polyinosini is dosage form (being pre-mixing agent, is not raw material form), and general polyinosinic acid solution and the poly solution of adopting after polymerization more, and directly add adjuvant and obtain, in this dry powder, the content of polyinosini is on the low side.
On the other hand, in the document now reported, the preparation of polyinosini preparation is generally after single step reaction, obtain intermediate polyinosini solution with polyinosinic acid, poly for raw material, then carries out the operation of preparation process.Its weak point is, first increases polymerization procedure in preparation production line, not only increases the complexity of operation, also add the difficulty of monitoring.Secondly, polyinosini intermediate exists in the form of a solution, holding conditions and the time limit stricter.
Therefore, this area is in the urgent need to providing that a kind of content is high, stability is high and preparing the preparation method of simple polyinosinic-polycytacidic acidic dry powder.
Summary of the invention
The object of the invention is to provide that a kind of content is high, stability is high and prepares the preparation method of simple polyinosinic-polycytacidic acidic dry powder.
The technical solution used in the present invention is:
A preparation method for polyinosinic-polycytacidic acidic dry powder, described method comprises:
(1) phosphate buffer of polyinosinic acid and each personal appropriate pH6.0 ~ 8.0 of poly is dissolved, add stabilizing agent after mixing, mix homogeneously, at 40 ~ 100 DEG C, be incubated 0.5 ~ 5 hour; Described stabilizing agent is one of following or wherein two or more mixture: kanamycin, oligochitosan, calcium chloride, poly-D-lysine (comprising the ε-poly-D-lysine of all size, L-poly-D-lysine etc.), calcium gluconate; The ratio of described polyinosinic acid, poly quality consumption is 1: 0.5 ~ 1.5, and stabilizing agent quality is 0.2 ~ 2.0 times of polyinosinic acid and poly quality sum; Described phosphate buffer consumption, to be fully dissolved as suitable by polyinosinic acid and poly, recommends consumption to be solution polyinosinic acid and poly being mixed with 10 ~ 100g/L;
(2) mix with the organic solvent of 0.5 ~ 20 times of volume after step (1) reactant liquor natural cooling, staticly settle 0.5 ~ 72 hour, taking precipitate 20 ~ 60 DEG C of cold drying, obtain described polyinosinic-polycytacidic acidic dry powder; Described organic solvent is one of following or wherein two or more mixture: ethanol, methanol, acetone, ether, isopropyl alcohol, normal propyl alcohol.
Polyinosinic-polycytacidic acidic dry powder in the present invention is raw material form, adopts base pair complementarity principle by poly, poly, and after adding stabilizing agent polymerization, then obtain high-quality polyinosinic-polycytacidic acidic dry powder with organic solvent deposit.This dry powder double center chain polyinosini content is high, can be directly used in the preparation of various dosage formulation, and the preparation process of preparation can be made more simple, and be easy to operation, existing market is in great demand.In addition, add stabilizing agent and not only can increase molecular weight, the stability of double-strand polyinosini (because PIC is macromole polyanion, the stability of PIC duplex structure can be made after adding stabilizing agent to increase) can also be made to strengthen, be better than solution form, be easy to preserve.After organic solvent deposit process, the hyperchromicity of sample (makes the double-spiral structure of double-strand polyinosini destroy by alkali treatment in addition, base fully exposes, the phenomenon causing uv absorption to increase is called hyperchromicity, and this index directly affects effect of product) index is more excellent.
Preferably, step (1) described stabilizing agent is chitosan, poly-D-lysine (comprising the ε-poly-D-lysine of all size, L-poly-D-lysine etc.) or kanamycin.
Preferably, step (2) described organic solvent is ethanol or methanol.
Preferably, in step (1), the ratio of described polyinosinic acid, poly quality consumption is 1: 0.8 ~ 1.2, and stabilizing agent quality is 0.2 ~ 1.5 times of polyinosinic acid and poly quality sum.
Described polyinosinic-polycytacidic acidic dry powder can make double-strand polyinosini aqueous solution further, therefore described method also can comprise step (A) after step (2): the phosphate buffer of preparation pH6.0 ~ 8.0, add appropriate polyinosinic-polycytacidic acidic dry powder and NaCl, being settled to polyinosinic-polycytacidic acidic dry powder final concentration is 0.01 ~ 1.0g/L, NaCl final concentration is 4.5g ~ 9.0g/L, obtains described double-strand polyinosini aqueous solution.According to said method obtained double-strand polyinosini aqueous solution, curative effect is high, safe and reliable, nontoxic, harmless, noresidue, and technique is simple, practical, can be used for prevention and therapy human and animal virus property disease, also can be used as immunological adjuvant.
