CN104706580A - Veterinary interferon inducer and preparation method thereof - Google Patents
Veterinary interferon inducer and preparation method thereof Download PDFInfo
- Publication number
- CN104706580A CN104706580A CN201510133896.5A CN201510133896A CN104706580A CN 104706580 A CN104706580 A CN 104706580A CN 201510133896 A CN201510133896 A CN 201510133896A CN 104706580 A CN104706580 A CN 104706580A
- Authority
- CN
- China
- Prior art keywords
- phosphate buffer
- sodium chloride
- calcium chloride
- aqueous solution
- animals
- 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
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a veterinary interferon inducer. The veterinary interferon inducer is prepared from the following components: a sodium chloride-phosphate buffer solution with the pH value of 7.2, a 10-80g/L calcium chloride aqueous solution, a 0.1-5g/L chitosan hydrochloride solution, polyinosinic acid and polycytidysic acid. The invention further provides a preparation method of the veterinary interferon inducer. The veterinary interferon inducer provided by the invention is few in side reaction, low in irritation and high in safety, and the preparation method can avoid precipitates generated by calcium chloride and other components.
Description
Technical field
The present invention relates to a kind of interferon inducers, be specifically related to a kind of interferon inducers base for animals and preparation method thereof.
Background technology
Interferon (Interferon, IFN) is a kind of cytokine, has the multiple effects such as T suppression cell division, immunity moderation, antiviral, antitumor.Interferon is as a kind of broad-spectrum disease resistance toxic agent, and direct killing or suppression virus, mainly do not make cell produce antiviral protein by cell surface receptor effect, thus suppress copying of virus; Also can strengthen natural killer cell (NK cell), macrophage and the lymphocytic vigor of T simultaneously, thus play immunoregulation effect, and strengthen anti-virus ability.They have on allogenic cell wide spectrum antiviral, affect Growth of Cells, and the multiple biological activity such as differentiation, immunity moderation function.
Polyinosini, also known as poly-cytidylic acid, being the double-stranded RNA polymer of synthetic, is the strong inducer of interferon, except the effect with broad-spectrum antiviral, also there is antibacterial, the various biological effect such as protozoacide, antitumor, radioprotective and immunomodulating simultaneously.In addition, polyinosini, because of the chemical constitution of its uniqueness, can be degraded in vivo, can not cause residual, and harm humans is healthy, has the feature of safety, environmental protection.The test of polyinosini in laboratory animal, the I phase of people doctor and the effect of II phase clinical treatment disease are proved.Current polyinosini is widely used in the control of the viral diseases such as herpesvirus, influenza virus, hepatitis virus and some tumor disease clinically people doctor.Veterinary clinic is mainly used in the research for the treatment of viral disease.
In current Polyinosinic injection product containing kanamycin as cationic stabilized agent, and the use of kanamycin easily causes the antibiotic problem such as residual.Although chitosan can replace kanamycin as cationic stabilized agent, but chitosan is a kind of aminopolysaccharide, because intermolecular active force is stronger, the acid solution that some is special can only be dissolved in, and easily degrade in chitosan acid solution storage process, being there is larger change and affect stay in grade in viscosity and molecular mass, strongly limit the extensive use of chitosan.Once report was had to utilize chitosan that polyinosini bag is formed nano-microcapsule, it is according to being the Tyndall phenomenon using laser pen to check final preparation, because chitosan dissolves the colloid namely forming viscosity in an acidic solution, inherently there will be Tyndall phenomenon under light illumination, polyinosini nano-microcapsule is contained without necessary connection with formation, therefore, this method is still not enough to prove that polyinosini is wrapped up by chitosan, in addition, because polyinosini is easily degraded in an acidic solution, polyinosini-chitosan acid solution is also not suitable for long-term preservation.
Calcium ion also has important effect to the secondary structure of stable polyinosini, prior art directly adds calcium chloride powder in building-up process in reactant liquor, because calcium chloride itself compares indissoluble, directly add powder and the compositions such as hydrogen phosphate, free polyinosinic acid and the poly of high concentration calcium ion easily and in reaction system can be caused to be formed precipitate and affect product quality and synthesize concentration.
