CN108309972A - Pogostone is preparing the application in preventing porcine reproductive and respiratory syndrome drug - Google Patents
Pogostone is preparing the application in preventing porcine reproductive and respiratory syndrome drug Download PDFInfo
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- CN108309972A CN108309972A CN201710055364.3A CN201710055364A CN108309972A CN 108309972 A CN108309972 A CN 108309972A CN 201710055364 A CN201710055364 A CN 201710055364A CN 108309972 A CN108309972 A CN 108309972A
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- pogostone
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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
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Abstract
The invention discloses Pogostones to prepare the application in preventing porcine reproductive and respiratory syndrome drug.Extraction obtains Pogostone from Chinese medicine Pogostemon cablin, by the methods of immunofluorescence microscopy analysis, RT PCR and Western blot confirmation Pogostone there is good anti-PRRSV activity on 145 cells of Marc, can be used as the drug that active ingredient prepares prevention porcine reproductive and respiratory syndrome.
Description
Technical field
The present invention relates to veterinary medicine fields, and it is comprehensive in preparation prevention pig breeding and breathing to be more particularly to a kind of Pogostone
The purposes of simulator sickness drug.
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) it is caused using breeding difficulty and respiratory symptom as one kind of main feature
Highly contagious disease.The disease can vertical infection but also horizontal transmission, pregnant sow fever, apocleisis, miscarriage, morning can be caused
Production, production the mummification of fetus and weak son, and induce piglet and be bred as a variety of diseases of respiratory diseases in pigs, it is brought to global pig breeding industry huge
Big loss.Active drug of the approval for treating the disease is had no mainly by vaccine immunity for the prevention and control of PRRS at present.But by
In the variability of PRRSV, the effect is unsatisfactory for the prevention ＆ protection of commercial vaccine clinically.Therefore, how PRRS is prevented
It is current urgent problem to be solved.The natural resources of Chinese medicinal materials in China is abundant, and utilization is with a long history, is found from natural resources of Chinese medicinal materials potential anti-
Virus drugs have become hot spot.
Pogostone (Pogostone) is a kind of monomer obtained from the volatile oil of labiate Pogostemon cablin, is changed
Scientific name is 4- hydroxyl -6- methyl -3- (4- methyl-1s-oxopentyl) -2H- pyran-2-ones, molecular formula C12H16O4, molecular weight
It is 224.254, molecular structure is as follows：
Pogostone has inhibiting effect (Yang get Po etc., the leaves of pulse plants to fungies such as Candida albicans, Cryptococcus neoformans, rhizopus nigers
The fragrant and anti-skin bacterium activity of Herba Pogostemonis Volatile oil and research [J] .1998. (4) of chemical composition：1-4.16)；To large intestine bar
Bacterium, Pseudomonas aeruginosa, hay bacillus, saccharomycete, Penicillium notatum etc. have inhibiting effect (hole magnitude in heptan, the separation of Pogostone and its
Application [J] Chinese Hospitals pharmacy in medicament contaminates will, 1986. (01)：32-33)；It is curved to rhizoctonia, Fusarlum moniliforme, corn
6 kinds of phytopathogens such as spore leaf spot fungi, sclerotinite, Chinese cassia tree stey and kind silly early epidemic germ also have inhibiting effect (not path
[J] Chinese medicines, 2004. (11) are studied to the bacteriostatic activity of plant pathogenic fungi Deng, Pogostemon cablin essential：805-807).Middle promulgated by the State Council
Bright patent CN201310141084.6 discloses purposes of the palchouli oil in preparing antiviral drugs, and treatment is caused by virus infection
Myocarditis and pneumonia.
The Effect study report of the anti-porcine reproductive and respiratory syndrome virus of Pogostone is had no at present, also not disclosed phase
Close patent.
Invention content
To solve the deficiencies in the prior art, the purpose of the present invention is to provide Pogostones on preparing anti-PRRSV drugs
Application；It is a further object of the present invention to provide Pogostones to prepare the application in preventing PRRS drugs.
