CN102274206B - Application of germacrone in preparation of medicament for treating or preventing influenza viruses - Google Patents
Application of germacrone in preparation of medicament for treating or preventing influenza viruses Download PDFInfo
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- CN102274206B CN102274206B CN 201110169121 CN201110169121A CN102274206B CN 102274206 B CN102274206 B CN 102274206B CN 201110169121 CN201110169121 CN 201110169121 CN 201110169121 A CN201110169121 A CN 201110169121A CN 102274206 B CN102274206 B CN 102274206B
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- methylethylidene
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Abstract
The invention discloses application of germacrone in preparation of a medicament for treating or preventing influenza viruses. The cell toxicity of the germacrone on madin-darby canine kidney (MDCK) cells is detected, and the result shows that CC50 is more than 250 microns. The influence of the germacrone on the expression of three antigens and the influence of germacrone on virus genome reproduction are detected, and the result shows that the germacrone formulation inhibits the expression of the virus antigens and the virus genome reproduction with dependency. The virus activity resistant mechanism of the germacrone is preliminarily researched. The virus activity resistance stage of the germacrone in a single reproduction period of the influenza viruses is researched by administration experiments of different time, and the result shows that the germacrone has inhibiting effect on adsorption, entry and early reproduction of the viruses. Moreover, the virus activity resistance of the germacrone to amantadine medicament resistant strains, B-type influenza virus strains and A-type influenza virus H3N2 subtype strains is detected, and the result shows that the germacrone has virus activity resistance to the three kinds of virus strains. The germacrone has the effects of relieving cough, relieving asthma and the like.
Description
Technical field
The invention belongs to medical technical field, more specifically relate to the application of a kind of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment or flu-prevention virus drugs.
Background technology
Influenza virus (influenza virus) has a strong impact on human health, can cause grippal pathogen.Different according to the antigenic characteristic of virion nucleoprotein (NP) and stromatin (M) and genetic characteristics, influenza virus is divided into A, B, C three types, also claims first, second, the third three types.All there is significant difference in aspects such as the genome structure of these three kinds of veriform viruses, polypeptide form, infectious and pathogenic.The full genome of A type influenza virus is made up of 8 sub-thread strand RNAs that differ in size, and names with sections 1 to sections 8 respectively.The about 13.6kb of viral genome total length, encode respectively 10 kinds of structural protein (PB2, PB1, PA, HA, NP, NA, M1, M2, PB1-F2 and NS2/NEP) and non-structural protein (NS1).Different according to virion surface glycoprotein hemagglutinin (HA) and neuraminidase (NA), A type influenza virus can be further divided into 16 H (H1-H16) and 9 N (N1-N9) hypotype.The human influenza virus mainly is H1, H2 and H3 hypotype, mostly is H5, H7 and H9 hypotype and endanger serious high pathogenic avian influenza at present, and is wherein the highest with the fatality rate of H5N1 hypotype.The Type B influenza virus often causes influenza localized epidemics, does not cause worldwide influenza great outburst, and does not find its existence so far in other animal except the people.C type influenza virus is many to be existed to be dispersed in form, mainly attacks infant, does not generally cause influenza pandemic, can infect the mankind and pig.
Influenza virus belongs to orthomyxoviridae family (Orthomyxoviridae), Influenza Virus at viral taxonomy.Influenza virus is segmented minus-stranded rna virus, so viral RNA does not have infectivity, each RNA sections must form vRNPs in conjunction with the back with RNA polymerase (PB2, PB1 and PA) and nucleoprotein (NP) just activity.That infects initially is the HA furcella identification on virion surface and contains sialic receptor in conjunction with host cell surface.The connecting key of sialic acid and time terminal galactose has determined viral host specificity, and this connecting key is α (2-3) in birds, is α (2-6) in the mankind.In the influenza virus HA protein molecular, indivedual amino acid whose replacements and the different glycosylation modified specificitys that can change its receptors bind.Behind the virus absorption onto cell, cell is taken in virus by the receptor-mediated pinocytosis of clathrin.In cell, the clathrin molecular dissociation, virus merges with endosome and forms phagosome, makes that the pH value around the virion drops to about 5.0.Under this pH condition, the conformation of viral HA albumen changes, and the fusogenic peptide that is positioned at light chain (HA2) N end is exposed, thereby causes that virus envelope and cell membrane merge.Low pH environment also causes a large amount of H
+Enter virion inside via the M2 ion channel, cause dissociating of M1 albumen and vRNPs.Both common results cause the vRNPs of virion to be discharged into the endochylema of infected cell.Different with other RNA viruses, the copying and transcribe in the nucleus that all occurs in host cell of influenza virus gene group.Transfer to endochylema, structural protein and the non-structural protein of synthetic virus after mRNA is synthetic in nuclear.Then begin to assemble progeny virus, the glycoprotein of neuraminidase hydrolyzable cell surface discharges the N-n acetylneuraminic acid n, impels virion to discharge from the site of sprouting.The final step of virus maturation is that HA is cracked into HA1 and HA2 polypeptide under the effect of host protein enzyme, and such virion just has infectivity, thereby begins copying of new round virus.