In addition, existing polyinosini technology of preparing also uses the material such as sodium pyrosulfite, excipient, as the application for a patent for invention file that publication number is CN101810638A, disclose a kind of injection of endogenous interferon inducer and the preparation method of nanometer microencapsulation solution, wherein specifically disclose: in injection water, add pyrophosphorous acid sodium, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, then 20-60 degree Celsius is heated, then add poly to stir, dissolve, insulation 10min, and then add polyinosinic acid, stir, dissolve, insulation 15-30min.The material such as sodium pyrosulfite, excipient is easily to body generation stimulation in various degree, and especially pyrosulfurous acid sodium toxicity very much not can be eaten, and touches people and animals' eyes and also can produce grievous injury, be also unfavorable for plant personnel production operation.The polyinosini product prepared of above-mentioned prior art to animal injection or dipping, mix bait administration and to occur now and then the risk of side reaction and contaminated environment, if also involve food-safety problem for meat economic animal, be difficult to promote.
Summary of the invention
The object of the present invention is to provide the interferon inducers for animals that a kind of side reaction is few, zest is low, safety is high, the present invention simultaneously also provides the preparation method of this interferon inducers for animals.
For solving the problem, the technical solution adopted in the present invention is as follows:
A kind of interferon inducers for animals, it is made up of following component: pH value is the sodium chloride-phosphate buffer of 7.2, concentration is the calcium chloride water of 10-80g/L, concentration is the chitosan hydrochlorate aqueous solution of 0.1-5g/L, polyinosinic acid, poly, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described poly quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described calcium chloride water and sodium chloride-phosphate buffer is 2-16:1000, the volume ratio of described chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 984-998:1000, the cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1.
PH value described in the present invention be 7.2 sodium chloride-phosphate buffer mixed by two hypophosphite monohydrate sodium dihydrogens, sodium hydrogen phosphate, sodium chloride and water; Containing 0.262g bis-hypophosphite monohydrate sodium dihydrogen, 1.546g sodium hydrogen phosphate, 8.768g sodium chloride in every 1L sodium chloride-phosphate buffer.
Interferon inducers for animals in the present invention, its preparation method comprises the steps:
A. get polyinosinic acid, then use the described sodium chloride-phosphate buffer (half of the sodium chloride namely in formula-phosphate buffer cumulative volume) of 1/2 volume to dissolve, then at the temperature of 40-70 DEG C, heat 5-10 minute;
B. get poly, then use the described sodium chloride-phosphate buffer (half of the sodium chloride namely in formula-phosphate buffer cumulative volume) of 1/2 volume to dissolve, then at the temperature of 40-70 DEG C, heat 5-10 minute;
C. by the liquid mixing that a step and b step obtain, then 3-10 minute is stirred with the speed of 10-40 rev/min; Then at the temperature of 40-70 DEG C, react 20-60 minute, keep the speed of 10-40 rev/min to stir in course of reaction, obtain reactant liquor;
D. question response liquid is cooled to room temperature, then dropwise calcium chloride water is dripped at interval of 5-20s, the cumulative volume of the calcium chloride water of described dropping and the volume ratio of sodium chloride-phosphate buffer are 2-16:1000, and the process that drips keeps the speed of 10-40 rev/min to stir;
E. in the reactant liquor through Step d process, slowly adding the chitosan hydrochlorate aqueous solution of formula ratio, (the chitosan hydrochlorate aqueous solution namely limited above and the volume ratio of sodium chloride-phosphate buffer are 984-998:1000; The cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1), adition process keeps the speed of 10-40 rev/min to stir;
F. by the reactant liquor membrane filtration of aperture 0.2-0.45 μm through step e process, collect filtrate, to obtain final product.
In the present invention, preferred scheme is that the calcium chloride water in described Step d is obtained by following steps: according to the concentration (i.e. 10-80g/L) of described calcium chloride water, get calcium chloride water-soluble, then use the membrane filtration of 0.2-0.45 μm, collect filtrate, to obtain final product.
In the present invention, preferred scheme is that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration (i.e. 0.1-5g/L) of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then the membrane filtration of 0.2-0.45 μm is used, collect filtrate, to obtain final product.