For achieving the above object, the present invention adopts the following technical scheme that：
The isolated Pogostone from wrinkled giant hyssop, and identify its structure.
Pogostone is measured to the toxicity of Marc-145 cells with CCK-8 methods, determines safe concentration.Then pass through foundation
PRRSV infection Marc-145 cell models, evaluation Pogostone is to infecting the protecting effect of Marc-145 cells of PRRSV and right
The inhibiting effect of virus replication.Evaluation means are mainly indirect immunofluorescence assay (IFA), real-time fluorescence quantitative PCR experiment
(Real-time PCR) and protein immunoblot are tested (Western blot) etc..
The present invention has found that Pogostone has apparent inhibition porcine reproductive and respiratory syndrome virus by cell assay in vitro
The effect of duplication, to provide strong support by medical treatment porcine reproductive and respiratory syndrome.
The present invention provides application and offer of the Pogostone on preparing anti-porcine reproductive and respiratory syndrome virus drug
Pogostone is preparing the application in preventing porcine reproductive and respiratory syndrome drug.
Further, porcine reproductive and respiratory syndrome is including infection highly pathogenic PRRSV
American type strain caused by disease.
Further, be equipped with using Pogostone as active ingredient pharmaceutical preparation made of adjuvant prevention pig breeding with
Application on respiration syndrome.
Further, the pharmaceutical preparation is acceptable on any types veterinary clinics such as injection, pulvis, granule
Dosage form.
Compared with prior art, the present invention has the following advantages and beneficial effects：
(1) for Chinese medicine Pogostemon cablin in tcm clinical practice and civil extensive use, biological safety is high.Pogostone is Pogostemon cablin
Main pharmacodynamics ingredient, content is higher in Pogostemon cablin, and mature preparation process is reliable, can be provided for the preparation of anti-PRRSV drugs
Sufficient raw material.
(2) present invention firstly discloses the effects of the anti-PRRSV viruses of Pogostone.It is confirmed by a variety of technique studies
Pogostone has good antiviral effect in cellular level to PRRSV viruses.The effective component of the anti-PRRSV viruses of Pogostone
And mechanism of action is clear, and it is quality controllable, it is safe and non-toxic, before there is application well in terms of the porcine reproductive and respiratory syndrome prevention
Scape.
Description of the drawings
Fig. 1 is the test result that the CCK-8 methods of embodiment 2 measure Pogostone to Marc-145 cytotoxicities；
Fig. 2 is that embodiment 3 is bred and exhaled to pig in cell with real-time fluorescence quantitative PCR measurement various concentration Pogostone
Inhale the inhibiting effect of syndrome virus RNA proliferation；
Fig. 3 is that embodiment 4 is bred and exhaled to pig in cell with the Pogostone of Western Blot analysis various concentrations
Inhale the inhibiting effect of syndrome virus N protein synthesis；
Fig. 4 be embodiment 5 with indirect immunofluorescence analyze the Pogostone of various concentration in cell to pig breeding with
The inhibiting effect of breath syndrome virus N protein synthesis.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Selected GD-XH plants of Marc-145 cells and highly pathogenic PRRSV be not right in the embodiment of the present invention
The included range of invention does any restriction.
Embodiment 1：The extraction of Pogostone
Pogostemon cablin herb 50kg obtains pale yellow oil 1.78kg through steam distillation, rectification under vacuum, at 1.3KPa,
159-163 DEG C of fraction 345g is obtained, silica gel column chromatography is carried out, is eluted with petroleum ether (30-60 DEG C)-ether gradient, is merged
10% ether-petroleum ether eluent, then through silica gel column chromatography, obtain colorless needles (260mg), 32.5-33 DEG C of fusing point, through matter
Spectrum and hydrogen nuclear magnetic resonance spectrum analysis are determined as Pogostone.
