CN108309973A - Qinghaosu and its derivative are preparing the application in preventing porcine reproductive and respiratory syndrome drug - Google Patents
Qinghaosu and its derivative are preparing the application in preventing porcine reproductive and respiratory syndrome drug Download PDFInfo
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- CN108309973A CN108309973A CN201710055389.3A CN201710055389A CN108309973A CN 108309973 A CN108309973 A CN 108309973A CN 201710055389 A CN201710055389 A CN 201710055389A CN 108309973 A CN108309973 A CN 108309973A
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- China
- Prior art keywords
- derivative
- qinghaosu
- porcine reproductive
- respiratory syndrome
- application
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
Abstract
The invention discloses qinghaosus and its derivative to prepare the application in preventing porcine reproductive and respiratory syndrome drug.Pharmacological testing shows that qinghaosu and its derivative have significant protective effect to the cell for infecting porcine reproductive and respiratory syndrome virus, has significant anti-porcine reproductive and respiratory syndrome virus activity.Qinghaosu and its derivative can prepare the new drug of prevention porcine reproductive and respiratory syndrome as active ingredient, have a extensive future.
Description
Technical field
The present invention relates to veterinary medicine fields, and in particular to is used to prepare the drug of prevention porcine reproductive and respiratory syndrome.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS it) is commonly called as " pig blue-ear disease ", is by porcine reproductive and respiratory syndrome virus (Porcine reproductive and
Respiratory syndrome virus, PRRSV) caused by a kind of new catastrophic disease occurred in swinery, it is clinical
The serious breeding difficulty of sow is shown as, wean pig generally occurs pneumonia, growth retardation and the death rate and increases, and is a kind of height
Communicable disease.PRRSV belongs to shell type virales, the single-stranded RNA virus of Arteriviridae, easy producer recombination, strain friendship
It is low to pitch protectiveness.Its strain is divided into European strain and America strain, caused by the pandemic blue otopathy in China is America strain variation.It should
Disease can vertical infection but also horizontal transmission, pregnant sow fever, apocleisis, miscarriage, premature labor, production the mummification of fetus and weak can be caused
Son, and induce piglet and be bred as a variety of diseases of respiratory diseases in pigs.
The end of the eighties in last century, PRRSV are found for the first time in the U.S., propagate to global most countries and ground rapidly later
Area is in endemic conditions in the whole world.China is isolated to PRRS pathogen in 1996 from pig aborted fetus；2006, China
The high-pathogenicity blue ear disease characterized by piglet high mortality has been broken out, so that China's pig breeding industry is sustained a great loss, is listed in me
State's one kind animal epidemic, and the effect is unsatisfactory for vaccine control.Currently, the prevention and control situation of high-pathogenicity blue ear disease is very tight
The problems such as weight, which easily occurs recombination, immune evasion escape, it is easy the secondary swine fever virus of excitation, pig circular ring virus, swine flu
The infection such as virus, still without effective control method, vaccine immunity is its main precautionary measures in the disease whole world.But clinically make
The problem of being primarily present with PRRS vaccines be：First, vaccine generates immune response and needs the long period；Secondly, PRRSV vaccines are simultaneously
Not having complete protection, especially inactivated vaccine, to be unable to activated cell immune；Third, PRRSV attenuated vaccines can exempt from
There are several weeks in epidemic disease pig body tissue, and are propagated from note seedling pig to susceptible pig.In recent years, the antiviral work of Chinese medicine preparation
With being increasingly valued by people.
Qinghaosu is a kind of colourless acicular crystal extracted from artemisia annua (Artemisia annua L.) leaf, is belonged to
Sesquiterpene lactone chemical combination object, derivative mainly have dihydroartemisinine, Artesunate, Artemether and arteether, and molecular structure is such as
Shown in lower：
Qinghaosu and its derivative are clinically mainly used for the treatment of malaria, also have been reported that other purposes, such as anti-inflammatory, anti-
The pharmacological activity such as tumour, antibacterial.The patent document of Publication No. CN102755316A discloses qinghaosu and its derivative and is making
Application in standby treatment hepatitis C virus cytotoxic drug, the patent document of Publication No. CN1711031A disclose qinghaosu, sweet wormwood
The crude extract of plain analog and some wormwood artemisias treatment flaviviridae (including hepatitis C virus, bovine viral diarrhea virus and
Swine fever virus) caused by infection in application.At present for using qinghaosu and its anti-porcine reproductive and respiratory syndrome of derivative
The effect of virus has not been reported, also not disclosed related patents.