Compared with prior art, tool of the present invention has the following advantages:
1, use soluble chitosan hydrochlorate to replace kanamycin and chitosan in the present invention, have the following advantages: 1) chitosan hydrochlorate all has no side effect to animal body and environment, and cost is low; 2) chitosan hydrochlorate has strong cation, can as polyinosini stabilizing agent, and effectively resist the Degradation of RNA enzyme in body or environment, antibiotic-free remains, and promotes that somatic cell is to the absorption of ingredient; 3) chitosan hydrochlorate can in neutral water rapid solution, the pH value of its aqueous solution is close to neutral, can with the sodium chloride of PH=7.2-phosphate buffer compatibility, be very beneficial for requiring harsher field application to it also avoid the degraded of acid solution to polyinosini to zest at Injection for animals etc.; 4) chitosan hydrochlorate properties of Aqueous Solution is stablized, and easily stores, must the now with the current thus restriction production in enormous quantities drawback of carrying out when avoiding the application of traditional shell polysaccharide;
2, without the need to adding sodium pyrosulfite, kanamycin, the materials such as excipient, while guaranteeing drug effect, the side reaction to body can be reduced, avoid antibiotic and poisonous substance to remain, without food-safety problem, free from environmental pollution, be particularly suitable for meat economic animal aquaculture and use;
3) preparation method of interferon inducers for animals of the present invention, optimize the adding technology of the adjuvants such as calcium chloride, avoid it when dissolving and other composition produces and precipitates, guarantee synthetic effect, the polyinosini solution of concentration between 0.1-20g/L all can be prepared smoothly.
Below in conjunction with detailed description of the invention, the present invention is described in detail.
Detailed description of the invention
Embodiment 1
A kind of interferon inducers for animals, it is made up of following component: volume to be the pH value of 500mL be 7.2 chitosan hydrochlorate aqueous solution, the polyinosinic acid of 0.5g, the poly of 0.5g of the calcium chloride water of sodium chloride-phosphate buffer, volume to be the concentration of 1mL be 44.4g/L, volume to be 499mL concentration be 0.65g/L.
The preparation method of this interferon inducers for animals comprises the steps:
A. get the polyinosinic acid of 0.5g, then dissolve with the sodium chloride-phosphate buffer of 250mL, then heat 5 minutes at the temperature of 45 DEG C;
B. get the poly of 0.5g, then dissolve with the sodium chloride-phosphate buffer of 250mL, then heat 5 minutes at the temperature of 45 DEG C;
C. by the liquid mixing that a step and b step obtain, then 3 minutes are stirred with the speed of 30 revs/min; Then react 30 minutes at the temperature of 45 DEG C, keep the speed of 30 revs/min to stir in course of reaction, obtain reactant liquor;
D. question response liquid is cooled to room temperature, then dropwise drips calcium chloride water at interval of 5s, and the cumulative volume of the calcium chloride water of described dropping is 1mL, and dropping process keeps the speed of 30 revs/min to stir;
E. in the reactant liquor through Step d process, slowly add the chitosan hydrochlorate aqueous solution of 499mL, adition process keeps the speed of 30 revs/min to stir;
F. by the reactant liquor membrane filtration in 0.22 μm, aperture through step e process, collect filtrate, to obtain final product;
Calcium chloride water in described Step d is obtained by following steps: according to the concentration (i.e. 44.4g/L) of described calcium chloride water, get calcium chloride water-soluble, then use the membrane filtration of 0.22 μm, collects filtrate, to obtain final product.
In the present invention, preferred scheme is that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration (i.e. 0.65g/L) of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then the membrane filtration of 0.22 μm is used, collect filtrate, to obtain final product.
The quality of the interferon inducers for animals obtained by the present embodiment and people's Polyinosinic injection of certain brand commercially available compares, and the foundation compared is State Drug Administration's national drug standards [WS1-XG-050-2000], and concrete testing result is as following table 1:
Table 1: interferon inducers prepared by this technology contrasts (1mg/ml) with similar sample quality testing result
As can be seen from table 1 testing result, the interferon inducers for animals obtained by the present invention meets State Drug Administration's national drug standards [WS1-XG-050-2000], and cleaning sterile, can directly use, effective content is high, constant product quality.
The for animals interferon inducers obtained to the present embodiment is tested opposing RNA enzymatic degradation stability; get the interferon inducers for animals that the present embodiment obtains, the people of certain brand commercially available uses Polyinosinic injection group; then do not add the obtained interferon inducers for animals of chitosan hydrochlorate as a control group according to the formula of the present embodiment and method; all samples being diluted to valid density is 0.08mg/ml; add the RNA enzyme of 20ul 10mg/ml; 37 DEG C of water-baths, measures a 248nm absorbance (testing result refers to table 2) every 15 minutes.
As can be seen from testing result, the ability of the interferon inducers opposing RNA enzymatic degradation for animals that the present invention obtains is better than matched group, is also better than commercially available people's Polyinosinic injection.Point out it to have good toleration to the RNA enzymatic degradation in environment and animal body, be applicable to being applied to the drinking-water in cultivation, mix bait administration and dipping.