Influenza virus is popular the propagation more than 300 year in the mankind, and the several years i.e. outbreak of epidemic once, and global flu outbreak then many decades breaks out once.Influenza pandemic can cause 250,000～500,000 examples dead every year, 3,000,000～5,000,000 severe disease examples, and it is infected that the whole world has 5～15% people approximately.For global flu outbreak, " vaccine and antiviral drugs are to reduce the most important counter-measure of M ﹠ M in epidemic period ".300,000,000 person-portion trivalent influenza vaccines-this only reaches the usefulness of Hesperian influenza prevention but can produce at present global every year, and can not satisfy the demand of global flu outbreak, and because the antigenic variation ability of influenza virus is strong, before occurring, novel strain can not develop vaccine.The extensive research and development of a common vaccine and manufacturing need six months at least, even till that time, because worldwide production is limited in one's ability and production facility concentrates on developed country, many do not have the country of production facility will use less than vaccine between the departure date at the 1st wave current.So the protective rate that current vaccine can provide is not high.Anti-influenza virus medicament not only comes out later, and medicines quick-acting, produce effects are also less clinically, belong to the little kind in the anti-infection drug.At present, the anti-influenza virus medicament through food and drug administration (FDA) approval official listing has two classes: (1) is the M2 ion channel blocking agent of representative with amantadine and rimantadine.This type of medicine only has preventive and therapeutic action to influenza A virus, studies show that medicine has toxic and side effects such as neurotoxicity, and owing to making the persister ubiquity, so CDC advises that this type of medicine is not used further to the flu-prevention viral infection.(2) neuraminidase inhibitor, the representative of this type of medicine are Ao Sitawei and Zha Lamiwei.This type of medicine is all effective to all known human influenza viruses and high pathogenic avian influenza virus.But the persister about Ao Sitawei really constantly has report in recent years.The active component of Chinese medicine is alkaloid, flavonoid, volatile oil, saponin etc., these components have diversified pharmacologically active, such as suppress virus replication, stop pathological changes caused by virus, regulate immunologic function, improve pulmonary circulation, comprehensive effect such as antalgic and inflammation relieving, and toxic and side effects is little, so Chinese medicine has special advantages and vast potential for future development aspect the influenza preventing and treating.
The Chinese medicine Rhizoma Curcumae is the rhizome of Zingiberaceae curcuma Rhizoma Curcumae, Guangxi zedoary and RADIX CURCUMAE.Rhizoma Curcumae has circulation of qi promoting, removing blood stasis, removing food stagnancy, the effect of pain relieving.Can treat the Zhi lumps in the chest and abdomen, blood stasis amenorrhea, traumatic injury, eating accumulation, abdominal distention.Main component in the Rhizoma Curcumae volatile oil has curcumenol, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and curdione etc., and volatile oil is to mice
Sarcoma-180 have stronger inhibitory action.Curcumenol has better curative effect to people's cervical cancer.Rhizoma Curcumae volatile oil also has antiinflammatory, hepatoprotective effect, and volatile oil has inhibitory action to staphylococcus aureus, Hemolytic streptococcus, escherichia coli.In addition, the bibliographical information 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has effects such as antitussive and antiasthmatic.But in the bibliographical information application of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-(Germacrone) aspect resisiting influenza virus do not appear also at present.