Embodiment 2：CCK-8 methods measure cytotoxicity of the Pogostone to Marc-145
After Marc-145 cells after 96 porocyte culture plates grow to single layer, culture medium is discarded, (phosphate is slow with PBS
Fliud flushing) it cleans 2 times, the wide leaves of pulse plants for the various concentration that 100 μ L have been diluted with the DMEM cell maintenance mediums containing 2% fetal calf serum is added
Ketone musk, each concentration do three multiple holes.Continue after cultivating 48h at 37 DEG C, 10 μ LCCK-8 (Japanese colleague's chemistry) examination is added per hole
Agent, 37 DEG C of culture 2h or so, detects light absorption value at 450nm wavelength.It will be seen from figure 1 that Pogostone is in the dense of 10 μ g/mL
The lower proliferation that will not inhibit Marc-145 cells of degree.
Embodiment 3：Real-time fluorescence quantitative PCR measures Pogostone and breeds to pig in Marc-145 cells and breathe synthesis
Levy the inhibiting effect of viral RNA proliferation
After Marc-145 cells after 6 porocyte culture plates grow to single layer, culture medium is discarded, with PBS (phosphate-buffereds
Liquid) it cleans 2 times, it is added and contains 100TCID with what the DMEM cell maintenance mediums containing 2% fetal calf serum had diluted50Pig breeding with
Breath syndrome virus (PRRSV), 600 holes μ L/, 37 DEG C of incubation 2h discard viral supernatants, and PBS is cleaned 2 times, is separately added into 12,
The Pogostone of 6,3 μ g/mL, the holes 2mL/.It tests while setting Normal group (being not added with test medicine, be not added with PRRSV) and PRRSV
Control group (is not added with test medicine), each tested concentration set three it is parallel.Cell continues 37 DEG C of cultures, until 48h is terminated after infection
Culture.After observing its disease, three times in -80 DEG C and 4 DEG C of multigelation cell plates, so that cell is fully cracked, cause cell
Interior virus all discharges into cell conditioned medium, then collects each hole supernatant.With the very fast extraction agent box of total serum IgE, (Shanghai flies
Prompt Bioisystech Co., Ltd) recommend operating method, by the cell supernatant being collected into carry out total serum IgE extracting.After extracting RNA
Reverse transcription is carried out immediately into cDNA, and using the cDNA as template, GAPDH is reference gene, Real Time PCR detections PRRSV's
The copy number of NSP9 genes；Reference is done with Normal group, evaluates the situation of change of NSP9mRNA.
PRRSVNSP9 gene upstream and downstream primer sequences：
NSP9-F：5′-CTAAGAGAGGTGGCCTGTCG-3′
NSP9-R：5’-GAGACTCGGCATACAGCACA-3’
GAPDH gene upstream and downstream primer sequences：
GAPDH-F：5’-GTCAGTGGTGGACCTGACCT-3’
GAPDH-R：5’-TGCTGTAGCCAAATTCGTTG-3’
Test result is as shown in Fig. 2, " * * * " indicates poor heteropolar notable (P ＜ 0.001) with virus-infected controls group.
The drug Pogostone of the present invention breeds the intracellular pigs of MARC-145 and breathes synthesis in the concentration range of 3~12 μ g/mL
The RNA proliferation of sign virus has significant inhibiting effect, and good dose-effect relationship is presented.
Embodiment 4：Western Blot analyze Pogostone in Marc-145 cells to porcine reproductive and respiratory syndrome
The inhibiting effect of viral N proteins synthesis
The step of Marc-145 cell infections PRRSV, addition drug, is same as Example 3, cell culture 48h to after infecting
Culture is terminated, liquid is discarded supernatant, is washed 2 times with PBS.Tissue culture plate is put on ice for, 150 holes μ L/ of RIPA lysates are added,
Liquid is sucked out into centrifuge tube after piping and druming, 12000 revs/min centrifuge 5 minutes, and supernatant is sucked out into another clean pipe
It is spare.After measuring each sample albumen concentration with BCA methods, the N protein and internal reference albumen of PRRSV are detected using Western Blot
The band of GAPDH.Test result as shown in figure 3, the present invention drug Pogostone in the concentration range of 3~12 μ g/mL, it is right
The N protein synthesis of the intracellular porcine reproductive and respiratory syndrome virus of Marc-145 has significant inhibiting effect, and presents good
Dose-effect relationship.