Table 2: anti-RNA enzymatic degradation test 248nm absorbance
The interferon-induced test of ɑ
30 21 age in days SPF chickens are divided into three groups at random, often organize 10, inject the interferon inducers for animals of the present embodiment respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection sodium chloride-phosphate buffer is as blank group.Every chicken presses the injection of 0.01mg/kg body weight, and interval is injected once for 3 days again, and the 12nd hour the last time after injection and blood sampling in the 36th hour, measure the ɑ interferon content in blood by ELISA method.Concrete outcome refers to table 3:
The interferon-induced result of the test of table 3: ɑ
Note: colleague ab represents significant difference compared with matched group, ac represents extremely remarkable with matched group difference, and alphabetical same difference is not remarkable.
As can be seen from testing result, the ability that the 0.01mg/kg body weight people that injects interferon inducers group for animals that the present embodiment obtains and certain brand commercially available produces ɑ interferon with the induction of Polyinosinic injection group is all higher than blank group and difference is extremely remarkable, and the interferon inducers group for animals that the present embodiment obtains uses Polyinosinic injection group higher than the people of certain brand commercially available, but difference is not remarkable.
Safety testing
30 14 age in days SPF chickens are divided into three groups at random, and often organize 10, inject the interferon inducers for animals of this enforcement respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection " sodium chloride-phosphate buffer " is as blank group.Every chicken presses the injection of 0.1mg/kg body weight, is used in conjunction 3 days, observes animal after injection and whether occurs abnormal response, within the 7th day after final injection, cut open and kill all animals, check injection site and visceral organ tissue.
Result shows: all non-abnormal response of each treated animal after injection, and off-test, cuts open and kill all animals, and injection site and visceral organ tissue are all normal, illustrate that this preparation is safe to chicken.
Embodiment 2
A kind of interferon inducers for animals, it is made up of following component: pH value is the sodium chloride-phosphate buffer of 7.2, concentration is the calcium chloride water of 10g/L, concentration is the chitosan hydrochlorate aqueous solution of 0.1g/L, polyinosinic acid, poly, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer is 0.5g/L, the volume ratio of described poly quality and sodium chloride-phosphate buffer is 0.5g/L, the volume ratio of described calcium chloride water and sodium chloride-phosphate buffer is 16:1000, the volume ratio of described chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 984:1000, the cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1.
Interferon inducers for animals in the present invention, its preparation method comprises the steps:
A. get polyinosinic acid, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then heat 10 minutes at the temperature of 40 DEG C;
B. get poly, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then heat 10 minutes at the temperature of 70 DEG C;
C. by the liquid mixing that a step and b step obtain, then 10 minutes are stirred with the speed of 10 revs/min; Then react 60 minutes at the temperature of 40 DEG C, keep the speed of 10 revs/min to stir in course of reaction, obtain reactant liquor;
D. question response liquid is cooled to room temperature, then dropwise drips calcium chloride water at interval of 10s, and the cumulative volume of the calcium chloride water of described dropping and the volume ratio of sodium chloride-phosphate buffer are 16:1000, and dropping process keeps the speed of 10 revs/min to stir;
E. in the reactant liquor through Step d process, slowly add the chitosan hydrochlorate aqueous solution of formula ratio, adition process keeps the speed of 10 revs/min to stir;
F. by the reactant liquor membrane filtration in 0.2 μm, aperture through step e process, collect filtrate, to obtain final product;
Calcium chloride water in described Step d is obtained by following steps: according to the concentration (i.e. 10g/L) of described calcium chloride water, get calcium chloride water-soluble, then use the membrane filtration of 0.2 μm, collects filtrate, to obtain final product.
In the present invention, preferred scheme is that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration (i.e. 0.1g/L) of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then use the membrane filtration of 0.2 μm, collect filtrate, to obtain final product.
The quality of the interferon inducers for animals obtained by the present embodiment and people's Polyinosinic injection of certain brand commercially available compares, and the foundation compared is State Drug Administration's national drug standards [WS1-XG-050-2000], and concrete testing result is as following table 4:
Table 4: interferon inducers prepared by this technology contrasts (1mg/ml) with similar sample quality testing result
As can be seen from table 4 testing result, the interferon inducers for animals that the embodiment of the present invention obtains meets State Drug Administration's national drug standards [WS1-XG-050-2000], and cleaning sterile, can directly use, effective content is high, constant product quality.