Summary of the invention
The objective of the invention is to be to provide the application of a kind of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment or flu-prevention virus (first type or B-mode) medicine.3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-claims big Mang cattle ketone, germacrone again, is the derivant of germacrane.Plant origin has the volatile oil of zingiberaceous plant Rhizoma Curcumae, the leaf of ericad Folium Cuculus polioephalus, various plants such as aristolochiaceae plant bilobate Herba Asari volatile oil.The content higher (20%～50%) of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in the plant volatile oil of source, and have various active, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has effects such as antimicrobial antiphlogistic, antitumor, antidepressant and antitussive and antiasthmatic according to the literature.3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is to A type strains of influenza viruses antiviral activity height, and suppression ratio reaches 88.4% when 50 μ M; Toxicity is little, and under 250 μ M 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-s were handled, MDCK behind the 48h (Madin-Darby canine kidney cell, influenza virus sensitive cells) survival rate was 100%, thereby provides a kind of safe and effective toxic and side effects little natural product for influenza virus property treatment of diseases clinically.
In order to realize above-mentioned purpose, the present invention by the following technical solutions:
The application of a kind of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment or flu-prevention virus (first type or B-mode) medicine the steps include:
At first, detected the cytotoxicity of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to mdck cell by mtt assay, the result is presented in the 250 μ M concentration, and medicine is to the cell avirulence.Secondly, after detecting virus infected cell and giving drug treating 48h, detected the viral load (detecting based on virus specific antigen neuraminidase and hemagglutinin) that discharges in the supernatant respectively, the result shows that medicine is in 1.56～50 μ M concentration, reduced the viral load that is discharged in the supernatant, suppression ratio is dose-dependent effect.We have also detected the influence of medicine to viral protein expression, the result shows that equally 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has suppressed the expression of virus protein, also detected simultaneously the influence that medicine copies viral nucleic acid, the result is shown to after the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-processing, and the synthetic of viral nucleic acid also obviously is suppressed.In addition, by the administration of viral infection different time, preliminary analysis the mechanism of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-antiviral activity, namely detected 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is brought into play antiviral activity in viral single-wheel replicative cycle stage, the results suggest 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has suppressed that viral absorption enters and the commitment of virus replication, but to not having activity or more weak activity the late period (assembling of virion and release) of virus replication.At last, we have detected the broad spectrum activity of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-anti-influenza virus activity, detected the antiviral activity of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to A type influenza virus H3N2 hypotype, A type influenza virus amantadine persister and Influenza B virus strain, experimental result prompting 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-all has activity to detecting Strain, and has dose-dependent effect.The Comprehensive Experiment interpretation of result determines that the medicine 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-can be used for the infection of prevention and treatment influenza virus.
The dosage of described 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is 1.56～50 μ M according to the cell experiment consumption.
Described 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is made the resisiting influenza virus compound medicine.
Described 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is made anti-influenza virus medicament separately.
Described influenza virus is first type or Influenza B virus.
Described influenza virus also has third type, and third type is less to the anthropogenic influence.
The present invention suppresses to detect by the neuraminic acid enzymatic activity and blood clotting suppresses experiment (HI) to viral infection and the viral load that is secreted in the cell culture fluid after giving drug treating detects, detect the expression of intracellular virus albumen simultaneously by indirect immunofluorescence (IFA), also detected the expression of viral nucleic acid by Semiquantitative reverse transcription PCR (Semi-quantitative RT-PCR).The result shows 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to the secretion of virion, and copying of the expression of virus protein and viral nucleic acid all has good inhibitory effect.By having detected medicine in the administration of viral infection different time to the effect of the different links of viral single-wheel replicative cycle, the result shows that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-all has an antiviral activity in early days to what the absorption of virus and invasion, viral gene copied.Experimental result shows that this medicine all has the active drug that is developed to resisiting influenza virus and is applied to clinically, and can be used as further open bright prospects of potential clinically antiviral drugs.
Utilize modern common drug preparation means, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-can be made capsule, granule, dispersant etc., thus adopt oral administration form eaily.