Embodiment 5：Indirect immunofluorescence analyzes Pogostone and breeds to pig in Marc-145 cells and breathe synthesis
Levy the inhibiting effect of viral N proteins synthesis
Marc-145 cells grow to single layer in 96 porocyte culture plates, the step of infecting PRRSV and drug is added and reality
It is identical to apply example 1.Cell culture to 48h terminations are cultivated after the Pogostone of various concentration is added.Liquid is discarded supernatant, PBS is washed 3 times,
The 500 μ L of paraformaldehyde that mass concentration is 4% are added per hole and fix 10min；PBS is cleaned 3 times, and it is 0.25% that mass concentration, which is added,
Triton X-100 permeable membranes, place 15min at room temperature；It is that 5%BSA closes foreign protein, room temperature closing that mass concentration, which is added,
2h；PBS is cleaned 3 times, and mouse source N monoclonal antibodies (1: 400 dilution), 4 DEG C of overnight incubations are added；PBS is cleaned 3 times, is protected from light addition secondary antibody
(Alexa488 label anti-mouse IgG, 1: 1000 dilution), 37 DEG C are protected from light and are incubated 1h, and PBS is cleaned；Under fluorescence microscope
Observation, green fluorescence represent the N protein of PRRSV.Test result as shown in figure 4, the present invention drug Pogostone in 3~12 μ
In the concentration range of g/mL, there is significant suppression to the N protein synthesis of the intracellular porcine reproductive and respiratory syndrome virus of Marc-145
It makes and uses, and good dose-effect relationship is presented.
Embodiment 6：Using Pogostone as the preparation of the anti-porcine reproductive and respiratory syndrome granule of active ingredient
Weigh Pogostone 1g, auxiliary material (dextrin: mannitol=1: 1) 20g is mixed, it is wetting that 95% ethyl alcohol 10mL, which is added,
Agent mixes well obtained softwood, crosses the sieve granulation of 10 mesh, 60 DEG C of drying, and Pogostone granule is made, through liquid phase in whole grain of being sieved
Chromatography content is 4.6%.
Claims (3)
1. a kind of Pogostone that structure is following is in the application for preparing prevention porcine reproductive and respiratory syndrome drug：
2. application according to claim 1, which is characterized in that porcine reproductive and respiratory syndrome includes infection high-pathogenicity porcine
The disease caused by american type strain including Reproductive and respiratory syndrome virus.
3. application according to claim 1, which is characterized in that be equipped with adjuvant using Pogostone as active ingredient and be made
Pharmaceutical preparation prevention porcine reproductive and respiratory syndrome on application.The pharmaceutical preparation is injection, pulvis, granule etc.
Acceptable dosage form on any type veterinary clinic.
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| CN201710055364.3A CN108309972B (en) | 2017-01-17 | 2017-01-17 | Application of patchoulenone in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome |
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| CN201710055364.3A CN108309972B (en) | 2017-01-17 | 2017-01-17 | Application of patchoulenone in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112336717A (en) * | 2020-12-09 | 2021-02-09 | 河南农业大学 | Application of yiquincotine in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome |
| CN117322422A (en) * | 2023-10-07 | 2024-01-02 | 华中农业大学 | Application of patchoulenone in preventing and treating false smut |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112336717A (en) * | 2020-12-09 | 2021-02-09 | 河南农业大学 | Application of yiquincotine in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome |
| CN112336717B (en) * | 2020-12-09 | 2022-07-29 | 河南农业大学 | Application of yiquincotine in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome |
| CN117322422A (en) * | 2023-10-07 | 2024-01-02 | 华中农业大学 | Application of patchoulenone in preventing and treating false smut |
| CN117322422B (en) * | 2023-10-07 | 2024-03-29 | 华中农业大学 | Application of patchoulenone in preventing and treating false smut |
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