The for animals interferon inducers obtained to the present embodiment is tested opposing RNA enzymatic degradation stability; get the interferon inducers for animals that the present embodiment obtains, the people of certain brand commercially available uses Polyinosinic injection group; then do not add the obtained interferon inducers for animals of chitosan hydrochlorate as a control group according to the formula of the present embodiment and method; all samples being diluted to valid density is 0.08mg/ml; add the RNA enzyme of 20ul 10mg/ml; 37 DEG C of water-baths, measures a 248nm absorbance (testing result refers to table 5) every 15 minutes.
As can be seen from testing result, the ability of the interferon inducers opposing RNA enzymatic degradation for animals that the present invention obtains is better than matched group, is also better than commercially available people's Polyinosinic injection.Point out it to have good toleration to the RNA enzymatic degradation in environment and animal body, be applicable to being applied to the drinking-water in cultivation, mix bait administration and dipping.
Table 5: anti-RNA enzymatic degradation test 248nm absorbance
The interferon-induced test of ɑ
30 21 age in days SPF chickens are divided into three groups at random, often organize 10, inject the obtained interferon inducers for animals of the present embodiment respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection sodium chloride-phosphate buffer is as blank group.Every chicken presses the injection of 0.01mg/kg body weight, and interval is injected once for 3 days again, and the 12nd hour the last time after injection and blood sampling in the 36th hour, measure the ɑ interferon content in blood by ELISA method.Concrete outcome refers to table 6:
The interferon-induced result of the test of table 6: ɑ
Note: colleague ab represents significant difference compared with matched group, ac represents extremely remarkable with matched group difference, and alphabetical same difference is not remarkable.
As can be seen from testing result, 0.01mg/kg body weight injects ability that interferon inducers group for animals that the present embodiment obtains and commercially available people produce ɑ interferon with the induction of Polyinosinic injection group all higher than blank group and difference is extremely remarkable, and the interferon inducers group for animals that the present embodiment obtains uses Polyinosinic injection group higher than commercially available people, but difference is not remarkable.
Safety testing
30 14 age in days SPF chickens are divided into three groups at random, often organize 10, inject the obtained interferon inducers for animals of the present embodiment respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection " sodium chloride-phosphate buffer " is as blank group.Every chicken presses the injection of 0.1mg/kg body weight, is used in conjunction 3 days, observes animal after injection and whether occurs abnormal response, within the 7th day after final injection, cut open and kill all animals, check injection site and visceral organ tissue.
Result shows: all non-abnormal response of each treated animal after injection, and off-test, cuts open and kill all animals, and injection site and visceral organ tissue are all normal, illustrate that this preparation is safe to chicken.
Embodiment 3
A kind of interferon inducers for animals, it is made up of following component: pH value is the sodium chloride-phosphate buffer of 7.2, concentration is the calcium chloride water of 80g/L, concentration is the chitosan hydrochlorate aqueous solution of 5g/L, polyinosinic acid, poly, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer is 10g/L, the volume ratio of described poly quality and sodium chloride-phosphate buffer is 10g/L, the volume ratio of described calcium chloride water and sodium chloride-phosphate buffer is 2:1000, the volume ratio of described chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 998:1000, the cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1.
Interferon inducers for animals in the present invention, its preparation method comprises the steps:
A. get polyinosinic acid, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then heat 6 minutes at the temperature of 70 DEG C;
B. get poly, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then heat 6 minutes at the temperature of 70 DEG C;
C. by the liquid mixing that a step and b step obtain, then 3 minutes are stirred with the speed of 40 revs/min; Then react 20 minutes at the temperature of 70 DEG C, keep the speed of 40 revs/min to stir in course of reaction, obtain reactant liquor;
D. question response liquid is cooled to room temperature, then dropwise drips calcium chloride water at interval of 10s, and the cumulative volume of the calcium chloride water of described dropping and the volume ratio of sodium chloride-phosphate buffer are 2:1000, and dropping process keeps the speed of 40 revs/min to stir;
E. in the reactant liquor through Step d process, slowly add the chitosan hydrochlorate aqueous solution (namely the volume ratio of chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 998:1000) of formula ratio, adition process keeps the speed of 40 revs/min to stir;
F. by the reactant liquor membrane filtration in 0.45 μm, aperture through step e process, collect filtrate, to obtain final product;
Calcium chloride water in described Step d is obtained by following steps: according to the concentration (i.e. 80g/L) of described calcium chloride water, get calcium chloride water-soluble, then use the membrane filtration of 0.45 μm, collects filtrate, to obtain final product.