The present invention compared with prior art has the following advantages and effect:
1. 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is a kind of natural origin micromolecular compound, no cytotoxicity in 1.35～50 μ M concentration, its CC
50More than 250 μ M.
2. 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-dosage relies on ground inhibition influenza infection, its EC
50Be 6 μ M.Selection index is about 40.
3. to H3N2, Type B influenza virus and amantadine persister all have the resisiting influenza virus effect.
4. Orally-administrable is easy to use.The result shows that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-all has antiviral activity to these three kinds of Strain.Effect such as have antitussive, relieving asthma.Has the prospect that is developed to anti-influenza virus medicament.
5, that influenza virus is copied suppression ratio is as follows for the variable concentrations 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-:
Description of drawings
Fig. 1 is a kind of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-chemical structural formula;
Fig. 2 is that the cytotoxicity of a kind of (embodiment 1) 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-detects sketch map.
After the variable concentrations medicine joins and cultivates 48h in the mdck cell, adopt the figure as a result that influences of tetramethyl azo azoles salt (MTT) method detection of drugs cell growth;
Fig. 3 is a kind of neuraminidase sketch map alive that detects in the culture fluid supernatant.
Embodiment 1 is with 100TCID
50Behind the viral infection mdck cell, give variable concentrations medicine (1.56～50 μ M) cultured cell 48h, the neuraminidase that detects in the culture fluid supernatant is lived.
Fig. 4 is a kind of virus titer result schematic diagram.Be followed successively by normal cell from top to bottom, untreated cell behind the viral infection is given 50 μ M ribavirin cultured cells behind the viral infection, give 50 μ M Ao Sitawei cultured cells behind the viral infection, gives 50 μ M 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-cultured cells behind the viral infection.
After embodiment 1 will adopt the hemagglutinative titer inhibition test to measure viral infection, the result that tires of the viral hemoagglutination in the supernatant behind the 50 μ M medicine cultured cell 24h.
Fig. 5 is a kind of detection viral protein expression result schematic diagram (last figure is that the nucleus of dyestuff DAPI dyes among A, B, the C figure, and figure below is to virus antigen NP dyeing).Wherein an A figure left side is normal cell, and right figure is no drug treating cell behind the viral infection; B figure gives ribavirin to cultivate the 24h testing result behind the viral infection, administration concentration is followed successively by 50,25,12.5,6.25,3.125 μ M from left to right; C figure gives 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to cultivate testing result behind the 24h behind the viral infection, administration concentration is followed successively by 50,25,12.5,6.25,3.125 μ M from left to right.
Behind 1 pair of virus infected cell of embodiment, give to detect the viral protein expression result by indirect immunofluorescence behind the variable concentrations medicine cultured cell 24h.
Fig. 6 copies the result schematic diagram that influences of carrying capacity to viral nucleic acid for a kind of detection of drugs.
Embodiment 1 behind virus infected cell, give 50 μ M medicine cultured cell 24h after, viral nucleic acid is copied the result that influences of carrying capacity by Semiquantitative reverse transcription PCR method detection of drugs.Be detection virus nucleoprotein (NP) fragment on the figure, figure is confidential reference items actin fragment down, be respectively the normal cell contrast from left to right, not dosing processing cell contrast behind the viral infection, Ao Sitawei cultured cell contrast behind the viral infection, ribavirin cultured cell contrast behind the viral infection, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-cultured cell behind the viral infection.
Fig. 7 is that a kind of detection of drugs is to the result schematic diagram that influences of viral single-wheel replicative cycle different phase.(A figure gives 50 μ M 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-s at the viral infection different time to handle cell 2h, detects virus antigen behind the 6h and expresses; B figure is at the viral infection different time, gives 50 μ M Ao Sitawei and handles cell 2h, detects virus antigen behind the 6h and expresses; C figure is different time behind the viral infection, gives 50 μ M ribavirins and handles cell 2h, detects virus antigen behind the 6h and expresses.)
Embodiment 2 is respectively at viral infection-2～0h, and 0～2h, 2～4h and 4～6h give 50 μ M medicines and cultivate, and the 6h fixed cell is gone forward side by side and connect immunofluorescence detection virus N P albumen in the ranks after infection.
