CN104887812B - A kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus and preparation method thereof and application method - Google Patents
A kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus and preparation method thereof and application method Download PDFInfo
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Abstract
The present invention proposes a kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus and preparation method thereof and application method, solves the problems, such as in the prior art in the market without such anti-porcine reproductive and respiratory syndrome virus drug.Such drug for effectively antagonizing porcine reproductive and respiratory syndrome virus, including the lyophilized preparation made from following raw material：65~75 parts by weight of enteromorpha powder, 6~9 parts by weight of Radix Astragali, 7~12 parts by weight of Radix Isatidis, 3~6 parts by weight of Schisandra chinensis, 2~5 parts by weight of Fructus Forsythiae, 8~13 parts by weight of Herba Andrographitis, 2~5 parts by weight of the coptis, it is respectively enteromorpha powder using raw material, Radix Astragali, Radix Isatidis, Schisandra chinensis, Fructus Forsythiae, Herba Andrographitis, the coptis, the lyophilized preparation of preparation can be substantially reduced the toxicity of pig breeding and respiratory virus, and using the extracting method of decocting boiling, the active constituent of each substance is more fully retained, when with the DMEM liquid mediums of 2 times of concentration by drug dilution to concentration range 40%, pretreatment cell, pig breeds to be suppressed with the proliferation of respiratory virus.
Description
Technical field
The present invention relates to veterinary medicine fields, particularly relate to a kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus
And preparation method thereof and application method.
Background technology
Porcine reproductive and respiratory syndrome (PRRS) is to cause pig by pig breeding and respiratory system syndrome viral (PRRSV)
Using breeding difficulty and respiratory symptom as a kind of highly contagious disease of main feature.The disease can vertical infection but also water
It flates pass and broadcasts, pregnant sow fever, apocleisis, miscarriage, premature labor, the mummification of fetus, young weak and induction piglet and bred pigs can be caused
Various respiratory systemic disease.Guo Baoqing in 1996 et al. determines that it has in China there are PRRS and propagates rapid, prevalence extensively
The strong feature of general and strain variation ability, which causes to seriously threaten to pig breeding industry and huge economic losses, and prevention is mainly by epidemic disease
Seedling prevents, and active drug, Shopping mall there is no to have great difficulty at present.
Invention content
The present invention proposes a kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus and preparation method thereof and uses
Method solves the problems, such as in the prior art in the market without the confrontation such drug of porcine reproductive and respiratory syndrome virus.
The technical proposal of the invention is realized in this way：A kind of medicine effectively antagonizing porcine reproductive and respiratory syndrome virus
Object, including the lyophilized preparation made from following raw material：65~75 parts by weight of enteromorpha powder, 6~9 parts by weight of Radix Astragali, plate are blue
7~12 parts by weight of root, 3~6 parts by weight of Schisandra chinensis, 2~5 parts by weight of Fructus Forsythiae, 8~13 parts by weight of Herba Andrographitis, 2~5 weight of the coptis
Part.
As a further preference, including the lyophilized preparation made from following raw material：70 parts by weight of enteromorpha powder, Huang
8 parts by weight of stilbene, 10 parts by weight of Radix Isatidis, 4 parts by weight of Schisandra chinensis, 3 parts by weight of Fructus Forsythiae, 12 parts by weight of Herba Andrographitis, 3 weight of the coptis
Part.
The present invention proposes a kind of preparation method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus, including with
Lower step：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) raw material is weighed by each parts by weight respectively, the water of 10~15 times of the raw material total amount is added afterwards, is decocted at 90 DEG C
4.2 hours, filter residue, first time boil liquid are obtained after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second afterwards
Boil liquid merges the first time boil liquid and second of boil liquid, is concentrated in vacuo to the 50% of boil liquid volume twice, through bacterium mistake
Filter filtration sterilization, freeze-drying to get.
The present invention proposes a kind of application method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus, feature
It is：Including operating procedure with the DMEM liquid mediums of 2 times of concentration by drug dilution to concentration range 20%~55%, to cell
It is pre-processed, rear Pigs Inoculated Reproductive and respiratory syndrome virus.
Include as a preferred technical solution, 2 times of concentration of operating procedure DMEM liquid mediums by drug dilution to dense
Range 40% is spent, cell is pre-processed, rear Pigs Inoculated Reproductive and respiratory syndrome virus.
The Enteromorpha that the present invention uses is rich in polysaccharide, and sea grass polysaccharide is the main active of Enteromorpha, is present in frond cell
In, having reduces many pharmacological actions such as cholesterol, antibacterial and antiviral, anti-oxidant, enhancing immune function, and external at present grinds
Studying carefully report algal polysaccharides has the function of anti-a variety of viruses.Early-stage study of the present invention confirms that Enteromorpha fights pig breeding and respiratory disease
Poison has apparent blocking and inhibiting effect.