In the present invention, preferred scheme is that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration (i.e. 5g/L) of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then use the membrane filtration of 0.45 μm, collect filtrate, to obtain final product.
The quality of the interferon inducers for animals obtained by the present embodiment and people's Polyinosinic injection of certain brand commercially available compares, and the foundation compared is State Drug Administration's national drug standards [WS1-XG-050-2000], and concrete testing result is as following table 7:
Table 7: interferon inducers prepared by this technology contrasts (1mg/ml) with similar sample quality testing result
As can be seen from table 7 testing result, the interferon inducers for animals that the present embodiment obtains meets State Drug Administration's national drug standards [WS1-XG-050-2000], and cleaning sterile, can directly use, effective content is high, constant product quality.
The for animals interferon inducers obtained to the present embodiment is tested opposing RNA enzymatic degradation stability; get the interferon inducers for animals that the present embodiment obtains, the people of certain brand commercially available uses Polyinosinic injection group; then do not add the obtained interferon inducers for animals of chitosan hydrochlorate as a control group according to the formula of the present embodiment and method; all samples being diluted to valid density is 0.08mg/ml; add the RNA enzyme of 20ul 10mg/ml; 37 DEG C of water-baths, measures a 248nm absorbance (testing result refers to table 8) every 15 minutes.
As can be seen from testing result, the ability of the interferon inducers opposing RNA enzymatic degradation for animals that the present invention obtains is better than matched group, is also better than commercially available people's Polyinosinic injection.Point out it to have good toleration to the RNA enzymatic degradation in environment and animal body, be applicable to being applied to the drinking-water in cultivation, mix bait administration and dipping.
Table 8: anti-RNA enzymatic degradation test 248nm absorbance
The interferon-induced test of ɑ
30 21 age in days SPF chickens are divided into three groups at random, often organize 10, inject the obtained interferon inducers for animals of the present embodiment respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection sodium chloride-phosphate buffer is as blank group.Every chicken presses the injection of 0.01mg/kg body weight, and interval is injected once for 3 days again, and the 12nd hour the last time after injection and blood sampling in the 36th hour, measure the ɑ interferon content in blood by ELISA method.Specific experiment is the results detailed in Table 9:
The interferon-induced result of the test of table 9: ɑ
Note: colleague ab represents significant difference compared with matched group, ac represents extremely remarkable with matched group difference, and alphabetical same difference is not remarkable.
As can be seen from testing result, the ability that the 0.01mg/kg body weight people that injects interferon inducers group for animals that the present embodiment obtains and certain brand commercially available produces ɑ interferon with the induction of Polyinosinic injection group is all higher than blank group and difference is extremely remarkable, and the interferon inducers group for animals that the present embodiment obtains uses Polyinosinic injection group higher than the people of certain brand commercially available, but difference is not remarkable.
Safety testing
30 14 age in days SPF chickens are divided into three groups at random, often organize 10, inject the obtained interferon inducers for animals of the present embodiment respectively, people's Polyinosinic injection of certain brand commercially available, other one group of injection " sodium chloride-phosphate buffer " is as blank group.Every chicken presses the injection of 0.1mg/kg body weight, is used in conjunction 3 days, observes animal after injection and whether occurs abnormal response, within the 7th day after final injection, cut open and kill all animals, check injection site and visceral organ tissue.
Result shows: all non-abnormal response of each treated animal after injection, and off-test, cuts open and kill all animals, and injection site and visceral organ tissue are all normal, illustrate that this preparation is safe to chicken.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (4)
1. an interferon inducers for animals, it is characterized in that being made up of following component: pH value is the sodium chloride-phosphate buffer of 7.2, concentration is the calcium chloride water of 10-80g/L, concentration is the chitosan hydrochlorate aqueous solution of 0.1-5g/L, polyinosinic acid, poly, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described poly quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described calcium chloride water and sodium chloride-phosphate buffer is 2-16:1000, the volume ratio of described chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 984-998:1000, the cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1.