Or described polyinosinic-polycytacidic acidic dry powder makes double-strand polyinosini pre-mixing agent further, therefore described method also can comprise step (B) after step (2): according to the mass percentage of pre-mixing agent double center chain polyinosini dry powder be 0.5 ~ 40%, Na
2hPO
412H
2o mass percentage is 0.05 ~ 10%, NaH
2pO
42H
2o mass percentage is 0.05 ~ 10%, surplus is the proportioning of adjuvant, quantitatively takes polyinosinic-polycytacidic acidic dry powder, and Na
2hPO
412H
2o and NaH
2pO
42H
2o, and add appropriate amount of auxiliary materials, fully pulverize, after crossing 60 mesh sieves, mix homogeneously, after 100 mesh sieves, after subpackage, sealing is preserved, and obtains described double-strand polyinosini pre-mixing agent; Described adjuvant is one of following or wherein two or more mixture: glucose, lactose, medical starch, soluble dextrins, compound nucleotide, sodium chloride.
Concrete, described adjuvant can be compound nucleotide, sodium chloride, and the mixture of glucose or medical starch, and adding proportion is to account for the mass percent of pre-mixing agent, and compound nucleotide is 0.5 ~ 10%, and sodium chloride is 1 ~ 10%, and surplus is glucose or medical starch.Namely pre-mixing agent mass percent is composed as follows: polyinosinic-polycytacidic acidic dry powder 0.5% ~ 40%, Na
2hPO
412H
2o0.05% ~ 10%, NaH
2pO
42H
2o0.05% ~ 10%, compound nucleotide 0 ~ 10%, sodium chloride 0 ~ 10%, surplus is glucose or medical starch.
Beneficial effect of the present invention is mainly reflected in: the preparation method that the invention provides a kind of polyinosinic-polycytacidic acidic dry powder, the polyinosini dry powder made has complete double-spiral structure characteristic sum physiologically active, and the quality of this polyinosinic-polycytacidic acidic dry powder and stability will well much than the polyinosini under solution state, enter polyinosini more exposed in organism and have stronger antienzyme performance, thus enhance curative effect.And to make in the process of dry powder at double-strand polyinosini solution purification has been carried out to it, eliminate a lot of small-molecule substance and impurity, reduce toxic and side effects, improve the quality of double-strand polyinosini.
(1) accompanying drawing explanation
Fig. 1 is product ultraviolet qualification result.
(2) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The content assaying method that following embodiment relates to is:
It is appropriate that precision takes double-strand polyinosini complex, is mixed with the aqueous solution of every 1ml containing 10mg, measures polyinosini phosphorus content according to phosphorus algoscopy.The phosphorus of every 1mg is equivalent to the polyinosini of 10.25mg.By (C
19h
23n
7o
14p
2)
ndry product calculates its content.
The hyperchromicity assay method that following embodiment relates to is:
Get double-strand polyinosini complex appropriate, make the solution about containing 0.08mg in every 1ml with sodium chloride-phosphate buffer (pH7.2), according to spectrophotography, measure trap A at the wavelength place of 248nm
1; Then this solution of accurate measuring 10ml puts in a test tube, adds 6mol/L sodium hydroxide solution 20 μ l, shakes up, and measures trap A immediately at Same Wavelength place
2, be calculated as follows and get final product:
A
2－A
1
Hyperchromicity=---------× 100%
A
1
The discrimination method that following embodiment relates to is:
Ultraviolet is differentiated: get this product, be mixed with the aqueous solution of 2mg/ml, add pH7.2 sodium chloride-phosphate buffer and [get sodium hydrogen phosphate 1.546g, sodium dihydrogen phosphate (NaH
2pO
42H
2o) 0.262g, sodium chloride 8.768g, be dissolved in water and be diluted to 1000ml, pH7.2] make the solution about containing 0.08mg in every 1ml, measure according to spectrophotography (Chinese Pharmacopoeia version in 2010 two annex IV A), there is absorption maximum at the wavelength place of 266nm ± 2nm, have minimal absorption at the wavelength place of 231nm ± 2nm.
Embodiment 1:
Take 5.2g polyinosinic acid and 5.5g poly respectively use 100mL phosphate buffer (pH6.4) to dissolve after mixing, add 10.7g oligochitosan mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the alcohol settling 2 hours of 400mL again, obtain polyinosini precipitate, 20 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 52.8%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 56.4%, and making the hyperchromicity after dry powder is 66.4%.