Invention content
To solve the deficiencies in the prior art, it is anti-in preparation that the purpose of the present invention is to provide qinghaosus and its derivative
Application on PRRSV drugs；It is a further object of the present invention to provide qinghaosus and its derivative in preparing prevention PRRS drugs
Application.
The specific experiment technology of the present invention is as follows：
Mtt assay is used to measure qinghaosu and its derivative to monkey embryo renal epithelial cell (Marc-145) and pig alveolar first
The toxicity of macrophage (PAM), determines safe concentration.Then by establishing PRRSV infection cell model, evaluate qinghaosu and its
Protecting effect of the derivative to the Marc-145 cells and PAM cells of infection PRRSV.Evaluation means are mainly indirect immunofluorescence
It tests (IFA), real-time fluorescence quantitative PCR experiment (Real-time PCR) and protein immunoblot and tests (Western
Blot) etc..
The present invention has found that qinghaosu and its derivative have apparent inhibition pig breeding and breathing by cell assay in vitro
The effect that syndrome virus replicates, to provide strong support by medical treatment porcine reproductive and respiratory syndrome.
The present invention provide the application on preparing anti-PRRSV drugs of qinghaosu and its derivative and offer qinghaosu and its
Derivative is preparing the application in preventing PRRS drugs.
Further, pharmaceutical preparation made of adjuvant is equipped with as active ingredient using qinghaosu and its derivative preventing
Application on porcine reproductive and respiratory syndrome.
Further, qinghaosu and its derivative are specially qinghaosu, dihydroartemisinine, Artesunate, Artemether and wormwood artemisia
Ether.
Further, the pharmaceutical preparation is the clinically acceptable dosage form such as injection, pulvis, granule.
The present invention has the following advantages that for preventing porcine reproductive and respiratory syndrome virus：
(1) qinghaosu and its derivative are the first-line drugs for clinically preventing malaria, to safety of human and livestock；Its preparation process at
It is ripe reliable, sufficient raw material can be provided for the preparation of anti-PRRSV drugs.
(2) first demonstration that disclosing the effect of the anti-PRRSV viruses of qinghaosu and its derivative.Pass through a variety of sides
Method research confirms that qinghaosu and its derivative have good protective effect to the cell in vitro that PRRSV viruses infect.Sweet wormwood
Element and its effective component and mechanism of action of the anti-PRRSV viruses of derivative are clear, securely and reliably, breed and exhale in exploitation prevention pig
Inhaling has potential value in syndrome virus new drug.
Description of the drawings
Fig. 1 is the toxicity test knot for measuring qinghaosu and its derivative to Marc-145 cells and PAM cells with mtt assay
Fruit；
Fig. 2 is right in Marc-145 cells with the qinghaosu and its derivative of indirect immunofluorescence analysis various concentration
The inhibiting effect of Porcine reproductive andrespiratory syndrome virus N protein synthesis；
Fig. 3 be Western Blot analysis various concentration qinghaosu and its derivative it is numerous to pig in Marc-145 cells
Grow the inhibiting effect (qinghaosu synthesized with breath syndrome virus N protein：Artemisinin；Dihydroartemisinine：
Dihydroarteannuin；Artesunate：Artesunate；Artemether：Artemether；Arteether：Arteether)；
Fig. 4 be with real-time fluorescence quantitative PCR measure various concentration Artesunate and dihydroartemisinine in cell to pig
The inhibiting effect of Reproductive and respiratory syndrome virus NSP9 gene mRNAs proliferation.
Specific implementation mode
Selected Marc-145 cells, PAM cells and high-pathogenicity porcine reproductive are integrated with breathing in the embodiment of the present invention
GD-XH plants of virus of sign does not do any restriction to inventing included range.The method of the present invention is as follows：
Embodiment 1：The influence of qinghaosu and its derivative to Marc-145 cells and PAM cell viabilities
By Marc-145 cells or PAM cell inoculations after 96 porocyte culture plates grow to single layer, culture medium is discarded,
PBS (phosphate buffer) is cleaned 2 times, is pressed drug dilution to concentration to be measured with the cell maintenance medium containing 2% fetal calf serum
100 holes μ L/ are added, and each concentration does three multiple holes.Continue after cultivating 48h at 37 DEG C, the MTT of 20 μ L 5mg/ml is added per hole
Reagent, 37 DEG C of culture 4h, abandons supernatant, and 150mL DMSO are added, after shaking 10min makes the first a ceremonial jade-ladle, used in libation of generation crystallize fully dissolving,
Light absorption value is detected at 490nm wavelength.It will be seen from figure 1 that qinghaosu and its derivative be under the concentration of 2.5 μ g/mL, Marc-
145 cells and PAM cells have 80% or more survival rate, do not influence the growth of cell.