The Radix Astragali that the present invention uses：Enhance immune function, promote organism metabolism, enhances body hypoxia-bearing and resisting stress is made
With, antibacterial and inhibit virus function.
The Radix Isatidis that the present invention uses：It is cold in nature, it is bitter after taste is slightly sweet, have effects that clearing heat and detoxicating, antibacterial anti-inflammatory.
The Schisandra chinensis that the present invention uses：Pulp gas is micro-, sour；After seed is broken, aromatic, acrid flavour, slight bitter can enhance machine
Defence capability and antibacterial action of the body to nonspecific stimulation.Early-stage study of the present invention confirms that Schisandra chinensis has apparent anti-pig
The blocking effect of breeding and respiratory virus.
The Fructus Forsythiae that the present invention uses：It is bitter in taste, cool, major function heat-clearing, removing toxic substances, dissipating bind, detumescence.
The Herba Andrographitis that the present invention uses：It is effective to various bacteria with relieving superficies by cooling, removing toxic substances antibacterial action, to influenza
There is inhibiting effect.Early-stage study of the present invention confirms that Herba Andrographitis confrontation pig breeding has apparent blocking with respiratory virus and inhibits
Effect.
The coptis that the present invention uses：The effect of its taste entrance is extremely bitter, heat-clearing and damp-drying drug, purging intense heat and detonicating.Early-stage study of the present invention
It confirms, the coptis has the blocking effect significantly to pig breeding and respiratory virus.
The present invention is respectively enteromorpha powder, Radix Astragali, Radix Isatidis, Schisandra chinensis, Fructus Forsythiae, Herba Andrographitis and the coptis using raw material, is prepared
Lyophilized preparation can be substantially reduced pig breeding and the toxicity of respiratory virus, and using the extracting method of decocting boiling, more protect completely
The active constituent for staying each substance, it is pre- to locate when with the DMEM liquid mediums of 2 times of concentration by drug dilution to concentration range 40%
Cell is managed, the pig breeding of inoculation and the proliferation of respiratory virus are suppressed.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without having to pay creative labor, may be used also for those of ordinary skill in the art
With obtain other attached drawings according to these attached drawings.
Fig. 1 is experimental group and the inhibition figure that control group is proliferated PRRSV；
Fig. 2 is the inhibition figure that different pharmaceutical concentration is proliferated PRRSV；
Fig. 3 is experimental group and the inhibition figure that control group is proliferated PRRSV.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts
Embodiment shall fall within the protection scope of the present invention.
A kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus, including freeze made from following raw material
Dry preparation：65~75 parts by weight of enteromorpha powder, 6~9 parts by weight of Radix Astragali, 7~12 parts by weight of Radix Isatidis, 3~6 parts by weight of Schisandra chinensis,
2~5 parts by weight of Fructus Forsythiae, 8~13 parts by weight of Herba Andrographitis, 2~5 parts by weight of the coptis.
The experiment material and reagent used in following embodiments：
145 cell lines of Marc are given by Shandong Agricultural University；Trypsase, DMSO are Invitrogen products, new born bovine
Serum is Hangzhou Sijiqing Biological Engineering Material Co., Ltd., and potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride etc. are
Domestic analysis is pure, and Enteromorpha is acquired by Huang Island marine site.
Embodiment one
A kind of preparation method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus, includes the following steps：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) 65 parts by weight of enteromorpha powder, 6 parts by weight of Radix Astragali, 7 parts by weight of Radix Isatidis, 3 parts by weight of Schisandra chinensis, Fructus Forsythiae 2 are pressed respectively
Parts by weight, 8 parts by weight of Herba Andrographitis, 2 parts by weight of the coptis weigh raw material, the water of 10~15 times of the raw material total amount are added afterwards, 90
It is decocted 4.2 hours at DEG C, obtains filter residue, first time boil liquid after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second afterwards
Boil liquid merges the first time boil liquid and second of boil liquid, is concentrated in vacuo to the 50% of boil liquid volume twice, through bacterium mistake
Filter filtration sterilization, freeze-drying to get.
The present invention proposes a kind of application method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus：Including behaviour
Make step with the DMEM liquid mediums of 2 times of concentration by drug dilution to concentration range 20%~55%, cell is pre-processed,
Pigs Inoculated Reproductive and respiratory syndrome virus afterwards.