Embodiment 2:
Mix after taking 3.5g polyinosinic acid and each 100mL phosphate buffer (pH7.2) dissolving of 5.2g poly (1:1.5), add 8.7g kanamycin mix homogeneously, 80 DEG C of insulation 60min, after natural cooling, use the alcohol settling 12 hours of 400mL again, obtain polyinosini precipitate, 35 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 51.2%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 55.3%, and making the hyperchromicity after dry powder is 57.9%.
Embodiment 3:
Take 5.4g polyinosinic acid and 5.1g poly respectively use 100mL phosphate buffer (pH7.5) to dissolve after mixing, add 10.5g kanamycin and 1.5g calcium chloride mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the methanol extraction 24 hours of 400mL again, obtain polyinosini precipitate, 60 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 45.8%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 65.5%, and making the hyperchromicity after dry powder is 68.6%.
Embodiment 4:
Take 5.2g polyinosinic acid and 5.5g poly respectively use 100mL phosphate buffer (pH7.2) to dissolve after mixing, add 10.7g kanamycin mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the alcohol settling 0.5 hour of 400mL again, obtain polyinosini precipitate, 55 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 51.9%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 65.5%, and making the hyperchromicity after dry powder is 67.9%.
Ultraviolet identification result following (Fig. 1 is shown in by collection of illustrative plates):
| Ultraviolet is differentiated | Maximum absorption wavelength | Minimal absorption wavelength |
| Critical field | 266nm±2nm | 231nm±2nm |
| Embodiment 4 | 264.9nm | 229.2nm |
Embodiment 5:
Take 5.1g polyinosinic acid and 5.3g poly respectively use 100mL phosphate buffer (pH7.2) to dissolve after mixing, add 10.7g kanamycin mix homogeneously, 100 DEG C of insulation 60min, after natural cooling, use the alcohol settling 72 hours of 600mL again, obtain polyinosini precipitate, 45 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 49.3%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 60.3%, and making the hyperchromicity after dry powder is 64.1%.
Embodiment 6:
Mix after taking 5.5g polyinosinic acid and each 100mL phosphate buffer (pH8.0) dissolving of 2.7g poly (1:0.5), add 8.2g kanamycin mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the alcohol settling 12 hours of 800mL again, obtain polyinosini precipitate, 30 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 50.0%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 57.0%, and making the hyperchromicity after dry powder is 59.1%.
Embodiment 7:
Get 5.2g polyinosinic acid and 5.5g poly respectively use 100mL phosphate buffer (pH7.2) to dissolve after mixing, add 2.2g ε-poly-D-lysine mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the alcohol settling 4 hours of 600mL again, obtain polyinosini precipitate, 55 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 82.7%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 66.8%, and making the hyperchromicity after dry powder is 70.2%.
Embodiment 8:
Take 5.2g polyinosinic acid and 5.5g poly respectively use 100mL phosphate buffer (pH7.2) to dissolve after mixing, add 5.3g ε-poly-D-lysine mix homogeneously, 60 DEG C of insulation 60min, after natural cooling, use the alcohol settling 2 hours of 400ml again, obtain polyinosini precipitate, 55 DEG C of dryings, obtain polyinosinic-polycytacidic acidic dry powder (its double-strand polyinosini content is 71.5%).
Detect the hyperchromicity under mixed liquor and dry powder 248nm respectively, result is as follows: hyperchromicity during solution is 73.4%, and making the hyperchromicity after dry powder is 76.2%.
Embodiment 9: Detection of Stability is tested
Polyinosinic-polycytacidic acidic dry powder prepared by Example 1 ~ 8, carry out Detection of Stability, method is as follows:
Sample is placed on 30 DEG C ± 2 DEG C, under the condition of relative humidity 65% ± 5%, places 6 months.Respectively 1,2,3,6 sampling at the end of month, investigate its project such as content, hyperchromicity.
Result is as shown in table 1:
Conclusion: adopt the inventive method, obtained polyinosinic-polycytacidic acidic dry powder good stability, quality is high.
Embodiment 10:
Accurately take 1.85gNa
2hPO
412H
2o and 1.06gNaH
2pO
42H
2o is made into the Diphosphonate buffer that pH value is 6.7, and add 0.2g polyinosinic-polycytacidic acidic dry powder successively, 8.50gNaCl dissolves, solution is finally settled to 1000ml.Solution aseptic filtration; Embedding; Quality inspection: product indices is all qualified.
Embodiment 11:
Accurately take 2.04gNa
2hPO
412H
2o and 0.05gNaH
2pO
42H
2o is made into the Diphosphonate buffer that pH value is 7.2, and add 2g polyinosinic-polycytacidic acidic dry powder successively, 8.77gNaCl dissolves, solution is finally settled to 1000ml.Solution aseptic filtration; Embedding; Quality inspection: product indices is all qualified.