Fig. 8 is that a kind of (embodiment 3) detect 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-anti-influenza virus activity broad spectrum activity detection sketch map.(A figure is amantadine persister A/WSN
Behind/33/S31N the infection cell, give variable concentrations 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-cultured cell, 48h detects neuraminidase slip-knot fruit in the supernatant; B figure gives variable concentrations 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-cultured cell after being H3N2 subtype virus strain A/human/Hubei/3/2005 infection cell, and 48h detects neuraminidase slip-knot fruit in the supernatant; After C figure is Type B strains of influenza viruses infection cell, give variable concentrations 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-cultured cell, 48h detects neuraminidase slip-knot fruit in the supernatant.)
Detected 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-respectively the antiviral activity of A type influenza virus amantadine persister, the H3N2 hypotype strain of A type influenza virus and Type B strains of influenza viruses has been influenced figure as a result.
The specific embodiment
In order to understand content of the present invention better, below in conjunction with specific implementation method content of the present invention is described further, but protection content of the present invention is not limited to following examples.
At present, the anti-influenza virus medicament in-vitro screening model is divided into the cell free system screening model of cell culture model and viral enzyme.The enzyme reaction screening model has high-throughout characteristics, but the chemical compound of screening still need carry out more cytology, histology and toxicity in vivo, effect experiment etc. to determine its effect.Cell culture model is the most frequently used screening model, its advantage is: can provide the identical cell of a large amount of hereditary characters to be object of study, and easy to operate, can eliminate the influence of other extraneous factor, and valid density and therapeutic index that can detection of drugs, for more how later stage mechanism research provide the basis.The present invention adopts the cell culture screening method to detect 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to the influenza infection mdck cell, to the different detection of antigens of virus, and quantitative analysis 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-anti-influenza virus activity.
Embodiment 1:
The application of a kind of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment or prevention first type or Influenza B virus medicine the steps include:
1. experiment material and method
1.1 cell, virus and medicine
Mdck cell is available from ATCC, and virus of A/PR8/34H1N1 cultivates amplification by Embryo Gallus domesticus and obtains, and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is purchased in Sichuan Wei Keqi bio tech ltd, and ribavirin is purchased in sky, Hubei medicine Pharmaceutical limited company;
1.2 experimental apparatus
Multi-tester PerkinElmer, inverted microscope Costar
2. experimental technique:
2.1 cell culture:
37 ℃, cultivate in 5% (volume ratio, below identical) CO2 humidification incubator.(volume ratio, below the identical) penicillin of FBS, 100U/mL and DMEM culture medium of streptomycin that use contains 10%.Go down to posterity the ratio that goes down to posterity 1/3-1/4 behind cell to 90% degree of converging.
2.2 Virus culture:
Get 9～11 age in days SPF Embryo Gallus domesticus, use the Egg checker inspection before the virus inoculation, and at the position mark away from the embryo, 0.2ml/ piece of inoculation 2 pressed in sterilization and punching back
4The viral liquid of hemagglutinative titer seals with nial polish, puts 37 ℃ of calorstats and hatches 48h (it is deadly that dead Embryo Gallus domesticus is generally misoperation behind the inoculation 24h, abandons it).Take out Embryo Gallus domesticus and put 4 ℃ of cold embryo 12h.75% alcohol disinfecting chick embryo air sac, the sterile working cuts off the air chamber shell, capillary pipette is drawn chick embryo allantoic liquid and amniotic fluid, 4 ℃ of centrifugal 30min of 3000rpm detect hemagglutinative titer, 200 μ l/ pipe packing and put-70 ℃ frozen standby.
(1) drug cell toxicity detects:
1.MDCK cell is inoculated in the 96 porocyte culture plates by 5000 cells/well (100 μ l), and is standby behind the cell attachment;
2. be initial concentration continuous 2 times gradient dilutions 6 gradients with medicine with 250 μ M with cell maintenance medium (DMEM+2% serum), 2 multiple holes of every gradient.In every hole, add 5mg/ml MTT 20 μ l after cultivating 48h, put and continue in the cell culture incubator to cultivate;
3. after cultivating 4h, abandon the culture fluid supernatant, every hole adds 100 μ l/ holes, three lysates, and (lysate is by SDS 10g, isobutanol 5ml, 10M HCl 0.1ml is made into 100ml with the distilled water dissolving), above or the back enzyme connection detector detection 570nm wavelength place light absorption value that spends the night of dissolving 4h in 37 ℃ of incubators, tuning wavelength is 630nm, and calculates each drug level cell survival rate, the results are shown in Figure 2.