Specific experimental procedure is as follows：
1) 145 cells of Marc are cultivated on 12 orifice plates to single layer, are arrived drug dilution with the DMEM culture solutions of 2 times of concentration
A concentration of 40% is spare, contrived experiment group A and control group a, and a concentration of 40% liquid is replaced culture solution in hole by experimental group A,
Control group a is not replaced, this two groups are respectively placed in a concentration of 5% carbon dioxide incubator and cultivate 2h；
2) liquid in experimental group A is discarded again, is replaced into maintaining liquid (containing 4% newborn bovine serum), control group equally replaces
Be changed to maintaining liquid, after to this two groups of difference Pigs Inoculated Reproductive and respiratory syndrome virus (0.01MOI), set carbon dioxide incubator
Continue to cultivate, observes cellular change, and harvest virus after 48h；
3) three times by cell plates freeze thawing, 12000rpm centrifuges 15min and collects supernatant, measures two groups of median tissue culture
Infective dose TCID50。
Experimental result as shown in Figure 1, experimental group A TCID50It is 3.89, the TCID of control group a50It is 5.63, experimental group A
Compared with control group a, virus titer reduces, and difference is apparent, and experimental group A is inhibited to the proliferation of PRRSV viruses.
Embodiment two
A kind of preparation method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus, includes the following steps：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) 70 parts by weight of enteromorpha powder, 8 parts by weight of Radix Astragali, 10 parts by weight of Radix Isatidis, 4 parts by weight of Schisandra chinensis, Fructus Forsythiae 3 are pressed respectively
Parts by weight, 12 parts by weight of Herba Andrographitis, 3 parts by weight of the coptis weigh raw material, the water of 10~15 times of the raw material total amount are added afterwards, 90
It is decocted 4.2 hours at DEG C, obtains filter residue, first time boil liquid after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second afterwards
Boil liquid merges the first time boil liquid and second of boil liquid, is concentrated in vacuo to the 50% of boil liquid volume twice, through bacterium mistake
Filter filtration sterilization, freeze-drying to get.
The present invention proposes a kind of application method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus：Including behaviour
Make step with the DMEM culture solutions of 2 times of concentration by drug dilution to concentration range 20%~55%, cell is pre-processed, after
It is inoculated with porcine reproductive and respiratory syndrome virus.
Specific experimental procedure is as follows：
1) 145 cells of Marc are cultivated on 12 orifice plates to single layer, with the DMEM liquid mediums of 2 times of concentration by drug dilution
Spare to a concentration of 10%, 20%, 30%, 50%, it is 20% drug concentration, experimental group C that five groups of design, which is experimental group B respectively,
For 30% drug concentration, experimental group D is 40% drug concentration, experimental group E is 50% drug concentration, control group F, experimental group B, C,
D, the liquid of each concentration is replaced culture solution in hole by E, and control group F is not replaced, this five groups are respectively placed in a concentration of 5% carbon dioxide
2h is cultivated in incubator；
2) liquid in experimental group B, C, D, E is discarded again, is replaced into maintaining liquid (containing 4% newborn bovine serum), control group F
Equally replace with maintaining liquid, after to this five groups of difference Pigs Inoculated Reproductive and respiratory syndrome virus (0.01MOI), set carbon dioxide
Incubator continues to cultivate, and observes cellular change, and virus is harvested after 48h；
3) three times by cell plates freeze thawing, 12000rpm centrifuges 15min and collects supernatant, measures five groups of median tissue culture
Infective dose TCID50。
Experimental result is as shown in table 1, Fig. 2, and four experimental groups are compared with control group F, and virus titer reduces, and difference is apparent,
Experimental group is inhibited to the proliferation of PRRSV viruses.
Table 1
| Experimental group B | Experimental group C | Experimental group D | Experimental group E | Control group F | |
| TCID50 | 4.89 | 4.50 | 3.07 | 3.49 | 5.61 |
Embodiment three
A kind of preparation method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus, includes the following steps：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) 75 parts by weight of enteromorpha powder, 9 parts by weight of Radix Astragali, 12 parts by weight of Radix Isatidis, 6 parts by weight of Schisandra chinensis, Fructus Forsythiae 5 are pressed respectively
Parts by weight, 13 parts by weight of Herba Andrographitis, 5 parts by weight of the coptis weigh raw material, the water of 10~15 times of the raw material total amount are added afterwards, 90
It is decocted 4.2 hours at DEG C, obtains filter residue, first time boil liquid after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second afterwards
Boil liquid merges the first time boil liquid and second of boil liquid, is concentrated in vacuo to the 50% of boil liquid volume twice, through bacterium mistake
Filter filtration sterilization, freeze-drying to get.
The present invention proposes a kind of application method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus：Including behaviour
Make step with the DMEM liquid mediums of 2 times of concentration by drug dilution to concentration range 20%~55%, cell is pre-processed,
Pigs Inoculated Reproductive and respiratory syndrome virus afterwards.