Embodiment 12:
Accurately take: 2.26gNa
2hPO
412H
2o and 0.06gNaH
2pO
42H
2o is made into the Diphosphonate buffer that pH value is 8.0, and add 2g polyinosinic-polycytacidic acidic dry powder successively, 8.70gNaCl dissolves, solution is finally settled to 1000ml.Solution aseptic filtration; Embedding; Quality inspection: product indices is all qualified.
Embodiment 13:
Accurately take 2.58gNaH
2pO
4.12H
2o, 0.44gNa
2hPO
4.2H
2o, 2.0gNaCl, 1.0g polyinosinic-polycytacidic acidic dry powder, 4.0g compound nucleotide, adds glucose to 100g.Above-mentioned former, adjuvant is fully pulverized, and carried out with 60 mesh sieves process of sieving; By former, adjuvant mix homogeneously after sieving.Subpackage after 100 orders sieve, sealing is preserved, and is double-strand polyinosini pre-mixing agent.
Embodiment 14:
Accurately take 3.54gNaH
2pO
4.12H
2o, 0.61gNa
2hPO
4.2H
2o, 4.0gNaCl, 2.0g polyinosinic-polycytacidic acidic dry powder, 2.0g compound nucleotide, adds medical starch to 100g.Above-mentioned former, adjuvant is fully pulverized, and carried out with 60 mesh sieves process of sieving; By former, adjuvant mix homogeneously after sieving.Subpackage after 100 orders sieve, sealing is preserved, and is double-strand polyinosini pre-mixing agent.
Embodiment 15:
Accurately take 2.90gNaH
2pO
4.12H
2o, 0.30gNa
2hPO
4.2H
2o, 5.0gNaCl, 4.0g polyinosinic-polycytacidic acidic dry powder, adds glucose to 100g.Above-mentioned former, adjuvant is fully pulverized, and carried out with 60 mesh sieves process of sieving; By former, adjuvant mix homogeneously after sieving.Subpackage after 100 orders sieve, sealing is preserved, and is double-strand polyinosini pre-mixing agent.
Embodiment 16:
Accurately take 3.39gNaH
2pO
4.12H
2o, 0.09gNa
2hPO
4.2H
2o, 1.0gNaCl, 8.0g polyinosinic-polycytacidic acidic dry powder, adds glucose to 100g.Above-mentioned former, adjuvant is fully pulverized, and carried out with 60 mesh sieves process of sieving; By former, adjuvant mix homogeneously after sieving.Subpackage after 100 orders sieve, sealing is preserved, and is double-strand polyinosini pre-mixing agent.
Embodiment 17: Detection of Stability is tested
Double-strand polyinosini aqueous solution prepared by Example 10 ~ 12 and double-strand polyinosini pre-mixing agent prepared by embodiment 13 ~ 16, carry out Detection of Stability, method is as follows: sample is placed on 30 DEG C ± 2 DEG C, under the condition of relative humidity 65% ± 5%, places 6 months.Respectively in sampling at 1,2,3,6 the end of month, investigate key project, concrete outcome in table 2,3.
The stability result of double-strand polyinosini aqueous solution is as shown in table 2:
The stability result of double-strand polyinosini pre-mixing agent is as shown in table 3:
Claims (5)
1. a preparation method for polyinosinic-polycytacidic acidic dry powder, described method comprises:
(1) phosphate buffer of polyinosinic acid and each personal appropriate pH6.0 ~ 8.0 of poly is dissolved, add stabilizing agent after mixing, mix homogeneously, at 40 ~ 100 DEG C, be incubated 0.5 ~ 5 hour; The ratio of described polyinosinic acid, poly quality consumption is 1: 0.5 ~ 1.5, and stabilizing agent quality is 0.2 ~ 2.0 times of polyinosinic acid and poly quality sum; Described stabilizing agent is chitosan, poly-D-lysine or kanamycin;
(2) mix with the organic solvent of 0.5 ~ 20 times of volume after step (1) reactant liquor natural cooling, staticly settle 0.5 ~ 72 hour, taking precipitate 20 ~ 60 DEG C of dryings, obtain described polyinosinic-polycytacidic acidic dry powder; Described organic solvent is ethanol or methanol.
2. the method for claim 1, is characterized in that in step (1), and the ratio of described polyinosinic acid, poly quality consumption is 1: 0.8 ~ 1.2, and stabilizing agent quality is 0.2 ~ 1.5 times of polyinosinic acid and poly quality sum.