The result shows that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-does not have obvious cytotoxicity to mdck cell in 1.56～250 μ M scopes, illustrates that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has the scope of application of comparison safety, and namely the dosage of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is 1.56～50 μ M according to the cell experiment consumption.
(2) activity of the anti-A/PR8/34H1N1 Strain of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-detects:
A. detect the effect of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-resisiting influenza virus based on the neuraminic acid enzymatic activity:
1. (4-methylumbelliferyl-α-N-acetyl-neuraminate) is the specific substrate of influenza neuraminidase to experimental principle: MUNANA, at the catalysate that produces under the neuraminidase effect under the 355nm excitation light irradiation, can produce 460nm fluorescence, the variation of fluorescence intensity, can sensitive reaction neuraminic acid enzymatic activity, thus but viral load in the reacting cells supernatant.
2. mdck cell is pressed 2*10
4Cells/well is inoculated in the 96 porocyte culture plates, cultivate 14～18h in 37 ℃ of cell culture incubators after, it is standby to treat that cell grows up to behind the monolayer.Culture medium in the orifice plate is discarded, PBS liquid is washed twice, add 100TCID50/ hole virus liquid inductance transfect cell, add simultaneously each Concentraton gradient medicine totally 200 μ l keep culture fluid (DMEM+0.3%BSA+2.5ug/L pancreatin) and in 37 ℃ of cell culture incubators, cultivate and get each experimental port supernatant behind the 48h and carry out the detection of neuraminic acid enzymatic activity.Experiment arranges blank group, positive controls (ribavirin), negative control group (not having drug treating behind the viral infection) and experiment medicine group.
3. (32.5mmol/L MES, pH 6.5,4mmol/L CaCl to add 40 μ l buffer in the black 96 hole micro plates
2) preparation substrate 20umol/L MUNANA, add each experimental port culture supernatant 20 μ l again, 37 ℃ of lucifuges are hatched 30min, add reaction terminating liquid (0.014 μ M NaOH, 83% ethanol) 100 μ l/ holes, measure fluorescent value (excitation wavelength 355nm, wavelength of transmitted light 465nm) on the multi-tester.
4. calculate and detect respectively that NA is suppressed rate in the hole.Suppression ratio (%)=100-(sample well-blank)/(enzyme contrast-blank alive) * 100% result shows: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has obviously suppressed influenza virus to be copied, and is the dose-dependence (see figure 3)
B. detect the effect of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-resisiting influenza virus based on the hemagglutinative titer inhibition test
1. mdck cell is pressed 1*10
6/ hole is inoculated in the 6 porocyte culture plates, treats behind the cell attachment standby.Culture medium in the orifice plate is discarded, PBS washing 2 times, the viral liquid inductance transfect cell of serum-free medium dilution adds 50 each drug treating of μ M simultaneously, puts 37 ℃, 5%CO
2Cultivate in the incubator and collect that supernatant carries out the blood clotting experiment in each experimental port behind the 24h.Experiment arranges blank hole, negative control hole, ribavirin control wells, Ao Sitawei control wells and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-medicine hole.
2. hemagglutination test operation: 25 μ l normal saline add each hole of V-type blood-coagulation-board, 96 hole; Each supernatant 25 μ l to be checked adds first hole of each row, and doubling dilution to the 12 holes discard 25 μ l then; Every hole adds 1% chicken erythrocyte suspension, 25 μ l, flicks brassboard, and erythrocyte is fully mixed with supernatant to be checked, and room temperature leaves standstill 30min, observes the blood clotting phenomenon, and Fig. 3-4 hemagglutinative titer detects.Be respectively from top to bottom: normal cell contrast, virus control, ribavirin contrast, Ao Sitawei contrast, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-(see figure 4).