Specific experimental procedure is as follows：
1) 145 cells of Marc are cultivated on 12 orifice plates to single layer, with the DMEM liquid mediums of 2 times of concentration by drug dilution
Spare to a concentration of 40%, a concentration of 40% liquid is replaced in hole and is cultivated by contrived experiment group G and control group g, experimental group G
Liquid, control group g are not replaced, this two groups are respectively placed in a concentration of 5% carbon dioxide incubator and cultivate 2h；
2) liquid in experimental group G is discarded again, is replaced into maintaining liquid (containing 4% newborn bovine serum), control group g is equally replaced
Be changed to maintaining liquid, after to this two groups of difference Pigs Inoculated Reproductive and respiratory syndrome virus (0.01MOI), set carbon dioxide incubator
Continue to cultivate, observes cellular change, and harvest virus after 48h；
3) three times by cell plates freeze thawing, 12000rpm centrifuges 15min and collects supernatant, measures two groups of median tissue culture
Infective dose TCID50。
Experimental result as shown in figure 3, experimental group G TCID50It is 3.96, the TCID of control group g50It is 5.71, experimental group G
Compared with control group g, virus titer reduces, and difference is apparent, and experimental group G is inhibited to the proliferation of PRRSV viruses.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Claims (2)
1. a kind of drug effectively antagonizing porcine reproductive and respiratory syndrome virus, which is characterized in that including former with following parts by weight
Material lyophilized preparation obtained：70 parts by weight of enteromorpha powder, 8 parts by weight of Radix Astragali, 10 parts by weight of Radix Isatidis, 4 parts by weight of Schisandra chinensis, Fructus Forsythiae
3 parts by weight, 12 parts by weight of Herba Andrographitis, 3 parts by weight of the coptis；
The preparation method of the drug, includes the following steps：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) raw material is weighed by each parts by weight respectively, the water of 10~15 times of the raw material total amount is added afterwards, it is small that 4.2 are decocted at 90 DEG C
When, filter residue, first time boil liquid are obtained after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second of boil liquid afterwards,
Merge the first time boil liquid and second of boil liquid, the 50% of boil liquid volume twice is concentrated in vacuo to, through biofilter
Filtration sterilization, freeze-drying to get；
Drug dilution pre-processes cell to concentration range 40% with the DMEM liquid mediums of 2 times of concentration, rear Pigs Inoculated
Reproductive and respiratory syndrome virus.
2. a kind of preparation method for the drug effectively antagonizing porcine reproductive and respiratory syndrome virus as described in claim 1,
It is characterized in that, includes the following steps：
1) fresh Enteromorpha is cleaned, is dried to constant, smashed it through 80 mesh and sieve to obtain enteromorpha powder；
2) raw material is weighed by each parts by weight respectively, the water of 10~15 times of the raw material total amount is added afterwards, it is small that 4.2 are decocted at 90 DEG C
When, filter residue, first time boil liquid are obtained after filtering respectively；
3) water of 10 times of the filter residue weight is added into the filter residue, repeat to decoct and extracts, filters to obtain second of boil liquid afterwards,
Merge the first time boil liquid and second of boil liquid, the 50% of boil liquid volume twice is concentrated in vacuo to, through biofilter
Filtration sterilization, freeze-drying to get；
70 parts by weight of enteromorpha powder, 8 parts by weight of Radix Astragali, 10 parts by weight of Radix Isatidis, 4 parts by weight of Schisandra chinensis, 3 parts by weight of Fructus Forsythiae, punching
12 parts by weight of lotus, 3 parts by weight of the coptis；
Drug dilution pre-processes cell to concentration range 40% with the DMEM liquid mediums of 2 times of concentration, rear Pigs Inoculated
Reproductive and respiratory syndrome virus.
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| CN111700892B (en) * | 2020-07-13 | 2022-08-02 | 江苏农牧科技职业学院 | Application of schisandrin B in resisting PRRSV |
| CN114558078A (en) * | 2022-03-28 | 2022-05-31 | 浙江大飞龙动物保健品股份有限公司 | Broad-spectrum antiviral traditional Chinese medicine injection and preparation method thereof |
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| CN103054999A (en) * | 2013-01-31 | 2013-04-24 | 山东方舟生物科技有限公司 | Traditional Chinese medicine preparation for preventing and treating highly pathogenic porcine reproductive and respiratory syndrome and preparation method |
| CN103478427A (en) * | 2013-09-30 | 2014-01-01 | 中国海洋大学生物工程开发有限公司 | Enteromorpha polysaccharide feed additive as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103054999A (en) * | 2013-01-31 | 2013-04-24 | 山东方舟生物科技有限公司 | Traditional Chinese medicine preparation for preventing and treating highly pathogenic porcine reproductive and respiratory syndrome and preparation method |
| CN103478427A (en) * | 2013-09-30 | 2014-01-01 | 中国海洋大学生物工程开发有限公司 | Enteromorpha polysaccharide feed additive as well as preparation method and application thereof |
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