3. the preparation method of a double-strand polyinosini aqueous solution, it is characterized in that polyinosinic-polycytacidic acidic dry powder claim 1 method obtained makes double-strand polyinosini aqueous solution further: the phosphate buffer of preparation pH6.0 ~ 8.0, add appropriate polyinosinic-polycytacidic acidic dry powder and NaCl, being settled to polyinosinic-polycytacidic acidic dry powder final concentration is 0.01 ~ 1.0g/L, NaCl final concentration is 4.5 ~ 9.0g/L, obtains described double-strand polyinosini aqueous solution.
4. a preparation method for double-strand polyinosini pre-mixing agent, is characterized in that polyinosinic-polycytacidic acidic dry powder claim 1 method obtained makes double-strand polyinosini pre-mixing agent further: according to the mass percentage of pre-mixing agent double center chain polyinosini dry powder be 0.5 ~ 40%, Na
2hPO
412H
2o mass percentage is 0.05 ~ 10%, NaH
2pO
42H
2o mass percentage is 0.05 ~ 10%, surplus is the proportioning of adjuvant, quantitatively takes polyinosinic-polycytacidic acidic dry powder, and Na
2hPO
412H
2o and NaH
2pO
42H
2o, and add appropriate amount of auxiliary materials, fully pulverize, after crossing 60 mesh sieves, mix homogeneously, after 100 mesh sieves, after subpackage, sealing is preserved, and obtains described double-strand polyinosini pre-mixing agent; Described adjuvant is one of following or wherein two or more mixture: glucose, lactose, medical starch, soluble dextrins, compound nucleotide, sodium chloride.
5. method as claimed in claim 4, is characterized in that described adjuvant is compound nucleotide, sodium chloride, and glucose or medical starch, adding proportion is to account for the mass percent of pre-mixing agent, compound nucleotide is 0 ~ 10%, and sodium chloride is 0 ~ 10%, and surplus is glucose or medical starch.
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| CN104434784B (en) * | 2014-11-15 | 2017-07-04 | 成都天台山制药有限公司 | Polyinosinic injection pharmaceutical composition and preparation method |
| CN104706580B (en) * | 2015-03-25 | 2017-11-10 | 华南农业大学 | A kind of interferon inducer for animals and preparation method thereof |
| ES2755418T3 (en) | 2015-11-17 | 2020-04-22 | Bioncotech Therapeutics S L | Novel pharmaceutical composition comprising particles comprising a complex of a double-stranded polyribonucleotide and a polyalkyleneimine |
| CN105434341B (en) * | 2015-12-22 | 2018-07-10 | 肇庆大华农生物药品有限公司 | A kind of polyadenylic-polyuridylic acid parenteral solution for animals and preparation method thereof |
| CN106265736A (en) * | 2016-08-12 | 2017-01-04 | 株洲市神农动物药业有限公司 | A kind of compound Radix Astragali polysaccharide injection preparation method and application |
| US11020419B2 (en) | 2016-09-30 | 2021-06-01 | Biosyngen Pte, Ltd | Use of polyinosinic-polycytidylic acid compositions in treatment of malignant effusion |
| CA3063805C (en) | 2017-05-17 | 2024-01-09 | Bioncotech Therapeutics Sl | Novel pharmaceutical composition comprising particles comprising a complex of a double-stranded polyribonucleotide and a polyalkyleneimine |
| CN108743938B (en) * | 2018-06-29 | 2019-04-09 | 信福(北京)医药科技有限公司 | For enhancing the preparation method of the compound of immune response |
| CN109078180B (en) * | 2018-06-29 | 2019-05-31 | 信福(北京)医药科技有限公司 | For enhancing the compound of immune response |
| WO2020001587A1 (en) * | 2018-06-29 | 2020-01-02 | 信福(北京)医药科技有限公司 | Complex for enhancing immune response |
| CN109939228A (en) * | 2019-03-25 | 2019-06-28 | 苏州博特龙免疫技术有限公司 | A kind of new type water-solubility immunologic adjuvant and preparation method thereof |
| CN110256516B (en) * | 2019-07-08 | 2020-07-03 | 河北韩美生物科技有限公司 | Preparation method of polyinosinic cells |
| CN110898014A (en) * | 2019-12-09 | 2020-03-24 | 美亚药业海安有限公司 | Preparation method of polyuridylic acid |
| CN115067318B (en) * | 2022-02-22 | 2023-07-14 | 深圳中检联新药检测有限责任公司 | Dental pulp mesenchymal stem cell preservation solution and application thereof |
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