3. the result is shown to 50 μ M 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-s and handles virus infected cell, and the hemagglutinative titer of virus is by 2
6Drop to 2
3, its suppression ratio is 87.5%.
C. indirect immunofluorescence detects 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to the effect of viral protein expression
1. cell is pressed 5*10
4Be inoculated in the 24 porocyte culture plates, treat that cell attachment is monolayer after, infected virus 1 hour, after the PBS washing, give to keep liquid and dilute various concentration medicines, behind the cultivation 24h, carry out the expression that the indirect immunofluorescence detection of drugs is handled the back virus protein according to the following step.Experiment arranges blank group, positive controls (ribavirin), negative control group (not having drug treating behind the viral infection) and experiment medicine group.
2. abandon the culture fluid supernatant, with fixative with cell at the fixing 15-20min of room temperature; Wash 3 times each 5min with PBS; With confining liquid room temperature sealing 1h; Abandon confining liquid, add primary antibodie (Mus source NP monoclonal antibody) the incubated at room 1h in conjunction with the liquid dilution; Wash 3 times each 5min with PBS; Adding is in conjunction with fluorescently-labeled two anti-and DAPI dye liquor incubated at room 1h of liquid dilution; Wash 3 times each 5min with PBS; Under inverted fluorescence microscope, observe.
Test used solution: fixative: 4% (mass volume ratio) paraformaldehyde is dissolved in PBS (pH7.4); In conjunction with liquid: 3% (mass volume ratio) BSA, 0.3% (mass volume ratio) TritonX-100 is dissolved in PBS (pH7.4);
Confining liquid: contain 10%FCS in conjunction with liquid.
3. experimental result is seen Fig. 5, and the result shows that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-dosage has suppressed the expression of virus protein with relying on.Through software analysis, its EC
50Be 20 μ l.
As can be seen from Figure, the negative cells of uninfecting virus does not have the specificity fluorescent signal; Do not add the about 100% generation fluorescence of cell in any drug treating group behind the viral infection; After giving the processing of ribavirin and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-behind the viral infection, fluorescence signal obviously lacks than untreated fish group, and is dose-dependence, illustrates that 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has suppressed the expression of virus protein.
D. sxemiquantitative RT-PCR analyzing and testing 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is to the effect of viral gene expression
1. mdck cell is pressed 1*10
6/ hole is inoculated in the 6 porocyte culture plates, treats behind the cell attachment standby.Culture medium in the orifice plate is discarded, PBS washing 2 times, the viral liquid inductance transfect cell of serum-free medium dilution adds 50 each drug treating of μ M simultaneously, puts 37 ℃, 5%CO
2Cultivate in the incubator and collect in each experimental port cell behind the 24h and carry out RNA and extract.Experiment arranges blank hole, virus control hole, ribavirin control wells, Ao Sitawei control wells and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-medicine hole.
2. design of primers
NP：
5’-TGCTGGATTCTCGTTCGGTC-3’；
5’-CCTTTATGACAAAGAAGAAATAAGGCG-3’
Canine?β-actin：
5’-GGCATCCTGACCCTGAAGTA-3’
5’-GGGGTGTTGAAAGTCTCGAA-3’
3. experimental implementation
Cell total rna extracts, and carries out according to Trizol reagent description.The total RNA that extracts carries out concentration determination with ultraviolet spectrophotometer.
(1) reverse transcription reaction
The RNA template adds random primer random primer (Promega) and ddH20, cumulative volume control is at 13 μ l, earlier at 70 ℃ of thermal denaturation 5min, put into ice bath rapidly, and then add dNTP, 5 * Reaction Buffer, reverse transcriptase and RNasin successively in 37 ℃ of reaction 60min, system is as follows: RNA template 1 μ g, Primer 1 μ l, 5 * Buffer, 5 μ l, dNTP (2.5mM/each) 6 μ l, RPI 1 μ l, M-MLV RT 1 μ l, ddH2O 10 μ l, last 70 ℃, 15min deactivation reverse transcriptase.
(2) PCR reaction
Preparation PCR mix, reaction system is: 10xPCR Buffer 5ul, r Taq 0.5ul, dNTP 10mM 0.5ul, Forward Primer10pM 1ul, Reverse Primer 10pM 1ul, cDNA template 3ul, ddH
2O 39ul.
(3) the PCR sample cell is placed on the PCR instrument, begins following program:
Agarose gel electrophoresis 20min test experience result.
4. experimental result: (swimming lane 1 is normal cell, and 2 is virus control, and 3 is the Ao Sitawei contrast from Fig. 6,4 are the ribavirin contrast, and 5 are 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-) can find out, give the medicine ribavirin behind the viral infection, the big 50uM treatment of Ao Sita can suppress the expression of viral gene fully; Give still can detect viral gene expression after same concentration 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is cultivated, but gene expression obviously reducing, is 77.1% (software I mageJ) through the software analysis suppression ratio.
Embodiment 2: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is to the inhibitory action of virus replication cycle different phase
1. cell is pressed 5*10
4Be inoculated in the 24 porocyte culture plates, treat that cell attachment is standby behind the monolayer.The about 6h of influenza virus particles biocycle is so we are respectively at viral infection-2～0h, 0～2h, 2～4h, 4～6h give the different pharmaceutical of same concentrations and cultivate 6h fixed cell after infection, carry out indirect immunofluorescence and detect virus N P protein expression, the results are shown in Figure 7.
2. experimental result shows: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-administration in-2～4h can suppress viral infection, but in that to infect-2～2h better than 2～4h administering effect, infers mainly to suppress virus absorption and the early stage event of viral infection such as enter.Ribavirin mainly suppresses the inosine dehydrogenase, blocking-up inosine-phosphoric acid is to the effects such as conversion of xanthosine-phosphoric acid, and it is synthetic to suppress nucleic acid, stops virus replication, this mainly occurs in the virus replication early metaphase stage, and this is consistent with our observed phenomenon; Ao Sitawei has suppressed the NP protein expression fully in 0～2h administration, and this may be to enter with the absorption of virus to need the effect of viral neuraminidase relevant.
Other step is identical with embodiment 1.
Embodiment 3: the research of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-anti-influenza virus activity broad spectrum activity
This experiment detects 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-to the antiviral activity of subtypes of influenza A virus H3N2 strain, influenza A virus amantadine persister and Influenza B virus strain.
1. experiment uses Strain to comprise: A/human/Hubei/3/2005 (H3N2), A/WS/33/S31N (H1N1), Influenza B virus
2. mdck cell is pressed 2*10
4Cells/well is inoculated in the 96 porocyte culture plates, cultivate 14～18h in 37 ℃ of cell culture incubators after, it is standby to treat that cell grows up to behind the monolayer.Culture medium in the orifice plate is discarded, PBS liquid is washed twice, add 100TCID50/ hole virus liquid inductance transfect cell, add simultaneously each Concentraton gradient medicine totally 200 μ l keep culture fluid (DMEM+0.3%BSA+2.5ug/L pancreatin) and in 37 ℃ of cell culture incubators, cultivate and get each experimental port supernatant behind the 48h and carry out the detection of neuraminic acid enzymatic activity.Experiment arranges blank group, positive controls (ribavirin), negative control group (not having drug treating behind the viral infection) and experiment medicine group.
3. (32.5mmol/L MES, pH 6.5,4mmol/L CaCl to add 40 μ l buffer in the black 96 hole micro plates
2) preparation substrate 20umol/L MUNANA, add each experimental port culture supernatant 20 μ l again, 37 ℃ of lucifuges are hatched 30min, add reaction terminating liquid (0.014 μ M NaOH, 83% ethanol) 100 μ l/ holes, measure fluorescent value (excitation wavelength 355nm, wavelength of transmitted light 465nm) on the multi-tester.
4. calculate and detect respectively that NA is suppressed rate in the hole.Suppression ratio (%)=100-(sample well-blank/(enzyme contrast-blank alive) * 100%
5. the result shows: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-has obviously suppressed other strains of influenza viruses to be copied, and is the dose-dependence (see figure 8).
Other step is identical with embodiment 1.
Claims (2)
1. a 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-is as the application of unique active component in preparation treatment or prevention influenza A virus medicament.
2. the application of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment or prevention Influenza B virus medicine.
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