2. a preparation method for interferon inducers for animals according to claim 1, is characterized in that comprising the steps:
A. get polyinosinic acid, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then at the temperature of 40-70 DEG C, heat 5-10 minute;
B. get poly, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then at the temperature of 40-70 DEG C, heat 5-10 minute;
C. by the liquid mixing that a step and b step obtain, then 3-10 minute is stirred with the speed of 10-40 rev/min; Then at the temperature of 40-70 DEG C, react 20-60 minute, keep the speed of 10-40 rev/min to stir in course of reaction, obtain reactant liquor;
D. question response liquid is cooled to room temperature, then dropwise calcium chloride water is dripped at interval of 5-20s, the cumulative volume of the calcium chloride water of described dropping and the volume ratio of sodium chloride-phosphate buffer are 2-16:1000, and the process that drips keeps the speed of 10-40 rev/min to stir;
E. in the reactant liquor through Step d process, slowly add the chitosan hydrochlorate aqueous solution of formula ratio, adition process keeps the speed of 10-40 rev/min to stir;
F. by the reactant liquor membrane filtration of aperture 0.2-0.45 μm through step e process, collect filtrate, to obtain final product.
3. the preparation method of interferon inducers for animals according to claim 2, it is characterized in that the calcium chloride water in described Step d is obtained by following steps: according to the concentration of described calcium chloride water, get calcium chloride water-soluble, then the membrane filtration of 0.2-0.45 μm is used, collect filtrate, to obtain final product.
4. the preparation method of interferon inducers for animals according to claim 2, it is characterized in that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then the membrane filtration of 0.2-0.45 μm is used, collect filtrate, to obtain final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510133896.5A CN104706580B (en) | 2015-03-25 | 2015-03-25 | A kind of interferon inducer for animals and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510133896.5A CN104706580B (en) | 2015-03-25 | 2015-03-25 | A kind of interferon inducer for animals and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104706580A true CN104706580A (en) | 2015-06-17 |
| CN104706580B CN104706580B (en) | 2017-11-10 |
Family
ID=53406630
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510133896.5A Active CN104706580B (en) | 2015-03-25 | 2015-03-25 | A kind of interferon inducer for animals and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104706580B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105396130A (en) * | 2015-11-10 | 2016-03-16 | 林海祥 | Polyriboinosinic polyribo-cytoidylic acid (PIC), ammonia and calcium adjuvant and vaccine containing same |
| CN105434341A (en) * | 2015-12-22 | 2016-03-30 | 肇庆大华农生物药品有限公司 | Veterinary polynosinic acid-polycyttdylic acid injection, and preparation method thereof |
| CN105535964A (en) * | 2016-01-27 | 2016-05-04 | 苏文全 | Double-strand polynucleotide-epsilon-polylysine-sulfuric acid glycan compound with immune regulating function and preparing and using method thereof |
| CN105664152A (en) * | 2016-01-27 | 2016-06-15 | 苏文全 | Double-strand oligonucleotide-epsilon-polylysine compound with immune regulation function and preparation and use method of double-strand oligonucleotide-epsilon-polylysine compound |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1759887A (en) * | 2004-10-11 | 2006-04-19 | 秦卫华 | Mucous membrane absorption ingestion type medicinal composition for drawing out interferon, and preparation |
| CN103599071A (en) * | 2013-11-08 | 2014-02-26 | 杭州美亚药业有限公司 | Preparation method of polyinosinic-polycytidylic acid dry powder |
-
2015
- 2015-03-25 CN CN201510133896.5A patent/CN104706580B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1759887A (en) * | 2004-10-11 | 2006-04-19 | 秦卫华 | Mucous membrane absorption ingestion type medicinal composition for drawing out interferon, and preparation |
| CN103599071A (en) * | 2013-11-08 | 2014-02-26 | 杭州美亚药业有限公司 | Preparation method of polyinosinic-polycytidylic acid dry powder |
Non-Patent Citations (1)
| Title |
|---|
| DANIELA OTT ET AL: "The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellularresponses in primary cultures from rat brain sites with an incompleteblood–brain barrier", 《NEUROSCIENCE LETTERS》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105396130A (en) * | 2015-11-10 | 2016-03-16 | 林海祥 | Polyriboinosinic polyribo-cytoidylic acid (PIC), ammonia and calcium adjuvant and vaccine containing same |
| CN105434341A (en) * | 2015-12-22 | 2016-03-30 | 肇庆大华农生物药品有限公司 | Veterinary polynosinic acid-polycyttdylic acid injection, and preparation method thereof |
| 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 |
| CN105535964A (en) * | 2016-01-27 | 2016-05-04 | 苏文全 | Double-strand polynucleotide-epsilon-polylysine-sulfuric acid glycan compound with immune regulating function and preparing and using method thereof |