Embodiment 2：Indirect immunofluorescence assay (IFA) analyzes the qinghaosu of various concentration and its derivative handles Marc-
The inhibiting effect that PRRSV viral N proteins synthesize after 145 cells
By Marc-145 cell inoculations after 6 porocyte culture plates grow to single layer, culture medium is discarded, with PBS (phosphate
Buffer solution) it cleans 2 times, it is added and contains 100 TCID with what the DMEM cell maintenance mediums containing 2% fetal calf serum had diluted50Pig it is numerous
It grows and breath syndrome virus (PRRSV), the holes 1mL/, 37 DEG C of incubation 2h abandons viral supernatants, PBS is cleaned 2 times, is separately added into
The drug of 2.5,1.25,0.6 μ g/mL, the holes 2mL/.Experiment simultaneously be arranged Normal group (being not added with test medicine and PRRSV) and
PRRSV control groups (are not added with test medicine), each tested concentration set three it is parallel.Continue to terminate culture after cultivating 48h at 37 DEG C.
Supernatant is abandoned, PBS is washed 3 times, and the paraformaldehyde that mass concentration is 4%, the holes 500ml/, fixed 10min is added；PBS is cleaned 3 times, is added
Enter the Triton X-100 permeable membranes that mass concentration is 0.25%, is placed at room temperature for 15min；Mass concentration is added to close for 5%BSA
Foreign protein, room temperature close 2h；PBS is washed 3 times, and mouse source N monoclonal antibodies (1: 400 dilution), 4 DEG C of overnight incubations are added；PBS is washed 3 times, is protected from light
Secondary antibody (Alexa is added488 label anti-mouse IgG, 1: 1000 dilution), 37 DEG C are protected from light and are incubated 1h, and PBS is cleaned；Fluorescence
Microscopically observation, green fluorescence represent the N protein of PRRSV.Test result as shown in Fig. 2, the present invention qinghaosu and its spread out
Biology is in the concentration range of 0.6~2.5 μ g/mL, to the N protein of the intracellular porcine reproductive and respiratory syndrome virus of Marc-145
Synthesis has different degrees of inhibiting effect, and dose-effect relationship is presented.
Embodiment 3：It is right after Western Blot analysis various concentration qinghaosus and its derivative processing Marc-145 cells
PRRSV viral N proteins expression quantity situations of change
The step of Marc-145 cell infections PRRSV, addition drug, is identical as step 2, after 37 DEG C of culture 48h of cell, abandons
Supernatant is washed 2 times with PBS.Tissue culture plate is put on ice for, 150 holes μ L/ of RIPA lysates are added, after piping and druming cracks
Liquid is sucked out into centrifuge tube, 12000 revs/min centrifuge 5 minutes, supernatant are sucked out spare into another clean pipe.It presses
Each sample albumen concentration is measured according to the method for BCA determination of protein concentration kits.The N eggs of PRRSV are detected with Western Blot
White and internal reference Protein G APDH band.Test result as shown in figure 3, qinghaosu and its derivative in the dense of 0.6~2.5 μ g/mL
It spends in range, there is different degrees of inhibition to make the N protein synthesis of the intracellular porcine reproductive and respiratory syndrome virus of Marc-145
With, and dose-effect relationship is presented.
Embodiment 4：To the inhibiting effect of PRRSV virus multiplications after Artesunate, dihydroartemisinine processing cell
By above-mentioned experiment, Artesunate and dihydroartemisinine are chosen as drug is represented and carries out the 48h tests of pesticide effectiveness.It will
Marc-145 cells or PAM cell inoculations discard culture medium after 6 porocyte culture plates grow to single layer, with PBS (phosphate
Buffer solution) it cleans 2 times, it is added and contains 100 TCID with what the DMEM cell maintenance mediums containing 2% fetal calf serum had diluted50Pig it is numerous
It grows and breath syndrome virus (PRRSV), the holes 1mL/, 37 DEG C of incubation 2h abandons viral supernatants, PBS is cleaned 2 times, is separately added into
The drug of 2.5,1.25,0.6 μ g/mL, the holes 2mL/.Experiment simultaneously be arranged Normal group (being not added with test medicine and PRRSV) and
PRRSV control groups (are not added with test medicine), each tested concentration set three it is parallel.Continue to terminate culture after cultivating 48h at 37 DEG C.
It observes its disease and keeps a record, three times in -80 DEG C and 4 DEG C of multigelation cell plates, cell is made fully to crack, intracellular
It is viral all to discharge into cell conditioned medium, then collect each hole supernatant.With the very fast extraction agent box of total serum IgE, (Shanghai flies prompt raw
Object Technology Co., Ltd.) step to specifications, the cell supernatant being collected into is subjected to total serum IgE extracting.Again reverse transcription at
CDNA, using the cDNA as template, GAPDH is reference gene, the copy number of the NSP9 genes of Real Time PCR detections PRRSV；
Reference is done with Normal group, evaluates the situation of change of virus N SP9 gene mRNAs.
As shown in figure 4, compared with virus-infected controls group, dihydroartemisinine and Artesunate are in a concentration of 0.6~2.5 μ
In the range of g/mL, have to the RNA proliferation of Marc-145 cells or the intracellular porcine reproductive and respiratory syndrome virus of PAM significant
Inhibiting effect, and good dose-effect relationship is presented.
Embodiment 5：Qinghaosu and its derivative prepare the drug of prevention porcine reproductive and respiratory syndrome as active constituent
Preparation：Tablet
By the qinghaosu of the present invention or any type derivative (1g) and lactose (20g) and microcrystalline cellulose (6g)
It is mixed with mixing machine.Gained mixture is pressed into flake with roller bearing compacting machine, is pressed the flake with beater grinder
Material processed is pulverized, and gained granular material is made to pass through 20 mesh sieves.By a light silicon dioxide (0.3g) and stearic acid
Magnesium (0.3g) is added in screened material, mixing.The pelleter tabletting of gained mix products, prepares tablet.
Claims (3)
1. qinghaosu and its derivative are in the application for preparing prevention porcine reproductive and respiratory syndrome drug.
2. application according to claim 1, which is characterized in that be equipped with auxiliary as active ingredient using qinghaosu and its derivative
Application of the pharmaceutical preparation made of agent on prevention porcine reproductive and respiratory syndrome.The pharmaceutical preparation be injection, pulvis,
Acceptable any dosage form on the veterinary clinics such as granula, tablet.
3. application according to claim 1, which is characterized in that the qinghaosu and its derivative be specially qinghaosu, double
Hydrogen arteannuin, Artesunate, Artemether and arteether.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710055389.3A CN108309973A (en) | 2017-01-17 | 2017-01-17 | Qinghaosu and its derivative are preparing the application in preventing porcine reproductive and respiratory syndrome drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710055389.3A CN108309973A (en) | 2017-01-17 | 2017-01-17 | Qinghaosu and its derivative are preparing the application in preventing porcine reproductive and respiratory syndrome drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108309973A true CN108309973A (en) | 2018-07-24 |
Family
ID=62892277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710055389.3A Pending CN108309973A (en) | 2017-01-17 | 2017-01-17 | Qinghaosu and its derivative are preparing the application in preventing porcine reproductive and respiratory syndrome drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108309973A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111714493A (en) * | 2020-07-09 | 2020-09-29 | 江苏农牧科技职业学院 | Application of dihydroartemisinin in preventing and treating porcine reproductive and respiratory syndrome |
| CN112294795A (en) * | 2020-11-17 | 2021-02-02 | 北京化工大学 | Application of artemether in treating and preventing coxsackie virus infection |
| CN114404439A (en) * | 2022-02-11 | 2022-04-29 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
-
2017
- 2017-01-17 CN CN201710055389.3A patent/CN108309973A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 刘 樱等: "３种中药及其提取物体外抗猪繁殖与呼吸综合征病毒作用的研究", 《中国畜牧兽医》 * |
| 田朝晖等: "板蓝根、黄芪和青蒿提取物抗PRRSV作用比较", 《中国饲料》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111714493A (en) * | 2020-07-09 | 2020-09-29 | 江苏农牧科技职业学院 | Application of dihydroartemisinin in preventing and treating porcine reproductive and respiratory syndrome |
| CN112294795A (en) * | 2020-11-17 | 2021-02-02 | 北京化工大学 | Application of artemether in treating and preventing coxsackie virus infection |
| CN112294795B (en) * | 2020-11-17 | 2022-03-29 | 北京化工大学 | Application of artemether in treating and preventing coxsackie virus infection |
| CN114404439A (en) * | 2022-02-11 | 2022-04-29 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
| CN114404439B (en) * | 2022-02-11 | 2023-07-11 | 山东农业大学 | Blocking agent for inhibiting different types of porcine reproductive and respiratory syndrome virus infection |
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Application publication date: 20180724 |
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