| CN105664152A (en) * | 2016-01-27 | 2016-06-15 | 苏文全 | Double-strand oligonucleotide-epsilon-polylysine compound with immune regulation function and preparation and use method of double-strand oligonucleotide-epsilon-polylysine compound |
| CN105664152B (en) * | 2016-01-27 | 2019-01-18 | 苏文全 | A kind of double stranded polynucleotide with immunoregulation effect-epsilon-polylysine compound and its preparation application method |
| CN105535964B (en) * | 2016-01-27 | 2019-01-18 | 苏文全 | A kind of double stranded polynucleotide-epsilon-polylysine-sulfuric acid glycan compound and its preparation application method with immunoregulation effect |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104706580B (en) | 2017-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11672756B2 (en) | Temperature sensitive hydrogel composition including nucleic acid and chitosan | |
| CN104706580A (en) | Veterinary interferon inducer and preparation method thereof | |
| CN105597146B (en) | Wound healing multi-functional temperature sensitive gel composite dressing and its preparation and application method | |
| CN105001434B (en) | Multi-functional hemostatic microsphere of one class based on chitosan | |
| CN102952275A (en) | Hyaluronic acid gel employing biphasic technology, and preparation method thereof | |
| CN103599071A (en) | Preparation method of polyinosinic-polycytidylic acid dry powder | |
| Yi et al. | Preparation and characterization of a Schiff-based chitosan-fructose quaternary ammonium salt for medical applications | |
| Priddy‐Arrington et al. | Characterization and Optimization of Injectable In Situ Crosslinked Chitosan‐Genipin Hydrogels | |
| US20160319059A1 (en) | Polyacrylamide Hydrogel-Based Material for Medical Purposes and Method for Producing Same | |
| Lan et al. | Investigation into the safety of perineural application of 1, 4‐butanediol diglycidyl ether‐crosslinked hyaluronan in a rat model | |
| CN103494780B (en) | Gamithromycin composition lyophilized powder for injection and preparation method | |
| CN106474053B (en) | A kind of composite high-molecular colloid surface of a wound thimerosal and its preparation method containing amino acid | |
| CN103495174A (en) | Multi-effect external preparation used for treating superficial skin injury and application thereof | |
| CN101766569B (en) | Long-acting antimicrobial medical oil emulsion and preparation method thereof | |
| CN104984354A (en) | Polyacrylic acid-calcium phosphate composite nano-drug carrier and preparing method and application thereof | |
| CN109303787A (en) | It is a kind of for repairing the pharmaceutical composition of wound | |
| CN105254780B (en) | A kind of bionical derivative of cation type chitosan and its application | |
| CN111234163B (en) | Nanogel with antibacterial repair performance and preparation method and application thereof | |
| CN104274494B (en) | A kind of American cockroach external preparation and preparation method thereof | |
| CN109431999B (en) | Nano self-microemulsion, veterinary drug nano self-microemulsion and preparation method and application thereof | |
| CN102106815A (en) | Florfenicol solution and preparation method thereof | |
| CN101879171A (en) | Oral liquid preventing and curing gallinaceous leucocyto zoonosis and preparation method thereof | |
| CN105434341B (en) | A kind of polyadenylic-polyuridylic acid parenteral solution for animals and preparation method thereof | |
| CN101703518A (en) | Pharmaceutical composition of dsRNA and astragalus polysaccharide and application thereof | |
| RU2611387C1 (en) | Method for production of antiparasitic long acting preparation for animals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160622 Address after: 526238 Guangdong Province Wang Zhaoqing City Economic Development Zone Industrial Park abundance Applicant after: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Applicant after: GUANGDONG WENS DAHUANONG BIOTECHNOLOGY CO., LTD. Address before: 526238 Guangdong Province Wang Zhaoqing City Economic Development Zone Industrial Park abundance Applicant before: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Applicant before: Guangdong Dahuanong Animal Health Products Co., Ltd. |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chen Ruiai Inventor after: Zhang Aiguo Inventor after: Liu Yupeng Inventor after: Lai Hanzhang Inventor before: Chen Ruiai Inventor before: Lai Hanzhang Inventor before: Liu Yupeng |
|
| COR | Change of bibliographic data | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20161026 Address after: 510000 Guangdong city of Guangzhou province Tianhe District Wushan Applicant after: South China Agricultural University Applicant after: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Applicant after: GUANGDONG WENS DAHUANONG BIOTECHNOLOGY CO., LTD. Address before: 526238 Guangdong Province Wang Zhaoqing City Economic Development Zone Industrial Park abundance Applicant before: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Applicant before: GUANGDONG WENS DAHUANONG BIOTECHNOLOGY CO., LTD. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |